Modulation of T-cell responsiveness during Trypanosoma cruzi infection: analysis in different lymphoid compartments

Spleen and lymph node cells of Trypanosoma cruzi-infected mice were studied for mitogen-induced responsiveness in terms of proliferation and lymphokine production (IL-2, IFN-gamma). Splenocyte (SP) as well as lymph node cell (LN) proliferation and IL-2 production were depressed during the acute phase of the infection. Proliferative capacity of LN cells recovered completely and that of SP partially during the chronic phase. In contrast to these suppressive effects, the mitogen-induced IFN-gamma response was enhanced. In vitro co-incubation of normal SP or LN cells with trypomastigotes resulted in a reduced mitogen-induced cell proliferation and IL-2 secretion, similar to those seen with cells taken from infected mice. In contrast, trypomastigotes exerted a stimulatory activity on the mitogen-induced IFN-gamma response of both SP and LN cells. Addition of lymph node cells from T. cruzi-infected mice (LN-I) to lymph node cells of control mice (LN-C) suppressed strongly the mitogen-induced responsiveness of such cocultures. A marginal level of suppression was recorded in cocultures of spleen cells from infected mice (SP-I) and control spleen cells (SP-C). The potent suppressive cells within LN-I populations were identified as macrophage-like and such cells were absent in SP-C and peritoneal exudate cells from T. cruzi infected animals.     

Rebamipide alleviates radiation‐induced colitis through improvement of goblet cell differentiation in mice

Background and Aim

Radiation‐induced colitis is a common clinical problem associated with radiotherapy and accidental exposure to ionizing radiation. Goblet cells play a pivotal role in the intestinal barrier against pathogenic bacteria. Rebamipide, an anti‐gastric ulcer drug, has the effects to promote goblet cell proliferation. The aim of this study was to investigate whether radiation‐induced colonic injury could be alleviated by rebamipide.

Methods

This study orally administered rebamipide for 6 days to mice, which were subjected to 13 Gy abdominal irradiation, to evaluate the therapeutic effects of rebamipide against radiation‐induced colitis. To confirm the effects of rebamipide on irradiated colonic epithelial cells, this study used the HT29 cell line.

Results

Rebamipide clearly alleviated the acute radiation‐induced colitis, as reflected by the histopathological data, and significantly increased the number of goblet cells. The drug also inhibited intestinal inflammation and protected from bacterial translocation during acute radiation‐induced colitis. Furthermore, rebamipide significantly increased mucin 2 expression in both the irradiated mouse colon and human colonic epithelial cells. Additionally, rebamipide accelerated not only the recovery of defective tight junctions but also the differentiation of impaired goblet cells in an irradiated colonic epithelium, which indicates that rebamipide has beneficial effects on the colon.

Conclusions

Rebamipide is a therapeutic candidate for radiation‐induced colitis, owing to its ability to inhibit inflammation and protect the colonic epithelial barrier.     

Active suppression of interleukin 2 secretion in mice infected with Trypanosoma brucei AnTat 1.1. E

Impairment of T cell proliferation in mice infected with the pleomorphic Trypanosoma brucei AnTat 1.1 E clone was found not to be related to a depletion of T cells or to an absence of functional accessory cells, but rather to an active suppression of interleukin 2 (IL-2) production. Lymph node cells derived from infected mice failed to produce IL-2 following Con A stimulation, whereas an exogenous supply of recombinant IL-2 could restore the impairment of the mitogen (Con A)-induced proliferative responses. Furthermore, lymph node cells derived from infected mice suppressed both secondary T-cell proliferative responses and IL-2 secretion, indicating that the trypanosome-induced suppression is mediated by a suppressive cell which interferes at the level of IL-2 secretion.     

肠系膜淋巴结Th1、Th17细胞在小鼠结肠炎模型发病中作用的研究

背景:大量研究表明细胞因子网络在溃疡性结肠炎(UC)的发病和疾病进程中起关键作用,但相关研究主要集中于肠黏膜免疫细胞方面。目的:探讨肠系膜淋巴结Th1、Th17细胞在模拟人类UC的小鼠DSS结肠炎模型发病中的作用。方法:C57BL/6小鼠饮用5%DSS溶液7 d诱导实验性结肠炎,实验过程中每天评估疾病活动指数(DAI)。于第8 d处死小鼠,ELISA法测定结肠组织IL-1β含量;分离肠系膜淋巴结细胞,以CD3/CD28单抗诱导淋巴细胞活化并以ELISA法测定细胞培养上清液中的Th1、Th17细胞因子含量,流式细胞术检测肠系膜淋巴结F4/80+CD11b+巨噬细胞和CD4+T细胞内Th1、Th17细胞因子表达。结果:结肠炎模型组DAI随实验进程而逐渐增加,于第7 d达峰值。与正常对照组相比,模型组结肠组织IL-1β蛋白表达显著上调(P<0.05),肠系膜淋巴结巨噬细胞浸润增加(P<0.001),淋巴细胞IL-17A分泌水平显著增高(P<0.05),IFN-γ分泌水平亦呈增高趋势(P>0.05),CD4+T细胞内IL-17A、IFN-γ表达显著上调(P<0.05)。结论:肠系膜淋巴结Th1、Th17细胞过度激活可能通过释放效应细胞因子诱导巨噬细胞等浸润、活化,参与介导小鼠DSS结肠炎模型的肠黏膜炎症反应和病理损伤。     

Comparative dynamics and phenotype of the murine immune response to Trichinella spiralis and Trichinella pseudospiralis

Infection of NIH mice with Trichinella spiralis and Trichinella pseudospiralis results in qualitatively comparable immune responses. Antigen-specific proliferation by mesenteric lymph node cells was transient and temporally associated with intestinal infection, but in contrast was sustained throughout infection by splenocytes. Early cytokine production by mesenteric lymph node cells was dominated by interleukin 10, but also IL-5 and IL-4, with rapid resolution following parasite expulsion from the gut. Splenocytes showed a mixed profile of cytokine production, although again dominated by IL-10 and sustained over 60 days of infection. All antibody classes were evident, with early production of IgA and IgG1, and subsequent secretion of other subclasses including IgG2a. Granulocytic infiltration of the spleen was significantly greater in T. spiralis infection. The concentration of serum corticosterone generally remained within normal boundaries, although was raised by day 60 in T. spiralis-infected mice. We conclude that the systemic suppression of inflammation reported for T. pseudospiralis does not result from selective induction of regulatory cytokines, or a major difference in the immune response to infection with T. spiralis.     

Pattern Recognition Scavenger Receptors, SR-A and CD36, Have an Additive Role in the Development of Colitis in Mice

The multifunctional pattern recognition scavenger receptors, SR-A and CD36, are predominantly expressed by lamina propria macrophages and considered important in innate immunity. We examined the role of these receptors in the pathophysiology of inflammatory bowel disease. Colitis was induced in wild type (WT), SR-A^−/−, CD36^−/−, and SR-A/CD36 double deficient mice by administering DSS. DSS-induced moderately severe colitis in WT mice was manifested by weight loss, reduced hematocrit, and pathology. SR-A/CD36 double deficient mice developed significantly more severe colitis as indicated by anemia (P < 0.01), decreased colonic length due to inflammation (P < 0.01), and lesions when compared with WT and single deficient animals. Serum amyloid A was significantly more elevated in SR-A/CD36^−/− mice (P < 0.01) compared with WT and single deficient animals. However, the spleens of WT mice (P < 0.05) were significantly enlarged. Inflammatory cytokine levels were considerably increased in WT mice (IL-6 P < 0.001, TNFα P < 0.01). In contrast, SR-A deficient mice maintained more normal body and splenic weight and developed less severe colonic lesions compared to other groups. In conclusion, our data indicate that SR-A/CD36 double deficiency leads to more severe colonic lesions and dysregulated inflammatory response as compared with single SR-A or CD36 deficiency in colitis, suggesting additive effects between these two receptors in this model.     

Oral administration of cyclosporin does not prevent expansion of antigen-specific,gut-associated,and spleen lymphocyte populations duringChlamydia trachomatis proctitis in nonhuman primates

To study the effects of oral cyclosporin (CsA) administration on immune responses in the gastrointestinal tract, humoral and cellular immune responses were studied in CsA-treated nonhuman primates having Chlamydia trachomatis proctitis (lymphogranuloma venereum, LGV). There was no apparent effect of CsA treatment on the gross or microscopic appearance of LGV proctitis, but CsA-treated animals, with or without LGV infection, had lymphoid hyperplasia of spleen and mesenteric lymph nodes. CsA treatment inhibited the primary antibody response to LGV, inhibited peripheral blood lymphocyte mitogen-induced proliferation and IL-2 production, and inhibited LGV-specific proliferation of peripheral blood lymphocytes. In contrast, mitogen-stimulated proliferation of spleen, mesenteric lymph node, and lamina propria lymphocytes was not significantly inhibited in CsA-treated animals. In addition, LGV-specific proliferation of spleen and mesenteric lymph node lymphocytes was not inhibited. High mitogen-stimulated IL-2 production of lamina propria lymphocytes was only partially inhibited in CsA-treated animals. In vitroCsA treatment had the expected inhibitory effects on mitogen- and antigen-induced proliferation of spleen and mesenteric lymph node lymphocytes. Thus, although oral cyclosporin inhibits the antibody and proliferative responses of peripheral blood lymphocytes to antigens and mitogens in animals having Chlamydia trachomatis proctitis, it does not prevent the expansion of antigen-specific, gut-associated, and spleen lymphocyte populations.Part of this work has been published as a preliminary report in abstract form (Clinical Research 34:446A, 1986).     

The Role of Potassium Channels in Relaxant Effect of Levosimendan in Rat Small Mesenteric Arteries

Summary We investigated both the effect of levosimendan and the role of various potassium channels in KCl-precontracted rat small mesenteric arteries. Levosimendan (10⁻⁶−10⁻³ M) or cromakalim (CRO, 10⁻⁷−10⁻⁴ M) produced concentration-dependent relaxation responses in small mesenteric arteries precontracted by 30 mM KCl. The relaxant responses to levosimendan in KCl-precontracted arteries did not differ significantly between endothelium-intact and endothelium-denuded preparations. Incubation of rat small mesenteric arterial segments with ATP-dependent potassium channel (KATP) blocker glibenclamide (GLI, 10⁻⁶ M) for 30 min significantly inhibited the relaxant responses to both levosimendan and CRO. Neither the Ca²⁺-activated potassium channel (KCa) blocker iberiotoxin (10⁻⁷ M) nor the voltage-dependent potassium channel (KV) blocker 4-aminopyridine (5 mM) incubation for 30 min caused significant alterations in relaxant responses to levosimendan in KCl-precontracted small mesenteric arteries. These findings suggested that levosimendan-induced relaxation responses in isolated rat small mesenteric arteries were neither depended on endothelium nor inhibited by the blockers of KV or KCa but, they rather seem to depend on the activation of KATP.     

Long-term treatment of NZB mice with anti-CD4 results in wasting disease,lymphoid atrophy and chronic diarrhea

In this paper, we have shown that long-term treatment of NZB mice with anti-CD4 antibody results in four major pathological effects. Firstly the development of a severe wasting disease; secondly lymphoid atrophy of the thymus, spleen, mesenteric lymph node and Peyers patches (PP); thirdly, severe chronic ulcerative colitis and fourthly a neutrophilia with neutrophil infiltration in the spleen, liver and mesenteric lymph nodes. At the same time, mice subjected to anti-CD4 treatment showed a reduction in the microbial diversity in ileal walls and contents, as well as in colonic contents, together with overgrowth of E. coli in the intestinal lumen and wall. In addition, there was the appearance of large numbers of spiral shaped bacteria on the mucosal surface often associated with colonic ulceration.     

Probiotics increase T regulatory cells and reduce severity of experimental colitis in mice

AIM:To investigate the effect of probiotics on regulating T regulatory cells and reducing the severity of experimental colitis in mice. METHODS:Forty C57/BL mice were randomly divided into four groups.Colitis was induced in the mice using 2,4,6-trinitrobenzene sulfonic acid(TNBS).After 10-d treatment with Bifico capsules(combined bifidobacterium,lactobacillus and enterococcus),body weight,colonic weight,colonic weight index,length of colon,and histological scores were evaluated.CD4+CD25+Foxp3+T cell in mesenteric lymph nodes were measured by flow cytometry,and cytokines in colonic tissue homogenateswere analyzed by a cytometric bead array. RESULTS:The colonic weight index and the colonic weight of colitis mice treated with Bifico were lower than that of TNBS-induced mice without treatment. However,colonic length and percent of body weight amplification were higher than in TNBS-induced mice without treatment.Compared with TNBS-induced mice without treatment,the level of CD4+CD25+Foxp3+T cells in mesenteric lymph nodes,the expression of interleukin(IL)-2,IL-4 and IL-10 in colonic tissues from colitis mice treated with Bifico were upregulated,and tumor necrosis factor-αand interferon-γwere downregulated. CONCLUSION:Probiotics effectively treat experimental colitis by increasing CD4+CD25+Foxp3+T cell and regulating the balance of Th1 and Th2 cytokines in the colonic mucosa.     

Serum from children with polyarticular juvenile idiopathic arthritis (pJIA) inhibits differentiation, mineralization and may increase apoptosis of human osteoblasts “in vitro”

We examined the effects of polyarticular juvenile idiopathic arthritis (pJIA) serum on proliferation, differentiation, mineralization, and apoptosis of human osteoblast cells (hOb) in culture. The hOb were cultured with 10% serum from active pJIA and healthy controls (CT) and were tested for DNA synthesis, alkaline phosphatase (AP) activity, osteocalcin (OC) secretion, calcium levels, caspase 3 activity, and DNA fragmentation. None of the patients had used glucocorticoids for at least 1 month before the study, or any other drug that can affect bone mineral metabolism. Human inflammatory cytokine levels (IL-6, IL-8, IL-10, IL-1β, TNF-α, and IL-12p70) were measured in pJIA and CT sera. Low levels of AP activity was observed in pJIA cultures compared with CT cultures (67.16 ± 53.35 vs 100.11 ± 50.64 μmol p-nitrophenol/h⁻¹ mg⁻¹ protein, P = 0.008). There was also a significant decrease in OC secretion (9.23 ± 5.63 vs 12.82 ± 7.02 ng/mg protein, P = 0.012) and calcium levels (0.475 ± 0.197 vs 0.717 ± 0.366 mmol/l, P = 0.05) in pJIA hOb cultures. No difference was observed in cell proliferation (323.56 ± 108.23 vs 328.91 ± 88.03 dpm/mg protein, P = 0.788). Osteoblasts cultured with JIA sera showed lower levels of DNA and increased fragmentation than osteoblasts cultured with CT sera. pJIA sera showed higher IL-6 values than CT (21.44 ± 9.31 vs 3.58 ± 2.38 pg/ml, P < 0.001), but no difference was observed related to IL-8, IL-10, IL-1β, TNF-α, and IL-12p70 between pJIA and controls. This study suggests that serum from children with pJIA inhibits differentiation, mineralization and may increase apoptosis of hOb cultures, and inflammatory cytokines such as IL-6 might be a mechanism in this find. These results may represent an alternative therapeutic target for prevention and treatment of bone loss in JIA.     

Copper addition prevents the inhibitory effects of interleukin 1-β on rat pancreatic islets

Summary Since copper [Cu(II)] is a necessary cofactor for both intra-mitochondrial enzymes involved in energy production and hydroxyl scavenger enzymes, two hypothesised mechanisms for action of interleukin-Iβ (IL-1β), we studied whether CU(II) addition could prevent the inhibitory effect of IL-1β on insulin release and glucose oxidation in rat pancreatic islets. Islets were incubated with or without 50 U/ml IL-1β, in the presence or absence of various concentrations of Cu(II)-GHL (Cu(II) complexed with glycyl-l-histidyl-l-lysine, a tripeptide known to enhance copper uptake into cultured cells). CuSO₄ (1–1000 ng/ml) was used as a control for Cu(II) effect when present as an inorganic salt. At the end of the incubation period, insulin secretion was evaluated in the presence of either 2.8 mmol/l (basal insulin secretion) or 16.7 mmol/l glucose (glucose-induced release). In control islets basal insulin secretion was 92.0±11.4 pg · islet⁻¹ h⁻¹ (mean ± SEM,n=7) and glucose-induced release was 2824.0±249.0 pg · islet⁻¹ h⁻¹. In islets pre-exposed to 50 U/ml IL-1β, basal insulin release was not significantly affected but glucose-induced insulin release was greatly reduced (841.2±76.9,n=7,p<0.005). In islets incubated with IL-1β and Cu-GHL (0.4 μmol/l, maximal effect) basal secretion was 119.0±13.1 pg · islet⁻¹ h⁻¹ and glucose-induced release was 2797.2±242.2, (n=7,p<0.01 in respect to islets exposed to IL-1β alone). In contrast to data obtained with Cu(II)-GHL, increasing concentrations of CuSO4 (up to 10 μmol/l) did not influence the inhibitory effect of IL-1β on glucose-stimulated insulin release. Glucose oxidation (in the presence of 16.7 mmol/l glucose) was 31.5±2.4 pmol · islet⁻¹·90min⁻¹ in control islets and 7.0±0.9 (p<0.01) in IL-1β-exposed islets. In islets exposed to IL-1β and Cu-GHL glucose oxidation was similar to control islets (31.9±1.9). In contrast, Cu-GHL did not prevent the IL-1β-induced increase in nitric oxide production. Nitrite levels were 5±1.7, 26±5 and to 29±4 pmol · islet⁻¹·48 h⁻¹ (mean ± SEM,n=5) in the culture medium from control IL-1β and IL-1β+Cu-GHL exposed islets, respectively. These data indicate that the Cu(II) complexed to GHL is able to prevent the inhibitory effects of IL-1β on insulin secretion and glucose oxidation, but not on NO production. The mechanism of action of Cu-GHL is still unclear, but it might restore the activity of the enzymatic systems inhibited by IL-1β. [Diabetologia (1995) 38∶39–45]     

Variable phenotypes of enterocolitis in interleukin 10-deficient mice monoassociated with two different commensal bacteria

BACKGROUND & AIMS: To explore the hypothesis that selective immune responses to distinct components of the intestinal microflora induce intestinal inflammation, we characterized disease kinetics and bacterial antigen-specific T-cell responses in ex germ-free interleukin 10 -/- and wild-type control mice monoassociated with Enterococcus faecalis , Escherichia coli , or Pseudomonas fluorescens . METHODS: Colitis was measured by using blinded histological scores and spontaneous interleukin 12 secretion from colonic strip culture supernatants. Interferon gamma secretion was measured from mesenteric or caudal lymph node CD4 + T cells stimulated with bacterial lysate-pulsed antigen-presenting cells. Luminal bacterial concentrations were measured by culture and quantitative polymerase chain reaction. RESULTS: Escherichia coli induced mild cecal inflammation after 3 weeks of monoassociation in interleukin 10 -/- mice. In contrast, Enterococcus faecalis-monoassociated interleukin 10 -/- mice developed distal colitis at 10-12 weeks that was progressively more severe and associated with duodenal inflammation and obstruction by 30 weeks. Neither bacterial strain induced inflammation in wild-type mice, and germ-free and Pseudomonas fluorescens-monoassociated interleukin 10 -/- mice remained disease free. CD4 + T cells from Enterococcus faecalis- or Escherichia coli-monoassociated interleukin 10 -/- mice selectively produced higher levels of interferon gamma and interleukin 4 when stimulated with antigen-presenting cells pulsed with the bacterial species that induced disease; these immune responses preceded the onset of histological inflammation in Enterococcus faecalis -monoassociated mice. Luminal bacterial concentrations did not explain regional differences in inflammation. CONCLUSIONS: Different commensal bacterial species selectively initiate immune-mediated intestinal inflammation with distinctly different kinetics and anatomic distribution in the same host.     

Interleukin-13 exerts autocrine growth-promoting effects on human pancreatic cancer, and its expression correlates with a propensity for lymph node metastases

Background and aims  Interleukin-13 (IL-13) is an anti-inflammatory cytokine produced in cells of hematopoetic origin. It is not known whether pancreatic cancer cells produce IL-13 or whether IL-13 can modulate pancreatic cancer cell growth and influence the frequency of lymph node metastases. Materials and methods  Cell growth and signaling were analyzed by cell counting, colorimetric proliferation assays, fluorescent-activated cell sorting, and in vitro kinase activity assays. IL-13 expression and secretion were determined by Northern blot analysis and enzyme-linked immunosorbent assay, respectively. Localization of IL-13 and its transmembrane receptor (IL-4R) in primary pancreatic ductal adenocarcinoma (PDAC) was characterized by immunohistochemistry. Results  IL-13 enhanced the growth of ASPC-1, CAPAN-1, and COLO-357 cells. This was associated with enhanced p44/42 mitogen-activated protein kinase (MAPK) phoshorylation. In contrast to p44/42 MAPK, phosphatidylinositol 3-kinase activity was also induced in IL-13-unresponsive MIA PaCa-2, PANC-1, and T3M4 cells. All cells expressed and secreted IL-13. Neutralizing IL-13 antibodies inhibited the growth of ASPC-1 and CAPAN-1 cells. Immunohistochemical analysis of resected primary ductal adenocarcinoma specimens revealed high levels of IL-13 in 30 of 70 cases and its transmembrane receptor (IL-4R) in 28 of 70 cases, respectively. Fifteen of 16 specimens (94%) exhibiting high IL-13 and IL-4R coexpression had lymph node metastases, while only 30 of the remaining 54 samples (56%) had positive lymph nodes (p = 0.0134). Conclusion  IL-13 can act as an autocrine growth factor in PDAC. Endogenous expression of IL-13 in conjunction with IL-4R in the cancer cells seems to facilitate lymph node metastasis. Andrea Formentini and Olga Prokopchuk have equally contributed to this work.     

Rebamipide, a gastro-protective and anti-inflammatory drug, promotes gastric ulcer healing following eradication therapy for Helicobacter pylori in a Japanese population: a randomized, double-blind, placebo-controlled trial

Background One week of Helicobacter pylori eradication therapy is insufficient for healing of gastric ulcers. We examined the efficacy of rebamipide in gastric ulcer healing following 1 week of eradication therapy in a randomized, double-blind, placebo-controlled trial. Methods Patients with H. pylori-positive gastric ulcer were enrolled and received 1 week of eradication therapy, followed by 100 mg of rebamipide or placebo for 7 weeks. The primary end point was the gastric ulcer healing rate. Results Of the 309 patients entered in the trial, 301 completed H. pylori eradication therapy; 154 patients took rebamipide, and 147 took placebo. The healing rate in the rebamipide group was higher than that in the placebo group in the per-protocol analysis—80.0% (104/130) versus 66.1% (82/124) [95% confidence interval (CI), 3.1–24.7; P = 0.013)—and in a full analysis—70.1% (108/154) versus 60.5% (89/147) (95% CI, −1.1 to 20.3; P = 0.080). Conclusions Compared with placebo, rebamipide significantly promoted gastric ulcer healing following 1 week of eradication therapy.     

Association of 22 cytokine gene polymorphisms with rheumatoid arthritis in population of ethnic Macedonians

To examine the possible role of 22 cytokine gene polymorphisms in host susceptibility to or protection against RA in Macedonians. In this study, 301 healthy unrelated individuals and 85 patients with RA were studied. Cytokine genotyping was performed by PCR with sequence-specific priming (PCR–SSP) (Heidelberg kit). Results showed susceptible association for four cytokine alleles, six cytokine genotypes, one haplotype, and four combinations of haplotypes, while protective associations were found for four cytokine alleles, three cytokine genotypes, three haplotypes, and only one combination of haplotypes. These results suggest that IL-4 −1098, IL-4 −590, IL-10 −1082, IL-10 −819, IL-2 −330, IL-6 −174, and TNF-α −238 cytokine gene polymorphisms might be significantly associated and affect host susceptibility and/or resistance to RA in Macedonians.     

Matrix metalloproteinase-3 secretion from human colonic subepithelial myofibroblasts: role of interleukin-17

Background. Subepithelial myofibroblasts (SEMFs) play a role in extracellular matrix (ECM) metabolism in the colon. In this study, we investigated the effects of interleukin (IL)-17, IL-1β, and tumor necrosis factor (TNF)-α on matrix metalloproteinase (MMP)-3 secretion in colonic SEMFs. Methods. MMP-3 secretion and MMP-3 mRNA expression were determined by Western and Northern blotting, respectively. The secretion of tissue inhibitor of matrix metalloproteinase (TIMP)-1 was determined by enzyme-linked immunosorbent assay (ELISA). Results. In human colonic SEMFs, MMP-3 secretion and MMP-3 mRNA expression were induced by IL-17, IL-1β, and TNF-α. The effect of IL-17 was observed, but this was weak as compared with those induced by IL-1β or TNF-α. A c-Jun/activating protein-1 (AP-1) inhibitor, curcumin, reduced the IL-17-, IL-1β-, and TNF-α-induced MMP-3 mRNA expression, and mitogen-activated protein (MAP) kinase inhibitors (U0126, PD098059, and SB203580) also blocked MMP-3 secretion. There findings indicate a role for AP-1 and MAP kinases in cytokine-induced MMP-3 secretion. Furthermore, costimulation by IL-17 + IL-1β and by IL-17 + TNF-α induced a marked increase in MMP-3 secretion. The costimulatory effects of these combinations were also observed for TIMP-1 mRNA expression and TIMP-1 secretion. Conclusions. Colonic SEMFs actively secreted MMP-3 in response to IL-17, IL-1β, and TNF-α. This was coupled with TIMP-1 secretion. Colonic SEMFs may play an important role in ECM turnover via MMP secretion. Received: July 2, 2002 / Accepted: December 18, 2002 RID="*" ID="*" Reprint requests to: A. Andoh     

Dual-association of gnotobiotic IL-10-/- mice with 2 nonpathogenic commensal bacteria induces aggressive pancolitis

BACKGROUND: Monoassociating gnotobiotic IL-10-deficient (-/-) mice with either nonpathogenic Enterococcus faecalis or a nonpathogenic Escherichia coli strain induces T-cell-mediated colitis with different kinetics and anatomical location (E. faecalis: late onset, distal colonic; E. coli: early onset, cecal). Hypothesis: E. faecalis and E. coli act in an additive manner to induce more aggressive colitis than disease induced by each bacterial species independently.METHODS: Germ-free (GF) inbred 129S6/SvEv IL-10-/- and wildtype (WT) mice inoculated with nonpathogenic E. faecalis and/or E. coli were killed 3-7 weeks later. Colonic segments were scored histologically for inflammation (0 to 4) or incubated in media overnight to measure spontaneous IL-12/IL-23p40 secretion. Bacterial species were quantified by serial dilution and plated on culture media. Mesenteric lymph node (MLN) CD4(+) cells were stimulated with antigen-presenting cells pulsed with bacterial lysate (E. faecalis, E. coli, Bacteroides vulgatus) or KLH (unrelated antigen control). IFN-gamma and IL-17 levels were measured in the supernatants.RESULTS: Dual-associated IL-10-/- (but not WT) mice developed mild-to-moderate pancolitis by 3 weeks that progressed to severe distal colonic-predominant pancolitis with reactive atypia and duodenal inflammation by 7 weeks. NF-kappaB was activated in the duodenum and colon in dual-associated IL-10-/- x NF-kappaB(EGFP) mice. The aggressiveness of intestinal inflammation and the degree of antigen-specific CD4(+) cell activation were greater in dual- versus monoassociated IL-10-/- mice.CONCLUSION: Two commensal bacteria that individually induce phenotypically distinct colitis in gnotobiotic IL-10-/- mice act additively to induce aggressive pancolitis and duodenal inflammation.     

Correlations between worm burden and markers of Th1 and Th2 cell subset induction in an inbred strain of mouse infected with Trichuris muris

Helper T cell subset induction was examined within a single inbred strain of mouse ( B10.D2/n) where individuals varied in their ability to expel the nematode parasite Trichuris muris. In this mouse strain approximately half of infected individuals resist infection whilst half are unable to expel the parasite and harbour chronic mature adult worm infections. We here assess various T cell and serological parameters in individual B10.D2/n mice infected with T. muris in relation to the number of parasites harboured. Worm burdens showed very significant negative correlations with five different parameters indicative of the selective expansion within the host of helper T cells of the Th2 subset. Thus, in vitro IL-5 and 1L-9 production by restimulated mesenteric lymph node cells, totallgE levels, the early parasite-specific IgG1 response (all P < 0·01) and intestinal eosinophilia (P < 0·05), were all significantly negatively correlated with worm burden. In addition, levels of IL-3 were significantly greater in mice resistant to infection (P < 0·01). In contrast there was a significant positive correlation between worm burden and parasite-specific IgG2a levels (P < 0·05), IgG2a production being under the tight control of the Th1-specific cytokine IFN-y and thus a reliable marker for in vivo Th1 cell activation. The data demonstrates that an individual infected with T. muris is capable of mounting either a protective Th2-type response or an inappropriate Th1-type response. Thus, under conditions where host genetic factors and route of antigen introduction into the host are identical, polarized helper T cell responses can arise and hence may be due to a parasite-derived influence rather than an intrinsic difference between hosts per se.     

Niflumic acid-sensitive ion channels play an important role in the induction of glucose-stimulated insulin secretion by cyclic AMP in mice

Aims/hypothesis  We have previously reported that glucose-stimulated insulin secretion (GSIS) is induced by glucagon-like peptide-1 (GLP-1) in mice lacking ATP-sensitive K⁺ (K_ATP) channels (Kir6.2 ^−/− mice [up-to-date symbol for Kir6.2 gene is Kcnj11]), in which glucose alone does not trigger insulin secretion. This study aimed to clarify the mechanism involved in the induction of GSIS by GLP-1. Methods  Pancreas perfusion experiments were performed using wild-type (Kir6.2 ^+/+ ) or Kir6.2 ^−/− mice. Glucose concentrations were either changed abruptly from 2.8 to 16.7 mmol/l or increased stepwise (1.4 mmol/l per step) from 2.8 to 12.5 mmol/l. Electrophysiological experiments were performed using pancreatic beta cells isolated from Kir6.2 ^−/− mice or clonal pancreatic beta cells (MIN6 cells) after pharmacologically inhibiting their K_ATP channels with glibenclamide. Results  The combination of cyclic AMP plus 16.7 mmol/l glucose evoked insulin secretion in Kir6.2 ^−/− pancreases where glucose alone was ineffective as a secretagogue. The secretion was blocked by the application of niflumic acid. In K_ATP channel-inactivated MIN6 cells, niflumic acid similarly inhibited the membrane depolarisation caused by cAMP plus glucose. Surprisingly, stepwise increases of glucose concentration triggered insulin secretion only in the presence of cAMP or GLP-1 in Kir6.2 ^+/+ , as in Kir6.2 ^−/− pancreases. Conclusions/interpretation  Niflumic acid-sensitive ion channels participate in the induction of GSIS by cyclic AMP in Kir6.2 ^−/− beta cells. Cyclic AMP thus not only acts as a potentiator of insulin secretion, but appears to be permissive for GSIS via novel, niflumic acid-sensitive ion channels. This mechanism may be physiologically important for triggering insulin secretion when the plasma glucose concentration increases gradually rather than abruptly.