Proteomics signature of autoimmune atrophic gastritis: towards a link with gastric cancer.

BACKGROUND: Autoimmune atrophic gastritis (AAG) is a chronic disease that can progress to gastric cancer (GC). To better understand AAG pathology, this proteomics study investigated gastric proteins whose expression levels are altered in this disease and also in GC. METHODS: Using two-dimensional difference gel electrophoresis (2D-DIGE), we compared protein maps of gastric corpus biopsies from AAG patients and controls. Differentially abundant spots (|fold change|≥ 1.5, P < 0.01) were selected and identified by LC-MS/MS. The spots were further assessed in gastric antrum biopsies from AAG patients (without and with Helicobacter pylori infection) and from GC patients and unaffected first-degree relatives of GC patients. RESULTS: 2D-DIGE identified 67 differentially abundant spots, with 28 more and 39 less abundant in AAG-corpus than controls. LC-MS/MS identified these as 53 distinct proteins. The most significant (adjusted P < 0.01) biological process associated with the less abundant proteins was "tricarboxylic acid cycle". Of the 67 spots, 57 were similarly differentially abundant in AAG-antrum biopsies irrespective of H. pylori infection status. The differential abundance was also observed in GC biopsies for 14 of 28 more abundant and 35 of 39 less abundant spots, and in normal gastric biopsies of relatives of GC patients for 6 and 25 spots, respectively. Immunoblotting confirmed the different expression levels of two more abundant proteins (PDIA3, GSTP gene products) and four less abundant proteins (ATP5F1A, PGA3, SDHB, PGC). CONCLUSION: This study identified a proteomics signature of AAG. Many differential proteins were shared by GC and may be involved in the progression of AAG to GC.

Quantitative Proteomic Approach Targeted to Fibrinogen ß Chain in Tissue Gastric Carcinoma.

Elevated plasma fibrinogen levels and tumor progression in patients with gastric cancer (GC) have been largely reported. However, distinct fibrinogen chains and domains have different effects on coagulation, inflammation, and angiogenesis. The aim of this study was to characterize fibrinogen ß chain (FGB) in GC tissues. Retrospectively we analyzed the data of matched pairs of normal (N) and malignant tissues (T) of 28 consecutive patients with GC at diagnosis by combining one- and two-dimensional electrophoresis (1DE and 2DE) with immunoblotting and mass spectrometry together with two-dimensional difference in gel electrophoresis (2D-DIGE). 1DE showed bands of the intact FGB at 50 kDa and the cleaved forms containing the fragment D at ~37-40 kDa, which corresponded to 19 spots in 2DE. In particular, spot 402 at ~50 kDa and spots 526 and 548 at ~37 kDa were of interest by showing an increased expression in tumor tissues. A higher content of spot 402 was associated with stomach antrum, while spots 526 and 548 amounts correlated with corpus and high platelet count (>208 × 108/L). The quantification of FGB and cleaved products may help to further characterize the interconnections between GC and platelet/coagulation pathways.

Molecular and Pathological Features of Gastric Cancer in Lynch Syndrome and Familial Adenomatous Polyposis.

Lynch syndrome (LS) and familial adenomatous polyposis (FAP) are autosomal dominant hereditary diseases caused by germline mutations leading to the development of colorectal cancer. Moreover, these mutations result in the development of a spectrum of different tumors, including gastric cancers (GCs). Since the clinical characteristics of GCs associated with LS and FAP are not well known, we investigated clinical and molecular features of GCs occurring in patients with LS and FAP attending our Institution. The Hereditary Tumor Registry was established in 1994 at the Department of Oncologic Gastroenterology, CRO Aviano National Cancer Institute, Italy. It includes 139 patients with LS and 86 patients with FAP. Patients were recruited locally for prospective surveillance. Out of 139 LS patients, 4 developed GC­3 in the presence of helicobacter pylori infection and 1 on the background of autoimmune diseases. All GCs displayed a high microsatellite instability (MSI-H) and loss of related mismatch repair (MMR) protein. One of the FAP patients developed a flat adenoma, displaying low-grade dysplasia at the gastric body, and another poorly differentiated adenocarcinoma with signet ring cells like Krukenberg without HP infection. LS carriers displayed a risk of GC. The recognition of HP infection and autoimmune diseases would indicate those at higher risk for an endoscopic surveillance. Regarding FAP, the data suggested the need of suitable endoscopic surveillance in long survivals with diffuse fundic gland polyps.

Loss of Multimerin-2 and EMILIN-2 Expression in Gastric Cancer Associate with Altered Angiogenesis.

Gastric cancer is a deadly tumor and a relatively common disease worldwide. Surgical resection and chemotherapy are the main clinical options to treat this type of disease, however the median overall survival rate is limited to one year. Thus, the development of new therapies is a highly necessary clinical need. Angiogenesis is a promising target for this tumor type, however clinical trials with the use of anti-angiogenic drugs have so far not met expectations. Therefore, it is important to better characterize the expression of molecules whose expression levels may impact on the efficacy of the treatments. In this study the characteristics of the gastric tumor associated blood vessels were first assessed by endomicroscopy. Next, we analyzed the expression of Multimerin-2, EMILIN-2 and EMILIN-1, three molecules of the EMI Domain ENdowed (EDEN) protein family. These molecules play important functions in the tumor microenvironment, affecting cancer progression both directly and indirectly impinging on angiogenesis and lymphangiogenesis. All the molecules were highly expressed in the normal mucosa whereas in a number of patients their expression was altered. We consider that better characterizing the gastric tumor microenvironment and the quality of the vasculature may achieve effective patient tailored therapies.

Low Pepsinogen I/II Ratio and High Gastrin-17 Levels Typify Chronic Atrophic Autoimmune Gastritis Patients With Gastric Neuroendocrine Tumors.

INTRODUCTION: Chronic atrophic autoimmune gastritis (CAAG) can lead to the development of gastric neuroendocrine tumors (gNETs) and can be accompanied by other autoimmune diseases. This study aimed to determine, in CAAG patients, the association of gNET development, the prevalence of autoimmune diseases other than CAAG, the association of autoimmunity, and gNET development with pepsinogen I, II, gastrin-17, and Helicobacter pylori infection analysis. METHODS: We determined the prevalence of gNETs and other autoimmune diseases and analyzed pepsinogen I and II, gastrin-17 serum levels, and H. pylori infection in all patients diagnosed with CAAG at our hospital between 2013 and 2017. RESULTS: A total of 156 patients were studied and in 15.4% was observed concomitant gNET. Approximately 68.6% had at least 1 other autoimmune disease at diagnosis of CAAG. Approximately 60.9% had autoimmune thyroiditis, followed by diabetes (19.9%) and autoimmune polyendocrine syndrome (12.8%). CAAG patients with and without gNET had similar rates of comorbidity with other autoimmune diseases, but the pepsinogen I/II ratio was lower in patients with gNET (1.6 vs 4.5, P = 0.018). Receiver operating characteristic curve analyses identified a pepsinogen I/II ratio <2.3 and gastrin-17 levels >29.6 pmol/L as cutoffs distinguishing CAAG patients with gNET from those without. The combined use of these cutoff correctly identified 16 of the 18 CAAG patients with gNET (P = 0.007). H. pylori infection was observed in 28.7% of cases tested but did not associate with gNET. DISCUSSION: This study suggests that a low pepsinogen I/II ratio and high gastrin-17 levels characterize patients with CAAG and gNET and confirms the frequent coexistence of CAAG with other autoimmune diseases.

Probe-based confocal laser endomicroscopy (pCLE) is a suitable method for extrapulmonary high grade neuroendocrine rectal carcinoma (HGNEC) evaluation.

The potential role of the probe-based confocal laser endomicroscopy (pCLE) has been analyzed in different pathologic conditions of the gastrointestinal tract. Here, we analyzed a case of extrapulmonary high grade neuroendocrine rectal carcinoma (HGNEC) using, for the first time, the pCLE system. A 72-year old man was diagnosed with an 8 cm diameter rectal HGNEC by standard colonoscopy integrated with the pCLE system. The diagnosis of neuroendocrine carcinoma was confirmed by immunohistochemical analyses. By using the pCLE system, we well defined and resolved vascular structures and mucosal architecture. An altered mucosal pattern and vascular defects, peculiar for HGNEC, were observed at high magnification, allowing the identification of a pattern which was quite different from that observed in poorly differentiated adenocarcinomas (PDA) where tissues appear darker, very irregular, even if glandular structures can still be recognized. This underlines the usefulness of pCLE in discriminating HGNECs from PDAs. In conclusion, pCLE could represent a valid and helpful method for in vivo HGNEC diagnosis, allowing prompt and careful management of the patient.

Differential Helicobacter pylori Plasticity in the Gastric Niche of Subjects at Increased Gastric Cancer Risk.

Helicobacter pylori (H. pylori) represents an independent risk factor for Gastric Cancer (GC). First Degree Relatives (FDR) of GC subjects and Autoimmune Gastritis (AG) patients are both at increased risk for GC. H. pylori genetic heterogeneity within the gastric niche of FDR and AG individuals has been little explored. To understand whether they exploit an increased H. pylori stability and virulence, 14 AG, 25 FDR, 39 GC and 13 dyspeptic patients (D) were investigated by a cultural PCR-based approach characterizing single colonies-forming-units. We chose three loci within the Cytotoxin-associated gene-A Pathogenicity Island (CagPAI) (cagA,cagE,virB11), vacA, homA and homB as markers of virulence with reported association to GC. Inflammatory/precancerous lesions were staged according to Sydney System. When compared to D, FDR, similarly to GC patients, were associated to higher atrophy (OR = 6.29; 95% CI:1.23-31.96 in FDR; OR = 7.50; 95% CI:1.67-33.72 in GC) and a lower frequency of mixed infections (OR = 0.16; 95% CI:0.03-0.81 in FDR; OR = 0.10; 95% CI:0.02-0.48 in GC). FDR presented also an increased neutrophil infiltration (OR = 7.19; 95% CI:1.16-44.65). Both FDR and GC carried a higher proportion of CagPAI+vacAs1i1mx+homB+ profiles (OR = 2.71; 95% CI: 1.66-4.41 and OR = 3.43; 95% CI: 2.16-5.44, respectively). Conversely, AG patients presented a lower frequency of subtypes carrying a stable CagPAI and vacAs1i1mx. These results underline different H. pylori plasticity in FDR and AG individuals, and thus, a different host-bacterium interaction capacity that should be considered in the context of eradication therapies.

Frontline Science: Mast cells regulate neutrophil homeostasis by influencing macrophage clearance activity.

The receptor tyrosine kinase cKit and its ligand stem cell factor are essential for mast cells (MC) development and survival. Strains with mutations affecting the Kit gene display a profound MC deficiency in all tissues and have been extensively used to investigate the role of MC in both physiologic and pathologic conditions. However, these mice present a variety of abnormalities in other immune cell populations that can affect the interpretation of MC-related responses. C57BL/6 KitW-sh are characterized by an aberrant extramedullary myelopoiesis and systemic neutrophilia. MC deficiency in KitW-sh mice can be selectively repaired by engraftment with in vitro-differentiated MC to validate MC-specific functions. Nevertheless, the impact of MC reconstitution on other immune populations has never been evaluated in detail. Here, we specifically investigated the neutrophil compartment in primary and secondary lymphoid organs of C57BL/6 KitW-sh mice before and after MC reconstitution. We found that, albeit not apparently affecting neutrophils phenotype or maturation, MC reconstitution of KitW-sh mice restored the number of neutrophils at a level similar to that of wild-type C57BL/6 mice. In vitro and ex vivo experiments indicated that MC can influence neutrophil clearance by increasing macrophages' phagocytic activity. Furthermore, the G-CSF/IL-17 axis was also influenced by the presence or absence of MC in KitW-sh mice. These data suggest that MC play a role in the control of neutrophil homeostasis and that this aspect should be taken into account in the interpretation of results obtained using KitW-sh mice.

The Probe Based Confocal Laser Endomicroscopy (pCLE) in Locally Advanced Gastric Cancer: A Powerful Technique for Real-Time Analysis of Vasculature.

Probe based confocal laser endomicroscopy (pCLE) is an advanced technique which provides imaging of gastrointestinal mucosa at subcellular resolution and, importantly, a valid tool for the evaluation of microvasculature during endoscopic examination. In order to assess intratumoral vascularization and the efficiency of blood flow in locally advanced gastric cancer, we examined 57 patients through pCLE imaging. The vascular alterations in gastric cancer were mainly characterized by leakage and by the presence of tortuous and large size vessels. Defects in blood flow were detected very rarely. No association between the angiogenic score and the gastric tumor site or histological type was observed. Interestingly, no correlation was also found with the tumor grading indicating that the vascular angiogenic anomalies in gastric cancer represent an early pathological event to be observed and detected. The majority of patients displayed unchanged vascular alterations following neoadjuvant chemotherapy and this positively correlated with stable or progressive disease, suggesting that an unaltered angiogenic score could per se be indicative of poor therapeutic efficacy. Different vascular parameters were evaluated by immunofluorescence using bioptic samples and the vessel density did not correlate with clinical staging, site or histologic type. Interestingly, only CD105, Multimerin-2 and GLUT1 were able to discriminate normal from tumoral gastric mucosa. Taken together, these findings indicate that functional and structural angiogenic parameters characteristic of tumor blood network were fully detectable by pCLE. Moreover, the evaluation of tumor vasculature by real-time assessment may provide useful information to achieve tailored therapeutic interventions for gastric cancer patients.

Polymorphism in Toll-Like Receptors and Helicobacter Pylori Motility in Autoimmune Atrophic Gastritis and Gastric Cancer.

Autoimmune atrophic gastritis (AAG) is associated with an increased risk of certain types of gastric cancer (GC). Helicobacter pylori (H. pylori) infection may have a role in the induction and/or maintenance of AAG and GC. Toll-like receptors (TLR) are essential for H. pylori recognition and subsequent innate and adaptive immunity responses. This study therefore aimed to characterize TLR polymorphisms, and features of bacterial flagellin A in samples from patients with AAG (n = 67), GC (n = 114) and healthy donors (HD; n = 97). TLR5 rs5744174 C/C genotype was associated with GC, lower IgG anti H. pylori response and a higher H. pylori flagellin A abundance and motility. In a subset of patients with AAG, H. pylori strains showed a reduction of the flagellin A abundance and a moderate motility compared with strains from GC patients, a prerequisite for active colonization of the deeper layers of the mucosa, host immune response and inflammation. TLR9 rs5743836 T allele showed an association with serum gastrin G17. In conclusion, our study suggests that alterations of flaA protein, moderate motility in H. pylori and two polymorphisms in TLR5 and TLR9 may favor the onset of AAG and GC, at least in a subset of patients. These findings corroborate the function of pathogen-host cell interactions and responses, likely influencing the pathogenetic process.

New Insights into the Pathogenesis of Celiac Disease.

Celiac disease (CD) is an autoimmune and multisystem gluten-related disorder that causes symptoms involving the gastrointestinal tract and other organs. Pathogenesis of CD is only partially known. It had been established that ingestion of gluten proteins present in wheat and other cereals are necessary for the disease and develops in individuals genetically predisposed carrying the DQ2 or DQ8 human leukocyte antigen haplotypes. In this review, we had pay specific attention on the last discoveries regarding the three cellular components mainly involved in the development and maintenance of CD: T-cells, B-cells, and microbioma. All of them had been showed critical for the interaction between inflammatory immune response and gluten peptides. Although the mechanisms of interaction among overall these components are not yet fully understood, recent proteomics and molecular studies had shed some lights in the pathogenic role of tissue transglutaminase 2 in CD and in the alteration of the intestinal barrier function induced by host microbiota.

Probe-based confocal laser endomicroscopy for in vivo evaluation of the tumor vasculature in gastric and rectal carcinomas.

Probe-based Confocal Laser Endomicroscopy (pCLE) is a powerful imaging technique that allows to perform gastrointestinal endomicroscopy at subcellular resolution. The aim of this study was to assess the use of pCLE to evaluate tumor angiogenesis in rectal and gastric cancers. A total of 35 consecutive patients with gastric and 91 with rectal carcinomas underwent endoscopy and pCLE during the same examination. Vascular assessment was based on vessel shape and size, vessel permeability and blood flow, and allowed the creation of an angiogenic score ranging from 0, for normal vasculature, to 4, for aberrant vasculature. A significant difference for the presence of vessels with large diameter and defective blood flow was found between rectal and gastric cancers. Overall, rectal cancers displayed a higher angiogenic score compared to gastric cancers. Conventional therapy induced a striking reduction in the angiogenic score only in rectal cancer patients. Taken together, our findings suggest that the pCLE technology is suitable for the evaluation of the tumor microvasculature abnormalities. Therefore, the real-time assessment of the vasculature status may represent a promising approach to predict the efficacy of the treatments and improve the clinical management of patients with gastric or rectal carcinomas.

Mast cells, basophils and B cell connection network.

It has been proven that both resting and activated mast cells (MCs) and basophils are able to induce a significant increase in proliferation and survival of naïve and activated B cells, and their differentiation into antibody-producing cells. The immunological context in which this regulation occurs is of particular interest and the idea that these innate cells induce antibody class switching and production is increasingly gaining ground. This direct role of MCs and basophils in acquired immunity requires cell to cell contact as well as soluble factors and exosomes. Here, we review our current understanding of the interaction between B cells and MCs or basophils as well as the evidence supporting B lymphocyte-MC/basophil crosstalk in pathological settings. Furthermore, we underline the obscure aspects of this interaction that could serve as important starting points for future research in the field of MC and basophil biology in the peculiar context of the connection between innate and adaptive immunity.