FC Fellowships - Call for Proposals 2016

SELECTED PROJECTS 2016

TOTAL FUNDING € 375,000

The Foundation for Celiac Disease FC and the Italian Society for Celiac Disease AIC are glad to inform you that the FC Fellowships - Call for Proposals 2016 is now closed.

AIC and FC are grateful to the Peer Reviewers for their high-expertise evaluation of the applicant projects, and their commitment on this task which is fundamental to sustain the best Italian Research on celiac disease and other gluten-related pathologies.

The FC Fellowships - Call for Proposals 2016 aimed at awarding up to 5 triennial fellowships to highly qualified young graduates and/or post-doctoral fellows who wish to broaden or acquire experience in scientific research within the field of celiac disease and dermatitis herpetiformis in public or private non-profit Italian research institutes/organizations.

The Peer Review Evaluation

Applications underwent a peer review exercise that ensures a fair, independent and expert evaluation of their scientific quality.

For the evaluation of Applications, FC relies on the expertise of a panel of well-established international investigators working in institutes outside Italy. Applications are independently reviewed by three (3) reviewers with expertise in the specific area of the research plan.

Reviewer assignments are made in compliance with disclosure of conflict of interest: reviewers disclose any conflict of interest toward the applicant and the project.


SELECTED PROJECTS
 

Dermatitis Hepetiformis - Immunology

Phenotipic and antigen characterization of the specific T cell response in celiac patients affected by dermatitis herpetiformis 

3-years Fellowship Project

Background: Dermatitis herpetiformis (DH) is the specific cutaneous manifestation of celiac disease (CD). However, the immune response in patients with DH is still unclear and it is not known why only a small subgroup of celiac patients will develop DH.

Hypothesis: The main hypothesis is that some differences are present in the immune response involved in patients with DH and CD (i.e. the skin and the gut), in terms of antigen-specificity, T cell polarization and immune regulation, homing chemokine receptors. This may lead to a different phenotype as well as duration of the organ damage, and may explain why only some patients with CD will develop DH and why, after the introduction of a gluten-free diet, the skin damage persists more than the intestinal one.

Aims:

1. To identify the antigen specificity of T helper cells of skin infiltrating T cells obtained from lesional DH specimens.

2. To investigate, the different T cells subtypes from skin, peripheral blood and gut of DH and CD patients by flow cytometry and by real time quantitative RT PCR at single cell level.

3. To investigate the variation of the immune response after gluten-free diet.

Experimental Design: The following investigations will be performed in skin, gut and blood of DH patients:

- Cytofluorimetric analysis: CD4 (including the T helper subtypes) and CD8 T cells, TCR

gamma/delta cells, B cells, monocytes, NK and NKT cells will be investigated

- Single cell mRNA expression analysis on fresh cells: for markers of Th1, Th2, Th9, Th17, and

Tregs, chemokine receptors.

- Confocal microscopy to investigate the most relevant T cell subtypes in the tissue

- Antigen-specific stimulation of T cell lines and T cell clones

Expected results and Impact on Dermatitis Herpetiformis: The main expected result is to find differences among the immune response between patients with DH and CD and between the skin and the gut. In particular, we expect to find TG3-specific T cells at the skin level but not at the intestinal level. Moreover, we expect to find differences in the T cell cytokines produced and in the chemokine receptors expressed in the gut compared to the skin. Finally, a possible expected result is that the gluten-free diet would affect in a different way the immune response in the gut and the skin.

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Maria Caterina Rossi
University of Florence, Italy

 

Celiac disease - Biology

Characterization of Circulating Microvesicles in the peripheral blood of Celiac’s Disease Patients as a potential prognostic tool of related complications

3-years Fellowship Project

Background: Celiac disease (CD) is an immune-mediated enteropathy caused by a permanent sensitivity to gluten. Microvesicles (MV), released as cellular response to different stimuli, have been detected in peripheral blood, where platelet-, leukocyte-, red blood cells-, endothelial- and epithelial-derived MV have been identified. MV, playing key roles in a variety of pathological conditions, represent a potential useful biomarker of disease progression and response to treatment. MV are also involved in cell-to-cell signaling, therefore emerging as key controller in a number of pathophysiological processes. Then, characterization of circulating MV may represent a potential prognostic tool of related complications in CD.

Hypothesis: Our preliminary data demonstrated, in CD patients, an increase of both epithelial- and leukocyte-derived MV, likely associated to the autoantibodies-induced impairment of the intestinal architecture and to the CD-associated inflammation, respectively. Therefore, we hypothesized that this may be also associated to an impairment of MV content (i.e. proteins, mRNA and miRNAs), possibly involved in the pathogenesis of CD-associated complications. Thus, the characterization of the MV content may provide an innovative CD diagnostic/prognostic tool, and it may also help to understand a possible role of MV in complications developed by CD patients.

Aims:

Aim 1. Characterize circulating MV in the peripheral blood of CD patients in order to determine whether such analysis may help to find specific diagnostic and/or prognostic tools, or biomarkers for the response to diet monitoring.

Aim 2. Characterize proteins, mRNAs and miRNAs contained in MVs, in order to identify patterns involved in organ-to-organ information exchange, or in further development of CD complications.

Experimental Design: In order to develop our proposal we will utilize the following experimental plan:

1. N = 40 healthy subjects, N = 40 CD and N = 40 Chron’s disease (ChD) patients will be enrolled and their peripheral blood samples will be collected, in order to determine, by flow cytometry, possible differences in total circulating MV and/or in platelet-, leukocyte-, endothelial- and epithelial-derived MV distributions among enrolled subjects.

2. MV proteins, mRNA and miRNA contents will be compared among CD, healthy subjects and ChD patients.

Clinical and blood chemical tests evaluations will be applied to all studied subjects. In addition, we will use polychromatic flow cytometry, cell sorting, molecular biology approaches within a three-year period.

Expected results and Impact on Dermatitis Herpetiformis: We here propose an experimental platform for MV characterization in the peripheral blood samples of CD patients; data might be reveal clinical information related to MV biogenesis along with their possible pathologic roles in the development of CD complications.

In detail, we will expected to demonstrate differences among patients affected by CD and relative controls (both healthy subjects and ChD patients) in terms of:

1. Total circulating MV and/or in platelet-, leukocyte-, endothelial- and epithelial-derived MV distribution.

2. MV proteins, mRNA and miRNA content, which might be unique for CD, therefore opening new perspectives for the comprehension of mechanism leading to CD complications.

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Giuseppina Bologna
University “G.d’Annunzio” Chieti-Pescara, Italy

 

Celiac Disease - Biology

Development of villous atrophy in potential celiac disease: search for new biomarkers

3-years Fellowship Project

Background: Potential CD is characterised by positive CD associated serum antibodies and normal duodenal architecture. During 9 years of follow-up on a gluten contacting diet, in a cohort of 200 potential CD subjects, only 37% of asymptomatic patients developed the villous atrophy. The prospectively collected biological samples (serum, blood monocytes, intestinal biopsies, DNA, RNA) from the potential CD patients that have developed the disease is unique and offers the opportunity to study the constitutive alterations, the mechanisms of the damage and the predictive factors of the disease.

Hypothesis: Try to identify constitutive alterations that precede the intestinal damage (villous atrophy)and biomarkers that will allow to predict the disease evolution in asymptomatic potential celiac disease patients on a gluten containing diet.

Aims: Aim of this fellowship is to study the biopsies from potential CD patients that have subsequently developed the disease. In particular, interest is on intestinal epithelium modifications before the mucosal damage is present.

Experimental Design: We will use biopsies for immunochemistry analysis to study the activation of the stress/innate immune pathways in the epithelium. In those transferred to a GFD (cases) the gluten-dependence of these alterations will be defined. Sequence of the total RNA(RNA sec) and epigenetic study of the DNA will give indications of the genetic alteration that can be present in the intestinal epithelium and in the lamina propria before the onset of the disease. The genes differentially expressed between the two groups will be validated on the whole biopsies. The effect of gliadin (PTG) and gliadin peptides (such as P31-49 or 33 mer) will be evaluated on intestinal organoids. The gliadin peptides effects will be evaluated by “omic” as well, both total RNA sequence and epigenomic analysis. Biological materials already available in the host lab: 20 Biopsies from potential celiac, who have still a normal mucosa(potential cases) and 20 biopsies from patients who developed villous atrophy (cases) (at least three biopsies per case). DNA/RNA from epithelium and lamina propria: 15 from potential cases and 5 from cases Organoids: 6 from potential cases and 2 from cases.

Expected results and Impact on Dermatitis Herpetiformis: The analysis of the epithelium both by immune-staining and by “omics” will allow the understanding of the pathways that are altered before the onset of the disease. The discovery of such pathways will be used to predict the onset of the disease and possibly to prevent the evolution to the villous atrophy.

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Monia Porpora
Università Federico II Napoli

 

Celiac Disease - Immunology

Functional characterization of the gut microbiota and associated immune response in patients with “Potential Celiac Disease” (PDC)

3-years Fellowship Project

Background: Celiac disease (CD) is an autoimmune disease of the small intestine that occurs in genetically predisposed individuals. CD is caused by a reaction to gliadin, a prolamin (gluten protein) found in wheat. The exposure to gliadin causes an inflammatory reaction, which leads to an atrophy of the intestinal villi that line the small intestine. Currently, the only known effective treatment is a lifelong gluten-free diet.

While, Potential Celiac Disease (PCD) is defined by the presence of specific antibodies for CD and HLA-compatible, but without histological duodenal changes. The patient may or not have symptoms and signs, and may or not develop a gluten enteropathy later.

Hypothesis: In addition to genetic factors, various studies indicate a potential involvement of the gut Microbiota in CD genesis, showing the CD patients an altered faecal microbiota composition. But, it is unclear if a different gut bacterial flora may be the cause or a CD consequence. Currently the composition and/or the function of microbiota and associated immune response in the early stages of the disease is uncharted. The working hypothesis is that the mutual interplay of gut microbiota and immune response might play a key role in the CD development and in the clinical “status of PCD.

Aims:

AIM 1: Metagenomic characterization of intestinal microbiota from fecal specimens and biopsies of CD / PCD patients.

AIM 2: Functional characterization of specific immune response in CD / PCD patients.

Aim 4: Correlation of quality of immune response and the composition of the gut microbiota with the clinical parameters of CD/PCD patients.

Experimental Design: We will use a multidisciplinary approach to achieve the proposed aims

Aim1: We will use the “Next Generation Sequencing” tecnology for the microbiota

evaluation

Aim 2: The cell markers will be evaluated by flow cytometry, the cytokine profile by

commercial kit Elisa and the functional activities by in vitro cellular assay.

Aim4: We will use multivariate statistical techniques, Pearson and Spearman correlation

matrices.

Expected results and Impact on Dermatitis Herpetiformis: We expected to give a more accurate identification of bacterial groups having discriminatory properties in the CD spectrum balance and contemporary to asses a correlated immunological profile, which may already be indicative and "predictive" of the future development of the PCD in CD. Moreover our findings could open new therapeutic strategies aimed at restoring the intestinal ecosystem in these patients.

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Federica Ricci
University of Florence, Italy

 

Celiac Disease - Immunology

Dissection of T cell mediated immunity to gluten in different phases of celiac disease

3-years Fellowship Project

Background: Celiac disease (CD) is associated with a broad spectrum of intestinal mucosal lesions. Full-blown CD is characterized by intestinal villous atrophy, HLA risk genes and serological markers, whilst potential CD is defined by the presence of risk genes, CD-serological markers but a histological normal mucosa. CD4+ T-cells reactive to gluten have a main role in CD pathogenesis. Inflammatory cytokines, as interferon(IFN)-γ and IL-21, are markedly produced by lamina propria gluten-reactive T-cells. However, very little is known about T-cell mediated response in potential CD. The molecular and cellular gluten-induced mechanisms leading to the switch from normal to damaged mucosa in CD patients are not completely understood.

Hypothesis: Gluten-reactive inflammatory T-cells could be present in potential CD intestinal mucosa and regulatory T-cells may prevent their activation and expansion. The perturbation of the fine balance between inflammatory and regulatory T-cell pathways may be one of the factors responsible of the switching from normal mucosa to villous atrophy in CD.

Aims: The aim of the present proposal is to investigate the gluten-reactive T cells, of either T-effector and T-regulatory lineages, in the small intestinal mucosa of subjects with: i) high genetic risk for CD (HLA DQ2.5/DQ8 and at least one first degree relative with CD), ii) potential CD, iii) full-blown CD, iiii) healthy mucosa. To this purpose, intestinal mucosa infiltrating T-cells will be dissected for: i) cell phenotype, ii) gluten-specificity, iii) functional characterization, iiii) T-cell receptor (TcR) repertoire, in the different CD stages.

Experimental Design: T-cell mediated immunity to gluten will be evaluated in small intestinal biopsies of CD subjects at different disease phases. Cellular phenotype, cytokine production pattern, and repertoire of immunogenic gluten peptides will be assessed either at the level of short-term T-cell lines in vitro raised against gluten, and of T-cell clones. The expression of CXCR3, CRTH2, and CCR6 will be analyzed by flow-cytometry to identify Th1, Th2 and Th17 lymphocytes, respectively. CD161 marker will allow to identify the classical (CD161-), and non-classical (CD161+) Th lineages. Regulatory (Tr1) cells will be also analysed using specific markers, CD49b and LAG-3. Both intracellular level and secreted amount of IL-21, IL-17, IL-4, IL-10, INF-γ will be measured. Known dominant gluten epitopes from α-, γ- and ω-gliadins will be assayed. TcR family of pathogenic gluten-reactive T-cells will be identified and correlated with the different disease stages. The gluten epitopes repertoire and the intensity of T-cell response will be correlated with the HLA DQA1*05/DQB1*02 gene expression of each enrolled subject.

Expected results and Impact on Dermatitis Herpetiformis: The availability of the different cohort of subjects (high risk, potential and acute CD patients) will let us to dissect the adaptive T-cell mediated immune response to gluten in the broad CD spectrum. To look at the anti-gluten T-cell immunity in the intestinal mucosa of subjects at different stages of CD will provide insights in the sequence of immune event cascade responsible of the changes from the early to full-blown CD lesion. The present proposal could provide tools to predict the development of acute CD in at risk individuals and support the diagnosis in doubt CD cases.

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Serena Vitale
National Research Council, CNR, Naples, Italy

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