ková*
Kv
to
*k Zídek
* Institutes of Microbiology and
Pharmacology, Academy of Sciences of the Czech Republic, Prague; and
Research Institute of Biopharmacy and Veterinary Drugs, Jílové, Czech Republic
Correspondence: Ludmila Tuková, Ph.D., Institute of Microbiology, Department of Immunology, Academy of Sciences of the Czech Republic, Vídeská 1083, 142 20 Prague 4, Czech Republic. E-mail: tuckova{at}biomed.cas.cz
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and IL-10 and a significant rise in NO production by peritoneal macrophages. The identity of the active fragments was established by separating the peptic digest of gliadin by RP-HPLC chromatography. The purest fraction with the highest activity was analyzed by mass spectrometry, and the gliadin peptide sequence was identified as VSFQQPQQQYPSSQ. This peptide (T) and its N- and C-terminally shortened forms (A, B, C and D, E, F) were synthesized. Peptide B (FQQPQQQYPSSQ) elicited the highest TNF-, IL-10, and RANTES secretion and increase in IFN-
-primed NO production by mouse macrophages. In contrast, C-terminally shortened peptides had a lower ability to stimulate macrophages than the native form.
Key Words: iNOS • celiac disease • IFN- • TNF-
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/
T cells. The mixed cellular infiltrate in lamina propria includes B and CD4+ and CD8+ T lymphocytes, macrophages, and/or dendritic cells (for review, see refs. [1
2
3
4
5
]). Mucosal B lymphocytes are triggered to produce antibodies (Ab) against the disease-inducing agent gluten (or its ethanol-soluble fraction gliadin) and against self-autoantigens [6
7
8
]. Gliadin-specific mucosal CD4+ /ß T lymphocytes seem to play a crucial role in the immunopathogenetic mechanisms. Activated T lymphocytes (peripheral blood- or gut-derived) and gluten-specific T-cell clones from celiac lesions are stimulated to increase production of cytokines of the T-helper cell type (Th)1/Th0 profile. A highly increased level of interferon- (IFN-) and lower but elevated levels of interleukin (IL)-2, IL-4, IL-6, tumor necrosis factor (TNF-), and transforming growth factor-ß (TGF-ß) expression were detected after gluten exposure. Secreted inflammatory cytokines participate again in cell infiltration and activation [9
, 10
]. In spite of intensive efforts to analyze the specific immune response to gliadin, experimental data concerning the role of cells of innate immunity in induction and effector mechanisms of the disease are very limited. Activated macrophages are present in intestinal mucosa and represent one of the main sources of cytokines, chemokines, and reactive oxygen and nitrogen inorganic intermediates [superoxide and nitric oxide (NO) radicals; 11]. Recently, it was shown that the activation of an inducible form of NO synthetase (iNOS) in jejunal biopsy samples is in keeping with the severity of the disease. The increased tissue expression of iNOS mRNA and elevated number of iNOS-positive cells were found to be reflected in a higher level of nitrite and nitrate in the plasma or urine of patients with an active form of the celiac disease (relative to patients on a gluten-free diet and healthy controls). The involvement of gluten molecules in iNOS generation was supported by the results of clinical examinations of all tested groups after gluten exposure [12 13 14 15 ].
We have investigated the direct effect of dietary gluten and gliadin and their peptic fragments on activation of macrophages. Unlike other food antigens tested, peptic fragments of gluten and gliadin increased NO production significantly when added to cultured peritoneal macrophages together with IFN-, a dominant cytokine produced by gut-derived T cells of celiac patients. The synergistic effect of gliadin peptides was confirmed on a iNOS mRNA level and was assumed to be mediated via direct stimulation of TNF- secretion [16
].
In this study, we tried to isolate and identify the amino acid sequence of active gliadin peptides that influences the activity of macrophages directly. We separated peptic fragments of gliadin by three-step reversed-phase high-pressure liquid chromatography (RP-HPLC) and evaluated the in vitro effect of single gliadin fractions on NO and cytokine production by mouse peritoneal macrophages. Furthermore, we identified the active gliadin peptide by mass spectrometry (MS). To confirm our findings, the active, native peptides and their modified forms were synthesized, and their activation effect on macrophages was analyzed.
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Isolation and cultivation of macrophages
Complete RPMI-1640 medium contained 10% heat-inactivated fetal bovine serum, 0.02 M L-glutamine, gentamicin (50 mg/L), and 5 x 10-5 M 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO).
Resident peritoneal macrophages were recovered from female C57/BL/6 and Balb/c/Ph mice (n=6–10 in each experiment) by instilling and withdrawing 8 mL sterile saline solution from the peritoneal cavity. Collected cells were washed, resuspended in culture medium, and seeded into 96-well round-bottom microplates (Costar, Cambridge, MA) in 100 µL volumes (2x105 cells/well). Adherent cells (macrophages) were obtained by incubating the cells for 2 h at 37°C in 5% CO2 and then vigorously shaking the plate and washing the wells three times to remove nonadherent cells. Cultures were maintained at 37°C in 5% CO2 in a humidified Heraeus incubator for 5 h (cytokine assay) and 24 h (NO assay). The cells were cultured in the presence or absence of IFN- and/or gliadin fragments or peptides. All experimental variants were performed in triplicate.
Gliadin fragments
Peptic fragments of gliadin (Sigma Chemical Co.) were prepared using pepsin-agarose gel (Sigma Chemical Co.) with an enzyme/protein ratio of 1/100 (wt/wt) in 0.1 M HCl, pH 1.8, at 37°C for 45 min. The enzymatic digestion was stopped by removing the pepsin-agarose gel by centrifugation (10 min; 1500 g). The stock solution (10 mg/mL) was neutralized by 0.4 M NaOH, diluted to a final concentration of 50 µg/mL in RPMI-1640 medium, tested for endotoxin contamination (all reagents used in the study were below the detection limit of E-toxate test; Sigma Chemical Co.), and frozen at -20°C.
HPLC purification of gliadin-peptic fragments
All separations were carried out using a HPLC system consisting of LKB-2150-pumps, a Hewlett-Packard 1040 diode-array detector, and a HP 79994A Analytical Workstation.
A solution of digested gliadin (10 mg/mL) in H2O (apyrogenic; Bieffe, Grosotto, Italy)/acetonitrile (HPLC grade; Aldrich Chemical Co., Milwaukee, WI)/trifluoroacetic acid (TFA; spectrophotometric grade; Aldrich) 9:1:0.01 was centrifugated for 10 min at 12,000 g. The supernatant was applied on a Vydac 218TP510 column [25.0x1.0 cm inner diameter (ID); 5 µm particles; 300 Å] and separated at a flow rate of 1 mL/min using linear gradient I (solvent A, 0.1% TFA/H2O; solvent B, 0.1% TFA/acetonitrile), 10–50% B/100 min and 50–80% B/10 min. The elution profile was monitored at 280 nm, and fractions were pooled and lyophilized.
Active peptides were separated on Dionex 218MS52 column (25.0 cmx2.1 mm ID) at a flow rate of 0.1 mL/min using linear gradient II (solvent A, 0.01% TFA/H2O; solvent B, 0.01% TFA/acetonitrile), 5–30% B/90 min, 30–50% B/10 min, and 50–90% B/10 min. The elution profile was monitored at 280 nm. Fractions were diluted to the same optical density before testing.
NO production by macrophages
NO production was measured as the concentration of nitrites in supernatants [17
]. The measurement was done in samples (50 µL) incubated for 10 min at 37°C with an aliquot of Griess reagent (1% sulfanilamide/0.1% naphthylethylenediamine/2.5% H3PO4). Absorbance at 540 nm was recorded using a Titertek Multiscan MCC/340 (Flow Lab., Irvine, Scotland). A nitrite calibration curve was used to convert absorbance into 1 x 10-3 M nitrite.
Cytokine assays
Recombinant mouse (rm) IFN- (specific activity, 1.1x107 U/mg) and rmTNF- (specific activity, 1.33x108 U/mL) were purchased from Genzyme Corporation (Cambridge, MA). The concentration of TNF- and IL-10 in murine macrophage supernatants was determined after a 5-h culture according to the manufacturer’s instructions, using enzyme-linked immunosorbent assay kit reagents from R&D Systems (Minneapolis, MN).
Synthetic peptides derived from gliadin sequence
Peptides were synthesized using the Fmoc/tBu protection strategy on aminomethyl copoly - (styrene-1% divinylbenzene) resin with a Knorr linker. After cleavage from the resin, the crude products were purified using preparative HPLC and characterized by amino acid analysis and liquid chromatography/MS (System Waters 2690 Separation Module and Waters 2487 Dual 
Absorbance Detector, connected to a Micromass Platform L.C.).
MS
Mass spectra were measured on matrix-assisted laser desorption/ionization reflectron time-of-flight (MALDI-TOF) mass spectrometer BIFLEX II (Bruker-Franzen, Bremen, Germany). Ion acceleration voltage was 19 kV, and the reflectron voltage was set at 20 kV. Spectra were calibrated externally using the monoisotopic [M+H]+ ion of the peptide standard somatostatin (Sigma Chemical Co.). A saturated solution of -cyano-4-hydroxy-cinnamic acid in 50% acetonitrile/0.2% TFA was used as a MALDI matrix. Post-source decay (PSD) spectra were recorded in 10 segments. About 50 shots were averaged per segment. Bruker-Franzen XMASS 5.0 software was used for data evaluation.
For further sequence analyses, 0.5 µl of the active fraction was applied on the PepMapTM C18 column (LP Packing, The Netherlands; 0.3x150 mm, 300 angstrom and 3 µm). Gradient elution (90 min) from 5% acetonitrile/0.5% acetic acid to 95% acetonitrile/0.4% acetic acid was performed at 20°C flow rate 4 µL/min and vacuum degassing. The column was linked on-line to the electrospray ionization (ESI) interface of the spectrometer.
Positive, full, and CID (collision-induced dissociation) mass spectra were recorded on an LCQDECA ion trap mass spectrometer (Thermoquest, San Jose, CA), equipped with an ESI ion source and interpreted by SEQUEST software. Spray voltage was held at 5.5 kV, and tube-lens voltage was -10 V. The sheath gas flow (nitrogen, 99.99%) was set at 55 arbitrary units, and heated capillary was kept at 350°C with a voltage of 32 V. Collision energy was kept at 42 units, and the activation time was 30 ms.
Statistics
The data were evaluated by means of analysis of variance and subsequent Dunnett’s test using the Prism program (Graph Pad Software, San Diego, CA).
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was analyzed [18
, 19
]. None of the gliadin fractions nor the crude gliadin digest induced significant in vitro NO production directly in Balb/c macrophages. However, when applied together with IFN- (25 U/mL), the entire gliadin digest and isolated fractions 2, 3, 6, and 10 increased IFN--stimulated NO secretion substantially (P<0.01 or P<0.05). As demonstrated recently, C57BL/6 mice belong to strains responding more readily to low doses of IFN-. Macrophages of C57BL/6 origin primed with IFN- (5 U/mL) synergized strongly with gliadin fractions 2 and 3 and also with fractions 1, 4, and 6 in stimulating NO production (P<0.01). Surprisingly, fractions 7 and 9 inhibited the response of cells to IFN- directly (P<0.01; Fig. 1 B and C
).
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Figure 1. RP-HPLC separation profile of gliadin-peptic digest monitored at 280 nm (A) using Vydac 218TP510 column in 10% acetonitrile/0.1% trifluoroacetic acid in a linear acetonitrile gradient and flow rate 1 mL/min. The capacity of aliquots of purified fractions (1–14) to stimulate peritoneal macrophages (5x106 cells/mL) from Balb/c mice in the presence of 25 U/mL IFN- (B) or from C57BL/6 mice cultured together with 5 U/mL IFN- (C) was tested after 24 h as NO secretion by Griess assay. Data are expressed as mean values from triplicate measurements ± SE (C1-induction by IFN-; C2-IFN-+crude gliadin-peptic digest, 25 µg/mL) and are representative of one of three independent experiments (*, P<0.005; **, P<0.001).
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was tested using C57BL/6 macrophages. A direct stimulation of TNF- secretion was found with gliadin fractions 2, 3, and 10 and to a lesser extent, with fractions 4, 5, and 6 (Fig. 2
). It is interesting that the direct stimulation of TNF- and the synergistic effect on IFN--stimulated NO production were detected in fractions 2 and 3 (Figs. 1
and 2
).
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Figure 2. Direct stimulation of TNF- production measured after a 5-h cultivation of C57BL/6 macrophages with aliquots of fractions 1–11 (see RP-HPLC profile in Fig. 1A
). Data are expressed as mean values from triplicate measurements ± SE. The control, i.e., the mean value of TNF- production by nonstimulated cells (48 pg/mL), was subtracted from all values.
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Figure 3. HPLC purification of gliadin digest in the next step. Samples eluted in the time interval 24–36 min (corresponding to active fractions 2 and 3; see Fig. 1
) were separated into 12 fractions (fraction/1 min). The elution profile of separated fragments recorded at 280 nm is documented (top). The stimulatory capacity of individual fractions, determined as significantly enhanced NO production by IFN--primed, cultured macrophages from Balb/c mice, is shown (bottom).
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Identification of active gliadin fragment
To purify the active gliadin fragments, fraction 11 was subjected to subsequent RP-HPLC fractionation on a Dionex 218MS52 column using linear gradient II. The collected fractions were denoted: 11/0 (time interval, 0–40 min); 11/1–6 (40–70 min, 6 equal fractions); and 11/late (70–90 min). All separated fractions displayed at least a low, enhancing effect on IFN--primed NO production. However, the highest activity was detected when fraction 11/5 was added to an in vitro macrophage culture together with IFN-, as shown in Figure 4
.
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Figure 4. Rechromatography of active gliadin peptides run on a Dionex 218MS52 column (25.0 cmx2.1 mm ID in a linear acetonitrile gradient containing only 0.01% TFA) at a flow rate of 0.1 mL/min. Samples collected in the time interval of 40–70 min were collected into six fractions, and their activity was tested as described in Figure 1B
.
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To test the ability of the synthetic peptides to stimulate in vitro iNOS activation and NO secretion, peritoneal macrophages from Balb/c or C57BL/6 mice were cultured with two doses of the peptides (25 or 100 µg/mL). None of the tested gliadin peptides induced direct NO production, but when added simultaneously with IFN-, they exhibited a costimulatory effect. The highest increase of NO production was elicited by the synthetic form of peptide T and peptide B in macrophage cultures of both strains. Also in this experiment, the response of Balb/c macrophages was less pronounced than that of C57BL/6 cells. Shortening the T peptide from the C-terminus reduced its stimulatory capacity (Fig. 5
).
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Figure 5. The costimulatory effect of synthetic gliadin peptides on NO production: T (VSFQQPQQQYPSSQ); N-terminally shortened peptide A: SFQQPQQQYPSSQ, B: FQQPQQQYPSSQ, and C: QQPQQQYPSSQ; and C-terminally shortened forms D: VSFQQPQQQYPSS, E: VSFQQPQQQYPS, and F: VSFQQPQQQYP added in two doses (25  or 100  µg/mL) to IFN--primed macrophages from Balb/c (open bars) and C57BL/6 (shaded bars) strains of mice. Production of nitrites stimulated by IFN- was measured as a control.
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and RANTES (regulated on activation, normal T expressed and secreted) chemokines directly in macrophages. A low stimulation was observed with peptides T, C, and A and a substantially higher one with peptide B. Similar results, i.e., a low response to peptides T, C, and A and a higher response to peptide B, were obtained when testing the production of the regulatory cytokine IL-10. However, in contrast to NO production, macrophages from Balb/c mice produced more IL-10 than cells from the C57BL/6 mouse strain (Table 1)
. The secretion of TNF- was elevated significantly by simultaneous addition of IFN- to cultured cells. The production of TNF- [picograms (pg)/mL] in the experiment was: IFN- = 175.6 ± 12.4; A + IFN- = 218.0 ± 23.6; B + IFN- = 1605.6 ± 5.6; C + IFN- = 238.0 ± 9.2; and T + IFN- = 489.2 ± 22.3. Again, these results show the unique, stimulatory activity of peptide B. |
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Table 1. Secretion of Cytokines TNF- and IL-10 and Chemokine RANTES Elicited by Synthetic Gliadin Peptides in Peritoneal Macrophages
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To characterize the active gliadin peptide(s), a mixture of peptic-gliadin fragments was separated by the RP-HPLC technique; stimulatory fractions were subjected to rechromatography and subsequent MS. Using this approach, the active gliadin fragment VSFQQPQQQYPSSQ (T peptide) was isolated and identified. The amino acid sequence of the T peptide was found in the primary sequence of /ß gliadins (it matches amino acid residues 246–259 of GDA5 WHEAT or residues 240–253 of GDA7 WHEAT, accession numbers P04725 and P04727, Swiss-Prot database) and also in other gliadin molecules (accession number AAA34275, AAA96522, CAA26385, or S07924, NCBI database). It is interesting that not only the whole peptide sequence but also a 2-amino acid-shortened form was found to be present in the sequence of /ß gliadins but not in other molecules. When comparing the sequence of peptide T with known gliadin epitopes recognized by specific T cells or Ab, similar motifs (QQPQQ, PQQQ, or VSFQQP) were found to be present in some of them [20
21
22
23
24
].
The synthesis of a sufficient quantity of peptide T in the original form and in forms shortened from the N- (peptides A–C) and C-terminus (peptides D–F) enabled us to study the relationship between the structure and biological activities of the gliadin peptides. When induction of TNF-, IL-10, RANTES, and nitrite production by synthetic peptides was measured in vitro, the dodecapeptide B with the sequence FQQPQQQYPSSQ (a 2-amino acid, N-terminally shortened T peptide) was found to be a potent gluten-derived, macrophage-stimulating peptide.
Activation of macrophages and dendritic cells is supposed to be accompanied by up-regulation of membrane molecules involved in antigen presentation. Together with genetic factors, it could lead to abrogation of oral tolerance and increased immune responses to this food component [3 ]. Presumably, the balance between Th1 and Th2 subpopulations in mucosal-associated lymphoid tissues can be diverted toward Th1 stimulation by the costimulatory activity of gliadin fragments.
Recently published data showed that macrophages from two mouse strains (C57BL/6 and Balb/c) respond in a quantitatively different manner to IFN-, the primary Th1 cytokine, and lipopolysaccharide. Macrophages from C57BL/6 mice responded to low IFN- doses [100 pg; 2 international units (IU)/mL] by NO production, whereas Balb/c macrophages required much higher IFN- doses (25–50 IU/mL) for stimulation [19
].
Based on these data, we used these two strains of mice to test the biological activities of gliadin fragments and synthetic peptides. Comparison of the response of macrophages from these two strains, primed with different stimulatory doses of IFN- and the same doses of gliadin fractions or peptides, revealed quantitative rather than qualitative differences.
The difference between the two strains can conceivably reflect an "increased" responsiveness of C57BL/6 macrophages and a "lower" response of Balb/c macrophages, the strain with higher production of IL-10 regulatory (inhibitory) cytokine. This situation can be likened to that in humans; innate-immunity cells of individuals susceptible to celiac disease development may respond to gliadin with various intensity, contributing however more vigorously to the cascade of immunopathological changes. The kinetics of individual steps of activation of innate-immunity cells needs to be studied in detail. IFN- is a crucial cytokine produced by gliadin-specific T cells in celiac disease pathogeny and also seems to be essential for modulating the intensity of the innate-immune response. The iNOS activation and NO production is IFN--dependent, and TNF- secretion is enhanced significantly by the presence of this cytokine.
It is interesting that the quantity of NO produced by macrophages from the two strains on induction by different doses of INF- was shown to be inversely proportional to the quantity of produced TGF-1ß, which down-regulates NO production. Recently, macrophages have been proposed to be important immunoregulatory cells in specific as well as nonspecific immune response. The different susceptibility of macrophages to stimuli such as bacterial antigens or IFN- is dependent on the genetic background and thus, could affect the characteristics of the immune response substantially [18
, 19
, 25
].
In conclusion, we confirmed our previous findings that the innate response of macrophages (cytokine and NO secretion) can be stimulated and/or enhanced directly by the unique food protein gliadin and/or its proteolytic fragments arising during digestion in the acid environment of the stomach. In the present study, we characterized the natural gliadin components responsible for these activities and confirmed this finding by using synthetic forms of active and modified gliadin peptides. Preliminary results of our ongoing studies with human monocyte cell lines (differing in cell-receptor expression) and human peripheral blood monocytes are supporting the stimulatory activity of gliadin peptides. Taking into account the stage of differentiation of monocyte/macrophages/dendritic cells [26 27 28 ], our data may provide a basis for further investigation of innate-immune response to defined gliadin fragments in celiac disease.
parová and Daniela Franková for excellent technical assistance. Received July 22, 2001; revised October 26, 2001; accepted December 10, 2001.
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ková, S., Tuková, L., Flegelová, Z., Michalak, M., Walters, J. R. F., Whelan, A., Harries, J., Vencovsk
, J., Tlaskalová-Hogenová, H. (1999) Identification of common epitopes on gliadin, enterocytes, and calreticulin recognised by antigliadin antibodies of patients with coeliac disease Gut 44,168-173, interleukin-6, and interleukin-2 mRNA in the jejunum of patients with coeliac disease Scand. J. Gastroenterol. 30,456-463[Medline]ková, L., Tlaskalová-Hogenová, H., Flegelová, Z., Zídek, Z. (2000) Activation of macrophages by food antigens: enhancing effect of gluten on nitric oxide and cytokine production J. Leukoc. Biol. 67,312-318[Abstract], IFN-, and LPS J. Mol. Cell. Cardiol. 29,1153-1165[Medline]This article has been cited by other articles:
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