Host-dependent control of early regulatory and effector T-cell differentiation underlies the genetic susceptibility of RAG2-deficient mouse strains to transfer colitis.

De novo differentiation of CD4(+)Foxp3(+) regulatory T cells (induced (i) Tregs) occurs preferentially in the gut-associated lymphoid tissues (GALT). We addressed the contribution of background genetic factors in affecting the balance of iTreg, T helper type 1 (Th1), and Th17 cell differentiation in GALT in vivo following the transfer of naive CD4(+)CD45RB(high) T cells to strains of RAG2-deficient mice with differential susceptibility to inflammatory colitis. iTregs represented up to 5% of CD4(+) T cells in mesenteric lymph nodes of less-susceptible C57BL/6 RAG2(-/-) mice compared with <1% in highly susceptible C57BL/10 RAG2(-/-) mice 2 weeks following T-cell transfer before the onset of colitis. Early Treg induction was correlated inversely with effector cell expansion and the severity of colitis development, was controlled primarily by host and not T-cell-dependent factors, and was strongly associated with interleukin-12 (IL-12)/23 production by host CD11c(+)CD103(+) dendritic cells. These data highlight the importance of genetic factors regulating IL-12/23 production in controlling the balance between iTreg differentiation and effector-pathogenic CD4(+) T-cell expansion in lymphopenic mice and indicate a direct role for iTregs in the regulation of colonic inflammation in vivo.

Radiation protection in digestive endoscopy: European Society of Digestive Endoscopy (ESGE) guideline.

This article expresses the current view of the European Society of Gastrointestinal Endoscopy (ESGE) about radiation protection for endoscopic procedures, in particular endoscopic retrograde cholangiopancreatography (ERCP). Particular cases, including pregnant women and pediatric patients, are also discussed. This Guideline was developed by a group of endoscopists and medical physicists to ensure that all aspects of radiation protection are adequately dealt with. A two-page executive summary of evidence statements and recommendations is provided. The target readership for this Guideline mostly includes endoscopists, anesthesiologists, and endoscopy assistants who may be exposed to X-rays during endoscopic procedures.

Increased expression of chemokine receptor CCR3 and its ligands in ulcerative colitis: the role of colonic epithelial cells in in vitro studies.

Human colonic epithelial cells express T helper type 1 (Th1)-associated chemoattractants, yet little is known about the production of Th2-associated chemoattractants. CCL11/eotaxin-1, CCL24/eotaxin-2 and CCL26/eotaxin-3 are known to attract CCR3-expressing, Th2-polarized lymphocytes. We studied constitutive and inflammation-induced expression and production of CCR3 together with its ligands in the colon and peripheral blood of patients with inflammatory bowel disease (IBD) by flow cytometry, reverse transcription­polymerase chain reaction (RT­PCR) and enzyme-linked immunosorbent assay (ELISA). We further defined the regulated expression of these chemokines by RT­PCR and ELISA using cultured human epithelial cell lines. A higher fraction of peripheral T lymphocytes were found to be positive for CCR3 in patients with ulcerative colitis (UC) compared to Crohn's disease (CD), while almost no CCR3(+) T cells were found in normal controls (NC). Similarly, higher and more frequent expression of CCR3 was observed in colonic biopsies from patients with UC, regardless of the disease activity, when compared to CD or NCs. Serum CCL11/eotaxin-1 was increased significantly in UC (306 ± 87 pg/ml) and less so in CD (257 ± 43 pg/ml), whereas CCL24/eotaxin-2, and CCL26/eotaxin-3 were increased only in UC. Colonic expression of the three chemokines was minimal in NCs but high in inflammatory bowel diseases (especially UC) and was independent of disease activity. Th2, and to a lesser extent Th1, cytokines were able to induce expression and production of all three eotaxins from colonic epithelial cells in culture. CCR3 and ligands over-expression would appear to be a characteristic of UC. The production of CCR3 ligands by human colonic epithelial cells suggests further that epithelium can play a role in modulating pathological T cell-mediated mucosal inflammation.

Bloodstream infections in patients with solid tumors: associated factors, microbial spectrum and outcome.

BACKGROUND: Although patients with malignant diseases are at increased risk for bloodstream infections (BSIs), limited data are available for those with solid tumors. PATIENTS AND METHODS: The etiology, clinical features and outcome of BSIs were retrospectively studied in patients with solid tumors treated at the Department of Medical Oncology at the University Hospital of Heraklion, Greece, from November 1995 through June 2000. RESULTS: A total of 157 episodes of BSIs was identified among 137 patients over the study period. The majority of the episodes (128; 82%) occurred in non-neutropenic patients. 80 of 157 (51%) of the episodes were healthcare-associated, 35% (55 of 157) were nosocomial and 14% (22 of 157) were community acquired. A single pathogen was isolated in 86% of the episodes. A total of 184 pathogens was isolated (51% gram-negative rods, 44% gram-positive cocci, 3% anaerobes and 3% fungi), while the portal of entry was identified in 104 of 157 (66%) of the episodes. The site of the primary tumor or the metastases were the source of BSI in 39 of 104 (37.5%) of the episodes with an identified source. The overall infectious mortality was 20% and was significantly higher when the initial empirical antibiotic therapy was inappropriate (39%; p < 0.001) and in the presence of shock (63%; p < 0.001). CONCLUSION: BSIs in patients with solid tumors are frequently healthcare associated and in a large percentage the portal of entry can be identified. Neutropenia is not as common as in patients with hematologic malignancies. Inappropriate initial empirical antibiotic therapy and shock are clinical factors associated with worse outcomes.

Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures.

We report a four-step procedure that optimizes the methodology for isolation of highly purified rat Kupffer cells (KC). We combined the previously reported techniques of enzymatic tissue treatment, density gradient centrifugation, centrifugal elutriation and selective adherence. ED-2 immunophenotyping and non-specific esterase histochemistry were used for cell identification. This combination resulted in a satisfactorily high yield of 80-100 x 10(6)KCs per liver, over 95% positive for ED-2 and 98% viable cells. Cultures of isolated KCs were functionally intact and exhibited a concentration and time-dependent LPS-induced TNF-alpha and nitric oxide production.

Matrix metalloproteinases and their inhibitors in acute viral hepatitis.

Matrix metalloproteases (MMPs) and their inhibitors are effector molecules involved in extracellular matrix remodelling. The serum profile for these proteolytic enzymes and their inhibitors during acute self-limiting viral hepatitis has not been studied. We therefore determined serum concentrations of MMP-1, MMP-3, MMP-2, MMP-9 and their inhibitors (tissue inhibitors of metalloproteinase) TIMP-1, TIMP-2 and alpha2 macroglobulin (AMG) in the serum of patients during the icteric stage of self-limiting acute viral hepatitis. Transforming growth factor-beta (TGF-beta) and interleukin (IL)-10, two cytokines involved in the regulation of MMPs and TIMPs were also assessed. Nineteen patients (12 men, seven women) with a mean age of 29.9 years (range 16-65 years) participated in the study. Fifteen had hepatitis B virus (HBV, two HCV and two HAV infection. The values of patients were compared with those obtained from 15 blood donor controls (eight men, seven women), mean age 36.2 years (range 18-55 years). Serum levels of TGF-beta, IL-10, MMP-1, MMP-3, MMP-2, MMP-9, TIMP-1 and TIMP-2 were assessed by ELISA. MMP-2 and MMP-9 were also measured by a zymogram protease assay. alpha2 macroglobulin (AMG) was measured by nephelometry. Compared with the healthy controls the mean serum concentrations of all MMPs were significantly decreased in the acute hepatitis patients. There was no difference in the serum concentration of TIMP-1 between patients and the controls. Serum levels of TIMP-2 (P < 0001), TGF-beta (P < 0.05), IL-10 (P < 0.001) and AMG (P < 0001) were increased in patients compared to healthy controls. A statistically significant negative correlation by linear regression analysis was found between AMG and MMP-1 (P=0003). The decreased levels of MMPs observed, together with normal and increased levels of TIMP-1 and TIMP-2, may indicate an attempt to limit matrix degradation at this stage of disease resolution. The increased levels of the anti-inflammatory cytokines IL-10 and TGF-beta might be the underlying mechanism responsible for the above effect. AMG inhibition especially for MMP-1 may play an additional important role.

Depletion of non specific esterase activity in the colonic mucosa of patients with ulcerative colitis.

BACKGROUND: Non specific esterases (NSE) are a group of cellular carboxylesterases, enzyme markers of monocytes/macrophages, whose tissue distribution in the human body and changes in various disease states have not been adequately studied. We investigate the presence and localization of NSE, in the normal and inflamed human colonic mucosa. DESIGN: NSE were studied histochemically and biochemically using alpha-naphthyl acetate as the substrate, in the colonic mucosa from 67 patients with colitis of various aetiologies and 10 normal controls. In addition, esterase activity was studied biochemically in serum from colitic patients and normal controls. RESULTS: Histochemical study of the colonic tissue demonstrated that NSE were localised in the epithelial brush border, the goblet cells of the glands and a macrophage population of the lamina propria in the colonic mucosa of normal controls and patients with non specific colitis. In active ulcerative colitis, esterase depletion and esterase negative macrophages were identified in parallel with goblet cell disappearance. Gradual reappearance of esterase activity was found after successful treatment. Biochemical study of NSE activity showed that serum and colonic tissue esterase levels were greatly (P < 0.001) reduced in active ulcerative colitis compared to the normal controls or non specific colitis patients and they were increased after successful treatment. Despite this increase, the esterase activity in the colonic tissue from ulcerative colitis patients after treatment was significantly reduced compared to the normal controls. Interestingly, the enzyme levels from non-inflamed areas of the bowel of patients with ulcerative colitis were also significantly (P < 0.01) decreased compared to the normal controls. CONCLUSIONS: These data suggest that esterase reduction in ulcerative colitis is not a simple result of the inflammatory process but rather it precedes its development. This enzyme depletion might have an important pathogenetic implication in the inflammatory process.

Apoptosis and apoptosis related proteins in chronic viral liver disease.

BACKGROUND: Apoptosis may be an important mechanism of hepatocyte death in chronic viral liver disease. METHODS: We studied apoptosis in liver biopsies from 30 patients with chronic viral hepatitis and 8 patients with viral cirrhosis by the TUNEL method. 12 cases of non-alcoholic steatohepatitis and 12 cases of primary biliary cirrhosis were used as non-viral disease controls. Immunohistochemical expression of p53, p21/waf1, bcl-2 and mdm-2 proteins was also studied in the same patients. RESULTS: A statistically significant increase of apoptotic liver cells was found in severe chronic viral hepatitis (5.3 +/- 0.3%), cirrhosis (3.4 +/- 0.5%) and PBC (4.4 +/- 0.4%) cases compared to patients with non-alcoholic steatohepatitis (0.8 +/- 0.3%). The expression of p53 protein was increased in the cases of viral cirrhosis and in chronic severe viral hepatitis whereas in the cases of chronic mild hepatitis, PBC and non-alcoholic steatohepatitis we found no expression of p53. P21/waf1 expression was increased in severe chronic hepatitis, cirrhosis and PBC cases compared to mild hepatitis and non-alcoholic steatohepatitis cases. However no induction of mdm-2 was observed in the subgroups of chronic liver disease. Bcl-2 was expressed only in epithelium of bile ducts and mononuclear cells of the portal tracts and liver lobules. A weaker Bcl-2 expression was noted in the epithelium of bile ducts of 7/12 PBC cases. CONCLUSION: Our results provide evidence of increased apoptosis in severe chronic viral liver disease, suggesting that apoptotic cell death might be involved in the pathogenesis of hepatocellular damage of viral hepatitis and cirrhosis. Furthermore we analysed part of the apoptotic pathways implicated in the above process and found an increased expression of p21/waf1, probably p53 mediated, without overexpression of the apoptosis inhibiting bcl-2 and mdm-2 proteins. By contrast p21/waf1 overexpression in PBC seems to be propagated by a p53 independent mechanism.

Bolus somatostatin but not octreotide reduces hepatic sinusoidal pressure by a NO-independent mechanism in chronic liver disease.

BACKGROUND: Evidence exists that somatostatin and octreotide might have different effects on hepatic haemodynamics. AIM: The investigation of the effects of somatostatin and its octapeptide analogue, octreotide, on sinusoidal pressure measured by the wedged hepatic venous pressure in patients with cirrhosis or chronic hepatitis and the correlation with the levels of hepatic vein NO. METHODS: Patients were randomly assigned to receive an injection of either 250 microg somatostatin (n=14: cirrhosis six, chronic hepatitis eight) or an injection of 125 microg octreotide (n=19: cirrhosis nine, chronic hepatitis 10) during hepatic vein catheterization. Baseline wedged hepatic venous pressure was measured, followed by measurements at 2, 5, 10 and 15 min after the injection of the drug. Nitrites/nitrates of the hepatic vein were measured before the injection and after 15 min. RESULTS: Both agents showed a similar qualitative but a different quantitative haemodynamic profile. No change in the wedged hepatic venous pressure was observed during the first 2 min after the injection of both drugs. This was followed by a decrease: 18% at 5 min (N.S.), 23% at 10 min (P < 0.01) and 24% at 15 min (P < 0.01) for somatostatin. Octreotide induced a relatively smaller decrease in the wedged hepatic venous pressure: 8% at 5 min (N.S.), 20% at 10 min (P < 0.01) and 16% at 15 min (N.S.). Further analysis of the sub-groups of cirrhotic and chronic hepatitis patients revealed a different effect. In the sub-group of cirrhotic patients, somatostatin caused a maximum decrease of 34% at 15 min post-injection (P < 0.01), but octreotide failed to produce a significant change on the wedged hepatic venous pressure. In contrast, no change was observed in chronic hepatitis patients with either drug. No change in the hepatic vein concentration of NO after treatment was observed with either somatostatin or octreotide. Moreover, no correlation of the levels of NO with the wedged hepatic venous pressure values was found. CONCLUSIONS: This study shows that somatostatin is more effective than octreotide in acutely reducing the wedged hepatic venous pressure after bolus injection and the observed change is probably mediated by a NO-independent mechanism.

Anemia of chronic disease is the more frequent type of anemia seen in patients with chronic idiopathic neutropenia of adults.

This study describes the frequency and the type of anemia seen in patients with nonimmune chronic idiopathic neutropenia of adults (NI-CINA). We found that NI-CINA patients had low hemoglobin levels and increased serum concentrations of erythropoietin (EPO), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). The hemoglobin levels correlated positively with the number of circulating neutrophils and inversely with the levels of EPO and TNF-alpha but not of IL-1beta. Anemia, defined as the reduction of the hemoglobin below 12.0 g/dl for women and 13.3 g/dl for men, was found in 23 out of 148 patients studied, a proportion of 15.5%. Two of the anemic patients had iron deficiency anemia (8.7%), 11 had anemia of chronic disease (ACD; 47.8%) presenting with normal or slightly reduced erythrocytic indices, low serum iron, and increased serum ferritin, and the remaining ten had anemia of undefined pathogenesis (AUP; 43.5%) with normal or slightly decreased erythrocytic indices, serum iron ranging from 43 to 88 microg/dl, and ferritin values ranging from 12 to 50 ng/ml. We conclude that ACD is the more frequent type of anemia seen in patients with NI-CINA, and that pro-inflammatory cytokines, notably TNF-alpha, may be involved in the pathogenesis of both ACD and AUP, given that serum levels of the cytokine were significantly increased and that the EPO response to anemia was blunted in these patients. These findings further support our previously reported suggestion for the possible existence, in NI-CINA patients, of an unrecognized low-grade chronic inflammatory process that may be involved in the pathogenesis of the disorder.

Levels of circulating endothelin-1 and nitrates/nitrites in patients with virus-related hepatocellular carcinoma.

A balance between endothelins (ET) and nitric oxide (NO) might interfere with liver haemodynamics and disease progression in various liver diseases. Increased levels of endothelin 1 (ET-1) and nitrites and nitrates (NOx, the end products of NO metabolism) have been reported in hepatocellular carcinoma (HCC), but the balance has not been studied. The purpose of this study was to assess the ratio of NOx to ET-1 in patients with virus-related hepatocellular carcinoma and to investigate its correlation with the extent of the disease. Eighteen patients with virus-related HCC (six Okuda stage I, six Okuda stage II and six Okuda stage III) were included in the study and were compared with 22 patients with viral cirrhosis (14 decompensated, eight compensated) and seven normal controls. ET-1 was measured with an ELISA assay and NOx with a modification of the Griess reaction. Patients with virus-related HCC had the highest levels of circulating ET-1 and NOx (13.24 +/- 0.82 pg/ml and 112.28 +/- 18.56 micromol/l) compared to compensated cirrhosis (9.47 +/- 0.50 pg/ml, P < 0.004 and 54.47 +/- 2.36 micromol/l, P < 0.01), decompensated cirrhosis (9.57 +/- 0.32 pg/ml, P < 0.001 and 90.20 +/- 11.23 micromol/l, NS) and normal controls (8.84 +/- 0.61 pg/ml, P < 0.001 and 51.17 +/- 6.18 micromol/l, P < 0.01). There was a significant increase of ET-1 and NOx at HCC stage III compared to HCC stages I and II, cirhotics and controls. HCC stage III patients also had a NOx/ET-1 ratio that was higher than HCC stages I and II patients, normal controls and patients with compensated cirrhosis. Virus-related HCC patients have high levels of circulating ET-1, compared to compensated or decompensated cirrhosis. Highest levels of ET-1 are produced in Okuda III tumours. NOx are also increased but only in Okuda stage III tumours. The NOx/ET-1 ratio is increased in virus-related HCC and DC. This increase may account for the known increase in tumour blood flow.