Plant Foods for Human Nutrition

https://doi.org/10.1007/s11130-020-00800-8

REVIEW ARTICLE

Oats as a Safe Alternative to Triticeae Cereals for People

Suffering from Celiac Disease? A Review
Klára Kosová 1 & Leona Leišová-Svobodová 1 & Václav Dvořáček 1

# Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Oats represent a promising alternative to small-grain cereals from Triticeae group (wheat, barley, rye) for persons suffering from
any form of gluten intolerance, especially celiac disease (CD), since oat-specific prolamins avenins reveal generally lower gluten
content and immunoreactivity. Recent studies on avenin molecular structure revealed large genetic variability in avenin se-
quences affecting the spectrum of gluten peptides produced by hydrolases in human digestive tract. The aim of the present
review is to summarise recent knowledge obtained in laboratory studies focused on the effect of avenin-derived peptides on
reactivity of crucial components of human immune system such as dendritic cells (DC) and T-cells. The other part of the review
summarises the results of clinical studies with CD patients including oat products in their diet. Since different clinical studies
revealed contradictory results regarding potential safety of oats for CD patients, the focus has to be directed at genetic variability
in oat avenins. Identification of avenin isoforms with minimum CD immunoreactivity will open up ways leading to designing
novel oat cultivars suitable for CD patients. Knowledge on immunoreactivity of gluten peptides together with breeding new oat
cultivars revealing minimum avenin immunoreactivity with respect to CD as well as application of food processing technologies
leading to gluten content reduction should result in development of gluten-free oats safe for celiacs.

Keywords Celiac disease . Avenins . Genetic variability . Immunoreactivity . Clinical studies . Oats (Avena sativa)

Abbreviations SCFA short chain fatty acid

AA amino acid tTG2 tissue transglutaminase 2
CD celiac disease γIFN interferon γ
DC dendritic cells
E glutamic acid
GFD gluten-free diet
GI gastrointestinal
Introduction
Glt glutenin
Celiac disease (CD) is an autoimmune enteropathy triggered
IL interleukin
by dietary gluten in genetically predisposed persons carrying
LMW low-molecular weight
HLA-DQ2.2/2.5/8 receptors on the membranes of dendritic
moAb monoclonal antibody
cells (DC). Gluten is a mixture of hardly-hydrolysable poly-
P proline
peptides rich in proline and glutamine which arises by hydro-
Q glutamine
lysis of grain storage proteins belonging to prolamin family in
wheat (glutenins and gliadins), barley (hordeins), rye
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11130-020-00800-8) contains supplementary
(secalins), and oats (avenins) [1]. Grain storage proteins in-
material, which is available to authorized users. cluding albumins, globulins, prolamins, glutelins, and
unextractable protein fractions are estimated to amount ap-
* Klára Kosová proximately 15% of total kernel weight in small-grain cereals
kosova@vurv.cz (wheat, barley, rye) out of which prolamins are estimated to
contribute roughly 15% of total grain storage protein weight,
1
Division of Crop Genetics and Breeding, Crop Research Institute, i.e., approximately 6–8% of total kernel weight. The name
Prague6-Ruzyně, Czechia prolamin is derived from proline and amine/amide referring

to amide groups in glutamine residues. The joint proline (P)
and glutamine (Q) content can reach up to 50–60% of total
amino acids in wheat gliadins [2]. Prolamins differ in their
solubility in ethanol and electrophoretic mobility as well as
their ability to form oligomers which is determined by the
number of cysteine residues enabling formation of intra-
and inter- molecular disulphide bonds. In wheat, prolamins
are distinguished into alcohol-insoluble oligomeric
glutenin fraction [high and low molecular weight (LMW)
glutenins] and alcohol-soluble monomeric gliadin fraction
containing S-rich α-, β, and γ-gliadins, and S-poor ω-gli-
adins. S-rich prolamins contain three conserved regions A,
B, and C interspersed by repetitive regions and variant
regions while S-poor prolamins lack the conserved re-
gions. S-poor prolamins such as wheat ω-gliadins and bar-
ley C hordeins were suggested to have evolved from S-rich
ones by deletion of the conserved regions and multiplica-
tion of repetitive regions [1].
Celiac disease represents a complex of human body re-
sponses to gluten intolerance which is underlied by the inabil-
ity of human small intestine enzymes to cleave peptide bonds
in P + Q rich sequences [1, 2]. CD is determined genetically
by the presence of HLA-DQ2.2, HLA-DQ2.5 or HLA-DQ8
haplotypes of receptors on the plasma membrane of dendritic
cells (DCs) which are important antigen-presenting cells [3].
A crucial role in CD biogenesis plays human enzyme tissue
transglutaminase 2 (tTG2) which deamidates glutamine (Q) to
glutamic acid (E) thus introducing a negative charge into QXP
motifs in gluten peptides and enhancing their interaction with
HLA-DQ2/8 receptors. The interaction of HLA-DQ2/8 with
deamidated gluten peptide leads to formation of HLA-DQ2/8-
gluten complex which interacts with T cell receptor (TCR) on
the surface of specific CD4+ T lymphocytes in lamina propria
of proximal small intestine. The interaction leads to T cell
proliferation and release of γ-interferon (γIFN) and interleu-
kin 21 (IL-21) leading to inflammatory processes resulting in
villous atrophy [1, 4]. Moreover, CD is also an autoimmune
disease due to tTG2 transamidation activity leading to a for-
mation of complexes between tTG2 and gliadin peptides
which can bind to HLA-DQ2.2/2.5. and induce specific
IgA-class autoantibodies against tTG2. [1, 4]. The strongest
immune response was found for 33-mer peptide (p57–89)
derived from wheat α2-gliadin repetitive region. Currently,
six different epitopes recognised by HLA-DQ2.5 receptors
were identified in α2-gliadin 33-mer [1, 5]. Besides the 33-
mer (p57–89), the 25-mer (p31–43) interacting with interleu-
kin 15 (IL-15) was identified in α2-gliadin [1]. Only three
epitopes recognised by HLA-DQ8 were identified; one in α-
gliadins, another one in γ-gliadins, and the third one in
glutenins [3]. Moreover, HLA-DQ8-mediated CD seems to
be independent on tTG2 mediated deamidation [6].
Molecular markers of CD include serum antibodies against
gluten-derived peptides (IgE, IgG); tTG2-targeted IgA class
Plant Foods Hum Nutr
autoantibodies, γIFN, interleukins IL-15, IL-21. Histological
symptoms of CD include damage to lamina propria (mucosa)
in duodenum leading to villous atrophy [1, 7, 8].
It is estimated that the amount of people sufferring from
gluten intolerance can reach up to 1% in Western countries
population. The only live-long efficient treatment lies in con-
summation of gluten-free diet (GFD) which is a serious prob-
lem in Western countries due to ubiqity of gluten as an addi-
tive in food industry. Contamination of gluten-free foods with
gluten represents another important problem. Oats can repre-
sent a promising alternative to foods prepared from wheat
(barley, rye) since the majority of celiacs can tolerate oats
without any clinical symptoms [4]. Currently, oats is included
into gluten-free ingredients, i.e., those ingredients which do
not exceed 20 mg gluten per 1 kg of food (20 ppm), by
European Commission Regulation 41/2009 [9] although its
safety for celiacs is still a matter of debate. In Canada, con-
sumption of pure and uncontaminated oats where gluten con-
tent does not exceed 20 mg/kg as detected by R5-ELISA test
was recommended by the Canadian Celiac Association,
Canadian Food Inspection Agency, and Health Canada [10].
In contrast, oat is not considered „gluten-free food “in
Australia and New Zealand [11]. Recent studies have indicat-
ed significant genetic variability within cultivated oats with
respect to oat avenin molecular structure, potential immuno-
reactivity and induction of CD-related clinical symptoms
(reviewed in [12, 13]).
The aim of the present review was to summarise recent
knowledge on oats avenins molecular structure, genetic vari-
ability and potential immunoreactivity with respect to CD.
Results of clinical studies aimed at determining the effects of
oats on key steps in induction of CD symptoms are
summarised. Currently, it is necessary to explore oat genetic
variability with respect to avenin molecular structure and their
potential immunoreactivity. Recommendations for oat
breeders with respect to desired avenin structure inducing no
(minimum) immunoractivity should be formulated. The
knowledge on the relationship between avenin molecular
structure and their potential immunoreactivity opens up new
ways for genetic modifications of avenin sequences.
Avenin Molecular Structure and Phylogenetic
Analysis
Oats (Avena sativa L.) belongs to gramineaceous group called
Avenae which is related but distinct from Triticeae group in-
cluding wheat, barley, and rye. The genus Avena includes up
to 30 species with diploid, tetraploid and hexaploid genomes;
cultivated oats (Avena sativa) is a hexaploid species with
AACCDD genomes. Oat grain represents a valuable source
of a wide array of nutrients including β-glucans as an impor-
tant source of fiber, lipids, avenanthramides, and grain storage
Plant Foods Hum Nutr
proteins including globulins, albumins, prolamins, and
glutelins [4, 14, 15]. Oat prolamins are called avenins. In
comparison to Triticeae prolamins reaching 6–8% of kernel
weight, avenins represent only about 1.2–2% of kernel weight
[2, 16]. Avenins also represent a lower fraction (around 13%)
of total oat grain proteins with respect to Triticeae prolamins
(up to 35% of total grain protein in wheat) since the major oat
grain storage proteins are globulins [4, 17].
Avenin Gene Number Currently, oat genome sequencing pro-
ject is run by people from Aberystwyth University (https://
avenagenome.org) using the diploid donors of the individual
genomes: A genome from Avena atlantica, C genome from
Avena eriantha, and a putative D genome. However, a
complete oat genome sequence has yet to be published, thus
the final number of avenin genes remains unknown. Chesnut
et al. [18] estimated around 25 avenin genes encoded by A.
sativa genome while Londono et al. [19] detected only 10 and
11 avenin genes expressed in A. sativa cvs Gigant and Dancer,
respectively. Currently, 54 avenin protein sequences (partial or
full-length sequences) from Avena sativa and 19 avenin se-
quences from other Avena species are available in NCBI da-
tabase (www.ncbi.nlm.nih.gov; downloaded on 30th
September, 2019).
Avenin Molecular Structure Similarly to Triticeae prolamins,
avenin molecules contain a signal peptide at N-terminal part
followed by the first repetitive region followed by conserved
A and B regions followed by second repetitive region and C
conserved region near to C-terminal part of the molecule. P +
Q rich repetitive regions can be deamidated by tissue
transglutaminase 2 (tTG2) to glutamic acid (E) to form immu-
noreactive epitopes in some types of avenin molecules. In
contrast to S-poor gliadins which have only one long repeti-
tive region, avenins have two shorter repetitive regions thus
their hydrolysis yields shorter P + Q rich gluten-like peptides
[1, 2, 19].
Phylogenetic Analyses of Avenin Proteins Cluster analysis
based on avenin protein sequences revealed three groups
termed avenins A, B, and C, respectively (Fig. 1; [2]).
Cluster corresponding to group C contains two sub-clusters
with high level of bootstrap values (Fig. 1). They differ by the
presence of avenin-specific T cell epitopes in the first con-
served region [19]. Avenins B and C are homologous to wheat
alcohol-soluble S-rich α- and γ-gliadins, barley B hordeins
and rye γ-secalins, respectively, while avenins A are
alcohol-insoluble proteins homologous to wheat LMW
glutenins which means that they form oligomers due to disul-
fide bridges between cysteine residues. However, A-class
avenins can be solubilised by using reduction agents indicat-
ing that their alcohol insolubility is underlied by intermolecu-
lar disulfide bonds. Oats does not contain S-poor prolamins
with large P + Q rich repetitive regions. Avenin sequences
belonging to groups B and C contain eight cysteine residues
while avenins of group A have nine cysteine residues [2].
However, in comparison to wheat prolamins, avenins can
form only intermolecular disulfide bonds [1]. Avenins re-
vealed a lower joint P + Q content of roughly 30% of total
amino acid content than compared to wheat α- and γ-gliadins
whose P + Q joint content can reach up to 50–60% of total
amino acid content [1, 2, 22]. Thus, it can be expected that
avenins are more easily hydrolyzable by duodenal enzymes.
Moreover, it is becoming evident that oat genotypes differ in
their avenin composition and that some oat genotypes could
lack some avenin structural types. In their study aimed at
avenin analysis in selected Spanish oat lines, Real et al. [2]
identified avenins belonging to all A, B and C classes in all
lines except for OP722 which lacks class A avenins.
Expression pattern of avenin proteins during oat kernel
development was also studied. Chesnut et al. [18] detected
avenin mRNAs and proteins between 4 and 6 days after an-
thesis (DPA) with a maximum at 8 DPA while Real et al. [2]
were able to detect avenin proteins at 8 DPA with a maximum
expression level between 20 to 28 DPA. Greater expression
levels were found for α- and γ-gliadin-like avenins of groups
B and C than for low-molecular-weight glutenins-like avenins
of group A.
Avenin CD Immunoreactivity
Avenin CD Reactive Epitopes and their Crossreactivity
with Anti-Gluten Antibodies
Sollid et al. [3] provided nomenclature of gluten epitopes
which react with the individual HLA-DQ receptors including
HLA-DQ2.2, HLA-DQ2.5, HLA-DQ8 and HLA-DQ8.5 re-
ceptor types. Two avenin-specific 9-mer epitopes named
DQ2.5-ave-1a (formerly known as Av-α9A) PYPEQQEPF
and DQ2.5-ave-1b (formerly known as Av- α9B)
PYPEQEQPF located in the first P + Q rich repetitive region
were identified as HLA-DQ2.5 immunoreactive epitopes in
some groups of avenin proteins based on the results of previ-
ous studies [7, 23]. In addition, 9-mer PFVQQQQPF se-
quence formerly known as Av-γ9B epitope is located down-
stream of DQ2.5-ave-1a in group III avenins [19].
Recently, it is becoming evident that avenin molecules
reveal differences in their CD reactivity determined by
various kinds of assays including immunoreactivity assays
based on crossreactivity of some avenin sequences with
primary antibodies raised against wheat gluten epitopes,
assays based on avenin-induced agglutination of K562
cells, as well as avenin potential stimulatory effects on
dendritic cells (DC), T cells, and other components of hu-
man immune system.
 Plant Foods Hum Nutr

Fig. 1 Molecular phylogenetic

analysis computed by Maximum
likelihood method based on the
Jones-Taylor-Thornton (JTT)
matrix-based model selected ac-
cording to AIC criterion [20]. The
tree with the highest log likeli-
hood (−1249.26) is shown. The
percentage of trees in which the
associated taxa clustered together
is shown next to the branches.
Initial tree(s) for the heuristic
search were obtained automati-
cally by applying Neighbor-Join
and BioNJ algorithms to a matrix
of pairwise distances estimated
using a JTT model, and then
selecting the topology with supe-
rior log likelihood value. A dis-
crete gamma distribution was
used to model evolutionary rate
differences among sites (5 cate-
gories (+G, parameter = 0.6862)).
The rate variation model allowed
for some sites to be evolutionarily
invariable ([+I], 0.00% sites).
Evolutionary analyses were con-
ducted in MEGA7 [21]

Immunoreactivity Assays Londono et al. [19] proposed a crossreactivity of wheat

Antibodies Used for Testing Anti-Gluten Immunoreactivity
Antibodies used for gluten peptides detection in oats in-
clude the two major kinds of anti-gluten monoclonal an-
tibodies R5 moAb and G12 moAb, respectively. The R5
moAb antibody binds to epitope variants QQPFP,
QQQFP, LQPFP, and QLPFP, and, to a lesser extent, to
QLPTF, QQSFP, QQTFP, PQPFP, and QQPYP, and is
mostly used for detection of gluten in wheat, barley and
rye [24]. Sandwich R5 ELISA assay was developed for a
rapid screening of gluten [25, 26].
Another kind of anti-gluten antibody represents G12 moAb
which binds to epitopes QPQLPY, QPQQPY, and QPQLPF
present in 33-mer (57–89) derived from α2 gliadin [27]. G12
mAb reveals limited cross-reactivity with avenins. An ELISA
assay using G12 moAb was developed for a rapid screening of
gluten [28].
G12 and R5 moAb with avenins since four R5-related epi-
tope variants and six G12-related epitope variants are pres-
ent in sequences present in avenin-specific epitopes
DQ2.5-ave-1a (formerly known as Av-α9A) and DQ2.5-
ave-1b (formerly known as Av-α9B), respectively. No per-
fect gluten epitopes were found in avenins; however, se-
quences revealing a substitution of one or two amino acids
were recognised by the antibodies raised against wheat gluten
peptides. Regarding wheat R5 responsive epitopes, avenin
epitopes QQPFL, QQPFV and QQPFM revealing a
crossreactivity with wheat epitope QQPFP and avenin se-
quence YQPFYP revealing a crossreactivity with wheat epi-
tope QQPYP were identified. Regarding wheat G12 respon-
sive epitopes, avenin sequence QPQLQ revealing a
crossreactivity to wheat QPQLPY, avenin sequences
QPQQQA, QQQQPF, QPQQQA, QQQQPF, QPQQLP and
QPQLPF revealing a crossreactivity to wheat QPQQPY, and
avenin sequence QPQLQL revealing a crossreactivity to
Plant Foods Hum Nutr
wheat QPQLPF were identified by Londono et al. [19] in
avenin protein sequences from cvs Dancer and Gigant.
Londono et al. [19] also reported the presence of one of the
two HLA-DQ2.5 responsive epitopes in 7 out of 11 avenin
genes in cv Dancer and in 4 out of 10 avenins in cv Gigant.
Londono et al. [19] also performed an analysis of avenin
proteins in 13 Avena species including 12 diploid and tetra-
ploid Avena species and a hexaploid Avena cultivar Gigant
with respect to gluten epitopes. A total of 717 avenin protein
sequences representing 78 different genes were obtained from
the 13 Avena species indicating that the individual oat species
encode 7 to 10 avenin genes; the following sequence analysis
revealed four avenin phylogenetic groups. Avenin sequence
analysis with respect to CD immunoreactivity revealed the
presence of two avenin-specific T cell recognized epitopes
which were present once only per protein molecules and were
located in the first repetitive region; the epitope DQ2.5-ave-1b
PYPEQQQPF was present in all avenin genes from group II
whereas the epitope DQ2.5-ave-1a PYPEQQEPF was present
in all genes from group III while avenin genes from groups I
and IV contained no T cell immunoreactive epitopes.
However, the authors reported that none of the oat species
studied lacked group II or III avenins, i.e., they all possess at
least one of the two avenin-specific T cell epitopes. However,
it should be borne in mind that the genes present in a given
genome can be expressed at differential rates resulting in dif-
ferent final protein levels; thus, the resulting levels of immu-
nogenic proteins cannot be estimated from the presence of
genes or gene copies in the genome due to various mecha-
nisms regulating transcription and posttranscription processes.
Later, Nassef et al. [29, 30] used electrochemical
immunosensors to detect gliadin peptides in raw and proc-
essed food using a monoclonal antibody against α-gliadin
56–75 region (CDC 5) or antigliadin Fab fragments (CDC5-
Fab). Results obtained by this method correlate with ELISA
determination and reveal high sensitivity, easy performance,
and short assay time.
In addition, moAb 401.21 also referred to as Skerritt anti-
body which binds to PQPQPFPQE and PQQPPFPEE epi-
topes can be used for gluten detection [31]. A brief overview
of studies aimed at detection of immunoreactive peptides oat
varieties is given in Supplementary Table S1.
Mujico et al. [32] compared avenin immunoreactivity to
anti-LMW glutenin moAb recognizing PFVQ sequence
known as Glt-156 LMW epitope [23] which is found in
two avenin epitopes QQPFVQQQPFVQQ and
QYQPYPEQQQPFVQ using immunoblot analysis of eth-
anol extracts and they found significant differences be-
tween the oat cultivars tested. Wodan and Zwarte
President revealed the strongest reaction while Mansholt
III revealed the faintest one which was in contrast with
the results of T cell stimulatory assay. Thus, different as-
says can reveal different results regarding anti-gluten
immunoreactivity in avenins. Similar differences were also
found in immunoreactivity of avenin extracts using a spe-
cific moAb against α20-gliadin epitope [33]. The obtained
results led the authors to conclusion that oat breeding pro-
grams aimed at selection of CD-safe oat varieties with a
minimum immunoreactivity may be a realistic goal; how-
ever, they also noted that true safety can be considered
only by the results of clinical trials.
Ballabio et al. [34] found out differential response of 36 oat
cultivars to anti-gliadin protein kit using RIDASCREEN
ELISA test based on R5 moAb. The majority of studied oat
cultivars revealed levels of gluten-like proteins well below the
limit of 20 mg/kg although some varieties such as Expander,
HJA76037N, Kynon and BG2 revealed concentration of
gluten-like proteins very close to 20 mg/kg, and a few oat
varieties such as commercial husked cv. Dakar, experimental
naked genotype BG3, naked cultivar Nave and genotype BG4
significantly exceeded the 20 mg/kg limit for gluten-free
products.
Similarly, Benoit et al. [35] tested 19 oat varieties given
their response to R5 moAb antibody assay; R5 epitope
LQPFP is present in highly immunogenic α-gliadin 33-mer
sequence. Most of the 19 oat cultivars tested revealed a neg-
ative response to R5 assay; however, three of them including
Brazilian Check, Kanota, and Curt, revealed a positive re-
sponse to R5 assay indicating presence of immunogenic pep-
tides. Moreover, the three oat cultivars also revealed a positive
response to Skerritt antibody which revealed even higher glu-
ten levels when compared with R5 antibody (Supplementary
Table S1).
Recently, Giménez et al. [36] studied variability in avenin
CD immunoreactivity in a set of 95 oat varieties of
Mediterranean origin using individual avenin protein resolu-
tion by reversed-phase high performance liquid chromatogra-
phy (RP-HPLC) and testing avenin immunoreactivity by
using G12 moAb based ELISA tests. The researchers reported
a high variability in avenin peak patterns within the studied oat
samples as well as differences in CD immunoreactivity among
different avenin peaks. Overall, gluten levels determined by
G12-based ELISA did not exceed 20 mg/kg in the individual
samples while only one sample exceeded 40 mg/kg.
Dendritic Cells (DC) Reactivity Assays
Comino et al. [37] studied avenin proteins, their gluten pep-
tides and their potential stimulatory effects on DC in a set of
oat cultivars from Spanish and Australian commercial re-
sources. They observed significant polymorphism patterns in
avenin fraction separated on 1D SDS-PAGE gels which was
even greater than in globulin fraction. The large differences in
avenin protein sequences whose size ranged between less than
20 up to 50 kDa on 1D gels were also reflected at the level of
their hydrolysis products. With respect to wheat gliadins

producing long 33-mer P + Q rich peptide inducing immune
response, oat avenins were found to yield a series of shorter
peptides (6, 10, 14, and 27 AA) which are easier to be cleaved
or internalised to endosomes in DCs than larger peptides from
wheat gliadins [37]. The short 6 AA avenin-derived gluten
peptides did not trigger any DC stimulatory effects. Only the
27 AA avenin-derived peptides EF27 and QM27 triggered T
cell proliferative response similar to wheat gliadin-derived 33-
mer peptide. However, a different way of endosomal internal-
ization of shorter (6 AA) and intermediate (10 and 14 AA)
peptides was proposed with respect to the larger ones. As a
result, these peptides do not induce T cell response leading to
development of CD molecular symptoms such as T cell pro-
liferation, γIFN release, tTG2 autoantibodies production, etc.
[37, 38].
Immunoreactive peptides interacting with anti-33mer
monoclonal antibody moG12 raised against 33-mer gluten
peptide from wheat α2-gliadin as detected in oat avenins by
Comino et al. [37]:
Immunoreactive peptides derived from gliadin-like avenins
(B- and C-type avenins): QL6 (QPQLQL), QQ6 (QPQLQQ),
P V 1 0 ( P Y P E Q Q E P F V ) , E F 2 7
(EQYQPYPEQQEPFVQQQPPFVQQEQPF).
Immunoreactive peptides derived from glutenin-like
avenins (A-type avenins):
QL14 (QQPFMQQQPFMQPL), QM27
(QYQPYPEQQPFMQQQQPFMQPLLQQQM).
Agglutination Assays
Silano et al. [39] compared avenins isolated from three oat
varieties given their ability to agglutinate K562 human mye-
logenous leukemia cells and to disrupt rat liver lysosomes
with respect to the activities of wheat gliadin and rice grain
proteins. A significant difference between the different oat
varieties was found as avenins from Italian variety Astra and
Australian variety Mortlock were much more active in K562
cells agglutination and lysosome disruption than avenins iso-
lated from Australian variety Lampton. Wheat gliadin (a pos-
itive control) revealed enhanced activity with respect to all
three avenins while rice protein (a negative control) did not
reveal measurable activity. Moreover, the same authors inves-
tigated the effect of avenins isolated from four oat cultivars
Astra, Ava, Lampton and Nave on proliferation and γIFN
release by peripheral lymphocytes isolated from ten celiac
children. They found out that avenins from Ava and
Lampton exhibited a relatively strong immunogenic reaction
comparable to wheat gliadin while avenins from Astra and
Nave induced significantly lower but still detectable immuno-
genic response [40].
Silano et al. [41] investigated the potential of different oat
varieties to elicit tTG2-derived events in some in vitro models
of CD. The following early gluten-induced TG2-dependent
Plant Foods Hum Nutr
events were studied: K562(S) cells agglutination,
transepithelial electrical resistance of T84-cell monolayers,
intracellular levels of TG2 and phosphorylated form of protein
42–44 in T84 cells. The tests revealed that Nave elicited these
events whereas Irina and Potenza did not. These results cor-
related with the extent of pepsin-mediated proteolysis of the
prolamin fraction. In addition, the ability of a cultivar to in-
duce the above-listed events correlated with avenin electro-
phoretic pattern and their reactivity to anti-gliadin polyclonal
antibodies.
T Cell Reactivity Assays
Mujico et al. [32] found a variation in stimulatory capacity of
specific T cell line from a celiac patient within a set of 26 oat
varieties of Dutch origin (Ascot, Astor, Charming, Charmoise,
Dalguise, Dominik, Fervente, Firth, Freddy, Gambo, Gele van
Timmermans, Gerald, Gigant, Leanda, Mansholt III, Markant,
Mustang, Ouderwetse Zeeuwse Partij, Panache de Roye,
Powys, Sang, Troshaver uit Besel, Valiant, Wodan, Zandster,
Zwarte President) compared to four synthetic peptides con-
taining two known avenin epitopes (QQPFVQQQQPFVQQ
and QYQPYPEQQQPFVQ). The results of the T cell prolif-
eration assay revealed that the majority of oat samples contain
avenins possessing epitopes with T cell stimulatory capacity
but some samples (Dalguise, Gambo, Markant, Wodan,
Zanster) revealed a minimum stimulatory capacity indicating
that they contain avenins with minimum gluten-like epitopes.
Arentz-Hansen et al. [7] studied T cell reactivity to synthet-
ic avenin-derived peptides and found recognition by two T
cell lines for peptide 1490 with the following sequence
SEQYQPYPEQQEPFVQQQQ which was dependent on pep-
tide deamidation by tTG2. A 9-mer core peptide Av-α9A with
sequence PYPEQEEPF arising from deamidation of gluta-
mine to glutamic acid (E) at 6th position and revealing se-
quence homology to wheat DQ2-α-I gliadin epitope
PFPQPELPY was predicted to be recognised as an epitope
for HLA-DQ2 receptors on the surface of dendritic cells.
Comino et al. [38] found a direct relationship between
avenin reactivity to monoclonal AbG12 antibody raised
against wheat α-2 gliadin-derived 33-mer (p57–89) and their
potential immunotoxicity determined as peripheral blood
mononuclear T cell proliferation and γIFN release.
Hardy et al. [16] found T cell reactivity to 20mers
encompassing 9mer sequences closely related to avenin-
derived epitopes DQ2.5-ave-1a (PYPEQEEPF where E re-
sults from deamidation of Q39 in several native avenin se-
quences) and DQ2.5-ave-1b (PYPEQEQPF, deamidation of
Q19 to E). Further immunogenic sequences include 9mer epi-
topes derived from partially deamidated avenin peptide
QYQPYPEQEQPILQQQ (deamidation of Q34 to E) that in-
cluded a 9mer amino-acid sequence differing from DQ2.5-
ave-1b by substitution of isoleucine for phenylalanine at
Plant Foods Hum Nutr
position 9. Moreover, they reported a crossreactivity between
hordein-derived epitope DQ2.5-hor-3b and avenin epitopes
DQ2.5-ave-1b and DQ2.5-ave-1c, respectively. Cross-
reactive T cells specific for DQ2.5-hor-3b and avenin epitopes
DQ2.5-ave-1b and DQ2.5-ave-1c are commonly mobilized in
HLA-DQ2.5+ CD patients when they eat barley, and also oats,
but less consistently and at lower frequencies than in barley. It
could be concluded that hordein-derived peptides functioned
as „priming “means, i.e., immunogenic response to avenin-
derived peptides was induced by hordein-derived peptides
since avenin peptides themselves had reduced binding stabil-
ity on HLA-DQ2.5 as compared to homologous hordein-
derived peptides. The researchers thus demostrated that food
antigen cross-reactivity has significant immunological rele-
vance in vivo which opens the possibility that other peptides
may induce pathogenic response in CD patients under some
circumstances.
Summary to Avenin CD Reactivity
Contradictory results obtained by different clinical stud-
ies probably reflect genetic variability with respect to
gluten content and its immunoreactivity in oats used in
food industry. Variability in avenin composition includ-
ing variability in avenin-derived gluten peptides and their
immunoreactivity to anti-gliadin antibodies was found
[37, 41]. Some recent studies revealed differential gluten
peptides with differential stimulatory capacity on DC
which indicates that people with CD who want to in-
clude oats in their diet should pay attention to oat genet-
ic resources used for food preparation [16]. It thus arises
a challenge for oat breeders to select oat genetic mate-
rials with no (minimum) CD reactivity without any neg-
ative effects on other oat qualities. Some celiac patients
can contain gluten-reactive or avenin-reactive intestinal T
cells without any clinical symptoms due to remission.
Oat genetic variability leads to a variability of avenin-
derived peptides which can potentially induce CD re-
sponse. A series of HLA-DQ2.5 interacting avenin-
derived peptides were characterised; however, only longer
peptides can induce inflammatory response similar to those
induced by wheat gliadin 33-mer, i.e., T-cells activation,
production of serum IgA anti-avenin and anti-tTG2 anti-
bodies, enhanced levels of γIFN [37]. It is becoming evi-
dent that besides sequence, conformation of gluten pep-
tides plays an important role in determining peptide immu-
noreactivity. Longer gluten peptides acquire β-sheet con-
formation [37, 42].
Besides qualitative aspects, i.e., structure of prolamin
molecules, and the length and sequence of gluten pep-
tides, respectively, recent studies also indicate a quanti-
tative aspect of gluten immunoreactivity, i.e., the fact
that potential immunoreactivity of gluten peptides
increases with their concentration. Recently, it was found
out that human body immune response is enhanced with
an increasing concentration of gliadin 33-mer due to a
formation of supramolecular oligomeric rod-like struc-
tures of polyproline II-type based on oligomerization of
type II β-sheets formed by monomeric 33-mers [42].
However, breeding for CD-safe oat varieties with reduced
gluten content can bring several obstacles due to the fact that
avenin genes are distributed in the whole oat genome includ-
ing a vicinity of several resistance genes. For example,
Satheeskumar et al. [43] detected three avenin loci in the vi-
cinity of Pc68 gene conferring resistance to Puccinia coronata
crown rust.
Lerner [44] postulated the major problems associated with
oat consumption as a part of GFD including genetic variability
in avenin-derived gluten peptides among oat varieties as well
as contamination of oat products by other gluten-containing
cereals resulting in exceeding the gluten limit of 20 mg/kg.
Regarding the potential breeding of gluten-safe oat varie-
ties, it is necessary to describe the variability of avenin alleles
with respect to the potential gluten peptides. In the next step, it
will be necessary to exclude potential unsuitable avenin alleles
from promising oat materials. Recently, a boom of gene
editing techniques including CRISPR/Cas, TALENs, and
others (reviewed in [45]) could facilitate the gain of prospec-
tive oat materials cumulating only suitable avenin alleles.
Clinical Studies on the Effects of Oats on CD Clinical
Symptoms
Clinical studies aimed at the effects of oat consumption
by persons sufferring from CD revealed contradictory
results with respect to the effect of oat-containing diet
on CD symptoms. A detailed overview of clinical studies
dealing with oats as a part of GFD in adults and children
is presented in the review of La Vieille et al. [46]. A
wide array of CD-related symptoms including gastroin-
testinal symptoms, malabsorption, mucosal changes, se-
rological responses and overall quality of life regarding
people using oat as a part of GFD were reviewed by
Cohen et al. [4]. Basic data on the clinical studies
discussed in the following text are provided in
Supplementary Table S2.
Clinical Studies in Adults
Kaukinen et al. [47] found no CD symptoms regarding the
levels of small-bowel mucosal CD3+, αβ + and γσ +
intraepithelial lymphocytes, small-bowel mucosal villous
damage, inflammation or other gastrointestinal CD symptoms
in a group of adult CD patients consuming a median of 20 g of
oats per day for up to eight years whereas the consumed oat
products come from general stores. Moreover, oats daily

consumption had beneficial effects since it provided enhanced
fiber intake to CD patients consuming oats with respect to
those who did not consume oats. No clinical symptoms at
serological (anti-gluten IgA or IgG antibodies and anti-
transglutaminase 2 antibodies) and histological (small-bowel
mucosal damage revealing either partial, subtotal or total
villous atrophy) were detected by Aaltonen et al. [48] in a
group of 869 adult celiacs consuming oats as part of GFD
for one year when compared to oat non-consuming celiacs.
Similarly, Janatuinen et al. [49] found no differences between
celiacs non-consuming oats and those consuming 46 g per day
for 12 months in histology of duodenal villi. No significant
differences in the levels of anti-avenin IgA antibodies were
found in adult groups on GFD who consume oats with non-
consuming ones [50]. Similarly, Sey et al. [51] found no sig-
nificant changes in symptoms scores (symptomatic recur-
rence, serological relapse, histological deterioration) and other
indicators such as weight, hemoglobin, ferritin, albumin and
tTG levels between CD patients consuming up to 350 g/week
oats as a part of their GFD (GFD-oat) with whose consuiming
standard GFD during a 12-week trial. Cooper et al. [52] found
no significant differences in tTG expression, intraepithelial
lymphocyte numbers or enterocyte proliferation in 46 CD pa-
tients consuming oat-containing GFD for one year.
In contrast, Lundin et al. [8] found enhanced levels of γIFN
mRNA after a challenge including 50 g of oats per day for
12 weeks in 19 adult CD patients who previously used GFD
only. One of the patients developed partial villous atrophy
after a challenge. Similarly, Tuire et al. [53] found persistent
intraepithelial lymphocytosis in small bowel while oats con-
sumption being the only significant factor contributing to this
state in adult CD patients although only minor effects of
intraepithelial lymphocytosis on villous architecture were
found. Similarly, Zanini et al. [54] reported persistent
intraepithelial lymphocytosis independent on the length of
GFD in CD patients.
Clinical Studies in Children
Koskinen et al. [55] studied the effect of 2-year-long diet
containing oats on 23 children with CD with respect to their
levels of jejunal mucosal tTG2-targeted IgA-class autoanti-
bodies deposits and found no significant change in the level
of anti-tTG2 autoantibodies in children with CD diagnosis.
Thus, they concluded that consumption of oats does not in-
duce anti-tTG2 autoantibody production at jejunal mucosal
level in children with CD.
In a double-blinded study, Gatti et al. [56] found no clinical
symptoms regarding gastronintestinal symptoms rate scale
(GSRS) and intestinal permeability tests (IPT) in a group of
Italian children after a 6-month diet containing pure oats.
Similarly, Lionetti et al. [57] found no differences in clinical
(GSRS), serologic (IgA anti-tTG2 and anti-avenin antibodies)
Plant Foods Hum Nutr
and intestinal permeability data after 6, 9 and 15 months of
double-blind, placebo-controlled, crossover design trials in
177 children who were more than two years on GFD.
Similarly, no significant differences were found between
groups of children consuming oat-enriched GFD with respect
to children consuming standard GFD regarding serology (an-
ti-gliadin, anti-tTG2 IgA antibodies) and small bowel mucosal
architecture by Högberg et al. [58]. Most children did not
report adverse effects of long-term oat consumption as part
of GFD in the study by Tapsas et al. [59]; however,
Tjellström et al. [60] reported enhanced levels of feacal short
chain fatty acids (SCFAs) including acetic acid and n-butyric
acid in GFD-oats group of children with respect to standard
GFD group which may present a potential risk for develop-
ment of gut mucosal inflammation.
In conclusion, de Souza et al. [61] and Pinto-Sánchez et al.
[62] provided a meta-analysis of clinical studies dealing with
safety of using pure oats as a part of GFD, and they concluded
that based on the results of 28 clinical studies comprising 661
patients, no adverse evidence of using pure oats were reported
regarding intestinal histology, serology, and immunology.
However, the authors pointed at limited quality of clinical
studies given their limited geographical distribution, and their
design indicating that more rigorous, double-blinded and
placebo-controlled studies are needed.
Conclusions and Future Perspectives
Oat avenins represent a relatively lower portion of grain stor-
age proteins when compared to wheat gliadins. In addition,
oat avenins reveal relatively lower P + Q content resulting in
shorter gluten peptides which can undergo endosomal
internalisation in dendritic cells (DCs) without any T cell-
mediated immune response. However, oat avenins reveal
large genetic variability resulting in a variety of gluten pep-
tides with differential immunoreactivity in human gastrointes-
tinal tract. The majority of avenin-derived gluten peptides
tested are relatively short and do not induce immunogenic
response; however, a few larger avenin-derived peptides re-
vealing immunogenic reactivity were also reported. Based on
the results of recent studies, it is becoming evident that some
oat varieties accumulate avenins yielding immunoreactive
peptides while many others seem to be safe for CD patients.
Given the genotypic variability in avenin-derived peptide se-
quences and their immunoreactivity, urgent need arises to test
the individual oat varieties used for preparation of oat-derived
foods for GFD. Therefore, the following aims for the future
research should be outlined:
1/ Clinical tests: The aim for clinical researchers
(immunologists) is to describe clinical symptoms induced by
various avenin-derived gluten peptides on CD patients in or-
der to identify immnuoreactive peptides.
Plant Foods Hum Nutr
2/ Oat breeding: The aim for oat breeders is to select those
oat genotypes accumulating avenins providing only short glu-
ten peptides with no or minimum immune response in CD
patients which would be safe for people suffering from CD.
The basis for achievements of these aims lies in detailed de-
scription of genetic variability in avenin molecular structure
among oat cultivars. Oat breeding can thus lead to develop-
ment of CD-safe oat varieties with well-characterised avenin
composition. Linkage of several prolamin genes to genes con-
ferring disease resistance could represent a serious obstacle;
however, precise gene mapping and utilization of modern
technologies of molecular breeding can resolve this problem.
3/ Oat food processing: Currently, technologies of food
processing based on dough fermentation using bacterial and
fungal proteases can significantly decrease gluten amounts in
gluten-containing foods such as wheat dough. For example,
Rizzello et al. [63] demonstrated efficient reduction of gluten
contents up to 12 mg/kg associated with hydrolysis of albu-
mins, globulins, glutenins as well as 33-mer peptide in wheat
dough fermented by Lactobacillus (L. brevis, L.
sanfranciscensis, L. hilgardii) and Aspergilus (A. niger, A.
oryzae) proteases.
4/ Oat purity: Last, but not least, oat contamination with
other cereals from Triticeae group often leads to enhanced
immunoreactivity of oat products. Therefore, the manufac-
turers of gluten-free oat products have to adhere strict purity
protocol to eliminate oat contamination with other cereals
[64].
In conclusion, given the genetic variability in avenin
protein sequences and their immunoreactivity in CD pa-
tients, it is necessary to carefully select and breed new
oat varieties with minimum CD reactivity for GFD. To
achieve this aim, characterization of variability and immu-
noreactivity of avenin-derived gluten peptides is a neces-
sary prerequisite. In the next step, programmes aimed at
breeding novel oat varieties carrying only those avenin
alleles providing gluten peptides with no (minimum) im-
munoreactivity should result in production of CD-safe oat
foods suitable for GFD. Companies producing oat foods
suitable for persons suffering from CD have to pay atten-
tion to the origin (genotype) of oats used for food produc-
tion. An alternative way can lie in gluten reduction during
food processing via bacterial and fungal fermentation.
Considering all these aspects of gluten reduction in oats,
CD-safe oat represents a matter of control.
Acknowledgements The work was supported by an institutional project
MZe CR RO0418 and by the project QK1810102 of the Ministry of
Agriculture of the Czech Republic.
Compliance with Ethical Standards
Conflict of Interest The authors declare no conflict of interest.
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