Diverse functions of the ecdysone receptor (EcR) in the panoistic ovary of the German cockroach.

Ecdysone regulates essential processes in insect life. Perhaps the most well-known of these are related to metamorphosis. However, ecdysone is also required to regulate the proliferation and differentiation of germ cells in the ovary. The role of ecdysone in insect oogenesis has been studied in depth in holometabolan species with meroistic ovaries, such as Drosophila melanogaster, while in hemimetabolan species with panoistic ovaries their functions are still poorly understood. In the present work, we studied the role of ecdysone in the ovary of the last nymphal instar of the cockroach Blattella germanica by using RNA interference to reduce the levels of the ecdysone receptor (EcR), and thereby deplete the expression of ecdysteroidogenic genes in the prothoracic gland. However, the expression of ecdysteroidogenic genes was upregulated in the ovary, resulting in cell overproliferation in the germarium, which appeared swollen. By analysing the expression of genes that respond to ecdysone, we found that when the source of 20E is the nymphal ovary, EcR appears to repress 20E-associated genes bypassing early genes signalling.

Paneth and intestinal stem cells preserve their functional integrity during worsening of acute cellular rejection in small bowel transplantation.

Graft survival after small bowel transplantation remains impaired due to acute cellular rejection (ACR), the leading cause of graft loss. Although it was shown that the number of enteroendocrine progenitor cells in intestinal crypts was reduced during mild ACR, no results of Paneth and intestinal stem cells localized at the crypt bottom have been shown so far. Therefore, we wanted to elucidate integrity and functionality of the Paneth and stem cells during different degrees of ACR, and to assess whether these cells are the primary targets of the rejection process. We compared biopsies from ITx patients with no, mild, or moderate ACR by immunohistochemistry and quantitative PCR. Our results show that numbers of Paneth and stem cells remain constant in all study groups, whereas the transit-amplifying zone is the most impaired zone during ACR. We detected an unchanged level of antimicrobial peptides in Paneth cells and similar numbers of Ki-67+ IL-22R+ stem cells revealing cell functionality in moderate ACR samples. We conclude that Paneth and stem cells are not primary target cells during ACR. IL-22R+ Ki-67+ stem cells might be an interesting target cell population for protection and regeneration of the epithelial monolayer during/after a severe ACR in ITx patients.

Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella.

Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS + FliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responses.

Characterization of the immune response induced by pertussis OMVs-based vaccine.

For the development of a third generation of pertussis vaccine that could improve the control of the disease, it was proposed that the immune responses induced by the classic whole cell vaccine (wP) or after infection should be used as a reference point. We have recently identified a vaccine candidate based on outer membrane vesicles (OMVs) derived from the disease etiologic agent that have been shown to be safe and protective in mice model of infection. Here we characterized OMVs-mediated immunity and the safety of our new candidate. We also deepen the knowledge of the induced humoral response contribution in pertussis protection. Regarding the safety of the OMVs based vaccine (TdapOMVsBp,) the in vitro whole blood human assay here performed, showed that the low toxicity of OMVs-based vaccine previously detected in mice could be extended to human samples. Stimulation of splenocytes from immunized mice evidenced the presence of IFN-γ and IL-17-producing cells, indicated that OMVs induces both Th1 and Th17 response. Interestingly TdapOMVsBp-raised antibodies such as those induced by wP and commercial acellular vaccines (aP) which contribute to induce protection against Bordetella pertussis infection. As occurs with wP-induced antibodies, the TdapOMVsBp-induced serum antibodies efficiently opsonized B. pertussis. All the data here obtained shows that OMVs based vaccine is able to induce Th1/Th17 and Th2 mixed profile with robust humoral response involved in protection, positioning this candidate among the different possibilities to constitute the third generation of anti-pertussis vaccines.

Pretreatment Combination Reduces Remote Organ Damage Secondary to Intestinal Reperfusion Injury in Mice: Follow-up Study.

BACKGROUND: Intestinal ischemia-reperfusion injury occurs after different surgical treatments, including intestinal transplantation. This harmful process may have an effect in remote organs, leading to multiple organ dysfunction syndrome and death. Therefore, to establish strategies to attenuate local and remote damage constitutes a challenge for experimental and clinical surgeons in the intestinal surgical field. METHODS: We evaluated the effect of ischemic preconditioning and tacrolimus pretreatment applied alone and in combination against local and remote damage caused by prolonged intestinal ischemia-reperfusion injury in a mouse model of warm ischemia. RESULTS: Ischemic preconditioning applied alone and in combination with tacrolimus decreased histological damage (P < .05), number of apoptotic cells (P < .05), nitrosative stress (P < .01), and serum lactate dehydrogenase activity (P < .05) and lowered uremia (P < .05) compared with untreated post-reperfused intestines. Regarding remote organ damage, combination therapy was the unique condition able to attenuate lung (mainly neutrophil infiltration and hemorrhage), liver (sinusoidal congestion and hepatic vacuolization), and kidney (acute tubular necrosis and hydropic degeneration) histological alterations (P < .05), compared with the untreated group. CONCLUSIONS: These results support the application of these strategies in combination to minimize the impact of ischemia-reperfusion injury in the whole organism as a strategy to prevent multiple organ dysfunction syndromes and minimize the clinical impact.

Probiotic yeast Kluyveromyces marxianus CIDCA 8154 shows anti-inflammatory and anti-oxidative stress properties in in vivo models.

Inflammatory bowel diseases (IBDs) are complex affections with increasing incidence worldwide. Multiple factors are involved in the development and maintenance of the symptoms including enhanced oxidative stress in intestinal mucosa. The conventional therapeutic approaches for IBDs are based on the use anti-inflammatory drugs with important collateral effects and partial efficacy. In the present work we tested the anti-inflammatory capacity of Kluyveromyces marxianus CIDCA 8154 in different models. In vitro, we showed that the pretreatment of epithelial cells with the yeast reduce the levels of intracellular reactive oxygen species. Furthermore, in a murine model of trinitro benzene sulfonic acid-induced colitis, yeast-treated animals showed a reduced histopathological score (P<0.05) and lower levels of circulating interleukin 6 (P<0.05). The capacity to modulate oxidative stress in vivo was assessed using a Caenorhabditis elegans model. The yeast was able to protect the nematodes from oxidative stress by modulating the SKN-1 transcription factor trough the DAF-2 pathway. These results indicate that K. marxianus CIDCA 8154 could control the intestinal inflammation and cellular oxidative stress. Deciphering the mechanisms of action of different probiotics might be useful for the rational formulation of polymicrobial products containing microorganisms targeting different anti-inflammatory pathways.

Immunological status of isolated lymphoid follicles after intestinal transplantation.

Intestinal transplantation (ITx) faces the challenge of grafting a high immunogenic organ, which is certainly one of the major obstacles for intestinal allograft acceptance. The allograft has to guarantee the proper functioning of the mucosal immune machinery under immunosuppressive conditions. Recently, it has been elucidated that isolated lymphoid follicles (ILFs) are an indispensable part of mucosal immunity to maintain IgA synthesis and consequently to control commensal microflora. No data about these follicular structures in the setting of ITx are available so far. Therefore, we addressed the question whether constitution, integrity and function of allograft ILFs are disturbed by immunosuppressive regimen. We compared allograft ILFs from terminal ileum of transplant patients with ILFs from nontransplant patients via flow cytometry, quantitative real-time polymerase chain reaction and immunohistochemistry. We found that host leukocytes rapidly repopulate allograft ILFs and that maintenance immunosuppressive regimen, tacrolimus and corticosteroids, does not affect their cellular integrity and function. However, allograft ILFs revealed a higher maturation state than control samples and IgA positive plasma cells were increased in number in allograft mucosa. Our results open the path for a better understanding of allograft mucosal immunity.

Airway structural cells regulate TLR5-mediated mucosal adjuvant activity.

Antigen-presenting cell (APC) activation is enhanced by vaccine adjuvants. Most vaccines are based on the assumption that adjuvant activity of Toll-like receptor (TLR) agonists depends on direct, functional activation of APCs. Here, we sought to establish whether TLR stimulation in non-hematopoietic cells contributes to flagellin's mucosal adjuvant activity. Nasal administration of flagellin enhanced T-cell-mediated immunity, and systemic and secretory antibody responses to coadministered antigens in a TLR5-dependent manner. Mucosal adjuvant activity was not affected by either abrogation of TLR5 signaling in hematopoietic cells or the presence of flagellin-specific, circulating neutralizing antibodies. We found that flagellin is rapidly degraded in conducting airways, does not translocate into lung parenchyma and stimulates an early immune response, suggesting that TLR5 signaling is regionalized. The flagellin-specific early response of lung was regulated by radioresistant cells expressing TLR5 (particularly the airway epithelial cells). Flagellin stimulated the epithelial production of a small set of mediators that included the chemokine CCL20, which is known to promote APC recruitment in mucosal tissues. Our data suggest that (i) the adjuvant activity of TLR agonists in mucosal vaccination may require TLR stimulation of structural cells and (ii) harnessing the effect of adjuvants on epithelial cells can improve mucosal vaccines.

Outer membrane vesicles derived from Bordetella parapertussis as an acellular vaccine against Bordetella parapertussis and Bordetella pertussis infection.

Bordetella parapertussis, a close related species of B. pertussis, can also cause the disease named pertussis or whooping cough. The number of cases caused by this related pathogen has risen sustained in the last years. The widely used cellular (wP) or acellular (aP) pertussis vaccines have little or no efficacy against B. parapertussis. In an effort to devise an effective acellular vaccine against B. parapertussis infection, outer membrane vesicles (OMVs) were obtained from B. parapertussis. Proteomic analysis of the resulting OMVs, designated OMVsBpp, evidenced the presence of several surface immunogens including pertactin. The characterized OMVsBpp were used in murine B. parapertussis intranasal challenge model to examine their protective capacity when administered by systemic route. Immunized BALB/c mice were challenged with sublethal doses of B. parapertussis. Significant differences between immunized animals and the negative control group were observed (p<0.001). OMVsBpp protected against B. parapertussis infection, whereas current commercial aP vaccine showed little protection against such pathogen. More interestingly, protection induced by OMVsBpp against B. pertussis was comparable to our previously designed vaccine consisting in OMVs derived from B. pertussis (OMVsBp). For these experiments we used as a positive control the current commercial aP vaccine in high dose. As expected aP offered protection against B. pertussis in mice. Altogether the results presented here showed that the OMVs from B. parapertussis are an attractive vaccine candidate to protect against whooping cough induced by B. parapertussis but also by B. pertussis.

Ischemic preconditioning and tacrolimus pretreatment as strategies to attenuate intestinal ischemia-reperfusion injury in mice.

The intestine is highly sensitive to ischemia-reperfusion injury (IRI), a phenomenon occurring in different intestinal diseases. Several strategies to mitigate IRI are in experimental stages; unfortunately, no consensus has been reached about the most appropriate one. We report a protocol to study ischemic preconditioning (IPC) evaluation in mice and to combine IPC and tacrolimus (TAC) pretreatment in a warm ischemia model. Mice were divided into treated (IPC, TAC, and IPC + TAC) and untreated groups before intestinal ischemia. IPC, TAC, and IPC + TAC groups were able to decrease postreperfusion nitrites levels (P < .05). IPC-containing groups had a major beneficial effect by preserving the integrity of the intestinal histology (P < .05) and improving animal survival (P < .002) compared with TAC alone or the untreated group. The IPC + TAC group was the only one that showed significant improvement in lung histological analysis (P < .05). The TAC and IPC + TAC groups down-regulated intestinal expression of interleukin (II)-6 and IL1b more than 10-fold compared with the control group. Although IPC and TAC alone reduced intestinal IRI, the used of a combined therapy produced the most significant results in all the local and distant evaluated parameters.

The stimulated innate resistance event in Bordetella pertussis infection is dependent on reactive oxygen species production.

The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can efficiently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 µg) was added before (48 h or 24 h), after (24 h), or simultaneously with the B. pertussis challenge (10(7) CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.

Defining the nonreturn time for intestinal ischemia reperfusion injury in mice.

Among the abdominal organs, the intestine is probably the most sensitive to ischemia reperfusion injury (IRI), a phenomenon that occurs in many intestinal disorders. Few studies have reported in detail the impact of intestinal ischemia time in mice. We evaluated the effect of various warm intestinal ischemia times in an intestinal IRI model in mice. Adult male Balb/c mice were divided into 4 groups that differed in intestinal ischemia time: G1, 30; minutes; G2, 35 minutes; G3, 40 minutes; and G4, 45 minutes. Histological evaluation showed average Park scores as follows: G1 0.6 ± 0.55; G2 1.8 ± 0.45; G3 4.8 ± 2.25; and G4 5 ± 1.79. All animals from G1 survived 30 hours. G2 animals showed intermediate behavior with all succumbing between 18 and 30 hours postprocedure. G3 and G4 displayed similar survival results with animals succumbing before 6 hours after intestinal reperfusion. These data showed that Park index scores of 3 or higher were related to early death. We concluded that the 5 minutes between 35 and 40 minutes is the critical limit, after which all mice die after reperfusion. This result may represent a valuable tool for future research in mice.

Analysis of immune cells draining from the abdominal cavity as a novel tool to study intestinal transplant immunobiology.

During intestinal transplant (ITx) operation, intestinal lymphatics are not reconstituted. Consequently, trafficking immune cells drain freely into the abdominal cavity. Our aim was to evaluate whether leucocytes migrating from a transplanted intestine could be recovered from the abdominal draining fluid collected by a peritoneal drainage system in the early post-ITx period, and to determine potential applications of the assessment of draining cellular populations. The cell composition of the abdominal draining fluid was analysed during the first 11 post-ITx days. Using flow cytometry, immune cells from blood and draining fluid samples obtained the same day showed an almost complete lymphopenia in peripheral blood, whereas CD3(+) CD4(+) CD8(-) , CD3(+) CD4(-) CD8(+) and human leucocyte antigen D-related (HLA-DR)(+) CD19(+) lymphocytes were the main populations in the draining fluid. Non-complicated recipients evolved from a mixed leucocyte pattern including granulocytes, monocytes and lymphocytes to an exclusively lymphocytic pattern along the first post-ITx week. At days 1-2 post-Itx, analysis by short tandem repeats fingerprinting of CD3(+) CD8(+) sorted T cells from draining fluid indicated that 50% of cells were from graft origin, whereas by day 11 post-ITx this proportion decreased to fewer than 1%. Our results show for the first time that the abdominal drainage fluid contains mainly immune cells trafficking from the implanted intestine, providing the opportunity to sample lymphocytes draining from the grafted organ along the post-ITx period. Therefore, this analysis may provide information useful for understanding ITx immunobiology and eventually could also be of interest for clinical management.

Mucosal innate response stimulation induced by lipopolysaccharide protects against Bordetella pertussis colonization.

Non-specific enhancement of the airways innate response has been shown to impair lung infections in several models of infection such diverse as influenza A, Streptococcus pneumoniae, and Aspergillus niger. Our aim was to evaluate whether a similar event could operate in the context of Bordetella pertussis respiratory infection, not only to enrich the knowledge of host-bacteria interaction but also to establish immunological basis for the development of new control strategies against the pathogen. Using a B. pertussis intranasal infection model and coadministration of different TLR agonists at the moment of the infection, we observed that the enhancement of innate response activation, in a TLR4-dependent way, could efficiently impair B. pertussis colonization (P < 0.001). While LPS from different microbial sources were equally effective in promoting this effect, flagellin and poly I:C coadministration, in spite of inducing expression of innate response markers TNFalpha, CXCL2, CXCL10 and IL6, was not effective to prevent B. pertussis colonization. Our results indicate that during the early stage of infection, specific anti-microbial mechanisms triggered by TLR4 stimulation are able to impair B. pertussis colonization. These findings could complement our current view of the role of TLR4-dependent processes that contribute to anti-pertussis immunity.

Evaluation of calprotectin level in intestinal content as an early marker for graft rejection.

INTRODUCTION: The diagnosis of rejection after intestinal transplantation is still performed by endoscopic biopsy monitoring. Less invasive diagnostic procedures are desirable, although they are not available so far. Calprotectin, a stable cytosolic granulocyte protein, which previously was used as a marker of inflammatory processes, has been proposed to be a biochemical marker for rejection. The aim of the present work was to analyze the concordance between calprotectin levels in intestinal content and the presence of graft rejection after small bowel transplantation. METHODS: Calprotectin level was measured using a commercial ELISA kit on 137 samples of intestinal content randomly collected during endoscopies performed on 11 intestinal transplantation patients during 2 years' follow-up. Calprotectin determinations were correlated with histological and clinical findings. The cut-off level was determined retrospectively by receiver-operator curve analysis. RESULTS: Based on histological findings and clinical records, samples were discerned as rejection positive (37 of 137), versus negative (35 of 137) samples or those with no clinical, endoscopic, or histological findings (65 of 137 samples). A cut-off value of 65 microg of calprotectin/g of intestinal content provided the best assay parameter according to the clinical findings: a 76% sensitivity and a 47% specificity. False positive results corresponded to patients with gastrointestinal infections (13%), systemic infections (13%), ulcers (10%), or nonspecific histological alterations of the mucosa (15%). The other false positive cases corresponded to postsurgical samples (4%), or patients with concomitant gastrointestinal symptoms (2%). Most false negative results (78%) were observed during recovery from severe acute rejection episodes, among successfully treated patients. In these cases, epithelial reconstitution and no mucosal infiltration was observed. If the latter group were discarded, sensitivity rose to 93%, and specificity, to 50% with a 96% negative predictive value. Furthermore, a weak correlation was observed between calprotectin levels and the severity of rejection. CONCLUSIONS: This study confirmed the results obtained by other groups: fecal calprotectin dosage showed a good sensitivity but low specificity for the diagnosis of intestinal rejection because high calprotectin levels can also be observed in other clinical conditions. Nevertheless, it might be used as a first line for continuous evaluation of intestinal transplantation status, like other biochemical parameters that are used in kidney or liver transplantation, before considering the need for a biopsy.

Isospora belli infection after isolated intestinal transplant.

Isospora belli is a protozoan that only affects humans, after ingestion of Isospora's oocysts. Immunocompetent patients usually do not develop the infection. Immunocompromised hosts may have profuse diarrhea with other gastrointestinal symptoms. Treatment is based on trimethoprim-sulfamethoxazole. In 2006 we performed an isolated intestinal transplantation in a patient with ultra-short bowel syndrome. Neither rejection nor clinical problems occurred after transplant, but signs of intestinal inflammation were seen in every protocol biopsy starting at the first month post transplant. Almost 3 months after the procedure, the patient was re-admitted with diarrhea. I. belli infection was diagnosed by detection of the oocysts in stool samples. Antibiotic treatment with trimethoprim-sulfamethoxazole was initiated with excellent outcome and without relapses. To the best of our knowledge, this is the first case of isosporosis in a small bowel recipient.

Ontogeny of intestinal epithelium immune functions: developmental and environmental regulation.

Intestinal mucosa integrates primary digestive functions with immune functions such as pathogen surveillance, antigen transport and induction of mucosal immunity and tolerance. Intestinal adaptive immunity is elicited in organized mucosa-associated lymphoid tissue (O-MALT) that is composed of antigen-presenting cells and lymphocytes and achieved by effector cells widely distributed in mucosa (diffuse MALT or D-MALT). Interaction between the intestinal epithelium, the O-MALT and the diffuse MALT plays a critical role in establishing an adequate immune response. In regions associated to O-MALT, lympho-epithelial cross-talks lead to acquisition of a specific epithelial phenotype that contributes to O-MALT organization and functionality. Beyond the expression of several innate immune functions, the intestinal epithelium may directly take up and present antigens due to the expression of major histocompatibility complex (MHC) and MHC-related molecules. A complex genetic program that will be outlined in the present review controls the development of immune functions of the intestinal epithelium. The effect of environmental signals on the modulation of this ontogenetic program during development and neonatal life, from bioactive components of amniotic fluid to lactation and bacterial colonization, will be discussed.

Evaluation of coeliac disease serological markers in Down syndrome patients.

BACKGROUND: The increased incidence of coeliac disease in patients with Down syndrome makes screening of coeliac disease in this population advisable. AIM: Evaluation of efficiency of different serological markers to detect coeliac disease in Down syndrome patients. PATIENTS: A total of 56 Down syndrome patients (aged: 1-17 years) were included in study. METHODS: Patients were evaluated for both IgG and IgA anti-transglutaminase antibodies and for anti-gliadin IgA and IgG antibodies using either purified omega-gliadin, wheat ethanol extract or commercial gliadin. Patients who had at least one positive result were evaluated for antiendomysium antibodies using either monkey oesophagus or human umbilical cord by indirect immunofluorescence. Coeliac disease was diagnosed by typical histological changes on duodenal mucosa. RESULTS: Increased levels of at least one anti-gliadin IgA and IgG antibody marker were found in 27 out of 56 cases (26 for IgG and 9 for IgA). 11/56 were positive for IgG anti-transglutaminase antibodies and two of them were also positive for IgA anti-transglutaminase antibodies and anti-endomysium antibodies. These two patients were finally diagnosed as coeliacs. Gliadin antigenic fractions employed produced differences in the performance of the anti-gliadin IgA and IgG antibody test. The use of commercial gliadin or wheat ethanol extract showed low sensitivity in IgA anti-gliadin IgA and IgG antibody determination, whereas good sensitivity and specificity were observed with omega-gliadins. IgG anti-transglutaminase antibodies showed a high proportion of false positive results (9 out of 56), whereas anti-endomysium antibodies and IgA anti-transglutaminase antibodies presented an excellent correlation with presence of active coeliac disease. CONCLUSIONS: Two out of 56 Down syndrome patients were diagnosed as coeliacs, corresponding to an incidence of 3.6%. The use of omega-gliadin presented the best efficiency in anti-gliadin IgA and IgG antibody determination whereas IgA anti-transglutaminase antibodies and anti-endomysium antibody determination showed an absolute correlation with presence of active coeliac disease.

Analysis of the effects of heat treatment on gliadin immunochemical quantification using a panel of anti-prolamin antibodies.

Antigen-labeled capture enzyme-linked immunosorbent assay with four different anti-gliadin monoclonal antibodies and an anti-gliadin serum and two different sample systems (purified gliadin fractions heat-treated in soluble phase and a model of dough simulating a baking process) were employed to study the effects of heat treatment on gliadin quantification. The analysis of purified gliadins showed that there is no particularly heat stable fraction. Remarkably, omega-gliadin did not present a differential heat stability. Reactivity varied depending on the time-temperature conditions of the treatment, the antibody employed, and the fraction analyzed. Heated dough samples showed an impairment of protein extraction depending on the intensity of the treatment. Capillary electrophoresis analysis of extracts showed that each gliadin group is affected to a different extent; omega-gliadin is less modified. Immunochemical analysis of the heat-treated samples using either of the five antibodies showed a decrease in the quantified gliadin, in concordance to the loss in the extracted proteins. Among the different sources of error in gliadin immunochemical quantification, the impairment in extraction efficiency in heat-treated samples appears as a major drawback to be overcome.

Determination of anti-omega-gliadin antibodies in serologic tests for coeliac disease.

BACKGROUND: Serologic detection of coeliac disease in the general population or in subjects belonging to risk groups implies the use of a test with high efficiency, large-scale use, and low cost. The enzyme-linked immunosorbent assay (ELISA) technique is the most appropriate assay for performing this kind of studies. Even though anti-gliadin determination has been the test most frequently used as the first step in screening procedures, many false-positive results produced a low-specificity test. In a previous work a selective recognition of omega-gliadins, mainly by IgA antibodies, was observed. Results also indicated that omega-gliadins can be useful as antigens in serologic detection of coeliac disease. We therefore wanted to analyse the anti-gliadin antibody reactivity by using purified gliadins and to evaluate the actual performance of the anti-omega-gliadin antibody test. METHODS: A population consisting of 105 coeliac patients, 81 healthy controls, and 73 subjects in a disease control group was analysed. Anti-endomysium (EMA), both IgG and IgA anti-omega-gliadins, and anti-tissue transglutaminase (tTG) antibodies were determined. RESULTS: Concordant results, positive and negative, in the EMA and IgG and IgA anti-gliadin determinations were observed in 220 of 259 samples from the total population analysed. The three assays showed high efficiency, being 96.9%, 90.7%, and 91.1% for EMA and anti-omega-gliadins IgG and IgA, respectively. Anti-tTG determination was performed on 103 samples (69 controls and 34 coeliac patients), finding 4 false results (2 false positive and 2 false negative), whereas anti-omega-gliadins showed 10 false results (5 false negative and 5 false positive), 3 of which were coincident with anti-tTG determination. To compare the reactivity of anti-gliadin antibodies, alpha-, beta-, gamma- and omega-gliadins were isolated under non-denaturing conditions by acid preparative electrophoresis and cation-exchange fast protein liquid chromatography (FPLC) and used in an indirect ELISA test. The composition of these fractions was analysed by means of capillary electrophoresis, showing no cross-contamination among them. CONCLUSIONS: The comparison of results using purified gliadins shows that omega-gliadins present a differential reactivity that has not previously been documented. Results using omega-gliadins isolated by either preparative electrophoresis or FPLC were similar. Tests using the purified omega-gliadin fraction present the best performance when anti-gliadin antibodies are evaluated.