3′-sulfated LewisA/C: An oncofetal epitope associated with metaplastic and oncogenic plasticity of the gastrointestinal foregut

Metaplasia, dysplasia, and cancer arise from normal epitheli

Interaction of Helicobacter pylori with glycosylated salivary proteins

Since the discovery of Helicobacter pylori (H. pylori) in 1983 enormous progress has been made in determining the pathogenesis of this microbe in gastric disease. While the way of transmission is still under dispute, it is generally accepted that H. pylori must reach the stomach via the oral cavity. During this passage it comes into contact with salivary components. However, there are only few studies about interactions of H. pylori with salivary components and no study about the influence of saliva on H. pylori exists so far. The immediate aim of this thesis was to search for possible interactions of H. pylori with glycosylated salivary proteins, by developing an appropriate methodological approach and to characterize the molecular basis of such interactions. In order to utilize two-dimensional gel electrophoresis (2-DE) and subsequent transfer of separated proteins onto nitrocellulose membrane (blotting) for a high resolution bacterial overlay, a proteome analysis of human whole saliva (WS), parotid, and submandibular-sublingual (SMSL) secretions was performed in the first part of this thesis. Salivary secretions were subjected to 2-DE and spots were analyzed by matrix-assisted laser desorption/ionization-MS. Altogether 131 protein spots were identified in WS, 53 spots were identified in SMSL secretion and 43 spots in the parotid secretion. New components identified in WS were cyclophilin-B and prolyl-4-hydroxylase. Also acidic and basic proline-rich proteins (PRPs), the proline-rich glycoprotein and mucin MUC7 could be mapped for the first time in a 2-D gel of human saliva. An intra-individual comparison (comparison of three different collection days) and an inter-individual comparison (comparison of four different individuals) of spot pattern showed moreover a sufficient reproducibility of 2-D salivary proteom maps for subsequent adhesion studies. Second part of the thesis was the characterization of the H. pylori wild-type strain J99A and isogenetic mutants, lacking either the BabA- and/or SabA-adhesins. This was performed by using a bacterial overlay technique with fluorescence-labeled bacteria on immobilized (neo)glycoproteins. Interaction between the adhesin BabA and the H-1-, Lewis b- and related fucose-containing antigens could be confirmed. The previously described interaction of H. pylori with terminal alpha-2,3-linked sialic acids could be shown too. The use of a sabA mutant and sialidase treatment of glycoconjugate arrays showed for the first time that the adherence of H. pylori to laminin is mediated by the sialic acid-binding adhesin, SabA. In addition, it could be shown that the adhesion to salivary mucin MUC5B is mainly associated with the BabA adhesin, and to a lesser extent with the SabA adhesin. It turned out that the adhesion of H. pylori to fibronectin and lactoferrin persisted in the babA/sabA double mutant. This binding could be abolished by denaturation but not by deglycosylation. Therefore, it was suggested that this interaction may depend on the recognition of unknown receptor moieties by one or more additional unknown bacterial surface components. The third and final part of the thesis was the performance of adhesion studies on human saliva. The characterized H. pylori mutants and the J99 wild-type were applied to blots of 1-D and 2-D gels of human saliva by bacterial overlay. Three receptor molecules of H. pylori detected by 1-D overlay could successfully be identified by MALDI-MS, confirming the binding of H. pylori to MUC5B, MUC7 and gp-340. With the help of the mutants also the responsible adhesins for binding could be determined. The 2-D overlay revealed novel salivary receptors for H. pylori. Identification of these receptors was achieved by comparison of the overlay membrane with the established proteome maps of human saliva. Binding of H. pylori to the proline-rich glycoprotein was detected for the first time and assigned to the activity of the BabA adhesin. The SabA adhesin was found responsible for binding to other newly detected receptor molecules, including carbonic anhydrase VI, secretory component (poly-Ig-receptor), parotid secretory protein and zinc-alpha-2-glycoprotein. In conclusion, this thesis combined for the first time successfully a proteomics approach with the bacterial overlay technique. This method showed not only the binding of H. pylori to salivary proteins but facilitated the identification of respective receptor molecules considerably. Because this technique is also suitable for other bacteria a widely applicable tool for studying adhesion of bacteria was developed

Vaccine Development

Vaccination is the most effective and scientifically based means of protection against infectious diseases, especially in this era of the COVID-19 pandemic. This book examines several issues related to the development of vaccines against viral, bacterial, and parasitic infections

The Helicobacter pylori HrcA repressor: Study of the Global Transcriptional Response during Heat Shock.

The ability to respond quickly to environmental changes and thus modulate gene expression is a crucial skill exploited by bacteria for their survival. In this context, the widespread human pathogen Helicobacter pylori, under stress conditions induces the synthesis of a class of highly conserved proteins, called Heat Shock Proteins. In this bacterium, the major heat-shock genes are negatively regulated by two transcriptional repressors, HspR and HrcA. Although the heat-shock regulatory circuit is well-studied, not enough is known about the global heat shock response in H. pylori. In order to identify other potential cellular process regulated by HrcA, we performed differential transcriptional analysis of heat shock regulation by RNA-seq, comparing the transcriptome of the H. pylori G27 wild type strain subjected and not subjected to heat shock stress versus the HrcA isogenic G27 mutant strain. This analysis showed that most of the differentially expressed genes were deregulated only under heat shock treatment. Moreover, several non-heat shock responsive genes were deregulated in the HrcA mutant. To further characterize the HrcA regulon in H. pylori we attempted setting up a ChIP-seq experiment. However, the HrcA protein appears to be poorly immunogenic, for this reason we used a strategy in which an epitope (3XFLAG), recognized by commercial antibodies, was fused to the N-terminal region of the HrcA protein. In addition, three levels of expression (weak, intermediate and strong) of the 3XFLA-HrcA were obtained by using three known H. pylori promoters. The immunoprecipitation assays performed showed a poor enrichment of DNA fragments bound by HrcA and hindered the identification of novel in vivo HrcA binding sites. Thereafter, we further analyzed the HrcA-DNA interactions on some putative targets through DNaseI footprinting assays on novel putative target promoters binding of the regulator could not be detected and possibly transcriptional regulation by HrcA of these genes is indirect

Untersuchung der immunstimulatorischen Eigenschaften des Helicobacter-cysteine-rich protein A (HcpA)

Helicobacter pylori (Hp) ist ein gramnegatives Bakterium, das den menschlichen Magen kolonisiert und dabei obligatorisch eine chronische Gastritis induziert. Trotz dieser Immunreaktion persistiert das Bakterium ohne antibiotische Behandlung im Normalfall lebenslang. Diese in der Regel asymptomatische Gastritis kann in Abhängigkeit von Wirtsfaktoren und Ernährungsgewohnheiten verschiedene klinische Manifestationen erreichen. Die Infektion mit Hp kann chronische aktive oder atrophische Gastritiden, peptidische Magengeschwüre und MALTome auslösen. Eine der Grundlagen der Entzündungsreaktion ist das sich im Magen ausbildende Zytokinmilieu, für dessen Polarisierung Interferon-gamma (IFN-gamma) als Leitzytokin der von T-Helfer-Zellen des Typs I (TH1) geprägten Immunantwort von großer Bedeutung ist. Helicobacter-cysteine-rich-protein A (HcpA) ist ein von Hp aktiv sezerniertes Protein, das als neuer, immunstimulatorisch wirkender Virulenzfaktor beschrieben wurde. HcpA gehört einer nur in Campylobacterales vorkommenden Familie paraloger Proteine an. Die Stimulation von Splenozyten-Einzelzellsuspensionen aus naiven Mäusen mit in E. coli rekombinant exprimiertem HcpA konnte eine der Ko-Kultur mit lebendem Hp vergleichbare Zytokinfreisetzung, insbesondere des entzündungsfördernd wirkenden IFN-gamma, induzieren. Neben dieser direkten Aktivierung des angeborenen Immunsystems konnten durch serologische Untersuchungen von Hp infizierten Patienten hohe anti-HcpA Antikörpertiter nachgewiesen werden. Zusammenfassend wurde in dieser Arbeit die Expression und Sekretion von HcpA unter physiologischen Bedingungen erstmals untersucht und beschrieben. Die erfolgreiche Etablierung einer stabil transfizierten Drosophila-Schneider-2 Zelllinie (DS-2) erlaubte die Expression großer Mengen von LPS-freiem rekombinantem HcpA, das im wesentlichen die gleichen immunstimulatorischen Eigenschaften wie in E. coli rekombinant exprimiertes HcpA aufwies. Die Aufklärung der durch HcpA induzierten Kaskade, die zur Aktivierung des angeborenen Immunsystems im Mausmodell als auch in humanen PBMC führt, konnte die Bedeutung von HcpA als immunologischem Virulenzfaktor weiter untermauern. Die Identifizierung von B- und T-Zellepitopen ist eine wichtige Vorraussetzung für sich jetzt anschließende Untersuchungen zur Entwicklung einer HcpA-basierten, protektiven Vakzinierungsstrategie. Der Befund, dass HcpA über den TLR4 seine Aktivierung des angeborenen Immunsystems direkt oder indirekt vermittelt, stellt ein weiteres zukunftsweisendes Ergebnis dieser Arbeit dar. Der Nachweis einer starken Immunaktivierung durch HcpA in humanen PBMCs zeigt erstmalig, dass die bisher nur im Maus-System beschriebenen immunmodulierenden Eigenschaften von HcpA voll auf den Menschen übertragbar zu sein scheinen. Insgesamt konnte die Arbeit damit einen wesentlichen Beitrag zum Verständnis eines neuen immunologisch wirkenden Virulenzfaktors von Hp leisten

ProInflam: a webserver for the prediction of proinflammatory antigenicity of peptides and proteins

Additional file 2: Table S2. Dipeptide composition distribution between proinflammatory and non proinflammatory data

Therapeutic IgY: Safe, Diverse, And Effective For Use Against Viral Targets

Passive antibody treatments are used to target infectious disease, toxins, venoms, and cancer antigens. Recently, there has been an increased interest in the use of avian-derived antibody treatments such as IgY. IgY is the primary serum antibody isotype present in the avian system, and IgY treatments have already been demonstrated to be effective against a variety of bacterial and viral infectious agents. There are two forms of IgY expressed in anseriformes birds, a full length IgY that is functionally similar to mammalian IgG, and an alternatively spliced IgY, IgY(ÃFc), that is comparable to the mammalian F(ab\u27)2 fragment. The difference in structure between IgY and mammalian IgG, prevents IgY from interacting with mammalian Fc receptors, complement, and other inflammatory factors. The phylogenetic distance between mammalian and avian species allows IgY to have a higher avidity for certain mammalian epitopes and a unique antibody repertoire is developed compared to mammals, further enhancing the therapeutic potential of IgY. Our current research is focused on developing goose IgY anti-viral treatments and ensuring the safety of these treatments in humans. The viral antigens of focus in this research are dengue virus type 2 (DENV-2) and the Andes virus (ANDV). In an attempt to generate monoclonal goose IgY antibodies, using modified mammalian hybridoma techniques, geese were immunized with DENV-2 antigen and blood was collected as a source of immune B-cells and fused with mammalian myeloma cells. Short lasting, virus- specific IgY producing hyrbidomas were created. To generate ANDV specific goose IgY antibodies geese were vaccinated with a DNA vaccine PWRG/AND-M, containing the full-length M genome segment of ANDV, via a needle-free device at two week intervals up to eight weeks and then at 12 weeks. One year later the same geese were booster vaccinated with either pWRG/AND(opt) or pWRG/AND(opt2) six times over a ten week time period. Average neutralizing titers of sera collected from geese six weeks after the primary vaccination was 10,000. Titers remained at this level for the one year in between vaccinations and then increased to nearly 100,000 after booster vaccination. Epitope mapping confirmed the specificity of the goose- derived antibodies and identified unique highly reactive epitopes. IgY from the initial vaccination recognized 11 epitopes across the M segment, and an additional 9 epitopes after booster vaccination. In vivo survival studies in a lethal challenge model of ANDV infection established the post-exposure treatment potential of the ANDV specific IgY. To test the safety of the anti-viral IgY treatments for use in humans in vivo and in vitro safety experiments were completed. In a single injection study, mice were injected with a single dose of IgY/IgY(ÃFc) or PBS, and in a multiple injection study, rabbits were injected with multiple doses of IgY/IgY(ÃFc), IgY(ÃFc), human immunoglobulin, or PBS. Organs were collected after injection, hematoxylin and eosin stained, and scored by a blinded pathologist for abnormal pathology and/or inflammation. There were no inflammatory manifestations in the organs from animals in either the single or multiple injection study receiving IgY/IgY(ÃFc) or IgY(ÃFc). PBMCs and neutrophils were isolated from fresh human blood and co-cultured with IgY/IgY(ÃFc), mammalian IgG, and other controls. Culture supernatants were collected at various time points and analyzed for the presence of IL-1â, TNF-á, IL-10, neutrophil elastase, and nitric oxide using kit-based assays. All assays reported less reactivity of goose IgY/IgY(ÃFc) with human PBMCs and neutrophils compared to mammalian IgG and positive control mitogens. These results further support the lack of reactivity of avian IgY in the mammalian system and the benefits of safely using IgY as a treatment in the mammalian system

Gut Microbial Antigenic Mimicry in Autoimmunity

The gut microbiota plays a major role in the developmental biology and homeostasis of cells belonging to the adaptive and innate arms of the immune system. Alterations in its composition, which are known to be regulated by both genetic and environmental factors, can either promote or suppress the pathogenic processes underlying the development of various autoimmune diseases, including inflammatory bowel disease, multiple sclerosis, systemic lupus erythematosus, type 1 diabetes and rheumatoid arthritis, to just name a few. Cross-recognition of gut microbial antigens by autoreactive T cells as well as gut microbe-driven alterations in the activation and homeostasis of effector and regulatory T cells have been implicated in this process. Here, we summarize our current understanding of the positive and negative associations between alterations in the composition of the gut microbiota and the development of various autoimmune disorders, with a special emphasis on antigenic mimicry

DEVELOPMENT OF A WEBTOOL FOR INTERACTOME­SEQUENCING DATA ANALYSIS AND IDENTIFICATION OF H. PYLORI EPITOPES RESPONSIBLE FOR HOST IMMUNO­RESPONSE MODULATION.

To elucidate the molecular mechanisms involved in persistency/latency of the H. pylori infection or in its progression towards serious diseases, it is necessary to analyse the host pathogen interaction in vivo. The circulating antibody repertoire represents an important source of diagnostic information, serving as biomarker to provide a \u201cdisease signature\u201d. The aim of this work is the identification of H. pylori epitopes responsible for host immuno\uadresponse modulation through: a discovery\uaddriven approach that couples \u201cphage display\u201d and deep sequencing (interactome-sequencing) and the development of a specific webtool for interactome-sequencing data analysis. We used this approach to identify novel antigens by screening gDNA libraries created from the pathogen\u2019s genome, directly with sera from infected patients. Two genomic phage display libraries from 26695 and B128 H. pylori strains have been constructed by using f\uadlactamase ORF selection vector. Genomic DNA was sonicated, fragments cloned into the filtering vector, after transformation libraries of 1x106 clones were obtained and sequenced by Illumina technology. More than 93% of Hp CDSs were represented in the phage genomic libraries therefore being representative of the whole H. pylori antigenic ORFeome. A webtool for interactome-sequencing data analysis was developed and used to identify the H. pylori antigens/epitopes which could be considered specific for infection progression towards three different pathological outcomes. Putative antigens were selected from libraries using sera from patients affected by: i) gastric adenocarcinoma; ii) autoimmune gastritis; iii) MALT lymphoma. The results, obtained thanks to the new interactome sequencing pipeline developed, show that the diversity of the libraries after selection is significantly reduced. Furthermore, individual ranks, for each infection condition, have been compared highlighting the pattern of putative antigens, shared by all the conditions, and some that can distinguish the different stages of infection. One of this new antigens, that seems to be specific for infection progression towards more serious diseases, has been successfully validated through ELISA assay on a wide number of sera from patients. Other more specific antigens identified by our approach and by the application of the new data analysis pipeline here described are in validation

CHARACTERISATION OF ¿-GLUTAMYL TRANSPEPTIDASE AND ELUCIDATING ITS ROLES IN THE PATHOGENESIS OF HELICOBACTER PYLORI

Ph.DDOCTOR OF PHILOSOPH