The MYTHS of De novo Crohn's Disease After Restorative Proctocolectomy with Ileal Pouch-anal Anastomosis for Ulcerative Colitis.

Background: 1.1.Inflammatory Bowel Disease (IBD) are the manifestation of overzealous dys-regulated immune response in the intestinal tract, directed primarily against the indigenous microbes combined with defective functioning of anti-inflammatory pathways. Finding a trustable lead to predicting de novo Crohn's Disease (CD) prior to performing "pouch surgery", Restorative Proctocolectomy (RPC) with Ileal Pouch-Anal Anastomosis (IPAA) for UC and/or Indeterminate Colitis (IC) is clinically important and remains debatable. De novo CD is a subsequent long-term postoperative complication in IBD patients with Ulcerative Colitis (UC) undergoing IPAA. Herewith we discuss this understanding in laboratory-based basic science research, with its molecular application as a possible corner stone tool for clinical progress and success in the IBD Clinic. Crypt Paneth cell (PCs) secreted enteroendocrine alpha-defensin 5 (DEFA5)" if developed properly is likely to solve diagnostic and prognostic difficulty in IBD Clinics. DEFA5 has shown the ability to differentiate the predominant subtypes of colonic IBD (CC vs. UC) at first endoscopy biopsy, avoiding diagnosis delay prior to colectomy. In addition, DEFA5 accurately circumvents indeterminate colitis (IC) patients into accurate IBD subtype (UC or CC). Further, DEFA5 can be used in selecting CC patients that may have positive outcomes after IPAA surgery [1]. Furthermore, likewise, DEFA5 can predict UC patients likely to have positive or poor outcome, e.g. those patients that are likely to transform/ convert and adhere to de novo Crohn's after IPAA can be picked up in endoscopy biopsy before surgery. Aim: 1.2.To assessed comprehensive state-of-the-art understanding domains on the de novo Crohn's disease subsequent to IPAA surgery for ulcerative colitis. Methods: 1.3.A literature search based on preferred reporting items for over-review and meta-analysis protocols (PRISMA-P) was performed. A comprehensive current search of PubMed, MEDLINE, CINAHL, Embase, Google® search engine and Cochrane Database of collected reviews was performed from January 1990 through December 2018. The search consists of retrospective studies and case reports of reporting postoperative de novo CD incidence and adverse events. Secondary and hand/manual searches of reference lists, other studies cross-indexed by authors, reviews, commentaries, books and meeting abstracts were also performed. Studies were included only if the diagnosis of de novo CD was established clinically and histologically based on inflammation of afferent limb(s) or perianal disease. The search excluded non-English language and non-human studies as well as editorials. Results: 1.4.Published data on de novo CD developing after RPC with IPAA are still limited. A total of three hundred and sixty-five (#365) patients in 13 publications reported de novo CD after a median follow-up of 66 (range: 3-236) months. All patients were diagnosed with clinically active pouch CD during follow-up surveillance after IPAA for UC or IC. A de novo CD diagnosis depended on either inflammation in the mucosa involving the small intestine proximal to the ileal pouch any time after IPAA surgery and/or when perianal complications developed after closure of a temporary diverting loop ileostomy. Successful management is facilitated by co-operation within a multidisciplinary team of gastroenterologists and colorectal surgeons and closely involving the patient in therapeutic decisions. Awareness of symptoms leads to timely consultation, diagnosis, treatment and restoration of intestinal continuity. Conclusion: 1.5.The nature history and risk of de novo CD after IPAA for UC remains debatable. Chronic pouchitis and/or pouch failure often precedes a diagnosis of de novo CD. A successful management is facilitated by a triad cooperation between gastroenterologists, colorectal surgeons and the patient.

Obesity, weight change, and risk of adenoma recurrence: a prospective trial.

BACKGROUND AND STUDY AIMS: Obesity is a risk factor for colorectal neoplasia. Lifestyle modifications, including weight loss, have been advocated to reduce the risk. However, no prospective study has evaluated whether weight loss actually affects adenoma recurrence. The aim of this study was to examine whether weight change (loss or gain) over 4 years is associated with adenoma recurrence. PATIENTS AND METHODS: A total of 1826 patients with colorectal adenoma in the Polyp Prevention Trial had their height and weight measured at baseline. Adenoma recurrence was determined by end of trial colonoscopy 4 years after study entry when patients' weights were re-measured. Poisson regression models were used to evaluate body mass index (BMI), weight change over 4 years, and the risk of any adenoma and advanced adenoma recurrence. RESULTS: Adenoma recurrence was observed in 723 patients (39.6%), 118 (6.5%) of whom had advanced adenoma recurrence. Among those with baseline BMI < 25 kg/m² (n = 466), BMI 25-29 kg/m² (n = 868), and BMI ≥ 30 kg/m² (n = 492), the recurrence rate was 34.5%, 41.0%, and 41.9%, respectively. Obesity was associated with an increased risk of adenoma recurrence (RR = 1.19; 95%CI 1.01-1.39) and advanced adenoma recurrence (RR = 1.62; 95%CI 1.01-2.57). However, when compared with those with relatively stable weight (weight change < 5 lb) over the 4-year trial, weight gain or loss was not associated with adenoma recurrence. This was consistent, regardless of the baseline BMI. CONCLUSIONS: Weight loss or gain over 4 years does not affect adenoma recurrence. This study does not support weight loss alone as an effective intervention for reducing adenoma recurrence.

2Am-DNT induces cell death and apoptosis in human cells.

During microbial or mammalian cell metabolism, TNT (2,4,6-tinitrotoluene) is reduced to 2Am-DNT (2-amino-4,6-dinitrotoluene), 4Am-DNT, or 2,4-diamino-NT (2,4-diaminonitrotoluelne) depending on the specific organism. The metabolite 2Am-DNT is the most common of the TBT biotransformation pathways in bacterial and fungal species studied to date. in the mammalian liver cells, TNT is metabolized to 2Am-DNT by the P450 enzyme system. Apoptosis is rapidly emerging as a relevant endpoint for detecting low-dose toxin exposure. We report in this study that 2Am-DNT treatment of mammalian cells causes cell death by apoptosis. Cell death was assayed by the Trypan Blue method. Apoptotic changes, such as DNA break down, were detected in treated cells by the production of a dark-brown DAB (diaminobenzidine) signal using the Fragel Klenow DNA fragment detection system, by immunohistochemical techniques with fluorescence microscopy, and by using a microplate reader for a single-stranded DNA binding assay. All of these results showed that 2am-DNT is toxic to mammalian cells and induces apoptosis.

Folate status and risk of colorectal polyps in African Americans.

Dietary folate status appears to influence risk for colorectal cancer possibly by alterations in DNA methylation and nucleotide precursor pools. Polymorphisms (677C-->T and 1298A-->C) in methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, determines enzyme activity. The frequency of polymorphisms in the gene varies extensively in different populations. We sought to determine the association between folate status, folate metabolism, DNA methylation, tobacco, alcohol consumption, and the risk of colorectal adenomas in African Americans. Among 58 patients who underwent a clinically indicated colonoscopy, 23 patients with histology confirmed colorectal polyps and 35 patients without were recruited for a case-control study. Blood samples were collected from fasting patients for determination of serum and red blood cell (RBC) folate, homocysteine, vitamin B(12), and methylation status. Polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) technique was performed to identify the MTHFR 677 C-->T polymorphism and specific PCR was used to analyze adenomatous polyposis coli (APC) gene-promoter sequence methylation. Among 23 cases, 49 polyps (adenomatous, n = 41 and hyperplastic, n= 8) were identified. Twenty-eight (57%) of the polyps were on the left side and 21 (42%) were on the right side of the colon. There was no association between the presence of colon polyps and levels of folate (serum, RBC), vitamin B(12), or homocysteine. Forty-eight individuals (84%) were homozygous for 677 CC. Of these individuals, 18 (37.5%) had >/=1 colorectal polyps, whereas 30 (62.5%) had no polyps. Nine individuals were heterozygous for 677 CT, and 4 (44%) of these individuals had colon polyps. Eighty-eight percent of the APC gene-promoter sequences tested using peripheral blood DNA from patients were unmethylated. Among the individuals who showed APC methylation, 66% had polyps; 33% were polyp free using their blood DNA. There was highly significant association between smoking and alcohol consumption with the presence of a colon polyp (P= .0006 and P= .05, respectively). In conclusion, the lack of the 677 TT may be a significant risk factor for colon neoplasm in the African-American population. Smoking and alcohol consumption were found to be risk factors for colon polyps. APC gene-promoter sequence methylation found in peripheral blood may be an indicator of risk for polyp formation and an important screening tool.

Bax translocation and mitochondrial fragmentation induced by Helicobacter pylori.

BACKGROUND AND AIMS: Previous in vitro and in vivo studies have revealed an association between Helicobacter pylori infection and apoptosis in gastric epithelial cells. Although involvement of the Bcl-2 family of proteins as well as cytochrome c release has been demonstrated in H pylori induced cell death, the exact role of the mitochondria during this type of programmed cell death has not been fully elucidated. Therefore, we sought to determine whether or not Bax translocation and mitochondrial fragmentation occur on exposure of gastric epithelial cells to H pylori, resulting in cell death. METHODS: Experiments were performed with human gastric adenocarcinoma (AGS) cells, AGS cells transfected with the HPV-E6 gene (which inactivates p53 function), AGS-neo cells (transfected with the backbone construct), mouse embryonic fibroblasts (MEFs), and p19(ARF) null (ARF(-/-)) MEFs. Cells were incubated with a cag positive H pylori strain for up to 24 hours, lysed, and cytoplasmic and mitochondrial membrane fractions were analysed by western blot for Bax translocation. RESULTS: Bax translocation was detected in AGS, AGS-neo, and normal MEF cells after exposure to H pylori for three hours, but not in ARF(-/-) MEFs cells. Translocation of Bax after H pylori incubation was also detected in AGS-E6 cells (inactive p53 gene) but to a lesser degree than in AGS-neo cells. In parallel studies, the mitochondrial morphology of living cells infected with H pylori was assessed by confocal microscopy. Mitochondrial fragmentation was detectable after 10 hours of H pylori incubation with AGS cells and after seven hours with MEF cells. In wild-type MEFs, mitochondrial fragmentation was significantly increased in comparison with ARF null MEFs (43% v 10.4%, respectively). Furthermore, mitochondrial depolarisation and caspase-3 activity were initiated within four hours in cells incubated with H pylori, and these events were inhibited by forced expression of Bcl-2. CONCLUSIONS: These data suggest that during H pylori induced apoptosis, Bax translocates to the mitochondria which subsequently undergo depolarisation and profound fragmentation. Functional ARF and p53 proteins may play an important role in H pylori induced mitochondrial modification.

Peptic ulcer disease.

Peptic ulcer disease is a common gastrointestinal disease whose management and treatment has changed dramatically over the last 25 years. Treatment of peptic ulcer disease has evolved from dietary modifications and antacids to gastric acid suppression with H2-receptor antagonists and proton pump inhibitors to eradication of Helicobactor pylori infection. Treatment of patients infected with H pylori using antibiotics has changed the natural history of peptic ulcer disease. As a result of H pylori treatment and other unknown factors ulcer disease is declining and complications from ulcer disease have diminished significantly.

Promoter methylation regulates Helicobacter pylori-stimulated cyclooxygenase-2 expression in gastric epithelial cells.

Cyclooxygenase (COX)-2, the inducible form of the rate-limiting enzyme for prostaglandin synthesis, is up-regulated in gastrointestinal cancers and is a key mediator of epithelial cell growth. Helicobacter pylori is causally linked to gastric cancer. In H. pylori gastritis, COX-2 expression localizes to the subepithelial region, with variable levels in the epithelium. In contrast, in gastric cancer, COX-2 strongly predominates in the epithelium, suggesting that the transition to consistent epithelial COX-2 overexpression may be a critical molecular event in gastric carcinogenesis. Because aberrant promoter methylation inhibits expression of a variety of genes in gastrointestinal cancers, we sought to determine whether methylation of the COX-2 promoter could regulate the response to H. pylori in gastric epithelial cells. We assessed COX-2 expression and promoter methylation status in six gastric epithelial cell lines. In all four of the cell lines that exhibited basal expression of COX-2 and a significant increase in expression in response to H. pylori, the COX-2 promoter was unmethylated, whereas in the two cell lines that did not express COX-2, the COX-2 promoter was methylated. Treatment of COX-2-methylated cells with the demethylating agent 5-azacytidine had a modest effect on COX-2 expression, but when 5-azacytidine-treated cells were subsequently stimulated with H. pylori, there was a significant, 5-10-fold enhancement of both COX-2 mRNA and protein expression and release of the COX-2 product, prostaglandin E2. In contrast, in COX-2-expressing cell lines that were unmethylated at the COX-2 promoter, 5-azacytidine had no effect on H. pylori-stimulated COX-2 expression. These findings suggest that loss of COX-2 methylation may facilitate COX-2 expression and promote gastric carcinogenesis associated with H. pylori infection.

Influence of Helicobacter pylori on reactive oxygen-induced gastric epithelial cell injury.

Risk factors for gastric cancer are receiving renewed attention in light of the recent positive association of Helicobacter pylori infection with gastric cancer. The effect of H.pylori on the balance between oxidants and antioxidants in the stomach is not well known. In this study, we investigated if exposure of gastric cells to H. pylori increases oxidant-associated gastric epithelial cell injury. A human gastric epithelial cell line (AGS) was grown on 96-well clusters, then exposed overnight to either live H.pylori (four cagA(+) and four cagA(-)) or broth culture supernatant from an isogenic H.pylori cagA(+) strain with and without vacA activity. Incubation of AGS cells with cagA(+) and cagA(-) H.pylori strains before exposure to reactive oxygen species (ROS) reduced cell viability on average to 73.7% and 39.5% of controls, respectively. The percent viability of cells exposed to ROS after incubation with control broth, vacA(-) broth and vacA(+) broth was 97.7%, 70.5% and 63.5%, respectively. Experiments were then performed to evaluate the effects of H.pylori exposure on the activities of ROS-scavenging enzymes [catalase, glutathione peroxidase and superoxide dismutase (SOD)] and formation of 8-hydroxy-2-deoxyguanosine (8-OH-dG) adducts in AGS cells. Overnight exposure to cagA(-) strains reduced catalase activity by 42%; in contrast, exposure to cagA(+) H.pylori strains increased catalase activity by 51%. Glutathione peroxidase activity increased with exposure to both cagA(-) and cagA(+) strains by 95% and 240%, respectively. Total SOD activity increased 156% after exposure to cagA(+) strains and was marginally increased (52%) with exposure to cagA(-) strains. CuZn-SOD protein levels, assayed by enzyme-linked immunosorbent assay, were not significantly altered by exposure to H.pylori strains; however, Mn-SOD concentrations were significantly increased (P: < 0.02) after exposure to both cagA(-) and cagA(+) H.pylori strains. Exposure of AGS cells to cagA(+) and cagA(-) H.pylori was associated with, on average, 44.5 and 99.0 8-OH-dG/10(6) dG, respectively. The increase in catalase, glutathione peroxidase and SOD activity is associated with fewer 8-OH-dG DNA adducts and reduced susceptibility of AGS cells to lethal injury from ROS after exposure to cagA(+) H.pylori strains when compared with exposure to cagA(-) H.pylori strains. Alteration in the activity of ROS-scavenging enzymes by the presence of H. pylori may in part be responsible for the increased risk of gastric cancer in persons infected with H.pylori.

Helicobacter pylori inhibits gastric cell cycle progression.

Helicobacter pylori infection of the gastric mucosa is associated with changes in gastric epithelial cell proliferation. In vitro studies have shown that exposure to H. pylori inhibits proliferation of gastric cells. This study sought to investigate the cell cycle progression of gastric epithelial cell lines in the presence and absence of H. pylori. Unsynchronized and synchronized gastric epithelial cell lines AGS and KatoIII were exposed to H. pylori over a 24-h period. Cell cycle progression was determined by flow cytometry using propidium iodide (PI), and by analysis of cyclin E, p21, and p53 protein expression using Western blots. In the absence of H. pylori 40, 45, and 15% of unsynchronized AGS cells were in G(0)-G(1), S, and G(2)-M phases, respectively, by flow cytometry analysis. When AGS cells were cultured in the presence of H. pylori, the S phase decreased 10% and the G(0)-G(1) phase increased 17% after 24 h compared with the controls. KatoIII cells, which have a deleted p53 gene, showed little or no response to H. pylori. When G1/S synchronized AGS cells were incubated with media containing H. pylori, the G(1) phase increased significantly (25%, P < 0.05) compared with controls after 24 h. In contrast, the control cells were able to pass through S phase. The inhibitory effects of H. pylori on the cell cycle of AGS cells were associated with a significant increase in p53 and p21 expression after 24 h. The expression of cyclin E was downregulated in AGS cells following exposure of AGS cells to H. pylori for 24 h. This study shows that H. pylori-induced growth inhibition in vitro is predominantly at the G(0)-G(1) checkpoint. Our results suggest that p53 may be important in H. pylori-induced cell cycle arrest. These results support a role for cyclin-dependent kinase inhibitors in the G(1) cell cycle arrest exerted by H. pylori and its involvement in changing the regulatory proteins, p53, p21, and cyclin E in the cell cycle.

A survey of internal medicine residents' knowledge about Helicobacter pylori infection.

OBJECTIVE: Despite recently published national guidelines, many physicians have only limited knowledge about Helicobacter pylori infection. We conducted this study to assess internal medicine residents' knowledge about H. pylori. METHODS: Two hundred and nineteen residents in seven accredited internal medicine training programs completed a self-administered questionnaire on personal demographics and practices related to testing for-and treating-H. pylori infection. RESULTS: Noon conferences (82%), ward teaching (66%), journals (70%), and sponsored symposia (27%) were their major sources of H. pylori-related information. Forty-eight percent had used office-based tests for the infection. Testing for (and treatment of) Helicobacter pylori infection was recommended by 97% (97%) for newly diagnosed duodenal ulcer, but by only 61% (63%) for a past history of duodenal ulcer. Many recommended testing in unproven conditions and might not have offered treatment to an infected patient. A proton pump inhibitor-based triple-drug regimen was the treatment of first choice of 55%; 20% recommended proton pump inhibitor-based dual regimens. Sixty-six percent and 80%, respectively, underestimated the rates of resistance to clarithromycin and metronidazole. In the absence of gastrointestinal symptoms, 22% would have ordered Helicobacter pylori testing but only 33% of these would undergo treatment if positive. CONCLUSIONS: Internal medicine residents usually test for Helicobacter pylori infection in appropriate conditions, but may not always treat the infection when the result is positive. Most use efficacious treatment regimens although many have inaccurate knowledge of resistance rates, which may adversely influence prescribing. Education should focus on practical issues surrounding Helicobacter pylori testing and treatment such as those contained in the American College of Gastroenterology's 1998 practice guidelines.

Helicobacter pylori, gastric MALT lymphoma, and adenocarcinoma of the stomach.

The discovery of Helicobacter pylori and its relationship to upper gastrointestinal tract diseases has emphasized the significance of infectious pathogens in clinical disease. Severe manifestations of H. pylori-associated diseases include gastric adenocarcinoma and the recently described gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Ongoing worldwide investigations of the interactions of H. pylori and the host response are rapidly clarifying the role of this bacterium in multiple gastrointestinal diseases. This review will address diagnosis, management, and follow-up of the patient presenting with gastric MALT lymphoma, including a discussion of the issues related to premalignant lesions associated with gastric adenocarcinoma. Prospective trials and long-term follow-up studies are in progress and will guide appropriate management of these diseases.

A method for establishing primary cultures of human gastric epithelial cells.

Long-term culture of human gastric epithelial cells has been difficult, and at present no normal human gastric epithelial cell lines are readily available. As part of our experiments to study pathogenesis of H. pylori, a bacterium that infects the stomach, we developed methods to culture normal human gastric epithelial cells. Primary cultures of human gastric epithelial cells can be established from gastric biopsies taken at upper G.I. endoscopy. Enzymatically isolated gastric epithelial-like cells are present in tight colonies on culture dishes within 24 hours of placing the cells in culture. Cells isolated stain positively for cytokeratin and produce neutral mucins, indicating that they are mucin secreting epithelial cells, consistent with gastric epithelial cells. Epithelial cells can be maintained up to 4 weeks in culture with evidence of DNA synthesis up through the first week of culture.

Effects of Helicobacter pylori on proliferation of gastric epithelial cells in vitro.

OBJECTIVE: H. pylori infection of the gastric mucosa has been associated with an increase in gastric epithelial cell proliferation. However, in vitro adherence of H. pylori to gastric epithelial cells is associated with reduced cell proliferation. Reduction of epithelial cell proliferation may contribute to ulcer formation and delay ulcer healing. The following study was undertaken to elucidate the ability of cagA-positive and -negative strains to impede gastric epithelial cell proliferation. METHODS: A human gastric adenocarcinoma cell line (AGS) was overlaid with either cagA-positive or cagA-negative H. pylori strains suspended in cell culture medium. Proliferation of AGS cells was analyzed by performing direct cell counts and by measuring metabolism of a soluble tetrazolium compound (MTS), after exposure to H. pylori for 24 h. RESULTS: When compared with control cells cultured in medium alone, AGS cell proliferation was reduced by 45.6% and 28.5% due to exposure to cagA-negative and cagA-positive strains, respectively. When bacterial-induced cytotoxicity was assessed by measuring release of lactose dehydrogenase (LDH) into the culture medium, cagA-positive strains were shown to induce significantly more cytotoxicity than cagA-negative strains. CONCLUSIONS: These experiments demonstrate that H. pylori exposure to AGS cells significantly reduces cell proliferation. However, cagA-positive strains that induce more cell injury reduce cell proliferation to a lesser extent than cagA-negative strains. Persistent replication of gastric epithelial cells injured by exposure to cagA-positive strains may be partially responsible for the stronger association with gastric cancer in persons infected with cagA-positive H. pylori strains.

Effectiveness of ranitidine bismuth citrate, clarithromycin, and metronidazole therapy for treating Helicobacter pylori.

OBJECTIVE: There are limited data available from the United States on the effectiveness of ranitidine bismuth citrate (RBC) plus two antibiotics to treat Helicobacter pylori. Therefore, the following study was undertaken to evaluate RBC with two antibiotics, which have been used successfully in combination, to treat H. pylori. METHODS: Adults with and without abdominal symptoms, who had never received H. pylori eradication therapy, were tested for the presence of H. pylori infection either by in-office rapid serology assays or histology. Positive subjects were administered the 13C-urea breath test. Subjects who had a positive urea breath test were then treated with RBC 400 mg b.i.d., clarithromycin 500 mg b.i.d., and metronidazole 500 mg b.i.d. for 10 days. Four to 6 wk after completing antibiotics all subjects were asked to return for a second urea breath test to assess treatment success. RESULTS: Forty-seven of the 50 subjects enrolled into this study completed the antibiotic regimen and returned for a repeat urea breath test. Thirty-seven subjects were negative for H. pylori by urea breath test and 10 were positive, resulting in a 79% eradication rate. Seven subjects (14%) stopped their medication because of side effects. When analysis was performed on the 40 subjects who took > or = 80% of their medication (per-protocol), the eradication rate was 90%. CONCLUSIONS: The combination of RBC with clarithromycin and metronidazole successfully treated H. pylori infection after only 10 days of therapy. The per-protocol eradication rate from this study was similar to that seen with Food and Drug Administration (FDA)-approved regimens. In conclusion, RBC plus clarithromycin and metronidazole should be considered as a first-line treatment regimen for H. pylori infection, and may only need to be taken for a period of 10 days, as opposed to 14 days for FDA-approved regimens.

Evidence for distinct intracellular signaling pathways in CD34+ progenitor to dendritic cell differentiation from a human cell line model.

Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC-->DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC-->DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.

How does Helicobacter pylori cause mucosal damage? Direct mechanisms.

Helicobacter pylori-associated gastritis is characterized by an abundant inflammatory response and gastric epithelial cell injury. Adherence of H. pylori to gastric epithelial cells seems to be required for bacterial colonization of the gastric mucosa. Attachment of the bacterium to polarized gastric epithelial cells causes damage to microvilli and stimulates actin polymerization, which is associated with adherence pedestal formation. Studies suggest that H. pylori directly contributes to the injury of gastric epithelial cells by the elaboration of cytotoxic factors. The first toxin identified from H. pylori strains, known as vacuolating cytotoxin, induces vacuole formation in eukaryotic cells. Elaborated enzymes by H. pylori may also contribute directly to epithelial cell injury. Ammonia produced through urease activity may be toxic to gastric epithelial cells. H. pylori protease and lipase degrade gastric mucus and disrupt the phospholipid-rich layer at the apical epithelial cell surface, allowing for cell injury from back diffusion of gastric acid. This cell injury may lead to cell death, believed to result from induction of apoptosis. There are sufficient data to suggest that H. pylori, through direct pathogenic mechanisms, contributes significantly to the gastric mucosal injury associated with this infection, and may enhance the susceptibility of gastric epithelial cells to carcinogenic conversion.

Mitogenic stimulation of T cells reveals differing contributions for B7-1 (CD80) and B7-2 (CD86) costimulation.

The requirement of accessory cells for concanavalin A (Con A) activation of T cells suggests delivery of a separate costimulatory signal. However, the costimulatory pathways involved have not been identified. These studies assess the role of CD28-B7-mediated costimulation during T-cell activation by Con A. The B7-1/B7-2 binding protein CTLA4-Ig inhibited the proliferative response of primary lymph node cells to either Con A or soluble anti-CD3 mAb. This suppression was dose dependent and could be reversed by CD28 cross-linking. CTLA4-Ig also completely suppressed induction of interleukin-2 (IL-2) mRNA by Con A. CTLA4-Ig-mediated suppression was not due to blockade of the Con A 'receptor(s)' or of the primary activation signal (as measured by the intracellular calcium response). Although both B7-1 and B7-2 were up-regulated following Con A activation, each played a different role in proliferation and cytokine production. Individually, anti-B7-2 Fab partially inhibited the Con A response whereas anti-B7-1 Fab had no effect. However, the combination of anti-B7-1 and anti-B7-2 Fab completely suppressed proliferation and IL-2 production. Therefore, while a part of the Con A response requires B7-2, the remainder of the response can utilize either B7-1 or B7-2. Together, these results demonstrate that Con A activation of T cells requires the delivery of a separate costimulatory signal that is mediated almost entirely by the B7 receptors.

Effects of Helicobacter pylori vacuolating cytotoxin on primary cultures of human gastric epithelial cells.

BACKGROUND: Many Helicobacter pylori strains produce a cytotoxin that induces cytoplasmic vacuolation in various types of eukaryotic cells. In contrast with the marked cell vacuolation that occurs in vitro in response to this cytotoxin, comparatively little epithelial vacuolation has been observed in the gastric mucosa of H pylori infected persons. AIMS: Experiments were performed to determine the susceptibility of human gastric epithelial cells in vitro to H pylori vacuolating cytotoxin activity. METHODS: Human gastric epithelial cells, harvested from upper gastrointestinal endoscopic biopsy specimens, were incubated overnight with broth culture supernatants from either a wild type cytotoxin producing (tox+) H pylori strain or an isogenic mutant strain that lacks cytotoxin activity. RESULTS: Prominent cytoplasmic vacuolation occurred in response to tox+ supernatant, but not supernatant from the isogenic mutant strain. Primary human gastric epithelial cells were significantly more sensitive to H pylori vacuolating cytotoxin activity than were either HeLa or AGS cells. Exposure of human gastric epithelial cells to high concentrations of tox+ supernatant for 48 hours caused lethal cell injury. CONCLUSIONS: These studies indicate that primary human gastric epithelial cells are highly sensitive to H pylori vacuolating cytotoxin activity.

Intact antigen receptor-mediated calcium signals in patients with early stage HIV-1 infection.

A variety of deficiencies in T cell activation have been described in HIV-1 infection. To determine whether one component of Ag receptor signal transduction might be impaired and contribute to the immunopathology of HIV infection, we tested CD4 cells from patients with early to mid-stage HIV infection for TCR-induced calcium mobilization. There was no detectable difference between patients and controls in the mean CD4 cell calcium response or in the fraction of responding CD4 cells after cross-linking the TCR with OKT3 Ab. In addition, in HIV-infected patients, there was no correlation between calcium mobilization and the CD4 cell count. These results indicate that there are no intrinsic impairments of Ag receptor calcium signaling in circulating CD4 cells from HIV-infected patients with more than 400 CD4 cells/mm3, although abnormalities in patients with later stage infections cannot be excluded.

Effects of sucralfate on vacuolating cytotoxin activity and adherence of Helicobacter pylori to human gastric epithelial cells.

Sucralfate inhibits activity of certain Helicobacter pylori enzymes, implying that this medication may limit gastric cell injury associated with H pylori infection. This study evaluates the ability of sucralfate and its two major structural components, sucrose octasulfate and aluminum hydroxide, to reduce the cytotoxic effects of H pylori and to inhibit binding of H pylori to human gastric epithelial cells. Experiments were performed using human gastric epithelial cells isolated from gastric biopsy tissue taken at upper gastrointestinal endoscopy. Primary cultures of human gastric epithelial cells, when exposed to broth-culture supernatant from a vacuolating cytotoxin-positive H pylori strain, were shown to form cytoplasmic vacuoles. Preexposing H pylori brothculture supernatant to sucralfate reduced vacuole formation in human gastric epithelial cells; however, preexposure of H pylori broth-culture supernatant to aluminum hydroxide or sucrose octasulfate did not reduce vacuolation in human gastric epithelial cells. H pylori binding to human gastric epithelial cells was significantly reduced when H pylori was exposed to sucralfate prior to incubating the bacterium with human gastric epithelial cells. These data show that sucralfate, but not its two major components, reduces the toxicity of an H pylori-produced cytotoxin (VacA) and decreases H pylori adherence to human gastric epithelial cells. This reduction in H pylori cytotoxicity may contribute to sucralfate's ulcerhealing properties and to the lower ulcer recurrence rates seen in patients treated with this medication.