A fine-tuned vector-parasite dialogue in tsetse's cardia determines peritrophic matrix integrity and trypanosome transmission success.

Arthropod vectors have multiple physical and immunological barriers that impede the development and transmission of parasites to new vertebrate hosts. These barriers include the peritrophic matrix (PM), a chitinous barrier that separates the blood bolus from the midgut epithelia and modulates vector-pathogens interactions. In tsetse flies, a sleeve-like PM is continuously produced by the cardia organ located at the fore- and midgut junction. African trypanosomes, Trypanosoma brucei, must bypass the PM twice; first to colonize the midgut and secondly to reach the salivary glands (SG), to complete their transmission cycle in tsetse. However, not all flies with midgut infections develop mammalian transmissible SG infections-the reasons for which are unclear. Here, we used transcriptomics, microscopy and functional genomics analyses to understand the factors that regulate parasite migration from midgut to SG. In flies with midgut infections only, parasites fail to cross the PM as they are eliminated from the cardia by reactive oxygen intermediates (ROIs)-albeit at the expense of collateral cytotoxic damage to the cardia. In flies with midgut and SG infections, expression of genes encoding components of the PM is reduced in the cardia, and structural integrity of the PM barrier is compromised. Under these circumstances trypanosomes traverse through the newly secreted and compromised PM. The process of PM attrition that enables the parasites to re-enter into the midgut lumen is apparently mediated by components of the parasites residing in the cardia. Thus, a fine-tuned dialogue between tsetse and trypanosomes at the cardia determines the outcome of PM integrity and trypanosome transmission success.

Evaluation of Barrett esophagus by multiphoton microscopy.

CONTEXT: Multiphoton microscopy (MPM) based on 2-photon excitation fluorescence and second-harmonic generation allows simultaneous visualization of cellular details and extracellular matrix components of fresh, unfixed, and unstained tissue. Portable multiphoton microscopes, which could be placed in endoscopy suites, and multiphoton endomicroscopes are in development, but their clinical utility is unknown. OBJECTIVE: To examine fresh, unfixed endoscopic biopsies obtained from the distal esophagus and gastroesophageal junction to (1) define the MPM characteristics of normal esophageal squamous mucosa and gastric columnar mucosa, and (2) evaluate whether diagnosis of intestinal metaplasia/Barrett esophagus (BE) could be made reliably with MPM. DESIGN: The study examined 35 untreated, fresh biopsy specimens from 25 patients who underwent routine upper endoscopy. A Zeiss LSM 710 Duo microscope (Carl Zeiss, Thornwood, New York) coupled to a Spectra-Physics (Mountain View, California) Tsunami Ti:sapphire laser was used to obtain a MPM image within 4 hours of fresh specimen collection. After obtaining MPM images, the biopsy specimens were placed in 10% buffered formalin and submitted for routine histopathologic examination. Then, the MPM images were compared with the findings in the hematoxylin-eosin-stained, formalin-fixed, paraffin-embedded sections. The MPM characteristics of the squamous, gastric-type columnar and intestinal-type columnar epithelium were analyzed. In biopsies with discrepancy between MPM imaging and hematoxylin-eosin-stained sections, the entire tissue block was serially sectioned and reevaluated. A diagnosis of BE was made when endoscopic and histologic criteria were satisfied. RESULTS: Based on effective 2-photon excitation fluorescence of cellular reduced pyridine nucleotides and flavin adenine dinucleotide and lack of 2-photon excitation fluorescence of mucin and cellular nuclei, MPM could readily identify and distinguish among squamous epithelial cells, goblet cells, gastric foveolar-type mucous cells, and parietal cells in the area of gastroesophageal junction. Based on the cell types identified, the mucosa was defined as squamous, columnar gastric type (cardia/fundic-type), and metaplastic columnar intestinal-type/BE. Various types of mucosa seen in the study of 35 biopsies included normal squamous mucosa only (n = 14; 40%), gastric cardia-type mucosa only (n = 2; 6%), gastric fundic mucosa (n = 6; 17%), and both squamous and gastric mucosa (n = 13; 37%). Intestinal metaplasia was identified by the presence of goblet cells in 10 of 25 cases (40%) leading to a diagnosis of BE on MPM imaging and only in 7 cases (28%) by histopathology. In 3 of 35 biopsies (9%), clear-cut goblet cells were seen by MPM imaging but not by histopathology, even after the entire tissue block was sectioned. Based on effective 2-photon excitation fluorescence of elastin and second-harmonic generation of collagen, connective tissue in the lamina propria and the basement membrane was also visualized with MPM. CONCLUSIONS: Multiphoton microscopy has the ability to accurately distinguish squamous epithelium and different cellular elements of the columnar mucosa obtained from biopsies around the gastroesophageal junction, including goblet cells that are important for the diagnosis of BE. Thus, use of MPM in the endoscopy suite might provide immediate microscopic images during endoscopy, improving screening and surveillance of patients with BE.

Inflammation in gastric adenocarcinoma of the cardia: how do EBV infection, Her2 amplification and cancer progression influence tumor-infiltrating lymphocytes?

Tumor-infiltrating lymphocytes (TILs) in gastric adenocarcinoma show a strong compartmentalization with high numbers of lymphocytes in the stroma and low intraepithelial lymphocyte counts. Our previous study has shown stromal regulatory T cells (Treg) to be associated with a beneficial outcome in intestinal type cancer of the cardia. We undertook the present study to further evaluate the immunogenic and inflammatory environment in intestinal-type gastric adenocarcinoma of the cardia. We assessed CXCR3 expression, Epstein-Barr virus (EBV) status, Her2/ERBB2 status and overexpression/amplification using tissue microarrays (immunohistochemistry and in situ hybridization) of 52 patients. The data were correlated to different TIL subset counts (CD3, CD8, GranzymeB, FoxP3 and CD20) and to infiltrating histiocytes (CD68) both in the tumor and the surrounding stromal tissue that were reported earlier. Her2/ERBB2 overexpression/amplification showed no correlation to tumor stage. Moreover, for the first time, we show here that Her2/ERBB2 overexpression/amplification has no correlation to overall or subset-specific TIL infiltration. EBV infection was seen in four cases and showed a strong association with intratumoral CD8(+) T cell infiltration as well as a moderate correlation to stromal CD8(+) T cell accumulation. Intratumoral CD8(+) T cell infiltration was significantly correlated to intratumoral FoxP3(+) Treg infiltration, and to a lesser extent, to stromal FoxP3(+) Treg counts. Stromal CXCR3(+) T cell infiltration showed an inverse correlation to T category. This highlights the importance of stromal immune processes for cancer growth and suggests a subversion of Th1 immunoresponse in cancer progression and underlines the important role of inflammation for early carcinogenesis.

Polymorphisms of transforming growth factor-ß1 associated with increased risk of gastric cardia adenocarcinoma in north China.

Transforming growth factor beta 1 (TGF-ß1) is a multifunctional cytokine that has been implicated in the oncogenesis and tumour progression. However, the association of TGF-ß1 polymorphism with gastric cardia adenocarcinoma (GCA) remains unclear. The aim of the study was to investigate the possible association of the polymorphisms of TGF-ß1 with susceptibility to GCA in a population of north China. A case-control analysis was performed to assess the association of six single nucleotide polymorphisms (SNPs) of TGF-ß1 and GCA risk. The genotype and allele distributions of TGF-ß1 G-800A, C-988A, G915C and C788T in GCA patients were not significantly different from that in healthy controls (P>0.05). The -509T and 869C allele significantly elevated the risk of developing GCA (adjusted OR=1.45 and 1.41; 95% CI=1.04-2.10 and 1.07-2.08, respectively). The CT and TT genotype of C-509T and the TC and CC genotype of T869C significantly elevated the risk of developing GCA. When stratified by tumour stage, the -509T and 869C allele carriers had an increased risk of TNM stage III+IV GCA as compared with noncarriers. The C-509T and T869C SNP are in a strong linkage disequilibrium (D'=0.94). Compared with C/T haplotype, T/C haplotype significantly increased the risk of developing GCA. The TGF-ß1 level and expression were higher in GCA patients with -509T or 869C allele than in those without T or C allele (P<0.05). GCA patients with -509TT and 869CC genotype had higher apoptotic tumour-infiltrating lymphocytes in their cancer tissues than those with -509CC and 869TT genotype. In all, TGF-ß1 C-509T and T869C polymorphisms may be associated with an increased risk of GCA in north China.

Naturally occurring regulatory T cells (CD4+, CD25high, FOXP3+) in the antrum and cardia are associated with higher H. pylori colonization and increased gene expression of TGF-beta1.

BACKGROUND: Helicobacter pylori causes gastric inflammation. Despite the induction of H. pylori-specific B- and T cells, the immune response is not sufficient to clear the infection. Regulatory T cells (Treg cells) suppress the activation and proliferation of antigen-specific T cells and mediate immunologic tolerance. FOXP3 was shown to be expressed in a subset of Treg cells known as 'naturally occurring Treg cells'. These cells have not been sufficiently studied in context to H. pylori-induced inflammation in human gastric mucosa. MATERIALS AND METHODS: The study included 76 patients stratified according to the presence of H. pylori. Gene expression levels of FOXP3, transforming growth factor (TGF)-beta1, and interleukin-10 were analyzed by quantitative real-time polymerase chain reaction in biopsies from gastric antrum, corpus, and cardia. FOXP3 expression was also analyzed by immunohistochemistry. Differences in expression levels were analyzed by comprehensive statistical analyses and correlated with clinical and histomorphologic parameters. RESULTS: H. pylori-positive patients revealed a 19- to 25-fold induction of FOXP3 transcript levels in antrum and cardia (p < .02). FOXP3 transcript levels correlated positively with inflammation (p < .04) and TGF-beta1 transcript levels (p < .001). Furthermore, a positive correlation between FOXP3(+) Treg cells and H. pylori colonization was demonstrated. CONCLUSION: This study demonstrates that H. pylori-induced gastritis is associated with a recruitment of naturally occurring FOXP3(+) Treg cells that correlates with the degree of bacterial colonization and mucosal TGF-beta1 expression. Together, these data support the hypothesis that naturally FOXP3(+) Treg cells play a role in the lifelong persistence of H. pylori infection in humans.