We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37 degrees C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham's F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number.(ABSTRACT TRUNCATED AT 250 WORDS)
Chronic (20-week) Schistosoma mansoni infections of CBA/J male mice present as two distinct forms of morbidity. Most mice develop moderate splenomegaly syndrome (MSS) resembling the intestinal form of chronic human schistosomiasis mansoni, while approximately 20% of mice develop hypersplenomegaly syndrome (HSS), more consistent with the severe hepatosplenic form of chronic human schistosomiasis mansoni. Here, we report the relative proportions of natural T regulatory cells (Treg) and activated CD4(+) T cells (Tact) for both splenic and granulomatous cell populations of MSS and HSS mice. Proportions of both Treg and Tact are greater in HSS than MSS mice. However, the ratios of Treg to Tact in both splenic and granulomatous cell populations from MSS mice are significantly higher than those of HSS mice. For both HSS and MSS mice, in vitro proliferation of their CD3(+) splenic cells induced by soluble egg antigens is inversely correlated with the ratio of Treg to Tact. Also, spleen or granuloma cells from MSS mice produced higher mean levels of IFN-gamma than those from HSS mice. Differential IFN-gamma productive capacities dictated by Treg : Tact ratios may contribute to the development of differential morbidities in this model of chronic experimental schistosomiasis mansoni.
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