FC Fellowships - Call for Proposals 2017

GRANTED PROJECTS 2017

TOTAL FUNDING € 425,000

 

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 2017 is now closed.

 

The FC Fellowships - Call for Proposals 2017 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.

 

Each Triennial FC Fellowship is granted for a total of € 85.000, of which € 75.000 for the triennial gross salary of the FC Fellow (€ 25.000 per year gross salary) and € 10.000 to be used during the Triennial FC Fellowship program exclusively for the purchase of Consumables and Supplies.

The total funding available for the ‘FC Fellowships - Call for Proposals 2017’ is € 425.000.

 

Applicants, who had a Scientific Master’s Degree or a Long Single Cycle Degree or an equivalent non Italian degree obtained after 31st December 2010, presented research plans fully focusing on celiac disease and/or dermatitis herpetiformis.

 

FC’s HIGH Funding Priorities

The following items were considered as high research priorities:

-   Proximity to find new pathogenic mechanisms
-   Proximity to cure
-   Natural history of the pathology by linking different phases of the disease to specific biological/genetic profiles
-   Interactions between environmental risk factors, genetic profiles and intermediate biomarkers. This also includes defining harmful components of gluten
-   Innovation of clinical methodologies
-   Evaluation in clinical practice of the efficacy of diagnostic and therapeutic approaches, in terms of outcome and quality of life. This also includes improvement of the quality and safety of the gluten-free diet, and innovation of food products
-   New therapeutic drugs, procedures or strategies (pilot clinical studies)
-   Critical evaluation of last generation drugs (their activity by mechanistic insights).

 

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.

 

 

FUNDED PROJECTS – GRANT FC 2017

 

Triennial Fellowship Project 007_FC_2017

Topic: Celiac Disease – Area: Genetics

LONG NON CODING RNA EFFECTS ON THE DEVELOPMENT OF CELIAC DISEASE 

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Donatella Cielo, Università Federico II Naples

Background: More than 90% of 112 CeD-associated SNPs are located outside protein-coding genes, suggesting they may have a regulatory role. A fine-mapping study in CeD identified 7 SNPs that affect long lncRNA.

Hypothesis: The analysis of eQTL CeD-associated Polymorphisms showed that they often affect the expression of nearby genes in different cells types, but up to now they have been explored in small intestinal biopsies only,which are composed of multiple cell types,leading to results difficult to interpret. Inasmuch the majority of published eQTL studies largely ignored the effects of SNPs on regulatory lncRNA.

Aims:

  1. To evaluate the effect of CeD-associated SNPs,putatively affecting lncRNAs function, in keys CeD cells types,observing directly their localization in FFPE sections of CeD small-intestinal biopsies,and then quantifying their expression by RealTime-PCR in epithelial and non-epithelial sorted intestinal cells.
  2. To explore the pathways regulated by the lncRNAs:indeed we plan to analyze the lncRNAs and their target gene expression, the relevant protein synthesis, and their effects on the downstream pathways.
  3. On the basis of their localization/function,we will then chose the specific read-out, such as epithelial organoid cultures exposed to gliadins peptides and/or cultures of stimulated immune cells.
  4. The modulation the expression of the lncRNAs by genetic engineering and,consequently, modulate their target pathways in specific CeD cell types, could provide the first evidence of the action of an epigenetic mechanism in the pathogenesis of CeD.

Experimental Design: We plan to conduct our research in 3 years according to the following steps:

  1. Selection of FFPE small-intestine biopsies from 20 CeD patients, 20 Controls(CTRs),and 20 positive inflammed controls(IBDs).
  2. Mapping lncRNAs expression in specific intestinal cells in situ by immunohistochemistry and RNA in-situ hybridization. We plan to use RNAscope®-assay that allows simultaneous single-molecule visualization while preserving tissue morphology.
  3. Bioinformatic prediction to identify the target genes and molecular pathways regulated by the lncRNA.
  4. Separation, by immuno-sorting, of the epithelial component with CD326(EpCAM) MicroBeads from non-epithelial component of small intestine biopsy of 20 CeD, 20 CTRs,and 20 IBDs, and analysis of the lncRNA and its target genes by Real-Time PCR,and by Western Blot.
  5. Development of epithelial organoid from 5 CeDpatients on a gluten free diet with normal histology, to explore the effect of gluten peptides on the function of the lncRNA through their target pathway.
  6. Confirmation of the causal effect of polymorphisms of lncRNAs in specific cell lines, modified by CRISPR/Cas9 technology, that is able to manipulate effectively the non-coding regions of the genomic DNA,to create or correct the specific mutations. Selection of appropriate read-out, among epithelial or immune cells, in relation to the role played by lncRNA and its target genes.

Expected results and Impact on the disease: Inherited epigenetic changes have been proposed as an explanation for the‘‘missing heritability’’ due to inherited causes of CeD. The investigation of epigenetic changes, such as the causal effect of CeD-associated SNPs on lncRNA,is conceptually novel in the field of CeD. The confirmed “epidemics”of CeD through the gluten consuming world is not explained by classical genetics neither by environmental factors:new, unknown, inheritable, elements are appearing in this unexpected scenario.

 

 

Triennial Fellowship Project 009_FC_2017

Topic: Celiac Disease – Area: Clinics

THE ROLE OF CB2 RECEPTOR IN THE PATHOGENESIS OF CELIAC DISEASE AND ASSOCIATED BONE LOSS 

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Chiara Tortora, Università della Campania “Luigi Vanvitelli”, Naples

Background: Celiac disease (CD) is an autoimmune small intestinal disorder with a systemic and chronic inflammatory immune response against gluten in genetically predisposed individuals. The gluten-free diet is the only available treatment. CD frequently predisposes to metabolic osteopathy.

The Cannabinoid Receptor type 2 (CB2), expressed by the immune cells and the bone, is involved in immune regulation and in bone cell differentiation, survival and activity. In a previous study we found a significant association of a common CB2 variant, Q63R, with CD and observed an increased number of CB2 receptor in CD small bowel biopsies. Moreover we demonstrated that stimulation of CB2 receptors is beneficial for reducing osteoclasts number and activity in osteoporosis associated with different disease.

Hypothesis: CB2 could be involved in the pathogenesis of CD and in associated bone loss.

Aims:

-   To characterize the CB2 receptor in CD
-  To evaluate the effect of a CB2 agonist, JWH-133, on the modulation of the inflammatory response in CD.
-   To characterize CB2 receptor in CD-associated bone loss.
-   To evaluate the effect of JWH-133 on osteoclast activity in CD.

 

Experimental Design:

-   CB2 expression will be evaluated in lymphocytes and macrophages isolated from bowel biopsies obtained from CD patients through Real Time PCR and Western Blot.

-   The effect of JWH-133 will be investigated:

  • on in vitro model of “immunocomponent gut” thorough co-culture of Caco-2 cells/lymphocyte or macrophages isolated from bowel biopsies obtained from CD patients. Will be performed ELISA assay, evaluating cytokine release by lymphocytes, macrophages and CaCo2 cells; Real Time PCR and Western Blot evaluating cytokine and molecules of inflammatory signalling pathways expression.
  • on CaCo2 cells Will be performed Cell Signaling Assay, evaluating PI3K/MAPK pathways; Real Time PCR and Western Blot evaluating ERK1/2, AKT/pERK1/2, pAKT
  • on ex vivo organ culture of small bowel biopsies from untreated patients with active CD. Will be performed Immunohistochemistry, evaluating lymphocytes and macrophages infiltration; RT-PCR and WB, evaluating IL17, IL15 expression.
  • On in vitro OCs obtained from PBMCs of CD patients. Will be performed TRAP assay evaluating TRAP activity; Bone resorption assay, Real Time PCR and Western Blot, evaluating Cathepsin K and TRAP expression.

Expected results and Impact on the disease: The project expects to demonstrate:

-   anti-inflammatory effect of JWH-133 on in vitro co-culture of Caco-2 cells/lymphocyte or macrophages isolated from bowel biopsies obtained from CD patients.
-   anti-proliferative effect of JWH-133 in Caco-2 cells.
-   anti-inflammatory effect of JWH-133 on ex vivo organ culture of small bowel biopsies from untreated patients with active CD.
-   anti-osteoporotic effect of JWH-133 on in vitro OCs isolated from PBMC of celiac patients.

 

This project could provide a target to modulate the immune components in CD providing an alternative to gluten-free diet not accepted by celiac patients.

In addition the same target could be used to reduce bone mass loss in CD patients.

 

 

Triennial Fellowship Project 014_FC_2017

Topic: Celiac Disease – Area: Immunology

IMMUNOMODULATORY FUNCTION OF γδ+ INTRAEPITHELIAL LYMPHOCYTES IN CELIAC DISEASE: IN VIVO AND IN VITRO RESPONSES TO GLIADIN STIMULI

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Vera Rotondi Aufiero, ISA-CNR  Istituto di Scienze Alimentari, Avellino

Background: An increase of γδ+ intraepithelial lymphocytes (IELs) has been observed in all stages of disease including those with latent CD and active CD (ACD) or those on GFD and in some first-degree relatives of CD patients. Although the reason for this increase has not been studied in depth, it has been suggested that TCRγδ+ IELs in vitro may have a regulatory potential by secreting TGF-β. The Cerf-Bensussan group showed that IL-15, overexpressed in active CD, was involved in the local downregulation of TGF-β signaling. In addition, our group showed that IL-15 impairs the functions of Treg cells in CD.

Hypothesis: Despite the increased frequency in vivo and suppressive activity in vitro, γδ+ IELs do not control the development of inflammation in the small intestinal mucosa with active CD, suggesting a defect in the activation of regulatory mechanisms.

Aims: The aim of the present proposal is to investigate the immunomodulatory function of γδ IEL:

-   In vivo, on the small intestinal mucosa of subjects in different CD histological stages (Marsh 0 to Marsh III);
-   In vitro on duodenal biopsy specimens from treated CD patients cultured for 24 h with or without IL-15.

In particular, the proposal will aim to determine if the regulatory capacity of γδ+ IELs could be impaired by IL-15, which upregulation in CD correlates with the degree of mucosal damage.

Experimental Design: Immunomodulatory function of γδ+ IELs will be evaluated in small intestinal biopsies of CD subjects that were adequately adhering to a strict gluten-free diet (histological stages Marsh 0-I), in small intestinal biopsies of CD subjects that were on diet containing gluten (histological stages Marsh II-III) and in small intestinal biopsies obtained from treated CD patients cultured in vitro for 24 h with gliadin, in presence or absence of IL-15. Anti-inflammatory IL-10 and TGF-β cytokines production, will be assessed on highly pure population of γδ+ IELs, isolated by immuno-laser capture microdissection. Moreover, also the eventual production of pro-inflammatory (INF-?, TNF-α, IL-17) cytokines will be analyzed.

Expected results and Impact on the disease: The availability of the different cohort of subjects in different CD histological stages (Marsh 0 to Marsh III) will let us to dissect the γδ+ IELs immune response. In particular, if IL-15 interferes with the regulatory activity of γδ+ IELs, we might expect that in inflammatory conditions ( Marsh II-III), where IL-15 is overexpressed, we can find a downregulation of anti-inflammatory cytokines production. This can also be proven in vitro by using the organ culture model, in presence of IL-15. Our study will provides insights into the immunomodulatory function of human small intestinal γδ+ IELs, which might help in the identification of potential therapeutic strategies for patients with CD.

 

 

Triennial Fellowship Project 015_FC_2017

Topic: Celiac Disease – Area: Genetics

microRNA PROFILING IN STOOL AND PLASMA OF SUBJECTS AFFECTED BY CELIAC DISEASE BY NEXT-GENERATION-SEQUENCING

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Antonio Francavilla, Italian Institute for Genomic Medicine, Turin

Background: Celiac disease (CD) is a complex immune-related disease with a genetic component occurring in about 1% of the population worldwide. However, many cases remain undiagnosed. Additional markers could be useful in CD detection/management. In this respect, molecular markers based on newly discovered non-coding RNA (ncRNA) species detectable in surrogate tissue may represent an interesting field of research. microRNAs (miRNAs) are the most studied class of ncRNAs, detectable in many different biospecimens and their altered expression has been observed in several diseases.

Only few studies analysed miRNA expression levels in duodenal tissue and blood of CD patients.

Hypothesis: CD implies a strong interplay between individual genetic background and the environment but, in recent years, the role of epigenetics has also emerged. An example is the involvement of ncRNAs in the immune response. I hypothesize that miRNA expression levels in surrogate biospecimens (stool and plasma) may reflect this altered status.

Aims:

The proposal aims at investigating:

  1. The effect of dietary changes on stool and plasma miRNA profiles of newly diagnosed CD patients over time, in comparison with those of subjects already on gluten-free-diet (GFD)and healthy subjects with no dietary restrictions.
  2. miRNA profiles of CD patients in comparison with those with gastrointestinal diseases (such as ulcerative colitis, inflammatory bowel disease, colorectal cancer and polyps) to highlight similar features in miRNA expression levels.
  3. Stool miRNA origin in CD patients, by mapping reads from deep sequencing on other miRNomes related to diet. Other human small ncRNAs of similar size of miRNAs will also be explored from deep-sequencing data from aim1.

Experimental Design: Blood and stool samples will be collected from newly diagnosed CD patients, those already on GFD and healthy subjects with no dietary restrictions. For at least 30 newly diagnosed CD subjects additional samples will be collected after 1 year of GFD. Small RNA-sequencing will be performed from total RNA isolated from stool and plasma exosome samples. Computational analyses will identify miRNAs differentially expressed among different categories or repeated samplings of investigated subjects. Expression levels of other human small ncRNAs of similar size of miRNAs (piRNAs, snoRNAs, tRNAs) will be also explored and compared among the different categories. Unmapped reads on the Human miRNome will be re-mapped on other miRNomes related to diet in CD patients.

Expected results and Impact on the disease: miRNA profiles in plasma and stool may represent non-invasive biomarkers to distinguish different forms of CD and patient status over time. Their concomitant analysis will allow to exhaustively investigate if they mirror target tissue profiles and, mostly, if they can clarify specific molecular pathways that characterize the disease development and changes after a specific diet has started.

The combination of up-to-date technologies should represent the best way to assess miRNA profiling in biomarker identification studies. Integration of molecular biology and methods of systems biology should provide an additional fundamental insight on the significance of miRNA alterations. Dietary and lifestyle factors will be comprehensively analysed for their potential influence on miRNA profiles.

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