Rapid identification of Mycobacterium spp. isolated from patients is important with increased isolation of mycobacteria other than tubercle bacilli (MOTT). DNA-DNA hybridization with streptavidin-peroxidase and tetramethylbenzidine (TMB) color reaction method was recognized as a useful tool for identification of various species of mycobacteria. In this study, optimum condition of the test was determined. The optimal concentrations of tetramethylbenzidine dihydrochloride and hydrogen peroxide for streptavidin-horseradish peroxidase were 0.3-0.6 ug/ ml and 0.16 mM, respectively. The TMB stock solution was stable when prepared in methanol and the dilution of TBM stock solution in 10 mM sodium citrate-10 mM EDTA solution (pH 5.0) gave highest peroxidase-TMB activity. The suitable composition of hybridization solution consisted of 2 x SSC, 10% dextran sulfate, 50 ug/ml salmon DNA, 5 x Denhardt's solution, and 50% formamide. The 5-minute heating at 100C of test DNA prior to photobiotin labeling significantly increased the reaction. In conclusion, DNA-DNA hybridization method with streptavidin-peroxidase and TMB color reaction method may be useful for rapid identification of Mycobacterium spp. isolated from patients.
Dextran Sulfate ; DNA ; Edetic Acid ; Heating ; Hot Temperature ; Humans ; Hydrogen Peroxide ; Methanol ; Mycobacterium* ; Peroxidase ; Salmon ; Sodium

Objective To explore whether aging increases severity of colitis in mice and its mechanism.Methods Young (6-8 weeks)and aged (56 weeks) C57Bl/6 mice were divided into the control and experimental group (n=5,each). Dextran sodium sulfate(DSS) was used to induce acute colitis mouse model in the experimental group.The mRNA expressions of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in colon were measured by RT-PCR. Tight junctions (TJs) of intestinal epithelial cells was examined by transmission electron microscopy (TEM). Protein expressions of E-cadherin and occludin were detected by Western blotting and immunohistochemistry in colon.Results Compared with the young DSS-induced mice,the aged DSS-induced mice had more weight loss(t=3.679,P=0.006),higher disease indexes (t=2.496,P=0.037),higher histologic scores(U=0.000,P=0.008) and higher colonic IL-6 level (U=4.000,P=0.191). The TJs of intestinal epithelial cells were discontinuous in old healthy rats,and the TJs were destroyed significantly in both young and aged DSS-induced mice. Compared with the young DSS-induced mice,the aged DSS-induced mice had decreased protein expressions of E-cadherin (t=0.184,P=0.863)and occludin (t=0.399,P=0.710).Conclusions Aging leads to more severe disease following DSS challenge. Age-related deterioration in the functions of the gastrointestinal barrier and integrity may be one of the possible mechanisms.
Animals ; Colitis ; Colon ; Dextran Sulfate ; Disease Models, Animal ; Intestinal Mucosa ; Mice ; Mice, Inbred C57BL ; Rats

OBJECTIVE: To investigate whether kirenol, the major pharmacologically active compound of the Chinese medicinal herb , can protect mice from dextran sulfate sodium (DSS)-induced ulcerative colitis (UC). METHODS: C57BL/6 mice with or without kirenol pretreatment were treated with DSS in drinking water for 7 days to induce UC. The symptoms of UC including weight loss, diarrhea and bloody stool were observed daily and graded using the disease activity index (DAI). Colon injury of the mice was assessed by measuring the length of the colon and HE staining of the colon tissue. The levels of inflammatory cytokines produced by the mesenteric lymph nodes (MLNs) lymphocytes were measured using enzyme-linked immunosorbent assay; the apoptosis of the lymphocytes and CD4 T cells was analyzed using flow cytometry. RESULTS: The mice receiving pretreatment with kirenol showed obviously ameliorated symptoms of UC and milder pathological changes in the colon as compared with the control mice. Kirenol treatment significantly down-regulated the secretion of IFN-γ, IL-17A, IL-6 and TNF-α by the MLNs lymphocytes and increased the apoptosis of lymphocytes, especially CD4 T cells in the DSS-treated mice. CONCLUSIONS Kirenol can protect against T cell-mediated colon injury in DSS-treated mice possibly by suppressing the secretion of inflammatory mediators and inducing apoptosis of the inflammatory lymphocytes.
Animals ; Apoptosis ; Colitis, Ulcerative ; Cytokines ; Dextran Sulfate ; Diterpenes ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes

Thalidomide is an immunomodulatory drug (IMiD) with proven therapeutic action in several autoimmune/inflammatory diseases; however, its inherent high toxicity has led to the development of more powerful and safer thalidomide analogs, including lenalidomide and pomalidomide. These are new generation IMiDs that exhibit direct antitumor activity as well as anti-inflammatory/immunomodulatory properties, and are FDA-approved for the treatment of several hematological malignances. Here we investigated the potential therapeutic effects of lenalidomide and pomalidomide in several experimental murine models of autoimmune/inflammatory diseases: 2,4,6-trinitrobenzene sulfonic acid- and dextran sulfate sodium-induced inflammatory bowel disease and type II collagen-induced arthritis. Lenalidomide displayed a strong therapeutic effect in all these models of autoimmune/inflammatory diseases, while the effect of pomalidomide was less pronounced. In vitro experiments confirmed the immunosuppressive effect of both IMiDs on the proliferative response of stimulated human lymphocytes and on the balance of secreted cytokines toward an anti-inflammatory profile. We conclude that lenalidomide may offer a therapeutic opportunity against autoimmune/inflammatory diseases.
Arthritis, Experimental ; Arthritis, Rheumatoid* ; Cytokines ; Dextran Sulfate ; Humans ; In Vitro Techniques ; Inflammatory Bowel Diseases* ; Lymphocytes ; Models, Theoretical* ; Thalidomide ; Therapeutic Uses

Inflamatory bowel disease is the general medical terminology on chronic inflammatory illness of unknown origin, but it is considered that environmental, genetical, immunological factors may develop this chronic disease. I examined the histological changes on the experimental ulcerative colitis induced by dextran sulfate sodium(DSS) in rats. Experimental colitis was induced with 5% DSS in the drinking water for 5days in rats (experimental group). And the repair group were treated with 5% DSS for 5days and with pure water after 7days in rats. In experimental group, there are many inflammatory finding in colon of rat which contained loss of body weight, crypt erosion, recruitment of inflammatory cells, submucosal edema. In repair group, the inflammation was recovered that the body weight was incerased, the crypt was recovered. And Ki 67 immunoreaction were restricted lower 1/3 crypt in normal group but positive Ki 67 reaction appeared in the repaired all region. In this study, DSS was induced experimental colitis and the colitis was repaired when we stoped DSS supply. And the Ki 67 during repaired period were overproduction.
Animals ; Body Weight ; Chronic Disease ; Colitis* ; Colitis, Ulcerative ; Colon ; Dextran Sulfate* ; Dextrans* ; Drinking Water ; Edema ; Immunologic Factors ; Inflammation ; Rats ; Water

The present study aimed to explore the effect of nuclear factor erythroid 2 related factor 2(Nrf2)/heme oxygenase-1(HO-1) signaling pathway in intestinal protection by Sishen Pills against ulcerative colitis(UC). After the UC model was induced by 3% dextran sodium sulfate(DSS), experimental animals were randomly divided into control group, model group, salazosulfapyridine(SASP) group, and low-and high-dose Sishen Pills groups. Drug intervention(ig) was performed for seven consecutive days during modeling. On the 7 th day, the mice were euthanized. The body weight and colon length were recorded, and the histopathological changes of the colon were observed by HE staining. Serum interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), total antioxidant capacity(T-AOC), malondialdehyde(MDA), and reactive oxygen species(ROS) were detected by ELISA. The protein and mRNA expression of Nrf2, HO-1, and NADPH quinine oxidoreductase-1(NQO-1) was determined by Western blot and reverse transcription-polymerase chain reaction(RT-PCR). Compared with the normal group, the model group exhibited reduced body weight, colon length, and T-AOC, increased IL-6, TNF-α, MDA, and ROS, and diminished protein and mRNA expression of Nrf2, HO-1, and NQO-1 in the colon tissues. Compared with the model group, the SASP group and high-dose Sishen Pills group showed elevated body weight, colon length, and T-AOC, lowered IL-6, TNF-α, MDA, and ROS levels, and increased protein and mRNA expression of Nrf2, HO-1, and NQO-1 in the colon tissues. As assessed by HE staining, Sishen Pills could improve the pathological changes of the colon. The findings suggested that Sishen Pills could protect the colon against UC induced by 3% DSS. The specific mechanism of action may be related to the anti-inflammatory and anti-oxidative stress effects by the activation of the Nrf2/HO-1 signaling pathway.
Animals ; Colitis, Ulcerative/genetics* ; Dextran Sulfate ; Heme Oxygenase-1/metabolism* ; Mice ; NF-E2-Related Factor 2/metabolism* ; Signal Transduction

BACKGROUND: Oxidative stress plays an important role in the pathogenesis of inflammatory bowel disease. The objective of this study is to investigate the protective effect of Sasa quelpaertensis leaf extract (SQE) against oxidative stress in mice with dextran sulfate sodium (DSS)-induced colitis. METHODS: Mice were treated with SQE (100 mg/kg or 300 mg/kg body weight) by gavage in advance two weeks before inflammation was induced. Then, the mice were administered with 2.5% DSS in drinking water for 7 days and normal drinking water for 7 days between two DSS treatment. Disease activity index values, gut motility, and severity of the resulting oxidative DNA damage were analyzed. The antioxidant effect of SQE was evaluated by measuring malondialdehyde (MDA) and superoxide dismutase (SOD) activity in plasma samples. Catalase activity and expressions levels of glutathione peroxidase 1 (Gpx1), SOD1, and SOD2 were also detected in colon tissues. RESULTS: Administration of SQE significantly reduced the severity of DSS-induced colitis compared to the control (Ctrl) group. Levels of 8-oxo-dG, an oxidative DNA damage marker, were significantly lower in the SQE group compared to the untreated DSS Ctrl group. In the SQE (300 mg/kg) group, MDA levels were significantly lower, while SOD and catalase activity levels in the plasma samples were significantly higher compared with the DSS Ctrl group. The expression levels of the antioxidant enzymes, SOD2 and Gpx1, were significantly higher, while the levels of SOD 1 expression were lower, in the colon tissues of the DSS Ctrl group compared with those of the Ctrl group. In contrast, administration of SQE significantly down-regulated SOD2 and Gpx1 expressions and up-regulated SOD1 expression. CONCLUSIONS: These results indicate that SQE efficiently suppresses oxidative stress in DSS-induced colitis in mice, and its action is associated with the regulation of antioxidant enzymes.
Animals ; Antioxidants ; Catalase ; Colitis* ; Colon ; Dextran Sulfate* ; Dextrans* ; DNA Damage ; Drinking Water ; Glutathione Peroxidase ; Inflammation ; Inflammatory Bowel Diseases ; Malondialdehyde ; Mice* ; Oxidative Stress ; Plasma ; Sasa* ; Superoxide Dismutase

Mesenchymal stem cells (MSCs) have been used experimentally for treating inflammatory disorders, partly owing to their immunosuppressive properties. The goal of the study was to determine whether TLR ligands can enhance the therapeutic efficacy of bone marrow-derived MSCs for the treatment of inflammatory bowel disease. Mice (C57BL6) were administered with 4% dextran sulfate sodium (DSS) in drinking water for 7 days and injected with MSCs on days 1 and 3 following DSS ingestion. Our results demonstrated that among various TLR ligands, MSCs treated with polyinosinic-polycytidylic acid [poly(I:C)], which is a TLR3 ligand, more profoundly induced IDO, which is a therapeutically relevant immunosuppressive factor, without any observable phenotype change in vitro. The poly(I:C)-treated MSCs attenuated the pathologic severity of DSS-induced murine colitis when injected i.p. but not i.v. In summary, preconditioning MSCs with poly(I:C) might improve their efficacy in treating DSS-induced colitis, and this effect at least partly depends on the enhancement of their immunosuppressive activity through increasing their production of IDO.
Animals ; Colitis* ; Dextran Sulfate ; Drinking Water ; Eating ; In Vitro Techniques ; Indoleamine-Pyrrole 2,3,-Dioxygenase* ; Inflammatory Bowel Diseases ; Ligands ; Mesenchymal Stromal Cells ; Mice ; Phenotype ; Poly I-C ; Toll-Like Receptors

It is well known that proteins present in the primary urine are reabsorbed in the renal proximal tubules, and that this reabsorption is mediated via the megalincubilin complex and the neonatal Fcgamma receptor. However, the reabsorption is also thought to be influenced by an electrostatic interaction between protein molecules and the microvilli of the renal proximal tubules. By analyzing the charge diversity of urinary IgG, we showed that this reabsorption process occurs in a cationic charge-preferential manner. The charge-selective molecular sieving function of the glomerular capillary walls has long been a target of research since Brenner et al. demonstrated the existence of this function by a differential clearance study by using the anionic dextran sulfate polymer. However, conclusive evidence was not obtained when the study was performed using differential clearance of serum proteins. We noted that immunoglobulin (Ig) A and IgG have similar molecular sizes but distinct molecular isoelectric points. Therefore, we studied the differential clearance of these serum proteins (clearance IgA/ clearance IgG) in podocyte diseases and glomerulonephritis. In addition, we studied this differential clearance in patients with Dent disease rather than in normal subjects because the glomerular sieving function is considered to be normal in subjects with Dent disease. Our results clearly showed that the charge-selective barrier is operational in Dent disease, impaired in podocyte disease, and lacking in glomerulonephritis.
Blood Proteins ; Capillaries ; Child Health ; Dent Disease ; Dextran Sulfate ; Glomerulonephritis ; Humans ; Immunoglobulin A ; Immunoglobulin G ; Immunoglobulins ; Isoelectric Point ; Microvilli ; Nephritis ; Podocytes ; Polymers ; Proteinuria*

Inflammatory bowel disease is a chronic inflammatory disorder occurring in the gastrointestinal track. However, the efficacy of current therapeutic strategies has been limited and accompanied by side effects. In order to eliminate the limitations, herbal medicines have recently been developed for treatment of IBD. Peuraria Lobata (Peuraria L.) is one of the traditional herbal medicines that have anti-inflammatory effects. Bioavailability of Peuraria L., which is rich in isoflavones, is lower than that of their fermented forms. In this study, we generated fermented Peuraria L. extracts (fPue) and investigated the role of fPue in inflammation and intestinal barrier function in vitro and in vivo. As the mice or intestinal epithelial cells were treated with DSS/fPue, mRNA expression of pro-inflammatory cytokines was reduced and the architecture and expression of tight junction proteins were recovered, compared to the DSS-treated group. In summary, fPue treatment resulted in amelioration of DSS-induced inflammation in the colon, and the disrupted intestinal barrier was recovered as the expression and architecture of tight junction proteins were retrieved. These results suggest that use of fPue could be a new therapeutic strategy for treatment of IBD.
Animals ; Biological Availability ; Colitis* ; Colon ; Cytokines* ; Dextran Sulfate* ; Dextrans* ; Epithelial Cells ; In Vitro Techniques ; Inflammation ; Inflammatory Bowel Diseases ; Isoflavones ; Mice ; Pueraria* ; RNA, Messenger ; Tight Junction Proteins