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1st International

Immunonutritio
n Workshop,
Valencia, 3–5
October 2007,
Valencia, Spain

Polyphenols and immunity

F. Sánchez de Medina and A. Zarzuelo

Immunomodulation and antioxidant capacity of

hydroxytyrosol present at oleic acid-rich sunflower oil

L. E. Díaz, S. Gómez, E. Nova, J. Romeo and A. Marcos

Arginine and the immune system

L. Tomás-Cobos, R. Miñambres, A. Rodrigo, M.

Navarro and D. Tomás

Effects of synbiotics on intestinal and immune function

E. Nova, B. Viadel, M. Blasco and A. Marcos

Exercise and the immune system

E. Ortega

Nutrition and immunity in the elderly

M. de la Fuente

The effects of dietary fish oil on chemokine secretion by

murine peritoneal and spleen cells

H. H. Arnardottir, D. H. Petursdottir and I.

Hardardottir
Immunomodulatory and anti-inflammatory potential of a

malleable protein matrix composed of concentrated

fermented whey proteins

J. Beaulieu, E. Trottier, R. Dubuc, C. Dupont and P.

Lemieux

Nutrition and inflammatory processes

P. C. Calder, R. Albers, J.-M. Antoine, S. Blum, R.

Bourdet-Sicard, G. A. Ferns, G. Folkerts, P. S.

Friedmann, G. S. Frost, F. Guarner, M. Løvik, S.

Macfarlane, P. D. Meyer, L. M'Rabet, M. Serafini, W.

van Eden, J. van Loo, W. vas Dias, S. Vidry, B. M.

Winklhofer-Roob and J. Zhao

Malnutrition, stress and immunodepression: their

interrelationship with fungal infections

I. Cesaroni, F. Negro and M. Guerrini

Impaired immune function in an animal model for cancer

cachexia

J. Faber, D. Kegler, P. Vos, A. van Helvoort and J.

Garssen

Impact of chia ( Salvia hispanica L.( on the immune

system: preliminary study

I. Fernandez, S. M. Vidueiros, R. Ayerza, W. Coates

and A. Pallaro

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In vitro and in vivo immunomodulating effects of

traditionally-prepared extract and purified compounds

from Cetraria islandica

J. Freysdottir, S. Omarsdottir, K. Ingolfsdottir, A.

Vikingsson and E. S. Olafsdottir


Oral lactoferrin and glycine display in vivo synergistic

anti-inflammatory activity

A. Hartog and J. Garssen

Interactions between dendritic cells, probiotic bacteria and

the adipokine leptin

S. C. Knight, N. R. English and A. I. F. Blakemore

A new n-3 PUFA-enriched formula for enteral nutrition

modulates oxidative LDL and inflammatory cytokines

J. Olza, R. Moreno, C. M. Aguilera, A. Pérez de la Cruz,

A. Gil and M. D. Mesa

Conjugated linoleic acid feeding during rat suckling period

enhances intestinal IgA production

F. J. Pérez-Cano, M. Molero, C. Ramírez-Santana, M.

Castell, C. Castellote and A. Franch

Detection of biological activities promoted by proteins from

supernatant fractions of Lactobacillus plantarum in a

promyelocytic cell line

E. Puertollano, M. A. Puertollano, L. Cruz-Chamorro,

G. Alvarez de Cienfuegos and M. A. de Pablo

Oral health and nutritional biochemical variables in adult

patients with AIDS

N. Slobodianik, P. Perris, A. Squassi, G. Sánchez,

M. S. Feliu and N. Bordoni

Gut microbes and obesity in adolescents

M. Sotos, I. Nadal, A. Marti, A. Martínez, M.


Martin-Matillas, C. Campoy, M. A. Puertollano, J.

Wärnberg, A. Marcos and Y. Sanz

PUFA in the pathogenesis and treatment of patients with

multiple sclerosis

L. S. Harbige, E. Pinto, M. Xiang, M. Leach and M. K.

Sharief

Potentiation of systemic humoral immune response in

suckling rats by conjugated linoleic acid (CLA)

C. Ramírez-Santana, F. J. Pérez-Cano, M. Castell, C.

Castellote and A. Franch

Effects of a diet with polyphenol-rich cereal

supplementation on the function and redox state of

peritoneal leucocytes from mice: differences between a

short (5 weeks) and long (20 weeks) period of

supplementation

P. Álvarez, C. Alvarado, L. Jimenez and M. de la

Fuente

Structure and antigenicity changes in 7S soyabean allergen

by enzymic deglycosylation

M. Amigo-Benavent, M. Villamiel and M. D. del

Castillo

Effects of IL-6 on amylase secretion and calcium signalling

in pancreatic AR42J cells: modulation by membrane fatty

acid composition

N. Audi, M. A. Martínez, M. D. Mesa, E.

Martínez-Victoria, M. Mañas and M. D. Yago

Effect of oil (sunflower oil) consumption with added


hydroxytyrosol (natural antioxidant) on antioxidant

variables in leucocytes from healthy adults

I. Baeza, N. M. de Castro, L. E. Díaz, A. Marcos and M.

de la Fuente

Expression of Toll-like receptors on peritoneal

macrophages and dendritic cells from old mice treated with

soyabean isoflavones and green tea

I. Baeza, N. M. de Castro, L. Arranz and M. de la

Fuente

The effects of organic v. conventional diets on immune

variables in rats

A. M. Baranska, E. Rembialkowska, E. Hallmann, L.

Lueck, J. M. Cooper and C. Leifert

Inflammatory mediators in overweight adolescents:

association with insulin sensitivity, body composition and

metabolic syndrome

R. Burrows, L. Leiva, A. M. Tong, J. Aldunate and E.

Díaz

Immunological response in coeliac disease is age related

M. Calzado, E. Donat, B. Polo, B. Baena and C. Ribes

Hormonal and immune changes with age: effect of energy

restriction

P. Cano, G. Segovia, M. P. Fernandez-Mateos, A. del

Arco, V. Jimenez, F. Mora and A. I. Esquifino

Use of inducible class II and MHC class I-related chain A/B

expression on T lymphocytes as an in vitro screen for the


immunomodulatory properties of dietary products

C. Carter, K. Varley and B. Clark

Dietary intake of nutrients of great interest in

immunonutrition to prevent muscle damage in soccer

players

C. Conejos, A. Giner, J. Mañes and J. M. Soriano

Total antioxidant capacity of refrigerated orange juice

treated with pulsed electric fields

C. Cortés, F. Barba, M. J. Esteve, R. González and A.

Frígola

Examination of host immune resistance against Listeria

monocytogenes infection in cyclophosphamide

(CPA)-treated mice after dietary lipid administration

L. Cruz-Chamorro, M. A. Puertollano, E. Puertollano,

C. Carazo, G. Alvarez de Cienfuegos and M. A. de

Pablo

Effect of dietary oils on oxidative stress and cytokine

production by murine macrophages

R. de la Puerta Vázquez, M. A. Fernández Arche, A.

Márquez Martín and V. Ruiz-Gutierrez

Seasonal changes in the neuroimmunoendocrine system

activity in young adult male rats administered with a

balanced controlled diet from weaning

A. I. Esquifino, M. P. Fernandez-Mateos, V. Jimenez,

J. Rios and P. Cano

Effect of long-chain fatty alcohols from orujo olive oil on


nitric oxide and eicosanoid generation

M. A. Fernández Arche, R. de la Puerta Vázquez, A.

Márquez Martín and V. Ruiz-Gutierrez

Fermented soyabean products as hypoallergenic food

J. Frias, Y. S. Song, C. Martínez-Villaluenga, E.

Gonzalez de Mejia and C. Vidal-Valverde

Immunomodulatory properties of Lactobacillus salivarius

are not limited to the intestine

J. Galvez, B. Arribas, M. E. Rodríguez-Cabezas, E.

Bailon, M. Comalada, D. Camuesco, J. Xaus and A.

Zarzuelo

Vitamin E as an IgE inhibitor: stability during cold storage

of human milk

G. García-Llatas, R. Lacomba, F. Ortega, A. Alegría, R.

Barberá and D. Silvestre

Melatonin supplementation modifies experimental chronic

colitis in mice

M. Golab, M. Dudziak and K. Skwarlo-Sonta

Effect of dietary supplementation on lymphocytes subsets

and liver-related variables in hospitalised patients with

anorexia nervosa

S. Gómez, L. E. Díaz, E. Nova, G. Morandé and A.

Marcos

Borage ( Borago officinalis) oil supplementation in relation

to monocyte chemoattractant protein 1 expression in

healthy subjects
M. Xiang, E. Pinto, M. A. Rahman, M. Leach and L. S.

Harbige

Vitamin D deficiency among older adults in England

remains a cause for concern!

V. Hirani, A. Ali and K. Tull

Evaluation of diet, anthropometry and immunocompetence

of young male athletes

D. Lacerda, V. Cotta-de-Almeida, A. Montero, W.

Savino and A. Marcos

Effect of inulin and probiotic bacteria on iron availability in

beans ( Phaseolus vulgaris L.)

J. M. Laparra, R. P. Glahn and D. Miller

Effect of different doses of creatine supplementation on

endogenous creatine synthesis

B. Li, M. Xiang, L. S. Harbige, X. Li and H. Ai

Effect of cessation of creatine supplementation on

endogenous creatine synthesis

X. Li, M. Xiang, L. S. Harbige and H. Ai

Activation of innate immunity by a probiotic bacterium and

a fermented milk containing this micro-organism

C. Maldonado Galdeano, A. de Moreno de Leblanc, C.

Dogi, S. Chaves, E. Carmuega, R. Weill and G.

Perdigón

Milk-derived supplement inhibits in vitro lymphocyte

proliferation and IL-2 production


S. Marín-Gallén, F. J. Pérez-Cano, M. Castell, C.

Castellote and A. Franch

Influence of fruit intake and antioxidants on the prevention

of opacities of the lens

M. I. Martínez, J. Martínez-Raga, J. Raga, A. Alegre,

M. C. Gimeno, E. Llusar and I. Alfonso-Sanchez

Alcohol consumption in secondary-school students: effects

on plasma total antioxidant capacity

M. I. Martínez, J. Martínez-Raga, A. Alegre, J. A.

Dominguez and I. Alfonso-Sanchez

Synthesis of galactooligosaccharides with prebiotic

potential during hydrolysis of lactose by Lactozym 3000 L

HP G

C. Martínez-Villaluenga, A. Cardelle-Cobas, N. Corzo,

A. Olano and M. Villamiel

Raffinose family oligosaccharides of lupin ( Lupinus albus L.

cv multolupa) as a potential prebiotic

C. Martínez-Villaluenga, J. Chicholoska, A. Kliber and

K. Gulewicz

Antioxidant and anti-inflammatory potency of different

wheat varieties and fractions

N. Mateo Anson, R. v. d. Berg, R. Havenaar, G.

Haenen and A. Bast

Elicitation of an allergic reaction in mice orally sensitized to

whey or casein proteins

A. J. Nauta, B. Schouten, B. C. A. M. van Esch, S. van


Doorn, G. A. Hofman, L. W. J. van den Elsen, L. E. M.

Willemsen, L. M. J. Knippels and J. Garssen

Effect of enteral nutrition on plasma soluble adhesion

molecules in an elderly population

J. Olza, M. D. Mesa, R. Moreno, A. Pérez de la Cruz, A.

Gil and C. M. Aguilera

Serum C3c and C4c concentrations and adenosine

deaminase activity of children with cystic fibrosis:

preliminary study

P. Perris, M. S. Feliu, S. Barbeito, I. Strasnoy, M.

Ferraro and N. Slobodianik

Introduction of complementary foods to the infant diet

within the first year of life: evaluation of general

recommendations using Achievable Benchmarks of Care®

N. Pastor, B. Soler, C. Lifschitz and and The

Generación Study Group

Plasma protein supplements modulate the activation of

gut-associated immune system induced by Staphylococcus

aureus enterotoxin B in rats

A. Pérez-Bosque, L. L. Miró, J. Polo, L. Russell, J.

Campbell, E. Weaver, J. Crenshaw and M. Moretó

Dietary fish oil increases IL-4 secretion by murine

splenocytes by an effect on accessory cells

D. H. Petursdottir and I. Hardardottir

Effects of borage ( Borago officinalis) oil supplementation

on the expression of monocyte and T-cell adhesion


molecules in healthy volunteers

E. Pinto and L. S. Harbige

Effect on lymphoproliferation and in vitro Ig production of

splenocytes from suckling rats when supplemented with

conjugated linoleic acid

C. Ramírez-Santana, F. J. Pérez-Cano, M. Castell, A.

Franch and C. Castellote

Anti-inflammatory effects of cocoa in rat

carrageenin-induced paw oedema

S. Ramos-Romero, E. Ramiro-Puig, F. J. Pérez-Cano,

C. Castellote, A. Franch and M. Castell

Lactobacillus fermentum exerts a beneficial effect in an

experimental model of rheumatoid arthritis in mice

M. E. Rodríguez-Cabezas, F. Fisac, E. Bailon, M.

Comalada, D. Camuesco, J. Xaus, A. Concha, P.

Talavera, A. Nieto, A. Zarzuelo and J. Galvez

Immunological changes after dehydration resulting from

physical effort in a hot environment

J. Romeo, D. Jimenez-Pavón, M. Cervantes-Borunda,

J. Wärnberg, L. E. Díaz and A. Marcos

Intake of some immunonutrients and ecoimmunonutrients

by young high-level basketball players

G. Santos, M. A. Buil, J. A. Ferrero, J. J. Delgado, J.

Silva and J. M. Soriano

Moderate ingestion of beer reduces inflammatory and

oxidative brain events induced by aluminium in mice


A. Schultz, R. Oliver, M. Bautista, M. J.

Gonzalez-Muñoz, I. Meseguer, A. Peña, M. I.

Sanchez-Reus, J. Benedí and F. J. Sánchez-Muniz

Impact of glycation on duodenal digestibility of

Bowman-Birk inhibitors

J. M. Silván and M. D. del Castillo

Lymphocyte subpopulations in Venezuelan preschool

children of high socio-economic status

L. Solano and D. Llovera

Timing of gluten introduction and quantity and nature of

gluten-containing foods consumed by infants in Valencia,

Spain

B. Soto, A. López and C. Ribes

Selenium deficiency in adults infected with HIV in the era of

highly-active antiretroviral therapy

M. Stambullian, M. S. Feliu, C. M. López, A. E. Piñeiro,

I. Cassetti and N. Slobodianik

Can dietary supplements reduce cell-surface expression of

molecules involved in organ transplantation?

K. Varley, B. Clark and C. Carter

Psychological stress and immune function: establishing

both acute and chronic psychological stress models in

balb/c mice

E. Vázquez, A. Barranco, M. Manzano, M. L. Jiménez

and R. Rueda
Effects of a synbiotic on intestinal and immune functions of

healthy adults

B. Viadel, E. Nova, J. E. Carreres and A. Marcos

Changes in nutritional status and their effects on serum

and thymus zinc levels and serum copper:zinc

S. M. Vidueiros, I. Fernandez, A. E. Piñeiro, N.

Slobodianik and A. Pallaro

Impact of a cereal-based diet on intestinal villi of growing

rats

S. M. Vidueiros, I. Fernandez, N. Slobodianik, M. E.

Roux and A. Pallaro

Breast-milk levels of long-chain PUFA in Kazakhstan and

Sweden

M. Xiang, L. S. Harbige and R. Zetterström

Time-course study of high-dose creatine supplementation

for endogenous creatine synthesis

M. Xiang, L. S. Harbige, X. Li, B. Li and H. Ai

The effect of moderate dose of EPA+DHA on lipid profile

and inflammatory markers in middle-aged men

H. M. Yusof and P. C. Calder

A specific mixture of short-chain galacto-oligosaccharides

and long-chain fructo-oligosaccharides induced an

anti-allergic Ig profile in infants at risk for allergy

A. J. Nauta, S. Arsnalognu, G. Boehm, G. Moro, J.

Faber, E. Knol, B. Ruiter, E. van Hoffen, L. M'Rabet

and J. Garssen
Immune-enhancing role of vitamin C and zinc and effect on

clinical conditions

S. Beveridge, E. S. Wintergerst, S. Maggini and D.

Hornig

Contribution of selected vitamins and trace elements to

immune function

S. Maggini, E. S. Wintergerst, S. Beveridge and D.

Hornig

Differential effect of Bifidobacterium species characteristic

of the gut microbiota of breast-fed and formula-fed infants

on in vitro cytokine production

E. Puertollano, T. Pozo, I. Nadal, A. Marcos, Y. Sanz

and E. Nova
Proceedings of the Nutrition Society (2008), 67 (OCE), E1 doi:10.1017/S0029665108006101

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Polyphenols and immunity

F. Sánchez de Medina and A. Zarzuelo


Department of Pharmacology, School of Pharmacy, University of Granada, Spain

Flavonoids are biologically-active polyphenolic compounds with antioxidative, antineoplasic, cardiovascular protective and anti-
inflammatory properties. Pharmacological therapy is essential in inflammatory bowel disease but has many adverse effects and does
not cure the disease. Flavonoids are excellent candidates because of their anti-inflammatory properties and their low toxicity. Several
flavonoids have been shown to exert intestinal anti-inflammatory activity in vivo, including (mg/kg) quercitrin 1–5(1), rutin 10–25(2), morin
25(3,4), hesperidin and diosmin 10–25(5). However, the mechanism of action is unclear. Since inflammation is associated by significant
oxidative stress, this mechanism may be relevant. Indeed, flavonoid treatment counters colitis-induced glutathione depletion. On the other
hand, quercitrin treatment reduces macrophage infiltration in the dextran sulfate sodium colitis model(6). The effects of flavonoids on
primary macrophages have been studied and their structure–activity relationship characterized(7). A number of flavonoids inhibit macro-
phage proliferation (but not cell viability) and some additionally reduce TNF and inducible NO synthase (iNOS) expression, probably
interfering with the NF-kB pathway. The structural determinants of activity include the C-2— —C-3 double bond, the catechol group in the
B ring and the 2-position of the B ring.
Most of these flavonoids are glycosides, which are known to be hydrolysed by bacterial enzymes in the gut. Since luteolin and quercetin
are not active in vivo and aglycone flavonoids are absorbed in the small intestine it is likely that glycosides act as prodrugs, releasing the
biologically-active aglycone in the lumen and preventing their premature absorption, which has been proven in the case of quercitrin(8). In
particular, a faecal homogenate was shown to mediate quercetin release from quercitrin in vitro, and the resulting aglycone retained TNF,
iNOS and IL-1b inhibitory activity in murine bone marrow-derived macrophages. This principle probably applies to the other heterosides
with known intestinal anti-inflammatory activity.
In conclusion, flavonoids have intestinal anti-inflammatory activity that is associated with macrophage inhibition and antioxidative
effects. Further investigation of the mechanistic aspects of flavonoid pharmacological action is underway.

1. Sanchez de Medina F, Galvez J, Romero JA & Zarzuelo A (1996) J Pharmacol Exp Ther 278, 771–779.
2. Galvez J, Cruz T, Crespo E et al. (1997) Planta Med 63, 409–414.
3. Galvez J, Coelho G, Crespo ME et al. (2001) Aliment Pharmacol Ther 15, 2027–2039.
4. Ocete MA, Galvez J, Crespo ME et al. (1998) Pharmacology 57, 261–270.
5. Crespo ME, Galvez J, Cruz T, Ocete MA & Zarzuelo A (1999) Planta Med 65, 651–653.
6. Camuesco D, Comalada M, Rodriguez-Cabezas ME et al. (2004) Br J Pharmacol 143, 908–918.
7. Comalada M, Ballester I, Bailon E et al. (2006) Biochem Pharmacol 72, 1010–1021.
8. Comalada M, Camuesco D, Sierra S et al. (2005) Eur J Immunol 35, 584–592.
Proceedings of the Nutrition Society (2008), 67 (OCE), E2 doi:10.1017/S0029665108006113

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Immunomodulation and antioxidant capacity of hydroxytyrosol


present at oleic acid-rich sunflower oil

L. E. Dı́az, S. Gómez, E. Nova, J. Romeo and A. Marcos


Instituto del Frı́o, Spanish Council for Scientific Research, Madrid, Spain

Typical components of the Mediterranean diet such as olive oil and red wine contain high concentrations of phenols that may have
important antioxidant and immunomodulation roles. The main phenols identified in extra-virgin olive oil belong to different classes:
simple phenols such as 3,4-dihydroxyphenylethanol and p-hydroxyphenylethanol; secoiridoids e.g. oleuropein, the aglycone of ligstroside
and their respective decarboxylated dialdehyde derivatives(1,2). These phenols have been suggested to prevent oxidative damage and
beneficially modify immune and inflammatory responses(3). The aim of the present study was to evaluate the effect of oil (sunflower oil)
containing added hydroxytyrosol (HT; ‘Oleoactive from Koipesol’; Sos Cuetara SA, Madrid, Spain) consumed at 45–50 mg/d, on the
immune cells and oxidation variables in healthy adults. Thus, twenty-two healthy subjects of both genders (20–45 years) were recruited
for a cross-over design study. The subjects were divided into two groups of eleven and assigned to one of two treatments for a period of
8 weeks: group A, 3 weeks of oil with added HT, 2 weeks of wash-out and 3 weeks of sunflower oil without HT; group B, 3 weeks of oil
without HT, 2 weeks of wash-out and 3 weeks of oil with added HT. Leucocytes were analysed using an automatic blood-cell counter.
T (CD3, CD4, CD8) and B (CD19) lymphocyte subsets and natural killer cells (CD56 + 16) were studied by flow cytometry using
peripheral blood marked with monoclonal antibodies. The oxidative and phagocytic capacities of polymorphonuclear leucocytes were
quantified in vitro after incubating lymphocytes with opsonised Escherichia coli. Finally, serum Ig levels were measured by nephelometry.
All variables were analysed at the beginning of the study and at 3, 5 and 8 weeks. No significant changes in leucocytes, differential cell
counts and lymphocyte subsets were observed in the two groups during the study. Nevertheless, the oxidative capacity showed a tendency
to increase in both groups after consuming oil with added HT. On the other hand, in group A the percentages of lymphocytes, monocytes,
eosinophils and basophils and the Ig levels showed a tendency to increase after the consumption of oil with added HT. However, in group
B leucocyte counts, the percentage of neutrophils and Ig levels showed a tendency to increase (Figure). In conclusion, HT could improve
the immune response, but further studies with increasing levels of intake or periods of consumption of HT are required to establish
whether the effects are significant.

IgG IgM IgA


2000 400

1500 300 300


(m g/d L)
(m g/d L)

(mg/dL)

1000 200 200


500 100 100

0 0 0
A B A B A B
Hydroxytyrosol Whitout hydroxytyrosol Hydroxytyrosol Whitout hydroxytyrosol Hydroxytyrosol Whitout hydroxytyrosol

WBC differential counts WBC differential counts (Neutrophils)


Group A Group B
50
80
40
30 60
(%)

(%)

20 40
10
20
0
Lymphocytes Monocytes Eosinophils Basophils 0
Hydroxytyrosol Whitout hydroxytyrosol
Hydroxytyrosol Whitout hydroxytyrosol

This study has been supported by Sos Cuetara SA.

1. Owen RW, Giacosa A, Hull WE, Haubner R, Wurtele GB Spiegelhalder & Bartsch H (2000) Lancet Oncol 1, 107–112.
2. Bonoli M, Montanucci M, Gallina, Toschi T & Lercker G (2003) J Chromatogr 1011A, 163–172.
3. Carliccio MA, Siculella L, Ancora MA et al. (2003) Arteroscl Tromb Vasc Biol 23, 622–629.
Proceedings of the Nutrition Society (2008), 67 (OCE), E3 doi:10.1017/S0029665108006125

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Arginine and the immune system

L. Tomás-Cobos, R. Miñambres, A. Rodrigo, M. Navarro and D. Tomás


Ainia Technological Centre, Parque Tecnológico de Valencia, c Benjamı́n Franklin 5-11, E 46980 Paterna, Valencia, Spain

L-Arginine (arg) has been classified as a semi-essential amino acid. In addition to participating in protein synthesis, L-arg has been shown
to be a powerful mediator of multiple biological processes, including the release of several hormones, collagen synthesis during wound
healing, antitumour activity and immune cell responses. L-Arg is metabolized in macrophages, endothelial cells, hepatocytes, kidneys cells
and certain tumour cells by three enzymic pathways: inducible NO synthase (iNOS); arginase I; arginase II. In macrophages L-arg is
metabolized by iNOS to produce citrulline and niticoxide, which is one of the principal cytotoxic mechanisms in these cells(1). The
availability of L-arg to modulate the immune system has lead to the this amino acid being considered to be an immunonutrient(2).
The almond (Prunus amygdalus) is a nut with a high energy and nutritional value. The main components are vitamin E, unsaturated
fatty acids, fibre and a high proportion of arg-rich protein(3). The aim of the present study was to investigate the effects of almond, as an
arg-rich food, on the immune system. Thus, NO production and genes encoding pro-inflammatory mediators such as cyclooxygenase-2
(COX-2), iNOS and TNFa were evaluated in a macrophage cell line of Raw 264.7 cells to test the effect of almond foods in the form of
almonds or a commercial almond cream (an almond-based product containing (g/kg): fat 177, protein 70, carbohydrate 360, water 370).
Based on the bioavailability of almonds in vivo, the almond foods were subjected to two pre-treatment procedures: (1) enzymic
treatment (trypsin and proteinase K); (2) simulated digestion (gastric digestion with pepsin and intestinal digestion with a pancreatin–
biliary extract). Raw 264.7 cells were treated with the pre-treated or non-pre-treated almond foods. As a positive control cells were
stimulated with lipopolysaccharide (LPS) and L-arg. After 6 h of treatment iNOS, TNFa and COX-2 gene expression were analysed by
RT–PCR (b-actin and 18S genes were used as an internal control) and after 24 h of treatment NO levels were determined using the Griess
reaction(4).
The data indicated that NO production was increased in cells treated with almond foods when compared with untreated cells. NO
production by cells stimulated with almonds was decreased in presence of iNOS inhibitor (1400W)(5). The production of NO requires a
higher concentration of digested samples than enzymic pre-treated samples. iNOS, TNFa and COX-2 gene expression was induced by the
almond product, although higher levels were found in LPS-induced cells (positive control).

iNOS expression by Raw 264.7 cells in presence of


NO production by Raw 64,7 in presence
enzymatic pretreated almond cream
1100
NO µ Molar of digested almond and almond cream

1000 16
900 14
800
12
700
10
600
8
500
6
400

300
4

200 2
100 0
0 Digested Digested Digested Digested LPS LPS+1400W L-arg 1mM L-arg
LPS L-arg Almond cream enzymatic inhibitor protease no treatment almond Almond+ Almond Almond 1mM+1400W
pretreated almond + enzymatic 1400W Cream Cream +
cream pretreated almond
cream 1400W

In conclusion, these preliminary results suggest that almonds can stimulate the activity of the macrophages in Raw 264.7 cells, and so
stimulate the immune system. Thus, almonds could be considered to be an ‘immunomodulator’(6). However, further investigation is
needed to establish the concentration at which almonds are immunomodulatory in vivo.

1. Rodriguez PC, Zea AH, Desalvo J et al. (2003) J Immunol 17, 1232–1239.
2. Grimble RF (2001) Proc Nutr Soc 60, 389–397.
3. Mataix J, Mañas M, Llopis J & Martinez de Victoria E (1998) Tabla de Composición de Alimentos Españoles (Spanish Food Composition Tables),
4th ed. Granada, Spain: Instituto de Nutrición y Tecnologı́a de Alimentos.
4. Kolb JP, Paul-Eugene N, Damais C, Yamaoka K, Drapier JC & Dugas B, (1994) J Biol Chem 269, 9811–9816.
5. Garvey EP, Oplinger JA, Furfine ES et al. (1997) J Biol Chem 272, 4959–4963.
6. Efron DT & Barbul A (2000) Nutrition 16, 73–74.
Proceedings of the Nutrition Society (2008), 67 (OCE), E4 doi:10.1017/S0029665108006137

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effects of synbiotics on intestinal and immune function

E. Nova1, B. Viadel2, M. Blasco2 and A. Marcos1


1
Immunonutrition Group, Metabolism and Nutrition Department, Instituto del Frı́o (CSIC), Madrid, Spain and
2
Department of New Products, AINIA,Technological Center, Valencia, Spain

A synbiotic results from the combination of a probiotic and a prebiotic in a single product that is used as a healthy dietary supplement in
the restoration and maintenance of colonic flora. The prebiotics most commonly used in the EU are fructo-oligosaccharides (FOS), inulin
and galacto-oligosaccharides (GOS). The prebiotic component (non-digestible carbohydrates) selectively increases the survival rate of a
particular probiotic or several probiotic strains during intestinal transit and thus their effect on the gastrointestinal tract. In this sense, FOS
has been reported to increase bile resistance in bifidobacteria. However, except for the prebiotic effect, the evidence to support the
purported effects of synbiotics on health is still scarce in man. There is also the need to establish what differences there are in the observed
effects of the synbiotic product v. the prebiotic alone. New prebiotics, other than FOS, GOS and inulin, are being assayed for novel
applications. b-Glucans, for example, have shown a lactobacillogenic effect(1) and biotechnology is being employed for the production of
human-milk oligosaccharides that might facilitate the development of a healthy microbiota in non-breast-fed infants.
The effects of synbiotic therapies on intestinal function of different types of critically-ill patients have been investigated in a few
studies(2). Different synbiotic preparations have also proved useful in preliminary studies of the management of patients with short bowel
syndrome(3), ulcerative colitis(4,5), acute pancreatitis(6) and allergy(7). A study has been carried out to evaluate the effects of the con-
sumption for 6 weeks of a synbiotic product containing Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus
paracasei ssp. paracasei, Streptococcus thermophilus, Bifidobacterium sp. (24 · 108 colony-forming units/d) and FOS on the intestinal
microbiota and self-reported intestinal function, as well as on immune function of generally healthy adults. Although no differences in
self-reported improvement were found with treatment of mild gastrointestinal symptoms present at baseline such as constipation, flatu-
lence, postprandial bloating or dyspepsia, there was a significant improvement overall in symptoms and in motility in the synbiotic group
compared with the placebo group. Intestinal microbiota did not change as a result of synbiotic consumption, and the only significant effect
of treatment (ANOVA; P=0.05) on immune variables was on the concentration of serum soluble L-selectin, which showed a decrease in
the synbiotic group. This outcome might lead to a more beneficial profile of the inflammatory markers in relation to the prevention of
atherosclerosis and CVD. A trend towards a decrease in CD3 - (CD56 + 16) + cells (natural killer cells) was found in the women in the
synbiotic group. This effect was not observed in men, probably because their values at baseline were at the lower limit of the normal
range. No effect of treatment was found in other lymphocyte subsets or other immune variables such as the phagocytic activity of
monocytes and granulocytes and inflammatory proteins such as C-reactive protein and caeruloplasmin.

1. Snart J, Bibiloni R, Grayson T et al. (2006) Appl Environ Microbiol 72, 1925–1931.
2. Jain PK, McNaught CE, Anderson AD, MacFie J & Mitchell CJ (2004) Clin Nutr 23, 467–475.
3. Kanamori Y, Sugiyama M, Hashizume K, Yuki N, Morotomi M & Tanaka R (2004) J Pediatr Surg 39, 1686–1692.
4. Haskey N & Dahl WJ (2006) Nutr Rev 64, 132–138.
5. Furrie E, Macfarlane S, Kennedy A, Cummings JH, Walsh SV, O’Neil D & Macfarlane GT (2005) Gut 54, 242–249.
6. Olah A, Belagyi T, Poto L, Romics L Jr & Bengmark S (2007) Hepatogastroenterology 54, 590–594.
7. Ogawa T, Hashikawa S, Asai Y, Sakamoto H, Yasuda K & Makimura Y (2006) FEMS Immunol Med Microbiol 46, 400–409.
Proceedings of the Nutrition Society (2008), 67 (OCE), E5 doi:10.1017/S0029665108006149

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Exercise and the immune system

E. Ortega
Department of Physiology, Faculty of Sciences, University of Extremadura, 06071-Badajoz, Spain

The immune system is a system for self-recognition and maintaining homeostasis. It is an extremely complex network that extends
throughout the body, and it can recognize and defend the organism against a theoretical infinity of challenges. The participants in innate
immune mechanisms are macrophages and neutrophils, along with natural killer (NK) cells, complement and defensins, and they con-
stitute the first line of defence. All its constituents need a basic capacity to distinguish between self and foreign, and danger or non-danger
signals with the involvement of Toll-like receptors (TLR). By engulfing, processing and presenting antigens, macrophages form the
critical link to the specific branch of the immune system that mainly comprises the various subpopulations of lymphocytes and their
products. While regular moderate exercise is very likely to be associated with decreased susceptibility to infection, excessive exhaustive
exercise has been associated with symptoms of transient immunosuppression, leading to increased susceptibility to infection. This
outcome is particularly the case for athletes in a competitive setting, who are frequently subjected to physical, psychological and
environmental stress as well as to inadequate nutrition that may cause immunosuppression.
However, recently there has been excessive generalization of the notion that moderate exercise is beneficial and intense exercise is
harmful for the immune system. The latest studies, however, have revealed that this general finding cannot be extended to phagocytosis.
Some stages of the phagocytic process, in particular chemotaxis and phagocytosis, are stimulated by both moderate and intense exercise,
which may counter some of the pronounced suppressive effects of intense exercise on lymphocytes and NK responses.
Exercise-induced changes in the immune system are mediated by the stress hormones, mainly glucocorticoids and catecholamines.
Exercise-induced stress also results in the release of the 72 kDa heat-shock protein (Hsp72), which also has marked effects on immunity.
Stress hormones and proteins may also be considered as ‘stress mediators’ in the exercise-induced stimulation of phagocytes and as
‘danger signals’ for the immune system during intense exercise. Stimulation of chemotaxis and phagocytosis of neutrophils and macro-
phages by glucocorticoids and noradrenaline at physiological exercise-induced concentrations has been reported(1). Post-exercise Hsp72
concentrations also stimulate neutrophil phagocytosis and chemotaxis through TLR-2 together with its cofactor CD14.
Chemotaxis and phagocytosis of neutrophils and macrophages are two important functions in the inflammatory response. However,
while inflammation plays an important role in host defence, uncontrolled inflammatory reactions are responsible for the initiation and
progression of autoimmune and inflammatory diseases. In this context, it has also been found that intense exercise also alters the pro-
inflammatory–anti-inflammatory cytokine balance, which is critical for inflammatory and autoimmune diseases; mainly in women, who
are more susceptible to these types of pathologies. It has been reported that glucocorticoids, noradrenaline and Hsp72 modulate the release
of pro-inflammatory cytokines by phagocytes and other immune cells(2,3). In addition pro-inflammatory cytokines, such as IL-1, IL-6, IL-
8, interferon-g and TNFa can inhibit food intake. IL-1, together with catecholamines and glucocorticoids, may regulate glucose home-
ostasis during an immune response; probably, as recently reported, serving to divert glucose to inflamed tissues to satisfy the high energy
cost of the immune response(4). During exercise muscle-derived IL-6 has been also shown to affect metabolic responses, such as increase
glucose metabolism, lipid oxidation and lipolysis(5).
Thus, it is necessary to bear in mind that the immune system is a homeostatic regulatory system that operates in situations of high
energy costs such as exercise-induced stress, and, moreover, it is involved in disease situations that also need high energy contributions.

This work has been partially supported by 2PR04A076 and DEP2006-56187-C04-03/PREV.

1. Ortega E (2003) Exerc Immunol Rev 9, 70–93.


2. Elenkov I & Chrousos GP (2002) Ann NY Acad Sci 996, 290–303.
3. Asea A, Kabingu E, Stevenson MA & Calderwood SK (2000) Nat Med 6, 435–442.
4. Besedovsk HO & del Rey A (2007) Brain Behav Immun. 21, 34–44.
5. Steenberg A (2003) Exer Immunol Rev 9, 41–47.
Proceedings of the Nutrition Society (2008), 67 (OCE), E6 doi:10.1017/S0029665108006150

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Nutrition and immunity in the elderly

M. de la Fuente
Department of Physiology, Faculty of Biology, Complutense University of Madrid, Madrid, Spain

Ageing is a universal process that may be defined as a progressive, endogenous and irreversible accumulation of adverse changes that
increase vulnerability to disease and finally to death. The aging process is very heterogeneous, showing different rates of physiological
changes in the various systems of the organism and in the diverse members of a population of the same chronological age. This diversity
justifies the introduction of the concept of ‘biological age’, which is very useful in assessing the level of aging experienced by each
individual and therefore his life expectancy. Currently, it is known that immune functions change with age. Since the functional capacity
of the immune system is a marker of health and longevity, several immune functions (Table) have been proposed as markers of biological
age and predictors of longevity. These immune functions have been standardized at different ages in mice and human subjects and show
similar changes with aging in leucocytes from both species. Moreover, a model of premature aging in mice as well as very high longevity
in mice and human populations (very old mice and centenarians) has been used. In the prematurely-aging mice the functional variables
investigated have been shown to have values characteristic of chronologically-older animals, and these mice also show a significantly
decreased lifespan. In very old mice and centenarians these immune functions have values similar to those for adults (6 months and 30
years respectively). The cause of immunosenescence has also been investigated, and since the oxidation theory is now the most widely
accepted explanation of the ageing process, the age-related imbalance between free radical production and antioxidant defences has been
analysed, with a higher production of the former, denominated oxidative stress, in the immune cells from subjects throughout ageing
(Table). Since free radicals are needed for many physiological processes including immune function, prevention of an imbalance is
required to maintain good health. Oxidative stress is responsible for a large number of pathologies, many of which occur more frequently
with ageing. The immune cells from prematurely-ageing mice show oxidative stress whereas leucocytes from very old mice and cen-
tenarians show values of oxidant and antioxidant compounds similar to those in cells from adults. One of the mechanisms involved is the
activation of NF-kB. A diet with adequate amounts of antioxidants neutralizes the oxidative stress of immune cells in ageing and therefore
the function of these cells improves, both in mice and human subjects, showing values close to those in adults (Table)(1–3). Mice
consuming a diet with antioxidants for only 5 weeks increase their lifespan. Thus, good nutrition with compounds rich in antioxidants can
prevent the age-related deterioration of the immune system and help to achieve a healthy longevity.

Aging Antioxidant supplementation


Functions
Chemotaxis, phagocytosis Decrease Increase (=adult)
Lymphoproliferation in response to mitogens Decrease Increase (=adult)
IL-2 release, NK activity Decrease Increase (=adult)
Adherence, TNFa release Increase Decrease (=adult)
Oxidant compounds
Extracellular superoxide anion, PGE2 Increase Decrease (=adult)
Oxidized gluthatione Increase Decrease (=adult)
Antioxidant defences
Reduced gluthatione Decrease Increase (=adult)
Superoxide dismutase and catalase activities Decrease Increase (=adult)
Gluthatione peroxidase and reductase activities Decrease Increase (=adult)

This work has been supported by the MEC (BFU 2005–06777) and RETICEF (RD06/0013/0003) (ISCIII) of Spain.

1. De la Fuente M, Hernanz A & Vallejo MC (2005) Antioxid Redox Signal 7, 1356–1366.


2. Alvarado C, Alvarez P, Puerto M, Gausserés N, Jimenez L & De la Fuente M (2006) Nutrition 22, 767–777.
3. Alvarez P, Alvarado C, Mathieu F, Jimenez L & De la Fuente M (2006) Eur J Nutr 45, 428–438.
Proceedings of the Nutrition Society (2008), 67 (OCE), E7 doi:10.1017/S0029665108006162

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

The effects of dietary fish oil on chemokine secretion by


murine peritoneal and spleen cells

H. H. Arnardottir, D. H. Petursdottir and I. Hardardottir


Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland

Dietary fish oil, rich in n-3 PUFA, affects immune function partly by its effects on cytokine secretion by peritoneal and splenic macro-
phages and T-cells(1,2,3). The effects of dietary fish oil on secretion of chemokines by these cells have been less studied. Macrophage
inflammatory protein 1a (MIP-1a) and monocyte chemotactic protein 1 (MCP-1) are chemokines that act on and are secreted by
monocytes, macrophages and T-cells(4). MIP-1a may promote a T-helper (Th) 1-type immune response(5), whereas MCP-1 is thought to
promote a Th2-type immune response(6). The aim of the present study was to determine the effects of dietary fish oil on MIP-1a and
MCP-1 secretion by murine peritoneal macrophages and splenic macrophages and T-cells ex vivo.
Mice were fed diets supplemented with (g/kg) 180 fish oil + 20 maize oil or 200 maize oil for 6 weeks (n 10). Resident peritoneal
macrophages were stimulated with lipopolysaccharide (LPS) with or without antibodies against TNFa or IL-10. Total spleen cells were
stimulated with LPS or antibodies against CD3 and CD28. Concentration of the chemokines MIP-1a and MCP-1 in the medium was
measured by ELISA.
Dietary fish oil did not affect LPS-induced MIP-1a secretion but decreased LPS-induced MCP-1 secretion by resident peritoneal
macrophages (329 v. 174 pg/ml respectively, P=0.01). The effect of dietary fish oil on MCP-1 secretion was not mediated by an effect on
TNFa or IL-10 production as it was not affected by antibodies against TNFa or IL-10. On the other hand, dietary fish oil increased LPS-
induced MIP-1a secretion by splenic macrophages (see Figure) but had little effect on MCP-1 secretion. Dietary fish oil decreased anti-
CD3/anti-CD28-induced MIP-1a secretion by splenic T-cells (see Figure) but had no effect on MCP-1 secretion.
These results demonstrate that dietary fish oil affects chemokine secretion by peritoneal and splenic macrophages as well as T-cells and
that its effects depend on the cell type and location. These effects of dietary fish oil on chemokine secretion may help explain its effects on
immune function in which chemokines play an important role.

MIP-1α secretion by total splenocytes


stimulated with LPS or anti-CD3/CD28

6000
KO
* FO
5000
4000
pg/ml

3000
2000
*
1000
0
LPS anti-CD3/CD28

1. Calder PC (1997) Ann Nutr Metab 41, 203–234.


2. Petursdottir DH, Olafsdottir I & Hardardottir I (2002) J Nutr 132, 3740–3743.
3. Petursdottir DH & Hardardottir I (2007) J Nutr 137, 665–670.
4. Rollins BJ (1997) Blood 90, 909–928.
5. Schrum S, Probst P, Fleischer B & Zipfel PF (1996) J Immunol 157, 3598–3604.
6. Gu L, Tseng S, Horner RM, Tam C, Loda M & Rollins BJ (2000) Nature 404, 407–411.
Proceedings of the Nutrition Society (2008), 67 (OCE), E8 doi:10.1017/S0029665108006174

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Immunomodulatory and anti-inflammatory potential of a malleable


protein matrix composed of concentrated fermented whey proteins

J. Beaulieu1, E. Trottier2, R. Dubuc1, C. Dupont1 and P. Lemieux1,2


1
Institut National de la Recherche Scientifique-Institut Armand-Frappier (INRS-IAF) 531 Blvd Des Prairies, Building 18,
Laval, Quebec H7V 1B7, Canada and 2Technologie Biolactis 500 Cartier West Suite 218, Laval, Quebec H7V 5B7, Canada

A novel nutraceutical ingredient, the malleable protein matrix (MPM), is obtained from the fermentation of pasteurized whey with a
unique probiotic strain isolated from kefir grains. It is also composed of capsular exopolysaccharides, vitamins, minerals (such as Ca) and
peptides generated during the fermentation process. MPM is a complex biological mixture in which synergistic effects between the
components are expected. Immunocompetent-animal studies have shown that MPM increases the polymorphonuclear cell counts(1), but
also modulates the overall level of glutathione in circulating cells(1)and cytokine production (Fig. 1). These results suggest that MPM
exerts a definitive stimulation of the innate immunity and that consumption of MPM could be either beneficial or detrimental on the
immune system.
In order to assess the resulting effect of MPM, an oxazolone-induced atopic contact dermatitis model (ACD) was used to induce
systemic inflammation. The results showed that MPM did not promote any detrimental side effects. On the contrary, MPM exhibited a
positive and significant anti-inflammatory effect comparable with that of hydrocortisone but without the side effects(2). Using this model a
marked reduction in ear inflammation was demonstrated in MPM-treated mice, and this effect was correlated with inhibition of neutrophil
extravasation in the tissue(2). The in vivo ‘air pouch’ model, which represents the pathological characteristics of arthritis, also demon-
strated a 50 % inhibition of neutrophil infiltration in MPM-treated mice, which was correlated with an important reduction in cytokine and
chemokine production following stimulation (J Beaulieu, D Girard, C Dupont and P Lemieux, unpublished results).

135 Water 140 Water


120 MPM MPM
120
Relative expression (%)

Relative expression (%)

105
100
90
75 80
60 60
45
40
30
20
15
0 0
IL-4 IL-12 IFNg TNFa GM-CSF IL2 IL4 IFNg TNFa GM-CSF

Fig. 1. Systemic cytokine production in healthy mice treated with water or Fig. 2. Systemic cytokine production in the ACD model in water- or MPM-treated mice.
MPM for 2 weeks. IFNg, interferon-g; TNFa, TNFa; GM-CSF, granulocyte- IFNg, interferon-g; TNFa, TNFa; GM-CSF, granulocyte-macrophage colony-stimulating
macrophage colony-stimulating factor. factor.

The evaluation of cytokine production in healthy animals indicates that the stimulation of immunity is not related to activation of
classical T-helper 1 or T-helper 2 pathways (Fig. 1). Furthermore, in the ACD model the cytokine production also indicates that these
pathways are not implicated in the anti-inflammatory effect of the MPM, and IL-2 appears to have a role in the mechanism of action
(Fig. 2). The modulation of cytokine production in animal models suggests that MPM regulates a newly-discovered subpopulation of
lymphocytes that acts specifically on neutrophils, which could explain the dual effects obtained with MPM, i.e. the capacity to stimulate
immunity as well as exhibiting important anti-inflammatory effects. This mechanism of action has not previously been associated with a
nutraceutical product, suggesting that MPM may become an alternative and a functional food of choice for those individuals suffering
from autoimmune diseases, as well as being able to stimulate infectious defence.

1. Beaulieu J, Dubuc R, Beaudet N, Dupont C & Lemieux P. (2007) J Med Food 10, 67–72.
2. Beaulieu J, Dupont C & Lemieux P. (2007) J Inflamm (Lond) 4, 6.
Proceedings of the Nutrition Society (2008), 67 (OCE), E9 doi:10.1017/S0029665108006186

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Nutrition and inflammatory processes

P. C. Calder1, R. Albers2, J.-M. Antoine3, S. Blum4, R. Bourdet-Sicard3, G. A. Ferns5, G. Folkerts6,


P. S. Friedmann1, G. S. Frost5, F. Guarner7, M. Løvik8, S. Macfarlane9, P. D. Meyer10,
L. M’Rabet11, M. Serafini12, W. van Eden13, J. van Loo14, W. vas Dias15, S. Vidry16,
B. M. Winklhofer-Roob17 and J. Zhao18
1
School of Medicine, University of Southampton, Southampton SO16 7PX, UK, 2Unilever Health Institute, 3130 AC,
Vlaardingen, The Netherlands, 3Danone Vitapole, Palaiseau 91767, France, 4Nestlé Research Centre, Lausanne 26,
Switzerland, 5School of Biomedical & Molecular Sciences, University of Surrey, Guildford GU2 7XH, UK, 6Department of
Pharmacology & Pathophysiology, University of Utrecht, 3508 TB, Utrecht, The Netherlands, 7Digestive System Research
Unit, Hospital General Vall d’Hebron, 08035 Barcelona, Spain, 8Division of Environmental Medicine, Norwegian Institute
of Public Health, Nydalen, 0403 Oslo, Norway, 9Division of Pathology and Neuroscience, Dundee University, Dundee DD1
9SY, UK, 10Sensus, 4804 RA, Roosendall, The Netherlands, 11Numico-Research, 6700 CA, Wageningen, The Netherlands,
12
Unit of Human Nutrition, National Institute for Nutrition, 00178 Roma, Italy, 13Faculty of Veterinary Medicine, University
of Utrecht, 5384 CL, Utrecht, The Netherlands, 14Raffinerie Tirlemontoise - Orafti, 3300 Tienen, Belgium, 15Seven Seas Ltd,
Marfleet, Hull HU9 5NJ, UK, 16ILSI Europe, 1200 Brussels, Belgium, 17Institute of Molecular Biosciences, University of
Graz, 8010 Graz, Austria and 18Yakult Europe, 1332 EN, Almere, The Netherlands

The ILSI Europe Task Force on ‘Nutrition and Immunity in Man’ aims to better understand the effects of diet or nutrients on various
aspects of immune function in essentially healthy individuals. In 2005 the Task Force commissioned an activity focusing on ‘the impact of
nutrition on inflammation’. The aim of this activity was to review current knowledge focusing on common mechanisms and markers of
inflammation, the role of inflammation in various diseases and conditions, and the potential for modulation of inflammation by nutrition.
The aim was addressed by establishing an Expert Group, drafting a document and holding a Workshop to discuss the draft document and
to finalise the conclusions. The finalised document will be published.
The Workshop was held in 2006 and gathered together clinicians, immunologists, pharmacologists and nutritionists in order to consider:
(a) the role of inflammation in a range of distinct pathological conditions (inflammatory bowel diseases, coeliac disease, asthma, chronic
obstructive pulmonary disorder, atopic dermatitis, psoriasis, rheumatoid arthritis, atherosclerosis, obesity) including the identification of
common and unique molecular and cellular responses and signalling pathway; (b) the mechanism of action of common anti-inflammatory
drugs; (c) the potential pro- and anti-inflammatory roles of specific dietary components (PUFA, vitamins C and E, carotenoids, flavonoids,
prebiotics, probiotics).
A number of conclusions were reached. Inflammation is a normal part of the host immune response to infection and to other insults; it
initiates pathogen killing as well as tissue repair processes and helps to restore homeostasis at infected or damaged sites. Normally, the
host is tolerant to microbes and other environmental components that do not pose a threat. This tolerance involves only a limited host
response or an active response that is tightly controlled. Where an inflammatory response does occur it is normally well regulated in order
that it does not cause excessive damage to the host, is self-limiting and resolves rapidly. Pathological inflammation involves a loss of
tolerance and/or of regulatory processes, although the reasons for this loss are not clear. Whatever the site of inflammation or the nature of
the trigger, common mediators of inflammation include certain cytokines (TNFa, IL-1b, IL-6, interferon-g), chemokines (IL-8, monocyte
chemoattractant peptide-1), eicosanoids (PGE2, 4-series leukotrienes), matrix metalloproteases and reactive oxygen species, and signalling
pathways often involve the activation of NF-kB. Several nutritional strategies, including n-3 PUFA, antioxidants vitamins, plant flavo-
noids, prebiotics and probiotics may be able to amelioriate chronic inflammatory processes. However, nutritional studies rely heavily on
cell culture and animal models, and more studies in human subjects are needed. Although nutritional studies have focused on therapy of
inflammatory conditions, appropriate nutrition may lower the risk of such conditions occurring, but strong evidence of this effect is
currently lacking.
Proceedings of the Nutrition Society (2008), 67 (OCE), E10 doi:10.1017/S0029665108006198

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Malnutrition, stress and immunodepression: their interrelationship


with fungal infections

I. Cesaroni, F. Negro and M. Guerrini


LABBCE, La Plata, Argentina

Prolonged stress, malnutrition and inappropriate nutrition represent 90 % of the causes of immunodepression in subjects without basal
pathologies, and are key to the entry into epithelia and endothelia of fungal species coexisting in the environment, as they provide by
immunological deficit an optimal and permissive host. Both persistent emotional and nutritional disorders converge in metabolic stress,
with hyperproduction of free radicals and an increase in cAMP. On the one hand, excessive production of free radicals, not neutralized
because of the antioxidant deficit, impacts on the skin, thus generating tissue microlesions. On the other hand, cAMP accumulation
produced by energy consumption associated with stress acts as a negative modulator of cellular immunity that decreases the activity of
T-cells and macrophages. Consequently, there is a decrease in levels of IL, interferon-g, TNFa and Ig, providing optimal conditions for
the entry of opportunistic pathogens. These outcomes are manifest in numerous cases as severe surface mycoses, and in some cases as
profound and systemic mycoses.
When patients with mycosis in different zones (n > 2400) were analysed 60 % showed onychomycosis (infection of finger and toenails)
and 40 % a dermal mycosis, some with genetic immunological restrictions similar to psoriasis(1) and vitiligo (20 % of those studied). In the
remainder of cases mycosis was coincident with immunodepression in patients such as badly-nourished and/or malnourished children and
elderly adults, breast-feeding mothers, postnatal mothers, alcohol- and drug-dependent patients, patients undergoing prolonged treatment
with quimiotherapeutics, radiotherapeutics and corticosteroids, with emotional stress and other less-frequent cases.
Analysis of observed causes and outcomes with an unexpected finding has led to the postulation of a treatment model based on 350
patients with psoriasis participating in a randomised double-blind study that included a natural probiotic solution based on four species of
Lactobacillus associated with Saccharomyces cerevisiae in oat solution. This product is a leading probiotic solution because of its topical
and oral use. Periodic consumption of the probiotic(2) allows recovery of the immunomodulating action of the intestinal mucosa, which
contains 70 % of the body’s immune cells. Furthermore, its topical application facilitates indispensable degradation of the hyperkeratosic
micaceous plaque stimulated by fungal toxins at the level of dermal receptors and growth factors, Rhodotorula and Malassezia(3) being the
effectors most involved.
Antifungal agents used in the form of cream, lotion or pills are selected according to results obtained by culture and antifungal tests of
isolated species following the norms of the Clinical and Laboratory Standards Institute for antifungal tablets. The therapy was com-
plemented with a diet based on fruits, vegetables, food containing n-3, n-6 and n-9 PUFA and a supplement of minerals and antioxidants
formulated to provide rapid reversal of acute cases. Some cases also required psychological support.
Compared with patients treated with placebo, patients treated with the probiotic and antifungal agent showed overall improvement in
their lesions. As the causes of immunodepression were controlled, mycotic growth could be stopped and the immunological deficit
reversed by dosing with IgA. In turn, this focused treatment allowed a specific action against the invasive fungal agent that resulted in
favourable responses, decreasing not only the cost but also the duration of the treatment.

1. Baroni A, Paoletti I, Ruocco E, Agozzino M, Tufano MA & Donnarumma G (2004) J Cutan Pathol 31, 35–42.
2. Weston S, Halbert A, Richmond P & Prescott SL (2005) Arch Dis Child 90, 892–897.
3. Baroni A, Orlando M, Donnarumma G, Farro P et al. (2006) Arch Dermatol Res 297, 280–288.
Proceedings of the Nutrition Society (2008), 67 (OCE), E11 doi:10.1017/S0029665108006204

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Impaired immune function in an animal model for cancer cachexia

J. Faber, D. Kegler1, P. Vos1, A. van Helvoort1 and J. Garssen1,2


1
Numico Research, Wageningen, The Netherlands and 2Department of Pharmacology & Pathophysiology,
Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, The Netherlands

In general it is assumed that 50 % of all patients with cancer have significant weight loss before treatment and many of them suffer from
cachexia(1,2). Malnutrition and inflammation occurring during cachexia directly affects the immune system. Thus, patients with cachexia
have a higher susceptibility towards infections, which significantly influences survival (3).
Several preclinical studies show the cachectic features of the colon-26 tumour model in mice in which the pro-inflammatory cytokines
IL-6 and TNFa are denoted as pivotal mediators(4). In contrast, fewer studies have investigated the effects of cachexia and nutrition on
immune function. The present study aims to develop a model to study nutritional effects on immune function in mice with cachexia.
Murine colon adenocarcinoma (C26) cells were inoculated in syngenic CD2F1 mice to induce cachexia. Body weight, skeletal-muscle
weight and weight of adipose tissue were measured to assess the cachectic status of the mice. To investigate effects on the immune system
contact hypersensitivity against oxazolone was determined, as an in vivo model for cellular (T-helper (Th) 1 dependent) immunity. In
addition, plasma cytokines, concanavalin A (ConA)-induced splenocyte proliferation and cytokine production, and lipo-polysaccharide
(LPS)-induced cytokine production in whole blood were measured.
Tumour inoculation resulted in a significant impairment of body weight and wasting of skeletal muscles and adipose tissue. In addition,
pro-cachectic cytokines IL-6 and TNFa in plasma demonstrated a substantial increase, whereas the anti-cachectic cytokine IL-4 was
significant lower in the tumour-bearing mice. Contact hypersensitivity showed a significant decrease in tumour-bearing animals, reflecting
a reduced Th1 immune response.
Furthermore, proliferation capacity, but also Th1 and Th2 cytokine production after ConA stimulation by splenocytes demonstrated a
significant reduction compared with the control group. Lower capacity of immune cells in whole blood to react on LPS was reflected in a
decreased production of both IL-1b and TNFa in the tumour-bearing group while IL-6 remained unchanged.
Present findings demonstrate an impaired immune function in tumour-bearing mice suffering from cachexia. Thus, it is a useful model
to study potential effects of nutritional interventions on immune function in cancer cachexia.

P<0.001
300
Ear swelling (um)

200

100

0
C TB
Fig. 1. Effect of tumour inoculation on contact hypersensitivity in a mouse model for cancer cachexia. Values are means (mm) with their standard errors represented by vertical
bars for control (C) group (n = 10) and tumour-bearing (TB) group (n = 20).

1. Tchekmedyian NS, Zahyna D, Halpert C & Heber D (1992) Oncology 49, Suppl. 2, 3–7.
2. Finley JP (2000) AACN Clin Issues 11, 590–603.
3. Dewys WD, Begg C, Lavin PT et al. (1980) Am J Med 69, 491–497.
4. Zhou W, Jiang ZW, Tian J, Jiang J, Li N & Li JS (2003) World J Gastroenterol 9, 1567–1570.
Proceedings of the Nutrition Society (2008), 67 (OCE), E12 doi:10.1017/S0029665108006216

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Impact of chia (Salvia hispanica L.) on the immune


system: preliminary study

I. Fernandez1, S. M. Vidueiros1, R. Ayerza2, W. Coates2 and A. Pallaro1


1
Department of Nutrition, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina and
2
Office of Arid Lands Studies, University of Arizona, Tucson, Arizona 85706, USA

Chia was one of the four basic foods of Central American civilizations in pre-Columbian times. Nowadays, this crop is being reintroduced
to Western diets to improve human health because it is an important source of n-3 fatty acids, antioxidants, dietary fibre, protein, vitamins
and minerals, and when added to animal diets it elicits a reduction in the SFA contents of the animal products and serum lipids(1,2). The
protein quality of chia has been demonstrated to be higher than that of common cereals, which could be important in thymus development
since previous studies have shown that protein quality affects thymus status(3,4). However, adverse reaction to food is frequently observed
among populations, and its symptoms may be localized in many organs and systems(5). The aim of the present study was to analyse the
effect of chia on some aspects of immune system such as the thymus and serum IgE concentration. Weanling male Wistar rats (23 d of
age) from the Department of Nutrition at the School of Pharmacy and Biochemistry of the University of Buenos Aires, were divided in
three groups (six rats each) that received for 1 month (g/kg diet): 150 ground chia seed (T1); 50 chia oil (T2); no chia (T3; control group).
Diets T1 and T2 were formulated to provide equal quantities of a-linolenic acid from the chia. All the experimental diets were iso-
energetic, contained (g/kg) 200 protein and 70 oil, and were prepared according to the American Institute of Nutrition guidelines(6). Food
intake was recorded (FI; g/d). At the end of the experimental period and after 4 h of fasting body weight (BW; g) was recorded. Animals
were anaesthetized in a CO2 chamber and blood was recollected by venous puncture and used to determined serum IgE levels (ng/ml)
by ELISA (Bethyl Labs, Montgomery, TX, USA). Thymuses were removed, weighed (TW; mg/P0.75) and total thymocyte number (TN;
no. of cells per organ) was determined using a Newbauer chamber. Statistical analysis was performed using ANOVA t test.

T1 T2 T3
Diet group . . . Mean SD Mean SD Mean SD

FI (g/d) 16.03 0.99 14.64 1.24 15.46 0.95


BW (g) 265.78 24.01 238.88 40.25 259.26 27.50
TW (mg/P0.75) 12.16 1.53 11.12 1.16 11.07 2.42
TN (cells · 10 - 7/ organ) 109.5 17.68 81.19 18.90 98.83 15.21
IgE (ng/ml) 6.77 4.06 4.67 1.05 3.18 1.05

No significant differences were observed in FI, BW, TW, TN and IgE levels when chia was added to experimental diets as seeds (T1) or
as oil (T2) when compared with the control (T3). Moreover, no symptoms such as dermatitis, diarrhoea and abnormal animal growth and
behaviour were observed. Adding chia seeds or oil to experimental diets did not produce any of the problems associated with other n-3
fatty acid sources such as flaxseed or marine products, e.g. fishy flavour, weight loss, digestive problems, diarrhoea and allergies, as has
been proposed(1).

1. Ayerza R & Coates W (2005) Nutr Res 25, 995–1003.


2. Fernandez I, Ayerza R, Coates W, Vidueiros SM, Slobodianik NH & Pallaro AN (2005) Medicina (Bs As) 65, Suppl. II, Abstr 259: 113.3.
3. Pallaro A, Feliu MS., Vidueiros S, Slobodianik N, Ayerza R, Coates W & Fernandez I (2004) Congreso Internacional de Ciencia y Tecnologı́a de los
Alimentos. Argentina. http://www.eatchia.com/pallarosp.htm
4. Pallaro A, Roux ME & Slobodianik NH (2001) Nutrition 17, 724–728.
5. Foschi FG, Marsigli L, Chiappelli F, Kung MA, Bernardi M & Stefanini GF (2000) Adverse reactions to foods. In Nutrition and Immunology,
pp. 233–246 [ME Gershwin, JB German and CL Keen, editors]. Totowa, NJ: Humana Press.
6. American Institute of Nutrition (1993) J Nutr 123, 1939–1951.
Proceedings of the Nutrition Society (2008), 67 (OCE), E13 doi:10.1017/S0029665108006228

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

In vitro and in vivo immunomodulating effects of traditionally-prepared


extract and purified compounds from Cetraria islandica

J. Freysdottir1,2, S. Omarsdottir3, K. Ingolfsdottir3, A. Vikingsson4 and E. S. Olafsdottir3


1
Centre for Rheumatology Research and 2Department of Immunology, Landspitali University Hospital, IS-101 Reykjavik,
Iceland, 3Faculty of Pharmacy, University of Iceland, Hagi, Hofsvallagata 53, IS-107 Reykjavik, Iceland and
4
Department of Rheumatology, Landspitali University Hospital, IS-108 Reykjavik, Iceland

Cetraria islandica has been used for centuries in folk medicine in many countries to treat a number of conditions, mainly as an aqueous
extract(1,2). C. islandica contains many compounds, some of which have shown biological activity(3–6). However, very little is known
about their effect on the immune system. Thus, the effect of traditionally-prepared aqueous extract and purified compounds isolated from
C. islandica (polysaccharides lichenan and isolichenan and secondary metabolites protolichesterinic acid (PLA) and fumarprotocetraric
acid (FPCA)) on the maturation of dendritic cells was assessed.
Human monocytes were isolated from healthy individuals and differentiated into immature dendritic cells by culturing them in the
presence of IL-4 and granulocyte–macrophage colony-stimulating factor. They were subsequently cultured in the presence of maturation
factors (IL-1b and TNFa), either alone (Neg) or with positive controls lipopolysaccharides (LPS), PGE2, cholecalciferol (VD3) or the
aqueous extract or purified compounds from C. islandica. Their effect on the maturation of the dendritic cells was assessed by measuring
secretion of IL-10 and IL-12 by ELISA and expression of the surface molecules CD86 and CD209 by flow cytometry. In addition, the
effect of the aqueous extract on antigen-induced arthritis in rats was investigated.
The aqueous extract up regulated secretion of both IL-10 and IL-12, with IL-10 secretion being more prominent (Figure). Lichenan had
similar effects, but not isolichenan or the secondary metabolites, suggesting that the effect observed with the aqueous extract was mainly
mediated by lichenan. Significantly less arthritis was observed for rats treated with the aqueous extract compared with rats treated with
saline (9 g NaCl/l) alone (data not shown).
These results suggest that the aqueous extract of C. islandica has an anti-inflammatory effect, possibly by changing the cytokine
secretion bias from IL-12 towards IL-10.

10
IL-12p40/IL-10 ratio (PI)

1
* * * *
* *

0.1
Neg. LPS PGE2 VD3 100 10 100 10 100 10 1 0.1 0.01 1 0.1 0.01

Extract Isolichenan Lichenan FPCA PLA

Figure. IL-12p40: IL-10 secretion by dendritic cells cultured with maturation factors alone (Neg); LPS; PGE2; VD3;extract, lichenan and isolichenan at 100 and 10 mg/ml; or
FPCA and PLA at 1, 0.1 and 0.01 mg/ml. Values are means and standard deviation represented by vertical bars. Mean values were significantly different from that for the control
(Neg): *P < 0.05.

1. Ingólfsdóttir K (2000) Bioactive compounds from Iceland moss. In Bioactive Carbohydrate Polymers – Occurrence, Function and Use. Proceedings of
the Phytochemical Society of Europe, vol. 44, pp. 25–36 [BS Paulsen, editor]. Dordrecht, The Netherlands: Kluwer Academic Publishers.
2. Olafsdottir ES & Ingólfsdottir K (2001) Planta Med 67, 199–208.
3. Ingolfsdottir K, Jurcic K, Fischer B & Wagner H (1994) Planta Med 60, 527–531.
4. Ingólfsdóttir K, Jurcic K & Wagner H (1998) Phytomedicine 5, 333–339.
5. Olafsdottir ES, Ingolfsdottir K, Barsett H, Paulsen BS, Jurcic K & Wagner H (1999) Phytomedicine 6, 33–39.
6. Omarsdottir S, Olafsdottir ES & Freysdottir J (2006) Int Immunopharmacol 6, 1642–1650.
Proceedings of the Nutrition Society (2008), 67 (OCE), E14 doi:10.1017/S002966510800623X

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Oral lactoferrin and glycine display in vivo synergistic


anti-inflammatory activity

A. Hartog and J. Garssen


Numico Research, Wageningen, The Netherlands and Department of Pharmacology & Pathophysiology,
Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, The Netherlands

There is a growing awareness of the interaction of food constituents with the immune system. The present study aims to evaluate
immunomodulatory effects of two of these nutritional components, i.e. glycine (Gly) and lactoferrin (LF)(1,2). Mice orally supplemented
with gly, LF or Gly + LF were injected intradermally in the ear with zymosan. Ear swelling, as a measure of inflammation, as well as IL-1,
TNFa and IL-6 levels in the ear and the number of TNFa-producing spleen cells were analysed. Gly and LF decreased the zymosan-
induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reduced
number of TNFa-producing spleen cells). Gly effects (20, 50 and 100 mg per mouse per d) were concentration dependent whereas for
LF only the lowest doses (0.1 and 1 mg per mouse per d) significantly inhibited the inflammatory response. Surprisingly, higher doses of
LF (5 and 25 mg per mouse per d) failed to influence the inflammatory reaction. A combination of both nutrients (LF 0.1 mg per mouse
per d + gly 20 or 50 mg per mouse per d) inhibited the zymosan-induced ear swelling synergistically (Figure). Additionally, an enhancing
effect of both components was seen on the number of TNFa-producing spleen cells.

110
Swelling (% of control at 6 hr)

80

50 Control
Gly 20
20
LF 0.1

Gly/LF
-10
0 2 4 6 23 24 25
Time (hr)

Figure. The effect of glycine, bovine LF and a combination of gly + LF on zymosan-induced ear inflammation. Vehicle (control), gly (20 mg per mouse per d), bovine
LF (0.1 mg per mouse per d) and gly + LF (Gly/LF) were orally administered for 3 d, at day 2 the ears were injected with zymosan. Ear thickness was measured before and 3, 6
and 24 h after the injection. The swelling of the ears of the vehicle-treated animals at 6 h (generally the maximal swelling) was set to 100 %. Results are shown as means with
their standard errors represented by vetical bars for six mice.

The present findings show anti-inflammatory activity of gly and LF using the zymosan-induced inflammation model. Moreover, a
combination of both components demonstrated a synergistic effect on inflammation of the skin and an enhancing effect on the number of
TNFa-producing spleen cells.

1. Zhong Z, Wheeler MD, Li X et al. (2003) Curr Opin Clin Nutr Metab Care 6, 229–240.
2. Legrand D, Elass E, Pierce A & Mazurier J (2004) Biometals 17, 225–229.
Proceedings of the Nutrition Society (2008), 67 (OCE), E15 doi:10.1017/S0029665108006241

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Interactions between dendritic cells, probiotic bacteria and


the adipokine leptin

S. C. Knight1, N. R. English1 and A. I. F. Blakemore2


1
Antigen Presentation Research Group, Imperial College London, Northwick Park & St Mark’s Campus, Watford Road,
Harrow HA1 3UJ, UK and 2Genomic Medicine Group, Imperial College London, Hammersmith Campus,
Du Cane Road, London W12 0NN, UK

Perinodal adipose tissue (PAT) has specialised properties; functional dendritic antigen-presenting cells (DC) are abundant in PAT but rare
in adipose tissues distant from nodes(1), and in human subjects, as previously reported in rodents, PAT normally has tissue-specific
properties, being high in PUFA(2). Several properties of PAT are altered in the inflammatory bowel disease (IBD) Crohn’s disease. It has
been shown first that DC in PAT lose MHC Class II expression and capacity to stimulate T-cells(1) and second that site-specific changes in
fatty acids are lost or altered in Crohn’s disease(2). Finally, hypertrophy of adipose tissue and increases in adipokines including leptin
occur in Crohn’s disease(2).
DC are found between gut epithelial cells with veils protruding into the lumen making direct contact with commensal bacteria. Gut DC
can then migrate via afferent lymphatics, entering lymph nodes via a perinodal sinus immediately below and connected with PAT.
Potential anti-inflammatory benefits of probiotic bacteria include the capacity to affect the function of DC. Thus, bacterial components can
decrease production of IL-12 and promote production of IL-10 in DC(3). By contrast, leptin, in addition to its widely-accepted role in
energy homeostasis and reproduction, acts as a pro-inflammatory cytokine promoting development of DC. In the present study, therefore,
effects of probiotic bacteria on the potential of DC to respond to stimulation with leptin have been investigated by examining their
expression of leptin receptors.
There are multiple leptin receptor splice forms, including a trans-membrane form that signals via the JAK/STAT pathway, and shorter
forms of receptor(4). DC were derived from blood monocytes and effects of probiotic bacteria on gene expression were examined for
different forms of receptor. DC expressed both long and short forms of leptin receptor but levels of expression varied in DC from different
individuals. Probiotic bacteria reduced relative expression of the long signalling form of receptor (Figure). Parallel studies of formation of
intracellular cytokine production showed increases in IL-10 and additional effects of probiotic bacteria on fat uptake into lipid bodies
in DC.
Relative gene expression

160
140 Short Long
120 isoform isoform
100
80
60
40
20
0
C PRO C PRO

Figure. Dendritic cells were grown from peripheral blood CD14 + monocytes for 7 d with granulocyte–macrophage colony-stimulating factor and IL-4. Over the last 2 d some
were exposed to a mixture of two probiotic bifidobacteria (B. longum and B. breve). Gene expression of the long and short forms of leptin receptor was measured by PCR in
relation to the housekeeping gene ubcH5B. DC expressed both long and short forms of receptor. The probiotic bacteria (PRO) caused preferential down-regulation of the long
signalling form of receptor. Values are means with their standard errors represented by vertical bars.

In conclusion, probiotic bacteria caused multiple changes in DC promoting anti-inflammatory effects; selected probiotic bacteria might
prove a beneficial therapy for IBD via modulating DC.

1. Bedford PA, TodorovicV, Westcott EDA, Windsor ACJ, English NR, Al-Hassi HO, Raju KS, Mills S & Knight SC (2006) J Leukoc Biol 80, 546–554.
2. Westcott E, Windsor A, Mattacks C, Pond C & Knight S (2005) Inflamm Bowel Dis 11, 820–827.
3. Hart AL, Lammers K, Brigidi P, Vitali B, Rizzello F, Gionchetti P, Campieri M, Kamm MA, Knight SC & Stagg AJ (2004) Gut 53, 1602–1609.
4. Quinton ND, Laird SM, Tuckerman EM, Cork BA, Li TC & Blakemore AIF (2003) Am J Reprod Immunol 50, 224–231.
Proceedings of the Nutrition Society (2008), 67 (OCE), E16 doi:10.1017/S0029665108006253

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

A new n-3 PUFA-enriched formula for enteral nutrition modulates


oxidative LDL and inflammatory cytokines

J. Olza1, R. Moreno2, C. M. Aguilera1, A. Pérez de la Cruz2, A. Gil1 and M. D. Mesa1,


1
Institute of Nutrition and Food Technology, Dept of Biochemistry and Molecular Biology II, University of Granada,
Granada, Spain and 2 Universitary Hospital Virgen de las Nieves, Unit of Clinical Nutrition and Dietetic, Granada, Spain

Aging is associated with chronic low-grade increases in circulating levels of inflammatory molecules. A wide range of factors including
obesity, metabolic syndrome, CVD, infection, smoking, genetic and declining function of sex hormones may contribute to the systemic
low-grade increase in inflammatory activity in the elderly. Circulating TNFa is a good predictor of mortality in the frail elderly population
and the oxidation of LDL can be a consequence of the secretion of cytokines and adhesion molecules during the inflammatory process.
Dietary interventions may be good strategies to decrease pro-inflammatory activity and improve human health. It is well known that long-
chain n-3 PUFA decrease the production of inflammatory cytokines. The aim of the present study was to compare the effect of T-Diet
Plus1 v. a standard control diet on plasma concentrations of inflammatory markers and oxidized LDL (ox-LDL) in elderly patients feed
total enteral nutrition (TEN) for 6 months.
Sixty-five patients aged 75 years feed TEN were divided into two groups (experimental and reference). The experimental group (n 32)
was fed a new enteral formula, T-Diet Plus1 (Vegenat SA), that contained (mg/l) 75 EPA and 35 DHA. Reference group (n 33) was fed a
standard enteral diet (Jevity1 , Abbot Laboratories) intended for nutrition in the elderly. At the end of the experimental period only sixteen
patients from the T-Diet Plus1 group and twenty from the reference group remained in the study. The daily intake was 5459 (SE 130) kJ,
with no difference between groups. Cytokines (basal, 3 months and 6 months) were measured using a human serum adipokine (panel B)
kit (LINCOplexTM ; Linco Research, St Charles, MO, USA) with the Luminex 200 System built on XMAP technology. Ox-LDL was
determined using an ELISA kit (Biomedica Medizinprodukte GmbH & Co KG, Vienna, Austria) and the ultra-sensitive C-reactive protein
(PCRus) with a turbidimetric immunoassay (Dade Behring Inc., Deerfield, IL, USA). Non-parametric tests were used for statistical
analysis: Mann-Witney test was performed to evaluate differences between independent groups (P £ 0.05); Wilcoxon test was used to
determine the effect of each diet after 3 or 6 months (mean values with unlike superscript letters were significantly different: P £ 0.05).

TNF-α ox-LDL
10 4000
b
8
3000
b
6 b
pg/ml

a
ng/ml

ab a 2000
4

2 1000

0 0
0 months 3 months 6 months 0 months 3 months 6 months
T-Diet Reference T-Diet Reference

T-Diet1 Reference
Initial 3 months 6 months Initial 3 months 6 months
Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE

PCRus (pg/ml) 1.87 0.35 1.91 0.56 0.90 0.25 2.13 0.42 1.94 0.55 1.88 0.55
IL-6 (pg/ml) 47.7 10.6 44.2 10.6 34.2 13.9 41.3 11.6 59.6 18.9 42.9 17.2
IL-8 (pg/ml) 9.24 1.44 9.24 1.80 10.55 4.13 8.69 1.69 7.83 1.22 6.91 1.45

The results demonstrate that ox-LDL decreased after 6 months of monitored nutrition in both groups (P £ 0.05), with no differences
between groups. No differences were found in other inflammatory markers, although both TNFa and IL-6 showed a tendency to decrease
after 6 months, which is important considering that the patients were frail elderly with different pathologies.

This study was financed by Vegenat SA.


Proceedings of the Nutrition Society (2008), 67 (OCE), E17 doi:10.1017/S0029665108006265

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Conjugated linoleic acid feeding during rat suckling period enhances


intestinal IgA production

F. J. Pérez-Cano, M. Molero, C. Ramı́rez-Santana, M. Castell, C. Castellote and A. Franch


Department of Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

Conjugated linoleic acid (CLA) is a mixture of isomers of linoleic acid, of which cis-9, trans-11 and trans-10, cis-12 are the major
constituents. These compounds induce beneficial effects on health and it has been suggested that they may modulate immune responses.
However, as in man, mucosal Ig production is poorly developed during the suckling period in rats. The aim of the present work was to
establish the effect of CLA on mucosal immunity during suckling by determining IgA levels in intestinal washes from 28-d-old Wistar
rats by an ELISA technique. IgA and PPAR-g (as a possible mediator of the CLA effect) mRNA expression in small intestine and colon
from 21- and 28-d-old Wistar rats was also evaluated by real-time PCR. Pregnant Wistar rats were obtained on day 7 of gestation. On the
day of birth pups were divided into eight different groups. Four groups were analysed on day 21: litters from mothers fed with CLA (80 %
cis-9, trans-11, 20 % trans-10, cis-12; Lipid Nutrition B. V. Wormerveer, The Netherlands;10 g/kg pellet chow) during gestation and
lactation (A); CLA only during gestation but litters received a CLA isomer mixture by daily oral supplementation during suckling (B);
exclusively receiving CLA by oral administration during the suckling period (C); a parallel-age reference group. Another four groups were
studied at 28 d old: litters receiving CLA from day 1 to 28 (D); from day 1 to 21 (X); from weaning to day 28 (E); a parallel-age reference
group (Z). On days 21 and 28 rats were killed and samples from the small intestine and colon were removed for RNA extraction for later
real-time PCR. Taqman1 specific probes and primers were used for each gene (Applied Biosystems, Foster City, CA, USA). Target gene
expression was normalised by GADH and b-actin endogenous controls in each sample. In all cases statistical analysis was performed by
conventional ANOVA, and when the experimental group variable had a significant effect on the dependent variable post hoc comparisons
(LSD test) were performed. Differences were considered to be significant at P < 0.05. The results provided evidence that mucosal pro-
duction of IgA increased in animals supplemented with CLA during suckling by increasing IgA mRNA expression in the small intestine
and colon and IgA protein in 28-d-old rats (see Table). PPARg gene expression was also up regulated in animals receiving CLA during
early life. In conclusion, dietary supplementation with CLA during suckling enhances the development of the immune system in Wistar
rats. Moreover, the effect of CLA supplementation is better when supplementation is given earlier and for longer.

IgA expression ( %) (normalized by b-actin)


Small intestine Colon
Age Group Mean SE Mean SE

Day 21 A 149.1 28.15 136.1 52.94


B 73.81 29.32 114.9 29.19
C 179.6 80.69 206.6 62.10
W 100.0 39.27 100.0 20.25
Day 28 D 545.8* 190.4 372.5* 122.2
X 15.91 5.04 40.32 10.82
E 49.85 23.22 79.30 15.75
Z 100.1 19.53 99.85 28.24
Mean values were significantly different from those for groups E, X and Z (n 4–5): *P < 0.05.
Proceedings of the Nutrition Society (2008), 67 (OCE), E18 doi:10.1017/S0029665108006277

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Detection of biological activities promoted by proteins from supernatant


fractions of Lactobacillus plantarum in a promyelocytic cell line

E. Puertollano1, M. A. Puertollano2, L. Cruz-Chamorro1, G. Alvarez de Cienfuegos1


and M. A. de Pablo1
1
Área de Microbiologı́a, Universidad de Jaén, Jaén, Spain and 2Instituto del Frı́o (CSIC), Madrid, Spain

Recently, probiotics have acquired considerable importance because of their beneficial properties in relation to human health(1). It is now
generally assumed that probiotics are capable of participating in the prevention and treatment of intestinal disorders, in the modification
of ‘immune response and in the reduction of incidence of cancer(2). In fact, lactic acid bacteria or soluble compounds synthesized by the
bacteria may interact with tumour cells in culture inhibiting their growth(3). The present study examined the effects promoted by proteins
from supernatant fractions of Lactobacillus plantarum on HL-60 cells (a promyelocytic cell line). Proteins were concentrated by cen-
trifugation (Centricon plus-20; Millipore, Madrid, Spain). After the culture of HL-60 cells for 24 h in the presence of different con-
centrations (5, 50 and 100 mg/ml) of protein from L. plantarum, cell viability was quantified by MTT assay, lactate dehydrogenase (LDH)
release, NO production, reactive oxygen species (ROS) generation and caspase-3 activity. In addition, the haemolytic activity of super-
natant fractions was determined. Cell viability was also determined visually by double staining with Hoechst 33342 and propidium iodide
dyes. Results indicated that proteins from L. plantarum produced a cytotoxic effect on this cell line, as well as a loss of cell viability after
the treatment. Similarly, a significant haemolytic effect was detected. The measurement of NO production, ROS generation and caspase-3
activity showed that proteins from L. plantarum did not promote apoptosis in HL-60 cells. In conclusion, it is suggested that proteins from
L. plantarum supernatant fractions exert necrotic activity rather than an apoptotic action on HL-60 cells.

Fig. 1. Measurement of cytotoxicity by LDH Fig. 2. Measurement of caspase-3 activity. HL-60 Fig. 3. Haemolytic activity of cell-free supernatant
release. Values, expressed as a percentage of cyto- cells were cultured in the presence (K) or absence fractions from L. plantarum. PBS and 0.1 % (v/v) Triton
toxicity, are means with their standard errors repre- ( ) of caspase-3 inhibitor. The values, expressed as X-100 were used as negative and positive controls
sented by vertical bars. Mean values were relative units of fluorescence, are means with their respectively. The values, expressed as a percentage of
significantly different from the control (CT) value: standard errors represented by vertical bars. Mean haemolysis, are means with their standard errors repre-
*P < 0.05. values were significantly different from the control sented by vertical bars. Mean values were significantly
(CT) value: *P < 0.05. different from the control (CT) value: *P < 0.05.

1. Lilly DM & Stillwell RH (1965) Science 147, 747–748.


2. Cross ML (2002) FEMS Immunol Med Microbiol 34, 245–253.
3. Hirayama K & Rafter J (2000) Microbes Infect 2, 681–686.
Proceedings of the Nutrition Society (2008), 67 (OCE), E19 doi:10.1017/S0029665108006289

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Oral health and nutritional biochemical variables in adult patients


with AIDS

N. Slobodianik1, P. Perris1, A. Squassi2,3, G. Sánchez2,3, M. S. Feliu1 and N. Bordoni3


1
Department of Nutrition, School of Pharmacy and Biochemistry, 2Oral Care Clinic for High Risk Patients (CLAPAR I)
and 3School of Dentistry, University of Buenos Aires, Argentina

High prevalence of oral disease has been observed in patients with AIDS(1–3). The aim of this preliminary study was to analyse oral health
and its relationship with nutritional biochemical variables in a group of adult patients with AIDS. Twenty-eight patients between 25 and
50 years old who were HIV + were included. Dental status, gingival status and presence of mucosal alterations were determined. The
DMFT index (decayed, missed and filled teeth) and its components (gingival index, bleeding sites and the presence of erosion on soft
tissues) were calculated and evaluated(4). Samples of whole blood and non-stimulated saliva were collected from fasting patients and
serum transthyretin (TTR), C3c fraction (C3c) and total saliva IgA levels were determined by quantitative radial immunodiffusion on
layers (The Binding Site, Birmingham, UK and Diffuplate; Biocientifica, Buenos Aires, Argentina)(5–7). The results were (mg/l): IgA
82 (SD 41); TTR 271 (SD 177); C3c 828 (SD 380). When results were compared with reference values (227 (SD 74), 337 (SD 92) and
1350 (SD 270) respectively) a reduction in levels was observed. Moreover, also observed were: (a) a negative correlation between the D
component of DMFT and the number of bleeding sites and TTR levels (r - 0.68, P < 0.001); (b) a positive correlation between the
presence of lesions on soft tissues and the concentration of total IgA in saliva (r 0.84, P < 0.001). No correlation between gingival index
and biochemical variables was observed. The results suggest a compromised nutritional status concomitant with alterations in oral health
in the study group.

16
14
8
12 6 R2 = 0,7103
Less ions on
10 4
soft tissues
CD 8 2
6 R2 = 0,4591 0
4
0 5 10 15 20
2
Ig A (mg/dl)
0
0 50 100 150 200 250
C3c (mg/dL)

This work was partially supported by the University of Buenos Aires (Grants B060 and O750).

1. MacPhail LA & Greenspan JS (1997) Oral Dis 3, Suppl. 1, S190–S193.


2. Aguirre-Urizar JM, Echebarria-Goicouria MA & Eguia del Valle A (2004) Med Oral Patol Oral Cir Bucal 9, Suppl., 148–157.
3. Fidel PL (2006) Adv Dent Res 19, 80–84.
4. Loe H (1967) J Periodontol 38, 610–618.
5. Mancini G, Carbonara AO & Heremans GF (1965) Immunochemistry 2, 235–254.
6. Feliu MS & Slobodianik N (1993) Acta Bioquim Clin Latinoam xxvii, 519–526.
7. Slobodianik N & Feliu MS (1992) Acta Bioquim Clin Latinoam xxvi, 101–102.
Proceedings of the Nutrition Society (2008), 67 (OCE), E20 doi:10.1017/S0029665108006290

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Gut microbes and obesity in adolescents

M. Sotos1, I. Nadal1, A. Marti2*, A. Martı́nez2*, M. Martin-Matillas3*, C. Campoy3*,


M. A. Puertollano4*, J. Wärnberg4*, A. Marcos4* and Y. Sanz1 (*on behalf of the
EVASYON study group)
1
Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), Valencia, Spain, 2Dpto Fisiologı́a y Nutrición,
Universidad de Navarra, Navarra, Spain, 3Dpto Pediatria, Universidad de Granada, Granada, Spain and
4
Grupo Inmunonutrición, Dpto Metabolismo y Nutrición, Instituto del Frio (CSIC), Madrid, Spain

The composition of the gut microbiota has been associated with obesity in animal models and considered to be a potential environmental
factor involved in this disorder(1). In the present preliminary study the composition of the faecal microbiota of obese and overweight
adolescents (aged 14.8 (SD 1.3) years) was investigated initially and after following a nutritional intervention strategy based on an energy-
restricted diet (30–40 %) and a physical activity programme (energy expenditure 3762–11 286 kJ/week) for 3 months. Eight obese and
overweight individuals were identified according to the International Obesity Task Force criteria(2) in the frame of the EVASYON project.
The microbiological analyses were carried out by fluorescent in situ hybridization, using oligonucleotide probes targeting the main
microbial groups colonizing the human distal gut (Archaea, Bacteroides, Bifidobacterium, Clostridium coccoides–Eubacterium rectale,
Clostridium leptum, Enterobacteriaceae, Enterococcus, Fusobacterium prausnitzii, Lactobacillus, Roseburia–Eubacterium, Ruminococcus
and sulphate-reducing bacteria). Enterobacteriaceae and sulphate-reducing bacterial counts were significantly reduced (P < 0.05) in faecal
samples of individuals (n 5) showing remarkable reductions in their weight (4–7 kg) but not in those (n 3) showing minor weight losses
( < 2 kg) after the intervention. In contrast, this last group of children showed significantly lower (P < 0.05) counts of Roseburia–Eubac-
terium populations. These gut microbes could play a role in obesity by affecting either the energy harvest from the diet(3) or the signalling
pathways that link inflammation with obesity(4). Overall, the present preliminary study shows that modifications in the gut microecology
are associated with corporal weight in adolescents under a similar energy-restricted diet. Investigations are in progress to confirm this
trend and assess whether the intentional manipulation of the gut microbiota could be envisaged as a strategy to combat obesity and
immune disorders resulting from obesity.
Table 1. Microbial composition of faeces from overweight and obese adolescents
Weight reduction . . . 4–7 kg < 2 kg
Initial 3 months Initial 3 months
No. of cells/g faeces (· 108) No. of cells/g faeces ( · 108)
Microbial group Mean SE Mean SE Mean SE Mean SE

Enterobacteriaceae 9.61 1.23 4.95* 0.96 7.44 2.97 6.78 1.28


Roseburia-Eubacterium 16.5 10.23 9.88 5.76 21.3 4.39 12.4* 5.96
Sulphate-reducing bacteria 3.53 3.24 1.09* 0.58 3.21 2.49 1.62 0.37
Mean values were significantly different from initial values (Student’s t test): *P < 0.05.

The EVASYON study was supported by grants from FIS (Spanish Ministry of Health) and this work was also supported by grant AGL2005–05788-C02–01
from the Spanish Ministry of Science and Education.

1. Ley RE, Backhed F, Turnbaugh P et al. (2005) Proc Natl Acad Sci USA 102, 11070–11075.
2. Cole TJ, Belizzy MC, Flegal KM et al. (2000) Br Med J 320, 1–6.
3. Turnbaugh PJ, Ley RE & Mahowald MA et al. (2006) Nature 444, 1027–1031.
4. Bleau C, Lamontagne L & Savard R (2005) Clin Exp Immuno 140, 427–435.
Proceedings of the Nutrition Society (2008), 67 (OCE), E21 doi:10.1017/S0029665108006307

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

PUFA in the pathogenesis and treatment of patients with


multiple sclerosis

L. S. Harbige1,2, E. Pinto1, M. Xiang1, M. Leach1,2 and M. K. Sharief3


1
Centre for Biosciences Research, School of Science, University of Greenwich, Kent, UK, 2Medway School of Pharmacy,
Universities of Kent and Greenwich, Kent, UK and 3Department of Neurology, King’s, Guy’s and St Thomas’ Hospital,
London, UK

Multiple sclerosis (MS) is a central nervous system-specific demyelinating disease, and most patients demonstrate a relapse–remitting
form of the disease characterised by attacks (relapses) and periods of recovery (remission). Although the aetiology of MS remains
unknown, much evidence suggests the presence of autoimmune mechanisms in the disease pathogenesis. Cytokine dysregulation has been
found in peripheral blood mononuclear cells (PBMC) during the relapse–remitting phases of MS, i.e. raised TNFa and IL-1b and lack of
regulatory transforming growth factor b (TGFb) production(1), and in an animal model of MS a protective effect of g-linolenic acid (GLA;
18: 3n-6)-rich borage oil associated with raised TGFb(2). The aim of the present study was to determine the effects of a selected GLA-rich
oil (BGC20-884) at two doses (a low dose (5 g/d) and a high dose (14 g/d)) and a placebo control (PEGA) on the clinical course and
PBMC cytokine and membrane fatty acid profiles of thirty-six patients with active MS in a randomized double-blind placebo-controlled
trial format over 18 months. Patients were diagnosed and assessed using international criteria for MS. Relapse rate and expanded disability
status scale (EDSS) were assessed every 3 months and blood taken and PBMC isolated for cytokine studies and membrane fatty acids.
High-dose BGC20-884 treatment markedly and significantly reduced the annualized mean of relapses per patient per year over 18
months (Figure) and disability progression (EDSS) compared with the placebo control and low-dose BGC20-884 treatment. PBMC
cytokine changes ran parallel with the clinical findings, i.e. high-dose BGC20-884 showed no changes in TGFb: TNFa and TGFb:IL-1b
and no changes in membrane fatty acids, whilst the placebo control and low-dose BGC20-884 group showed significant decreases in
TGFb:TNFa and TGFb: IL-1b and loss of n-6 fatty acids, particularly linoleic acid (18: 2n-6) and arachidonic acid (20: 4n-6), over time.
It is concluded that high-dose BGC20-884 markedly affects the clinical course of MS. Furthermore, the EDSS improvement in the high-
dose group suggests there maybe a beneficial effect on neuronal lipids and neural function in MS. The present study supports the
hypothesis of dysregulation of fatty acid metabolism and cytokines in MS(1,2).

p<0.01
2.0
p<0.05
Relapses/patient/year

1.5
Mean Number of

1.5 1.25 1.3


1.1

1.0

0.35
0.5

0.0
Placebo Both Active Placebo Low Dose High Dose
Arms

Prior to Study Entry

1. Hollifield, R, Harbige L, Pham-Dinh D & Sharief MK (2003) Autoimmunity 36, 133–144.


2. Harbige L, Layward M, Morris-Downes M, Dumonde D & Amor A (2000) Clin Exp Immunol 122, 445–452.
Proceedings of the Nutrition Society (2008), 67 (OCE), E22 doi:10.1017/S0029665108006319

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Potentiation of systemic humoral immune response in suckling rats by


conjugated linoleic acid (CLA)

C. Ramı́rez-Santana, F. J. Pérez-Cano, M. Castell, C. Castellote and A. Franch


Department of Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

Cis-9, trans-11 (c9, t11) and trans-10, cis-12 (t10, c12) are the predominating molecules in the positional and geometrical isomers mixture
termed CLA. Although CLA has shown positive effects on human health and seems to be associated with immunomodulatory activities,
its effect in the developing immune system has not been studied. Thus, the present study was designed to establish the effect of CLA
supplementation during gestation and/or lactation on humoral immune response, i.e. by analysing sera Ig levels during the suckling period.
Wistar rats were allocated to four groups (A, B, C and W) on day 7 of pregnancy. From day 7 and throughout the study period group C
and W gestating mothers were fed standard pellet chow. Group A and B dams were fed 10 g CLA (80% cis-9, trans-11, 20 % trans-10,
cis-12; Lipid Nutrition B. V. Wormerveer, The Netherlands)/kg pellet chow during gestation. Moreover, group A mothers were also fed
the CLA-supplemented chow until the litters were 21 d old, the end of suckling period. Group B and C litters received the CLA mixture of
isomers by daily oral administration while their respective dams were fed standard pellet chow during lactation. In all cases litters were
equalised to ten rats per lactating mother. Pups from each experimental group were killed at the end of the second week of life (day 14)
and at the end of the suckling period (day 21), and blood samples were collected. Serum IgA, IgG and IgM levels were quantified by the
ELISA sandwich technique. ANOVA and post hoc comparisons (LSD test) were performed. Differences were considered to be significant
at P < 0.05. Animals receiving CLA passively from their mothers (group A) during gestation and the suckling period exhibited the highest
concentrations of IgG and IgM at 14 d old (P < 0.05; see Table). At the end of suckling period the serum IgG concentration in this group
was also increased, up to three times more than in the other groups (P < 0.05). Those animals supplemented with CLA only during
suckling period (group C) showed no difference in relation to those receiving no supplement. Thus, these results demonstrate that CLA
supplementation during gestation and lactation promotes systemic humoral immune response.

Serum IgG (mg/ml) Serum IgM (mg/ml)


Age Group Mean SE Mean SE

Day 14 A 11.5†‡ 0.84 79.8†‡ 4.67


C 2.09 0.34 64.4 2.56
W 1.71 0.30 59.5 4.19
Day 21 A 22.1*†‡ 2.0 88.1 7.61
B 7.73 0.7 100.7 12.0
C 3.94 0.28 98.7 6.70
W 4.79 0.21 94.8 3.58
n 10. Mean values were significantly different from those for group B: *P < 0.05. Mean values were significantly different
from those for group C: †P < 0.05. Mean values were significantly different from those for group W: ‡P < 0.05.
Proceedings of the Nutrition Society (2008), 67 (OCE), E23 doi:10.1017/S0029665108006320

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effects of a diet with polyphenol-rich cereal supplementation on the


function and redox state of peritoneal leucocytes from mice: differences
between a short (5 weeks) and long (20 weeks) period of supplementation

P. Álvarez, C. Alvarado, L. Jimenez and M. de la Fuente


Department of Animal Physiology, Faculty of Biology, Complutense University of Madrid, 28040 Madrid, Spain

Although intake of natural products is a matter of great importance in relation to health, little attention is focused on the key role of cereal
polyphenols in this respect. It has been shown that many of these compounds, especially as a mixture, exert a high antioxidant activity and
synergistic effects. However, the protective role of a short-term and long-term intake of polyphenol-rich cereals on immune function
and cellular redox state is still poorly understood. Thus, the aim was to investigate the effects of 5 and 20 weeks of supplementation
(200 g/kg) with four different cereal fractions on a population of female CD1 mice. Control animals received the standard diet without
supplementation. Several indices of the function and redox state of peritoneal leucocytes were evaluated. The cereals, designated B (wheat
germ), C (buckwheat flour), D (fine rice bran) and E (wheat middlings), contained a complex mixture of gallic acid, p-hydroxybenzoic
acid, vanillic acid, sinapic acid, p-coumaric acid, ferulic acid, quercetin, catechin, rutin and oryzanol as the major polyphenolic com-
pounds. In general, the polyphenol-rich cereals improved the immune function (by increasing chemotaxis capacity, lymphoproliferative
response to mitogens and IL-2 levels) and the redox state (by increasing catalase antioxidant activity and decreasing oxidised glutathio-
ne:reduced glutathione, TNFa and malondialdehyde levels) of peritoneal leucocytes from mice. The control animals from the 20 weeks
supplementation study (middle-age) showed typical age-related changes in their leucocyte functions and redox state. In several cases
the antioxidant supplement administration for 20 weeks to middle-aged mice restored values to those of the adult controls. Similar effects
of the cereal fractions were observed, but these effects were quantitatively more marked following long-term (20 weeks) compared with
short-term (5 weeks) supplementation.

80 ††† †††
70 *** †††
***
60 ** †† Control
* ***
U/10 cells

50 B
40
6

C
30 D
20 E
10
0
5 weeks 20 weeks
Figure. Catalase activity in peritoneal leucocytes from mice supplemented for 5 weeks and 20 weeks with polyphenol-rich cereal fractions (B, C, D or E). Values are means and
standard deviations represented by vertical bars for eight (5 weeks) and seven (20 weeks) animals. Mean values were significantly different from those for the corresponding
control group: *P < 0.05, **P < 0.01, ***P < 0.001. Mean values were significantly different from those for the adult control group: ††P < 0.01, †††P < 0.001.

In conclusion, the present results suggest that polyphenol-rich cereal supplementation, especially designed for long-term intake, may be
an important tool for optimizing the immune function and the redox state of leucocytes. Further studies are needed to determine the
optimal doses and to establish which are the key compounds involved in mediating the observed beneficial effects before their regular
consumption can be recommended.

This work was supported by MEC (BFU 2005–06777), RETICEF (RD06/0013/0003) (ISCIII) of Spain and DANONE VITAPOLE (France).
Proceedings of the Nutrition Society (2008), 67 (OCE), E24 doi:10.1017/S0029665108006332

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Structure and antigenicity changes in 7S soyabean allergen by


enzymic deglycosylation

M. Amigo-Benavent, M. Villamiel and M. D. del Castillo


Instituto de Fermentaciones Industriales (CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain

Soyabean allergy is one of the most common food allergies. The marked rise in allergenic reactions to soyabean is related to the
increasing use of soya products in processed foods, which convert it in a major public health and soyabean industry concern. Different
processes have been used to mask soyabean allergenicity, including heat treatment, fermentation, enzymic proteolysis, carbohydrate
conjugation, genetic modification, extrusion and agronomic practices(1). It is still unclear which of the components of soyabean cause
allergenic reactions. Some authors have identified 7S as a major soybean allergen(2); 7S is a N-glycoprotein that constitutes about 25 % of
the total soyabean proteins(3). Glycans forming glycoproteins have been described as being responsible for the allergenicity of some plants
and insects(4) and their removal has been proposed to reduce allergenicity(5). PNGase F is an enzyme used to deglycosylate different N-
glycans such as those present in ribonuclease B, among others. No studies focused on deglycosylation of 7S by PNGase F activity for
reducing its allergenicity have been found in the literature. The aim of the present research was to assess enzymic deglycosylation as
a strategy for reducing soyabean protein allergenicity.
7S was isolated by isoelectric precipitation and the protein purity checked by SDS–PAGE. Enzymic deglycosylation was carried out on
the isolated 7S (1 mg/ml) with PNGase F at pH 8 and 37 C for 24 h. Deglycosylation was followed by reversed-phase HPLC, SDS–PAGE
and capillary zone electrophoresis analyses, which provided information relating to changes in protein structure. Antigenicity was assayed
by immunoblotting and indirect ELISA with polyclonal rabbit anti-soyabean sera and horseradish peroxidase-labelled goat anti-rabbit IgG.
The Figure shows SDS–PAGE (A) and immunoblotting (B), corresponding to the 7S control (c) and its deglycosylated form (d). As can
be observed, the electrophoretic mobility of 7S changed as a result of deglycosylation, indicating a decrease in protein molecular mass.
Immunoblotting and ELISA data suggest that the carbohydrate moiety forming 7S does not have a key role in its in vitro antigenic
response as assayed in the present study. Further studies should be conducted in order to confirm the results by employing digestion and
absorption assays in vitro and in vivo as well as an immunological test using serum from patients allergic to soyabean.

c d c d

67 kDa
64 kDa
A
A B
These studies were supported by projects AGL2004-05031 and ALIBIRD-CM-S0505/AGR-0153.

1. Wilson S, Blaschek K & de Mejia E (2005) Nut Rev 63, 47–58.


2. Breiteneder H & Radauer C (2004) J Allergy Clin Immunol 113, 821–830.
3. Garcı́a MC, Torre M, Marina ML & Laborda F (1997) Crit Rev Food Sci Nut 37, 361–391.
4. Fötisch K & Vieths S (2001) Glycoconj J 18, 373–390.
5. Ahrazem O, MD, Lopez-Torrejon G, Sanchez-Monge R, Sastre J Lombardero M, Barber D & Salcedo G (2006) Int Arch Allergy Immunol 139, 96–103.
Proceedings of the Nutrition Society (2008), 67 (OCE), E25 doi:10.1017/S0029665108006344

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effects of IL-6 on amylase secretion and calcium signalling in pancreatic


AR42J cells: modulation by membrane fatty acid composition

N. Audi1, M. A. Martı́nez1, M. D. Mesa2, E. Martı́nez-Victoria1, M. Mañas1 and M. D. Yago1


1
Institute of Nutrition and Food Technology, Departments of Physiology and 2Biochemistry and Molecular Biology,
University of Granada, Spain

Oleic acid is a typical component of the Mediterranean diet. The type of dietary fat strongly influences the fatty acid (FA) composition of
rat pancreatic cell membranes, and this effect is associated with changes in the functionality of viable pancreatic acini(1,2). The AR42J cell
line is a useful tool for assessing the effect of membrane compositional changes on acinar cell function(3). The aim was to study the effects
of chronic treatment with IL-6 (400 pM for 48 h) on amylase secretion and Ca2 + homeostasis in AR42J cells, and to establish whether
these effects are influenced by different membrane FA composition.
The membrane FA composition of AR42J cells was modified by growing them in medium enriched with oleic or linoleic acid, as
described previously(3). Amylase activity was determined and expressed as a percentage of the initial total amylase content that was
released into the extracellular medium during incubation. Ca2 + mobilization (expressed as F340:F380) was determined by epifluorescence
microscopy. Cells were loaded with the fluorescent ratiometric Ca2 + indicator fura-2. For quantification of fluorescence, cells were
alternately excited at 340 and 380 nm using a high-speed monochromator. ANOVA was performed to compare amylase secretion between
groups treated with or without IL-6. The Ca2 + decay constant for each group was calculated and mean values were compared
by ANOVA.

25


amylase release (% of total)

† †
20
† Oleic
Oleic+IL-6
15 † Linoleic
Linoleic+IL-6

10
*

5
0 0.01 0.1 1 10 100
CCK-8 (nM)

Figure. Amylase release (% total content) evoked by different concentrations of cholecystokinin octapeptide (CCK-8). Values are means with their standard errors represented
by vertical bars (n 16 for all groups). Mean value for the oleic acid group was significantly different from that for the oleic + IL-6 group: *P < 0.05. Mean values for the linoleic
acid group were significantly different from those for the linoleic acid + IL-6 group: †P < 0.05.

Table. Ca2 + response evoked by perfusion with CCK-8 (1 nm) in AR42J cells with different membrane FA profiles treated for 48 h with IL-6 or vehicle
Peaks (340 : 380 intensity) Ca2 + decay constants (/s)
Mean SE Mean SE

Oleic acid (n 44) 2.2070 0.0861 0.0034* 0.0002


Oleic acid + IL-6 (n 39) 2.1297 0.1037 0.0028 0.0002
Linoleic acid (n 42) 2.1930 0.1112 0.0029 0.0003
Linoleic acid + IL-6 (n 29) 2.1457 0.0978 0.0032 0.0002
n, No. of cells measured in each group. Mean value for the oleic acid group was significantly different from that for the oleic + IL-6 group: *P < 0.05.

Membranes rich in oleic acid were not affected by the action of IL-6 at different concentrations of CCK-8, while membranes rich in
linoleic acid were more sensitive to the effects of IL-6 in relation to basal and stimulated amylase secretion. Dietary fat and IL-6 did not
affect Ca2 + peaks elicited by 1 nM-CCK-8. It would be useful to know whether the consumption of olive oil (rich in oleic acid) can
prevent and/or attenuate the effects of pancreatic inflammatory processes compared with sunflower oil (rich in linoleic acid).

1. Martinez MA, Lajas AI, Yago MD et al. (2004) Nutrition 20, 536–541.
2. Yago MD, Diaz RJ, Ramirez R, Martinez MA, Mañas M & Martinez-Victoria E (2004) Br J Nutr 9, 1227–1234.
3. Audi N, Mesa MD, Martinez MA, Martinez-Victoria E, Mañas M & Yago MD (2007) Exp Biol Med 232, 532–541.
Proceedings of the Nutrition Society (2008), 67 (OCE), E26 doi:10.1017/S0029665108006356

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effect of oil (sunflower oil) consumption with added hydroxytyrosol


(natural antioxidant) on antioxidant variables in leucocytes
from healthy adults

I. Baeza1, N. M. de Castro1, L. E. Dı́az2, A. Marcos2 and M. de la Fuente1


1
Departamento de Fisiologı́a Animal, Facultad de Ciencias Biológicas, Universidad Complutense de Madrid, Spain and
2
Grupo Inmunonutrición, Departamento de Metabolismo y Nutrición, Consejo Superior de Investigaciones Cientı́ficas,
Madrid, Spain

Hydroxytyrosol (3,4-dihydroxyphenylethanol; HT), also known as dihydroxyphenylethanol, is a natural phenolic antioxidant(1,2) found in
olives and olive oil that is responsible for their antioxidant properties(3). Although HT is known to exert an antioxidant effect, the
mechanism of its action and the identity of the reactive oxygen molecule(s) targeted are not known(4). The aim of the present study was to
evaluate the effect of oil (sunflower oil) consumption with ‘Oleoactive from Koipesol’ (Sos Cuetara SA, Madrid, Spain), which contains
added HT and is consumed at the level of 45–50 mg/d, on two antioxidant variables (total glutathione levels and glutathione peroxidase
(GPx) activity) in leucocytes (neutrophils and lymphocytes) from healthy adults. The design was a randomized cross-over study with
twelve healthy subjects of both gender (20–45 years of age). The subjects were divided into two groups. Group A (n 6): 3 weeks of oil
(sunflower oil) with added HT; 2 weeks of wash-out; 3 weeks of oil (sunflower oil) without added HT. Group B (n 6): 3 weeks of oil
(sunflower oil) without added HT; 2 weeks of wash-out; 3 weeks of oil (sunflower oil) with added HT. The antioxidant variables (total
glutathione in lymphocytes and neutrophils and GPx activity in lymphocytes) were analysed before starting oil (sunflower oil) intake and
at the three other time-points during the study. In both groups tested the total glutathione levels in lymphocytes did not show significant
changes. Nevertheless, in group A glutathione levels in neutrophils and GPx activity in lymphocytes increased in the subjects after the
intake of oil (sunflower oil) with added HT (Figure), however no changes were found in group B. In conclusion, HT could protect against
oxidative damage since it increases the levels and the activity of two very important antioxidants in the immune cells.

Lymphocytes Neutrophils
Glutathione peroxidase (mU/10exp6

600
Total Glutathione (pmol/106 cells)

*** 2000
***
500 1800
1600
400
1400
cells)

1200
300
1000
200 800
600
100 400
200
0
0
Basal Hydroxytyrosol
Basal Hydroxyt yr osol

Figure. GPx activity in lymphocytes and total glutathione levels in neutrophils from subjects in group A before (basal) and after 3 weeks of oil (sunflower oil) intake with added
HT. Values are means and standard deviations represented by vertical bars. Mean values were significantly different from basal values: ***P < 0.001.

This work has been supported by Sos Cuetara of Spain.

1. Papadopoulos G & Boskou D (1991) J Am Oil Chem Soc 78, 669–671.


2. Baldioli M, Servili M, Perretti G & Montedoro GF (1996) J Am Oil Chem Soc 73, 1589–1593.
3. Carliccio MA, Siculella L, Ancora MA, Massaro M, Scoditti E, Storelo C, Visioli F, Distante A & De Caterina R (2003) Arteroscler Thromb Vasc Biol
23, 622–629.
4. ODowd Y, Driss F, Dang PM, Elbim C, Gougerot-Pocidalo MA, Pasquier C & El-Benna J (2004) Biochem Pharmacol 68, 2003–2008.
Proceedings of the Nutrition Society (2008), 67 (OCE), E27 doi:10.1017/S0029665108006368

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Expression of Toll-like receptors on peritoneal macrophages and


dendritic cells from old mice treated with soyabean isoflavones
and green tea

I. Baeza, N. M. de Castro, L. Arranz and M. de la Fuente


Complutense University of Madrid, Faculty of Biology, Department of Physiology, Madrid, Spain

Cells of the immune system recognize pathogens via Toll-like receptors (TLR), which are the basic signalling receptors of the innate
immune system and direct the course of acquired immunity by recognizing specific microbial products that activate immune cells for
effector functions(1). It is well known that ageing correlates with a decline in immunity, an increase in the inflammatory state and an
impaired production of several hormones, such as oestrogens(2). In recent years it has been shown that oestrogens play an essential role in
modulating immune function and pro-inflammatory cell signalling(2,3). Soyabean isoflavones, since they show a structural similarity to
oestradiol, are being investigated in order to determine whether they can mimic oestrogen actions(4). Furthermore, the possible effects of
the green tea plant on human health, because of its content of polyphenolic antioxidants (especially catechins), has been studied with
increasing interest in recent years(5). Since the expression of TLR on cell membranes constitutes the first step in the inflammatory
signalling cascade, the aim of the present study was to determine the role of isoflavones and green tea as modulators of such TLR
expression on peritoneal macrophages and dendritic cells (those that mediate innate immunity) from old mice.
ICR-CD1 female mice aged 16 months were fed a diet supplemented with soyabean or green tea + soyabean (Diviser-Aquilea SL,
Barcelona, Spain; 1 mg soyabean per mouse per d; 3.75 mg green tea per mouse per d) for 25 weeks. The control groups were fed standard
diet (A04; Panlab LS, Barcelona, Spain). Then, at the age of 22 months peritoneal leucocytes were obtained from the animals and the
expression of TLR 2 and 4 was determined by flow cytometry in macrophages and dendritic cells.
Previous studies have detected an increased expression of TLR2 and TLR4 with advancing age (L. Arranz, I. Baeza, N. M. De Castro,
and M. De la Fuente, unpublished results). In the present experiment the combined diet (soyabean isoflavones + green tea) significantly
decreased the presence of TLR2 and TLR4 on cell membranes in the old animals. In conclusion, adequate treatment with isoflavones and
green tea could be useful in slowing down the effects of the ageing process through a mechanism of control of the age-related inflam-
matory state.

Macrophages
120 † 60
100 50
80 ** ††
% TLR 2

% TLR 4

40
60 30
40 20 **
20 10
0 0
Cont rol Soybean Soy bean + Green Cont rol Soybean Soybean + Gr een
t ea t ea
Dendritic cells
TLR4
TLR2
100
100
95
90
80 *** **
90
* 70
% TLR 2

% TLR4

60
85 50
40
80 30
75 20
10
70 0

Control Soybean Soybean + Green Control Soybean Soybean + Green tea

tea

Figure. Expression of TLR2 and TLR4 on macrophages and dendritic peritoneal cells. Values are means and standard deviations represented by vertical bars for groups of four
animals. Mean values were significantly different from those for control animals: *P < 0.05, **P < 0.01, ***P < 0.001. Mean values were significantly different from those for the
soyabean group: †P < 0.05, ††P < 0.01.

This work has been supported by Uriach-Aquilea OTC SL, the MEC (BFU 2005–06777) and RETICEF (RD06/0013/0003) (ISCIII) of Spain.

1. Mukhopadhyay S, Herre J, Brown GD & Gordon S (2004) Immunology 112, 521–530.


2. Rehman HU & Masson EA (2005) Gend Med 2, 41–56.
3. Lesmeister MJ, Jorgenson LR, Young SL & Misfeldt ML (2005) Reprod Biol Endocrinol 3, 74.
4. Gris Martı́nez JM (2006) Med Clin (Barc) 127, 352–356.
5. Friedman M (2007) Mol Nutr Food Res 51, 116–134.
Proceedings of the Nutrition Society (2008), 67 (OCE), E28 doi:10.1017/S002966510800637X

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

The effects of organic v. conventional diets on immune variables in rats

A. M. Baranska1, E. Rembialkowska2, E. Hallmann2, L. Lueck3, J. M. Cooper3 and C. Leifert3


1
Department of Animal Physiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02–096 Warsaw, Poland,
2
Division of Organic Foodstuffs, Faculty of Human Nutrition and Consumer Sciences, Warsaw Agricultural University,
Nowoursynowska 159C, 02–776 Warsaw, Poland and 3Nafferton Ecological Farming Group (NEFG), University of
Newcastle, Nafferton Farm, Stocksfield NE43 7XD, UK

Organic food production is gaining increasing interest worldwide. In Europe the regulations for organic crop cultivation prohibit the use of
chemosynthetic pesticides, mineral fertilizers, growth promoters and genetic engineering or GM organisms. Despite a small number of
studies comparing potential effects of organic and conventional foodstuffs consumption on human and animal health, there are few reports
of in vitro immunosuppressive or no effects of the presence of chemical contaminants (e.g. pesticides) in conventional food(1). The aim of
the present study was, therefore, to assess the effect of diets, based on organically, low-input and conventionally-grown crops on selected
immune variables of rats in an experimental feeding trial. Experimental diets were prepared according to the nutritional recommendations
for rats as pellets containing dried wheat, potatoes, carrots and onions and enriched with lactoalbumin, casein, rapeseed oil, minerals and
vitamins, and were characterized by the content of total flavonoids, polyphenols, b-carotene and lutein, pesticides, and antioxidant
activity(2). Adult male and female Wistar rats were kept for 3 weeks under controlled conditions with free access to water and experi-
mental feeds (without pesticides and mineral fertilizers, ORG-ORG; without pesticides, ORG-CV; without mineral fertilizers, CV-ORG;
with pesticides and mineral fertilizers, CV-CV; control group fed Labofeed (Andrzej Morawski Feed Production Plant, Kcynia, Poland),
LF), and paired and bred. Twelve young males from each experimental group were fed each of the diets for 3 and 12 weeks. Animals were
killed with Thiopental overdose, blood was collected from the heart and used for haematological analysis, spleens were isolated asepti-
cally and used immediately for in vitro studies of lymphocyte proliferation.
There were no significant differences in haematological variables of rats from all dietary groups. The effect of experimental diets on
spontaneous lymphocyte proliferation was similar after 3 and 12 weeks of feeding; however, the differences were significant only after 12
weeks (ORG-ORG v. CV-CV, ORG-ORG v. ORG-CV, CV-ORG v. ORG-CV, CV-ORG v. CV-CV; P < 0.001). A higher proliferation was
found in rats on both diets based on products from farming systems without the use of mineral fertilizers (ORG-ORG and CV-ORG) and
lower proliferation in ORG-CV and CV-CV dietary groups. These results correspond with the flavonoids and polyphenols content found
in the experimental feeds. The mitogen-stimulated proliferation index was lower in ORG-ORG and CV-ORG groups. It can only be
speculated whether the alteration in spontaneous lymphocyte proliferation can result from immunostimulatory effect of flavonoids and
polyphenols found in feed or from the immunosuppressive influence of some dietary components in products cultivated with mineral
fertilizers. Further studies are in progress.

The study was supported under the Quality Low Input Food Project: FOOD-CT-2003–506358.

1. Baranska A & Skwarlo-Sonta K (2006) Pol J Food Nutr Sci 56, 13–16.
2. Rembialkowska E, Hallmann E, Rusaczonek A, Bennett RN, Brandt K, Lueck L & Leifert C (2007) In Proceedings of 3rd International Congress of the
European Project Quality Low Input Food (QLIF), pp. 112–117 [U Niggli, C Leifert, T Alföldi, L Lück and H Willer, editors]. Frankfurt, Germany:
Research Institute of Organic Agriculture.
Proceedings of the Nutrition Society (2008), 67 (OCE), E29 doi:10.1017/S0029665108006381

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Inflammatory mediators in overweight adolescents: association with


insulin sensitivity, body composition and metabolic syndrome

R. Burrows, L. Leiva, A. M. Tong, J. Aldunate and E. Dı́az


Institute of Nutrition and Food Technology, University of Chile, Chile

The insulin-resistance (IR) metabolic syndrome (MetS) leads to high CVD risk in adult life and has been associated with an early state of
chronic low–grade inflammation (CLGI). The aim of the present study was to determine the association between a state of CLGI and
insulin sensitivity, body composition and MetS prevalence in overweight adolescents. The study included 158 adolescents (BMI ‡ 85th
percentile; eighty-six men) between 13 and 16 years of age, who were assessed for BMI, body composition ( % body fat mass (BFM) and
% free-fat mass (FFM) by pletysmography, prevalence of MetS for three or more variables (waist circumference (WC) ‡ 90th percentile;
HDL-cholesterol £400 mg/l; TAG ‡ 1100 mg/l; blood arterial pressure ‡ 90th percentile; fasting glucose ‡100 mg/d), HOMA-IR
(glucose · insulin/22.5) and the state of inflammation by C-reactive protein (CRP). Pearson correlation and y2 were used to study
associations between variables, OR to calculate risk and ANOVA and Tukey test to compare averages between groups. The median and
ranges of CRP levels (mg/l) were 0.7 (range 0.04–9.1) in males and 0.7 (range 0.04–6.2) in females. CRP showed a correlation with BMI
(P < 0.05), WC (P < 0.02), % BFM (P < 0.05), % FFM (P < 0.05), fasting insulin (P < 0.001) and HOMA (P < 0.001).
Table 1. Pearson correlation for CRP with variables of metabolic and cardiovascular risk
CRP
Variable r P
BMI 0.158 < 0.05
WC 0.199 < 0.02
% BFM 0.168 < 0.05
% FFM 0.169 < 0.05
Fasting insulin 0.269 < 0.001
HOMA 0.259 < 0.001

CRP was significantly (P < 0.01) associated with an anthropometric and metabolic cardiovascular risk profile. The prevalence and the
risk of abdominal obesity (WC ‡ 90th percentile), IR (HOMA ‡ 3.3) and MetS were significantly higher (63%, OR 3.0; 43 %, OR 4.1;
26 %, OR 4.1 respectively) in adolescents with CRP levels ‡1.12 mg/l (‡ tertile 2).
Table 2. Anthropometric, cardiovascular and metabolic profile across the CRP tertile distribution
CRP . . . < Tertile 1 ( < 0.43 mg/l) Tertile 1–2 (0.43–1.12 mg/l) > Tertile 2 ( > 1.12 mg/l)
Mean SD Mean SD Mean SD P
BMI (kg/m2) 2.0 0.5** 2.4 0.8 2.5 1.1 < 0.01
WC (cm) 88.1 6.5** 92.5 7.4 95.4 10.8 < 0.01
TBF (%) 35.4 6.4 37.6 7.6 40.4 7.0††‡ < 0.01
FFM (%) 64.3 6.9 62.5 7.9 59.7 6.9††‡ < 0.01
HDL (mg/l) 505 101 467 104 459 113† < 0.05
TAG (mg/l) 995 495 1129 687 1271 697†† < 0.01
HOMA 1.9 1.2 2.3 1.3† 3.4 2.6‡‡ < 0.001
Mean values were significantly different from those for tertile 1–2 and > tertile 2: **P < 0.05. Mean values were
significantly different from those for < tertile 1: †P < 0.05, ††P < 0.01. Mean values were significantly different from those
for tertile 1–2: ‡P < 0.05, ‡‡P < 0.05.

These results confirm that (1) PCR levels in overweight adolescents are associated with a greater cardiovascular and metabolic risk and
(2) IR involves inflammatory processes that may play an early role in the development of cardiovascular lesions.
Proceedings of the Nutrition Society (2008), 67 (OCE), E30 doi:10.1017/S0029665108006393

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Immunological response in coeliac disease is age related

M. Calzado, E. Donat, B. Polo, B. Baena and C. Ribes


Pediatric Gastroenterology and Hepatology Section, La Fe Hospital, Valencia, Spain

Anti-tissue transglutaminase antibodies (t-TG), mostly IgA class, is considered to be the most specific serological immune response in
coeliac disease, and is associated with enteropathy, specific Ig A class antibody against t-TG being found at the intestinal level(1,2).
The objective was to evaluate the serological immune response in coeliac disease according to age by measuring antibodies against the
autoantigen, i.e. t-TG and if t-TG can be used as a screening method. A retrospective study was done reviewing the files of all patients
who had undergone a small intestinal biopsy in the previous 3 years for diagnostic purposes. IgA t-TG (VarelisaTM , Phadia, cut off 6 IU/
ml) and IgA anti-gliadin antibodies (AGA; home method, cut off 0.3 arbitrary units) were determined when the biopsy was performed. In
patients with negative IgA t-TG, IgG t-TG (VarelisaTM ; Phadia, Freiburg, Germany; cut off 10 IU/ml) was determined. A total of 280
patients were included, 160 of them showed Marsh 3 lesions and one of them showed Marsh 2 lesions. All of them were consuming gluten
at the time the biopsy was performed. Diagnosis of coeliac disease was established in all of them. No patient had IgA deficiency. All
children with t-TG > 100 (upper detection limit) were classified as Marsh 3. Specificity for AGA was lower than that for t-TG. Eighty-eight
patients (55 %) were < 3 years of age and sixty-one were < 2 years of age. Ten of the eighty-eight patients (11.4%) were IgA t-TG
negative, only one being > 2 years of age. All ten subjects had elevated IgA AGA and no Ig G class antibodies against t-TG were detected.
Clinical symptoms in patients who were t-TG positive and t-TG negative were the same.

< 3 years old (n 88) > 3 years old (n 73)


Ig A t-TG positive 78 82
Ig A t-TG negative 10 1
IgA AGA positive 88 75

The results show negative IgA class t-TG is expected in about 10 % of patients with coeliac disease who are < 2 years of age.
Furthermore, no Ig G response is to be expected in those patients. It can be speculated that the immature development of the immune
system slowly responding to gluten exposure delays the appearance of autoimmunity. This notion would be in keeping with reports of no
IgA t-TG antibodies deposited at the intestinal level in younger patients(3–5). Moreover, the data confirm the response against gliadin is the
immunological trigger in coeliac disease.

1. Salmi TT, Collin P, Jarvinen O et al. (2006) Aliment Pharmacol Ther 24, 541–552.
2. Salmi TT, Collin P, Korponay-Szabó IR et al. (2006) Gut 55, 1746–1753.
3. LC Stene, MC Honeyman, EJ Hoffenberg et al. (2006) Am J Gastroenterol 101, 2333–2340.
4. Norris JM, Barriga K, Hoffenberg EJ et al. (2005) JAMA 293, 2343–2351.
5. Tosco A, Maglio M, Paparo F, Colicchio B, Sannino A, Granata V, Rapacciulo L & Troncone R (2007) Proceedings of the 40th Meeting of the
European Society of Paediatric Gastroenterology, Hepatology and Nutrition, Barcelona 2007, Abstr (In the Press).
Proceedings of the Nutrition Society (2008), 67 (OCE), E31 doi:10.1017/S002966510800640X

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Hormonal and immune changes with age: effect of energy restriction

P. Cano, G. Segovia, M. P. Fernandez-Mateos, A. del Arco, V. Jimenez, F. Mora and A. I. Esquifino


Departamento de Bioquı́mica y Biologı́a Molecular III y Fisiologı́a, Facultad de Medicina, Universidad Complutense,
Madrid, Spain

The effects of chronic 40 % energy-restriction on age-related changes in plasma prolactin, luteinising hormone (LH), testosterone, thyroid-
stimulating hormone (TSH) and corticosterone levels as well as the distribution and activity of T and B lymphocytes in the spleen were
determined. Male rats (3 months old) were subjected to 40 % energy restriction up to the age of 6, 15 and 24 months. Hormones were
measured by specific RIA. Lymphocyte subsets were measured by flow cytometry (FASC-Vantage; BD Biosciences, San Jose, CA,
USA)(1,2). Plasma LH and testosterone levels decreased with age, and energy restriction did not change this pattern. However, plasma
prolactin and TSH levels, which increased with age in the controls, were significantly decreased in energy-restricted animals. The
percentage of CD4 + lymphocytes, which increased with age in the controls, was further increased in energy-restricted rats. Although the
percentage of CD8 + lymphocytes decreased with age both in the control and energy-restricted rats, the values were higher in energy-
restricted rats at all ages studied. The percentage of T-cells was not affected by age and was increased by energy restriction at all ages
studied. B lymphocytes decreased with age and were not modified by energy restriction. The CD4 + :CD8 + was greatly increased in the
oldest control group, while energy restriction partially decreased this ratio to normal values. These data suggest that energy restriction can
prevent some of the deleterious effects that aging induces on the endocrine immune system.

CD4 Lymphocytes CD8 Lymphocytes CD4/CD8 INDEX

60 30 6

50 25 5
40 20 4

30 15 3
20 10 2

10 5 1

0 0 0
6 months 15 months 24 months 6 months 15 months 24 months 6 months 15 months 24 months

control caloric restriction control caloric restriction control caloric restriction

T Lymphocytes B Lymphocytes

50 30

40
20
30

20
10
10

0 0
6 months 15 months 24 months 6 months 15 months 24 months

control caloric restriction control caloric restriction

Figure. Values are means with their standard errors represented by vertical bars for six animals per group.

This work was supported by SAF2003–0448 of Spain.

1. Esquifino AI, Szary A, Brown-Borg HM & Bartke A (1996) Proc Soc Exp Biol Med 211, 87–93.
2. Esquifino AI, Chacon F, Cano P, Marcos A, Cutrera RA & Cardinali DP (2004) J Neuroimmunol 156, 66–73.
Proceedings of the Nutrition Society (2008), 67 (OCE), E32 doi:10.1017/S0029665108006411

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Use of inducible class II and MHC class I-related chain A/B expression
on T lymphocytes as an in vitro screen for the immunomodulatory
properties of dietary products

C. Carter, K. Varley and B. Clark


Transplant and Cellular Immunology, St James’ University Hospital, Beckett Street, Leeds LS9 7TF, UK

Immune-mediated rejection following renal transplantation is essentially centred around two primary mechanisms. The presence of
preformed antibodies against donor human leucocyte antigen (HLA) in sensitised recipients results in hyperacute rejection. Non-self HLA
can also trigger a cell-mediated immune response in which a key event is the direct activation of CD4 + T-cells by non-self HLA class II
molecules expressed on antigen-presenting cells present in the donor organ. Furthermore, recent reports have highlighted an association
between increased MHC class I-related chain A/B (MICA/B) expression in graft tissue and immunological tissue damage and rejection
following renal transplantation(1). Protocols that decrease the expression of MICA/B or class II HLA proteins either pre- or post trans-
plantation would be a potentially useful approach in a live donor setting. Whilst considerable efforts are made to control the recipient’s
immune system post transplant little attention is paid to this aspect in the donor pre-transplant. There are, however, a number of reports
suggesting that pharmacological agents (e.g. statins)(2)as well as dietary products (most notably fish oils)(3) reduce the expression of HLA
class II expression. Whilst administering statins to healthy donors before the transplant may be difficult to justify, the use of dietary
products such as fish oils may be both beneficial and appropriate. The aim of the present study was to develop an in vitro assay in which
the effects of potential immunomodulators on MICA/B and HLA-DR expression were measured. To this end, human T lymphocytes were
stimulated with phytohaemagglutinin (PHA) and HLA-DR and MICA/B expression were measured by antibody staining and two-colour
flow cytometry. Incubation of peripheral blood mononuclear cells with PHA for 3–5 d led to consistently increased HLA-DR expression.
This was found to be the case both in the frequency (% positive) of CD3 + T-cells expressing HLA-DR and in the mean channel
fluorescence (MCF) that estimates antigen density (Table). The expression of MICA/B following PHA stimulation was found to be
considerably lower than HLA-DR and was considered to be too inconsistent for further consideration in this assay (Table). The kinetics of
expression and the required concentration of PHA to give optimal expression as well as alternative stimulators (anti-CD3 stimulation)
were also investigated. This in vitro system is now being used to assess the ability of dietary products to down regulate the expression of
class II HLA-DR expression on T-cells following stimulation.
Table. Expression of HLA-DR and MICA/B on T-cells following in vitro PHA stimulation
CD3/HLA-DR CD3/MICA/B
% Positive MCF % Positive MCF
Unstimulated 8.62 1.32 0.04 3.05
+ PHA 64 407 16.4 8.00

1. Hankey KG, Drachenberg CB, Papadimitriou JC, Klassen DK, Philosophe B, Bartlett ST, Groh V, Spies T & Mann DL (2002). Transplantation 73,
304–306.
2. Kuipers HF, Biesta PJ, Groothuis TA, Neefjes JJ, Mommaas AM & van den Elsen PJ (2005) Hum Immunol 66, 653–665.
3. Hughes DA, Pinder AC, Piper Z, Johnson IT & Lund EK (2000) Am J Clin Nutr 71, Suppl., 343S–348S.
Proceedings of the Nutrition Society (2008), 67 (OCE), E33 doi:10.1017/S0029665108006423

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Dietary intake of nutrients of great interest in immunonutrition to


prevent muscle damage in soccer players

C. Conejos1,2, A. Giner2, J. Mañes1 and J. M. Soriano1


1
Laboratory of Nutrition, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100,
Burjassot, Spain and 2Valencia C,. Ciudad Deportiva, 46980 Paterna, Spain

Soccer is a sport in which players are exposed to long-term periods of training and players need to augment strength and fat-free mass
gains by resistance training, minimize muscle damage and soreness and help to improve endurance. Recently, the incidence of injuries has
increased during and preceding soccer matches, which influences the effectiveness of the team. One study(1)has reported that muscle
injury resulting from intensive exercise triggers the immune response of Ig and complement in serum, and induces the inflammatory
reaction. Although neutrophil reactive oxygen species (ROS) production is thought to play a part in clearance of phagocyte-damaged host
tissue, such as muscle tissue, by exercise(2), oxidative stress has been associated with decreased physical performance, muscular fatigue,
muscle damage, and overshooting inflammation(3). In a well-trained player a good diet can enhance physical and athletic performance and
decrease the incidence of injuries during soccer matches. The repetition of intense exercise diminishes the neutrophil inflammatory
reaction, and the recovery from physical damage may be delayed(4). However, it has been observed that carbohydrate ingestion has only a
minimal influence on the immune response to exercise(5). It has been suggested that 4 weeks of Fe supplementation significantly increases
body Fe stores and inhibits the decrease in Hb concentration induced by soccer training(6). The objective of the present study was to
evaluate the dietary intake of nutrients of relevance to immunonutrition in relation to muscle damage in young high-level soccer players
(group A: n 20, age 17–18 years; group B: n 22, age 20–24 years). The 24 h diet-record method was used by the dietitian of the soccer
club to analyse the dietary intake of arginine, branched-chain amino acids (valine, leucine and isoleucine), Zn, Cu, Fe, Se, Mg and
vitamins A, C and E.
The results showed that 65 % of the players between 20 and 24 years of age had intakes below the Spanish and European recommended
intakes (SERI) for Zn and Mg, whereas approximately 60 % of the youngest players were below the SERI only for Zn. Nevertheless, if
results were compared with recommended values for sportsmen (SRV), > 65 % of both groups failed to achieve the SRV for Fe, Zn and
vitamins A, C and E.
In conclusion, the results show that the group of players studied need to include in their diet foods containing antioxidant vitamins, Fe
and Zn in order to prevent possible muscle damage. Currently, a nutritional education programme is being developed to promote an
appropriate food pattern to aid the reduction of lesions in soccer players.

The work was supported by University of Valencia (UV-AE-20070219).

1. Mashiko T, Umeda T, Yamamoto Y & Sugawara K (2004) Br J Sports Med 38, 617–621.
2. Peake J & Suzuki K (2004) Exerc Immunol Rev 10, 129–141.
3. König D, Wagner KH, Elmadfa I, Berg A (2001). Exercise and oxidative stress: significance of antioxidants with reference to inflammatory, muscular,
and systemic stress. Exerc Immonol Rev 7: 108–33.
4. Rebelo AN, Candeias JR, Fraga MM, Duarte JA, Soares JM, Magalhaes C & Torrinha JA (1998) J Sports Med Phys Fitness 38, 258–261.
5. Bishop NC, Blannin AK, Robson, PJ, Walsh NP & Gleeson M (1999) J Sports Sci 17, 787–796.
6. Kang HS & Matsuo T (2004) Asia Pac J Clin Nutr 13, 353–358.
Proceedings of the Nutrition Society (2008), 67 (OCE), E34 doi:10.1017/S0029665108006435

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Total antioxidant capacity of refrigerated orange juice treated


with pulsed electric fields

C. Cortés, F. Barba, M. J. Esteve, R. González and A. Frı́gola


Nutrition and Food Chemistry, Faculty of Pharmacy, University of Valencia, Avd. Vicent Andres Estelles s/n, Burjassot,
Valencia, Spain

Orange juice is an important source of carotenoids and ascorbic acid, a nutrient that besides its vitamin action is valuable for its
antioxidant effect, stimulation of the immune system and other health benefits that are being actively investigated and reported, such as
inhibition of the formation of cancer-causing N-nitroso compounds in the stomach(1). During processing and/or storage orange juice
undergoes an important number of undesirable effects on some nutrients, antioxidant compounds, colour, flavour and texture. The use of
pulsed electric fields (PEF) is an emerging technology in the field of food preservation(2). The aim of the present work was to study the
antioxidant capacity of the orange juice treated with PEF (30 kV/cm, 100 ms) in comparison with orange juice subjected to conventional
thermal treatments (90 C during 20 s), as well as changes in orange juice stored at 2 and 10 C, using a method adapted from that of Miller
et al.(3). The Trolox equivalent antioxidant capacity (TEAC) of the samples was (mmol Trolox/l) 4.03 (SD 0.04), 3.51 (SD 0.04) and 2.49
(SD 0.20) for untreated, PEF-treated and pasteurized orange juice respectively. Thus, TEAC decreased significantly (P < 0.05) after the
orange juice was processed by both types of treatment; the decrease being greater in the pasteurized juice (38.2%) than in the PEF-treated
juice (12.9 %). Thus, PEF treatment of orange juice had an antioxidant capacity more similar to that of the untreated juice. The total
antioxidant capacities of samples during refrigerated storage are shown in the Table. The results show a decrease with time at both
temperatures, although the decrease was higher in the samples stored at 10 C. Compared with conventional pasteurization, PEF treatment
led to a higher total antioxidant activity of orange juice immediately after processing, as well as during storage at 2–10 C.

TEAC (mmol Trolox/l)


Temperature ( C) Period of storage (weeks) 0 1 2 3 4 6 7
2 PEF juice 3.51 3.22 2.98 2.94 2.33 2.23 2.21
Pasteurized juice 2.49 1.93 2.10 2.40 1.83 1.80 1.80
10 PEF juice 3.51 2.375 2.10 2.37 1.54 1.53 –
Pasteurized juice 2.49 2.02 2.15 2.52 1.74 1.70 –

This study was carried out with funds from the Spanish Ministry of Science and Technology and European Regional Development Funds (ERDF) (Project
AGL-2003–05236-C02–02 and AGL-2006–13320-C03–03).

1. Valkoa M, Rhodesb CJ, Moncola J, Izakovica M & Mazur M (2006) Chem Biol Interact 160, 1–40.
2. Min S, Jin ZT, Yeom H, Min SK & Zhang QH (2003) J Food Sci 68, 1265–1271.
3. Miller NJ, Diplock AT, Rice-Evans C, Davies MJ, Gopinathan V & Milne A (1993) Cli Sci 84, 407–412.
Proceedings of the Nutrition Society (2008), 67 (OCE), E35 doi:10.1017/S0029665108006447

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Examination of host immune resistance against Listeria monocytogenes


infection in cyclophosphamide (CPA)-treated mice after
dietary lipid administration

L. Cruz-Chamorro1, M. A. Puertollano2, E. Puertollano1, C. Carazo3, G. Alvarez de Cienfuegos1


and M. A. de Pablo1
1
Área de Microbiologı́a, Universidad de Jaén, Jaén, Spain, 2Instituto del Frı́o (CSIC), Madrid, Spain and 3Área
de Microbiologı́a y Parasitologı́a Clı́nica, Hospital Médico-Quirúrgico, Jaén, Spain

Numerous investigations have clearly established that the administration of diets containing fish oil (FO) may produce important immu-
nosuppressive effects in both animals and human subjects(1). In fact, this property has been utilized in the reduction of typical symptoms
caused by diseases characterized by an overactivation of the immune response. Nevertheless, prolonged utilization and the administration
of high levels of n-3 PUFA may result in a severe reduction in host immune resistance to infectious micro-organisms(2). The action of
different dietary lipids in severely-immunosuppressed mice was evaluated using a model of chemotherapy in which cyclophosphamide
(CPA) injections, which cause a delay in the onset of acquired cellular resistance, were followed by an enhanced and slightly prolonged
response in Listeria monocytogenes-infected mice(3). Balb/c mice were fed one of four diets, which contained olive oil (OO; 200 g/kg diet;
n 5), FO (200 g/kg diet; n 5), hydrogenated coconut oil (HCO; 200 g/kg diet; n 5) or low fat (LF) for 4 weeks. After the feeding period,
mice were treated with CPA or PBS (control), before infection with L. monocytogenes. Survival analysis and measurement of viable
bacteria counts for spleens and livers and serum pro-inflammatory cytokine levels were carried out. The FO-rich diet reduced survival,
particularly in CPA-treated mice. CPA was responsible for a significant increase in viable bacteria recovery from spleens and livers within
each group fed high-fat diets, which was exacerbated in mice fed an FO diet. In addition, significant increases in both TNFa and IL-12p70
levels were detected in this group. The application of CPA moderately aggravates the immunosuppressive state in FO-fed animals.
Table. Recovery of viable bacteria from spleens and livers of mice fed dietary lipids after a 48 h challenge with L. monocytogenes
L. monocytogenes (log10 CFU )
Spleen Liver
- CPA + CPA - CPA + CPA
Diets Mean SE Mean SE Mean SE Mean SE

LF 3.5c 1.6c 3.8c 1.1 3.0d 0.9 3.2d 1.2


OO 4.3b 2.0 4.6b 1.8 4.5c 2.1 5.0c 1.8
FO 6.3a 2.5 6.7b 1.8 5.9a 1.1 7.0a 2.1
HCO 6.0a 2.1 6.5a 2.6 5.3b 2.0 6.2b 2.3
Values are means with their standard errors represented by vertical bars for three independent experiments after logarithmic (log10)
transformation (five mice per dietary group). CFU, colony-forming units. a,b,c,dMeans within a column with unlike superscript letters
were significantly different (P < 0.05; two-way ANOVA).

These results may be of crucial relevance in clinical nutrition, particularly when n-3 PUFA are administered to patients who are
immunocompromised and at risk of sepsis.

1. Calder PC (2000) Lipids 36, 1007–1024.


2. de Pablo MA, Puertollano MA & Alvarez de Cienfuegos G (2002) Clin Diagn Lab Immunol 9, 945–950.
3. Kerckhaert JA, Hofhuis FM & Willers JM (1977) Immunology 32, 1027–1032.
Proceedings of the Nutrition Society (2008), 67 (OCE), E36 doi:10.1017/S0029665108006459

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effect of dietary oils on oxidative stress and cytokine production


by murine macrophages

R. de la Puerta Vázquez1, M. A. Fernández Arche1, A. Márquez Martı́n2 and V. Ruiz-Gutierrez2


1
Dpto Farmacologı́a, Facultad de Farmacia, Universidad de Seville, Spain and 2Grupo de Nutrición y Metabolismo
Lipı́dico, Instituto de la Grasa, (CSIC), Seville, Spain

Many studies have shown that the nature of the lipid consumed in the diet significantly affects the prevalence of coronary, inflammatory or
autoimmune diseases(1,2). The present study was conducted to compare the impact of feeding mice on diets enriched with edible oils
(150 g/kg diet; fish oil (FO), olive oil (OVO) and orujo olive oil (ORO)) with that of a basal diet (BD) enriched with 20 g maize oil/kg on
the ability to modulate oxidative reactive species and pro-inflammatory mediator generation by stimulated murine macrophages. Swiss
male mice were fed on the different diets for 8 weeks. Diets were formulated according to American Institute of Nutrition (AIN)
recommendations(3). Peritoneal macrophages were isolated from these mice and stimulated. Reactive oxygen (O2– and H2O2) and nitrogen
(NO2–) species, PGE2, TNFa and IL-6 were measured in the supernatant fractions from 106 cells. All test diets down regulated NO
generation compared with the BD (Fig.1); in contrast, FO increased H2O2 generation whereas OVO and ORO group diets significantly
inhibited this ROS production compared with the BD. In addition, ORO was able to decrease O2– formation compared with the BD group
(Fig.1). Finally, both OVO and FO groups significantly decreased PGE2 and cytokine production (Fig. 2). These results are in agreement
with those of other authors in that a diet enriched in olive oil was found to show a protective effect against oxidative stress and
inflammation(4,5)and they confirm the preventive anti-inflammatory properties of FO(6). Moreover, the results provide important additional
data about the ability of ORO to prevent oxidative damage to cells.

75 75 ** 40

nmoles O /2h/1x10 cell


nmol H2O2/2h/1x10 cell

6
6

30
*
NO2 (ng/ml)

50 50
* *
* 20
-2
-

**
*** 25
25
10

0 0 0
BD OVO ORO FO BD OVO ORO FO BD OVO ORO FO

Fig. 1. Reactive oxygen and nitrogen species generated by stimulated murine peritoneal macrophages isolated from mice fed on different oil-enriched diets. Values are means
with their standard errors represented by vertical bars for five animals fed on each diet. Mean values were significantly different from those for BD (ANOVA and Student’s t test):
*P < 0.05, **P < 0.01, ***P < 0.001.

4000 150000
75000
α ] pg/ml

[IL-6] (pg/ml)

3000
PGE2 (pg/ml)

100000
50000 *
[TNF-α

2000
*
* 50000 **
25000
1000
** ***

0 0 0
BD OVO ORO FO BD OVO ORO FO BD OVO ORO FO

Fig. 2. PGE2 and cytokines generated by lipopolysaccharide-stimulated murine peritoneal macrophages isolated from mice fed on different oil-enriched diets. Values are means
with their standard errors represented by vertical bars for five animals fed on each diet. Mean values were significantly different from those for BD (ANOVA and Student’s t test):
*P < 0.05, **P < 0.01, ***P < 0.001.

This study is part of the project AGL2005–00572/AlI, financially supported by the Comision Interministerial de Ciencia y Tecnologia (CICYT).

1. James MJ, Gibson RA & Cleland LG (2000) Am J Clin Nutr 71, 343–348.
2. Yaqoob P (2003) Curr Opin Clin Nutr Metab Care 6, 133–150.
3. Bieri JG, Stoewsand GS, Briggs GM, Philips RW, Woodward JC & Knapka JJ (1977) J Nutr 107, 1340–1348.
4. Moreno JJ, Carbonell T, Sanchez T, Miret S & Mitjavila MT (2001) J Nutr 31, 2145–2149.
5. Perona JS, Arcemis C, Ruiz-Gutierrez V & Catala A (2005) J Agric Food Chem 53, 730–735.
6. Calder PC (2006) Am J Clin Nutr 86, 1505–1519.
Proceedings of the Nutrition Society (2008), 67 (OCE), E37 doi:10.1017/S0029665108006460

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Seasonal changes in the neuroimmunoendocrine system activity in


young adult male rats administered with a balanced controlled diet
from weaning

A. I. Esquifino1, M. P. Fernandez-Mateos2, V. Jimenez1, J. Rios1 and P. Cano1


Departamentos de 1Bioquimica y Biologia Molecular III and 2Biologia Celular de Medicina, Universidad Complutense,
Madrid, Spain

Temporal changes in the activity of the neuroimmunoendocrine system maintain homeostasis in living organisms(1,2). The present work
analyses the 24 h changes in the activity of the neuroimmunoendocrine system in young adult male rats (3 months old) kept under
standard conditions of controlled light (fluorescent cool white bulbs providing 100 lux intensity at the level of the cages) with 12 h light
- 12 h dark (light on at 08:00 hours) at 22°2 C and ad lbitum access to a balanced diet (AIN-93G; Diets Inc., Pennsylvania, USA) and
water. The conditions were identical for all seasons studied. Animals were killed by decapitation at six different time intervals (every 4 h
throughout a single 24 h period) during spring, summer, autumn or winter time periods taking as an index the regulatory mechanism of
prolactin, a hormone that is involved in the development and maintenance of immune function. The median eminence dopamine con-
centration, plasma prolactin levels and submaxillary lymph node lymphocyte proliferative capacity were studied. Globally, dopamine
concentration in the median eminence showed specific 24 h variation depending on the season studied. Changes in plasma prolactin levels
were in accordance with variations in dopamine. Likewise, proliferative capacity of lymphocytes from the submaxillary lymph nodes
exhibited specific 24 h variation according to the season (Figure). There were specific seasonal correlations between dopamine, prolactin
and the proliferative capacity of the lymphocytes. This outcome may suggest the existence of a seasonal signal that could allow indivi-
duals to adapt their physiological functions to the annual environmental changes.
120 300
Spring Autumn
Proliferation Index/Number of

Proliferation Index/Number of

250

80 200
Cells x 10 -5

Cells x 10 -5

150

40 100

50

0 0
9:00 13:00 17:00 21:00 1:00 5:00 9:00 9:00 13:00 17:00 21:00 1:00 5:00 9:00
Hou rs Hours

10
Summer
Proliferation Index/Number of

60
Winter
Proliferation Index/Number of

8
50
Cells x 10 -5

6 40
Cells x 10 -5

30
4
20
2
10

0 0
9:00 13:00 17:00 21:00 1:00 5:00 9:00 9:00 13:00 17:00 21:00 1:00 5:00 9:00
Hours Hou rs
Figure. Proliferative capacity of T lymphocytes stimulated with concanavalin A during spring, summer, autumn and winter measured as proliferative index (stimulated cpm/
unstimulated cpm).Values are means with their standard errors represented by vertical bars for eight animals per group.

1. AI Esquifino, D Pazo, RA Cutrera & DP Cardinali (1999) Chronobiol Int 16, 451–460.
2. N Vazquez, E Dı́az, C Fernández, V Jiménez, AI Esquifino & B Dı́az (2007) Physiol Res 56, 79–88.
Proceedings of the Nutrition Society (2008), 67 (OCE), E38 doi:10.1017/S0029665108006472

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effect of long-chain fatty alcohols from orujo olive oil on nitric oxide
and eicosanoid generation

M. A. Fernández Arche1, R. de la Puerta Vázquez1, A. Márquez Martı́n1 and V. Ruiz-Gutierrez2


1
Department of Pharmacology, School of Pharmacy, University of Seville, C/ Profesor Garcı́a Gonzalez No. 2
and 2Instituto de la Grasa (CSIC), Av. Padre Garcı́a Tejero No. 4, 41012 Seville, Spain

Olive pomace oil (‘orujo’ oil) is an olive oil product suitable for human consumption that is traditionally produced in Spain(1). The non-
acylglycerol component of this oil is a good source of interesting minor components, e.g. triterpenes(2), or fatty alcohols, derived from
waxy materials. Tetracosanol (C24OH; 30 %), hexacosanol (C26OH; 37 %) and octacosanol (C28OH; 15 %) are the major constituents of
the long-chain fatty alcohol (LCFA) fraction isolated from orujo olive oil(3). A similar mixture of long-chain alcohols, termed ‘polico-
sanol’ and purified from waxy materials of different sources such as sugar cane, bees wax, rice bran or spinach, have shown many
beneficial physiological activities(4,5). The present study focused on the effect of LCFA isolated from orujo olive oil on NO, PGE2 and
TNFa release by a lipopolysaccharide (LPS)-stimulated murine macrophage cell line (RAW-264.7) as well as the effect on thromboxane
B2 (TXB2) generation by A-23187-stimulated rat peritoneal neutrophils (PMN). Nitrite (as an index of NO generation) levels were
determined by a fluorometric method. PGE2, TNFa and TXB2 production were quantified by sandwich immunoassay.
LCFA significantly and dose-dependently decreased the NO production in LPS-stimulated RAW-264.7 cell line macrophages (Fig. 1).
Western-blot analysis for inducible NO synthase (iNOS) showed that NO reduction was a consequence of the 100% inhibition of iNOS
expression at a dose of 100 mg/ml (Fig. 2). By contrast, LCFA scarcely affected PGE2 levels (Fig. 1). TNFa production was also
significantly decreased by LCFA at the highest dose assayed (100 mg/ml; Fig. 1). LCFA significantly reduced TXA2 production in rat
PMN stimulated with A-23187 (Fig. 3).

750 30 500

 400

TNF-α (pg/ml)
 cells alone cells alone
PGE 2 (ng/ml)

cells alone
NO2 (ng/ml)

500 20  300
LP S LPS

LP S
200
250 10 25 LCHFA 25
25 LC H FA 100 LCHFA
5 0 (µg/ml)  50
(µg/ml)
 50 ( µg/m l) 1 00
10 0 100 0

25

50

100
cells alone

LPS
0 0
25

50

25

50
cells alone

cells alone
100

100
LPS

LPS

Fig. 1. Effect of LCFA on NO, PGE2 and TNFa produced by LPS (10 mg/ml)-stimulated RAW-264.7 murine macrophages (1 · 106 cells/ml). Mean values were significantly
different from those for LPS control group: **P < 0.01, ***P < 0.001.

2 0 0
pg/ml TXB2

*
*
1 0 0 **

0
A 2 3 1 8 7 25 50 100
(1 µ M )

L C F A
( µ g /m l)

Fig. 2. Effect of LCFA subfraction on iNOS expression Fig. 3. Effect of LCFA on TXB2 produced by A-23187-stimulated rat PMN. Mean
and densitometric analysis in RAW 264.7 cells. DEX, values were significantly different from the control value: *P < 0.05, ***P < 0.001.
dexamethasone; OD, optical density.

These results showed that LCFA isolated from ‘orujo’ oil has a protective effect on some mediators implicated in the development of
inflammatory damage in these experimental models and suggest its potential value as a functional component of the olive pomace oil.

This study is part of the project AGL2005–00572/ALI, financially supported by the Comision Interministerial de Ciencia y Tecnologia (CICYT).

1. Perona JS, Aracemis C, Ruiz-Gutierrez V & Catalá A (2005) J Agric Food Chem 53, 730–735.
2. Perez Camino MC & Cert A (1999) J Agric Food Chem 47, 1558–1562.
3. Marquez A (2007) Doctoral Thesis, Universidad de Sevilla.
4. Taylor JC, Rapport L & Lockwood GB (2003) Nutrition 19, 192–195.
5. Singh DK, Li L & Porter TD (2006) J Pharmacol Exp Ther 318, 1020–1026.
Proceedings of the Nutrition Society (2008), 67 (OCE), E38 doi:10.1017/S0029665108006472

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effect of long-chain fatty alcohols from orujo olive oil on nitric oxide
and eicosanoid generation

M. A. Fernández Arche1, R. de la Puerta Vázquez1, A. Márquez Martı́n1 and V. Ruiz-Gutierrez2


1
Department of Pharmacology, School of Pharmacy, University of Seville, C/ Profesor Garcı́a Gonzalez No. 2
and 2Instituto de la Grasa (CSIC), Av. Padre Garcı́a Tejero No. 4, 41012 Seville, Spain

Olive pomace oil (‘orujo’ oil) is an olive oil product suitable for human consumption that is traditionally produced in Spain(1). The non-
acylglycerol component of this oil is a good source of interesting minor components, e.g. triterpenes(2), or fatty alcohols, derived from
waxy materials. Tetracosanol (C24OH; 30 %), hexacosanol (C26OH; 37 %) and octacosanol (C28OH; 15 %) are the major constituents of
the long-chain fatty alcohol (LCFA) fraction isolated from orujo olive oil(3). A similar mixture of long-chain alcohols, termed ‘polico-
sanol’ and purified from waxy materials of different sources such as sugar cane, bees wax, rice bran or spinach, have shown many
beneficial physiological activities(4,5). The present study focused on the effect of LCFA isolated from orujo olive oil on NO, PGE2 and
TNFa release by a lipopolysaccharide (LPS)-stimulated murine macrophage cell line (RAW-264.7) as well as the effect on thromboxane
B2 (TXB2) generation by A-23187-stimulated rat peritoneal neutrophils (PMN). Nitrite (as an index of NO generation) levels were
determined by a fluorometric method. PGE2, TNFa and TXB2 production were quantified by sandwich immunoassay.
LCFA significantly and dose-dependently decreased the NO production in LPS-stimulated RAW-264.7 cell line macrophages (Fig. 1).
Western-blot analysis for inducible NO synthase (iNOS) showed that NO reduction was a consequence of the 100% inhibition of iNOS
expression at a dose of 100 mg/ml (Fig. 2). By contrast, LCFA scarcely affected PGE2 levels (Fig. 1). TNFa production was also
significantly decreased by LCFA at the highest dose assayed (100 mg/ml; Fig. 1). LCFA significantly reduced TXA2 production in rat
PMN stimulated with A-23187 (Fig. 3).

750 30 500

 400

TNF-α (pg/ml)
 cells alone cells alone
PGE 2 (ng/ml)

cells alone
NO2 (ng/ml)

500 20  300
LP S LPS

LP S
200
250 10 25 LCHFA 25
25 LC H FA 100 LCHFA
5 0 (µg/ml)  50
(µg/ml)
 50 ( µg/m l) 1 00
10 0 100 0

25

50

100
cells alone

LPS
0 0
25

50

25

50
cells alone

cells alone
100

100
LPS

LPS

Fig. 1. Effect of LCFA on NO, PGE2 and TNFa produced by LPS (10 mg/ml)-stimulated RAW-264.7 murine macrophages (1 · 106 cells/ml). Mean values were significantly
different from those for LPS control group: **P < 0.01, ***P < 0.001.

2 0 0
pg/ml TXB2

*
*
1 0 0 **

0
A 2 3 1 8 7 25 50 100
(1 µ M )

L C F A
( µ g /m l)

Fig. 2. Effect of LCFA subfraction on iNOS expression Fig. 3. Effect of LCFA on TXB2 produced by A-23187-stimulated rat PMN. Mean
and densitometric analysis in RAW 264.7 cells. DEX, values were significantly different from the control value: *P < 0.05, ***P < 0.001.
dexamethasone; OD, optical density.

These results showed that LCFA isolated from ‘orujo’ oil has a protective effect on some mediators implicated in the development of
inflammatory damage in these experimental models and suggest its potential value as a functional component of the olive pomace oil.

This study is part of the project AGL2005–00572/ALI, financially supported by the Comision Interministerial de Ciencia y Tecnologia (CICYT).

1. Perona JS, Aracemis C, Ruiz-Gutierrez V & Catalá A (2005) J Agric Food Chem 53, 730–735.
2. Perez Camino MC & Cert A (1999) J Agric Food Chem 47, 1558–1562.
3. Marquez A (2007) Doctoral Thesis, Universidad de Sevilla.
4. Taylor JC, Rapport L & Lockwood GB (2003) Nutrition 19, 192–195.
5. Singh DK, Li L & Porter TD (2006) J Pharmacol Exp Ther 318, 1020–1026.
Proceedings of the Nutrition Society (2008), 67 (OCE), E39 doi:10.1017/S0029665108006484

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Fermented soyabean products as hypoallergenic food

J. Frias1, Y. S. Song2, C. Martı́nez-Villaluenga1, E. Gonzalez de Mejia2 and C. Vidal-Valverde1


1
Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain and 2Department of Food
Science and Human Nutrition, University of Illinois Urbana-Champaign, 228 ERML, MC-051, 1201 W Gregory Drive,
Urbana, IL 61801, USA

Food allergy has become a public health problem that continues to challenge both the public and the food industry, and soyabean is
causative of food allergy. The allergenicity of food proteins has been shown to be modified by food-processing technologies such as
fermentation. The objective of the present research was to ferment soyabeans as flour (liquid fermentation) or as cracked seeds (solid
fermentation) using different micro-organisms in order to study the effect on immunoreactivity after fermentation. Immunochemical
methods have been developed and validated for the detection and quantification of the major human allergenic soyabean proteins, and a
comparison with fermented soyabean proteins has been carried out. ELISA and Western blot were used to quantify IgE antibody response
and HPLC to identify and quantify total amino acids. The extractable protein concentration decreased after fermentation (from 31 % for
cracked seeds fermented with Bacillus subtilis to 74 % with Aspergillus oryzae). Lactobacillus plantarum- and naturally-fermented
soyabean flour showed a higher reduction in IgE immunoreactivity (£ 92 % and 97 %) in comparison with the corresponding fermented
products of cracked seeds (92 % and 88 % reduction respectively). Among the solid fermented products, the lowest performance was given
by mould strains, Rhizopus oryzae and A. oryzae, which showed a reduction in allergenic values of 66 % and 71 % respectively. Although
B. subtilis showed the highest soluble protein concentration (221.5 mg protein/g product), this fermented soyabean product yielded a 75 %
and 86 % reduction in immunoreactivity against human plasma and human pooled plasma samples respectively.
The raw soyabean flour presented the highest complexity on protein profile and immunoreactivity. Human plasma samples presented an
intense immunoreactivity towards specific immunodominant proteins. Fermentation with B. subtilis or L. plantarum and natural fermen-
tation yielded peptides of smaller size and less-intense immunoreactivity. Moreover, soyabeans subjected to fermentation processes
showed rises for most of the non-essential and essential amino acids (P £ 0.05). In conclusion, fermentation applied to soyabean induces a
noticeable variation in protein profile and allergenicity and there is potential for developing nutritious hypoallergenic soya products.

120
LIQUID STATE
Ig E immunoreactivity reduction (%)

100 L. plantarum (soy flour)


L. plantarum (cracked soy)
80 Natural (soy flour)
Natural (cracked soy)
60

40 SOLID STATE
A. oryzae (cracked soy)
20
R. oryzae (cracked soy)
0 B. subtilis (cracked soy)
Soybean Liquid State Solid State
seed fermentation fermentation
Figure 1. Effect of fermentation of soybean seeds and soya fermented products on immune response by ELISA.

This work was partly funded by AGL2004-0886ALI and by USDA-Future Foods Initiative.
Proceedings of the Nutrition Society (2008), 67 (OCE), E40 doi:10.1017/S0029665108006496

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Immunomodulatory properties of Lactobacillus salivarius are


not limited to the intestine

J. Galvez1, B. Arribas1, M. E. Rodrı́guez-Cabezas1, E. Bailon1, M. Comalada1, D. Camuesco1,


J. Xaus2 and A. Zarzuelo1
1
Department of Pharmacology, University of Granada, Granada, Spain and 2Puleva Biotech, SA, Granada, Spain

Previous studies have shown the beneficial effects exerted by probiotics on inflammatory bowel disease(1), an intestinal condition char-
acterized by an altered intestinal immune response(2). However, it would be interesting to know whether the immunomodulatory prop-
erties of probiotics are restricted to a local effect in the intestine or whether their effect can also be extrapolated to other systemic immune
alterations. The aim of the present study was to test the effect of a probiotic, Lactobacillus salivarius CECT5713, in two experimental
models of local or systemic altered immune response, i.e. the trinitrobenzenesulfonic acid (TNBS) model of rat colitis and the lipopo-
lysaccharide (LPS)-induced septic shock in mice. For this purpose, mice or rats (n 10) were given the probiotic (5 · 108 colony-forming
units/ml drinking water), starting 2 weeks before damage induction. A control group (n 10) without probiotic was also used for reference.
Colitis was induced in rats by intracolonic administration of TNBS (10 mg) and after 1 week was evaluated both histologically and
biochemically (myeloperoxidase activity, glutathione content, inducible NO synthase (iNOS) expression)(3). Septic shock was induced in
mice by administering LPS (40 mg/kg, intraperitoneally) and the mice killed 24 h later, when colon and spleen were removed. Colonic
iNOS expression was determined by Western blot, and activated T-cells were obtained from spleens by concanavalin A incubation and the
immune response evaluated by RT–PCR or ELISA for different cytokines (IL-2, -5, -6 and -10). The results showed that L. salivarius was
able to ameliorate both the local and systemic altered immune response. The probiotic exerted intestinal anti-inflammatory activity, since
it significantly reduced the extension of the colonic damage induced by TNBS in comparison with non-treated colitic rats; this effect was
accompanied by a 42 % reduction in myeloperoxidase activity (P < 0.05), a 44 % increase in glutathione content (P < 0.05) and a reduction
in colonic iNOS. Moreover, the probiotic treatment significantly prevented the increase in colonic weight (mg/cm) induced by septic
shock (264 (SE 15) v. 322 (SE 15); P < 0.05), without showing differences from normal mice (246 (SE 14)). Similarly, the LPS-induced
colonic iNOS expression was lower in the probiotic-treated mice (30 %). LPS also stimulated the expression of different cytokines assayed
in the splenocytes, while the probiotic-treated mice showed a reduction in cytokine expression of 80 % for IL-5 and 100% for both IL-2
and IL-6 (Figure 1). IL-10 secretion was reduced in control mice (603 (SE 102) pg/ml; P < 0.05) in comparison with the normal group
(1064 (SE 80) pg/ml), which was increased in probiotic-treated mice (1034 (SE 150 pg/ml; P < 0.05) when compared with the LPS control
group. In conclusion, the immunomodulatory properties of the probiotic L. salivarius are not restricted to the intestine, since it is also able
to ameliorate the alteration in the systemic immune response derived from LPS administration to mice.

Figure 1. Lactobacillus salivarius administration reduced cytokine expression (RT-PCR) in LPS-induced septic shock in mice.

1. Ewaschuk JB & Dieleman LA (2006) World J Gastroenterol 12, 5941–5950.


2. Hanauer SB (2006) Inflamm Bowel Dis 12, S3–S9.
3. Peran L, Sierra S, Comalada M et al. (2007) Br J Nutr 97, 96–103.
Proceedings of the Nutrition Society (2008), 67 (OCE), E41 doi:10.1017/S0029665108006502

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Vitamin E as an IgE inhibitor: stability during cold storage of


human milk

G. Garcı́a-Llatas1, R. Lacomba1, F. Ortega1, A. Alegrı́a1, R. Barberá1 and D. Silvestre2


1
Nutrition and Food Chemistry, University of Valencia, Avda. Vicente Andrés Estellés s/n, 46100 Burjassot, Valencia, Spain
and 2Food Science, Cardenal Herrera-CEU University, Avda. Seminario s/n, 46113 Moncada, Valencia, Spain

Human breast milk is recommended as the unique food for neonates based on its known properties. When the production of milk by the
mother is not sufficient or the mother is not able to feed her child for professional reasons, milk banks or the mothers’ practice of
collecting their own milk are the existing alternatives for breast-feeding. In both situations cold storage (refrigeration or freezing) can be
used in neonatal units, at home and in human milk banks(1,2).
Many micronutrients with immunological properties are found in breast milk. In addition to its antioxidant activity, vitamin E has been
shown to be associated with a decrease in the frequency of allergen sensitisation by the inhibition of IgE responses to allergic stimuli;
asthma, rhinitis and hayfever are atopic disorders characterised by raised serum IgE and skin sensitisation to common environmental
allergens(3).
The stability of vitamin E contents can be influenced by temperature and time of storage. Thus, the aim of the present study was the
evaluation of vitamin E contents during cold storage of human milk. Fourteen samples of mature human milk from healthy and well-
nourished women were collected from a breast-feeding workshop. Each sample was divided in three aliquots, one of which was analysed
just after collection (F) and the others were stored under the conditions used at home (refrigerating at 2–4 C for 48 h; R) and at milk banks
(freezing at - 18 C for 15 d; Fz).
a-, b-, g- and d-Tocopherols were quantified simultaneously in the milk samples by a normal-phase HPLC method with fluorescence
detection(4) (four determinations per sample). The results (ranges) are shown in the Table.

Tocopherols
(mg/100 ml human milk) F R Fz
a 209.9–264.0 169.3–223.4 193.2–247.3
b 34.8–57.5 44.1–68.1 25.0–47.7
g 54.2–66.0 49.6–61.4 55.9–67.8
d 9.25–18.0 14.1–22.8 9.43–18.2

One-way (storage temperature) ANOVA applied to the results showed no significant differences (P < 0.05) between the treatments
studied. Thus, the inhibition of IgE responses provided by vitamin E from human milk is maintained after it is refrigerated or stored
frozen.

This study is part of the project PRUCH 03/36 (2003–2004) supported financially by Cardenal Herrera-CEU University.

1. Human Milk Banking Association of North America (2006) Best Practice for Expressing, Storing and Handling Human Milk in Hospitals, Homes and
Child Care Settings, 2nd ed. [F Jones and MR Tully, editors]. Raleigh, NC: HMBANA.
2. Lindemann PC, Foshaugen I & Lindemann R (2004) Arch Dis Child Fetal Neonatal Ed 89, F440–F441.
3. Fogarty A, Lewis DS, Weiss S & Britton J (2000) Lancet 356, 1573–1574.
4. Rodrigo N, Alegrı́a A, Barberá R & Farré R (2002) J Chromatogr 947A, 97–102.
Proceedings of the Nutrition Society (2008), 67 (OCE), E42 doi:10.1017/S0029665108006514

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Melatonin supplementation modifies experimental chronic colitis in mice

M. Golab, M. Dudziak and K. Skwarlo-Sonta


Department of Animal Physiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland

Melatonin (Mel) has been adopted by most living organisms as a circadian timing signal, but is also known to be a potent free radical
scavenger and immunomodulatory agent(1). Mel is not only synthesized in the pineal gland, but also in the gastrointestinal tract (GIT),
where it does not depend on the light–dark cycle and may be induced by food intake or withdrawal. GIT-derived Mel may also have
paracrine and autocrine functions, modifying the intestinal blood flow or mucosal regeneration(2). The potent free radical scavenging
activity of Mel may give rise to its protective and therapeutic application in chronic inflammation of the GIT(3).
The aim of the present study was to examine the immunomodulatory properties of Mel when administered differently in experimental
colitis in mice. Experiments were conducted on 6–8-week-old male BALB/c mice (five to six individuals per group) housed in standard
laboratory conditions (22 C, 12 h light–12 h dark). Chronic colitis was induced by administration of dextran sodium sulfate (DSS; 0.06 g
per mouse per d) in the drinking water for 2 or 3 weeks. From the beginning of the experiment some groups of mice were simultaneously
(preventively) treated with Mel (10 mg/kg body weight) by oral administration (Expt 1) or in the drinking water during the night (Expt 2).
Another group of mice received Mel therapeutically after showing the first clinical symptoms of colitis (positive occult blood tests, rectal
bleeding). Mice were killed by CO2 asphyxia, colons were isolated and proximal and distal fragments used for histological tests.
Additionally, spleens were isolated and splenocytes cultured in the presence of mitogens. Colon mucosa was gently scraped into a
protease-inhibitor solution, centrifuged and supernatant fractions used for the measurement of cytokine concentrations using flow cyto-
metry with a mouse T-helper (Th1)/Th2 cytokine cytometric bead array kit (Becton Dickinson, Franklin Lakes, NJ, USA).
In control mice (without colitis) Mel caused immune cell proliferation in gut lymphatic nodules without any change in cytokine
concentration and crypt structure. DSS administration resulted in an elevation in mucosal TNFa, a decrease in IL-2, subsequent shortening
and crypt loss as well as intensive mononuclear infiltration to the mucosa. Preventive Mel supplementation caused no changes in TNFa
concentration in DSS-treated mice, but completely reversed the DSS-induced decrease in mucosal IL-2 content. Mel did not prevent
inflammatory infiltration, but crypt loss was located either in the proximal or distal part of the colon, depending on the period of Mel
treatment. Therapeutic Mel administration did not change IL-2 concentration but caused a significant decrease in TNFa and interferon-g
concentrations. No significant changes in Th2-specific cytokines (IL-4, IL-5) after preventive or therapeutic administration of Mel were
observed. In Expt 1 possible Mel-induced systemic changes in immunity were perturbed by the stress associated with oral administration.
Splenocyte proliferation was changed by colitis and Mel in Expt 2, as a result of the less-invasive method of Mel treatment. Both B- and
T-cell proliferation were decreased by Mel in mice without colitis. Splenocyte proliferation was elevated in the DSS-treated animals and
decreased by Mel only if administered preventively.
These results show a possible protective action of Mel in the GIT, but its effects are dependent on the method and period of
administration. Further experiments are in progress.

Supported by MNiN grant 2P04C 117 29 and by Faculty of Biology, University of Warsaw intramural grant BW #1720/20 to M.G.

1. Altun A, Ugur-Altun B (2007) Int J Clin Pract 61, 835–845.


2. Bubenik GA (2002) Dig Dis Sci 47, 2336–2348.
3. Nosál’ová V, Zeman M, Cerná S, Navarová J, Zakálová (2007) J Pineal Res 42, 364–370.
Proceedings of the Nutrition Society (2008), 67 (OCE), E43 doi:10.1017/S0029665108006526

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effect of dietary supplementation on lymphocytes subsets and


liver-related variables in hospitalised patients with anorexia nervosa

S. Gómez1, L. E. Dı́az1, E. Nova1, G. Morandé2 and A. Marcos1


1
Departamento de Nutrición y Bromatologı́a, Instituto del Frı́o (CSIC) 28040 Madrid, Spain and
2
Servicio de Psiquiatrı́a, Hospital Niño Jesús, 28009 Madrid, Spain

Medical complications are common and often serious in patients with eating disorders such as anorexia nervosa (AN)(1). Several studies
have described an increase in serum liver enzymes in severely-malnourished patients who are affected by AN(2)and in the refeeding phase
of therapeutic intervention(3,4). It is also considered that, the lymphocyte subset ratios CD4:CD8 and CD2:CD19 are indexes of mal-
nutrition(5). Thus, a study was carried out that aimed to evaluate the effect of a dietary supplement (Pentadrink fibra; Nutricia SA, Madrid,
Spain; energy density 6.3 kJ/ml) on some liver-related and immunological variables. The supplement was used in order to increase the
energy content of the refeeding diet administered to the patients with AN during their admission to the hospital. Sixty patients with AN
were divided into three groups: (1) without oral supplementation (ANWP; n 21); (2) with 200 ml oral supplementation (ANP; n 19); (3)
with 400 ml oral supplementation (ANPP; n 19). The results were compared with those obtained from a control group matched for gender,
age and socio-economic status. Energy intake and energy profile were evaluated for each group. Liver-related variables (glutamic–
oxaloacetic transaminase (GOT), glutamic–pyruvic transaminase (GPT), g-glutamyltransferase (GGT), alkaline phosphatase (AP) and
bilirubin) and immunological variables (CD4 + , CD8 + , CD2 + , CD19 + cell numbers and ratios) were determined. All variables were
assessed on admission to (time 0) and discharge from hospital (time 1).
The mean initial energy intake (kJ/d) of the patients was 6145 (SD 560, 6947 (SD 594), 11520 (SD 878) in the ANWP, ANP and ANPP
groups respectively. At discharge the energy intake (kJ/d) was: ANWP, 12218 (SD 1150); ANP, 13188 (SD 1283); ANPP, 14154 (SD 1317).
CD8 + in ANPP, CD2 + in ANP and ANPP, and CD19 + in all three groups showed a significant decrease during the study. These
changes led to an increase in the CD2:CD19; however, the increase was only significant in the ANPP group (see Figure). Both CD4:CD8
and CD2:CD19 were always within normal values. Although GOT and AP showed a significant increase in the ANPP group, the levels
were also maintained within normal values. The results show that the inclusion of the energy supplement in the refeeding therapy of
patients with AN does not produce adverse effects on the liver enzymes or immunological variables measured. However, special care
should be taken with those patients with AN at risk of suffering hepatic disease, particularly if the treatment is prolonged.

ratio
12

10
C
8 ANWP (0)
ANWP (1)
6 *
ANP (0)
4 ANP(1)
ANPP(0)
2 ANPP(1)
0
CD4/CD8 CD2/CD19

Figure. Mean values were significantly different from those for the controls (C) (Student’s t test): *P < 0.05. Mean values were significantly different from those for time 1
(Student’s t test): †P < 0.05. (0), admission to hospital; (1), discharge from hospital.

This work has been supported by Nutricia (Spain).

1 Mitchell JE & Crow S (2006) Curr Opin Psychiatry 19, 438–443.


2 Jones SC, Pieri LF & Losowsky MS (1999) Eur Eat Disord Rev 7, 28–36.
3 Ozawa Y, Shimizu T & Shishiba Y (1998) Intern Med 37, 32–39.
4 De Caprio C, Alfano A, Senatore I, Zarrella L, Pasanisi F & Contaldo F (2006) Nutrition 22, 572–575.
5 Marcos A (2007) Nutrition 13, 853–862.
Proceedings of the Nutrition Society (2008), 67 (OCE), E44 doi:10.1017/S0029665108006538

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Borage (Borago officinalis) oil supplementation in relation to monocyte


chemoattractant protein 1 expression in healthy subjects

M. Xiang1, E. Pinto1, M. A. Rahman1, M. Leach1,2 and L. S. Harbige1,2


1
Centre for Biosciences Research, School of Science, University of Greenwich, Kent ME4 4TB, UK and 2Medway School of
Pharmacy, University of Kent and University of Greenwich, Kent ME4 4TB, UK

Essential fatty acids (EFA) have unique roles as precursor molecules of chemical regulators of inflammatory cell function(1–3). In
comparison with linoleic acid (18: 2n-6), g-linolenic acid (GLA; 18: 3n-6) may have superior biopotency because the GLA bypasses the
D6 desaturation, which is regarded as a key regulatory rate-limiting enzymic step controlling the formation of long-chain (LC) PUFA(4,5).
The present study investigated GLA-rich borage oil supplementation in relation to the monocyte chemoattractant protein 1 (MCP-1;
CCL2) expression from peripheral blood mononuclear cells (PBMC) at the gene and protein levels in human subjects. Seven healthy
volunteers who ingested 14 g borage oil/d consecutively for 13 weeks were studied. It was found that the MCP-1 production from both
unstimulated and phytohaemagglutinin (PHA)-stimulated PBMC was reduced during the time-course of the intervention. Furthermore,
MCP-1 from the PHA-stimulated PBMC decreased significantly during the 13 weeks of the intervention period. In addition, the level of
PBMC MCP-1 gene expression was reduced significantly during the supplementation. A significant positive correlation was found
between the expression of MCP-1 gene and MCP-1 production from both unstimulated (r 0.40, P < 0.05; Figure (A)) and PHA-stimulated
PBMCs (r 0.66, P < 0.001; Figure (B)).
The study has, for the first time, revealed that GLA-rich borage oil supplementation in human subjects results in the inhibition of PBMC
MCP-1 expression at the gene and protein levels. The suppressive effect of GLA-rich borage oil on PBMC MCP-1 expression may be
beneficial to chronic inflammatory diseases.
800
MCP-1 from unstimulated PBMCs (pg/ml)

A
600

400

200

0
0 5 10 15 20 25 30 35

1200
MCP-1 from PHA-stimulated PBMCs (pg/ml)

1000 B

800

600

400

200

0 10 20 30 40

MCP-1 gene expression

1. Harbige LS (2003) Lipids 38, 323–341.


2. Kast RE (2001) Int Immunopharmacol 1, 2197–2199.
3. Belch JJ & Hill A (2000) Am J Clin Nutr 71, Suppl., 352S–356S.
4. Xiang M, Harbige LS & Zetterstrom R (2007) Acta Paediatr 96, 387–390.
5. Xiang M, Rahman MA, Ai H, Li X & Harbige LS (2006) Ann Nutr Metab 50, 492–498.
Proceedings of the Nutrition Society (2008), 67 (OCE), E45 doi:10.1017/S002966510800654X

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Vitamin D deficiency among older adults in England remains


a cause for concern!

V. Hirani, A. Ali and K. Tull


Department of Epidemiology and Public Health, Royal Free and University College London Medical School,
University College London, London, UK

The importance of vitamin D in Ca absorption and metabolism for bone health is well known. Vitamin D deficiency in adults clinically
manifests as osteomalacia and osteoporosis. There is also now emerging evidence on how vitamin D may modify the risk of several
chronic diseases, such as cancers and CVD and is protective against type 1 diabetes mellitus, multiple sclerosis and rheumatoid arthritis.
These effects may be related to newly-proposed biological roles for vitamin D such as having a modulating function for immunity(1) and
regulating the growth of many tissues other than bone(2). Concerns about vitamin D deficiency, its role and importance, have been raised
by the WHO. In the UK since 1998 government agencies have emphasised the importance of vitamin D in the elderly(3). In 2000 the
Health Survey for England (HSE) showed the prevalence of vitamin D deficiency (serum concentrations of £ 25 nmol 25-hydro-
xycholecalciferol/l) to be 15 % in women and 10 % in men living in private households.The aim of the present study was to assess vitamin
D status of individuals aged ‡ 65 years, and make comparisons with earlier surveys (HSE 2000 and the National Diet and Nutrition survey
(NDNS) data collected in 1994) and examine associations between vitamin D deficiency and risk factors.
A valid vitamin D sample was obtained from 2070 informants (950 men and 1120 women) as part of the HSE 2005, a nationally-
representative survey of individuals aged ‡ 65 years living in private households in England. Analysis included 1160 informants (524 men
and 636 women), excluding those on medication that would affect the vitamin D status and those taking vitamin supplements.
The prevalence of vitamin D deficiency (£ 25 nmol 25-hydroxycholecalciferol/l) was 20 % in women and 12 % in men. When a higher
threshold of < 50 nmol/l was used (defined as vitamin D insufficiency) 62 % of women and 57 % of men were vitamin D insufficient.
Regression analyses showed that women were more vitamin D deficient than men (OR 1.7) and that vitamin D deficiency increased with
age (OR 1.7 for those aged 70–74 years and OR 3.2 for those aged ‡ 85 years), was more likely for those who smoked cigarettes (OR
3.1), was more prevalent in the winter and autumn (OR 2.7) and was associated with poor general health (OR 1.9). Separate analysis for
each gender showed that among men vitamin D deficiency was also associated with vitamin B12 deficiency (OR 1.9) and was more likely
in those with cancer (OR 2.2). In women deficiency was also 50 % more likely among those in manual social classes and 90 % more likely
in obese women (BMI > 30 kg/m2).
Overall, the results show no significant improvements in vitamin D status in comparison with earlier HSE 2000 results or the NDNS
results. Low vitamin D status shows an association with many risk factors and poor general health outcomes. Further action and guidance
is required to actively address vitamin D deficiency among the elderly.

1. Hayes CE, Nashold FE, Spach KM & Pedersen LB (2003) Cell Mol Biol (Noisy-le-Grand) 49, 277–300.
2. Moan J, Lagunova Z & Porojnicu A (2005) Sunlight, Vitamin D and Health, p. 38 [O Gillie, editor]. http://www.healthresearchforum.org.uk/reports/
sunbook.pdf
3. Department of Health (1998) Nutrition and Bone Health with Particular Reference to Calcium and Vitamin D. Report on Health and Social Subjects
no. 49. London: The Stationery Office.
Proceedings of the Nutrition Society (2008), 67 (OCE), E46 doi:10.1017/S0029665108006551

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Evaluation of diet, anthropometry and immunocompetence of


young male athletes

D. Lacerda1, V. Cotta-de-Almeida2, A. Montero3, W. Savino1 and A. Marcos4


1
Laboratory on Thymus Research, Dept of Immunology and 2Dept of Ultrastructure and Cell Biology, Institute Oswaldo
Cruz–Fiocruz, Rio de Janeiro, Brazil, 3Dpto Nutrición, Bromatologı́a y Tecnologı́a de los Alimentos, Facultad de Farmacia,
Universidad San Pablo-CEU, Madrid, Spain and 4Grupo inmunonutrición, Dpto Metabolismo y Nutrición, Instituto Frı́o,
Consejo Superior de Investigaciones Cientı́ficas, Madrid, Spain

Currently, it is well accepted that nutrition is an important factor for the development of the immune response. Epidemiological and
clinical results suggest that any nutritional deficiency alters immunocompetence and increases the susceptibility to infections(1,2). thus, the
purpose of the present study was to compare the nutritional status of ten young male elite track and field athletes (20–48 h training/week)
aged 13–18 years at a baseline state (48 h relaxation after training) with a control group consisting of ten volunteer students, matched by
age and socio-economic status, who were doing < 8 h physical exercise/week. Nutritional status was assessed by dietary intake for 3 d,
anthropometry (BMI and triceps skinfold thickness) and immunocompetence (total counts of leucocytes, lymphocytes and lymphocyte
subsets (CD3, CD4, CD8, CD19 and CD16 + 56)).
The energy intake was similar for athletes and controls (8327 v. 8235 kJ/d respectively), and lower than that recommended by the Food
and Nutrition Board, Institute of Medicine(3), the American Dietetic Association(4)and the American Academy of Pediatrics(5). BMI and
triceps skinfold thickness were significantly lower for the athletes than for the controls. Total leucocyte and total lymphocyte counts were
similar for both groups. However, a decrease in CD3, CD8, CD19 and CD16 + 56 lymphocyte subset counts was found for athletes in
comparison with controls.

Controls (n 10) Athletes (n 10)


Mean SD Mean SD

Age (years) 16.3 1.38 17.1 0.80


Energy intake (kJ) 8235 672 8327 584
Anthropometric variables
Weight (kg) 68.7 6.63 63.7 7.63
Height (m) 1.75 0.68 1.76 0.77
BMI (kg/m2) 22.5 1.18 20.7* 2.00
Triceps skinfold thickness (mm) 11.9 2.78 5.67* 1.1
Immunological variables
Total leucocytes (cells/mm3) 6287 1533 5786 1026
Total lymphocytes (cells/mm3) 3413 1003 3527 918
Lymphocyte subsets (cells/mm3):
CD3 1380 418 919* 380
CD8 659 224 432* 151
CD19 248 54 179* 60
CD16 + 56 (NK cells) 257 73 185* 59
NK, natural killer. Mean values were significantly different from those of the controls (Student’s t test): *P < 0.05.

These results suggest that the young athletes assessed in the current study could show subclinical malnutrition, with a leaner body and
an impaired immunocompetence compared with the control group, although the dietary intake was similar for both groups but below
recommendations. A higher-energy diet should be recommended for these high-performance athletes in order to avoid further risk of
malnutrition and to avoid more serious complications, such as infections.

1. Gleeson M, Nieman DC & Pedersen BK (2004) J Sport Sci 22, 115–125.


2. Moreira A, Kekkonen RA, Delgado L, Fonseca J, Korpela R & Haahtela T (2007) Eur J Clin Nutr 61, 443–460.
3. Food and Nutrition Board, Institute of Medicine (2002) Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol,
Protein and Amino Acids (Macronutrients). Washington, DC: National Academy Press.
4. American Dietetic Association (2000) J Am Diet Assoc 100, 1543–1556.
5. American Academy of Pediatrics (2006) Pediatrics 11, 544–559.
Proceedings of the Nutrition Society (2008), 67 (OCE), E47 doi:10.1017/S0029665108006563

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effect of inulin and probiotic bacteria on iron availability in


beans (Phaseolus vulgaris L.)

J. M. Laparra1, R. P. Glahn2 and D. Miller1


1
Department of Food Science, Cornell University, Ithaca, NY 14853, USA and 2US Plant, Soil, and Nutrition Laboratory,
Agricultural Research Service, US Department of Agriculture, Ithaca, NY 14853, USA

Anaemia is the most common nutritional disorder in the world and Fe deficiency (ID) is implicated in a majority of these cases. Clinical
studies have emphasized the importance of Fe in the integrity of the immune system(1). One of the keys is poor bioavailability of Fe in the
diet. Beans constitute a good source of protein for large groups of the population around the world, and can also be a good source of
essential minerals such as Fe. Nevertheless, legumes also contain phytates, polyphenols, etc., which impair Fe bioavailability. Among the
phytochemicals, polyphenols have been suggested to have a beneficial effect on immune cell function(2). Prebiotics such as inulin have
been suggested to have an enhancing effect on Fe absorption(3), and can also modulate the immune system(4). The objectives were to
evaluate the effects of inulin and probiotic bacteria on Fe availability from white and red beans.
White and red beans, with and without supplemental 40 ginulin/kg, were subjected to an in vitro gastrointestinal digestion (pepsin,
pH 2; pancreatin, pH 7.2). The digests were incubated overnight with Bifidobacterium infantis (ATCC 15697) or Lactobacillus acid-
ophilus (ATCC 11974). Aliquots were then placed in the upper chamber of cell-culture plates containing monolayers of Caco-2 cells(5).
Cell associated Fe and ferritin formation were used as estimates of Fe bioavailability.
Inulin by itself increased white-bean Fe uptake in Caco-2 cultures; however, no differences in ferritin formation were detected. This
observation may be explained by the capacity of each ferritin molecule to bind 4000 Fe atoms. Fermentation of both white and red beans,
with or without inulin, with probiotic bacteria caused differences in Fe availability only as a function of the bacteria tested. In fermented
samples higher growth rates for B. infantis than L. acidophilus were quantified, probably as a result of a faster bacteria metabolic
adaptation to the media and use of nutrients in the digests. Following fermentation with B. infantis Fe uptake from both white and red
beans was decreased in Caco-2 cells. This observation correlated with an increased total phenolic content in the digests. However, the
fermentation with L. acidophilus resulted in a lower total phenolic content and subsequent higher Fe uptake values than controls.
These results show that inulin may enhance Fe bioavailability, and suggest that probiotic bacteria caused differences in soluble
polyphenols in the digests that may explain the contrasting results from B. infantis and L. acidophilus.

Bean Iron in the ferritin 1 Total phenols


sample upper chamber (mg) (ng mg - 1 protein) (mg Gallic acid g - 1)
No treatment with bacteria
White 2.07°0.02 62.88°2.29 3.03°0.08
Red 2.65°0.11 3.43°1.33 4.17°0.33
White + Inulin 2.08°0.29 61.43°4.17 3.08°0.24
Red + Inulin 2.79°0.19 3.56°1.09 4.05°0.48
Incubated with B. infantis
White 2.07°0.01 44.15°5.21 3.48°0.12
Red 2.72°0.10 1.75°1.99 5.45°0.33
White + Inulin 2.10°0.05 50.80°8.56 4.25°0.19
Red + Inulin 2.63°0.03 1.61°0.41 5.35°0.12
Incubated with L. acidophilus
White 2.04°0.01 75.55°6.11 1.85°0.28
Red 2.75°0.04 6.31°1.89 3.04°0.25
White + Inulin 2.13°0.03 86.50°7.38 1.80°0.14
Red + Inulin 2.64°0.01 6.15°0.51 2.95°0.32

1. Mullick S, Rusia U, Sikka M & Faridi MA (2006) Indian J Med Res 124, 647–654.
2. Álvarez P, Alvarado C, Puerto M, Schlumberger A, Jimenez L & De la Fuente M (2006) Nutrition 22, 913–921.
3. Yeung CK, Glahn RE, Welch RM & Miller DD (2005) J Food Sci 70, R88–R92.
4. Watzl B, Girrbach S & Roller M (2005) Br J Nutr 93, S49–S55.
5. Glahn RP, Lee OA, Yeung A, Goldman MI & Miller DD (1998) J Nutr 128, 1555–1561.
Proceedings of the Nutrition Society (2008), 67 (OCE), E48 doi:10.1017/S0029665108006575

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effect of different doses of creatine supplementation on


endogenous creatine synthesis

B. Li1, M. Xiang2, L. S. Harbige2,3, X. Li1 and H. Ai1


1
Division of Nutrition and Biochemistry, Institute of Sports Medicine, The Third Hospital, Peking University, Beijing,
People’s Republic of China, 2Centre for Biosciences Research, School of Science, University of Greenwich, Kent ME4 4TB,
UK and 3Medway School of Pharmacy, University of Kent and University of Greenwich, Kent ME4 4TB, UK

Creatine (Cre) has been widely used as a nutritional ergogenic aid among athletes since the middle of the 1990s(1) but has not been
categorised as a stimulant. Athletes often take a greater dose of Cre for a longer period in order to obtain a rapid, high and longer
ergogenic effect(2). L-Arginine:glycine amidinotransferase (L-AGAT) in the kidney is regarded as a key enzyme in endogenous Cre
synthesis in mammals, and guanidinoacetic acid (GAA) is the precursor in this synthesis(3). The present study has investigated the effect
of supplementation with 0, 0.75, 1.5, 3.0 and 6.0 g Cre/kg body weight per d for 4 weeks on L-AGAT activity and GAA concentration in
the kidneys and total Cre in the gastrocnemius muscle in adult male Sprague-Dawley rats. L-AGAT activity and GAA concentration in the
kidneys decreased ( %) by 19.5, 42.6, 61.9 and 66.5, and 5.9, 13.7, 23.8 and 24.5 respectively in the groups supplemented with 0.75, 1.5,
3.0 and 6.0 g Cre/kg per d for 4 weeks. The total Cre in the gastrocnemius in the supplemented groups increased ( %) by 7.1, 8.5, 12.6 and
13.7 respectively. The weight of the gastrocnemius in the groups receiving 0.75, 1.5 and 3.0 g Cre/kg per d also increased significantly (see
Table). No significant differences were found in the body weights among the groups supplemented with 0, 0.75, 1.5 and 3.0 g Cre/kg per d
for 4 weeks (Table). However, the body weight of the group receiving 6.0 g Cre/kg per d was significantly less than that of the control
group (Table). After 3 d of supplementation diarrhoea was observed in all the rats in the group receiving 6.0 g Cre/kg per d, and persisted
for the remainder of the supplementation period, which probably explains the loss in body weight. No significant differences were found
in the serum Cre kinase activity and serum creatinine among the five groups. L-AGAT activity was positively correlated with the
concentration of GAA (n 50, r 0.80, P < 0.001). The Cre intake was, however, negatively correlated with the L-AGAT activity (n 50,
r 0.87, P < 0.001) and the concentration of GAA (n 50, r 0.75, P < 0.001).

Body weight (g) Gastrocnemius weight (g)


Cre (g/kg per d) n Mean SD Mean SD

0 10 365.2 36.0 2.02 0.17


0.75 10 379.4 23.7 2.33** 0.24
1.5 10 374.8 19.6 2.19* 0.10
3.0 10 371.8 21.8 2.27** 0.20
6.0 10 340.6** 31.0 2.05 0.19
Mean values were significantly different from that for the control group (without Cre supplementation): *P < 0.05,
**P < 0.01.

These results indicate that in rats L-AGAT activity and GAA concentration could be rapidly reduced by supplementation with 0.75–
3.0 g Cre/kg per d, which is equivalent to 10–40 g Cre/d (67 kg body weight) in human subjects(4), suggesting that high-dose Cre sup-
plementation may result in depression of endogenous Cre metabolism and may have potential adverse effects on the body. Little is known
about the effect of high-dose Cre supplementation on the immune system, the results of a follow-up study of immune function will be of
interest.

1. Bemben MG & Lamont HS (2005) Sports Med 35, 107–125.


2. Brudnak MA (2004) Toxicol Lett 150, 123–130.
3. Wyss M & Kaddurah-Daouk R (2000) Physiol Rev 80, 1107–1213.
4. Calabrese EJ (1991) Principles of Animal Extrapolation, pp. 499–527. Chelsea, MI: Lewis Publishers Inc.
Proceedings of the Nutrition Society (2008), 67 (OCE), E49 doi:10.1017/S0029665108006587

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effect of cessation of creatine supplementation on


endogenous creatine synthesis

X. Li1, M. Xiang2, L. S. Harbige2,3 and H. Ai1


1
Division of Nutrition and Biochemistry, Institute of Sports Medicine, The Third Hospital, Peking University, Beijing,
People’s Republic of China, 2Centre for Biosciences Research, School of Science, University of Greenwich, Kent ME4 4TB,
UK and 3Medway School of Pharmacy, University of Kent and University of Greenwich, Kent ME4 4TB, UK

Creatine (Cre) can be obtained exogenously from either the diet or supplementation(1–3). It can also be endogenously synthesised in man
and other mammals(4,5). One of the adverse effects of Cre supplementation is that L-arginine:glycine amidinotransferase (L-AGAT), a key
enzyme in the endogenous Cre synthesis in mammals, may be severely inhibited(6). The present study investigated the time-course of
recovery of L-AGAT activity and guanidinoacetic acid (GAA) concentration after 1 week of supplementation with 3.0 g Cre/kg body
weight per d Cre in adult male Sprague-Dawley rats. On days 1, 2, 4, 8, 16 and 32 (ten treatment and ten control rats per time point) after
the 7 d supplementation period L-AGAT activity and GAA concentration in the kidney recovered ( %) by 32.8, 51.5, 76.1, 94.4, 100.2 and
102.0 (Figure (A)) and 77.9, 86.2, 96.8, 100.6, 101.1 and 108.0 (Figure (B)) respectively when compared with their respective controls.
The total Cre in the gastrocnemius muscle decreased rapidly and reached its lowest level on day 4 after the supplementation period, and
then increased gradually. No significant differences were found in serum Cre kinase activity and serum creatinine in the experimental
groups when compared with their respective control groups.
These findings indicate that in rats the reduction in L-AGAT activity and GAA concentration associated with supplementation with 3.0 g
Cre/kg per d for 1 week could be reversed, suggesting that short-term and high-dose Cre supplementation does not result in depression of
endogenous Cre metabolism. As little is known about the effect of Cre supplementation on the immune system, investigation of the effect
of long-term and high-dose Cre supplementation on endogenous Cre metabolism in the immune cells of rats will be of interest.

120
A
Relative L-AGAT activity

100
%)
in kidneys (%

80
*
60
**
40
**
**
20
0 4 8 12 16 20 24 28 32

B
%)

120
Relative GAA in kidneys (%

100

*
80
**
**

60
0 4 8 12 16 20 24 28 32
Time (days)

1. Pan JW & Takahashi K (2007) Am J Physiol 292, R1745–R1750.


2. Candow DG & Chilibeck PD (2007) J Nutr Health Aging 11, 185–188.
3. Silva AJ, Machado Reis V, Guidetti L, Bessone Alves F, Mota P, Freitas J & Baldari C (2007) J Sports Med Phys Fitness 47, 58–64.
4. Derave W, Marescau B, Vanden Eede E, Eijnde BO, De Deyn PP & Hespel P (2004) J Appl Physiol 97, 852–857.
5. Braissant O, Henry H, Loup M, Eilers B & Bachmann C (2001) Brain Res Mol Brain Res 86, 193–201.
6. Wyss M & Kaddurah-Daouk R (2000) Physiol Rev 80, 1107–1213.
Proceedings of the Nutrition Society (2008), 67 (OCE), E50 doi:10.1017/S0029665108006599

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Activation of innate immunity by a probiotic bacterium and


a fermented milk containing this micro-organism

C. Maldonado Galdeano1,2, A. de Moreno de Leblanc1, C. Dogi1, S. Chaves1, E. Carmuega3,


R. Weill4 and G. Perdigón1,2
1
CERELA, Chacabuco 145, 4000 Tucumá, Argentina, 2Cát Inmunologı́a, Fac Bqca, Qca y Fcia, Universidad Nacional
de Tucumán, Argentina, 3Nutritia, Buenos Aires, Argentina and 4Dto Investigación y Desarrollo, Danone SA, Argentina

Improvement of the immune status of the host is one of the beneficial properties attributed to probiotics. Previous results have shown that
the effect induced by a probiotic strain Lactobacillus casei CRL 431 is through innate immunity(1,2,3). The present work investigated the
behaviour of another probiotic bacterium Lactobacillus casei DN 114001 in relation to the interaction between the epithelial and immune
cells, using mice as the experimental model. Using electron microscopy and fluorescent bacteria it was demonstrated that this bacterium
interacts with the intestinal epithelial cells and the small fragments of the bacterium can internalize and make contact with the immune
cells (macrophages and dendritic cells) present in Peyer patches and in the lamina propria of the small and large intestine. These antigenic
particles remain in the gut for 72 h, similar to other particulate antigens. A subsequent study investigated the effect of the inclusion of this
probiotic bacterium in fermented milk on the mucosal innate immune system of the gut in BALBc mice. The fermented milk was
administered to the mice for five consecutive days, which was previously determined to be the optimal dose to stimulate the immune
response. At the end of the administration period the animals were killed and the small and large intestine was removed for the
determination of: (a) the number of IgA- and cytokine (IL-10, interferon g (IFN-g), TNFa)-producing cells in histological slices of both
intestines; (b) the expression in the lamina propria of the small intestine of the different receptors present in the immune cells involved in
the innate immunity, e.g. mannose receptor CD206 present in macrophages or on the dendritic cell surface (this receptor participates
mainly in the internalization process and in antigen clearance); (c) Toll-like receptor 4 (TLR4), which is involved in the adhesion and pro-
inflammatory signals induced by pathogenic bacteria, as a measure of possible adverse effects. Increases were found in the number of
IgA-, TNFa- and IFNg-producing cells in both intestines. The anti-inflammatory cytokine IL-10 also showed a significant increase in
relation to the control (without fermented milk). This increase may have been induced to down regulate the mucosal response. The
receptor CD206 was slightly increased, but TLR4 was unchanged. These results showed that innate immunity is involved in the gut
mucosal immune activation observed, and led to an investigation of the effect of the consumption of this fermented milk in early period of
the life, when the maturation of the innate immune cells occurs. After weaning, newborn mice received the fermented milk until the
adulthood (45 d of age). The number of cells positive for maturation markers, such as F4/80 + cells (macrophages) and 33D1 + cells
(dendritic cells) were determined in the small intestine. The expression of these markers increased in treated animals compared with the
control group (newborn mice not receiving fermented milk).
These results demonstrate that the probiotic bacterium or its fragments interact with epithelial and phagocytic immune cells associated
with the gut. This observation is reflected in the results obtained with the fermented milk, which show an increase in the number of
CD206 receptors and activation of the immune cells associated with the gut, with an increase in cytokine- and IgA + -producing cells. The
results for the specific markers of maturation of the macrophages and dendritic cells in adult mice (45 d of age) that received the
fermented milk after weaning are in agreement with those of previous studies.
It has been demonstrated that fermented milk containing the probiotic strain L. casei DN 114001 can modulated the gut immune
response mainly through the innate immune system by increasing the receptors related to maturation of the macrophages and dendritic
cells associated with the gut, by activating these cells and by increasing the number of IgA + B-cells.

1. Galdeano CM & Perdigón G (2004) J Appl Microbiol 97, 673–681.


2. Maldonado Galdeano C, de Moreno de Leblanc A, Vinderola G, Bibas Bonet ME & Perdigón G (2007) Clin Vaccine Immunol 14, 485–492.
3. Maldonado Galdeano C & Perdigón G (2006) Clin Vaccine Immunol 13, 219–226.
Proceedings of the Nutrition Society (2008), 67 (OCE), E51 doi:10.1017/S0029665108006605

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Milk-derived supplement inhibits in vitro lymphocyte proliferation


and IL-2 production

S. Marı́n-Gallén, F. J. Pérez-Cano, M. Castell, C. Castellote and A. Franch


Department of Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

Recently, significant progress has been made in the characterisation of milk components affecting the function of the immune system. The
aim of the present report was to study the effect of a premium ultra-grade freeze-dried bovine whey-protein concentrate (WPC) on the
functional capacity of lymphocytes. Thus, the immunomodulating properties of this dairy extract were evaluated in relation to the
proliferative response of spleen T and B lymphocytes. Splenocytes from adult Wistar rats were incubated in a medium supplemented with
WPC (60–480 mg protein/ml), which contains (g/kg) < 0.1 fat, 750–900 proteins and 35–50 carbohydrates, and also lactoferrin 9.2; IgG
300–600, IgA 50–70 and IgM 70–90 and high proportions of active compounds, e.g. natural growth factors and hormones, vitamins and
amino acids. Standard commercial infant formula (SIF; 40–415 mg protein/ml) and BSA (250–500 mg protein/ml) were also added as milk-
derived and inert control proteins respectively. Concanavalin A (ConA; specific stimulus for T-cells) or pokeweed (Phytolacca amer-
icana) mitogen (specific for B-cells) were added to the cell culture for 72 h. Proliferating cells were quantified by means of the BioTrakTM
cell proliferation system (BioTrak, Carlsbad, CA, USA) based on 5-bromo-20 -deoxyuridine incorporation. IL-2 levels were quantified by
ELISA in 24 h-culture supernatant fractions obtained from ConA-stimulated lymphocytes. Statistical analysis was performed by con-
ventional ANOVA and when an experimental group variable had a significant effect on the dependent variable post hoc comparisons
(LSD test) were performed. Significant differences were accepted at P < 0.05.

100 T proliferation
In vitro response (%)

B proliferation
80 IL-2 production

60

40

20

0
0 100 200 300 400 500

Protein concentration of WPC (µg/mL)

Results showed a dose–dependent inhibitory effect of WPC on both spleen B- and T-lymphocyte proliferative response induced by
mitogen stimulation (see Figure). The inhibitory effect on T- and B-cell proliferation was approximately 55 % and approximately 40 %
respectively. In parallel, a WPC dose-dependent inhibition of IL-2 production was also found (approximately 80 %). Cell viability was not
modified by WPC addition. SIF produced similar inhibitory effects. However, non-milk-derived proteins such as BSA did not modify
these lymphocyte responses. WPC and SIF, both milk-derived components, inhibited lymphocyte proliferation and IL-2 production
in vitro. This immunomodulatory effect may prevent the newborn from over-reacting immunologically to the environmental antigens
during early life.
Proceedings of the Nutrition Society (2008), 67 (OCE), E52 doi:10.1017/S0029665108006617

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Influence of fruit intake and antioxidants on the prevention of


opacities of the lens

M. I. Martı́nez1, J. Martı́nez-Raga1,2, J. Raga1, A. Alegre3, M. C. Gimeno1, E. Llusar1


and I. Alfonso-Sanchez4
1
Instituto CEU Sobre Drogas y Conductas Adictivas (IDYCA), Universidad Cardenal Herrera – CEU, Moncada,
Valencia, Spain, 2Unidad de Conductas Adictivas de Gandı́a, Valencia, Spain, 3IES Pobla de Vallbona (Valencia),
Conselleria d’Educació, Generalitat Valenciana, Spain and 4Consorcio Hospital General Universitario and
Facultat de Medicina, Universitat de Valencia, Spain

The WHO considers cataracts to be the leading cause of avoidable blindness worldwide. Cataracts develop as a result of protein glycation,
which takes place when proteins react with sugars, by cross-link formation and by oxidative processes(1). Such end products of glycation
alter proteins, DNA and lipids, modifying their chemical properties and causing a colour loss of the lens to yellow or brown, which
impairs vision. Given the importance of oxidative processes in the development of cataracts, the putative role of antioxidant agents in its
development have been investigated.
The aim of the present study was to assess the possible protective effect of the different antioxidant agents present in fruit on the
development of opacities of the lens relative to age.
The subjects were a random sample (one in every three individuals attending a community pharmacy) of seventy-four male and female
patients with a mean age of 59 (range 45–80) years. Subjects completed a self-administered questionnaire to assess lifestyle status, as well
nutritional habits with a particular focus on the fruit intake.
N-acetyl-serotonin, a precursor of melatonin, appears to be effective in preventing damage to the lens caused by UVA radiation, both
for its ability to capture free radicals and for its antioxidant properties. Vitamin E also has a protective effect against radiation-induced
cataracts by reducing oxidative stress. In addition, a similar effect has also been observed in relation to cataracts induced by tobacco
smoking. N-acetyl-carnosine plays an important role as an antioxidant against free radicals (superoxide, hydroxide) both in the lipid phase
of the cell membranes of the lens and in the aqueous membrane. It has also been suggested that vitamin C decreases cataract risk relative
to age among middle-aged Japanese. After oral administration of a combination of antioxidant micronutrients (b-carotene, vitamin C and
vitamin E) for a 3-year period a slight deceleration of cataract progression relative to age was observed. A majority of patients were
consuming fruit regularly (93.2 % v. 6.8 %; Table). Of those consuming fruit, 18.8% showed opacities of the lens (P=0.949), with a
relative risk of 0.94. Thus, it could be considered that consuming fruit may provide a mild protective effect against developing opacities of
the lens. To date, there are very few studies showing the protective effect of antioxidants on the development of cataracts in human
subjects(2). However, it has been suggested that both natural and artificial antioxidants could have a protective effect in the prevention of
cataracts in the early stages of oxidative damage to the lens.
Table. Relationship between fruit intake and development of opacities of the lens
Lens opacity
No Yes Total
Fruit intake No n 4 1 5
% No-fruit consumers 80.0 20.0 100
% Total sample 5.4 1.4 6.8
Yes n 56 13 69
% Fruit consumers 81.2 18.8 100
% Total sample 75.6 17.6 93.2

The present results suggest that antioxidants from fruit could provide some protective effects in the prevention of opacities of the lens.

1. Moeller SM, Taylor A, Tucker KL, McCullough ML, Chylack LT, Hankinson SE, Willett WC & Jacques PF (2004) J Nutr 134, 1812–1819.
2. Gritz DC, Srinivasan M, Smith SD, Kim U, Lietman TM, Wilkins JH et al. (2006) Br J Ophthalmol 90, 847–851.
Proceedings of the Nutrition Society (2008), 67 (OCE), E53 doi:10.1017/S0029665108006629

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Alcohol consumption in secondary-school students: effects on


plasma total antioxidant capacity

M. I. Martı́nez1, J. Martı́nez-Raga1,2, A. Alegre3, J. A. Dominguez1 and I. Alfonso-Sanchez4


1
Instituto CEU sobre Drogas y Conductas Adictivas (IDYCA), Universidad Cardenal Herrera – CEU, Moncada, Valencia,
Spain, 2Unidad de Conductas Adictivas de Gandı́a, Valencia, Spain, 3IES Pobla de Vallbona (Valencia), Conselleria
d’Educació, Generalitat Valenciana, Spain and 4Consorcio Hospital General Universitario and Facultat de Medicina,
Universitat de Valencia, Spain

National Household Surveys have been carried out biannually in Spain since 1994 to establish the patterns and trends of substance use
among students in secondary education (age 14–18 years). Alcohol has been consistently, although to a different extent, the psycho-active
substance of abuse most commonly used by students. In 2004 42.7 % of 14–18 year olds had had a drink at least once in their lives, the
prevalence being higher among boys than among girls (45.3% and 40.2% respectively).
The aim of the present study was to compare the prevalence of alcohol consumption in a random sample of students in secondary
education (age 14–18 years) from Valencia (Spain) with that of the National Household Survey, and to relate alcohol use to antioxidant
status.
An anonymous self-administered questionnaire designed to collect information on alcohol consumption was completed during school
hours by a sample of 291 Valencian secondary school students between January and June 2006. A blood sample was collected from a
subgroup of volunteers (n 53) and plasma total antioxidant capacity (TAC) was determined in 1 ml serum using spectrophotometry(1) in
order to establish its relationship with alcohol consumption. Both TAC and alcohol consumption could have a negative impact on the
present and future health of the subjects since excessive production of free radicals and lipid peroxidation may be implicated in the
development of chronic diseases, such as atherosclerosis and cancer, and are responsible for cellular aging(2).
Among this sample of adolescents the percentage of students surveyed in 2006 that reported using alcohol and cannabis was much
lower (36.7%) than that in the 2004 Spanish National Household Survey (65.6%) and showed no significant gender differences. Reference
values for plasma TAC were in the range 1.30–1.77 mmol/l, and did not appear to be affected by alcohol use (1.37 (SD 0.20) mmol/l v.
1.37 (SD 0.18) mmol/l). The Figure shows the percentage distribution of values for plasma TAC and alcohol use according to age.

50,0%
Alcohol
NO
Percentage YES

40,0%

30,0%

20,0%

10,0%

0,0%
12 13 14 15 16 17 18 19
AGE

Alcohol consumption does not significantly affect plasma TAC, probably because of its cardioprotective effects at moderate doses.
Within this age-group no significant differences were observed.

1. Rice-Evans C & Miller NJ (1994) Methods Enzymol 234, 279–293.


2. Soardo G, Donnini D, Varutti R et al. (2005) Alcohol Clin Exp Res 29, 1889–1898.
Proceedings of the Nutrition Society (2008), 67 (OCE), E54 doi:10.1017/S0029665108006630

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Synthesis of galactooligosaccharides with prebiotic potential during


hydrolysis of lactose by Lactozym 3000 L HP G

C. Martı́nez-Villaluenga, A. Cardelle-Cobas, N. Corzo, A. Olano and M. Villamiel


Instituto de Fermentaciones Industriales (CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain

Galactooligosaccharides (GOS) are non-digestible oligosaccharides consisting of two to twenty-20 molecules of galactose and glucose that
are recognized as prebiotics because they can stimulate the proliferation of lactic bacteria and bifidobacteria in the human intestine(1). The
stimulation of the immune system is among the possible benefits of prebiotics that are being investigated(2). Although no definitive
conclusions have been reached, the possibility that prebiotic oligosaccharides could influence the immune response would make prebiotic
dietary intervention a more attractive choice than probiotics in the reduction of atopic diseases in infants(3) and the chronic inflammatory
bowel disease in human subjects(4).
The role of human milk in the stimulation of the immune response in newborn babies during the first months of life is well known. One
of the carbohydrates in human milk for which the immune effect has been established is 60 -galactosyl lactose. This trisaccharide and other
carbohydrates have potential use as ingredients in the development of different infant formulas. Recently, considerable attention has been
paid to improving GOS production; enzymic transgalactosylation from lactose being one of the most promising alternatives. Lactozym
3000 L HP G is a commercial preparation in which b-galactosidase from Kluyveromyces lactis is the most active enzyme, although
transgalactosylation can also occur at the same time; the transferase:b-galactosidase activity depends on the different reaction conditions
used. The present study is an exhaustive investigation of the optimal conditions for GOS (60 -galactosyl lactose, galactobiose and allo-
lactose) formation during the hydrolysis of lactose with Lactozym 3000 L HP G. The effect of the reaction conditions (temperature, pH,
time, substrate and enzyme concentrations) was different for the formation of di- and trisaccharides. Thus, the best conditions for
producing galactobiose and allolactose were 50 C, pH 6.5, 250 mg lactose/ml, 3 U enzyme/ml and 300 min, while the optimal conditions
for the synthesis of 60 -galactosyl lactose were 40 C, pH 7.5, 250 mg lactose/ml, 3 U enzyme/ml and 120 min.

This work has been financed under an R & D programme of the Spanish Ministry of Education and Science, project AGL-2004–07227-C02, and project
ALIBIRD S-0505/AGR/000153 from the Comunidad de Madrid. A.C.-C. thanks MEC for an FPU grant and C.M.-V. is the recipient of a I3P-CSIC
contract.

1. Sako T, Matsumoto K & Tanaka R (1999) Int Dairy J 9, 69–80.


2. Maning TS & Gibson GR (2004) Best Pract Res Clin Gastroenterol 18, 287–298.
3. Moro M, Arslanoglu S, Stahl B, Jelinek J, Wahn U & Boehm GA (2006) Arch Dis Child 91, 814–819.
4. Macfarlane S, Macfarlane GT & Cummings JH (2006) Aliment Pharmacol Ther 24, 701–714.
Proceedings of the Nutrition Society (2008), 67 (OCE), E55 doi:10.1017/S0029665108006642

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Raffinose family oligosaccharides of lupin


(Lupinus albus L. cv multolupa) as a potential prebiotic

C. Martı́nez-Villaluenga1, J. Chicholoska2, A. Kliber2 and K. Gulewicz2


1
Instituto de Fermentaciones Industriales, CSIC, C/ Juan de la Cierva 3, 28006 Madrid, Spain and 2Department of
Animal Physiology and Biochemistry, Agricultural University, Wolynska 35, Poznan 60-637, Poland

The beneficial effects on health attributed to prebiotics are directly related to the stimulation of the growth and activities of lactic bacteria
and bifidobacteria in the human intestine(1). Moreover, proliferation of these beneficial bacteria contributes indirectly to the stimulation of
the immune system through IgA and IL (IL1, IL6 and g-IL) production(2–6).
Raffinose family oligosaccharides (a-galactosides) are non-digestible oligosaccharides with an extent of polymerization of three to six
molecules. These compounds are a(1!6)galactosides linked to the C-6 of the glucose moiety of sucrose, which are abundant in legume
seeds of which lupins are the richest source (120 g/kg seed dry weight)(7). These oligosaccharides can be easily extracted from the seeds
and further purified(8) to be used as ingredients during the manufacture of different functional foods. In ovo studies have demonstrated the
potential prebiotic effect of a-galactosides derived from lupin seeds(9).
In the present work the evaluation of a-galactosides as prebiotics was carried out in vivo using an animal model. These results were
compared with those obtained for pure raffinose, and also commercial fructo-oligosaccharides (FOS) and inulin. Doses of 15 mg/100 g
body weight were administered orally once daily to Wistar rats for 23 d. The numbers of faecal bifidobacteria were estimated at days 0, 10
and 23. Oligosaccharide administration for all experimental groups showed gradual increases (P £ 0.05) in faecal bifidobacteria with the
duration of the experiment, reaching the highest value after the longest time period. The numbers of faecal bifidobacteria in rats after
administration of raffinose family oligosaccharides (RFOS) from lupin seeds were comparable with those found with pure raffinose and
commercial fructo-oligosaccharides and inulin (Figure).

12
Control
10
log cfu/g faeces

8 Raffinose

6 RFOS
4
FOS
2

0 Inulin
0d 10 d 23 d
Time of treatment
Figure. Effect of oligosaccharide administration (15 mg/100 g body weight) on numbers of faecal bifidobacteria.

Thus, this in vivo study has shown that a-galactosides from lupin stimulate bifidobacterial growth and indirectly immune response.
Nevertheless, subsequent intervention studies are needed to establish definitive conclusions.

This work is a result of a bilateral scientific cooperation joint project between Poland (Polish Academy of Sciences) and Spain (CSIC).

1. Gibson GR & Roberfroid MB (1995) Br J Nutr 125, 1401–1412.


2. McCraken VJ & Gaskins HR (1997) In Probiotics: A Critical Review, pp. 85–111 [GW Tannock, editor]. Norwich, Norfolk: Horizon Scientific Press.
3. Kato I (1997) Healthist Special anniversary ed., 60–66.
4. Ishikawa H. (1997) Healthist Special anniversary ed. 787–789.
5. Ballongue J (1993) Bifidobacteria and Probiotic Action, pp. 357–428 [S Salminen and A von Wright, editors] New York: Marcel Dekker.
6. Isoulari E (2000) Hosp Med 61, 6–7.
7. Martı́nez-Villaluenga C, Frı́as J & Vidal-Valverde C (2005) Food Chem 91, 645–649.
8. Martı́nez-Villaluenga C, Frı́as J, Gulewicz K & Vidal-Valverde C (2004) J Agric Food Chem 52, 6920–6922.
9. Martı́nez-Villaluenga C, Wardeńska M, Pilarski R, Bednarczyk M & Gulewicz K (2004) Folia Biol (Krakow) 52, 135–142.
Proceedings of the Nutrition Society (2008), 67 (OCE), E56 doi:10.1017/S0029665108006654

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Antioxidant and anti-inflammatory potency of different wheat varieties


and fractions

N. Mateo Anson1,2, R. v. d. Berg1, R. Havenaar1, G. Haenen2 and A. Bast2


1
TNO Quality of Life, PO Box 360, 3700 AJ Zeist, The Netherlands and 2University of Maastricht, PO Box 616,
6200 MD Maastricht, The Netherlands

Risk factors for diet-related disorders are oxidative stress and chronic inflammation. Wheat is a source of phytochemicals with antioxidant
activity that might play a role in the observed protection of whole-grain diets against metabolic disorders.
The aims of the present study were first to investigate the bioaccessibility of antioxidant and anti-inflammatory activity from different
wheat fractions during gastrointestinal (GI) transit and second to demonstrate the contribution of wheat compounds to these health factors.
Experiments were performed in the TIM system, which is a dynamic computer-controlled model consisting of gastric, duodenal, jejunal
and ileal compartments simulating conditions in the human GI tract(1,2). Samples were collected in 1 h aliquots for 6 h from the dialysates
of the jejunal and ileal compartments to measure the kinetics of bioaccessibility of antioxidant capacity and anti-inflammatory responses.
Antioxidant capacity (Trolox equivalent antioxidant capacity assay) and ferulic acid, polyphenol and protein contents were determined.
Anti-inflammatory effects were measured in extracts and TIM samples using a human macrophage cell system with lipopolysaccharide
(LPS)-induced TNFa and IL-6 secretion.
Antioxidant capacity was unevenly distributed within the wheat fractions (aleurone fractions > bran fractions > flour fractions), without
differences between cultivars (Tiger and Crousty). Antioxidant capacity was correlated with the ferulic acid content (R 0.96, P < 0.00001).
Ferulic acid was the major contributor to the antioxidant capacity in bran and aleurone fractions (50–60 %). However, ferulic acid did not
reduce TNFa and IL-6 levels in a dose-dependent manner. The TIM experiments showed that the antioxidant capacity was bioaccessible
during GI digestion. In the case of the bran and aleurone fractions the bioaccessibility was significantly lower (20–30 % initial antioxidant
capacity) than that from the flour fraction. The bioaccessibility of ferulic acid was similarly low from the bran and aleurone fractions. In
experiments with flour ferulic acid was below the detection limit in the dialysate samples, because of the low initial level at oral intake.
The dialysates samples collected from TIM showed reduced levels of TNFa for the bran and aleurone fractions, reaching maximal
inhibition at the 2nd hour and 3rd hour (Figure). These data corresponded with the maximum levels of the bioaccessibility of the
antioxidant capacity from these fractions.

250 Control
(LPS)
200
% TNF-α (LPS=100%)

Starch

150
Flour

100
Bran

50
Aleurone

0
LPS 1 2 3 4

Figure. Variations in TNFa induced by dialysate samples from the jejunal compartment from 1st hour to 4th hour (1–4). The dialysate samples represent the absorption from the
in vitro digestion of starch and the wheat fractions of the variety Tiger (flour, bran and aleurone). Values are means and standard deviations represented by vertical bars for three
different days.

This publication is financially supported by the European Commission in the Communities 6th Framework Programme, Project HEALTHGRAIN
(FOOD-CT-2005–514008). It reflects the authors’ views and the EC is not liable for any use that may be made of the information contained in this
publication.

1. Minekus M & Havenaar R (1998) Reactor system. European Patent no. 0642382. Eur Patent Bull 98/07, Art. 97(4) and (5) EPC, dated 11.02.98.
2. Minekus M, Marteau P, Havenaar R & Huis in ’t Veld JHJ (1995) Altern Lab Anim 23, 197–209.
Proceedings of the Nutrition Society (2008), 67 (OCE), E57 doi:10.1017/S0029665108006666

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Elicitation of an allergic reaction in mice orally sensitized to whey or


casein proteins

A. J. Nauta1, B. Schouten2, B. C. A. M. van Esch2, S. van Doorn1, G. A. Hofman2,


L. W. J. van den Elsen2, L. E. M. Willemsen2, L. M. J. Knippels1 and J. Garssen1,2
1
Immunology, Numico-Research BV, Wageningen, The Netherlands and 2Pharmacology and Pathophysiology,
UIPS, Utrecht University, The Netherlands

Cow’s-milk allergy (CMA) is the most common food allergy in children, affecting 1–2% of all infants. So far, the pathogenesis of the
disease is incompletely understood, and no effective treatment is available to cure or actively prevent food allergy. Animal models may
provide a useful tool to explore the mechanisms underlying the development of CMA and to identify new therapeutic strategies. The
purpose of the present study was to develop a murine model of IgE-mediated cow’s milk hypersensitivity that closely mimics the clinical
features of human CMA.
Female C3H/HeOuJ mice (5 weeks old; n 6) were sensitized by intragastric administration of casein or whey, using cholera toxin (CT)
as an adjuvant (methods adapted from Li et al. 1999 and Frossard et al. 2004(1,2)) and boosted five times at weekly intervals. At week 7
the mice were challenged subcutaneously (ear) and orally. Serum levels of mouse mast cell protease-1 (mMCP-1), total IgE and allergen-
specific IgE, IgG1 and IgG2a were measured. The acute allergic skin reaction was determined by measuring ear swelling.
An antigen-specific acute allergic skin response was induced in casein- and whey-sensitized mice (mm; 71.2 (SD 8.4) and 137.9 (SD 21.7)
respectively v. - 4.6 (SD 4.7) for CT controls; P < 0.01). Total IgE and mMCP-1 serum concentrations were enhanced in both the casein-
and whey-sensitized mice. In whey-sensitized mice whey-specific IgE, IgG1 and IgG2a serum levels were enhanced, while in casein-
sensitized mice only casein-specific IgG1 was increased (Table). In casein-sensitized mice the number of mast cells per villus–crypt unit
was enhanced compared with whey-sensitized and CT control mice (1.0 (SD 0.2) v. 0.1 (SD 0.1) v. 0.3 (SD 0.0); P < 0.01).
Taken together these results suggest that the oral administration of whey and casein elicit an allergic reaction in mice that mimics
immediate CMA in human subjects. Differential pathophysiological changes were observed in whey-sensitized and casein-sensitized
mice. The serology of whey-sensitized mice more closely resembles the human situation, whereas casein-sensitized mice develop gas-
trointestinal symptoms similar to the clinical features of human CMA. These mouse models of CMA provide a useful tool to examine the
mechanism underlying the development of CMA and to explore new therapeutic strategies for the treatment of food allergy.

Whey Ig Casein Ig
Sensitized Control* SD Whey* SD Control* SD Casein* SD

IgE 0.28 0.02 1.67 0.33 0.11 0.01 0.26 0.32


IgG1 0.22 0.09 0.99 0.17 0.09 0.04 0.54 0.33
IgG2a 0.25 0.07 0.59 0.20 0.13 0.01 0.23 0.23
* Presented as OD values.

1. Frossard CP, Hauser C & Eigenmann PA (2004) J Allergy Clin Immunol 114, 377–382.
2. Li XM, Schofield BH, Huang CK, Kleiner GI & Sampson HA (1999) J Allergy Clin Immunol 103, 206–214.
Proceedings of the Nutrition Society (2008), 67 (OCE), E58 doi:10.1017/S0029665108006678

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effect of enteral nutrition on plasma soluble adhesion molecules in


an elderly population

J. Olza1, M. D. Mesa1, R. Moreno2, A. Pérez de la Cruz2, A. Gil1 and C. M. Aguilera1


1
Institute of Nutrition and Food Technology, Dept Biochemistry and Molecular Biology II, University of Granada, Granada,
Spain and 2Universitary Hospital Virgen de las Nieves, Unit of Clinical Nutrition and Dietetic, Granada, Spain

The entry of inflammatory cells into the arterial wall is mediated by adhesion molecules expressed on the endothelial surface by binding to
their counter-ligands on the leucocytes. The origin of soluble adhesion molecules such as endothelial selectin (sE-selectin), intercellular
adhesion molecule 1 (sICAM-1) and vascular cell adhesion molecule 1 (sVCAM-1) is mainly the activated endothelium. Thus, they
are considered to be biomarkers of the inflammatory state of the arterial wall(1). sICAM-1 appears to be a general marker of a pro-
inflammatory status, whereas sVCAM-1 emerges as a predictor for future cardiovascular events in patients with pre-existing disease. Both
DHA and EPA have been found to decrease the expression of soluble adhesion molecules in studies of the cytokine-activated endothelium
in vitro(2). Enteral nutrition may influence inflammatory molecule secretion in patients under pathological conditions. Feeding long-chain
n-3 PUFA results in partial replacement of arachidonic acid in cell membranes by EPA. A number of experimental and human trials have
demonstrated immunomodulatory properties of n-3 PUFA, by suppressing different variables of immune function, including the produc-
tion of inflammatory cytokines and eicosanoids(3). There are no reports of studies that have evaluated the influence of dietary EPA and
DHA on soluble adhesion molecules in elderly patients fed an enteral diet, which is the aim of the present study.
Thirty-two patients aged 75 years were fed a complete formula for enteral nutrition (T-Diet Plus1 ; Vegenat SA, Madrid, Spain) with
added EPA (75 mg/l) and DHA (35 mg/l), as compared with standard diets. Patients were monitored for 6 months, and blood samples were
taken at the beginning and after 3 and 6 months of feeding. At the end of the experimental period only sixteen patients remained in the
study. Mean daily intake was 5459 (SE 130) kJ/d. Plasma EPA and DHA were measured by GC in order to establish fatty acid incor-
poration; EPA increased by approximately 41 % while DHA increased by 6 %. The plasma soluble adhesion molecules sE-selectin,
sICAM-1 and sVCAM-1 were measured by immunoassay with a LINCOplexTM kit (Millipore Corp., Billerica, MA, USA) using the
Luminex 200 System built on XMAP technology (Luminex Corp., Austin, TX, USA). A non-parametric Wilcoxon test was used to
determine statistical differences between dietary interventions after 3 or 6 months (P £ 0.05).

sE-selectin (ng/ml) sICAM-1 (ng/ml) sVCAM-1 (ng/ml)


T-Diet Plus1 Mean SE Mean SE Mean SE

Initial 35.0 3.37 238.9 29.0 1161 91.8


3 month 34.0 2.34 254.6 34.8 1134 70.2
6 month 29.5 3.87 286.6 59.3 1288 90.4

sE-selectin tended to decrease over the 6-month period after dietary intervention, although differences were not significant, probably
because of the great variation between patients with a number of underlying pathologies. sICAM-1 and sVCAM were not modified after
the experimental period. The decrease in some plasma adhesion molecules may indicate an improved outcome for elderly patients fed
enteral diets, which may have the potential to be complementary to pharmacological treatments.

This study was financed by Vegenat SA.

1. Paoletti R, Gotto AM Jr & Hajjar DP (2004) Circulation 109, 20–26.


2. Yaqoob P & Calder P (2003) Eur J Med Res 8, 337–354.
3. Mesa MD, Aguilera CM & Gil A (2006) Nutr Hosp 21, 105–118.
Proceedings of the Nutrition Society (2008), 67 (OCE), E59 doi:10.1017/S002966510800668X

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Serum C3c and C4c concentrations and adenosine deaminase activity of


children with cystic fibrosis: preliminary study

P. Perris1, M. S. Feliu1, S. Barbeito2, I. Strasnoy2, M. Ferraro2 and N. Slobodianik2


1
Department of Nutrition, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina and
2
Nutrition Service, Pedro Elizalde Hospital, Buenos Aires, Argentina

Nutrition is a critical component of the management of cystic fibrosis (CF), and nutritional status is directly associated with both
pulmonary status and survival(1). Previous results have shown that several serum fractions (apoB, transferrin) related to the nutritional
status of children with CF are diminished(2). The present preliminary study measured in the same group of children specific serum proteins
associated with the immune system and the activity of adenosine deaminase (ADA), an enzyme associated with T lymphocytes(3,4).
Sixteen children of both genders with CF, between 5 months and 11 years of age, were evaluated between September 2005 and February
2007,with assistance from the Nutrition Service. Samples of whole blood were collected from fasting patients. C3 and C4 complement
fractions (C3c, C4c) were measured by single radial immunodiffusion techniques using commercially-available kits (Diffuplate; Bio-
cientifica, Buenos Aires, Argentina)(5). The activity of ADA was determined by the method of Giusti & Galanti(6). The results were
compared with reference values obtained from healthy children matched for age and gender.

C3c (mg/l) C4c (mg/l) ADA (U/l)


Group Mean SD Mean SD Mean SD

CF 882* 356 183* 42 36.3* 15.8


Reference 1263 455 275 77 23.0 5.6
Mean values were significantly different from those for the reference group: *0.001 < P < 0.02.

Significantly decreased C3c and C4c values with a concomitant increase in the activity of ADA were observed in patients with CF. The
increase in the activity of ADA would be an alternative mechanism to avoid the accumulation of high levels of deoxynucleotides, which
would be toxic for T-cell lymphocytes(4). These preliminary results suggest that the immune system, evaluated using serum levels of C3c
and C4c and the activity of ADA, is altered. Specific nutritional support should be established and adjusted to individual needs.

Partially supported by University of Buenos Aires (Grant B-060).

1. Dodge JA & Turck D (2006) Best Pract Res Clin Gastroenterol 20, 531–556.
2. Perris P, Barbeito S, Strasnoy I, Ferraro M, Ramos O & Slobodianik N (2007) Proceedings of the AACC Annual Meeting. Poster-B-56.
http://www.aacc.org/AACC/events/ann_meet/annual2007/
3. Kurashige S, Akusawa X, Yoshida T, Teshima C, Kodama K & Mitsuhashi S (1982) Microbiol Immunol 26, 87–92.
4. Feliu MS & Slobodianik NH (2000) Nutrition 16, 1082–1083.
5. Mancini G, Carbonara AO & Heremans GF (1965) Immunochemistry 2, 235–254.
6. Bergmeyer HU (1965) Methods of Enzymatic Analysis. New York: Academic Press.
Proceedings of the Nutrition Society (2008), 67 (OCE), E60 doi:10.1017/S0029665108006691

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Introduction of complementary foods to the infant diet within the


first year of life: evaluation of general recommendations using
Achievable Benchmarks of CareÕ

N. Pastor1, B. Soler2, C. Lifschitz1,3 and The Generación Study Group


1
Mead Johnson Nutritionals, Madrid, Spain, 2E-C-BIO, SL Estudios Cientı́ficos, Spain and
3
Division of Gastroenterology and Nutrition, Texas Children’s Hospital and Children’s Nutrition Research Center,
Baylor College of Medicine, TX, USA

The primary objective of the present study was to assess the effect of DHA and arachidonic acid (ARA) supplementation of infant
formulas on the incidence of respiratory illnesses during the first year of life. Enrolment for this multicentre prospective open-label
12-month observational study was conducted from 2002 to 2003 and included 1392 children from 357 Spanish paediatricians (the
GENERACIÓN Study Group). Infants were assigned in the proportion 4.4 :1 to receive a formula supplemented with 3.2 g DHA and 6.4 g
ARA/kg or a low or non-supplemented control formula. Eligible infants were healthy, born at a gestational age that exceeded 36 weeks
and non-breast-fed. Infants were to visit the paediatrician at baseline and months 1, 3, 5, 7, 9, and 12. At each subsequent visit records
were taken of: anthropometric measurements; month of introduction for gluten-free cereal, gluten, fruits, vegetables, meat, fish, egg yolk,
whole egg, cow’s milk and legumes; the occurrence of clinical symptoms associated with common ailments in infancy. Results of the
main objective have been published elsewhere(1,2), and showed a significantly lower incidence of bronchitis or bronchiolitis in
DHA + ARA-fed children. The secondary objective was to determine the adherence to paediatricians’ recommendations to guidelines(3,4)
on the introduction of complementary foods to the infant’s diet. Achievable Benchmarks of Care (ABC)1 ratios were also calculated to
determine the standards of excellence attained by the 10 % of top performers(5) and identify areas for improving adherence to guidelines
on the introduction of complementary foods(6). The Table summarizes the findings from the study. Overall, the adherence was appropriate
for most recommendations, but some recommendations need to be reinforced, such as the introduction of fruits, fish, cow’s milk and
legumes.

Recommendation n % Adherence ABC1 (%)


Cereal without gluten from 4 months of age 999 97.2 98.9
Cereal with gluten from 6 months of age 829 94.5 100
Fruits from 6 to 7 months of age 1001 16.1 30.3
Vegetables from 6 to 7 months of age 989 82.9 94.3
Meat from 6 to 7 months of age 974 92.4 100
Fish from 9 to 10 months of age 921 75.5 97.8
Egg yolk from 9 to 10 month of age 910 91.1 100
Whole egg from 12 months of age 790 84.4 100
Cow’s milk from 12 months of age 530 55.5 79.4
Legumes from 12 months 679 59.6 83.1

1. Pastor N, Soler B, Mitmesser S, Ferguson P & Lifschitz C (2006) Clin Pediatr 45, 850–855.
2. Pastor N, Soler B, Ferguson P & Lifschitz C (2005) J Pediatr Gastroenterol Nutr 40, 698–699.
3. Ministry of Health (2001) Consejo de buen Provecho para tus Hijos. Alimentación infantil (Guide to Recommended Intakes. Infant Nutrition). Dirección
General de Salud Pública M-52.662–2001; www.msc.es
4. European Society of Paediatric Gastroenterology, Hepatology and Nutrition Committee on Nutrition (1982) Acta Paediatr Scand Suppl 302, 61–95.
5. Weissman NW, Allison JJ, Kiefe CI et al. (1999) J Eval Clin Pract 5, 269–281.
6. Kiefe CI, Allison JJ, Williams OD, Person SD, Weaver MT & Weissman NW (2001) JAMA 285, 2871–2879.
Proceedings of the Nutrition Society (2008), 67 (OCE), E61 doi:10.1017/S0029665108006708

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Plasma protein supplements modulate the activation of gut-associated


immune system induced by Staphylococcus aureus enterotoxin B in rats

A. Pérez-Bosque1, L. L. Miró1, J. Polo2, L. Russell3, J. Campbell3, E. Weaver3, J. Crenshaw3


and M. Moretó1
1
Departament de Fisiologia, Facultat de Farmàcia, Universitat de Barcelona, Spain, 2APC Europe, Granollers,
Spain and 3APC Inc, Ankeny, IA 50021, USA

Supplementation of diets with plasma protein has been shown to prevent the activation of lymphocyte populations of Peyer’s patches and
mesenteric lymph nodes(1) and improve the intestinal epithelial barrier function in a rat model of intestinal inflammation(2). The present
study investigated the effects of porcine plasma proteins (SDAP) and Ig concentrate (IC) supplements on diffuse gut-associated lymphoid
tissue in a model of mild intestinal inflammation. The different populations of lamina propria and intraepithelial lymphocytes, as well as
mucosal expression of cytokines (interferon-g (IFN-g), TNFa, IL-6 and IL-10) and pro-inflammatory mediators (inducible NO synthase
(iNOS) and leukotriene B4 (LTB4)), were investigated. Wistar-Lewis rats were fed diets supplemented with SDAP (80 g/kg; n 9), IC (15 g/
kg; n 9) or milk proteins (control die; n 9) from weaning (day 21) to day 33 or 34 after birth. On days 30 and 33 rats were administered S.
aureus enterotoxin B (SEB; 0.5 mg/kg). Experimental groups were control, SEB, SEB-SDAP and SEB-IC. Lymphocyte populations were
analysed by immunohistochemistry on day 34 (i.e. 24 h after SEB administration). The markers used were: CD3 (T lymphocytes), CD25
(activated T lymphocytes), CD4 (T-helper lymphocytes), CD8 (T-suppressor/cytotoxic lymphocytes), TCRgd (T-gd lymphocytes) and
NKPR1A (NK cells). Cytokines were determined by a cytometric bead array assay, LTB4 by commercial ELISA and iNOS by real-time
PCR in mucosal homogenates, all at 6 h after SEB administration.
In both lamina propria and epithelium compartments SEB increased the lymphocyte cytotoxic populations (T-gd 40 % and 70 %; NK
cells 60 % and 75 % respectively, all P < 0.05) and doubled the number of activated T lymphocytes (P < 0.001). Both SDAP and IC
prevented the SEB effects on the lamina propria, while in the epithelium only SDAP reduced the extent of T-cell activation (P < 0.05).
SEB increased mucosal iNOS expression by 28 % (P < 0.05) and both plasma protein supplements prevented SEB effects on iNOS
expression in the intestinal mucosa (both P < 0.05).
In the mucosa SEB doubled IFN-g and LTB4 concentrations and increased TNFa and IL-6 concentrations by 20–30%; P < 0.05). SDAP
partially prevented these effects on IFN-g, IL-6 and LTB4 (P < 0.05). IC was also effective in reducing the expression of TNFa and LTB4
in the mucosa (P < 0.05). It is concluded that dietary supplementation with plasma proteins can attenuate the intestinal inflammatory
effects induced by SEB.

1. Pérez-Bosque A, Pelegrı́ C, Vicario M et al. (2004) J Nutr 134, 2667–2672.


2. Pérez-Bosque A, Amat C, Polo J, Campbell JM, Crenshaw J, Russell L & Moretó M (2006) J Nutr 136, 2838–2843.
Proceedings of the Nutrition Society (2008), 67 (OCE), E62 doi:10.1017/S002966510800671X

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Dietary fish oil increases IL-4 secretion by murine splenocytes by


an effect on accessory cells

D. H. Petursdottir and I. Hardardottir


Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland

Dietary fish oil has immunomodulatory effects(1). It decreases T-cell proliferation, reduces macrophage eicosanoid synthesis and has
different effects on cytokine secretion by T-cells and macrophages. The present study examined the effects of dietary fish oil on secretion
of T-helper (Th) 1 and Th2 type cytokines by splenocytes and the involvement of accessory cells in the effects of dietary fish oil on
cytokine secretion.
Mice were fed diets supplemented with (g/kg) 180 fish oil + 20 maize oil or 200 maize oil for 6 weeks (n 10). Spleen cells, isolated
T-cells, and splenocytes depleted of T-cells were stimulated with concanavalin A (ConA), anti-CD3 or anti-CD3/anti-CD28. Secretion of
the Th1 and Th2 cytokines interferon-g (IFN-g) and IL-4 and the pro- and anti-inflammatory cytokines TNFa and IL-10 were measured
by ELISA.
Dietary fish oil decreased ConA-, anti-CD3-, and anti-CD3/anti-CD28-induced secretion of IFN-g and TNFa by total splenocytes and
isolated T-cells (P < 0.05). On the other hand, dietary fish oil increased secretion of IL-4 by total splenocytes (P < 0.05) without an effect
on IL-4 secretion by isolated T-cells (Figure). When isolated T-cells were cultured with CD11b + cells, T-cells from mice fed the fish oil
diet secreted more IL-4 than T-cells from mice fed the maize oil diet (P < 0.05; Figure).

IL-4 secretion following stimulation


with anti-CD3/anti-CD28

1200
C orn oil
Fish oil
1000

800
ng/mL

600

400

200

Total T cells T + CD11b+ cells

The results from the present study demonstrate that dietary fish oil directs the immune response of splenocytes towards a Th2 phenotype
and that the effects of dietary fish oil on secretion of Th2 type cytokines are mediated by its effect on CD11b + accessory cells.

1. Calder PC (1997) Ann Nutr Metab 41, 203–234.


Proceedings of the Nutrition Society (2008), 67 (OCE), E63 doi:10.1017/S0029665108006721

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effects of borage (Borago officinalis) oil supplementation on the


expression of monocyte and T-cell adhesion molecules in
healthy volunteers

E. Pinto1 and L. S. Harbige1,2


1
Centre for Biosciences Research, School of Science, University of Greenwich, Kent, UK and 2Medway School of
Pharmacy, University of Kent and University of Greenwich, Kent, UK

Essential fatty acids (EFA) have unique roles in that they can affect inflammatory cell functions directly or as precursors to molecules that
can regulate their functions(1). In comparison with linoleic acid (LA; 18: 2n-6), g-linolenic acid (GLA; 18: 3n-6) has superior biopotency
because it bypasses D6 desaturation, which is regarded as a key regulatory rate-limiting enzymic step controlling the formation of long-
chain PUFA and is beneficial in some inflammatory disorders(1,2). In the present study seven healthy volunteers ingested 14 g GLA-rich
borage oil/d consecutively for 13 weeks. Peripheral blood was obtained at 0, 4, 7 and 13 weeks and the expression of the adhesion
molecules CD11a + , CD11b + , CD36 + , CD54 + and CD62L + on monocytes (CD14 + ) and CD11a + , CD54 + and CD62L + on T-cells
(CD3 + ) was investigated using specific conjugated monoclonal antibodies and flow cytometry. Cell surface expression of CD62L + ,
CD36 + and CD54 + on monocytes after 4, 7 and 13 weeks was significantly lower (P < 0.001, P < 0.005 and P < 0.01 respectively)
compared with baseline expression. CD62L + and CD54 + expression on T-cells after 4 and 7 weeks of supplementation was also
significantly lower (P < 0.05 and P < 0.01 respectively) compared with baseline expression. The Figure illustrates flow cytometric analysis
of CD14 + CD62L + expression before and after 13 weeks of borage oil supplementation.

(A) Before (B) After

These results demonstrate that GLA can significantly decrease cell surface expression of certain adhesion molecules particularly on
monocytes, suggesting that this may be one of the mechanisms by which it is beneficial in some human and experimental autoimmune
inflammatory disease states(1,2).

1. Harbige LS (2003) Lipids 38, 323–341.


2. Harbige LS, Yeatman N, Amor S & Crawford MA (1995) Br J Nutr 74, 701–715.
Proceedings of the Nutrition Society (2008), 67 (OCE), E64 doi:10.1017/S0029665108006733

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effect on lymphoproliferation and in vitro Ig production of splenocytes


from suckling rats when supplemented with conjugated linoleic acid

C. Ramı́rez-Santana, F. J. Pérez-Cano, M. Castell, A. Franch and C. Castellote


Department of Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

Conjugated linoleic acid (CLA) refers to a mixture of positional and geometric isomers of linoleic acid that contain two conjugated double
bonds, of which cis-9, trans-11 and trans-10, cis-12 predominate. Besides the health effects described in human subjects, it has been
reported that CLA modulates immune cell functions in animal models, although there are no reports on this effect during early life. Thus,
the objective of the present work was to establish the effect of CLA supplementation during gestation and/or lactation on ability of spleen
lymphocytes to produce Ig and proliferate under ex vivo conditions. Pups were divided into four groups (A, B, C and W), with each group
comprising ten rats per lactating mother. Mothers of C and W group pups were fed standard pellet chow from day 7 of gestation and
throughout the study period. Mothers of group A and B pups were fed pelleted chow with 10 g CLA (80 % cis-9, trans-11, 20 % trans-10,
cis-12; Lipid Nutrition B.V. Wormerveer, The Netherlands)/kg during gestation and mothers of group A pups continued with this
supplemented chow until weaning. Groups B and C received the CLA isomer mixture by daily oral supplementation while dams were fed
standard pelleted chow during lactation. Animals were killed and their spleens were removed at day 21, the end of suckling period.
Splenocytes were isolated and cultured. After incubation for 7 d, in vitro IgG and IgM production was quantified using the ELISA
sandwich technique. Cells stimulated with phorbyl myristate acetate–ionomycin (250 ng/ml) were used to: (1) quantify IL-2 and inter-
feron-g (IFN-g) levels in culture supernatant fractions after 24 h incubation using ELISA; (2) to measure cell proliferation after incubation
for 72 h (see Table). Cell viability was also evaluated. Conventional ANOVA and post hoc comparisons (LSD test) were performed.
Significant differences were accepted at P < 0.05. No differences among groups were seen in in vitro IL-2 and IFN-g production or
lymphocyte proliferation (see Table), irrespective of life period (gestation or suckling), duration and route of supplementation. However,
spontaneous IgM production by splenocytes from group B animals was higher compared with that of groups C and W (P < 0.05;
see Table). These results suggest that supplementation with CLA during the gestation and suckling periods increases spontaneous Ig
production by immunocompetent cells.

IgM production (ng/ml) Proliferation (%)


Group Mean SE Mean SE

A 937.9 144.0 196.9 37.60


B 1225.6*† 193.5 257.2 29.33
C 539.5 139.7 234.5 57.89
W 534.9 157.3 191.5 33.23
n 10. Mean value was significantly different from that for group C: *P < 0.05. Mean value
was significantly different from that for group W: †P < 0.05.
Proceedings of the Nutrition Society (2008), 67 (OCE), E65 doi:10.1017/S0029665108006745

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Anti-inflammatory effects of cocoa in rat carrageenin-induced


paw oedema

S. Ramos-Romero, E. Ramiro-Puig, F. J. Pérez-Cano, C. Castellote, A. Franch and M. Castell


Department of Physiology, Faculty of Pharmacy, University of Barcelona, Spain

Acute inflammation is the fastest immune system response to damage. Normally, short-lasting and limited inflammation develops beneficial
effects. However, permanent inflammation becomes harmful and painful. Carrageenin-induced paw oedema is a reproducible acute
inflammatory model used in the screening of anti-inflammatory drugs. Cocoa, a product from Theobroma cocoa seeds, contains flavonoids
with potential anti-inflammatory properties. Previous studies have shown that in vitro cocoa extract inhibits some pro-inflammatory
cytokines (TNFa, monocyte chemotactic protein 1 (MCP-1) and IL-6)(1). The aim of the present study was to establish the in vivo anti-
inflammatory effect of cocoa. Female Wistar rats (Harlan Iberica SA, Barcelona, Spain) received water (reference animals; n 10), quercetin
(3.1 g/kg body weight orally; n 10) or cocoa at doses of 4.6 (n 10) or 9.6 (n 10) g/kg body weight orally for 7 d. On day 8 some reference
animals were injected with quercetin (10 mg/kg rat intraperitoneally (ip)). After 1 h all animals were injected with 0.1 ml carrageenin l
(1%, w/v) into subplantar area of the right hind paw. Paw volume was measured by plethysmometry at 30 min post carrageenin injection
and every hour for 6 h. Peritoneal macrophages (PM) and paw inflammatory exudates were obtained at the end of the study. PM were
stimulated with lipopolysaccharide (LPS; 10 mg/ml) and supernatant fractions were obtained after 24 h incubation. TNFa levels in exudates
and supernatant fractions were determined by an ELISA technique. A significant reduction in paw oedema (P < 0.05) was observed in both
groups treated with cocoa from 4 h after carrageenin injection (Figure). At 6 h paw volumes from cocoa-treated animals were about 35 %
lower than that of the reference group. Quercetin treatment did not modify paw volume. TNFa levels in paw inflammatory exudates were
lower in animals treated with the highest cocoa dose (Table). However, TNFa secreted by PM was reduced in the group treated with 4.6 g
cocoa/kg and in animals injected with quercetin ip. In summary, a cocoa-rich diet could play a role as an anti-inflammatory adjuvant. This
immunomodulatory action can be partially attributed to a reduction in cytokine release by pro-inflammatory cells such as macrophages.

100

Reference
Quercetin i.p.
80 Quercetin p.o.
Cocoa (4.8 g/kg)
Cocoa (9.6 g/kg)
volume increase (%)

*
60 * *

*
40 *

20

0
0,5 1 2 3

time from induction (h)

TNFa (pg/ml )
Inflammatory exudate Non-stimulated PM LPS-stimulated PM
Mean SE Mean SE Mean SE

Reference 82.4 12.5 5991.3 685.2 11780.9 1444.6


Quercetin: ip 106.6 18.3 327.6** 87.7 1810.3** 410.6
Orally 63.4 15.1 4395.6 1147.3 7925.2 1954.4
Cocoa: 4.8 g/kg 65.4 10.1 1801.2* 1037.7 2444.0** 1217.7
9.6 g/kg 38.5* 12.2 6719.7 2896.3 12309.7 4489.7
Mean values were significantly different from those for the reference group: *P < 0.05, **P < 0.001.

1. Ramiro E, Franch A, Castellote C, Pérez-Cano F, Permanyer J, Izquierdo-Pulido M & Castell M (2005) J Agric Food Chem 53, 8506–8511.
Proceedings of the Nutrition Society (2008), 67 (OCE), E66 doi:10.1017/S0029665108006757

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Lactobacillus fermentum exerts a beneficial effect in


an experimental model of rheumatoid arthritis in mice

M. E. Rodrı́guez-Cabezas1, F. Fisac1, E. Bailon1, M. Comalada1, D. Camuesco1, J. Xaus2,


A. Concha3, P. Talavera3, A. Nieto4, A. Zarzuelo1 and J. Galvez1
1
Department of Pharmacology, University of Granada, Spain, 2Puleva Biotech SA, Granada, Spain, 3Department of
Pathology, Hospital Universitario ‘Virgen de las Nieves’, Granada, Spain and 4Health and Progress Foundation,
Granada, Spain

Previous studies have shown the intestinal anti-inflammatory activity of the probiotic Lactobacillus fermentum CECT 5716 acting as a
modulator of the intestinal immune response(1). The aim of the present study was to determine the efficacy of this probiotic as a systemic
immune modulator in the experimental model of rheumatoid arthritis induced by Mycobacterium butyricum–Freund’s adjuvant in mice(2).
Male Balb/C mice (n 10) were given the probiotic in the drinking water (108 colony-forming units (CFU)/ml) for 2 weeks and a control
group (n 20) not receiving probiotic was also used for reference. After this period 0.1 ml of a suspension of 10 mg Mycobacterium
butyricum in 10 ml Freund’s adjuvant was administered subcutaneously in the right paw in treated mice and in half the control group,
whereas Freund’s adjuvant was administered in the left paw of the mice; normal mice (n 10) only received Freund’s adjuvant in both
paws. Animals were assessed for clinical arthritis for 4 weeks, and after this period all mice were killed and their arthritic status evaluated
by histological analysis of the paws. In addition, the spleens were removed and T-cells were obtained from spleens and activated with
concanavalin A and the lymphocyte response evaluated by RT–PCR for the cytokines IL-2 and IL-10.
The results showed that the probiotic treatment ameliorated the severity of the disease since it significantly improved the clinical
arthritis score (6.0 (SE 0.7) in control mice with arthritis v. 3.3 (SE 0.3) in probiotic-treated mice; P < 0.05) (Figure 1). Similar findings were
obtained by histological analysis. Mycobacterium-induced arthritis also stimulated the expression of IL-2 in the splenocytes, which was
also reduced in the probiotic-treated mice. Although IL-10 expression by splenic T-cells was not modified during the course of arthritis in
control mice, treatment with the probiotic stimulated the expression of this regulatory cytokine in mice with arthritis. In conclusion,
probiotic treatment ameliorated the disease outcome in the Mycobacterium butyricum model of rheumatoid arthritis in mice, showing
systemic immunomodulatory properties.

Figure 1: CLINICAL ARTHRITIS SCORE

8,0

6,0

4,0

2,0

0,0
0 2 3 5 8 9 11 12 14 15 17 20 27 30
Days

Non arthritic Control arthritic L.fermentum

1. Peran L, Camuesco D, Comalada M et al. (2006) Int J Colorectal Dis 21, 737–746.
2. Mihara M, Nishimoto N, Yoshizaki K & Suzuki T (2002) Immunol Lett 84, 223–229.
Proceedings of the Nutrition Society (2008), 67 (OCE), E67 doi:10.1017/S0029665108006769

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Immunological changes after dehydration resulting from physical effort


in a hot environment

J. Romeo1, D. Jimenez-Pavón2,3, M. Cervantes-Borunda4, J. Wärnberg1, L. E. Dı́az1 and A. Marcos1


1
Immunonutrition Research Group, Department of Metabolism and Nutrition, Consejo Superior de Investigaciones
Cientı́ficas, Madrid, Spain, 2Research Group EFFECTS 262, Department of Medical Physiology, School of Medicine,
University of Granada, Granada, Spain, 3Facultad de Ciencias de la Actividad Fı́sica y del Deporte, Universidad
Politécnica de Madrid, Madrid, Spain and 4Facultad de Educación Fı́sica y Ciencias del Deporte, Universidad
de Chihuahua, México

Intensive exercise is well known to affect the immune function, leading to higher risk of infections and inflammatory processes in
sportsmen(1–3). Several factors are involved in the immune modulation, such as the intensity and duration of the physical activity as well
as inter-individual variations(4,5). Thus, the aim of the present study was to confirm the effects of intensive physical effort in a relatively
short time period and high environmental temperature on variables of immune function and inflammatory markers. A total of twenty-two
young male adults (age 21.2 (SD 1.7) years; VO2max 55.4 (SD 3.6) ml/kg per min) volunteered to participate in an exercise session of 60 min
on a treadmill ergometer at moderate speed (60 % maximum aerobic speed) in hot environmental conditions (35 C and humidity 65 %).
Total leucocyte and lymphocyte counts, absolute values of T-lymphocyte CD8 + , CD4 + , CD3 + , natural killer and CD19 + subsets and
the capacity of cytokine production (IL-2, IL-4, IL-5, IL-10, interferon-g and TNFa) were evaluated before and after exercise without
rehydration. Several inflammation-related proteins (caeruloplasmin, C-reactive protein, C3 and C4) were also measured. The results show
that absolute numbers of leucocytes and neutrophils increased while eosinophils decreased after the intensive exercise. In addition,
caeruloplasmin, C3 and C4 levels also increased after exercise. In contrast, no changes in T-lymphocyte subsets or cytokine production
were observed.

Before exercise After exercise


Mean SD Mean SD

Leucocytes (·109/l) 5.73 1.48 8.84* 2.39


Neutrophils (·109/l) 3.56 1.10 6.83* 2.17
Lymphocytes (·109/l) 1.68 0.44 1.59 0.30
Eosinophils (·109/l) 0.17 0.13 0.08* 0.05
Caeruloplasmin (mg/l) 325.8 49.6 342.8* 56.3
C3 (g/l) 0.99 0.14 1.04* 0.13
C4 (g/l) 0.23 0.07 0.25* 0.07
Mean values were significantly different from those before exercise (Student’s t test): *P < 0.05.

Since prolonged bouts of intensive exercise cause a temporary reduction in various aspects of immune function that usually lasts 3–24 h
after exercise, the results confirm those from previous studies and suggest that 60 min exercise in a hot environment is enough to cause an
immune system adaptation to these conditions leading to an increase in some immune cells and promoting inflammatory processes.
Interestingly, these changes in immune cells have not altered the T-lymphocyte subsets and the capacity for cytokine production;
therefore, immune cell function seems to be preserved in relatively-short-term acute exercise. Further research is necessary to determine to
what extent habitual intensive exercise under high environmental temperature can impair the immune system.

1. Gleeson M (2007) J Appl Physiol 15 (Epublication ahead of print version).


2. Nieman DC (1997) J Appl Physiol 82, 1385–1394.
3. Hoffman-Goetz L & Pedersen BK (1994) Immunol Today 15, 382–387.
4. Shephard RJ (1997) Physical Activity, Training and the Immune Response. Carmel, IN: Cooper Publishing Group.
5. Nieman DC & Pedersen BK (1999) Sports Med 27, 73–80.
Proceedings of the Nutrition Society (2008), 67 (OCE), E68 doi:10.1017/S0029665108006770

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Intake of some immunonutrients and ecoimmunonutrients by


young high-level basketball players

G. Santos1,2, M. A. Buil2, J. A. Ferrero2, J. J. Delgado2, J. Silva2 and J. M. Soriano1


1
Laboratory of Nutrition, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n 46100,
Burjassot Spain and 2Pamesa Valencia Basketball Club, Av. Germans Maristes s/n 46013, Valencia, Spain

An adequate nutritional intake is essential to preserve the health of basketball players. Several studies have demonstrated a low energy
intake by student basketball players on training days, as well as a daily diet with an unbalanced composition of nutrients (too much fat and
a low intake of Ca and Fe)(1–3). A new approach to the nutrition of basketball players is being developed, according to a project of the
University of Valencia (Project UV-AE-20070219), that aims to study immunonutrition and ecoimmunonutrition as the key factors in
order to enhance physical and athletic performance and to decrease the incidence of physical damage.
The aim of the present study was to determine the consumption of ecoimmunonutritional nutrients (fibre) and some immunonutritional
nutrients (Zn, Fe, Mg, vitamins A, C and E) by young basketball players.
The diet of ten male basketball players from Pamesa Valencia’s 12–13-year-old team (weight range 47.4–81.5 kg; height range 1.63–
1.95 m) and eight male basketball players of Pamesa Valencia’s 16–17-year-old team (weight range 75.6–103.0 kg; height range 1.79–
1.995 m) was studied. Three 24 h recalls per player (two weekdays and one day of a weekend) were used for data collection. DIAL version
1.01 (Alce Ingenierı́a, Madrid, Spain) was used to evaluate the dietary intake of the ecoimmunonutrients and immunonutrients investi-
gated.
The youngest basketball players had an intake of Mg, Fe and vitamin C that exceeded the Spanish recommended daily intakes (RDI)(4)
by 20.7, 81.6 and 84.3 % respectively. The consumption of vitamin A and vitamin E was lower than the RDI (12.3 and 28 %) and the Zn
intake was adequate. The intake of fibre was lower than the Spanish final nutritional objective(5). The older group of basketball players had
an intake of Mg, Fe, Zn, vitamin C and vitamin E that exceeded the RDI by 38.6, 65.3, 29.3, 186.5 and 26.6% respectively. The
consumption of vitamin A was 10.9% lower than the RDI and the intake of fibre was the adequate.
In view of the inadequate intake for some of the ecoimmunonutrients and immunonutrients, the basketball players, their relatives and
their trainers and condition coaches are being educated to improve their knowledge of nutrition.

1. Dubnov G & Constantini NW (2004) Int J Sport Nutr Exerc Metab 14, 30–37.
2. Leinus K & Ööpik V (1998) Nutr Res 18, 683–691.
3. Martinchik AN, Baturin AK, Petukhov AB, Baeva VS, Zemlianskaia TA, Sokolov AI, Peskova EV & Tysiachnaia EM (2003) Vopr Pitan 72, 35–40.
4. Instituto de Nutrición (1994) Ingestas de Energı́a y Nutrientes Recomendadas para la Población Española (Recommended Energy and Nutrient Intakes
for the Spanish Population). Madrid, Spain: Departamento de Nutrición, Universidad Complutense.
5. Serra Majem L & Aranceta Bartrina J (2001) Sociedad Española de Nutrición Comunitaria. Guı́as Alimentarias para la Población Española (Spanish
Society of Community Nutrition. Nutrition Guidelines for the Spanish Population). Madrid, Spain: SENC.
Proceedings of the Nutrition Society (2008), 67 (OCE), E69 doi:10.1017/S0029665108006782

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Moderate ingestion of beer reduces inflammatory and oxidative


brain events induced by aluminium in mice

A. Schultz1, R. Oliver1, M. Bautista1, M. J. Gonzalez-Muñoz4, I. Meseguer4, A. Peña4,


M. I. Sanchez-Reus2, J. Benedı́3 and F. J. Sánchez-Muniz1
1
Dpto Nutrición, 2Dpto Bioquı́mica y Biologı́a Molecular II, 3Dpto Farmacologı́a, Facultad de Farmacia,
Universidad Complutense, Madrid, Spain and 4Dpto Nutrición, Bromatologı́a y Toxicologı́a, Facultad de Farmacia,
Universidad de Alcalá, Madrid, Spain

Al is a highly neurotoxic element and has been suggested to participate in the degeneration of cells in the brain of human subjects and
experimental animals. Although the exact mechanism by which the metal may influence degenerative brain processes in unknown, there is
evidence that exposure to Al causes an increase in both oxidative stress and inflammatory events(1). On the other hand, Si intake affects
the bioavailability of Al at the gastrointestinal level, and therefore the Al accessibility to the brain. The aim of the present study was to
examine the effect of supplementing Si in the diet, as silicic acid or by drinking beer, to prevent inflammation and oxidative stress in brain
of mice exposed to oral Al. Four groups of male adult NMRI mice with an initial body weight of 30 g were studied: (1) negative control
administered with deionised water; (2) positive control receiving 450 mg Al(NO3)3/kg body weight per d; (3) positive treatment group
with Si (diet 2 plus 50 mg silicic acid/l; (4) positive treatment group with beer (diet 2 plus beer, equivalent to 1 litre/d for human subjects).
The Al and Si contents of beer and brain tissue were measured by inductively-coupled plasma MS and atomic emission spectrometry
respectively following wet ashing of the organic matter. Whole right hemibrains were dissected, frozen, homogenized and analysed for
mineral content and mRNA for superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and TNFa.
Brain Al levels were significantly lower (P < 0.01) for the negative control animals than for those of groups 2–4. Si intake in the form of
beer lowered, although not significantly (2.45 mg/g v. 3.85 mg/g), the brain Al levels of intoxicated mice (group 2). Quantitative RT–PCR
showed that administration of Al significantly decreased levels of both SOD and catalase mRNA for group 2, which were normal for the
brains of mice receiving silicic acid or beer. Furthermore, for group 2 TNFa and GPx mRNA levels were significantly increased (P < 0.05;
Figure) suggesting that Al induced inflammation and brain damage. These levels were approximately normal for groups 3 and 4 after
consuming Si as silicic acid and beer respectively (P < 0.05). There was a significant correlation between brain Si and Al levels and the
expression of some brain enzymes.
The results suggest that moderate beer consumption, by means of its Si content, effectively protects against the neurotoxic effects of Al.
However, consumption of strong beers should be avoided because of the well-known negative effect of alcohol on the brain.

B
1400 c

1200
mRNA levels of GPx (% control)

1000

800

600
b

400 b

200 a

0
Cont r ol Al Al/ Beer Al/ Si( OH) 4

Figure. The GPx values, expressed in arbitrary units, are means with their standard errors represented by vertical bars for determinations for twelve mice. a,b,cMeans with unlike
superscript letters were significantly different (P < 0.05; Kruskal-Wallis and non-parametric multiple comparison test).

This work has been supported by the Asociación de Cerveceros Españoles and project AGL-2005–07204-C02–01.

1. Campbell A (2006) J Alzheimers Dis 10, 165–172.


Proceedings of the Nutrition Society (2008), 67 (OCE), E70 doi:10.1017/S0029665108006794

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Impact of glycation on duodenal digestibility of Bowman-Birk inhibitors

J. M. Silván and M. D. del Castillo


Instituto de Fermentaciones Industriales (CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain

Adverse effects of soyabean-containing diets include poor digestibility and allergy to soyabean proteins. To improve the nutritional
quality of soya foods inhibitors are generally inactivated by heating or eliminated by fractionation during food processing(1). These
inhibitors could generate an immune response(2). Bowman-Birk (BBI) and Kuntiz (KTI) inhibitors are heat stable and they may be
inactivated by glycation(3), which also reduces soyabean-protein allergenicity(4). The aim of the present study was to investigate the
impact of the BBI glycation on the activity of duodenal digestive enzymes in order to optimise the beneficial effects of soyabean proteins.
The BBI was glycated with glucose, fructose and fructooligosaccharides (FOS; Raftilose P95; Orafti España SL, Barcelona, Spain) in
KOH (0.2 %, w/v) at 60 C for 20 min. The protein glycation was confirmed by isoelectric focusing (IEF) and SDS–PAGE gel electro-
phoresis. In vitro duodenal digestion was performed using the method described by Moreno et al.(5). Samples were incubated with
trypsin–chymotrypsin for 120 min.
Fig. 1 shows the IEF gel (pH 3–10) stained with Comassie Blue. Different electrophoretic profiles were obtained for native and heated
BBI samples (lanes 1 and 2) compared with BBI glycated with glucose, fructose and FOS (lanes 3–5). A decrease in positive charges
was observed as a result of the involvement of basic amino acids (Lys and Arg) in the Maillard reaction (glycation). This analysis
also confirmed BBI stability to heat treatment at 60 C for 20 min. Heating had no effect on the isoelectric point of BBI. Fig. 2 shows the
SDS–PAGE gel (10%, w/v) stained with Comassie Blue. The molecular mass (8 kDa) of native BBI (lane 1) was increased as a
consequence of glycation with glucose, fructose and FOS (lanes 2–4; Fig. 2(a)). All samples inhibited trypsin and chymotrypsin activities.
Samples did not change their electrophoretic profiles after enzymic digestion (Fig. 2(b); lane 1, native BBI; lane 2, heated BBI; lanes 3–5
BBI glycated with glucose, fructose and FOS respectively). Further studies are needed to establish the glycation conditions that cause a
reduction in BBI inhibitory activity against digestive enzymes and to identify the glycation product responsible for this change. Both IEF
and SDS–PAGE analyses are suitable for following glycation of BBI and other soyabean proteins.

Fig. 1 Fig. 2
KDa M
1 2 3 4 5 M 11 22 33 44 11 22 33 44 55
14

6
3,5
2,5
a b

Authors thank ORAFTI for kindly supplying commercial FOS. These studies were funded by projects AGL2004-05031 and ALIBIRD-CM-S0505/
AGR-0153.

1. Friedman M & Brandon DL (2001) J Agric Food Chem 49, 1069–1086.


2. Kaayla TD (2004) Nexus Magazine 11; available at www.nexusmagazine.com
3. Kato,Y & Matsuda T (1997) J Agric Food Chem 45, 3826–3831.
4. van de Lagemaat J, Silván JM, Moreno J, Olano A & Del Castillo MD (2006) Food Res Int 40, 153–160.
5. Moreno FJ, Mellon FA, Wickham MS. Bottrill AR & Mills EN (2005) FEBS J 272, 341–352.
Proceedings of the Nutrition Society (2008), 67 (OCE), E71 doi:10.1017/S0029665108006800

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Lymphocyte subpopulations in Venezuelan preschool children


of high socio-economic status

L. Solano and D. Llovera


Centro de Investigaciones en Nutrición, Facultad de Ciencias de la Salud, Universidad de Carabobo, Valencia, Edo,
Carabobo 2001, Venezuela

Nutrition is a critical determinant of the immune response, thus malnutrition is the most common cause of immunodeficiency in the world.
Micronutrient deficiencies, especially Zn deficiency, have already been shown to be associated with modification of the immune response
in undernourished children, mostly those living in poverty; however, data on children of high socio-economic classes is lacking. The aim
of the present study was to establish lymphocyte population values and to investigate possible interactions with nutritional status,
Zn deficiency and socio-economic conditions in a group of children from high socio-economic classes.
The subjects were 104 preschool children attending a private nursery. Their nutritional status was measured by the weight-for-height
anthropometric indicator(1), lymphocyte subpopulations (CD3 + , CD4 + , CD8 + , CD20) by flow cytometry and serum Zn by atomic
absorption spectrophotometry. Socio-economic data were collected using the Graffar-Méndez method(2). Data are presented as means and
standard deviations, frequency and percentile distribution. Statistical analysis was by Student’s t test, ANOVA and Bonferroni test, with a
significance level of P < 0.05.
A normal nutritional status was found in 74.3% of the children, 9.5 % were undernourished and 16.2 % were overweight. Lymphocyte
subpopulations (CD3 + 65 (SD 6) %, CD4 + 34 (SD 6) %, CD8 + 28 (SD 6) %, CD20 18 (SD 5) %) and serum Zn (889 (SD 150) mg/l) were
within normal values. There was no significant difference by gender, age or nutritional status. Low concentrations of serum Zn were
present in 6.7% of the children. There was a tendency to lower (NS) values for the T lymphocyte population for those children with serum
Zn < 730 mg/l (10th percentile for the population), as shown in the Table.

Serum Zn £730 mg/l Serum Zn > 730 mg/l


Lymphocytes Mean SD Mean SD

T CD3 (%) 62 5 65 6
T CD4 (%) 33 5 35 6
T CD8 (%) 25 6 28 6
T CD4:CD8 1.34 0.68 1.30 0.39
B CD20 (%) 19 7 18 6

Since there are no reference values for the lymphocyte subpopulation in Venezuelan children and these results are in agreement with
previous reports relating to other population groups(3), the up-to-date data from the present study could be used as a reference for future
studies.
These children from high socio-economic classes showed lymphocyte populations within the normal range and a low prevalence of Zn
deficiency. Although a significant association between the lymphocyte population and serum Zn was not observed, the data provide
information on the association between Zn concentrations and cell-mediated immunity expressed by T lymphocytes, as previously
reported(4).
Poverty, infectious diseases and poor sanitation contribute to the development of undernutrition and specific micronutrient deficiencies,
and to modifications of the cellular immune response. Maintenance of an adequate nutritional and immune status in the children evaluated
could be associated with a protective environment related to their socio-economic status.

Project Sponsor: Consejo de Desarrollo Cientı́fico y Humanı́stico de la Universidad de Carabobo, Venezuela.

1. World Health Organization (1995) Physical Status: The Use and Interpretation of Anthropometry. Technical Report Series no. 854. Geneva: WHO.
2. Méndez-Castellano H & Méndez MC (1994) Sociedad y Estratificación. Método Graffar-Méndez-Castellano (Society and Stratification. The Graffar-
Méndez Method). Caracas, Venezuela: Fundacredesa.
3. Comans-Bitter WM, de Groot R, van den Beernd R & Neijens HJ (1997) J Paediatr 130, 388–393.
4. Long KZ & Nanthakumar N (2004) Am J Hum Biol 16, 499–507.
Proceedings of the Nutrition Society (2008), 67 (OCE), E72 doi:10.1017/S0029665108006812

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Timing of gluten introduction and quantity and nature of


gluten-containing foods consumed by infants in Valencia, Spain

B. Soto, A. López and C. Ribes


Pediatric Gastroenterology and Hepatology Section, La Fe Hospital, Valencia, Spain

Currently, there is no information on the level of gluten intake for Spanish children. Recent studies have shown that the timing of gluten
introduction in relation to breast-feeding as well as the amount of gluten could be related to a higher incidence of coeliac disease(1,2). The
food questionnaire estimates how frequently certain foods are eaten in a specific period of time. The aim of the present study was to
develop and validate a food questionnaire for the assessment of gluten consumption of children aged 3–12 months in the Valencia area.
Information on the gluten-containing food intake of healthy children aged 3–12 months was collected prospectively, including the brand
names. For the study the children were assigned to two groups: 3–6 months; 7–12 months. Information was compiled by interviewing
parents to establish how and when foods other than breast milk or formula milk were introduced into their children’s diet. The most-
frequently-used gluten-containing food products were selected and included in the FFQ, which covered a period of 7 d. To calculate the
amount of gluten in these products the vegetable protein content was multiplied by 0.8(3). To validate the FFQ the results of this FFQ were
compared with those from a 7 d food record(4). If gluten had been introduced before the study was conducted, the exact timing of
introduction was recorded.
A total of 100 children were included in the study. In the age-category 3–6 months none of the thirty babies had started consuming
gluten. Moreover, when asked about the timing of gluten introduction the thirty mothers with children aged between 7 and 24 months all
reported that gluten had been introduced after the sixth month of life.

Age (Months) <7 7–8 8–9 >9


% children consuming gluten 0 60 70 100

In the 7–12 months age-group (n 70) the most important gluten-containing food products consumed were muffins and croissants,
cereals, chocolate, ready-to-eat fruit and ready-to-eat meals, biscuits, bread, pasta (macaroni, spaghetti etc.) and crumbed products. All
children > 9 months were consuming gluten-containing food products. In a preliminary study of the gluten intake in this group the mean
gluten intake obtained from the FFQ was 2.55 mg/d.
The current amount of gluten consumed by children in Spain, and more specifically in Valencia, has been established. The variety of
gluten-containing food is limited and controlled, and the mean daily gluten intake in this preliminary study is 2.55 mg/d. Moreover, the
timing of gluten introduction for infants in Valencia is between 7 and 9 months of age. It has been shown previously that gluten
introduction after 7 months of age in at-risk infants is associated with a higher risk of coeliac disease, i.e. 1.87 as compared with gluten
introduction between 4 and 6 months (OD 1)(2). It is possible that this late introduction of gluten could be related to the higher prevalence
of coeliac disease observed in the Valencia area in the last 5 years.

1. Ivarsson A, Persson LA, Nyström L et al. (2000) Acta Paediatr 89, 165–171.
2. Norris JM, Barriga K & Hoffenberg EJ (2005) JAMA 293, 2343–2351.
3. Overbeek FM, Uil-Dieterman IG, Mol IW, Köhler-Brands L, Heymans HS & Mulder CJ (1997) Eur J Gastroenterol Hepatol 9, 1097–1099.
4. Hopman EG, Kiefte-de Jong JC, le Cessie S, Moll H, Witteman J, Bleeker S & Mearin M (2007) Clin Nutr 26, 264–271.
Proceedings of the Nutrition Society (2008), 67 (OCE), E73 doi:10.1017/S0029665108006824

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Selenium deficiency in adults infected with HIV in the era of


highly-active antiretroviral therapy

M. Stambullian1, M. S. Feliu2,4, C. M. López3,4, A. E. Piñeiro3,4, I. Cassetti1 and N. Slobodianik2,4


1
Helios Salud Health Center, 2Department of Nutrition and Food Science, 3Department of Toxicology and 4School of
Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina

The introduction of highly-active antiretroviral therapy (HAART) has produced a minor incidence of malnutrition and an improvement in
the survival and immunological functions of patients infected with HIV. On the other hand, there are still reports of micronutrient
deficiency in the early stage of the disease(1,2,3). Se is particularly relevant in HIV because it may modulates immune cells(1,2,3). The aim
of the present study was to evaluate Se status in adults infected with HIV. The cross-sectional study included fifty-one adults infected with
HIV: 72.5% males; mean age 37.1 (SD 6.9) years; 80.4 % under HAART treatment; median CD4 count 426 (20–1029) cells/mm3; median
viral load < 50 (< 50-273000) copies/ml. Samples of whole blood were collected from fasting patients. Plasma Se was determined in
haemolysis-free plasma by flame atomic absorption spectrometry. A calibration curve was established using commercial standards.
Reference values were taken from the literature (60–160 mg/l)(4–6). The Ethics Committee of the University of Buenos Aires approved the
study. All participants gave informed consent before recruitment. Statistical analysis was by Student’s t test, with significance at P < 0.05,
using SPSS version 13.0 (SPSS, Chicago, IL, USA). Although nutritional status evaluated by BMI was adequate (median 24.02 (inter-
quartile range 22.3–25.5) kg/m2), 82.4% of the patients presented with Se below reference levels (median 42.4 (SD 17.9; 95 % CI 37.5,
47.3) mg). Plasma Se levels were not significantly different between subjects grouped according to the stage of the disease (HIV or AIDS);
the same outcome was observed with and without HAART (P=0.35 and P=0.83 respectively). The results showed a high proportion
of patients with Se deficiency, which could reduce the number of immune cells and/or their function. These results are in agreement with
a previous study performed in children(7). Supplementation with Se may delay the development of the disease and may improve the
prospects of survival and quality of life.

The research was partially supported by University of Buenos Aires (Grant B-060).

1. Carr A & Cooper D (2000) Lancet 356, 1423–1429.


2. Lai H, Lai S, Shor-Posner G, Ma F, Trapido E & Baum MK (2001) J Acquir Immune Defic Syndr 27, 56–62.
3. Baum MK, Shor-Posner G, Lai S, Zhang G, Lai H, Fletcher MA, Sauberlich H & Page JB (1997) J Acquir Immune Defic Syndr Hum Retrovirol 15,
370–374.
4. Varian (1989) Analytical Methods. Flame Atomic Absorption Spectrometry. Publication no. 85-100009-00. Mulgrave, Victoria: Varian Australia Pty
Ltd.
5. Varian (1988) Analytical Methods. Graphite Tube Atomizers. Publication no. 85-100848-00. Mulgrave, Victoria: Varian Australia Pty Ltd.
6. Lockitch G, Halstead A & Wadsworth L (1988) Clin Chem 34, 1625–1627.
7. Pallaro A, Barbeito S, Strasnoy I, Franchello A, Giraudi V, Ramos O & Slobodianik NH (1998) Acta Bioquim Clin Latinoam XXXII, 555–557.
Proceedings of the Nutrition Society (2008), 67 (OCE), E74 doi:10.1017/S0029665108006836

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Can dietary supplements reduce cell-surface expression of


molecules involved in organ transplantation?

K. Varley, B. Clark and C. Carter


Transplant Immunology Department, St James Hospital, Leeds LS9 7TF, UK

Preformed donor-relevant antibodies are a contraindication to successful renal transplantation, causing irreversible damage to the
endothelial cells on the donor organ that leads to vessel obstruction and swift rejection. Such sensitised patients represent a challenge to
successful renal transplantation and individual treatment plans have become necessary in order to obtain a negative cross-match. Current
methods used to treat the recipient before transplant are based on the removal of donor-relevant antibodies and include the use of
high-dose intravenous Ig, immunoadsorption and plasmapheresis. All methods of reducing antibody levels require immunosuppression
therapies in order to halt antibody re-synthesis. Whilst significant efforts are made to control the recipient’s immune system, scant regard
is given to direct modulation of the donor antigens present on the transplanted organ.
In the present study a strategy was explored in which pre-conditioning of the donor, in a live donor situation, could lead to a transient
reduction of human leucocyte antigen (HLA) expression at the time of transplant, thus reducing organ antigenicity. Several studies have
shown that specific compounds can act as immunomodulatory agents. Whilst the use of prescription drugs in a healthy donor would be
obviously inappropriate, n-3 fish oil (a widely-used food supplement) has been shown to have a direct effect on peripheral monocytes by
inhibiting the expression of surface molecules involved in antigen presentation(1). Although controversial, the relatively innocuous nature
of fish oils indicates their potential as an acceptable immune-response modifier for use in the diet of the organ donor before transplant.
In order to quickly screen a number of potential compounds, work was undertaken to establish an in vitro screen for HLA class II
expression and other relevant cell-surface molecules on human monocytic cell lines. This procedure will be used to test potential
compounds (health food or dietary supplements) to assess their ability to down regulate molecules involved in antigen presentation. The
pre-monocytic cell line THP-1 was analysed by two-colour flow cytometry to measure baseline expression of CD14/HLA-DR. Cells were
subsequently stimulated with lipopolysaccharide (LPS) for 24 h or 48 h and analysed for CD14/HLA-DR up-regulation. A representative
example showing CD14 (FL-1)/HLA-DR(FL-2) expression from unstimulated and LPS-stimulated THP-1 cells is shown in the Figure.

Unstimulated Stimulated

It was found that resting (unstimulated) THP-1 cells did not express CD14, although they expressed variable levels of HLA class II
HLA-DR (Figure). The levels of class II showed a marginal increase following stimulation with LPS. CD14 expression was also detected
on significant numbers of THP-1 cells following stimulation with LPS (Figure).

1. Hughes DA & Pinder AC (2000) Am J Clin Nutr. 71, Suppl., 357S–760S.


Proceedings of the Nutrition Society (2008), 67 (OCE), E75 doi:10.1017/S0029665108006848

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Psychological stress and immune function: establishing both acute


and chronic psychological stress models in balb/c mice

E. Vázquez, A. Barranco, M. Manzano, M. L. Jiménez and R. Rueda


Science & Technology, R&D Dept, Abbott Nutrition, Granada, Spain

Stress-induced immunological alterations have been considered to be a major cause of increased risk for immune-related diseases such
as cancer, autoimmune disorders, food allergy, alteration of mucosal barrier function and susceptibility to infection. Stressors trigger
physiological responses involving the hypothalamic–pituitary–adrenal axis (HPA), which is closely associated with the immune system.
It is well known that some foods, in addition to contributing essential nutrients, also contain additional components such as prebiotic,
probiotic and other functional ingredients that induce a balanced immune system and improve disease resistance and general healthy
status. There is a need for appropriate animal models as a tool for testing the potential benefit of some of these functional foods on the
HPA–immune system axis.
The aim of the present work was to establish both acute and chronic mouse models for the study of cellular immunity modulation by
psychological stress. Female 6-week-old Balb/c mice were used in both stress models (n=12 for each experimental group). The acute
stress model comprised for four consecutive days overnight 16 h-restraint with food and water deprivation (RST model). On the other
hand, the chronic stressor was based on the natural panic of mice in relation to water; thus, animals were placed on a very small water-
surrounded platform for 1 h/d for 10 d (the water-avoidance stress model; WAS). At the end of both experimental schedules mice were
killed and serum, small intestine, spleen and thymus were removed. Physiological variables such as body weight, intestinal length and
weight, intestinal mucosa and serum corticosterone levels were recorded. The immunological variables determined were spleen and
thymus weights, intestinal secretory IgA content, serum ovoalbumin-specific antibody production, number of peritoneal macrophages,
number of spleen cells, number of spleen natural killer cells and the expression of the surface spleen cell markers CD45, CD3, CD4, CD8,
CD45R/B220, CD16/CD32 and Ia/Ie. Both stress models were found to impair the normal animal status, affecting most of the selected
physiological and immunological variables (table 1). In the RST model the body, thymus and small intestine weights, the levels of
corticosterone and ovoalbumin-specific antibody in serum and the number of spleen B-, T-, T-helper, CD3–CD4 + and CD3–CD8 + cells
were significantly lower in the stressed animals than in the controls (P < 0.05). Similarly, in the WAS model the stressed animals showed
a lower numbers of spleen T-helper, T-cytotoxic and total T-cells, although the numbers of CD3–CD4 + and CD3–CD8 + cells were
significantly higher. In addition, the level of serum corticosterone and thymus weight were significantly lower in stressed animals than in
controls.
In conclusion, both psychological stress models significantly affected the normal status of the mice, the RST model (acute) being much
more detrimental to the animals than the WAS model, which seems to be an appropriate tool to mimic the psychological stress damage
experienced by populations in Western countries.
Table 1. Data of anthropometric measurements and cell counts
WAS model RST model
Control Stress Control Stress
Body weight (g) 19,76°1.07 19,86°1,28 18,95°0,85 15,91°1,24*
Thymus weight (g) 0,059°0,011 0,039°0,006* 0,038°0,009 0,016°0,006*
Spleen weight (g) 0,131°0,02 0,135°0,017 0,118°0,02 0,090°0,0180
Spleenocytes (n cells) 93,11 · 106°15,91 · 106 98,54 · 106°16,35 · 106 87,87 · 106°25,5 · 106 50,53 · 106°25,83 · 106*
NK (n cells) 1,68 · 106°0,37 · 106 2,703 · 106°0,82 · 106* 3,05 · 106°0,71 · 106 1,26 · 106°0,22 · 106*
Small intestine mucose weight (g) 0,662°0,092 0,659°0,071 0,759°0,078 0,562°0,091*
Mean°SD. *P < 0.001.
Proceedings of the Nutrition Society (2008), 67 (OCE), E76 doi:10.1017/S002966510800685X

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Effects of a synbiotic on intestinal and immune functions of


healthy adults

B. Viadel1, E. Nova2, J. E. Carreres1 and A. Marcos2


1
Department of New Products, AINIA, Technologic Center, Valencia, Spain and 2Immunonutrition Group,
Department of Metabolism and Nutrition, Instituto del Frı́o, CSIC, Madrid, Spain

Synbiotic preparations contain a combination of probiotics and prebiotics (fermentable fibre). The principal purported health-promoting
effect of probiotics is the enhancement of mucosal immune defences, while fructose oligosaccharides have a bifidogenic effect on human
colonic endogenous flora. The main objective of the present study was to evaluate the effects of a 6-week intervention with a synbiotic
product (Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus paracasei ssp. paracasei, Streptococcus
thermophilus, Bifidobacterium sp. (2.4 · 109 colony-forming units/d) and fructo-oligosaccharides; SYMBYO1 : Laboratorios Phergal SA,
Madrid, Spain) on the intestinal and immune functions of thirty-six healthy adults aged 25–45 years (sixteen males, twenty females).
Subjects were randomly assigned to the symbiotic (S; seven males, eleven females) or placebo (C; nine males, nine females) groups.
Faecal and blood samples were collected at baseline and on completion of the study. Total aerobic and anaerobic micro-organisms,
enterobacteria, enterococci, pseudomonas, yeasts, lactobacilli, bacteroids, Clostridium spp. and bifidobacteria were quantified in faeces.
Phagocyte function was quantitatively assessed (Phagotest; BD Biosciences, Madrid, Spain) and highly-sensitive C-reactive protein,
caeruloplasmin (immunoturbidimetry) and soluble adhesion molecules intercellular adhesion molecule-1 (sICAM-1), vascular cell adhe-
sion molecule-1 (sVCAM-1) and sL-selectin (ELISA) were determined in serum. Intestinal motility and improvement in any previous
gastrointestinal symptoms were assessed through a self-administered questionnaire. A non-parametric Wilcoxon test was used to assess
changes in faecal flora after treatment, repeated-measures ANOVA for the effect of treatment on immune variables and Monte Carlo exact
test for differences in symptom improvement.
The improvement in intestinal motility was significantly more reported in the S group than in the C group (P < 0.003). Also, the
cumulative self-reported improvement in any symptom was significantly more frequent in the S group than in the C group (P < 0.001),
although asymptomatic volunteers were significantly more frequent in the S group in the baseline evaluation (P < 0.01). No significant
interaction of time · group was observed for any immunological variable except sL-selectin, which showed a significant decrease in the
S group (see Table). When correlations were analysed in the groups defined by treatment, it was observed that in the S group baseline L-
selectin levels were correlated with final sICAM-1 levels (r 0.468; P=0.050) and baseline sICAM-1 levels were negatively correlated with
final L-selectin concentration (r - 0.457; P=0.056). In contrast, no significant correlations were observed between sL-selectin and
sICAM-1 in the C group. The microbiological analysis of faecal samples showed no differences between the C and S groups, probably
because a low bacterial load was administered.

C group (n 18) S group (n 18)


T1 T2 T1 T2
Mean SD Mean SD Mean SD Mean SD P (ANOVA)
sICAM-1 (ng/ml) 469 85 433 82 434 78 406 50 0.674
sVCAM-1 (ng/ml) 615 212 575 198 545 126 521 148 0.732
sL-selectin (ng/ml) 2586 555 2560 626 2724 600 2389* 420 0.050
T1, baseline; T2, after 6 weeks of treatment. Mean value was significantly different from that for T1 (Student’s test):
*P < 0.05.

The present study shows that the daily intake of the synbiotic supplement can have beneficial effects on the intestinal motility of healthy
subjects and might improve the serum levels of L-selectin, possibly leading to a more beneficial profile of the inflammatory markers in
relation to the prevention of atherosclerosis and CVD.

This work has been supported by LABORATORIOS PHERGAL SA.


Proceedings of the Nutrition Society (2008), 67 (OCE), E77 doi:10.1017/S0029665108006861

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Changes in nutritional status and their effects on serum and


thymus zinc levels and serum copper:zinc

S. M. Vidueiros1, I. Fernandez1, A. E. Piñeiro2, N. Slobodianik1 and A. Pallaro1


1
Department of Nutrition and 2Department of Toxicology, School of Pharmacy and Biochemistry,
University of Buenos Aires, Argentina

Previous studies have shown that the intake of low-quality dietary protein causes a decrease in plasma Zn levels and an uptake by other
organs(1,2). It is known that nutritional disorders affect the immune system and that Cu and Zn play a critical role in its integrity.
Moreover, an increase in Cu :Zn is associated with a higher risk of morbidity and mortality in patients who are immunodeficient(3,4). In the
present work the effect of feeding a cereal-based diet and a recovery diet on serum Cu and Zn levels, Cu :Zn and thymus Zn concentration
was investigated in weaning rats.
Wistar rats at weaning (21–23 d) were fed a diet containing 65 g precooked maize protein (M) or casein (Cas)/kg for18 d. Group M was
refed with a 200 g casein/kg diet (MC) for 20 d. Age-matched control groups received stock diet (C1 (40 d) and C2 (60 d)). Body weight
(BW; g) and body-weight gain (BWG; g/d per 100 g BW) were determined. Serum Cu (mg/ml) and Zn (mg/ml) concentrations were
determined by atomic absorption spectrophotometry and Cu:Zn was calculated. Thymuses were removed and weighed (TW; mg) and the
Zn content (ng/ g organ) was determined by atomic absorption spectrophotometry.
Serum Zn was decreased in M and Cas groups compare with the age-matched controls (group C1); thus, Cu: Zn was higher in these
experimental groups. This ratio, as well as serum Zn level, returned to normal in group MC. Thymus Zn level in group M was higher
compared with groups Cas and C1, which had similar values. Thymus Zn was normal in group MC and no significant differences were
found between groups MC and C2.

Serum Zn (mg/ml) Serum Cu (mg/ml) Cu : Zn Thymus Zn (mg/g organ)


Group (n 6–10) Mean SD Mean SD Mean SD Mean SD

M (40 d) 1.22a 0.4 1.32a 0.3 1.25b 0.4 65.7a 18.8


Cas (40 d) 1.32a 0.3 1.37a 0.1 1.07b 0.2 21.4b 1.3
C1 (40 d) 2.18b 0.7 1.30a 0.1 0.65a 0.2 30.1b 4.3
C2 (60 d) 2.18b 0.6 1.57b 0.2 0.75a 0.2 25.6b 2.0
MC (60 d) 1.94b 0.3 1.16a 0.2 0.61a 0.1 26.3b 4.9
a,b
Mean values within columns with unlike superscript letters were significantly different (0.05 > P > 0.001).

The increase in serum Zn level and the reduction in Cu :Zn during recovery was concomitant with a higher BWG (MC 5.48 (SD 0.66) v.
M - 0.15 (SD 0.37); P < 0.001) and TW (MC 625.4 (SD 157.2) v. M 88.6 (SD 26.2); P < 0.001).
These results indicate that serum Zn and Cu: Zn are dependent on the quantity and/or quality of dietary protein. The reduced serum Zn
concentration could be caused by a higher uptake of this mineral by the thymus, which was observed to have an increased Zn content.
This outcome may represent a compensatory mechanism to overcome the low concentration and/or activity of thymic hormones described
in malnourished status, as it is known that Zn could be an immunomodulator of thymocyte proliferation and maturation(4). These data
suggest that it would be not necessary to determine the Cu:Zn, since data for serum Zn and for Cu :Zn lead to the same conclusion.

1. Pallaro A & Slobodianik N (1999) Nut Res 19, 1089–1095.


2. Pallaro A, Roux ME & Slobodianik N (2001) Nutrition 17, 724–728.
3. Fraker P (2000) Nutritional Immunology: Principles and Practice, pp. 147–156 [M Gershwin, JB German and CL Keen, editors], Totowa, NJ; Humana
Press Inc.
4. Dardenne M, Pleau JM, Nabarm B et al. (1982) Proc Natl Acad Sci USA 79, 5370–5373.
Proceedings of the Nutrition Society (2008), 67 (OCE), E78 doi:10.1017/S0029665108006873

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Impact of a cereal-based diet on intestinal villi of growing rats

S. M. Vidueiros, I. Fernandez, N. Slobodianik, M. E. Roux and A. Pallaro


Department of Nutrition, School of Pharmacy and Biochemistry, University of Buenos Aires, Argentina

Malnutrition has an adverse impact on cell-mediated, secretory and humoural immunity, as well as on non-specific host defences.
Although generalized malnutrition can affect all aspects of host immunity, the impact is greater on T-cell functions and cell-mediated
immunity and smaller on B-cell functions as well as humoural immunity. Effects on secretory immunity are intermediate, mainly affecting
IgA B-cells. Recent studies have shown that certain chemokines play an important role in regulating homeostatic lymphocyte recirculation
through secondary lymphoid organs. The chemokine thymus-expressed chemokine (TECK) is a chemoattractant involved in the ability of
lymphocytes to localize in the gastrointestinal tract(1).
The aim of the present work was to study the effect on B and T lymphocytes as well as the TECK + cell population on the intestinal
villi of growing rats after the administration of a cereal-based diet as the only source of protein.
Wistar rats were fed a 65 g precooked maize protein/kg diet for 18 d (M). An age-matched control group received a stock diet (C). Body
weight (BW; g) was determined, ponderal growth rate (PGR; g/d per 100 g) was calculated and intestines were removed and processed by
the Saint Marie technique. Tissue sections were studied by an indirect immunofluorescence technique. CD5 + T-cells and the subsets
TCRab+ , TCRgd+ , CD4+ , CD8aa+ , CD8ab+ in the lamina propria (LP) and intraepithelium (iIEL) and IgA-B+ cells in LP were
determined (number of cells per thirty fields). Also, the presence of TECK + cell population was assessed.
There were significant differences between the M and C groups in BW (44.7 (SD 7.83) v. 149.1 (SD 17.7); P < 0.0001) and PGR (–0.30
(SD 0.39) v. 5.27 (SD 0.53); P < 0.0001). As shown in the Table, the numbers of IgA + B-cells in the LP and CD5+ T-cells and CD4+ ,
CD8aa+ , CD8ab+ , TCRab+ , and TCRgd+ T subpopulations in the LP and iIEL of the gut villi for the M group were significantly
decreased (P < 0.001).

Phenotye . . . IgA B+ T CD5+ TCRab+ TCRgd + CD8aa+ CD8ab+ CD4+


Group (n 6) Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

Gut LP
Maize 57.5 16.5 39.3* 13.4 35.7* 7.6 49.7* 15.2 46.9* 13.8 52.6* 17.6 59.5* 20.47
Control 123.5 18.7 107.8 16.2 106.7 37.5 100.9 17.8 110.1 25.1 131.7 20.8 97.4 19.3
Gut iIEL
Maize 2.2† 1.6 1.6† 1.4 2.8† 1.3 0.4† 0.6 0.4† 0.7 0.5† 1.1
Control 7.6 4.3 3.3 2.3 5.4 3.8 2.8 2.2 2.0 2.0 1.7 1.9
Mean values were significantly different from those for corresponding age-matched C group: *P < 0.0001.
Mean values were significantly different from those for corresponding age-matched C group: †0.03 > P > 0.001.

Differences in the size and cellularity of the gut villi and in the distribution of TECK were also observed in the experimental groups.
When histological slides were analysed, the C group showed TECK+ cells in the villi and crypt epithelium, while the M group showed a
decreased intensity.
The results show that the intake of a low-quality dietary protein as the only source of protein produces an important disorder in mucosal
immunity that could explain the incidence of gastrointestinal infections observed in malnourished children.

1. Kunkel EJ, Campbell JJ, Haraldsen G et al. (2000) J Exp Med 192, 761–767.
Proceedings of the Nutrition Society (2008), 67 (OCE), E79 doi:10.1017/S0029665108006885

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Breast-milk levels of long-chain PUFA in Kazakhstan and Sweden

M. Xiang1, L. S. Harbige1,2 and R. Zetterström3


1
Centre for Biosciences Research, School of Science, University of Greenwich, Kent ME4 4TB, UK, 2Medway School of
Pharmacy, University of Kent and University of Greenwich, Kent ME4 4TB, UK and 3Department of Paediatrics,
Karolinska Hospital, Karolinska Institute, Stockholm, Sweden

The deterioration of human health with increasing infant mortality rate, declining life expectancy at birth and increasing prevalence of
serious infectious diseases in Russia and other former Soviet Republics is thought to be the result of a combination of several factors such
as inadequate nutrition, poor sanitation, collapse of the healthcare system and pollution from Soviet agriculture and industries(1,2).
Environmental pollutants, which may occur in breast milk and in various food products and drinking water, and which are also transferred
to the foetus, constitute a severe threat to the health of infants and children. In Kazakhstan, which is the second largest area of the former
Soviet Republics with only eighteen million inhabitants, the situation is particularly alarming. Long-chain (LC) PUFA are essential dietary
nutrients required for the optimal growth and development of infants, particularly of the brain and retina. It is important for exclusively-
breast-fed infants to receive milk of a correct balance between n-6 and n-3 fatty acids(3–5). In the present study the composition of LC
PUFA in the colostrum of Kazakh and Swedish mothers and the growth rate of their foetuses were compared. Ten and nineteen
mother - term infant pairs from a countryside located about 600 km from Almaty, Kazakstan and Stockholm, Sweden, who were 1 - 5 d
old and exclusively breast-fed, were studied. The colostrum of the Kazakh mothers had significantly higher linoleic acid (LA; 18: 2n-6)
and lower a-linolenic acid (LNA; 18: 3n-3), arachidonic acid (AA; 20: 4n-6), EPA (20: 5n-3) and DHA (22: 6n-3) levels than those of the
Swedish mothers. The recommended ranges for LA: LNA and AA : DHA in human milk are 5 - 10 and 0.5 - 1 compared with 32.4 and
3.22 in the Kazakh breast milk, and 8.98 and 1.35 in the Swedish breast milk respectively (Table).

Kazakh (n 10) Swedish (n 19)


Ratio Mean SE Mean SE P value
LA : LNA 32.4 2.01 8.98 0.47 < 0.001
AA :DHA 3.22 0.24 1.35 0.08 < 0.001
Sn-6/Sn-3 PUFA 24.6 1.16 6.13 0.26 < 0.001
Sn-6/Sn-3 LC PUFA 9.78 0.60 3.12 0.14 < 0.001

The milk of the Kazakh mothers is less balanced in relation to the levels of n-6 and n-3 PUFA than that of the Swedish mothers.
Whether environmental pollutants and/or PUFA might affect the developing immune and nervous systems in this Kazakh population
could be followed up by a study of immune and neuro-functions of Kazakh and Swedish infants.

1. Zetterstrom R (1999) Acta Paediatr 429, 49–54.


2. Jensen S, Mazhitova Z & Zetterstrom R (1997) Sci Total Environ 206, 187–193.
3. Xiang M, Harbige LS & Zetterstrom R (2007) Acta Paediatr 96, 387–390.
4. Xiang M, Rahman MA, Ai H, Li X & Harbige LS (2006) Ann Nutr Metab 50, 492–498.
5. Xiang M, Harbige LS & Zetterström R (2005) Acta Paediatr 94, 1543–1549.
Proceedings of the Nutrition Society (2008), 67 (OCE), E80 doi:10.1017/S0029665108006897

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Time-course study of high-dose creatine supplementation for


endogenous creatine synthesis

M. Xiang2, L. S. Harbige2,3, X. Li1, B. Li1 and H. Ai1


1
Division of Nutrition and Biochemistry, Institute of Sports Medicine, The Third Hospital, Peking University, Beijing,
People’s Republic of China, 2Centre for Biosciences Research, School of Science, University of Greenwich, Kent ME4 4TB,
UK and 3Medway School of Pharmacy, University of Kent and University of Greenwich, Kent ME4 4TB, UK

Creatine (Cre) is a popular nutritional supplement in many population groups, and has potential therapeutic effects in some diseases(1–4).
Cre supplementation may increase the Cre content of muscle(5), and improve muscle strength(6). L-Arginine:glycine amidinotransferase
(L-AGAT) in the kidney is the rate-limiting enzyme of endogenous Cre synthesis in mammals, and guanidinoacetic acid (GAA) is
the precursor in this synthesis. The aim of the present study was to determine the effect of high-dose Cre supplementation on L-AGAT
activity and endogenous Cre synthesis. A time-course of repression of L-AGAT activity and GAA concentration by 3.0 g supplemented
Cre /kg per d was studied in adult male Sprague-Dawley rats. In the comparison between groups supplemented with 3.0 g Cre/kg body
weight per d for 0.5, 1, 2, 4 and 8 d respectively and the control group (without Cre supplementation; n 10), the L-AGAT activity in the
kidney in the supplemented groups decreased (%) by 31.7, 41.4, 48.9, 61.4 and 73.5 respectively (Fig. 1(A)). Furthermore, the kidney
concentration of GAA for the groups receiving 3.0 g Cre/kg per d for 0.5, 1, 2, 4 and 8 d decreased ( %) by 4.9, 20.4, 31.7, 37.9 and 40.8
respectively (Fig. 1(B)). The total Cre in gastrocnemius muscle increased gradually with time and was 109.3% above the control levels
after 8 d. In the groups receiving 3.0 g Cre/kg per d for 0.5, 1 and 2 d the serum creatinine increased significantly and, then declined
gradually until day 8. No significant differences were found in the serum creatine kinase activity among the six groups.
These results indicate that for rats L-AGAT activity and GAA concentration could be repressed rapidly by 3.0 g Cre/kg per d in 8 d,
suggesting that high-dose Cre supplementation may quickly result in the depression of endogenous Cre metabolism. These results may be
relevant to the immune system and this aspect is currently being investigated in lymphocytes, as little is known about the effect of Cre
supplementation on immune cells.
120
A
Relative L-AGAT activity

100
%)
in kidneys (%

80
**
60 **
**
40 **
**
20

0
0 1 2 3 4 5 6 7 8

120
B
Relative GAA in kidneys (% )

110
100
90
80 **

70 **
**
60 **

50
0 1 2 3 4 5 6 7 8
Time (days)

1. Prass K, Royl G, Lindauer U, Freyer D, Megow D, Dirnagl U, Stockler-Ipsiroglu G, Wallimann T & Priller J (2007) J Cereb Blood Flow Metab 27, 452–459.
2. Kley RA, Vorgerd M & Tarnopolsky MA (2007) Cochrane Database Syst Rev 1, CD004760.
3. McMorris T, Harris RC, Swain J, Corbett J, Collard K, Dyson RJ, Dye L, Hodgson C & Draper N (2006) Psychopharmacology (Berl) 17, 1–11.
4. Bender A, Auer DP, Merl T et al. (2005) J Neurol 252, 36–41.
5. Rockwell JA, Rankin JW & Toderico B (2001) Med Sci Sports Exerc 33, 61–68.
6. Becque MD, Lochmann JD & Melrose DR (2000) Med Sci Sports Exerc 32, 654–658.
Proceedings of the Nutrition Society (2008), 67 (OCE), E81 doi:10.1017/S0029665108006903

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

The effect of moderate dose of EPA+DHA on lipid profile


and inflammatory markers in middle-aged men

H. M. Yusof and P. C. Calder


Institute of Human Nutrition, School of Medicine, University of Southampton, Developmental Sciences Building,
Mailpoint 887, Southampton General Hospital, Tremona Road, Southampton SO16 6 YD, UK

The aim of the present study was to investigate the effects of a moderate dose of long-chain (LC) n-3 PUFA (2.3 g EPA + DHA/d; 6 : 1)
in the form of fish oil capsules on cardiovascular risk factors, particularly the plasma lipid profile and plasma inflammatory markers
(C-reactive protein, IL-6, soluble adhesion molecules). In a double-blind placebo-controlled study twenty healthy middle-aged men were
randomised to fish oil providing 2.3 g/d EPA + DHA or medium-chain TAG-rich oil (placebo) for 8 weeks. Blood was collected after an
overnight fast.
Plasma total cholesterol (TC) concentration was significantly increased in the fish oil group but HDL-cholesterol and LDL-cholesterol
concentrations were not significantly affected following supplementation with fish oil, although they were influenced by the placebo oil
(see Table). Fish oil supplementation did not exhibit a favourable TAG-lowering effect previously shown(1). Likewise, positive effects
of LC n-3 PUFA on inflammatory markers were not observed, except that there was a significant difference between fish oil and placebo
in terms of percentage change from baseline for soluble intercellular adhesion molecule 1 (sICAM-1; P=0.05). It was demonstrated that in
the fish oil group plasma sICAM-1 concentration decreased by 9.5 % compared with a 9.9% increase in the placebo group. Correlation
analyses, however, did not show any relationships between the changes in the fatty acid profiles, particularly of LC n-3 PUFA, and the
reduction in sICAM-1.

Fish oil (n 9) Placebo (n 11)


Baseline 8 weeks Baseline 8 weeks
Outcome Mean SE Mean SE P Mean SE Mean SE P
TC (mmol/l) 5.2 0.4 5.6 0.4 < 0.05 4.8 0.3 5.6 0.3 < 0.001
LDL-cholesterol (mmol/l) 3.1 0.3 3.3 0.3 NS 3.0 0.3 3.5 0.3 < 0.001
HDL-cholesterol (mmol/l) 1.4 0.1 1.5 0.2 NS 1.1 0.1 1.3 0.1 < 0.001
TAG (mmmol/l) 1.3 0.2 1.7 0.3 NS 1.1 0.1 1.4 0.2 NS
TC : HDL 4.1 0.5 4.2 0.5 NS 4.5 0.3 4.5 0.3 NS
LDL : HDL 2.5 0.4 2.6 0.4 NS 2.8 0.3 2.8 0.2 NS

The results of the current study are consistent with a limited effect of modest doses of LC n-3 PUFA on the outcomes studied here.
Reasons for the lack of TAG lowering by fish oil may be too low an intake of LC n-3 PUFA, insufficient DHA or the inclusion of subjects
who were normotriacylglycerolaemic. The finding of the current study in relation to sICAM-1 is in general accordance with some previous
studies(2–5) and it can be concluded that some aspects of the inflammatory response are sensitive to modest-dose LC n-3 PUFA and that
EPA + DHA may exert similar modest anti-inflammatory effects in middle-aged men and in the elderly. In summary, findings from the
present study indicate that a dose of 2.3 g/d EPA + DHA is not sufficient to demonstrate maximal effects on CVD risk factors, including
inflammatory markers, in fairly-healthy middle-aged men.

1. Harris WS (1996) Curr Opin Lipidol 7, 3–7.


2. De Caterina R, Liao JK & Libby P (2000) Am J Clin Nutr 71, Suppl., 213S–223S.
3. Abe Y, El Masri B, Kimball KT, Pownall H, Reilly CF, Osmundsen K, Smith, CW & Ballantyne CM (1998) Arterioscler Thromb Vasc Biol 18,
723–731.
4. Berstad P, Seljeflot I, Veierod MB, Hjerkinn EM, Arnesen H & Pedersen JI (2003) Clin Sci (Lond) 105, 13–20.
5. Hjerkinn EM, Seljeflot I, Ellingsen I, Berstad P, Hjermann I, Sandvik L & Arnesen H (2005) Am J Clin Nutr 81, 583–589.
Proceedings of the Nutrition Society (2008), 67 (OCE), E82 doi:10.1017/S0029665108006915

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

A specific mixture of short-chain galacto-oligosaccharides and


long-chain fructo-oligosaccharides induced an anti-allergic Ig profile in
infants at risk for allergy

A. J. Nauta1, S. Arsnalognu2, G. Boehm3, G. Moro2, J. Faber1, E. Knol1, B. Ruiter4,


E. van Hoffen4, L. M’Rabet5 and J. Garssen1,2
1
Immunology, Numico-Research BV, Wageningen, The Netherlands, 2Macedonio Melloni Maternity Hospital, Center for
Infant Nutrition, Milano, Italy, 3Numico-Research, Friedrichsdorf, Germany, 4Academic Hospital Utrecht, Dermatology,
Utrecht, The Netherlands and 5Utrecht Institute for Pharmaceutical Sciences, Pharmacology and Pathophysiology,
Utrecht, The Netherlands

In a prospective study in infants with a family history of atopy a specific prebiotic oligosaccharide mixture (90 % short-chain galacto-
oligosaccharides and 10 % long-chain fructo-oligosaccharides (GOS/FOS; IMMUNOFORTIS) reduced the cumulative incidence of atopic
dermatitis at 6 months of age(1). In a subgroup of these infants (n 84) it was possible to obtain a blood sample at 6 months of age to
analyse the potential effect of these dietary oligosaccharides on the Ig profile.
In this prospective double-blind randomized placebo-controlled study the infants received a hypoallergenic formula with either 8 g
GOS/FOS/l or 8 g/ maltodextrin (placebo)/l for 6 months. At 3 months of age children were vaccinated against diphtheria, tetanus and
polio (DTP). At 6 months of age total plasma levels of IgE, IgG1, IgG2, IgG3 and IgG4 as well as cow’s-milk protein (CMP)- and DTP-
specific Ig were measured by ELISA.
Supplementation with GOS/FOS led to a significant reduction in plasma levels of total IgE (P=0.007), IgG2 (P=0.029) and IgG3
(P=0.0343) whereas no significant effect on IgG4 was observed. The plasma levels of CMP-specific IgG1 was significantly decreased
(P=0.015) in the GOS/FOS group. The levels of CMP-specific IgE were very low and no effect of GOS/FOS supplementation was
observed. CMP-specific IgG4 was not detectable in the samples. No effect of GOS/FOS supplementation on any vaccine-specific antibody
isotype levels was found.
Evidently, GOS/FOS supplementation induced an anti-allergic Ig profile in infants at high risk for allergic diseases while the
desired specific immune responses were unaffected, indicating the potential role of oral GOS/FOS exposure for primary prevention of
allergies.

Placebo (median) GOS/FOS (median) P


Total IgE (kU/ml) 10.0 4.00 0.008
Total IgG1 (g/L) 3.09 2.26 0.005
Total IgG4 (mg/L) 187.7 427.2 0.728
CMP IgE (ng/ml) 2.50 1.80 0.348
CMP IgG1 (AU/ml) 3.40 1.00 0.015
DTP IgE (AU/ml) 0.40 0.37 0.884
DTP IgG1 (AU/ml) 441.3 329.6 0.748

1. Moro G, Arslanoglu S, Stahl B, Jelinek J, Wahn U & Boehm G (2006) Arch Dis Child 91, 814–819.
Proceedings of the Nutrition Society (2008), 67 (OCE), E83 doi:10.1017/S0029665108006927

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Immune-enhancing role of vitamin C and zinc and effect on


clinical conditions

S. Beveridge1, E. S. Wintergerst2, S. Maggini1 and D. Hornig3


1
Bayer Consumer Care, Basel, 2Bayer Diabetes Care, Basel, Switzerland and 3 Bayer Diabetes Care, Reinach, Switzerland

The present paper is intended to give an overview on the roles of vitamin C and Zn in immune functions. Vitamin C concentrations in the
plasma and leucocytes rapidly decline during infections and stress. Supplementation of vitamin C improves components of the human
immune system such as antimicrobial and natural killer (NK) cell activities, lymphocyte proliferation, chemotaxis and delayed-type
hypersensitivity. Vitamin C contributes to maintaining the redox integrity of cells and thereby protects them against reactive oxygen
species generated during the respiratory burst and in the inflammatory response. Similarly, Zn deficiency impairs cellular mediators of
innate immunity such as phagocytosis, NK cell activity, and the generation of oxidative burst. Thus, both nutrients play important and
complementary roles in immune function and the modulation of host resistance to infectious agents, reducing the risk, severity and
duration of infectious diseases. A deficiency in one of these essential nutrients weakens immunity since vitamin C is crucial for cellular
immunity and Zn for the production of antibodies.A large number of randomized controlled intervention trials with intakes of £1 g
vitamin C and £30 mg Zn are available. These trials show that adequate intakes of vitamin C and Zn ameliorate symptoms and shorten the
duration of respiratory tract infections including the common cold. Natural defences can only provide full protection when the body has
sufficient Zn, as well as high levels of vitamin C.The physiological effects of vitamin C provide clear evidence and rationale for a number
of ways in which it might help to protect against infection. This evidence is termed mechanistic evidence because it stems from
knowledge of the chemical reactions and biochemical processes in which vitamin C is known to play an important role (Table). The
actions of Zn not only complement the actions of vitamin C to provide ‘double protection’ (Table), but may even have a synergistic effect.
Like vitamin C, in recent years research has proved that Zn is essential for effective immune defence at several different levels.
Table. Summary of the role of vitamin C and Zn in body defences(1,2,3)
Defence Vitamin C Zn
Skin and mucosal barriers Collagen synthesis Cellular proliferation
Improved strength Maintains thickness
Neutrophils and macrophages Improved motility and chemotaxis
Enhanced killing
Overall improvement in phagocytosis
Lymphocytes Proliferation of stem cells
B- and T-cell differentiation
B- and T-cell interaction
B lymphocytes Antibody production
T lymphocytes Proliferation Proliferation and appropriate response
Destruction of infected tissue cells and tumours
Interferon Production enhanced

Adequate intakes of vitamin C and Zn are essential for health.This is of special importance in populations in which insufficient intake of
these nutrients is prevalent. The current belief is that regular prophylactic intakes of vitamin C at doses of ‡ 200 mg daily have no effect
on the incidence of the common cold, but may be beneficial in the reduction of the severity and duration of the symptoms, suggesting that
vitamin C plays some role in the respiratory defence mechanisms. However, the elderly, who have been shown to have a lowered vitamin
C status and may therefore be more prone to infections, individuals exposed to continuous oxidative stress, such as chronic smokers, and
individuals exposed to heavy physical exercise and/or cold environment may benefit from a moderate continuous vitamin C intake. Other
vulnerable population groups include children. As a result of the high prevalence of Zn deficiency, especially in children in developing
countries, and the impaired immune status, susceptibility to infectious diarrhoea, malaria and pneumonia is found to be substantially
increased. Large intervention trials with daily intakes of 10–30 mg Zn have shown that Zn supplementation could be an important
adjuvant therapy for treating these infectious diseases in children in developing countries. Given that both vitamin C and Zn have
an important and synergistic effect on immune function and the modulation of host resistance to infectious agents it is hence appropriate
and beneficial to combine the trace element Zn with a high dose of vitamin C in one supplement.

1. Wintergerst ES, Maggini S & Hornig DH (2006) Ann Nutr Met 50, 85–94.
2. Wintergerst ES, Maggini S & Hornig DH (2007) Ann Nutr Met 51, 301–323.
3. Maggini S, Wintergerst ES, Beveridge S & Hornig DH (2007) Br J Nutr 98, Suppl. 1, S29–S35.
Proceedings of the Nutrition Society (2008), 67 (OCE), E84 doi:10.1017/S0029665108006939

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Contribution of selected vitamins and trace elements to immune function

S. Maggini1, E. S. Wintergerst2, S. Beveridge1 and D. Hornig3


1
Bayer Consumer Care, Basel, Switzerland, 2Bayer Diabetes Care, Basel, Switzerland and 3Bayer Diabetes Care,
Reinach, Switzerland

The immune system requires essential nutrients to function efficiently. Inadequate intake and status of vitamins and trace elements may
lead to suppressed immunity, which predisposes to infections and aggravates undernutrition. Available data indicate a role for vitamins A,
B6, B12, C, D and E and folate and for the trace elements Se, Zn, Cu and Fe in the immune response. Evidence has accumulated that in
human subjects these nutrients selectively influence the immune response, induce dysregulation of a coordinated host response to infec-
tions in the case of deficiency and oversupply, and that deficiency may impact on the virulence of otherwise harmless pathogens. Thus,
micronutrients are required at appropriate intakes for the immune system to function optimally and contribute to the body’s natural
defences on three levels by supporting the physical barriers (skin and mucosa), cellular and humoral immunity. Vitamins A, C and E and
the trace element Zn assist in enhancing the skin barrier function. The vitamins A, B6, B12, C, D and E and folic acid and the trace
elements Fe, Zn, Cu and Se work in synergy to support the protective activities of immune cells. Finally, all these micronutrients, except
vitamin C and Fe, are essential for antibody production.
The table summarises the most important roles of selected vitamins and trace elements in immune function(1–3).

Micronutrient Main roles in the immune system


Vitamin A Normal differentiation of epithelial tissue and gene expression
Important role both in humoral antibody response and cell-mediated immunity
Deficiency impairs innate immunity, induces inflammation, potentiates existing inflammatory conditions and impairs ability
to defend against extracellular pathogens
Supplementation is of benefit in reducing the morbidity and mortality from infectious diseases (especially children)
Vitamin D Potent immune system modulator when metabolized to 1,25-dihydroxycholecalciferol, is involved in cell proliferation and enhances
innate immunity by increasing the differentiation of monocytes to macrophages
Deficiency correlates with higher susceptibility to infections as a result of impaired localized innate immunity and defects in antigen
specific cellular immune response
Supplementation together with a high-Ca diet inhibits progression of autoimmune disorders
Vitamin E Optimizes and enhances immune response (T-helper (Th) 1 response)
Supplementation in healthy adults increases T-cell proliferation, improves the CD4 + :CD8 + , and decreases indices of oxidative stress
Supplementation of elderly individuals improves overall immune function
Vitamins B6 and B12 Interfere with immune function through involvement in nucleic acid and protein biosynthesis
and folic acid Maintain Th1 immune response (vitamin B6), innate immunity (natural killer (NK) cell activity; folate) and act as
immunomodulatory for cellular immunity, especially with effects on cytotoxic cells (NK; CD8 + T lymphocytes; vitamin B12)
Vitamin C Maintains redox integrity of cells and protection against reactive oxygen species (ROS) generated during respiratory burst and inflammatory response
Stimulates leucocyte functions (neutrophil, monocytes movement)
Role in antimicrobial and NK cell activities, lymphocyte proliferation, chemotaxis and delayed-type hypersensitivity response
Decreases duration and severity of common cold
Se Antioxidant essential for optimal immune response, influencing both the innate and the acquired immunity
Deficiency impairs antibody production and causes viruses to undergo mutations to more virulent forms
Supplementation normalizes age-related decline in immune response
Zn Influences both innate and acquired immunity, supports Th1 response, helps to maintain skin and mucosal integrity
Deficiency leads to thymic atrophy, decreased cell-mediated cytotoxicity, helper T-cell and NK cell activities
Cu Part of Cu/Zn-superoxide dismutase, a key enzyme in the defence against ROS
Maintains intracellular antioxidant balance, suggesting an important role in inflammatory response
Adequate intake supports a Th1 response and both deficiency and oversupply modulate the immune response
Fe Essential for cell differentiation and growth, component of enzymes critical for functioning of immune cells
Involved in the regulation of cytokine production and action

Insufficient intake of micronutrients occurs in individuals with eating disorders, in smokers (both active and passive), in individuals
with chronic alcohol abuse, in patients with certain diseases, during pregnancy and lactation, and in the elderly. With aging a variety of
changes are observed in the immune system, which translate to less-effective innate and adaptive immune responses and increase
susceptibility to infections. Overall, inadequate intake and status of vitamins A, B6, B12, C, D and E and folate and of the trace elements
Se, Zn, Cu and Fe may lead to suppressed immunity, which predisposes to infections and aggravates malnutrition. Thus, supplementation
with a combination of these selected micronutrients can support the body’s natural defence system by enhancing all three levels of
immunity: epithelial barriers; cellular immunity; antibody production.

1. Wintergerst ES, Maggini S & Hornig DH (2006) Ann Nutr Met 50, 85–94.
2. Wintergerst ES, Maggini S & Hornig DH (2007) Ann Nutr Met 51, 301–323.
3. Maggini S, Wintergerst ES, Beveridge S & Hornig DH (2007) Br J Nutr 98, Suppl. 1, S29–S35.
Proceedings of the Nutrition Society (2008), 67 (OCE), E85 doi:10.1017/S0029665108006940

1st International Immunonutrition Workshop, Valencia, 3–5 October 2007, Valencia, Spain

Differential effect of Bifidobacterium species characteristic of the


gut microbiota of breast-fed and formula-fed infants on
in vitro cytokine production

E. Puertollano1, T. Pozo1, I. Nadal2, A. Marcos1, Y. Sanz2 and E. Nova1


1
Immunonutrition Group, Metabolism and Nutrition Department, Instituto del Frı́o (CSIC), Madrid, Spain and
2
Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), Valencia, Spain

The importance of the bacterial colonization process of the newborn intestine in the maturation of the immune system has been
recognized. The presence of a healthy microflora (adequate composition of species and their frequency) in the gut is thought to play a role
in the balance of T-helper (Th) 1–Th2 immune responses. Some differences have been observed in the composition of bifidobacterial
species in relation to the type of milk fed. While B. breve is one of the predominant species of the gut microbiota of breast-fed (BF)
babies, B. catenulatum and B. adolescentis are characteristic of that of formula-fed (FF) infants(1). The aim of the current study was to
assess the differential effect of the bifidobacterial species identified in the intestinal microbiota of BF and FF infants on cytokine
production by peripheral blood mononuclear cells (PBMC). The effects of different bifidobacterial species were assessed individually and
in combinations representing their proportions in infants under both feeding types. Live bacterial cell suspensions (107 colony-forming
units/ml) were incubated with PBMC in the proportion 10 :1 for 48 h (5% CO2, 37 C) and cytokine concentrations were measured in the
supernatant fraction by flow cytometry (CBA; BD Biosciences, Madrid, Spain). The experiments were performed with blood from four
volunteers and duplicates were done of each experimental condition and analysed separately. Statistical analyses were carried out using
ANOVA and Mann-Whitney tests. Results for representative cytokines of Th1 and Th2 responses (interferon-g (IFN-g) and IL-4
respectively) and the anti-inflammatory cytokine IL-10 are presented. Significant differences were found among different bifidobacterial
species for the induction of IFN-g and IL-10 production (P < 0.001 and P=0.003). B. catenulatum was the strongest enhancer of IFN-g
production, followed by B. breve. B. catenulatum is more frequent in FF infants and B. breve is more frequent in BF infants(1), but no
differences were found in the induction of IFN-g production by BF and FF mixtures of bifidobacteria used. B. catenulatum also induced
significantly higher levels of IL-4 than B. adolescentis and B. infantis (P=0.029). B. infantis was a mild cytokine inducer, showing the
lowest effect among the assayed species for all three cytokines. B. longum, which is the third most frequent bifidobacterial species in
infant flora of both BF and FF infants, showed the strongest IL-10-inducing capacity. The effects of BF and FF bifidobacterial species
combinations on cytokine production were not significantly different.

IL4 (pg/ml) IL10 (pg/ml)


IFN-gamma (pg/ml)
2000
1500
6000 1500
1000
4000 1000
500
2000 500

0 0 0

Mean values and standard deviations represented by vertical bars.


Bifidobacterium adolescentis Bifidobacterium angulatum Bifidobacterium breve Bifidobacterium catenulatum
Bifidobacterium infantis Bifidobacterium longum Breast-fed Formula-fed
Phytohaemaglutinin Lipopolysaccharide Control, non-stimulated

These results suggest that the presence or absence of particular bifidobacterial species as well as the overall composition of
the bifidobacterial population in the infant gut could be key factors defining the immunomodulatory effect of the gut microbiota in
early life.

This work received financial support from CSIC; project references 200570F0091 and 200570F0093.

1. Haarman M & Knol J (2005) Appl Environ Microbiol 71, 2318–2324.