Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on gastric epithelium: implication for a role of TREM-1 in Helicobacter pylori infection.

In Helicobacter pylori gastritis gastric epithelium plays a central role in the innate immunity to H. pylori. However, epithelial receptors interacting with H. pylori have been poorly characterized so far. Recently a new triggering receptor expressed on myeloid cells-1 (TREM-1) has been identified on human neutrophils and monocytes. On these cells TREM-1 triggers innate immunity by stimulating the secretion of interleukin (IL)-8 and tumour necrosis factor (TNF)-alpha and thus amplifies bacterial-induced inflammation. In this study expression and function of TREM-1 in gastric epithelium exposed to H. pylori has been investigated. TREM-1 mRNA and protein were expressed on gastric epithelial cell lines as demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence activated cell sorter analysis. Gastric epithelial TREM-1 expression was up-regulated directly by H. pylori and was independent of epithelial IL-8 induced by H. pylori. Immunohistochemistry and tissue RT-PCR demonstrated significantly stronger TREM-1 expression in H. pylori gastritis compared with the non-inflamed gastric mucosa supporting in vivo that epithelial TREM-1 is up-regulated during H. pylori infection. Stimulation of gastric epithelial TREM-1 receptor resulted in IL-8 up-regulation on mRNA and protein level, as shown by real-time PCR and immunoassay. This is the first study localizing TREM-1 on gastric epithelium. Functional data suggest that TREM-1 expressed on gastric epithelium amplifies inflammation of the underlying gastric mucosa by up-regulation of IL-8.

Gastric extranodal marginal zone B-cell lymphomas of MALT type exclusively express Toll-like receptor 4 in contrast to other lymphomas infiltrating the stomach.

BACKGROUND: Development and growth of extranodal marginal zone B-cell lymphomas (eMZBCLs) of mucosa-associated lymphoid tissue (MALT) type are thought to be highly dependent on Helicobacter pylori and autoantigens. Receptors mediating these effects are not characterised so far. Toll-like receptors (TLRs) recognise bacterial proteins and autoantigens, which results in inflammatory reactions and influences tumour development and growth. MATERIALS AND METHODS: TLR4, 5 and 9 expressions were evaluated by immunohistology and confocal microscopy in gastric eMZBCL in comparison to other lymphomas infiltrating the stomach. RESULTS: TLR4 was exclusively expressed on the cell surface in all eMZBCL (n = 19) and not in chronic lymphocytic leukaemia (CLL, n = 12) or mantle cell lymphoma (MCL, n = 10). TLR5 was strongly expressed in CLL and weak in some eMZBCL (15 of 19), but not in MCL. TLR4, 5 and 9 were negative in all the three lymphoma entities. CONCLUSIONS: Exclusive TLR4 expression may enable eMZBCL to interact with H. pylori and autoantigens. Blockade of TLR4 might be a new approach for therapy of eMZBCL of MALT type.

The chemokine receptor CCR7 is expressed on epithelium of non-inflamed gastric mucosa, Helicobacter pylori gastritis, gastric carcinoma and its precursor lesions and up-regulated by H. pylori.

CCR7 chemokine-receptor expression on tumour cells of gastric carcinoma has been associated with lymph-node metastasis and is thought to play an important role in metastasis. However, so far it is unknown whether CCR7 is newly up-regulated on gastric carcinoma or already expressed in non-neoplastic gastric epithelium. Therefore, epithelial CCR7 expression was investigated in the process of gastric carcinogenesis: non-inflamed mucosa --Helicobacter pylori gastritis -- intestinal metaplasia/dysplasia -- gastric carcinoma. CCR7 was expressed by gastric epithelium in non-inflamed gastric mucosa (n = 5), H. pylori gastritis (n = 17), intestinal metaplasia (n = 10), dysplasia (n = 3) and on tumour cells in 20 of 24 patients with gastric carcinoma (13/14 intestinal-type; 7/10 diffuse-type) as tested by immunohistochemistry. As CCR7 expression by gastric epithelium was significantly stronger in H. pylori gastritis than in non-infected mucosa, the influence of H. pylori on CCR7 receptor expression of gastric epithelial cells was investigated by fluorescence activated cell sorter analysis. H. pylori strains up-regulated the CCR7 chemokine-receptor in CCR7-positive cell lines. No difference in CCR7 up-regulation between cag(+) and cag(-)H. pylori strains was found. Epithelial CCR7 up-regulation by H. pylori may alter the metastatic fate of gastric carcinoma. Additionally, CCR7 expression not only on gastric carcinoma, but also on non-neoplastic gastric epithelium, suggests a novel biological function.

Expression and subcellular distribution of toll-like receptors TLR4, TLR5 and TLR9 on the gastric epithelium in Helicobacter pylori infection.

Toll-like receptors (TLRs) expressed by mucosal epithelium play an essential role in the defense against microbes by recognizing conserved bacterial molecules. For the first time TLR4, TLR5 and TLR9 have been microanatomically localized in patients with noninflamed gastric mucosa and Helicobacter pylori gastritis by immunohistochemistry. Because polarized expression of TLRs in apical and basolateral epithelial compartments is thought to modulate mucosal immunity, subcellular TLR distribution by gastric epithelium was investigated using confocal microscopy. TLR4, TLR5 and TLR9 were expressed by gastric epithelium in antrum and corpus of all patients with H. pylori gastritis (n = 14) and with noninflamed gastric mucosa (n = 5). TLR4 was expressed at the apical and the basolateral pole of the gastric epithelium as well in noninflamed gastric mucosa as in H. pylori gastritis. TLR5 and TLR9 expression in the noninflamed gastric mucosa was identical to that of TLR4 with localization at the apical and the basolateral epithelial pole. However, in H. pylori gastritis TLR5 and TLR9 expression on the gastric epithelium changed to an exclusive basolateral localization without detectable expression at the apical pole. In the human stomach, the gastric epithelium expressed TLR4, TLR5 and TLR9, which gives it the possibility to interact with H. pylori. Furthermore, gastric epithelial TLR4 expression is highly polarized in an apical and a basolateral compartment, whereas TLR5 and TLR9 polarization seems to be a process dynamically influenced by H. pylori infection. This polarized and dynamically regulated gastric epithelial expression of TLRs supports a sentinel role for these receptors in the mucosal immunity to H. pylori.

Pleiotropic effects of CXC chemokines in gastric carcinoma: differences in CXCL8 and CXCL1 expression between diffuse and intestinal types of gastric carcinoma.

CXC chemokines modulate host immunity, neovascularization, growth and invasive behaviour of tumours. Despite their relevance in tumour biology, chemokine expression in intestinal- and diffuse-type gastric carcinoma, which exhibit a completely different growth pattern, has not been investigated in detail. In this study, expression of the CXC chemokines CXCL8 [interleukin (IL)-8], CXCL1 [growth-related oncogene alpha (Gro alpha)], CXCL9 [monokine induced by interferon (IFN)-gamma] and CXCL10 [IFN-gamma-inducible protein-10 (IP-10)] and the corresponding chemokine receptors CXCR1-3 was investigated by immunohistochemistry in intestinal- and diffuse-type gastric carcinoma. Tumour cells of all patients expressed CXCL8. CXCL8 expression was significantly stronger in tumour cells of diffuse- rather than intestinal-type gastric carcinoma (P < 0.01) as determined by a semiquantitative score. CXCL1 was expressed almost exclusively by diffuse- but not intestinal-type carcinoma cells. The corresponding chemokine receptors, CXCR1 and CXCR2, were found on carcinoma cells. Furthermore, CXCL8 expression correlated with number of tumour vessels (P < 0.01), suggesting an angiogenetic function in gastric carcinoma not only in vitro but also in vivo. CXCL10 and CXCL9, attractants for T cells, were expressed by peritumorous macrophages in close proximity to IFN-gamma-producing CXCR3-positive T cells in both tumour types. These chemokines may attract gastric carcinoma-infiltrating T cells via an IFN-gamma-mediated pathway and enhance host immunity against the tumour. In gastric carcinoma a complex interplay between CXC-chemokine signals derived from both tumour cells and tumour-infiltrating immune cells may exhibit pleiotropic effects in tumour biology that go far beyond their originally described functions as leucocyte chemoattractants. Because CXCL8 and CXCL1, which are known to increase growth and invasive behaviour of malignant tumours, are significantly stronger expressed in diffuse- than intestinal-type gastric carcinoma, one may speculate that these chemokines influence the different growth pattern of gastric carcinoma types.

Regulation of the new coexpressed CD55 (decay-accelerating factor) receptor on stomach carcinoma cells involved in antibody SC-1-induced apoptosis.

The human monoclonal antibody SC-1 was isolated from a patient with a diffuse-type adenocarcinoma of the stomach using somatic cell hybridization. The immunoglobulin (Ig)M antibody reacts specifically with diffuse- (70%) and intestinal-type (25%) gastric adenocarcinoma and induces apoptosis in vitro and in vivo. When used in clinical trials with stomach carcinoma patients, significant apoptotic and regressive effects in primary tumors have been observed with the antibody SC-1. The SC-1 receptor is a new 82 kd membrane-bound isoform of glycosylphosphatidylinositol (GPI)-linked CD55 (decay-accelerating factor, DAF). CD55 is known to protect cells from lysis through autologous complement and is coexpressed with the ubiquitously distributed 70 kd isoform. The SC-1-specific CD55 isoform is up-regulated shortly after antibody binding, followed by an internalization of the antibody/receptor-complex, whereas the membranous expression of wild-type CD55 remains unchanged. The apoptotic process is marked by cleavage of cytokeratin 18, indicating the involvement of caspase-6 in the apoptotic process. In contrast to other apoptotic pathways, a cleavage of poly(ADP-ribose)polymerase (PARP) is not observed. The expression of the cell-cycle regulator c-myc becomes up-regulated, whereas expression of topoisomerase IIalpha is down-regulated. Induction of apoptosis leads to an increase in the internal Ca(2+) concentration, which is not necessary for the apoptotic process but for the transport of newly synthesized SC-1-specific CD55 isoform to the membrane.

CXC chemokines Gro(alpha)/IL-8 and IP-10/MIG in Helicobacter pylori gastritis.

Infection with Helicobacter pylori causes chronic active gastritis, which is characterized by neutrophils infiltrating the gastric epithelial layer and the underlying lamina propria as well as by T, B lymphocytes and macrophages accumulating in the lamina propria. In this study, the chemokine profile responsible for the recruitment of these inflammatory cells is investigated. Using both RNA/RNA in situ hybridization and immunohistochemistry, the expression of the neutrophil and/or lymphocyte-attractant CXC chemokines growth-related oncogene alpha (Gro(alpha)), IL-8, interferon-gamma (IFN-gamma)-inducible protein-10 (IP-10), monokine induced by IFN-gamma (MIG) and the CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), -1beta, regulated on activation normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein-1 (MCP-1) is studied and microanatomically localized in the gastric mucosa. Macrophages in the lamina propria at sites with neutrophil infiltration and gastric epithelium infiltrated by neutrophils highly expressed the neutrophil-attractant chemokines Gro(alpha) and IL-8. Additionally, Gro(alpha) and IL-8 were expressed by neutrophils themselves localized within gastric epithelium, in the foveolar lumen and in the cellular debris overlying mucosal erosion. IP-10 and to a lower extent MIG, both selectively chemotactic for inflammatory T cells, were expressed by endothelial cells of gastric mucosal vessels and by mononuclear cells at sites with T cell infiltration. Expression of all other CC chemokines tested was significantly lower. These in vivo data indicate that a set of predominantly CXC chemokines modulates the inflammation in H. pylori gastritis. Gro(alpha) and IL-8 may play an important role in neutrophil trafficking from the mucosal vessel into the gastric epithelium, whereas IP-10 and MIG contribute to the recruitment of inflammatory T cells into the mucosa.

Identification of immunogenic antigens of Helicobacter pylori via the Escherichia coli hemolysin secretion system(1).

We describe a new procedure allowing the generation and detection of immunogenic antigens from Helicobacter pylori via the hemolysin secretion apparatus of Escherichia coli. The gene (or gene fragment) encoding the H. pylori protein (or protein domain) is inserted in-frame into a residual portion of the hemolysin gene (hlyA), encoding the HlyA secretion signal (HlyA(s)). These fusion proteins are secreted efficiently by E. coli. This new approach allows the identification of immunodominant antigens by using sera derived from H. pylori-infected patients suffering from different gastroduodenal pathologies. Three immunodominant antigens bearing the ureB (urease B-subunit), flaA (flagellin A-subunit), and an unknown ORF (HP0888) encoding an E. coli FecE analogous protein fused to hlyA(s) were identified and characterized.

Helicobacter pylori in gastric mucosa-associated lymphoid tissue type lymphoma.

Infection with Helicobacter pylori triggers the acquisition of gastric mucosa-associated lymphoid tissue (MALT) and provides the background for MALT-type lymphoma development. This concept has been supported by a high association of H. pylori infection and MALT-type lymphoma and by the regression of most lymphomas after eradication therapy. In almost all patients with MALT-type lymphoma, serum antibodies to H. pylori were detectable. However, H. pylori was found only in 78% of the patients on histological examination. In addition to other effects, changes in the gastric micro-milieu caused by tumor infiltration of the gastric mucosa may be responsible for the loss of the bacterium. The discrepancy of high seroprevalence and lower histological yield has been already described in other gastric diseases, e.g. atrophic gastritis or gastric carcinoma with extensive destruction of the gastric mucosa. H. pylori strains expressing the CagA protein have been associated with duodenal ulceration and gastric carcinoma. A very high percentage of patients with MALT-type lymphoma is also infected by CagA+ strains of H. pylori as tested by immunoblotting. Antibodies directed to CagA were detectable in the serum as well as in micro-cultured gastric mucosa. Infection with H. pylori may be a precondition for the development of gastric MALT-type lymphoma. In particular, CagA+ strains of H. pylori may, together with additional up to now unknown factors, play a role in the development of gastric MALT-type lymphoma.

Helicobacter pylori induces DNA damage in vitro.

A close association between Helicobacter pylori infection and the development of gastric adenocarcinoma in humans has been demonstrated. Therefore, the direct induction of DNA damage by H. pylori was investigated here using the in vitro micronucleus assay. After 5 days of incubation with bacterial lysate a dose-dependent formation of micronuclei was found, which was not limited to cytotoxic protein concentrations and was not observed after treatment with Escherichia coli lysate (control). This induction of DNA damage may be a link between chronic H. pylori infection and development of adenocarcinoma of the stomach.

Mucosal humoral immune response to CagA shows a high prevalence in patients with gastric MALT-type lymphoma.

In the pathogenesis of gastric mucosa-associated lymphoid tissue (MALT)-type lymphoma, CagA-positive Helicobacter pylori strains have been suspected of making a significant contribution. To investigate this hypothesis in more detail, the mucosal humoral immune response of 15 patients with gastric MALT-type lymphoma was examined in the tumor and in the tumor-free gastritis of the same patient. Mononuclear cells from different sites (antrum, corpus, lymphoma) were cultured. Culture supernatant and serum of the same patient were used for immunodetection of CagA. All patients displayed an immune response to CagA in the tissue-culture supernatants. Although the humoral immune response in the tumor was restricted to a very few H. pylori antigens, antibodies directed against CagA protein were found in most patients. The immune response to CagA in nearly all lymphoma patients--not only in the serum, but also in the mucosa, including the tumor site--support the hypothesis that CagA is involved in the pathogenesis of gastric MALT-type lymphoma.

Evaluation of Helicobacter pylori in gastric MALT-type lymphoma: differences between histologic and serologic diagnosis.

Helicobacter pylori provides the pathogenic background for the development of gastric MALT-type lymphoma. The assessment of H. pylori is limited by the accuracy of the detection method used. Especially in patients with low-grade MALT-type lymphoma, the H. pylori status is the crucial question for therapeutic management. In this study, 60 patients with gastric MALT-type lymphoma. (lowgrade, 22; high-grade, 38) were investigated for the presence of H. pylori by histologic and serologic means. In 98% of the patients with MALT-type lymphoma, H. pylori-specific IgG serum antibodies were detected. In contrast, on histologic examination, H. pylori was found only in 78% of the patients (low-grade, 77%; high-grade, 79%). In this study, a discrepancy between serologic and histologic evaluation of the H. pylori status in gastric MALT-type lymphoma was found. Therefore, a H. pylori eradication therapy in low-grade MALT-type lymphoma, which often leads to a complete tumor regression, should not be excluded as a first line therapy because of a negative H. pylori status on histologic examination.

[Diagnostic value of stomach biopsy in comparison with surgical specimen in gastric B-cell lymphomas of the MALT type]. / Diagnostische Aussagekraft der Magenbiopsie im Vergleich zum Resektat bei primären gastralen B-Zell-Lymphomen vom MALT-Typ.

Diagnostic und therapeutic management of gastric lymphomas of the mucosa-associated lymphoid tissue type (MALT-type lymphomas) is often based exclusively on the evaluation of biopsy material. To evaluate the diagnostic value of gastric biopsies in gastric MALT-type lymphomas, biopsies--on average six per patient--and subsequent surgical specimens of 64 patients were compared at the Institute of Pathology, University of Würzburg. Tumor diagnosis and tumor gradind were assessed. Using biopsy specimens, primary gastric MALT-type lymphomas were correctly diagnosed by local pathologists in 69% of cases, but correctly graded as low-grade, high-grade or secondary high-grade lymphomas in only 41%. When immunohistochemistry and molecular biological techniques were applied in addition to conventional histology, diagnosis of gastric MALT-type lymphoma was achieved in biopsies in 95% of cases at the Institute of Pathology Würzburg, but correct grading in only 73%. In secondary high-grade MALT-type lymphomas, both components--the high-grade and the low-grade component--were identified in gastric biopsies in only 33% of cases. Diagnostic accuracy in gastric lymphomas based on biopsies is limited by biopsy artefacts, but improved by using immunohistochemistry and molecular biological techniques. Particularly in secondary high-grade MALT-type lymphomas the correct diagnosis is often missed when using biopsies, due to a low number of biopsy specimens.

Active fulminant myocarditis characterized by T-lymphocytes expressing the gamma-delta T-cell receptor: a new disease entity?

Lymphocytic myocarditis is thought to be a virus-induced disease. T cells expressing the alpha-beta T-cell receptor seem to play a central role in the pathogenesis and to mediate tissue injury in this disease. A case of active fulminant myocarditis is described, which was analyzed by immunohistochemical, molecular biologic, and serologic methods. Infiltration of the heart tissue predominantly by gamma-delta T cells was detected by immunohistochemistry. No evidence of viral disease could be obtained by in situ hybridization with different enterovirus-specific DNA probes; by reverse-transcriptase polymerase chain reaction using specific primers for enteroviruses, adenoviruses, herpes simplex viruses, influenza A and B viruses, and cytomegaloviruses; or by enzyme-linked immunosorbent assay and electron microscopy. Because gamma-delta T cells may have an autoimmune capacity, we propose that these cells may trigger autoimmune myocarditis. These findings may be important in order to identify subgroups of patients who may benefit from immunosuppressive therapy.

MALT-type lymphoma of the stomach is associated with Helicobacter pylori strains expressing the CagA protein.

BACKGROUND & AIMS: Helicobacter pylori is considered to be involved in the pathogenesis of gastric lymphoma of mucosa-associated lymphoid tissue (MALT) type. Strains expressing the CagA protein (CagA+ strains) have been strongly associated with severe gastritis, duodenal ulceration, and gastric adenocarcinoma. The aim of this study was to determine the presence of H. pylori as well as incidence of CagA+ strains in gastric MALT-type lymphoma. METHODS: Sera of 68 patients with gastric MALT-type lymphoma (22 with low grade, 36 with high grade, and 10 with secondary high grade) were obtained, and the serological response to CagA was studied by immunoblotting using a purified recombinant CagA protein, a CagA+ strain, and the corresponding isogenic CagA- mutant. RESULTS: Of the patients with MALT-type lymphoma, 98.5% (67 of 68 patients) were H. pylori seropositive. In the only seronegative patient, the bacterium was detected histologically by Warthin-Starry staining. Of the seropositive patients, 95.5% had serum immunoglobulin G antibodies to CagA compared with 67% of an H. pylori-positive control group (33 of 49 patients; P = 0.000037) with chronic active gastritis. CONCLUSIONS: These results indicate infection of almost all patients with MALT-type lymphoma by CagA+ H. pylori strains. Strains expressing the CagA protein seem to play a crucial role in the pathogenesis of gastric MALT-type lymphoma.

[Laboratory diagnosis in iron deficiency anemia]. / Labordiagnostik bei Eisenmangelanämie.

The diagnosis of iron deficiency is based, in first line, on clinical and hematological findings. Clinical-chemical laboratory investigations lend support to these findings, and may also provide differential diagnostic information. In particular with respect to the question whether an iron deficiency or an iron distribution disorder is presenting, clinical-chemical parameters can be of value. For the diagnosis of an iron deficiency and for the evaluation of the iron metabolism as a whole, determination of ferritin, transferrin and iron is adequate.

Isolated dihydroxyacetonephosphate-acyl-transferase deficiency in rhizomelic chondrodysplasia punctata: clinical presentation, metabolic and histological findings.

UNLABELLED: Rhizomelic chondrodysplasia punctata (RCDP) is clinically characterized by symmetrical shortening of the proximal limbs, contractures of joints, a characteristic dysmorphic face, and cataracts. In the classical form an impairment of several peroxisomal functions and enzymes (plasmalogen synthesis, phytanic acid oxidation, 3-oxoacyl-CoA thiolase) has been repeatedly shown. Recently a variant involving only the peroxisomal dihydroxyacetonephosphate acyltransferase (DHAP-AT) has been described. We present a patient with isolated DHAP-AT deficiency and all clinical, radiological and pathological features of classical RCDP. For the first time, microscopy and immunocytochemistry of hepatocytes could be performed. CONCLUSION: In contrast to studies on classical rhizomelic chondrodysplasia punctata which have shown enlarged peroxisomes in numbers varying from hepatocyte to hepatocyte, the peroxisomes in our patient seem to be normal in size, number and shape.

[Meckel-Gruber syndrome]. / Das Meckel-Gruber-Syndrom.

The lethal Meckel-Gruber-Syndrome can be diagnosed prenatally during ultrasound screening between 16 and 20 weeks of pregnancy. Two case reports of Meckel-Gruber-Syndrome are given, which demonstrate the importance of a reliable ultrasound examination. The results supply the basis for an adequate counseling of the patient with the option of pregnancy termination in case of the lethal syndrome.

Immortalized B-lymphocytes from rheumatoid synovial tissue show specificity for bacterial HSP 60.

Several studies indicate a pathogenetic role of T-lymphocytes with specificity for heat shock proteins (HSP) in rheumatoid arthritis (RA). Surprisingly, there are no experimental data for B-lymphocytes with specificity for HSP. To investigate whether B-lymphocytes from rheumatoid synovial tissue show a specificity for HSP 60 we immortalized synovial tissue B-lymphocytes by the electrofusion technique and tested the specificity of the B-cell clones for HSP 60 by ELISA. Tissue samples from four patients with classic, active RA were used in this study. The isolated cells were electrofused in strongly hypo-osmolar medium with cells either of the mouse strain X63-Ag8-653 (Ag8) or the heteromyeloma strain HAB-1. Clones positive for IgG, the IgG fraction of the supernatant of the isolated synovial cells and the IgG of the serum of the patients were tested in an ELISA for reactivity to the recombinant HSP 60 or Yersinia enterocolitica, which shows great homology with mycobacterial HSP 65 and human HSP 60. The expression of this HSP 60 was studied in normal and rheumatoid synovial tissue using a polyclonal rabbit serum against HSP 60 from Y. enterocolitica (Ye HSP 60). In this way we investigate differences in the expression of HSP 60 and compared the pattern of this HSP60 with the pattern of mycobacterial HSP65 and human HSP 60 described by others. In three of four patients 10 IgG secreting B-cell clones showing a specificity for HSP 60 were detected. IgG specific for HSP 60 was also detected in the supernatant of the isolated synovial cells before fusion and in the serum of these patients. HSP 60 was demonstrated immunohistochemically within the rheumatoid synovial tissue and showed stronger expression with a different distribution when compared with the expression in normal synovial tissue. B-cell clones from rheumatoid synovial tissue thus exhibit a specificity for bacterial HSP 60, and a monospecific rabbit serum against this HSP shows strong reactivity within the rheumatoid synovial tissue. It may be postulated that a humoral HSP 60 response, initially directed against an infectious agent, could react with cross-reactive epitopes of rheumatoid synovial tissue or with self-HSP perpetuating the local inflammatory process.