Non-lymphoid and lymphoid cells in acute, chronic and relapsing experimental colitis.

In rodents, intracolonic administration of ethanol 30% induces an acute colitis, while administration of 2,4,6-trinitrobenzene sulphonic acid (TNBS) in ethanol induces a longer lasting colitis. In the acute and chronic stages of experimental colitis, lymphoid and non-lymphoid cells were studied in the colon by immunohistochemistry. During the acute inflammation a high damage score of the colon was observed, which was related to an increase in the number of macrophages and granulocytes. Also a change in distributional patterns of macrophage subpopulations was found. The chronic stage of TNBS-ethanol-induced colitis was characterized by an increase in the number of lymphocytes, especially T cells. These data suggest that macrophages and granulocytes are important in the acute phase of experimental colitis, while lymphocytes play a pivotal role in the chronic stage. As most inflammatory bowel disease (IBD) patients have relapses during the chronic disease, we attempted to induce a relapse during experimental colitis by giving a second i.p. or s.c. dose of TNBS. This resulted in increased damage scores of the colon, new areas of ulceration and a further increase in macrophage numbers. No effect on the number of granulocytes was seen. These results indicate that it is possible to mimic relapses in experimental colitis by a second administration of TNBS, and suggest that the rats had been sensitized by the first dose of TNBS, given into the colon.     

A method for the accurate determination of anti-hapten cytotoxic T lymphocyte precursors: correction for apparent 'anti-self' reactivity

A method is described for accurately determining the frequency of precursors of hapten specific cytotoxic T cells. The method is based on a standard Poisson analysis of limit dilution cultures, but makes a correction of 'anti-self' reacting clones and for spontaneously arising clones that recognise modified self. These corrections are shown to be especially important when low hapten densities are used, where there may be more than a 10-fold difference between the corrected and uncorrected frequency estimates. Determined levels of antigen specificity and of H-2 restriction are significantly enhanced by application of this method.     

Characterization of major histocompatibility antigens on trinitrophenyl-modified cells.

Cell membrane proteins encoded for by the major histocompatibility complex (MHC)¹ are associated with the antigenic determinant(s) recognized on trinitrophenyl (TNP)-modified cells by syngeneic murine cytotoxic T lymphocytes and by hapten-reactive guinea pig T cells. To investigate the relationship of the TNP moiety on TNP-modified cells to these major histocompatibility antigens, peritoneal exudate cells or splenocytes from two inbred guinea pig strains and one inbred murine strain were TNP-modified, radioiodinated and lysed in detergent. TNP-derivatized proteins were then isolated using an anti-TNP immunoabsorbent, and the presence on TNP-derivatized histocompatibility antigens in the eluted proteins was determined by immunoprecipitation experiments and SDS-polyacrylamide gel electrophoretic analysis. Whereas most of the various histocompatibility antigens examined were found to be TNP-derivatized in amounts proportional to the degree of membrane protein derivation as a whole, only small amounts of TNP-modified strain 2 guinea pig Ia antigens were found, and no hapten-modified strain 13 guinea pig Ia antigens were detected. It is concluded that, in contrast to most MHC gene products, strain 13 Ia antigens are not derivatized on TNP-modified cells and, thus, represent an important exception demonstrating that histocompatibility antigens need not be directly TNP-derivatized for T cell recognition and activation.     

己酮可可碱对2,4,6-三硝基苯磺酸肠炎模型的影响

目的 :观察己酮可可碱 (PTX)对 2 ,4 ,6 三硝基苯磺酸 (TNBS)肠炎模型影响。方法 :通过直肠给予雄性BALB/c小鼠TNBS诱导结肠炎 ,应用PTX对其进行治疗 ,6d后收集结肠标本评价结肠炎症程度 ;采用半定量RT PCR检测肠黏膜IL 18mRNA表达 ,采用免疫组织化学染色显示IL 18阳性细胞。结果 :直肠内给予BALB/c小鼠TNBS后可造成小鼠结肠炎性改变 ;PTX治疗可使小鼠疾病活动指数、结肠重量和炎症指数均显著下降 (P <0 .0 5 ) ,结肠IL 18mRNA和肠黏膜固有层表达IL 18细胞数显著下降 (P <0 .0 5 )。结论 :PTX治疗可使TNBS肠炎模型小鼠肠黏膜局部IL 18表达显著下降 ,肠炎得以减轻。     

Anti-inflammatory effect of taurocholate on TNBS-induced ulcerative colitis in mice

Taurocholate is a natural conjugated bile acid. The aim of this study was to evaluate the anti-inflammatory effect of taurocholate in TNBS-induced ulcerative colitis in mice. The colitis were induced by rectal administration of TNBS. After 24 h, the experimental animals were treated with sulfasalazine (SASP, 500 mg/kg/day) and taurocholate (20, 40 and 60 mg/kg) for 7 consecutive days. The anti-inflammatory effects of taurocholate for colitis were assessed by body weight, colonic weight and length, macroscopic scores, and histopathological examinations. In addition, the colonic tissue levels of myeloperoxidase (MPO) activity, interleukin (IL)-1β, interferon (IFN-γ) and tumour necrosis factor-α (TNF-α) were also determined to assess the effect of taurocholate. Compared with the model group, treatment with taurocholate (20, 40 and 60 mg/kg) significantly inhibited the body weight loss, improved colonic weight and length, and decreased macroscopic and histopathological scores. Furthermore, the activity accumulation of MPO and the colonic tissue levels of IL-1β, IFN-γ and TNF-α were also decreased by administration of taurocholate. All the findings of this study suggested that taurocholate has the anti-inflammatory effect in ulcerative colitis in mice and indicated it as a good candidate to treat inflammatory bowel disease.     

Preventative effects of a probiotic, Lactobacillus salivarius ssp. salivarius, in the TNBS model of rat colitis

AIM: To investigate the intestinal anti-inflammatory effect and mechanism of a probiotic Lactobacillus salivarius ssp. salivarius CECT5713 in the TNBS model of rat colitis. METHODS: Female Wistar rats (180-200 g) were used in this study. A group of rats were administered orally the probiotic L. salivarius ssp. salivarius(5×108 CFU suspended in 0.5 mL of skimmed milk) daily for 3 wk. Two additional groups were used for reference, a non-colitic and a control colitic without probiotic treatment, which received orally the vehicle used to administer the probiotic. Two weeks after starting the experiment, the rats were rendered colitic by intracolonic administration of 10 mg of TNBS dissolved in 0.25 mL of 500 mL/L ethanol. One week after colitis induction, all animals were killed and colonic damage was evaluated both histologically and biochemically. The biochemical studies performed in colonic homogenates include determination of myeloperoxidase (MPO) activity, glutathione (GSH) content, leukotriene B4 (LTB4) and tumor necrosis factor a (TNF-α) levels, as well as inducible nitric oxide synthase (iNOS) expression. In addition, the luminal contents obtained from colonic samples were used for microbiological studies, in order to determine Lactobacilli and Bifidobacteria counts. RESULTS: Treatment of colitic rats with L salivarius ssp. salivarius resulted in amelioration of the inflammatory response in colitic rats, when compared with the corresponding control group without probiotic treatment. This anti-inflammatory effect was evidenced macroscopically by a significant reduction in the extent of colonic necrosis and/or inflammation induced by the administration of TNBS/ethanol (2.3±0.4 cm vs 53.4±0.3 cm in control group, P<0.01) and histologically by improvement of the colonic architecture associated with a reduction in the neutrophil infiltrate in comparison with non-treated colitic rats. The latter was confirmed biochemically by a significant reduction of colonic MPO activity (105.3±26.0 U/g vs 180.6±21.9 U/g, P<0.05) a marker of neutrophil infiltration. The beneficial effect was associated with an increase of the colonic GSH content (1 252±42 nmol/g vs 1 087±51 nmol/g,P<0.05), which is depleted in colitic rats, as a consequence of the oxidative stress induced by the inflammatory process. In addition, the treatment of colitic rats with L. salivarius resulted in a significant reduction of colonic TNF-α levels (509.4±68.2 pg/g vs 782.9±60.1 pg/g, P<0.01) and in a lower colonic iNOS expression, when compared to TNBS control animals without probiotic administration. Finally, treated colitic rats showed higher counts of Lactobacilli species in colonic contents than control colitic rats, whereas no differences were observed in Bifidobacteria counts. CONCLUSION: Administration of the probiotic L. salivarius ssp. salivarius CECT5713 facilitates the recovery of the inflamed tissue in the TNBS model of rat colitis, an effect associated with amelioration of the production of some of the mediators involved in the inflammatory response in the intestine, such as cytokines, including TNF-α and NO. This beneficial effect could be ascribed to its effect on the altered immune response that occurs in this inflammatory condition.     

Sensory and inflammatory colonic changes induced by vincristine in distinct rat models of colitis

Preclinical and clinical studies show that gastrointestinal (GI) inflammation can evoke sensory changes occasionally far from the original inflammatory site. Animal models of colitis with either trinitrobenzenesulphonic acid (TNBS) or mustard oil (MO) produce distinct patterns of somatic and visceral sensory changes. We evaluated the effects of four doses of i.v. vincristine 150 μg kg^?1 (total of 600 μg kg^?1) treatment on the somatic (thermal nociceptive threshold) and colonic (morphological) changes induced by TNBS or MO in rats. TNBS and MO groups were further submitted to vincristine or saline pretreatments. TNBS induced somatic hypersensitivity, while MO induced somatic hyposensitivity (P < 0.05) when compared to the saline and ethanol control groups. Vincristine per se induced somatic hypersensitivity (P < 0.05). This effect was enhanced by TNBS and reversed by MO treatments. Although vincristine increased the colitis area (colonic weight length^?1 ratio) and the Morris' score in TNBS‐treated rats, it did not alter the colitis area and even lowered the Morris' score in MO‐treated rats. Compared to the saline (control) group, vincristine did not alter the colonic microscopic pattern. However, such lesions scores are higher (P < 0.05) in colitis groups induced by TNBS and MO, pretreated or not with vincristine. In conclusion, the somatic changes induced by different models of experimental colitis are diverse and modulated differently by vincristine.     

Immunostimulatory DNA ameliorates experimental and spontaneous murine colitis

BACKGROUND & AIMS: Impaired mucosal barrier, cytokine imbalance, and dysregulated CD4(+) T cells play important roles in the pathogenesis of experimental colitis and human inflammatory bowel disease. Immunostimulatory DNA sequences (ISS-DNA) and their synthetic oligonucleotide analogs (ISS-ODNs) are derived from bacterial DNA, are potent activators of innate immunity at systemic and mucosal sites, and can rescue cells from death inflicted by different agents. We hypothesized that these combined effects of ISS-DNA could inhibit the damage to the colonic mucosa in chemically induced colitis and thereby limit subsequent intestinal inflammation. METHODS: The protective and the anti-inflammatory effect of ISS-ODN administration were assessed in dextran sodium sulfate-induced colitis and in 2 models of hapten-induced colitis in Balb/c mice. Similarly, these effects of ISS-ODN were assessed in spontaneous colitis occurring in IL-10 knockout mice. RESULTS: In all models of experimental and spontaneous colitis examined, ISS-ODN administration ameliorated clinical, biochemical, and histologic scores of colonic inflammation. ISS-ODN administration inhibited the induction of colonic proinflammatory cytokines and chemokines and suppressed the induction of colonic matrix metalloproteinases in both dextran sodium sulfate- and hapten-induced colitis. CONCLUSIONS: As the colon is continuously exposed to bacterial DNA, these findings suggest a physiologic, anti-inflammatory role for immunostimulatory DNA in the GI tract. Immunostimulatory DNA deserves further evaluation for the treatment of human inflammatory bowel disease.     

Improved cisplatin delivery in cervical cancer cells by utilizing folate-grafted non-aggregated gelatin nanoparticles

PurposeCisplatin is highly effective in the treatment of cervical cancer. However, in therapeutic doses, cisplatin induces several adverse effects due to undesirable tissue distribution. Therefore, it is worth targeting cisplatin in cervical cancer cells by implicating non-aggregated ligand-modified nanotherapeutics.Methods and resultsHere, we report the preparation of non-aggregated folic acid-conjugated gelatin nanoparticles of cisplatin (Cis-GNs-FA) by two-step desolvation method with mean particle size of 210.6 ± 9.6 nm and 140.5 ± 10.9 nm for Cis-GNs to improve the drug delivery in cervical cancer, HeLa cells. FTIR and DSC spectra confirmed the presence and stability of cisplatin in gelatin matrix. Furthermore, amorphization of cisplatin in nanoparticles was ascertained by PXRD. Drug release followed a first-order release kinetic at both pH ∼ 5.6 (cervical cancer pH) and pH ∼ 7.4. In addition, a significant (P < 0.05) decrease in IC₅₀ value (8.3 μM) and enhanced apoptosis were observed in HeLa cells treated with Cis-GNs-FA as compared to Cis-GNs (15.1 μM) and cisplatin solution (40.2 μM). In contrast, A549 lung cancer cells did not discriminate between Cis-GNs-FA and Cis-GNs due to the absence of folate receptors-α (FR-α). Consistently, higher cellular uptake, 80.54 ± 7.60% was promoted by Cis-GNs-FA significantly (two-way ANOVA, P < 0.05) greater than 51.68 ± 9.78%, by Cis-GNs. This was also illustrated by CLSM images, which indicated that Cis-GNs-FA preferably accumulated in the cytoplasm of HeLa cells nearby nucleus by following receptor-mediated endocytosis pathway as compared to Cis-GNs.ConclusionTherefore, Cis-GNs-FA warrants further in-depth in vitro and in vivo investigations to scale up the technology for clinical translation.     

溃肠宁对TNBS模型大鼠肠形态、血清及结肠组织IL-6和IL-10影响的观察

[目的]观察中药溃肠宁对三硝基苯磺酸（TNBS）致溃疡性结肠炎（UC）大鼠模型肠黏膜形态、大鼠血清及结肠黏膜组织中白介素-6（IL-6）、白介素-10（IL-10）表达的影响,探讨溃肠宁抗UC的相关作用机制以及疗效.[方法]采用TNBS复合50％乙醇法复制UC大鼠模型,分别采用中药溃肠宁和柳氮磺胺毗啶（SASP）作为阳性对照组进行治疗,对比模型组和空白对照组采用ELISA法对各组大鼠肠血清以及肠黏膜中IL-6、IL-10进行检测,并对其检测结果进行分析.[结果]模型组大鼠结肠和血清中的IL-6含量明显高于正常组（P＜0.01）,IL-10的含量则明显低于正常组（P＜0.01）;SASP组结肠及血清中IL-6、IL-10含量与模型组比较差异有统计学意义（P＜0.01）;中药组和模型组相比,结肠和血清中IL-6含量明显低于模型组（P＜0.01）,IL-10的含量则明显高于模型组（P＜0.01）;中药组与西药SASP组比较,中药组结肠和血清中IL-6含量低于西药组,IL-10的含量高于西药组,可见2组之间差异有统计学意义（P＜0.01）.[结论]溃肠宁对TNBS复合50％乙醇法UC大鼠疗效显著,其作用机制可能与调节机体的免疫有关.