The sentinel node technique is useful for studies of intestinal immunology in inflammatory bowel disease patients

OBJECTIVE: Mesenteric lymph nodes may play a crucial role in the pathogenesis of human inflammatory bowel disease. We have used the sentinel node technique to analyze mesenteric lymph nodes draining inflammatory lesions sentinel nodes and corresponding site of inflamed bowel in patients with ulcerative colitis or Crohn's disease. METHODS: Thirty-two patients undergoing surgical treatment of inflammatory bowel disease were included. Sentinel nodes were identified intraoperatively. The T cells were harvested from the mesenteric lymph nodes and characterized by flow cytometry. RESULTS: The distribution of CD4CD62L (homing-marked) and CD4CD69 (activated) T cells was studied in mesenteric lymph nodes draining inflammation, nodes draining normal intestine, blood, and mucosa. The turnover of T cells was markedly increased in the lymph nodes connected to inflammatory segments. The immunologic activity of the sentinel lymph nodes correlated with the degree of intestinal inflammation. CONCLUSION: Mesenteric sentinel nodes provide important information about locoregional immunology and pathogenesis in inflammatory bowel disease.     

Telomerase activity in B and T lymphocytes of patients with systemic lupus erythematosus

OBJECTIVE: To evaluate telomerase activity as a marker of lymphocyte proliferation in systemic lupus erythematosus (SLE). METHODS: CD19+, CD4+, and CD8+ lymphocytes were isolated from the peripheral blood of nine patients with SLE and nine healthy controls by means of magnetic bead-coupled antibodies and tested for telomerase activity with the TRAP assay. RESULTS: Telomerase activity was significantly increased in CD19+ B cells from patients with SLE. CD4+ and CD8+ T cells from lupus patients displayed increased mean telomerase activity, although the difference from normal controls did not reach statistical significance. CONCLUSIONS: Increased telomerase activity in the B and the T cell lineage might indicate activation and proliferation of these lymphocytes.     

免疫功能受损宿主外周血单个核细胞端粒酶活性研究

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Activation of NF-κB in intestinal epithelial cells by<Emphasis Type="Italic"> E. coli</Emphasis> strains isolated from the colonic mucosa of IBD patients

Background and aims The involvement of bacteria in the pathogenesis of inflammatory bowel disease has been discussed for several years. In this study we evaluated the ability of E. coli isolates from inflamed and noninflamed colonic mucosa to activate NF-B.Materials and methods Fifteen bacterial strains from inflamed and six from noninflamed colonic tissues from IBD patients. Their ability to induce NF-B activation was examined in vitro by gel-shift assays. The activation of the TNF- promoter was determined by reporter gene assays. Bacterial isolates were characterized by invasion assays, electron microscopy, and PCR.Results Four of 15 E. coli bacterial isolates from inflamed IBD tissues induced NF-B activity in intestinal epithelial cells as determined by gel-shift assays. NF-B activation was only seen with living bacteria but not with heat-inactivated cells. Isolates from noninflamed tissues and a wild-type E. coli control strain induced a weaker or no activation. Reporter gene assays with a construct comprising a luciferase gene driven by the TNF- promoter revealed that isolates from Crohns disease patients induced a stronger activation of the TNF- gene than isolates from ulcerative colitis patients. The isolated bacteria invaded HT-29 cells, although typical virulence genes for enteropathogenic, enterhemorrhagic, or enteroinvasive E. coli, i.e., eae, tir, EspA, Per (A-C), ipaC, were not detected in these cells. Bacterial invasion was additionally confirmed by electron microscopy examination.Conclusion Our results indicate that E. coli strains can be found in the mucosa of some IBD patients which are able to activate NF-B similar to known pathogenic strains. The absence of several virulence genes in these cells suggests that they are members of the luminal flora which acquire as yet unidentified virulence determinants and are therefore involved in the pathophysiology of IBD.     

Expression of platelet-derived endothelial cell growth factor in inflammatory bowel disease

Background: Platelet-derived endothelial cell growth factor (PD-ECGF) is reported to be highly expressed in tumors and inflammatory tissues, but its expression and role in inflammatory bowel disease (IBD) are still unclear. In this study we examined the location and tissue density of cells immunoreactive for PD-ECGF in the colonic mucosa of IBD. Methods: Paraffin-embedded sections of colonic tissue from patients with ulcerative colitis (UC) or Crohn's disease (CD) were immunostained for PD-ECGF. As controls, noninflamed mucosa of IBD, as well as normal colonic mucosa from patients with colorectal cancer, were used. Also, cancer tissues were evaluated. In addition, changes in the expression of PD-ECGF in human umbilical vein endothelial cells (HUVEC) after treatment with inflammatory cytokines and angiogenic factors, as well as after coculture with colon cancer cell lines, were evaluated by flow cytometry. Results: In normal colonic mucosa and noninflamed mucosa of IBD, PD-ECGF expression was negligible. In inflamed colonic mucosa, strong expression was observed, predominantly in macrophages and fibroblasts. Vascular endothelial cells of the inflamed colonic mucosa, but not of normal colonic mucosa or of neoplastic tissues, stained for PD-ECGF, and the microvessel density was significantly increased in the severely inflamed mucosa. Flow cytometry demonstrated that PD-ECGF was constitutively expressed in HUVEC. Inflammatory cytokines and vascular endothelial growth factor (VEGF) increased its expression, whereas basic fibroblast growth factor (bFGF) decreased it. Coculture with colon cancer cell lines in direct contact, but not in those without contact, also resulted in an important decrease in the expression of PD-ECGF in HUVEC. Conclusions: Autocrine production of PD-ECGF by endothelial cells may be a mechanism of inflammatory angiogenesis, but not tumor angiogenesis, and may be particularly important for the maintenance of damaged vasculature in IBD. Received: September 17, 2001 / Accepted: September 6, 2002 Acknowledgments. This study was supported partly by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and partly by a grant from the Ministry of Health, Labour, and Welfare of Japan. Reprint requests to: S. Saito, Present address: Department of Surgery, Chigasaki Municipal Hospital, 5-15-1 Honson, Chigasaki 253-0042, Japan     

Characterization of colonic and mesenteric lymph node dendritic cell subpopulations in a murine adoptive transfer model of inflammatory bowel disease

Ulcerative colitis and Crohn's disease, collectively termed inflammatory bowel diseases (IBD), are chronic inflammatory diseases of the intestine that afflict more than 4 million people worldwide. Intestinal inflammation is characterized by an abnormal mucosal immune response to normally harmless antigens in the gut flora. In Crohn's disease, the pathogenic mucosal immune response is a typical T helper (TH1) type cell response, whereas ulcerative colitis is predominantly associated with a TH2 response. We are interested in the role of dendritic cells in early immunologic events leading to T cell activation and chronic intestinal inflammation. Using a murine adoptive transfer model of IBD, we found an accumulation of dendritic cells in colon and mesenteric lymph nodes during the early stage of IBD before the appearance of epithelial lesions and tissue degradation. In situ immunostaining and flow-cytometric analysis revealed that approximately 50% of colonic dendritic cells were CD11b B220 myeloid dendritic cells and 50% expressed the CD11b B220 plasmacytoid phenotype. In corresponding mesenteric lymph nodes, approximately 16% were plasmacytoid dendritic cells. Colonic myeloid dendritic cells were shown to express the co-stimulatory molecule CD40. Both, colonic myeloid and plasmacytoid dendritic cells released interferon-alpha in situ and stimulated T cell proliferation ex vivo. Our results show that dendritic cells can mature in the intestine without migrating to mesenteric lymph nodes. Mature intestinal dendritic cells may form a nucleation site for a local T cell response and play an important role in the pathogenesis of IBD.     

Telomerase activity in normal and malignant hematopoietic cells.

Bone marrow and peripheral blood leukocytes from 19 leukemia patients were found to contain telomerase activity detectable by a PCR-based assay. Telomerase was also detectable in nonmalignant bone marrow and peripheral blood leukocytes from normal donors, including fractions enriched for granulocytes, T lymphocytes, and monocytes/B cells. Semiquantitative comparison revealed considerable overlap between telomerase activities in samples from normal subjects and leukemia patients, confounding evaluation of the role of telomerase in this disease. These data indicate that human telomerase is not restricted to immortal cells and suggest that the somatic expression of this enzyme may be more widespread than was previously inferred from the decline of human telomeres.     

High Prevalence of Mycoplasma pneumoniae in Intestinal Mucosal Biopsies from Patients with Inflammatory Bowel Disease and Controls

Intestinal microflora are believed to play an important role in the pathogenesis of inflammatory bowel disease (IBD). Mycoplasma have been suggested previously as organisms of ubiquitous distribution with the potential to cause inflammatory diseases, including IBD in susceptible individuals. The aim of this study was to determine the frequency of the presence of M. pneumoniae DNA in intestinal biopsies from patients with IBD and non-IBD controls using a microplate polymerase chain reaction–hybridization assay (PCR-ELISA). A total of 260 endoscopic biopsies (49 from 19 patients with Crohn's disease, 76 from 27 patients with ulcerative colitis, and 135 from 43 non-IBD controls) were used in this study. Overall, M. pneumoniae-specific DNA was detected in 100 endoscopic biopsy samples (38.5%). Among them, the detection rate of M. pneumoniae DNA was significantly higher in biopsies from patients with CD (59.2%) than in those from patients with UC (26.3%) or non-IBD controls (37.7%) (² = 13.65, P 0.001). The high prevalence of M. pneumoniae in both IBD patients and controls suggest this organism is ubiquitous and may persist in the intestinal mucosa. Epidemiological studies in IBD suggest acquisition of some agents early in life probably during epidemics in temperate latitudes. M. pneumoniae could be one of the ubiquitous agents implicated in the pathogenesis of IBD.     

Gut Lavage Fluid Proteins as Markers of Activity of Inflammatory Bowel Disease

Intestinal secretions may be obtained by gut lavage using a polyethyleneglycol-based electrolyte lavage solution; concentrations of immunoglobulins and other proteins are readily measured in processed gut lavage fluid. As patients with inflammatory bowel disease (IBD) have greatly increased numbers of IgG-producing intestinal immunocytes, we measured gut lavage fluid IgG levels in 44 patients with IBD with various degrees of disease activity to determine whether total IgG in gut lavage fluid reflects disease activity. We also measured levels of albumin in gut lavage fluid, to determine the degree of plasma leakage. Both IgG and albumin levels in the patients with active IBD were significantly higher than those in controls and patients with inactive IBD (all p < 0.00001). IgG is a more specific index of disease activity than albumin, with no overlap between levels in controls and patients with active IBD. There was a positive correlation (r = 0.68, p < 0.0001) between IgG and albumin levels, suggesting that gut lavage fluid IgG is mainly plasma-derived.     

Colonic Ornithine Decarboxylase in Inflammatory Bowel Disease: Ileorectal Activity Gradient,Guanosine Triphosphate Stimulation,and Association with Epithelial Regeneration but Not the Degree of Inflammation and Clinical Features

The role of colonic mucosal ornithine decarboxylase (ODC) in inflammatory bowel disease (IBD) remains controversial. This study assessed mucosal ODC activity in IBD patients segment by segment with regard to patient characteristics, disease activity/duration, medication, degree of mucosal inflammation, and presence/absence of epithelial regeneration and guanosine triphosphate (GTP) stimulation. Mucosal ODC activity was determined in biopsy specimens from the terminal ileum, cecum/ascending, transverse, and descending colon, and the sigmoid/rectum of 35 patients with IBD (18 with Crohn’s disease, 17 with ulcerative colitis) and 29 controls, using the amount of ¹⁴CO₂ liberated from (carboxyl-¹⁴C)ornithine hydrochloride. GTP-stimulatable activity was expressed as the ratio of ODC activity in the presence and absence of GTP (70 μmol/L). Mucosal inflammation was assessed endoscopically/microscopically with previously described criteria. Presence/absence of mucosal regeneration also was determined by predefined criteria. Mucosal ODC-activity did not significantly differ in IBD patients and controls. There was a 4.4-fold activity gradient from the ileum to the rectum. Mucosal ODC activity was significantly higher in areas with epithelial regeneration compared to those without regeneration, and was stimulated by GTP by a factor of 1.42 in Crohn’s disease and 1.19 in ulcerative colitis patients compared to controls (p < 0.004). On the other hand, there was no significant association/relationship of mucosal ODC activity with disease activity/duration and the endoscopic/histologic degree of mucosal inflammation. The observation of unchanged mucosal ODC activity in patients with IBD and the absence of a significant relationship with clinical and endoscopic/histologic disease characteristics speaks against a major role of ODC in IBD as a major disease marker. The role of the ileorectal gradient, the enhanced activity in areas with epithelial regeneration, and the GTP-stimulatable form, however, need further investigation with regard to a possible involvement in carcinogenesis in IBD.     

Perihilar lymph nodes in patients with primary sclerosing cholangitis with and without cholangiocellular carcinoma

Objective. Enlarged perihilar lymph nodes have been described in patients with primary sclerosing cholangitis (PSC). The aim of the study was to determine the clinical relevance of perihilar lymph nodes in PSC patients with and without cholangiocellular carcinoma (CCC). Material and methods. The status of perihilar lymph nodes was investigated in 117 patients with PSC using “high-end” ultrasound. Thirty-five of the 117 PSC patients had histologically proven CCC. Lymph node status was correlated with the presence of CCC and inflammatory bowel disease (IBD). Results. Seventy-three percent of PSC patients without CCC and 86% of patients with CCC had enlarged perihilar lymph nodes (NS). In CCC patients, the width of lymph nodes was significantly larger (12±6 mm versus 8±4 mm; p=0.0001), and the length:width ratio (2.15±0.7:1 versus 2.5±0.6:1; p=0.004) of the lymph nodes was significantly lower. Thirty-seven percent of PSC patients without CCC and 57% of patients with PSC and CCC had multiple perihilar lymph nodes (p=0.04). In all patients, the presence versus absence of IBD had no influence on the number (84% versus 74%,) and size of perihilar lymph nodes (length: 21±10 mm versus 19±7 mm). Lymph node status did not correlate with the number of episodes of cholangitis. Conclusions. Enlarged perihilar lymph nodes are characteristic of patients with PSC. Since perihilar lymph nodes are not predictive of the presence of complicating CCC, such patients should not be excluded from liver transplantation.     

Inflammatory response in patients with malignant obstructive jaundice

Objective. Surgery in patients with malignant obstructive jaundice is associated with increased risks for postoperative septic complications. The aim of this study was to investigate the inflammatory and the local cellular immune response in patients accepted for surgery because of tumours in the hepatic-pancreatic-biliary (HPB) tract. Material and methods. Patients with obstructive jaundice (group HPB⁺) were compared with those without (HPB^?). Patients undergoing surgery for benign abdominal disorders served as controls. Obstructive jaundice was present in 18 out of 33 HPB patients. Preoperatively, blood was analysed for bacteria, endotoxins and cytokines (TNF-α, IL-6 and IL-10). At operation, mesenteric lymph nodes (MLNs) were excised for bacterial cultures using standard microbiological techniques, and immunohistochemistry, using antibodies CD4 and CD8 (mainly staining T lymphocytes), CD68 (macrophages), and anti-caspase-3 (to determine the rate of apoptosis). Results. Bacterial translocation was not demonstrated in any of the patients. Increased preoperative concentrations of endotoxins were found in group HPB⁺. The number of macrophages and the rate of apoptosis in MLNs were increased in jaundiced patients, while the number of T lymphocytes was decreased. Conclusions. Malignant obstructive jaundice causes increased blood concentrations of endotoxins and cytokines, an increased number of macrophages in MLNs, a higher rate of apoptosis in MLNs, but a decreased number of T lymphocytes in MLNs. The lymphocyte depletion is probably due to the increased rate of apoptosis, and might reduce the ability of jaundiced patients to eradicate infection.     

Lamina Propria and Circulating Interleukin-8 in Newly and Previously Diagnosed Pediatric Inflammatory Bowel Disease Patients

Dysregulation of interleukin-8 (IL-8) production has been proposed to contribute to intestinal inflammation in inflammatory bowel disease (IBD) patients. Previous studies, which evaluate adult patients with long-standing or steroid-modulated disease, have reported conflicting results regarding the role of IL-8 in IBD pathogenesis. The present study evaluates IL-8 in colonic organ cultures and sera of newly and previously diagnosed pediatric IBD patients with various degrees of histopathologic activity. Colon and terminal ileum biopsies were obtained from 26 patients with Crohn’s disease, 12 with ulcerative colitis, 4 with indeterminate colitis, and 12 age-matched normal controls. IBD patients were additionally characterized as newly or previously diagnosed. Supernatants from organ-cultured lamina propria biopsies and sera were evaluated by ELISA for IL-8 protein. IL-8 increased with degree of histologic inflammation regardless of diagnosis (no pathologic diagnosis, 62.6 ng/ml, interquartile range [IQR] 30.4–94.6 ng/ml; mild, 92.0 ng/ml, IQR 21.9–170.0 ng/ml; moderate, 676.2 ng/ml, IQR 46.4–2967.7 ng/ml; severe, 585.6 ng/ml, IQR 149.7–1602.2 ng/ml; P < 0.01). Lamina propria IL-8 was significantly elevated in moderately and severely inflamed tissue segments (603.26 ng/ml; IQR, 72.15–2240.4 ng/ml) compared to noninflamed and mildly inflamed segments (67.70 ng/ml; IQR, 30.38–124.1 ng/ml; P = 0.0009). There was no significant trend in IL-8 concentration when compared by clinical diagnosis. No significant difference was found in IL-8 concentrations in organ cultures from newly diagnosed patients versus those from previously diagnosed patients. There was no significant correlation between serum IL-8 concentration and organ culture IL-8 concentration. We conclude that higher concentrations of IL-8 are found in more histologically inflamed tissue segments from pediatric IBD patients. IL-8 does not appear to be associated with clinical IBD subtype. IL-8 appears to be an integral part of both early and established mucosal inflammation in pediatric IBD patients. These findings suggest that IL-8-specific therapies may universally modify inflammatory activity in IBD patients.     

Perinodal Adipose Tissue and Mesenteric Lymph Node Activation During Reactivated TNBS-Colitis in Rats

Background  

Colitis induced by trinitrobenzene sulfonic acid (TNBS) with reactivation is a good experimental model for studying inflammatory bowel disease pathogenesis and appropriate therapeutics. This experimental model allows the induction of colitis relapse and remission periods and the establishment of chronic disease features, such as the mesenteric adipose tissue alterations observed in Crohn’s disease. Lymph node activation and the role of perinodal adipose tissue (PAT) have been poorly studied in this model. Thus, a study of the interactions of lymph nodes and PAT could help to elucidate the mechanisms behind IBD pathogenesis.     

Intestinal colonization with phylogenetic group B2 Escherichia coli related to inflammatory bowel disease: a systematic review and meta-analysis

Abstract

Background and objectives. Increased numbers of Escherichia coli and, furthermore, specific subtypes of E. coli, such as E. coli of the phylogenetic groups B2 and D have been found in the intestine of patients with inflammatory bowel disease (IBD). In this review, we wanted to evaluate the relationship between B2 and D E. coli intestinal colonization and IBD. Methods. A systematic review with meta-analyses. We included studies comparing colonization with B2 and D E. coli in IBD patients and in controls. Random-effects and fixed-effect meta-analyses were performed. Results. We included 7 studies on 163 patients with IBD and 89 controls. Among IBD patients, 57 patients had ulcerative colitis (UC) and 95 Crohn’s disease (CD). Random-effects meta-analysis showed that IBD patients were more likely to have B2 E. coli intestinal colonization compared with controls (odds ratio [OR]: 2.28; 95% confidence interval [CI]: 1.25–4.16). There was little between-study heterogeneity (I² = 0). The result was confirmed in subgroup analyses of patients with UC (OR: 3.58; 95% CI: 1.62–7.90), but not CD (OR: 1.94; 95% CI: 0.98–3.82). Intestinal colonization with phylogenetic group D E. coli was not found to be related to IBD, UC or CD. Conclusions. Our study reveals that intestinal colonization with phylogenetic group B2 E. coli is associated with UC. Due to the design, we are unable to determine if the colonization with B2 E. coli leads to the development of the disease or the disease increases the risk of colonization with B2 E. coli.     

Correlation of telomerase activity to apoptosis and survival in adult acute lymphoblastic leukemia: an Egyptian single-center study

Telomerase is activated in most tumors, but suppressed in normal human somatic cells. Current evidence indicates that telomerase reactivation is a critical step in carcinogenesis, with a close relationship to apoptosis. The goal of this study was to investigate the levels and relationship of telomerase activity to apoptosis and its impact on the survival of Egyptian adult acute lymphoblastic leukemia patients. Telomerase activity was quantified by polymerase chain reaction (PCR) and detected by enzyme-linked immunosorbent assay (ELISA), while apoptosis was measured at the single-cell level by fluorescence in situ detection using flow cytometry in 15 control subjects and 40 acute lymphoblastic leukemia (ALL) patients at presentation. Telomerase activity in ALL patients was negatively correlated to apoptosis [percent and mean fluorescence intensity (MFI)] (p < 0.001 for percent and p < 0.001 for MFI) and to the 4-year survival rate (p < 0.05), to which apoptosis (percent and MFI) was consequently positively correlated (p < 0.001 for percent and p < 0.05 for MFI). For telomerase, the highest positive predictive value (PPV) for mortality (93.3%) was at a cut-off value of 13 amol/ml, while those for apoptosis (85% for percent of apoptotic cells and 90.9% for MFI) were at a cut-off of 8% and 0.19 MFI. This makes the measurement of telomerase activity in ALL patients a potential tool to predict disease with unfavorable outcome and a candidate tumor marker.     

Peripheral blood and mesenteric lymph node lymphocytes in Crohn's disease.

Analysis of peripheral blood lymphocytes from 44 patients with Crohn's disease showed no difference in the proportions of T- and B-cells from those in 38 healthy controls. Analysis revealed no disturbances in relation to duration or to activity of disease or to drug treatment. Lymphocytes from 18 patients with rheumatoid arthritis also showed normal proportions of T- and B-cells. Lymphocytes taken from gut lymph nodes were studied in five patients with Crohn's disease. On comparison with peripheral blood lymphocytes, significantly decreased proportions of T-cells and significantly increased proportions of B-cells were found in lymph nodes draining areas of diseased bowel. No differences were seen in the proportions of T- and B-cells from lymph nodes taken from apparently healthy bowel of the Crohn's patients and of four control subjects without inflammatory bowel disease, though these were different from those in the peripheral blood in both the Crohn's patients and control subjects.     

Telomerase activity in peripheral blood mononuclear cells of systemic connective tissue diseases

OBJECTIVE: Telomerase activity has been detected in a large number of cancers, as well as human germline tissue, but is absent in most normal somatic tissue. It has been reported that telomerase is also expressed at a low level in normal peripheral blood lymphocytes and that its activity is increased by antigen processing. We investigated if the telomerase activity in peripheral blood mononuclear cells (PBMC) from patients with systemic connective tissue diseases reflects disease activity. METHODS: We examined the enzyme activity of PBMC from patients with systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), primary Sj?gren's syndrome (SS), and systemic sclerosis (SSc) using the telomeric repeat amplification protocol. In SLE and SS patients, the telomerase activity level was assessed for correlations with clinical disease activity. RESULTS: Telomerase activity was detected in 64.7% (11/17) of SLE patients, 63.6% (7/11) of MCTD, 54.5% (6/11) of SS, and 44.4% (4/9) of SSc. There was a significant correlation between SLE Disease Activity Index scores and telomerase activity in patients with SLE (p < 0.01). In patients with SS, telomerase activity was detected predominantly in patients with extraglandular manifestations, but was less detectable in patients without extraglandular manifestations (p < 0.05). CONCLUSION: Our data show that telomerase activity of PBMC is an indicator of disease activity, and may play a role in pathogenesis of systemic connective tissue diseases.