S from either syngeneic (BALB/c) or allogeneic (B10.D2) donor

S from either syngeneic (BALB/c) or K162 allogeneic (B10.D2) donor mice. Transplant parameters were chosen to avoid excessive acute GVHD-related mortality in allogeneic recipients so that sufficient animals would be available for analysis at day +100 after 22948146 HCT. Survival was monitored daily until day +100 after HCT, and clinical GVHD scores were assessed weekly using a commonly used acute GVHD scoring system incorporating following five clinical parameters:, weight loss, posture (hunching), mobility, fur texture and skin integrity [18]. Each parameter was graded between 0 and 2. Once an animal reached a cumulative score of more than 6.5 or a weight loss of more than 30 , it was sacrificed and counted as death due to transplantation related mortality.MCMV reactivationDNA was extracted from spleen by use of the DNeasy Blood Tissue kit (QIAGEN, Valencia, CA). Primers for transcription of IE-1 were used as described [23]. DNA was amplified in the following condition: 1 cycle at 94uC for 3 min; 35 cycles of 30 sec at 94uC, 30 sec at 53uC and 30 sec at 72uC; and 1 cycle at 72uC for 7 min; the reaction was performed in total volume of 50 ul with Promega enzyme and reagents. Amplified products were separated by electrophoresis in 1 agarose gels, and gels were stained with ethidium bromide.HistopathologyAt 100 days after transplantation, animals were sacrificed for analysis. Organs were removed and fixed in formalin for 48 hrs, then transferred into 70 ethanol, paraffin-embedded and sectioned. Hematoxylin osin-stained lung, liver and colonCMV and GVHDMeasurement of cytokine and chemokine levels by ELISALung, liver and colon samples were homogenized in cold PBS with complete protease inhibitor cocktail (Roche Diagnostics, IN, USA). The levels of TNF, IFN- c, CXCL1 and CXCL9 were detected using specific standard sandwich ELISA. TNF and IFNc were detected using mouse cytokine ELISA kit from BD Biosciences, CXCL1 and CXCL9 were detected using mouse chemokine ELISA kit from R D Systems Inc. Minneapolis, MN, USA according to manufacturer’s protocol. Absorbance was measured at 450 nm using an ELISA plate reader (MULTISKAN FC, Thermo Scientific, Asheville, NC USA).MCMV latency increases clinical GVHD severity and mortality after allogeneic HCTFollowing a waiting period of 25 week after MCMV or mock infection, animals underwent HCT from either syngeneic (BALB/ c) or allogeneic (B10.D2) donors. Hypothesizing, that MCMV potentially exacerbates GVHD, a conditioning K162 site regimen of 15755315 750cGy TBI and a relatively low dose of 36106 splenocytes were chosen as transplant parameters. Hereby it was aimed to achieve donor engraftment and at least mild GVHD pathology in allogeneic controls, and at the same time to allow for a sufficient number of allogeneic MCMV treated recipients reaching the planned time endpoint for analysis at day +100. Mice were divided into four experimental groups: group 1 – mock infected syngeneic; group 2 MCMV latent syngeneic; group 3 – mock infected allogeneic; group 4 – MCMV latent allogeneic. Following HCT, survival and clinical GVHD were monitored daily or weekly, respectively. As expected, all mock treated syngeneic recipients survived and were clinically healthy. MCMV latent syngeneic recipients showed slightly elevated clinical scores initially, but after 8 weeks did not differ from syngeneic Mock controls. Allogeneic controls developed moderate clinical symptoms of GVHD and were evidently sick when compared to syngeneic groups, yet as intend.S from either syngeneic (BALB/c) or allogeneic (B10.D2) donor mice. Transplant parameters were chosen to avoid excessive acute GVHD-related mortality in allogeneic recipients so that sufficient animals would be available for analysis at day +100 after 22948146 HCT. Survival was monitored daily until day +100 after HCT, and clinical GVHD scores were assessed weekly using a commonly used acute GVHD scoring system incorporating following five clinical parameters:, weight loss, posture (hunching), mobility, fur texture and skin integrity [18]. Each parameter was graded between 0 and 2. Once an animal reached a cumulative score of more than 6.5 or a weight loss of more than 30 , it was sacrificed and counted as death due to transplantation related mortality.MCMV reactivationDNA was extracted from spleen by use of the DNeasy Blood Tissue kit (QIAGEN, Valencia, CA). Primers for transcription of IE-1 were used as described [23]. DNA was amplified in the following condition: 1 cycle at 94uC for 3 min; 35 cycles of 30 sec at 94uC, 30 sec at 53uC and 30 sec at 72uC; and 1 cycle at 72uC for 7 min; the reaction was performed in total volume of 50 ul with Promega enzyme and reagents. Amplified products were separated by electrophoresis in 1 agarose gels, and gels were stained with ethidium bromide.HistopathologyAt 100 days after transplantation, animals were sacrificed for analysis. Organs were removed and fixed in formalin for 48 hrs, then transferred into 70 ethanol, paraffin-embedded and sectioned. Hematoxylin osin-stained lung, liver and colonCMV and GVHDMeasurement of cytokine and chemokine levels by ELISALung, liver and colon samples were homogenized in cold PBS with complete protease inhibitor cocktail (Roche Diagnostics, IN, USA). The levels of TNF, IFN- c, CXCL1 and CXCL9 were detected using specific standard sandwich ELISA. TNF and IFNc were detected using mouse cytokine ELISA kit from BD Biosciences, CXCL1 and CXCL9 were detected using mouse chemokine ELISA kit from R D Systems Inc. Minneapolis, MN, USA according to manufacturer’s protocol. Absorbance was measured at 450 nm using an ELISA plate reader (MULTISKAN FC, Thermo Scientific, Asheville, NC USA).MCMV latency increases clinical GVHD severity and mortality after allogeneic HCTFollowing a waiting period of 25 week after MCMV or mock infection, animals underwent HCT from either syngeneic (BALB/ c) or allogeneic (B10.D2) donors. Hypothesizing, that MCMV potentially exacerbates GVHD, a conditioning regimen of 15755315 750cGy TBI and a relatively low dose of 36106 splenocytes were chosen as transplant parameters. Hereby it was aimed to achieve donor engraftment and at least mild GVHD pathology in allogeneic controls, and at the same time to allow for a sufficient number of allogeneic MCMV treated recipients reaching the planned time endpoint for analysis at day +100. Mice were divided into four experimental groups: group 1 – mock infected syngeneic; group 2 MCMV latent syngeneic; group 3 – mock infected allogeneic; group 4 – MCMV latent allogeneic. Following HCT, survival and clinical GVHD were monitored daily or weekly, respectively. As expected, all mock treated syngeneic recipients survived and were clinically healthy. MCMV latent syngeneic recipients showed slightly elevated clinical scores initially, but after 8 weeks did not differ from syngeneic Mock controls. Allogeneic controls developed moderate clinical symptoms of GVHD and were evidently sick when compared to syngeneic groups, yet as intend.

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