Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-induced STAT6 Activation and STAT6-dependent Host Arginase 1 Expression

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection

Differentially expressed genes in aortic cells from atherosclerosis-resistant and atherosclerosis-susceptible pigeons

Representational Difference Analysis (RDA) was used to identify genes that were differentially expressed between White Carneau (WC) and Show Racer (SR) pigeon aortic smooth muscle cells. The gene(s) responsible for atherosclerotic resistance in cultured SR smooth muscle cells (SMC) were hypothesized to be silent or down regulated in the WC. In the reciprocal experiments, it was hypothesized that the gene(s) contributing to the spontaneous atherosclerotic phenotype in cultured WC SMC would not be expressed in the SR. Total RNA was extracted from primary cultured cells of each breed, converted to cDNA, and compared in four reciprocal RDA experiments. Seventy-four transcripts were identified exclusively in the WC cells, and 63 were unique to the SR. Genes representing several biochemical pathways were distinctly different between aortic cells from susceptible (WC) and resistant (SR) pigeons. The most striking genetic differences were observed in energy metabolism and smooth muscle contractility. The WC cells derived their energy from glycolysis, while the SR cells utilized oxidative phosphorylation to produce energy. Myosin light chain kinase and alpha actin were exclusively expressed by the SR SMC, whereas beta actin and collagen were dominant in the WC. Because of the compressed in vitro time frame compared with in vivo development, it was not obvious whether insufficient ATP synthesis is preventing the WC aortic cells from performing their contractile function or if the lack of functional contractile elements in the WC causes the mitochondrial ATP synthesis to down regulate. Either way, energy production was successfully coupled to muscle contraction in the SR, but not in the WC. This difference was observed prior to lipid accumulation in the WC cells, and appears to be a major contributing factor in pigeon atherogenesis. One hundred forty five pigeon transcripts were homologous to the chicken. However, the mitochondrial genes expressed in the pigeon were more closely related to non-domestic birds such as the turkey vulture and oriental stork. Despite this categorical exception, the recently published chicken genome was an ideal resource for identifying differentially expressed genes in the pigeon. The results were interpreted in the context of current hypotheses of human atherogenesis. The pigeon transcripts can also be used in comparative studies of avian genomics

Experimental methodology for non-thermal effects of electromagnetic radiation on biologics

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2006.Includes bibliographical references (p. 133-137).Appropriate equipment is needed for research on the effects of radio-frequency radiation from radio-frequency identification (RF-ID) systems on biological materials. In the present study, a complete test system comprising assembled hardware and custom-built software was developed for a research project investigating whether RFID radiation produces significant effects in biologics. Furthermore, we document a method for determining specific absorption rate (S.A.R.) using vials containing 1.5 cc of saline to represent biological samples. This methodology yielded S.A.R. values of approximately 30 W kg-1(-9 dBm) and 150 W kg-1(-6.5 dBm) for 915 MHz and 320 W kg-1 (-9 dBm) and 450 W kg-1 (-6.5 dBm) for 2.45 GHz. Finally, the key system components - the transverse electromagnetic (TEM) cell, vial and saline solution -- were modelled using CST Microwave Studio®. Through modelling, we obtained values for the absorption as a percentage of the incident power - 0.2% for 915 MHz and 7.32% for 2.45 GHz. These values approximately matched those calculated - 0.36% for 915 MHz and 5.0% for 2.45 GHz. Errors in the calculated absorption were due primarily to the precision of the power meters relative to the power levels being detected, and indicate that further study may be required.by Felicia C.A.I. Cox.M.Eng

Arg1 expression and polyamine content in <i>L. donovani</i> infected macrophages.

<p> <b>A</b>) Arginase activity in macrophages isolated by adherence from single-cell suspensions from the spleens of control hamsters (0 days post-infection) and hamsters infected with <i>L. donovani</i> for 7, 14, 21, 42, and 56 days. The mean and SD (error bars) of the arginase activity in 100,000 cells, determined by assay of urea production, is shown from a single experiment that is representative of 2 independent experiments. <b>B</b>) Log₁₀ ratio of host arg1 to NOS2 mRNA in splenic macrophages of 4-week <i>L. donovani</i> infected mice and hamsters. The mRNA expression was determined by real time RT-PCR in groups of 5 animals and used to calculate the log ratios, shown as the mean and SD (error bars) from a single experiment that is representative of 2 independent experiments. The ratios were determined from the following raw data: Arg1 mRNA fold-increase with reference to the BHK cell calibrator (mean ± SD): uninfected mice, 5.7±5.9; uninfected hamster, 3,367±2,858, infected mice, 8.5±10; infected hamster, 131,984±56,407; iNOS mRNA fold-increase with reference to BHK cell calibrator (mean ± SD): uninfected mice, 133.34±182.4; uninfected hamster, 1.03±0.3, infected mice, 2,984±4,535; infected hamster, 19.56±10.87). <b>C</b>) Arginase activity in peritoneal macrophages isolated from mice and hamsters that were uninfected (open bars) or infected in vitro with <i>L. donovani</i> for 48 hrs (filled bars). The mean and SD (error bars) of the arginase activity, determined by assay of urea production, is shown from a single experiment that is representative of 2 independent experiments. <b>D</b>) Arginase activity in hamster peritoneal macrophages that were uninfected (Un) or infected in vitro with <i>L. donovani</i> (1∶5 macrophage:parasite ratio). Parasites were either unopsonized (None), opsonized with normal fresh complement-containing hamster serum (Comp), or opsonized with freeze-thawed hamster serum containing anti-Leishmania antibody (Ab). The mean and standard deviation (error bars) of the arginase activity in 200,000 cells of 6 different samples determined by assay of urea production, is shown from a single experiment that is representative of 2 experiments. Statistical comparisons are made to the control group. <b>E</b>) Expression of hamster arginase mRNA in hamster peritoneal macrophages that were uninfected (Un) or infected in vitro with <i>L. donovani</i> stationary-phase promastigotes for 24 hrs (Inf). The mean and SEM (error bars) of the fold-increase of arg1 mRNA relative to BHK cells, determined by real time RT-PCR in groups of 6 samples per experiment, is shown as data pooled from 4 independent experiments. <b>F</b>) The concentration of polyamines in uninfected (open bars) and 48-hour in vitro infected hamster peritoneal macrophages (filled bars) (n = 6 per group) is expressed as the mean and SD (error bars) of nmol polyamine per mg protein. The data shown are from a single experiment that is representative of 2 independent experiments. The statistical significance of differences in each of the panels is identified by asterisks (*, p<0.05; **, p<0.01; ***, p<0.001).</p

Kinetics of arginase expression in spleen tissue during progressive visceral leishmaniasis. A

<p>) Parasite burden in the spleens of hamsters (n = 6 per group) infected with <i>L. donovani</i> for 14, 28, 42, and 56 days. The mean and SEM (error bars) of the parasite burden, determined by luminometry and interpolation from an amastigote standard curve, is shown from a single experiment that is representative of 2 independent experiments. <b>B</b>) Arginase activity in the serum (open bars) and spleen tissue (filled bars) of uninfected hamsters (Day 0) and hamsters infected with <i>L. donovani</i> (n = 6 per group) for 7, 14, 28, 42, and 56 days. The mean and SD of the tissue arginase activity, determined by assay of urea production, is shown. <b>C</b>) Hamster arg1 protein expression determined by western blot in spleens of control hamsters (0 days post-infection) and hamsters infected with <i>L. donovani</i> for 7, 14, and 28 days. The expression of GAPDH is shown as a control for protein loading. Each lane contains splenic lysate from a single hamster, with two lanes per time point. The anti-arginase antibody did not react with parasite arginase by immunoblot. <b>D</b>) Time course of expression of hamster arg1 mRNA (filled bars), <i>L. donovani</i> arginase mRNA (empty bars), hamster NOS2 (iNOS) mRNA (hatched bars), and hamster arg2 mRNA (hatched bars) in spleens of control hamsters (0 days post-infection) and hamsters infected with <i>L. donovani</i> for 7, 14, 28, 42, and 56 days. The mean and standard deviation (error bars) of the fold-increase of arginase mRNA relative to BHK cells, determined by real time RT-PCR in groups of 6 animals, is shown from a single experiment that is representative of 3 independent experiments. The statistical significance of differences in each of the panels is identified by asterisks (*, p<0.05; **, p<0.01; ***, p<0.001).</p