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

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

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

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

Dysfunction of gut immune regulation is involved in mucosal damage in inflammatory bowel disease (IBD). However, there is still no efficacious immune-regulator for the treatment of IBD. Alloferon is a novel immune-modulatory peptide that was originally isolated from infected insects. It shows anti-inflammatory effects by the regulation of cytokine production by immune cells and their activities. Therefore, we investigated the effect of alloferon in a mouse model of colitis using dextran sulfate sodium (DSS). Colitis was induced by administration of DSS in drinking water for 7 consecutive days. It was confirmed by the presence of weight loss, diarrhea, hematochezia, and colon contraction. Alloferon was injected 4 days after DSS administration. We found that alloferon improved the pathogenesis of IBD based on the reduced disease activity index (DAI) and colon contraction. Edema, epithelial erosion, and immune cell infiltration were found in mice administered DSS, but the phenomena were reduced following alloferon treatment. The plasma level of IL-6, a classical pro-inflammatory cytokine in colitis, was also decreased by alloferon. Moreover, alloferon inhibited the TNF-alpha-induced degradation and phosphorylation of IkappaB in Colo205 colon cancer cells. Taken together, these results show that alloferon has anti-inflammatory effects and attenuates DSS-induced colitis.
Animals ; Colitis* ; Colon ; Colonic Neoplasms ; Dextran Sulfate* ; Diarrhea ; Drinking Water ; Edema ; Gastrointestinal Hemorrhage ; Inflammatory Bowel Diseases ; Insects ; Interleukin-6 ; Mice ; Phosphorylation ; Plasma ; Weight Loss

BACKGROUND: In 2001, the third report the National Cholesterol Education Program (NCEP) has concluded that LDL cholesterol levels should be a major goal for preventing coronary artery disease and atherosclerotic events. Those in the higher risk groups should then have lipoprotein analysis to determine LDL cholesterol levels. LDL cholesterol has traditionally been estimated by the Friedwald forrmula : LDL-C=total cholesterol-[high density lipoprotein cholesterol (HDL-C)+trigryceride/5]. However, when trigryceride level is >400 mg/dL, this formula is inaccurate. Therefore, We have compared the direct LDL cholesterol immunoseparation method with Friedwald formula from both normotriglyceridemic (triglyceride400 mg/dL). METHODS: The direct LDL cholesterol immunoseparation method was performed on 53 sera with triglyceride levels 61 to 1,684 mg/dL (classified as400 mg/dL : 31). Total cholesterol was measured enzymatic colorimetry. HDL cholesterol was measured in the supernatant after precipitating LDL by HDL cholesterol precipitating reagent containing dextran sulfate and magnesium chloride for serum. Direct LDL cholesterol was measured by immunoseparation method (Sigma Diagnostics, St Louis, Mo) that is based on selective absorption of HDL cholesterol and VLDL cholesterol by polystylene beads coated with goat polyclonal antibodies to human apolipoproteins. The cholesterol was measured by an enzamatic method on Hitachi 747. The linear regression and paird t-test were performed to evaluate the differences of data from Immunoseparation method and Friedwald formula. RESULTS: In triglyceride400 mg/dL, the LDL cholesterol value obtained by the direct LDL-C assay on the 31 frozen sera studied was significantly different from that of Friedwald formula (103+/-38.4; 50.4+/-56.2 mg/dL). Therefore, Friedwald formula is unreliable in triglyceride>400 mg/dL. CONCLUSION: Guidelines for treatment decisions, including diet and drug initiation, and therapeutic goals for high risk groups are based entirely on the LDL cholesterol level. At triglyceride levels >400 mg/dL, Friedwald fomula is inaccurate. Immunoseparation method is more rapid, higher specific, precise and helps monitor LDL lowering drugs and diets in triglyceride level>400 mg/dL.
Absorption ; Antibodies ; Apolipoproteins ; Cholesterol* ; Cholesterol, HDL ; Cholesterol, LDL ; Cholesterol, VLDL ; Colorimetry ; Coronary Artery Disease ; Dextran Sulfate ; Diet ; Education ; Goats ; Humans ; Linear Models ; Lipoproteins* ; Magnesium Chloride ; Triglycerides

BACKGROUNDDisrupted Ca2+ homeostasis contributes to the development of colonic dysmotility in ulcerative colitis (UC), but the underlying mechanisms are unknown. This study aimed to examine the alteration of colonic smooth muscle (SM) Ca2+ signaling and Ca2+ handling proteins in a rat model of dextran sulfate sodium (DSS)-induced UC.

METHODSMale Sprague-Dawley rats were randomly divided into control (n = 18) and DSS (n = 17) groups. Acute colitis was induced by 5% DSS in the drinking water for 7 days. Contractility of colonic SM strips (controls, n = 8 and DSS, n = 7) was measured in an organ bath. Cytosolic resting Ca2+ levels (n = 3 in each group) and Ca2+ transients (n = 3 in each group) were measured in single colonic SM cells. Ca2+ handling protein expression was determined by Western blotting (n = 4 in each group). Differences between control and DSS groups were analyzed by a two-sample independent t-test.

RESULTSAverage tension and amplitude of spontaneous contractions of colonic muscle strips were significantly enhanced in DSS-treated rats compared with controls (1.25 ± 0.08 g vs. 0.96 ± 0.05 g, P= 0.007; and 2.67 ± 0.62 g vs. 0.52 ± 0.10 g, P= 0.013). Average tensions of carbachol-evoked contractions were much weaker in the DSS group (1.08 ± 0.10 g vs. 1.80 ± 0.19 g, P= 0.006). Spontaneous Ca2+ transients were observed in more SM cells from DSS-treated rats (15/30 cells) than from controls (5/36 cells). Peak caffeine-induced intracellular Ca2+ release was lower in SM cells of DSS-treated rats than controls (0.413 ± 0.046 vs. 0.548 ± 0.041, P= 0.033). Finally, several Ca2+ handling proteins in colonic SM were altered by DSS treatment, including sarcoplasmic reticulum calcium-transporting ATPase 2a downregulation and phospholamban and inositol 1,4,5-trisphosphate receptor 1 upregulation.

CONCLUSIONSImpaired intracellular Ca2+ signaling of colonic SM, caused by alteration of Ca2+ handing proteins, contribute to colonic dysmotility in DSS-induced UC.

Animals ; Colitis ; chemically induced ; metabolism ; Colon ; cytology ; metabolism ; Dextran Sulfate ; toxicity ; Male ; Muscle, Smooth ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology