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Pathological and immunological profiles of rat tuberculosis

Pathological and immunological profiles of rat tuberculosis. of tuberculosis. Intro All-is significantly restricted in THP-1 macrophages exposed to chenodeoxycholic acid/RA (13). Taken together, these findings suggest that RA may have restorative potential for tuberculosis. In this study, we examined the therapeutic effectiveness of orally given RA on development of rat tuberculosis after aerosol illness with H37Rv (ATCC27294) was produced in Middlebrook 7H9 broth with 0.05% Tween 80 for 2 wk. Then, the broth was filtered with an Acrodisk filter no. 4650 (pore size 5.0 m, Pall) to disperse bacillary clumps. The filtered bacillary answer was then stored freezing at ?80C until use. The rats were infected via the aerosol route using a Glas-Col aerosol generator, in which the nebulizer compartment was filled RK-33 with 5 mL of a suspension comprising 107 colony-forming models (CFU) of H37Rv bacilli under conditions that would allow ~500 bacilli to be inhaled by each rat. Related experiments were performed twice with similar results and 1 experiment is demonstrated for the 2 2 studies. The number of viable bacteria in the lungs was identified at specific time points by plating 10-fold serial dilutions of individual partial organ homogenates on 1% Ogawas medium and counting bacterial colonies after 4 wk of incubation at 37C, as previously explained (14). Dental administration of vitamin A as RA After aerosol illness with tubercle bacilli, all-= 15). For control rats (= 15), the vehicle dose was given in the same manner as that for RA-treated rats. Dental administration was performed throughout the experimental period inside a security cabinet placed in a level 3 biosafety facility without illumination inside the cabinet, but with space illumination, to avoid degradation of the RA. CFU assay At 1, 3, and 5 wk after aerosol illness, groups of 3 rats were anesthetized with pentobarbital sodium, the abdominal cavities RK-33 were incised, and exsanguination was carried out by splenectomy and transection of the remaining renal artery and vein. The lungs, spleens, and livers were excised and weighed. Part of the right lower lobes of the lungs and part of the spleen were weighed separately and used to evaluate the in vivo growth of (14). The lung and spleen samples for in vivo CFU assay were each homogenized having a mortar and pestle and then placed in test tubes and 1 mL of sterile saline was added to each sample. After homogenization, 100 RK-33 L of the homogenate was plated in 10-collapse serial dilutions on 1% Ogawa slant medium. Colonies within the medium were counted after 4 wk of incubation at 37C (14,15). RNA extraction and real-time PCR Another portion of the remaining right lower lobe of the lungs and the spleen were utilized for RT-PCR analysis to RK-33 examine the manifestation levels of several cytokine messenger RNA (mRNA) in these samples during illness. These samples were snap-frozen in liquid nitrogen and stored at ?85C until use. RNA extraction was performed as explained previously (14). Briefly, the frozen cells were homogenized inside a microcentrifuge tube with an autoclaved disposable 1000-L tip cooled by dipping in liquid nitrogen. Then the homogenates were treated with 1 mL of TRIzol reagent (Invitrogen Japan, K.K.), as specified by the manufacturer. After RNA isolation, total RNA concentration was measured having a spectrophotometer and the agarose gel electrophoresis pattern of the total RNA was examined. The total RNA were reverse transcribed into cDNA with Moloney murine leukemia computer virus RT (Invitrogen). ABI Taqman Gene Manifestation Assay was utilized for relative quantitative measurement of the mRNA manifestation of IFN (Rn00594078_m1), TNF (Rn00562055_m1), inducible nitric oxide synthetase (iNOS) (Rn00561646_m1), IL-1 (Rn00580432_m1), IL-2 (Rn00587673_m1), IL-4 (Rn01456866_m1), IL-6 (Rn00561420_m1), IL-10 (Rn00563409_m1), IL-12 p40 (Rn00575112_m1), IP-10 (CXCL-10, Rn00594648_m1), and TGF (Rn00572010_m1). A TaqMan Rodent GAPDH Control Reagents arranged was utilized for normalization for data analysis. Real-time RT-PCR was performed according to the instructions for the ABI PRISM 7900HT Sequence Detection system (Applied BioSystems). Data were analyzed from the CT method using to the ABI PRISM Sequence Detection system software package (version 2.1; Applied BioSystems) working on a Windows GABPB2 2000 OS. The results from RA-treated and control rats were indicated relatively, with manifestation in the focuses on compared with those of uninfected rats that were calibrated with the manifestation of an internal control gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (16). Effect of in vitro RA treatment on bacterial burden and cytokine mRNA manifestation Alveolar macrophages were prepared from uninfected rats.


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