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During recent years, the identification of monogenic mutations that cause sterile inflammation has expanded the spectrum of autoinflammatory diseases, clinical disorders characterized by uncontrolled systemic and organ-specific inflammation that, in some cases, can mirror infectious conditions. Early studies support the concept of innate immune dysregulation with a predominance of myeloid effector cell dysregulation, particularly neutrophils and macrophages, in causing tissue inflammation. However, recent discoveries have shown a complex overlap of features of autoinflammation and/or immunodeficiency contributing to severe disease phenotypes. Here, we describe the first Argentine patient with a newly described frameshift mutation in SAMD9L c.2666delT/p.F889Sfs*2 presenting with a complex phenotypic overlap of CANDLE-like features and severe infection-induced cytopenia and immunodeficiency. The patient underwent a fully matched unrelated HSCT and has since been in inflammatory remission 5 years post-HSCT., Instituto de Estudios Inmunológicos y Fisiopatológicos

Patients with inborn errors of immunity (IEI) in Argentina were encouraged to receive licensed Sputnik, AstraZeneca, Sinopharm, Moderna, and Pfizer vaccines, even though most of the data of humoral and cellular responses combination on available vaccines comes from trials conducted in healthy individuals. We aimed to evaluate the safety and immunogenicity of the different vaccines in IEI patients in Argentina. The study cohort included adults and pediatric IEI patients (n = 118) and age-matched healthy controls (HC) (n = 37). B cell response was evaluated by measuring IgG anti-spike/receptor binding domain (S/RBD) and anti-nucleocapsid(N) antibodies by ELISA. Neutralization antibodies were also assessed with an alpha-S protein-expressing pseudo-virus assay. The T cell response was analyzed by IFN-γ secretion on S- or N-stimulated PBMC by ELISPOT and the frequency of S-specific circulating T follicular-helper cells (TFH) was evaluated by flow cytometry.No moderate/severe vaccine-associated adverse events were observed. Anti-S/RBD titers showed significant differences in both pediatric and adult IEI patients versus the age-matched HC cohort (p 0.05). Neutralizing antibodies were also significantly lower in the patient cohort than in age-matched HC (p 0.01). Positive S-specific IFN-γ response was observed in 84.5% of IEI patients and 82.1% presented S-specific TFH cells. Moderna vaccines, which were mainly administered in the pediatric population, elicited a stronger humoral response in IEI patients, both in antibody titer and neutralization capacity, but the cellular immune response was similar between vaccine platforms. No difference in humoral response was observed between vaccinated patients with and without previous SARS-CoV-2 infection.In conclusion, COVID-19 vaccines showed safety in IEI patients and, although immunogenicity was lower than HC, they showed specific anti-S/RBD IgG, neutralizing antibody titers, and T cell-dependent cellular immunity with IFN-γ secreting cells. These findings may guide the recommendation for a vaccination with all the available vaccines in IEI patients to prevent COVID-19 disease.

Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is characterized by chronic, relapsing intestinal inflammation. Galectin-1 (Gal-1) is an endogenous lectin with key pro-resolving roles, including induction of T-cell apoptosis and secretion of immunosuppressive cytokines. Despite considerable progress, the relevance of Gal-1-induced T-cell death in inflamed tissue from human IBD patients has not been ascertained. Intestinal biopsies and surgical specimens from control patients (n = 52) and patients with active or inactive IBD (n = 97) were studied. Gal-1 expression was studied by RT-qPCR, immunoblotting, ELISA and immunohistochemistry. Gal-1-specific ligands and Gal-1-induced apoptosis of lamina propria (LP) T-cells were determined by TUNEL and flow cytometry. We found a transient expression of asialo core 1-O-glycans in LP T-cells from inflamed areas (p

Background: Manifestations of food allergies vary among patients, being the most common gastrointestinal, skin and respiratory symptoms. This study aimed to identify and characterize polyp Th2 cells as a critical component of the allergic reaction. We previously characterized colorectal polyps in patients sensitized to food allergens as inflammatory, with a Type 2-dominated stroma, with elevated IL-13, IL-4 and locally synthesized IgE. Methods: We isolated milk-specific T cells from tissue and blood of food-sensitized patients (n=10) and obtained cell lines and cell clones. Proliferation, specificity, cytokine secretion, gut homing markers and TCR nature were analyzed after antigen recall; CCL25 was assessed in polyps and surrounding tissue. Results: Lamina propria and peripheral T cells responded similarly to cow´s milk proteins, showing similar cell proliferation index and Th2 cytokine release in vitro. All CD4 T cells expressed the membrane T receptor and secreted higher IL-13 amounts than unstimulated cells, whereas IFN-γ secretion remained unchanged. Remarkably, the gut homing chemokine receptor CCR9 was augmented in cow’s milk-specific peripheral and lamina propria T cells, and CCL25 was found to be expressed in the inflammatory polyp tissue and not in the adjacent mucosa. Conclusion: We isolated and characterized cow´s milk-specific lamina propria CD4 TCRαβ Th2 cells from colonic inflammatory polyps. The CCR9 expression on polyp cells and the increased production of tissue CCL25 might facilitate T cells’ recruitment, which are key players in the allergic reaction to promote local IgE synthesis. Our findings may be critical to proposing rational and novel T cell-targeted immunotherapies.

Purpose Proteases play an essential role in the pathophysiology of inflammatory bowel disease (IBD), contributing to the intestinal mucosal lesions through the degradation of the extracellular matrix and alteration of the barrier function. Ulcerative colitis (UC) is characterized by an extensive infiltrate of neutrophils into the mucosa and hence, increased proteolytic activity. Human neutrophil elastase (HNE) is a serine protease that has been reported to be increased in UC patients' intestinal mucosa. Based on our previous studies, we hypothesized that HNE might induce proteolytic degradation and loss of function of therapeutic monoclonal antibodies in IBD patients. Patients and Methods Elastase expression and elastinolytic activity were determined in mucosal explants from ulcerative colitis patients (n=6) and cultured ex vivo in the presence or absence of recombinant elafin. Enzymatic digestions of therapeutic monoclonal antibodies were performed using recombinant HNE and elafin. The integrity of the therapeutic antibodies was evaluated by immunoblotting and protein G binding assay, whereas their TNF-neutralizing activity was assessed with a reporter cell line. Results We found that HNE and its elastinolytic activity were increased in the gut mucosa of UC patients. We also demonstrated that HNE cleaved biological drugs, impairing the TNF-α neutralizing capacity of anti-TNF monoclonal antibodies. This proteolytic degradation was inhibited by the addition of the specific inhibitor, elafin. Conclusion Our results suggest that the high level of proteolytic degradation by mucosal neutrophil elastase, along with a potential imbalance with elafin, contributes to the loss of function of biologic agents, which are currently used in patients with IBD. These findings might explain the non-responsiveness of UC patients to therapeutic monoclonal antibodies and suggest the potential beneficial concomitant use of elafin in this treatment.

Ulcerative colitis and Crohn’s disease, the two main forms of inflammatory bowel disease (IBD), are immunologically mediated disorders. Several therapies are focused on activated T cells as key targets. Although Lactobacillus kefiri has shown anti-inflammatory effects in animal models, few studies were done using human mucosal T cells. The aim of this work was to investigate the immunomodulatory effects of this bacterium on intestinal T cells from patients with active IBD. Mucosal biopsies and surgical samples from IBD adult patients (n = 19) or healthy donors (HC; n = 5) were used. Lamina propria mononuclear cells were isolated by enzymatic tissue digestion, and entero-adhesive Escherichia coli-specific lamina propria T cells (LPTC) were expanded. The immunomodulatory properties of L. kefiri CIDCA 8348 strain were evaluated on biopsies and on anti-CD3/CD28-activated LPTC. Secreted cytokines were quantified by ELISA, and cell proliferation and viability were assessed by flow cytometry. We found that L. kefiri reduced spontaneous release of IL-6 and IL-8 from inflamed biopsies ex vivo. Activated LPTC from IBD patients showed low proliferative rates and reduced secretion of TNF-α, IL-6, IFN-γ and IL-13 in the presence of L. kefiri. In addition, L. kefiri induced an increased frequency of CD4+FOXP3+ LPTC along with high levels of IL-10. This is the first report showing an immunomodulatory effect of L. kefiri CIDCA 8348 on human intestinal cells from IBD patients. Understanding the mechanisms of interaction between probiotics and immune mucosal cells may open new avenues for treatment and prevention of IBD., Instituto de Estudios Inmunológicos y Fisiopatológicos

La alergia a la leche de vaca es la causa más común de alergia alimentaria en lactantes y niños, probablemente porque la LV constituye uno de los primeros antígenos alimentarios a los que resultan expuestos los niños en la primera infancia. Si bien generalmente esta situación patológica resulta transitoria, ya que la mayoría de los niños afectados desarrollan tolerancia con el tiempo, los pacientes son sometidos a tratamientos con dietas libres de LV y sus derivados. En pacientes con edades menores a los dos años se emplean sustitutos lácteos, siendo los más empleados los hidrolizados extensivos de proteínas de leche. También suelen incluirse fórmulas alternativas como las formuladas a base de proteínas de soja o sus derivados. Sin embargo, se ha reportado que un cierto porcentaje de pacientes con alergia a leche de vaca muestran una intolerancia clínica a las mismas. Descartando la existencia de proteínas de leche como contaminantes o adulterantes, y considerando que los niños afectados probablemente no han estado en contacto con antígenos de soja, dado que no es un alimento común en la dieta de un lactante, la intolerancia clínica podría explicarse por la presencia de epitopes de reactividad cruzada entre proteínas de leche bovina y de soja. Sobre la base de estas observaciones, en el presente trabajo de tesis se planteó dilucidar las bases moleculares de este fenómeno y desarrollar herramientas y conocimientos que permitan en un futuro desarrollar terapias alternativas. Los objetivos generales planteados fueron los siguientes: o Caracterizar la inmunorreactividad in vitro e in vivo de proteínas de soja empleando distintas herramientas inmunológicas. o Identificar la presencia de epitopes inmunorreactivos presentes en proteínas de soja que compartan homologia secuencial o estructural con caseínas bovinas. o Validar un modelo murino de alergia alimentaria a proteínas de leche bovina para ser empleado como herramienta biológica para el estudio de la reactividad cruzada entre soja y proteínas de leche de vaca, para analizar la alergenicidad de nuevas fórmulas hipoalergénicas y para el desarrollo de nuevas vacunas mucosales a ser empleadas en el tratamiento de la alergia alimentaria., Facultad de Ciencias Exactas

The mucosal immune system constitutes a physical and dynamic barrier against foreign antigens and pathogens and exerts control mechanisms to maintain intestinal tolerance to the microbiota and food antigens. Chronic alterations of the intestinal homeostasis predispose to inflammatory diseases of the gastrointestinal tract, such as Inflammatory Bowel Diseases (IBD). There is growing evidence that the frequency and severity of these diseases are increasing worldwide, which may be probably due to changes in environmental factors. Several stromal and immune cells are involved in this delicate equilibrium that dictates homeostasis. In this review we aimed to summarize the role of epithelial cells and fibroblasts in the induction of mucosal inflammation in the context of IBD. It has been extensively described that environmental factors are key players in this process, and the microbiome of the gastrointestinal tract is currently being intensively investigated due to its profound impact the immune response. Recent findings have demonstrated the interplay between dietary and environmental components, the gut microbiome, and immune cells. “Western” dietary patterns, such as high caloric diets, and pollution can induce alterations in the gut microbiome that in turn affect the intestinal and systemic homeostasis. Here we summarize current knowledge on the influence of dietary components and air particulate matters on gut microbiome composition, and the impact on stromal and immune cells, with a particular focus on promoting local inflammation., Facultad de Ciencias Exactas, Instituto de Estudios Inmunológicos y Fisiopatológicos, Departamento de Ciencias Biológicas

Crohn's disease is an idiopathic disorder of the gut thought to be caused by a combination of environmental and genetic factors in susceptible individuals. It is characterized by chronic transmural inflammation of the terminal ileum and colon, with typical transmural lesions. Complications, including fibrosis, mean that between 40 and 70% of patients require surgery in the first 10 years after diagnosis. Presently, there is no evidence that the current therapies which dampen inflammation modulate or reverse intestinal fibrosis. In this review, we focus on cytokines that may lead to fibrosis and stenosis and the contribution of experimental models for understanding and treatment of gut fibrosis., Instituto de Estudios Inmunológicos y Fisiopatológicos

Allergies are complex inflammatory diseases with a not fully understood etiology. Several factors, including genetic, environmental, age of exposition, diet, etc., are associated with the induction of these diseases. The incidence of allergies has increased during the last decades and constitutes the most common immune-based disease worldwide. According to the hygiene hypothesis, a lower exposure to pathogens and commensal microbes that reside in the intestinal lumen is responsi‐ ble for the rapid rise of the prevalence of atopic and allergic disorders, specifically food allergy. To overcome this tendency, the immunological mechanisms underly‐ ing this pathology should be better understood, which will undoubtedly impact the development of novel therapies. A large body of evidence demonstrates that immunotherapies constitute corrective treatments of the impaired regulation of the immune system in allergic patients. The aim of this chapter is to present an overview about allergic diseases and food allergies, mechanisms involved, differences with toxic reactions and food intoleran‐ ces, a brief description of the main milk allergenic proteins, and the new therapeu‐ tic strategies derived from the old immunotherapies to restore oral tolerance, and finally, to describe different pathologies associated with milk allergy.

Reactions to soy have been reported in a proportion of patients with IgE-mediated cow’s milk allergy (CMA). In this work, we analyzed if Gly m Bd 28K/P28, one of the major soybean allergens, is a cross-reactive allergen with cow milk proteins (CMP). We showed that P28 was recognized by IgE sera from CMA patients and activated human peripheral basophils degranulation. Moreover, IgE sera of mice exclusively sensitized to CMP recognized P28. Splenocytes from sensitized animals secreted IL-5 and IL-13 when incubated with CMP or soy proteins, but only IL-13 when treated with P28. In addition, a skin test was strongly positive for CMP and weakly positive for P28. Remarkably, milk-sensitized mice showed hypersensitivity symptoms following sublingual challenge with P28 or CMP. With the use of bioinformatics’ tools seven putative cross-reactive epitopes were identified. In conclusion, using in vitro and in vivo tests we demonstrated that P28 is a novel cross-reactive allergen with CMP., Centro de Investigación y Desarrollo en Criotecnología de Alimentos, Instituto de Estudios Inmunológicos y Fisiopatológicos

Background: Cow’s milk allergy (CMA) is an important problem worldwide and the development of an in vivo system to study new immunotherapeutic strategies is of interest. Intolerance to soybean formula has been described in CMA patients, but it is not fully understood. In this work, we used a food allergy model in BALB/c mice to study the cross-reactivity between cow’s milk protein (CMP) and soy proteins (SP). Methods: Mice were orally sensitized with cholera toxin and CMP, and then challenged with CMP or SP to induce allergy. Elicited symptoms, plasma histamine, humoral and cellular immune response were analyzed. Th1- and Th2-associated cytokines and transcription factors were assessed at mucosal sites and in splenocytes. Cutaneous tests were also performed. Results: We found that the immediate symptoms elicited in CMP-sensitized mice orally challenged with SP were consistent with a plasma histamine increase. The serum levels of CMP-specific IgE and IgG1 antibodies were increased. These antibodies also recognized soy proteins. Splenocytes and mesenteric lymph node cells incubated with CMP or SP secreted IL-5 and IL-13. mRNA expression of Th2-associated genes (IL-5, IL-13, and GATA-3) was upregulated in mucosal samples. In addition, sensitized animals exhibited positive cutaneous tests after the injection of CMP or SP. Conclusions: We demonstrate that CMP-sensitized mice, without previous exposure to soy proteins, elicited hypersensitivity signs immediately after the oral administration of SP, suggesting that the immunochemical cross-reactivity might be clinically relevant. This model may provide an approach to further characterize cross-allergenicity phenomena and develop new immunotherapeutic treatments for allergic patients.

Purpose: Soy-based formulas are widely used as dairy substitutes to treat milk allergy patients. However, reactions to soy have been reported in a small proportion of patients with IgE-mediated milk allergies. The aim of this work was to explore whether P34, a mayor soybean allergen, is involved in this cross-reactivity. Methods: In vitro recognition of P34 was evaluated by immunoblotting, competitive ELISA and basophil activation tests (BAT) using sera from allergic patients. In vivo cross-reactivity was examined using an IgE-mediated milk allergy mouse model. Results: P34 was recognized by IgE antibodies from the sera of milk allergic patients, casein-specific monoclonal antibodies, and sera from milk-allergic mice. Spleen cells from sensitized mice incubated with milk, soy or P34 secreted IL-5 and IL-13, while IFN-γ remained unchanged. In addition, the cutaneous test was positive with cow's milk proteins (CMP) and P34 in the milk allergy mouse model. Moreover, milk-sensitized mice developed immediate symptoms following sublingual exposure to P34. Conclusions: Our results demonstrate that P34 shares epitopes with bovine casein, which is responsible for inducing hypersensitivity symptoms in milk allergic mice. This is the first report of the in vivo cross-allergenicity of P34., Centro de Investigación y Desarrollo en Criotecnología de Alimentos, Instituto de Estudios Inmunológicos y Fisiopatológicos, Centro de Investigación y Desarrollo en Fermentaciones Industriales

Exposure to cow’smilk constitutes one of the most common causes of food allergy. In addition, exposure to soy proteins has become relevant in a restricted proportion ofmilk allergic pediatric patients treated with soy formulae as a dairy substitute, because of the cross-allergenicity described between soy and milk proteins. We have previously identified several cross-reactive allergens between milk and soy that may explain this intolerance. The purpose of the present work was to identify epitopes in the purified αS1-casein and the recombinant soy allergen Gly m 5.0101 (Gly m 5) using an α-casein-specific monoclonal antibody (1D5 mAb) through two different approaches for epitope mapping, to understand cross-reactivity between milk and soy. The 1D5 mAb was immobilized onto magnetic beads, incubated with the peptide mixture previously obtained by enzymatic digestion of the allergens, and the captured peptides were identified by MALDI-TOFMS analysis. On a second approach, the peptidemixture was resolved by RP-HPLC and immunodominant peptides were identified by dot blot with the mAb. Finally, recognized peptides were sequenced by MALDI-TOF MS. This novel MS based approach led us to identify and characterize four peptides on α-casein and three peptides on Gly m 5 with a common core motif. Information obtained from these cross-reactive epitopes allows us to gain valuable insight into the molecular mechanisms of cross-reactivity, to further develop new and more effective vaccines for food allergy., Facultad de Ciencias Exactas, Instituto de Estudios Inmunológicos y Fisiopatológicos, Instituto Multidisciplinario de Biología Celular

Despite their abundance at gastrointestinal sites, little is known about the role of galectins in gut immune responses. We have therefore investigated the Citrobacter rodentium model of colonic infection and inflammation in Galectin-1 or Galectin- 3 null mice. Gal-3 null mice showed a slight delay in colonisation after inoculation with C. rodentium and a slight delay in resolution of infection, associated with delayed T cell, macrophage and dendritic cell infiltration into the gut mucosa. However, Gal-1 null mice also demonstrated reduced T cell and macrophage responses to infection. Despite the reduced T cell and macrophage response in Gal-1 null mice, there was no effect on C. rodentium infection kinetics and pathology. Overall, Gal-1 and Gal-3 play only a minor role in immunity to a gut bacterial pathogen., Centro de Investigación y Desarrollo en Criotecnología de Alimentos

IL-13 has been implicated in the pathogenesis of ulcerative colitis (UC), and may have a role in animal models of gut fibrosis. We studied the involvement of IL-13 in inflammation and fibrosis in UC and Crohn's disease (CD). Intestinal biopsies and anti-CD3/CD28- or anti-CD2/CD28-stimulated lamina propria mononuclear cells from UC and CD patients and control subjects were cultured, and IL-13, IL-4, IL-5, IL-17A and IFN-γ production was measured. Mucosal IL-13-producing cells were characterised by flow cytometry. Gut explants from strictured CD, non-strictured CD and healthy donors were cultured ex vivo, and secreted IL-13, IL-1β and collagen were measured. IL-13 production by mucosal explants and activated lamina propria mononuclear cells did not differ between CD, UC and control subjects, and was at least a log lower than IFN-γ and IL-17A. IL-13-producing cells, and in particular natural killer T cells, were uniformly low in all groups. IL-4 and IL-5 were undetectable in culture supernatants. Explants of CD strictures produced low amounts of IL-13, whereas IL-1β and collagen were elevated. We could not confirm that UC or strictured CD are associated with elevated IL-13 production. These data suggest that an anti-IL-13 Ab would not be an appropriate therapeutic strategy in inflammatory bowel disease.

Background: Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity. Methods: Cow's milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy. Results: Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice. Conclusions: Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide., Centro de Investigación y Desarrollo en Fermentaciones Industriales, Centro de Investigación y Desarrollo en Criotecnología de Alimentos, Laboratorio de Investigaciones del Sistema Inmune, Facultad de Ciencias Exactas

In all, 1,702 unselected pregnant women from the city of La Plata were tested for gestational diabetes mellitus (GDM) and evaluated to determine GDM prevalence and risk factors. In women with GDM, we evaluated compliance with guidelines for GDM management, and perinatal complications attributable to GDM. GDM prevalence was 5.8%, and its risk factors were pre-gestational obesity, previous hyperglycaemia, age > 30 years, previous GDM (and its surrogate markers). In primi-gravida (PG) subjects, GDM was equally prevalent in the presence (4.2%) or absence (4.0%) of risk factors. In multi-gravida (MG) women, although risk factors doubled the prevalence of GDM (8.6%), in the absence of risk factors GDM prevalence was similar to that of PG women (3.9%). Half of all women with GDM received inadequate post-diagnosis obstetric control, and this induced a fourfold increase in infant perinatal complications. In conclusion, all non-hyperglycaemic 24-28-week pregnant women should be tested for GDM, although particular attention must be paid to MG women with risk factors., Facultad de Ciencias Exactas

Summary Background Cows’ milk allergy (CMA) is the most common cause of food allergy in infancy. The only proven treatment is the complete elimination of cows’ milk proteins (CMPs) from the diet by means of hypoallergenic formulas. Soybean-based formulae are widely used although intolerance to soy has been reported to occur in 15–40% of infants with CMA. Objective The aim of this work was to analyse the in vitro reactivity of the soybean cultivar Raiden, which naturally lacks glycinin A4A5B3, to evaluate whether this genotype could be a safe CMP substitute for CMA patients. Methods The reactivity of conventional soybean (CS) and Raiden soybean (RS) genotypes and also recombinant glycinin A4A5B3 and ab-conglycinin with casein-specific monoclonal antibodies and CMP-specific polyclonal serum was evaluated by immunoblotting and ELISA. A sequential competitive ELISA with the polyclonal antiserum and different soluble inhibitors was performed. In addition, an indirect ELISA with sera of atopic children with CMA was carried out to analyse the IgE-binding capacity of the different soybean components. Results We have shown that CS contains four components that cross-react with CMP, while RS has only one. The remaining cross-reactive component in RS was identified as a-subunit b-conglycinin. By means of inhibitory ELISA, we demonstrated that CS, RS and the a-subunit b-conglycinin extracts inhibited the binding of CMP-specific antibodies to the CMP-coated solid phase. Finally, we showed that CS, RS and the recombinant proteins were recognized by human CMP-specific IgE antibodies. Conclusion This work shows that although Raiden has fewer cross-reactive components than conventional soybean, it still has a residual cross-reactive component: the a-subunit b-conglycinin. This reactivity might make this genotype unsuitable to treat CMA and also explains adverse reactions to soybean in CMA infants.

Introduction Interleukin (IL)-17A, which is up-regulated in inflammatory bowel disease (IBD) mucosal lesions, and IL-17F are normally present as IL-17AA and IL-17FF homodimers and may occasionally form IL-17A/F heterodimers. The role of each IL-17 dimer in IBD is currently unknown. Therefore, we studied the effects of IL-17AA, IL-17FF and IL-17-A/F in ulcerative colitis (UC) and Crohn’s disease (CD) mucosa. Methods Inflamed colonic biopsies from 17 IBD patients (6 UC and 11 CD) were cultured ex vivo for 24 h with IL-17AA, IL-17FF or IL-17A/F (1 ng/ml). Mucosal myofibroblasts isolated from the inflamed colon of 4 CD and 4 UC patients were cultured for 24 h with tumour necrosis factor (TNF)-α 20 ng/ml or with increasing concentrations (1–100 ng/ml) of IL-17AA, IL-17FF or IL-17A/F. IL-6 and IL-8 were measured in culture supernatants by ELISA. Results IL-17AA, but not IL-17FF, significantly reduced both IL-6 and IL-8 production by inflamed IBD biopsies cultured ex vivo , whereas IL-17A/F decreased IL-8 release by IBD mucosa. No difference was observed between CD and UC. Neither IL-17AA, nor IL-17FF, nor IL-17A/F exerted any effect on IL-6 and IL-8 production by IBD myofibroblasts. As expected, TNF-α stimulation significantly increased IL-6 and IL-8 production by both CD and UC myofibroblasts in vitro . No difference was observed between CD and UC myofibroblasts. Conclusion IL-17AA exerts an anti-inflammatory action on inflamed IBD biopsies cultured ex vivo . The action of IL-17AA is not mediated by myofibroblasts, therefore further studies are underway to ascertain which cell type is the main target of IL-17AA in IBD mucosa. Disclosure of Interest None Declared.

Introduction Interleukin (IL)-1b, IL-6, IL-8 and tumour necrosis factor (TNF)-a, are elevated in the inflamed mucosa of patients with ulcerative colitis and play a central role in driving the pro-inflammatory immune response in this condition. TNF-a is cleaved from the cell surface by TNF-a converting enzyme (TACE), which is inhibited by tissue inhibitor of Metalloproteinase (TIMP)-3. We studied whether the addition of TIMP-3 to inflamed colonic biopsies from ulcerative colitis patients reduced the release of the pro-inflammatory cytokines TNF-a, IL-1b, IL-6 and IL-8. Methods Colonic biopsies were obtained from macroscopically inflamed areas of 4 patients with ulcerative colitis undergoing colonoscopy for a disease flare. Biopsies were then cultured ex vivo for 24 h in 300 ul of serum free HL-1 medium in the absence or presence of recombinant human TIMP-3 (100 ng/ml). The concentration of TNF-a, IL-1β, IL-6 and IL-8 were measured in culture supernatants by ELISA. Results Culture with TIMP-3 significantly reduced TNF-a production by inflamed ulcerative colitis biopsies cultured ex vivo (from 1365 ug/ml in the absence of TIMP-3 to 45 ug/ml after TIMP-3 addition). Furthermore, the addition of TIMP-3 significantly reduced IL-1b production by inflamed ulcerative colitis biopsies (from 776 to 261 ug/ml). There was a trend in the reduction of IL-6 (from 3018 to 2702 ug/ml), which did not reach statistic significance, and no significant change in IL-8 production (from 30812 to 29114.5 ug/ml). Conclusion TIMP-3 administration not only causes a reduction in TNF-a via TACE inhibition but also IL-1β. This raises the possibility of its use therapeutically in the treatment of ulcerative colitis. Disclosure of Interest None Declared.

depends on the cell type, where it is produced and the time and location of release. IL-10 distribution in the intestine is not well known due to the methodological difficulty of cytokine staining in human biopsies. Aims: To evaluate the distribution and intensity of production of IL-10: (1) in healthy and inflamed intestine and (2) in Crohn’s disease (CD) in relation to the corticosteroid response. Methods: 26 patients with intestinal inflammation [16 Crohn’s disease (CD), 6 ulcerative colitis (UC) and 4 infectious colitis (IC)] and 10 healthy controls were included. Based on the steroid response, CD patients were classified as responders (steroid sensitive) and non-responders (steroid dependent and refractory). Samples in healthy controls were taken from ileum, left and right colon, whereas in patients samples were taken from inflamed areas. The protein expression of IL-10 was evaluated by immunofluorescence and the images obtained were processed with the ImageJ software [area (A) and mean fluorescence intensity (FI)]. We evaluated the distribution patterns and the cell type morphology. Nonparametric tests were used to compare IL-10 values between groups. Results: No significant differences in the IL-10 production were found between ileum, right and left colon in the healthy intestine (A: p = 0.589; IF: p = 0.513). No significant differences were neither found between controls and the different conditions with intestinal inflammation (A: p = 0.869; IF: p = 0.366). In CD there were no significant differences between steroid responders and non-responders (A: p = 0.913; IF: p = 0.913) In both healthy and inflamed intestine the IL-10 was distributed making a subepithelial band or making cell clusters predominantly of dendritic cells in the lamina propria. No differences in the IL-10 distribution pattern were found depending on the location in the healthy intestine (ileum or colon), nor depending on the steroid response in CD. Conclusions: The good IL-10 staining achieved by immunofluorescence reveals that this anti-inflammatory citokine is always present in along the intestine and regardless of health or disease status. The IL-10 disposition as a subepithelial band suggests an important role in defense against luminal antigens.

Introduction Mucosal inflammation in ulcerative colitis (UC) is characterised by an influx of neutrophils which secrete large amounts of human neutrophil elastase (HNE), causing matrix degradation. They also produce the elastase-specific inhibitor, elafin. The aim of this study is to evaluate the relative production of elastase and elafin in active UC, and to investigate the modulatory effect of elafin on mucosal proteolytic activity ex vivo . Methods We utilised intestinal biopsies from 18 patients with active UC and 12 non-UC healthy controls. Biopsies were homogenised and lysed to extract mucosal proteins. Proteolytic activity, using elastin as a substrate, was determined. Concentrations of elafin were measured using ELISA. The effect of protease inhibitors on proteolytic activity were determined in vitro using elafin, marimastat (matrix metalloproteinase inhibitor) and the synthetic elastase inhibitor, AAPV [N-(Methoxysuccinyl)-Ala-Ala- Pro-Val Chloromethyl Ketone]. The effect of elafin on proteolytic activity ex vivo was assessed by 24 hour organ culture in the presence and absence of elafin. Unpaired Student’s t-test was used for statistical analyses. Results Mucosal protein homogenates from patients with active UC displayed higher proteolytic activity in comparison to healthy controls ( p = 0.002). Elafin levels were increased in mucosal homogenates from active UC ( p = 0.007). The addition of elafin, marimastat or AAPV, in vitro , each diminished proteolytic activity. Organ culture of UC biopsies in the presence of elafin reduces the proteolytic activity of active UC ex vivo (n.s.). Conclusion Colonic mucosal tissue from UC patients displays significantly higher elastinolytic activity in comparison to healthy controls. The addition of elafin has a restorative effect on the elastinolytic activity of UC mucosal homogenates, with the most notable effect in those tissues that had highest proteolytic activity. This occurs in the presence of significantly higher quantities of elafin in active UC mucosa. These data also show a beneficial modulatory effect of elafin on human gut tissue, suggesting a possible role for supplementary elafin in the treatment of UC. Disclosure of Interest None Declared.

ACTIVATION OF TRIGGERING RECEPTOR EXPRESSED ON MYELOID CELLS 1 INCREASES THE PRODUCTION OF PRO-INFLAMMATORYCYTOKINES IN INFLAMMATORY BOWEL DISEASE MUCOSA P. Biancheri∗ ,1, A. Di Sabatino1 , F. Ammoscato2, R. Curciarello 2 , L. Kruidenier 3 , T.T. Macdonald2 , G.R. Corazza1 1First Department of Medicine, Fondazione IRCCS Policlinico S. Matteo, University of Pavia, Pavia, Italy; 2CIID, Blizard Institute, Barts and the London School of Medicine and Dentistry, London, United Kingdom; 3Immuno-Inflammation CEDD, GlaxoSmithKline, Stevenage, United Kingdom

The miR-29 family is involved in fibrosis in multiple organs, including the intestine where miR-29b facilitates TGF-β-mediated up-regulation of collagen in mucosal fibroblasts from Crohn’s disease (CD) patients. Myeloid cell leukemia-1 (MCL-1), a member of the B-cell CLL/Lymphoma 2 (BCL-2) apoptosis family, is involved in liver fibrosis and is targeted by miR-29b via its 3’-UTR in cultured cell lines. We investigate the role of MCL-1 and miR-29b in primary intestinal fibroblasts and tissue from stricturing CD patients. Transfection of CD intestinal fibroblasts with pre-miR-29b resulted in a significant increase in the mRNA expression of MCL-1 isoforms [MCL-1Long (L)/Extra Short (ES) and MCL-1Short (S)], although MCL-1S was expressed at significantly lower levels. Western blotting predominantly detected the anti-apoptotic MCL-1L isoform, and immunofluorescence showed that staining was localised in discrete nuclear foci. Transfection with pre-miR-29b or anti-miR-29b resulted in a significant increase or decrease, respectively, in MCL-1L foci. CD fibroblasts treated with IL-6 and IL-8, inflammatory cytokines upstream of MCL-1, increased the total mass of MCL-1L-positive foci. Furthermore, transfection of intestinal fibroblasts with pre-miR-29b resulted in an increase in mRNA and protein levels of IL-6 and IL-8. Finally, immunohistochemistry showed reduced MCL-1 protein expression in fibrotic CD samples compared to non-stricturing controls. Together, our findings suggest that induction of MCL-1 by IL-6/IL-8 may surmount any direct down-regulation by miR-29b via its 3’-UTR. We propose that an anti-fibrotic miR-29b/IL-6 IL-8/MCL-1L axis may influence intestinal fibrosis in CD. In the future, therapeutic modulation of this pathway might contribute to the management of fibrosis in CD. Fil: Nijhuis, Anke. Queen Mary University of London; Reino Unido Fil: Curciarello, Renata. Queen Mary University of London; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mehta, Shameer. Queen Mary University of London; Reino Unido Fil: Feakins, Roger. The Royal London Hospital; Reino Unido Fil: Bishop, Cleo L.. Queen Mary University of London; Reino Unido Fil: Lindsay, James O.. Queen Mary University of London; Reino Unido Fil: Silver, Andrew. Queen Mary University of London; Reino Unido