JAB-40; No.

of Pages 6

journal of applied biomedicine xxx (2014) xxx–xxx

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: http://www.elsevier.com/locate/jab

Original Research Article

Caffeine downregulates antibody production in a

mouse model
a,b,
Miroslav Pohanka *
a
Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic
b
Karel English College in Brno, Brno, Czech Republic

article info abstract

Article history: Caffeine is a secondary plant metabolite found in coffee and tea. Its major pathway is
Received 13 June 2014 interaction with adenosine receptors. Minor pathways are also known. An effect of caffeine
Received in revised form on immunity has been proposed. In this paper, the role of caffeine on the immune system
3 September 2014 was studied, using a BALB/c mouse model. The animals received saline (controls), keyhole
Accepted 4 September 2014 limpet hemocyanin (KLH) 1 mg/kg or caffeine (alone or in combination with KLH) in doses of
Available online xxx 1–16 mg/kg. The mice were sacrificed 1–7 days later and plasma levels of interleukin (IL) 2, 4,
6, 10, 12 and antibodies against KLH were determined by enzyme linked immunosorbent
Keywords: assay (ELISA). Caffeine caused a significant decrease in KLH-stimulated antibody produc-
Caffeine tion. The effect was dose dependent. There were similar findings for IL-2 and IL-4 but not for
Cholinergic anti-inflammatory IL-6, IL-10 and IL-12. The significance of the findings is discussed with extrapolation to
pathway humans based on caffeine doses used in the study and the amount of caffeine in available
Antibody beverages.
Interleukin # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske
Keyhole limpet hemocyanin Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Vaccination
Adjuvants
Coffee
Immunity

accessibility is due not only to its popularity but also because

Introduction
There are few legally available stimulants on the market.
Caffeine (proper chemical name 1,3,7-trimethyl-1H-purine-
2,6(3H,7H)-dione; also designated trimethylxanthine) is con-
tained in numerous foods, drugs and drinks without legal
restriction making it an exception to other stimulants. Easy
it has minimal adverse health effects (Cizkova et al., 2008;
Seifert et al., 2013). In the body, caffeine competitively
antagonizes adenosine at adenosine receptors. This is
considered its major pharmacodynamic mechanism (Ribeiro
and Sebastiao, 2010; Chen et al., 2013). Neurons with adenosine
receptors are implicated in pro-inflammatory responses
and caffeine can inhibit the initiation of inflammation

* Correspondence to: Faculty of Military Health Sciences, University of Defence, Trebesska 1575, CZ-50001 Hradec Kralove, Czech Republic.
Tel.: +420 973253091.
E-mail address: miroslav.pohanka@gmail.com
1214-021X/$ – see front matter # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published
by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
http://dx.doi.org/10.1016/j.jab.2014.09.001

Please cite this article in press as: Pohanka, M., Caffeine downregulates antibody production in a mouse model. J. Appl. Biomed. (2014),
http://dx.doi.org/10.1016/j.jab.2014.09.001
JAB-40; No. of Pages 6
2 journal of applied biomedicine xxx (2014) xxx–xxx
(Antonioli et al., 2013; Peacock et al., 2013; Samsel et al., 2013;
Sun et al., 2013).
Beside the major pathway, caffeine is known to involve
minor pathways as well. For example, it can interfere with
secondary messengers via inhibition of phosphodiesterase
(Bhaskara et al., 2008; Herman and Herman, 2013) causing
alteration to intracellular Ca2+ levels via ryanodine receptors
(Hotta et al., 2007; Puttachary et al., 2010; Skiteva et al., 2012;
Gerasimova et al., 2013; Gaburjakova and Gaburjakova, 2014).
Caffeine interacts with a number of enzymes too. Weak
inhibition of acetylcholinesterase (AChE) is an example (Okello
and Leylabi, 2012). In some papers, a link between caffeine and
the immune response has been proposed; however, the
mechanism of this effect remains obscure. Immunomodula-
tion via the cholinergic system has been extensively reviewed
(Pohanka, 2014a). This review was aimed at the effect of
caffeine on multiple targets in the body and the cholinergic
anti-inflammatory pathway was considered one possible
mechanism of the action of caffeine. However, direct proof
was missing. Anti-inflammatory effects such as reduction in
tumor necrosis factor a (TNFa), pyrogenic interleukin (IL) 1 and
pro-inflammatory acting IL-6 were described in some studies
(Horrigan et al., 2004, 2006; Bessler et al., 2012; Gordillo-
Bastidas et al., 2013). However, the molecular mechanisms
underlying these effects were not revealed. Some researchers
have proposed that it can be mediated by toll-like receptors
(Tunc et al., 2013), adenosine receptors (Ohta et al., 2006; Ohta
and Sitkovsky, 2009), cAMP pathway (Rosenthal et al., 1992a),
and adenosine-deaminase (Bandyopadhyay and Poddar, 1994;
Tunc et al., 2013). Conclusions drawn on the effects of caffeine
on immunity can be affected by congeners in natural products
containing caffeine. For example, Nosal'ova et al. (2011) found
a significant effect of arabinogalactan-protein in instant coffee
powder. This should be taken into consideration when testing
natural products containing caffeine. In the following experi-
ment, pure caffeine was used, to obviate confounding by any
other biologically active congeners.
One hypothesis tested here is that caffeine could modulate
vaccination efficacy. To date, there is scant literature on the
subject. For this reason, the experiment below was carried out
to respond to the question whether caffeine can modulate
immunity in a mouse model with keyhole limpet hemocyanin
(KLH) as a standard antigen.
Experimental
Laboratory animals and immunization
Laboratory BALB/c female mice (Velaz, Unetice, Czech Repub-
lic) were used for the experiment. Female mice were chosen
Table 1 – Experimental groups and time of euthanasia after the start of the experiment.
Controls (saline) 1 day (group 1)
Caffeine 16 mg/kg 1 day (group 2)
KLH 1 mg/kg 1 day (group 3)
Caffeine 1 mg/kg + KLH 1 mg/kg 1 day (group 4)
Caffeine 4 mg/kg + KLH 1 mg/kg 1 day (group 5)
Caffeine 16 mg/kg + KLH 1 mg/kg 1 day (group 6)
Please cite this article in press as: Pohanka, M., Caffeine downregulates antibody production in a mouse model. J. Appl. Biomed. (2014),
http://dx.doi.org/10.1016/j.jab.2014.09.001
because of their low aggressiveness and better social tolerance
compared to males (Kuchiiwa and Kuchiiwa, 2014; Tomaz
et al., 2014). The results then can be more easily attributed to
the caffeine variable than any other undesired effect of social
stress in the animals.
In total, 180 mice were kept as 18 groups of 10 animals. The
mice weighed 19
2 g and they were 8 weeks old at the
beginning of experiment. The animals were kept in standard
conditions for breeding of laboratory rodents: temperature 22

2 8C, humidity 50
10% and 12 h light cycle. The experiment
was permitted by the Ethics Committee at the Faculty of
Military Health Sciences, University of Defense (Hradec
Kralove, Czech Republic; license number CZ 52760099).
Caffeine and KLH were purchased from Sigma–Aldrich
(Saint Louis, MO, USA) and dissolved in saline. 100 ml of the
compounds were intramuscularly injected into the hind limb.
KLH solution was injected into the right limb followed by
application of caffeine into left limb 30 min later. Controls
received saline in the same volume. The experimental design
and time of euthanasia are shown in Table 1. 1 mg/kg reflects
amount of caffeine in one cup of coffee that is reported to be
approximately 100 mg (Yubero-Lahoz et al., 2012). The doses 4
and 16 mg are approximately 2.5% and 10% of median lethal
dose for rodents (Warszawski et al., 1978). The dose range was
chosen to cause physiological effects and no toxicity. At the
end of experiment, the animals were sacrificed by severing the
jugular vein using surgical scissors. The animals were under
CO2 narcosis when sacrificed.
The time intervals were chosen to allow for innate
immunity stimulation (first and second day after application)
and the interval when antibody production can be expected
(seventh day) because of antigen administration to BALB/c
mice (Pohanka, 2007, 2009). Controls receiving caffeine with no
KLH received 16 mg of caffeine. The control group was chosen
in order neglect pertinent initiation of immunity without
presence of an antigen. Lower doses of caffeine were not
applied for this purpose because of effort to limit number of
used laboratory animals.
Blood was collected in a lithium heparin tube (Dialab,
Prague, Czech Republic) and centrifuged at 1000  g for 5 min.
Plasma was separated and stored at 80 8C until assay (see
next chapter).
Enzyme linked immunosorbent assay (ELISA) of antibodies
against KLH
100 ml per well of a 96 well microplates Maxisorp KLH solution
(10 mg/ml) was injected (Nunc, Thermo Fisher Scientific,
Waltham, USA) and allowed to incubate at 4 8C overnight
and then twice washed by 400 ml of phosphate buffered saline
with Tween 20. The wells were blocked by 100 ml of bovine
2 days (group 7) 7 days (group 13)
2 days (group 8) 7 days (group 14)
2 days (group 9) 7 days (group 15)
2 days (group 10) 7 days (group 16)
2 days (group 11) 7 days (group 17)
2 days (group 12) 7 days (group 18)
JAB-40; No. of Pages 6

journal of applied biomedicine xxx (2014) xxx–xxx 3

albumin 5%, w/w for 2 h and washed by the buffer with Tween.
Finally, 100 ml of 100 times diluted plasma samples were
applied per one well and allowed to incubate at 37 8C for 2 h.
After the washing as described above, anti-mouse immuno-
globulins antibody specific against IgG, IgA and IgM labeled by
peroxidase (Sigma–Aldrich) diluted 10,000 times was injected
in a volume of 100 ml per well and kept at 37 8C for 2 h. The
plates were washed and a fresh solution of 0.5 mg/ml 3,30 ,5,50 -
tetramethylbenzidine and 5 mmol/l H2O2, allowed to react for
15 min, and then stopped by 2 mol/l H2SO4 in a dose 100 ml per
well. Optical density was measured by ELISA reader Sunrise
(Salzburg, Austria) at 650 nm. Replacement of plasma sample
by bovine albumin (1 mg/kg) was used for control purposes.

ELISA of IL-2, IL-4, IL-6, IL-10, IL-12

The tested cytokines (IL-2; IL-4; IL-6; IL-10; IL-12) were assayed
by indirect ELISA kit from Sigma–Aldrich. The 96-well
microplates with immobilized recognition antibody were used
in compliance with the manufacturer's protocol. Calibration
was processed in the same way. In these tests, ELISA reader
Sunrise was used for optical density measurement.
Experimental data processing
Statistical software Origin 8 Pro (OriginLab Corporation,
Northampton, MA, USA) was used to analyze the data using
two way, ANOVA with Bonferroni correction (two-sided tests)
to determine whether the markers differed between groups.
The alpha level was 0.05. The tests of significance are
expressed for controls being sacrificed in the same interval
like the exposed animals (i.e. groups 5–6 to group 1; groups 8–
12 to group 7, and groups 14–16 to group 13).
Fig. 1 – Results from ELISA for total antibodies against KLH.
Error bars indicate standard error of mean and the number
inside bars indicates number of days (1, 2 or 7) following
stimulation by caffeine at which euthanasia was made.
Symbol * respective ‘‘a’’ is expression for statistical
difference against controls (the first three columns marked
as ‘‘control’’) respective animals exposed to KLH alone (the
three columns marked as ‘‘KLH’’) at the significance level
2alpha = 0.05. When the significance tested, the same time
interval (days) from either controls or the KLH groups taken
into consideration.

Results and discussion

No animal perished or showed any pathology. The animals

had normal behavior and no differences in the behavior were
observed when the controls compared to the other animals. It
should be emphasized that the controls have the tested
immunochemical markers in a narrow interval and no
significant differences between the control groups (1, 2 and
7 days i.e. groups 1, 7 and 13) were observed. Production of
antibodies against KLH is depicted in Fig. 1. As can be seen
caffeine reduced antibodyproduction triggered by KLH. The
effect was significant and in a dose response manner. The data
negatively correlated with coefficient of determination 0.856.
The highest dose of caffeine (16 mg/kg) caused reduction in
antibody level to values indistinguishable from controls.
Antibody production appeared seven days after antigen
application. Production of antibodies in the controls corre- Fig. 2 – Results from ELISA for IL-2 level in plasma. Error
sponded with expectations based on cited laboratory experi- bars indicate standard error of mean and the number
ments where production of antibodies was stimulated by inside bars indicates number of days (1, 2 or 7) following
disparate antigens (Pohanka, 2007, 2009). However, caffeine stimulation by caffeine at which euthanasia was made.
was not used in these studies. Symbol * is expression for statistical difference against
Plasma levels of IL-2 are shown in Fig. 2. In the animals controls (the first three columns marked as ‘‘controls’’) at
exposed to KLH only, IL-2 was significantly increased in all the significance level 2alpha = 0.05. When the significance
KLH-treated animals compared to controls. The increase was tested, the same time interval (days) from either controls or
significant. The controls and animals exposed to caffeine only the KLH groups taken into consideration.

Please cite this article in press as: Pohanka, M., Caffeine downregulates antibody production in a mouse model. J. Appl. Biomed. (2014),
http://dx.doi.org/10.1016/j.jab.2014.09.001
JAB-40; No. of Pages 6
4 journal of applied biomedicine xxx (2014) xxx–xxx
Fig. 3 – Results from ELISA for IL-4 level in plasma. Error
bars indicate standard error of mean and the number
inside bars indicates number of days (1, 2 or 7) following
stimulation by caffeine at which euthanasia was made.
Symbol * respective ‘‘a’’ is expression for statistical
difference against controls (the first three columns marked
as ‘‘control’’) respective animals exposed to KLH alone (the
three columns marked as ‘‘KLH’’) at the significance level
2alpha = 0.05. When the significance tested, the same time
interval (days) from either controls or the KLH groups taken
into consideration.
had levels of IL-2 under 18 pg/ml. Caffeine caused small
decrease in IL-2 in the animals immunized by KLH. However,
the decrease in IL-2 was minor compared to the effect of
caffeine on antibody production.
Plasma IL-4 levels are presented in Fig. 3. Caffeine did not
cause any alteration in IL-4. However, animals which two time
intervals (2 and 7 days) than the first. In the seventh day, IL-4
level in plasma was approximately doubled over controls. IL 4
levels in the KLH and the KLH plus caffeine 1 and 4 mg/kg were
not significantly different. However, the upper dose of caffeine,
16 mg/kg, reduced IL-4 production in animals stimulated with
KLH. IL-4 is responsible for induction of naive T cells
differentiation into Th2 cells (Caubet et al., 2014; Xiang
et al., 2014). It can be inferred that caffeine enhances Th2
cell maturation and the increased antibody production was a
consequence of the effect on the T cells.
IL-6, IL-10 and IL-12 were assayed as well. However, no
significant difference in the tested cytokines was found when
the markers from animals exposed to KLH and/or caffeine
were compared to the controls (data not shown). The IL-6 level
ranged from around 1 to 3 pg/ml, IL-10 from 0.7 to 1 pg/ml and
IL-12 from 0.90 to 1.4 pg/ml, respectively. The range was equal
to the expected values found in the literature (Cift et al., 2013;
Brunoni et al., 2014; Kuvibidila et al., 2014). The fact that no
significant response for the IL-6, IL-10 and IL-12 markers was
revealed may be that assayed values were on the lower end of
calibration plots in the ELISA kits. Limit of detections of the kits
Please cite this article in press as: Pohanka, M., Caffeine downregulates antibody production in a mouse model. J. Appl. Biomed. (2014),
http://dx.doi.org/10.1016/j.jab.2014.09.001
were not provided by the manufacture; however, it is
estimated that the limits were around 0.5 pg/ml for calibration
plots and confidence intervals of the calibration. The minor
changes would disappear in value dispersions (analytical
noise). On the other hand, model chosen here was not
proposed to cover inflammation. Major focus antibody
production. Effect of caffeine on inflammation was described
by some researchers including effect on TNFa, IL-6, and IL-6
(Horrigan et al., 2004, 2006; Bessler et al., 2012; Gordillo-
Bastidas et al., 2013).
From the results, caffeine proved to be significantly
involved in the regulation of the immune response to a
protein antigen. As the antibodies as well as IL-2 and IL-4 were
reduced by caffeine in a dose response manner in the animals
immunized by KLH, a significant role of caffeine inhibition of
specific immunity maturation can be inferred. The effect on
innate immunity is in a good compliance with the cited papers
(Saxena et al., 1984; Mandal and Poddar, 2008) where
suppression of the innate immune response was revealed.
The suppression probably precedes maturation of specific
immunity response and caffeine interferes in the primary
initiation of immunity. A decrease of antigen-stimulated CD4+
cells caused by caffeine is also mentioned in the literature
(Fletcher and Bishop, 2012). The effect would be based on
inhibition of T cell proliferation as proposed by Rosenthal et al.
(Rosenthal et al., 1992b). Decline in antibody production is the
consequence.
The role of caffeine in immunity cannot be explained by a
simple mechanism. Fletcher and Bishop stated that caffeine
initiates increase of circulating NK cells and number of
activated NK cells (Fletcher and Bishop, 2011a,b). Caffeine
would act in immunity regulation even by inhibition of the
enzyme AChE and consequent activation of the cholinergic
anti-inflammatory pathway (Pohanka, 2012, 2013, 2014b). The
link to the anti-inflammatory pathway should be however,
further researched.
The results showed that caffeine may have e impact on
immunity and could affect the health of everyone drinking
coffee or consuming caffeine in another form. However, the
amount of caffeine should be taken into account when
considering caffeine implication in immune response. The
amount of caffeine consumed depends on personal habits,
marketed products, etc. In the literature, the content of
caffeine in a cup of coffee is approximately 90 mg (Lloret-
Linares et al., 2012) or 200 mg in another source (Howards et al.,
2012). If we consider the upper value, an 80 kg man will receive
2.5 mg/kg of caffeine in one cup of coffee. This value lies
between the lowest and medium dose of caffeine used here. It
can be inferred that the man may have e.g. less effective
vaccination if he regularly takes coffee. Role of caffeine in
sensitivity to infectious diseases is also highly probable but the
effect is hardly predictable.
Conclusions
Caffeine modifies the immune response to a protein antigen. A
significant effect of caffeine would be expected in humans if
the findings here apply to people drinking coffee or other
caffeine containing products if the doses used here are
JAB-40; No. of Pages 6
journal of applied biomedicine xxx (2014) xxx–xxx
extrapolated to humans and content of caffeine in beverages
considered.
The impact of caffeine should be taken into account in
vaccinations and during infectious diseases. On the other
hand, the extrapolation should be confirmed by a separate
clinical test in humans.
Conflict of interest statement
None.
Acknowledgements
The Ministry of Education, Youth and Sports of the Czech
Republic is gratefully acknowledged for project LH11023. A
long-term organization development plan 1011 (Faculty of
Military Health Sciences, University of Defence, Czech Repub-
lic) is acknowledged as well.
references
Antonioli, L., Blandizzi, C., Pacher, P., Hasko, G., 2013. Immunity,
inflammation and cancer: a leading role for adenosine. Nat.
Rev. Cancer 13, 842–857.
Bandyopadhyay, B.C., Poddar, M.K., 1994. Caffeine-induced
increase of adenosine-deaminase activity in mammalian
lymphoid organs. Methods Find. Exp. Clin. Pharmacol. 16,
731–733.
Bessler, H., Salman, H., Bergman, M., Djaldetti, M., 2012. Caffeine
alters cytokine secretion by PBMC induced by colon cancer
cells. Cancer Invest. 30, 87–91.
Bhaskara, S., Chandrasekharan, M.B., Ganguly, R., 2008. Caffeine
induction of cyp6a2 and cyp6a8 genes of drosophila
melanogaster is modulated by camp and d-jun protein
levels. Gene 415, 49–59.
Brunoni, A.R., Machado-Vieira, R., Zarate, C.A., Valiengo, L.,
Vieira, E.L., Bensenor, I.M., Lotufo, P.A., Gattaz, W.F., Teixeira,
A.L., 2014. Cytokines plasma levels during antidepressant
treatment with sertraline and transcranial direct current
stimulation (TDCS): results from a factorial, randomized,
controlled trial. Psychopharmacology 231, 1315–1323.
Caubet, J.C., Masilamani, M., Rivers, N.A., Mayer, L., Sampson, H.
A., 2014. Potential non-t cells source of interleukin-4 in food
allergy. Pediatr. Allergy Immunol. 25, 243–249.
Cift, T., Uludag, S., Aydin, Y., Benian, A., 2013. Effects of amniotic
and maternal CD-146, TGF-beta 1, IL-12, IL-18 and INF-
gamma, on adverse pregnancy outcome. J. Matern.-Fetal
Neonatal Med. 26, 21–25.
Cizkova, H., Voldrich, M., Mlejnecka, J., Kvasnicka, F., 2008.
Authenticity evaluation of tea-based products. Czech. J. Food
Sci. 26, 259–267.
Fletcher, D.K., Bishop, N.C., 2011a. Effect of a high and low dose
of caffeine on antigen-stimulated activation of human
natural killer cells after prolonged cycling. Int. J. Sport Nutr.
Exerc. Metab. 21, 155–165.
Fletcher, D.K., Bishop, N.C., 2011b. Effect of a single and repeated
dose of caffeine on antigen-stimulated human natural killer
cell CD69 expression after high-intensity intermittent
exercise. Eur. J. Appl. Physiol. 111, 1329–1339.
Please cite this article in press as: Pohanka, M., Caffeine downregulates antibody production in a mouse model. J. Appl. Biomed. (2014),
http://dx.doi.org/10.1016/j.jab.2014.09.001
5
Fletcher, D.K., Bishop, N.C., 2012. Caffeine ingestion and
antigen-stimulated human lymphocyte activation after
prolonged cycling. Scand. J. Med. Sci. Sports 22, 249–258.
Gaburjakova, J., Gaburjakova, M., 2014. Coupled gating modifies
the regulation of cardiac ryanodine receptors by luminal ca(2
+). Biochim. Biophys. Acta 1838, 867–873.
Gerasimova, E.V., Yakovleva, O.V., Zefirov, A.L., Sitdikova, G.F.,
2013. Role of ryanodine receptors in the effects of hydrogen
sulfide on transmitter release from the frog motor nerve
ending. Bull. Exp. Biol. Med. 155, 11–13.
Gordillo-Bastidas, D., Oceguera-Contreras, E., Salazar-Montes,
A., Gonzalez-Cuevas, J., Hernandez-Ortega, L.D.,
Armendariz-Borunda, J., 2013. Nrf2 and snail-1 in the
prevention of experimental liver fibrosis by caffeine. World J.
Gastroenterol. 19, 9020–9033.
Herman, A., Herman, A.P., 2013. Caffeine's mechanisms of
action and its cosmetic use. Skin Pharmacol. Physiol. 26, 8–
14.
Horrigan, L.A., Kelly, J.P., Connor, T.J., 2004. Caffeine suppresses
TNF-alpha production via activation of the cyclic amp/
protein kinase a pathway. Int. Immunopharmacol. 4, 1409–
1417.
Horrigan, L.A., Kelly, J.P., Connor, T.J., 2006. Immunomodulatory
effects of caffeine: friend or foe? Pharmacol. Ther. 111, 877–
892.
Hotta, S., Morimura, K., Ohya, S., Muraki, K., Takeshima, H.,
Imaizumi, Y., 2007. Ryanodine receptor type 2 deficiency
changes excitation–contraction coupling and membrane
potential in urinary bladder smooth muscle. J. Physiol. 582,
489–506.
Howards, P.P., Hertz-Picciotto, I., Bech, B.H., Nohr, E.A.,
Andersen, A.M.N., Poole, C., Olsen, J., 2012. Spontaneous
abortion and a diet drug containing caffeine and ephedrine:
a study within the danish national birth cohort. PLoS ONE 7.
Chen, J.F., Eltzschig, H.K., Fredholm, B.B., 2013. Adenosine
receptors as drug targets – what are the challenges? Nat.
Rev. Drug Discov. 12, 265–286.
Kuchiiwa, S., Kuchiiwa, T., 2014. A novel semi-automated
apparatus for measurement of aggressive biting behavior in
mice. J. Neurosci. Methods 228, 27–34.
Kuvibidila, S., Porretta, C., Baliga, S., 2014. Aneuploidy assessed
by DNA index influences the effect of iron status on plasma
and/or supernatant cytokine levels and progression of cells
through the cell cycle in a mouse model. Cytokine 65, 175–
183.
Lloret-Linares, C., Lafuente-Lafuente, C., Chassany, O., Green, A.,
Delcey, V., Mouly, S., Bergmann, J.F., 2012. Does a single cup
of coffee at dinner alter the sleep? A controlled cross-over
randomised trial in real-life conditions. Nutr. Diet. 69, 250–
255.
Mandal, A., Poddar, M.K., 2008. Long-term caffeine consumption
reverses tumor-induced suppression of the innate immune
response in adult mice. Planta Med. 74, 1779–1784.
Nosal'ova, G., Prisenznakova, L., Paulovicova, E., Capek, P.,
Matulova, M., Navarini, L., Liverani, F.S., 2011. Antitussive
and immunomodulating activities of instant coffee
arabinogalactan-protein. Int. J. Biol. Macromol. 49, 493–497.
Ohta, A., Sitkovsky, M., 2009. The adenosinergic
immunomodulatory drugs. Curr. Opin. Pharmacol. 9, 501–
506.
Ohta, A., Gorelik, E., Prasad, S.J., Ronchese, F., Lukashev, D.,
Wong, M.K.K., Huang, X.J., Caldwell, S., Liu, K.B., Smith, P.,
Chen, J.F., Jackson, E.K., et al., 2006. A2a adenosine receptor
protects tumors from antitumor t cells. Proc. Natl. Acad. Sci.
U. S. A. 103, 13132–13137.
Okello, E.J., Leylabi, R.J.M.G., 2012. Inhibition of
acetylcholinesterase by green and white tea and their
simulated intestinal metabolites. Food Funct. 3, 651–661.
JAB-40; No. of Pages 6
6 journal of applied biomedicine xxx (2014) xxx–xxx
Peacock, A., Bruno, R., Martin, F.H., Carr, A., 2013. The impact of
alcohol and energy drink consumption on intoxication and
risk-taking behavior. Alcoholism 37, 1234–1242.
Pohanka, M., 2007. Evaluation of immunoglobulin production
during tularaemia infection in balb/c mouse model. Acta Vet.
Brno 76, 579–584.
Pohanka, M., 2009. Monoclonal and polyclonal antibodies
production–preparation of potent biorecognition element. J.
Appl. Biomed. 7, 115–121.
Pohanka, M., 2012. Alpha7 nicotinic acetylcholine receptor is a
target in pharmacology and toxicology. Int. J. Mol. Sci. 13,
2219–2238.
Pohanka, M., 2013. Hi-6 modulates immunization efficacy in a
balb/c mouse model. Environ. Toxicol. Pharmacol. 36, 801–
806.
Pohanka, M., 2014a. The effects of caffeine on the cholinergic
system. Mini Rev. Med. Chem. 14, 543–549.
Pohanka, M., 2014b. Inhibitors of acetylcholinesterase and
butyrylcholinesterase meet immunity. Int. J. Mol. Sci. 15,
9809–9825.
Puttachary, S., Robertson, A.P., Clark, C.L., Martin, R.J., 2010.
Levamisole and ryanodine receptors. II: an
electrophysiological study in ascaris suum. Mol. Biochem.
Parasitol. 171, 8–16.
Ribeiro, J.A., Sebastiao, A.M., 2010. Caffeine and adenosine. J.
Alzheimers Dis. 20, S3–S15.
Rosenthal, L.A., Klyczek, K.K., Blank, K.J., 1992a. Interferon-
alpha/beta, pentoxifylline, and caffeine synergize with
interferon-gamma to induce major histocompatibility
complex class-I expression on a constitutively class I-
negative murine tumor-cell line. J. Interferon Res. 12, 403–
410.
Rosenthal, L.A., Taub, D.D., Moors, M.A., Blank, K.J., 1992b.
Methylxanthine-induced inhibition of the antigen-specific
and superantigen-specific activation of t-lymphocyte.
Immunopharmacology 24, 203–217.
Samsel, M., Dzierzbicka, K., Trzonkowski, P., 2013. Adenosine,
its analogues and conjugates. Postep. Hig. Med. Dosw. 67,
1189–1203.
Please cite this article in press as: Pohanka, M., Caffeine downregulates antibody production in a mouse model. J. Appl. Biomed. (2014),
http://dx.doi.org/10.1016/j.jab.2014.09.001
Saxena, A.K., Singh, K.P., Srivastava, S.N., Khanna, S., Shukla, L.
J., Shanker, R., 1984. Immunomodulating effects of caffeine
(1,3,7-trimethylxanthine) in rodents Indian. J. Exp. Biol. 22,
298–301.
Seifert, S.M., Seifert, S.A., Schaechter, J.L., Bronstein, A.C.,
Benson, B.E., Hershorin, E.R., Arheart, K.L., Franco, V.I.,
Lipshultz, S.E., 2013. An analysis of energy-drink toxicity in
the national poison data system. Clin. Toxicol. (Phila.) 51,
566–574.
Skiteva, O.I., Lapteva, V.I., Balezina, O.P., 2012. Role of stored
calcium in the regulation of neurotransmitter quantum size.
Bull. Exp. Biol. Med. 152, 392–396.
Sun, L.Y., Tian, X., Gou, L.S., Ling, X., Wang, L., Feng, Y., Yin, X.X.,
Liu, Y., 2013. Beneficial synergistic effects of concurrent
treatment with theanine and caffeine against cerebral
ischemia–reperfusion injury in rats. Can. J. Physiol.
Pharmacol. 91, 562–569.
Tomaz, B., Aljaz, M., Ales, J., Gregor, M., 2014. Deletion of the
prion gene Prnp affects offensive aggression in mice. Behav.
Brain Res. 266, 216–221.
Tunc, T., Aydemir, G., Karaoglu, A., Cekmez, F., Kul, M., Aydinoz,
S., Babacan, O., Yaman, H., Sarici, S.U., 2013. Toll-like
receptor levels and caffeine responsiveness in rat pups
during perinatal period. Regul. Pept. 182, 41–44.
Warszawski, D., Gorodischer, R., Kaplanski, J., 1978.
Comparative toxicity of caffeine and aminophylline
(theophylline ethylenediamine) in young and adult rats. Biol.
Neonate 34, 68–71.
Xiang, L., REhm, K.E., Marshall, G.D., 2014. Effects of strenuous
exercise on th1/th2 gene expression from human peripheral
blood mononuclear cells of marathon participants. Mol.
Immunol. 60, 129–134.
Yubero-Lahoz, S., Pardo, R., Farre, M., Mathuna, B.O., Torrens,
M., Mustata, C., Perez-Mana, C., Langohr, K., Carbo, M.L., de la
Torre, R., 2012. Changes in cyp1a2 activity in humans after
3,4-methylenedioxymethamphetamine (MDMA, ecstasy)
administration using caffeine as a probe drug. Drug Metab.
Pharmacokinet. 27, 605–613.