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Droplet digital PCR for the precise quantification of HTLV-1
76
Abstracts
resident cells and recruitment of immune cells from the periphery. We were interested to analyze the phenotype, function and dynamics of myeloid cell-subsets. Here we show, that depletion of CCR2+ Ly6Chi inflammatory monocytes, a subset of recruited myeloid cells, results in decreased survival, suggesting their critical importance in host defense against T. gondii infection. Moreover, CCR2+ Ly6Chi monocytes produce pro-inflammatory mediators, such as IL-1a, IL-1b, iNOS, TNF and ROS, confirming their anti-parasitic role. Furthermore, we demonstrate by adoptive transfer that inflammatory monocytes develop into two distinct subpopulations: CCR2+ Ly6Cint F4/80int and CCR2+ Ly6Cneg F4/80hi. The CCR2+ Ly6Cint F4/80int cells perform dendritic cell like functions such as interacting with T cells via MHC I and MHC II molecules. The CCR2+ Ly6Cneg F4/80hi cell subset displays elevated phagocytic capacity and upregulates TREM2. Finally, we show that recruitment of Ly6Chi monocytes to the CNS is mediated by selectins. These results indicate the crucial role of recruited Ly6Chi monocytes upon cerebral toxoplasmosis, unraveling the dynamics and function of further differentiated mononuclear cell subsets in parasite control and immune regulation in the CNS. doi:10.1016/j.jneuroim.2014.08.199
361 Peripheral sympathectomy affects the course and severity of Friend retrovirus infection in mice Dominique Bloemkera, Kathrin Gibbertb, Adriana Del Reyc, Ulf Dittmerb, Manfred Schedlowskia, Harald Englera a Institute of Medical Psychology and Behavioral Immunobiology, University Hospital Essen, Essen, Germany; bInstitute of Virology, University Hospital Essen, Essen, Germany; cDepartment of Immunophysiology, Institute of Physiology and Pathophysiology, Philipps University of Marburg, Marburg, Germany
The regulation of immune responses is not exclusively mediated by factors intrinsic to the immune system. In fact, a variety of important immune processes such as leukocyte trafficking, antigen presentation, cell activation, and clonal expansion were shown to be affected by noradrenaline (NA), the main neurotransmitter of the sympathetic nervous system. Here we demonstrate that also a lack in sympathetic output can have profound effects on immune responses and can influence the severity and course of an acute retroviral infection. Adult C57BL/6 mice were chemically sympathectomized with two injections (150 mg/kg) of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) and subsequently infected with Friend retrovirus (FV), a murine erythroleukemia virus that induces a strong immune response and causes a transient enlargement of the spleen during the acute infection. Neurotoxin treatment effectively destroyed peripheral noradrenergic nerve terminals and led to a substantial reduction (N85%) in splenic NA levels compared to vehicle-treated controls. More importantly, sympathectomized mice developed a significantly more pronounced splenomegaly and exhibited higher splenic viral loads on days 5, 10 and 15 post-infection compared to vehicle-treated mice, indicating a more severe disease course in animals lacking peripheral NA. This suggests that NA and/or certain sympathetic tone are required for normal immune functioning during acute FV infection. The underlying mechanisms are currently under investigation and will provide new insights into the relevance of SNS–immune interactions during viral infections.
doi:10.1016/j.jneuroim.2014.08.200
422 Droplet digital PCR for the precise quantification of HTLV-1 Breanna Caruso, Giovanna Brunetto, Raya Massoud, Emily Leibovitch, Steven Jacobson NINDS, NIH, Bethesda, United States Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that is the etiologic agent in a clinical neuromyelopathy known as HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/ TSP). Quantification of the amount of virus in patient samples is critical for monitoring infection, evaluating the efficacy of therapeutic agents, and assisting with clinical diagnosis. Real-time PCR (qPCR) is the standard method for determining HTLV-1 proviral load (PVL). However, qPCR is dependent upon well-defined standards and calibration to a standard curve, precluding precise and accurate quantification particularly at low cell concentrations. In this study, we use a third generation PCR technique, droplet digital PCR (ddPCR), which allows for direct quantification of PVL. DNA samples are partitioned into thousands of nanoliter-sized droplets, amplified on a thermocycler, analyzed for fluorescent signal, and normalized to a housekeeping gene. Poisson distribution statistics are used to determine absolute copy numbers independently of a standard curve. We found that the intra-assay and inter-assay reliability of ddPCR are robust and more consistent than those of qPCR. Moreover, ddPCR yields similar patient PVL by quantifying different viral genes. Our preliminary data suggests ddPCR can also be used to examine viral gene expression, a key player in the pathogenesis of HAM/TSP and an important parameter to monitor during therapeutic interventions in this condition. The reliability and precision of ddPCR for HTLV-1 quantification should be widely acknowledged as it might be an important tool to further understand the clinical correlates of the viral load in the context of HAM/TSP and other HTLV-1 related conditions; however, further investigation is required to determine the efficacy of ddPCR for mRNA analysis. doi:10.1016/j.jneuroim.2014.08.201
582 Risk factors for immunosuppression-associated infections in aquaporin-4 antibody mediated neuromyelitis optica spectrum disorders Fan Chenga, Jithin Georgea, J. Rochab, George Tackleya, John Revisa, Paddy Watersa, John Elstonc, Jacqueline Palacea, Nicola Jonesd, Maria Isabel Leitea a Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, United Kingdom; bDepartment of Clinical Neurology, Hospital S. Marcos, Braga, Portugal; cThe Oxford Eye Hospital, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom; dDepartment of Infectious Diseases, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom
Introduction: Opportunistic, atypical and severe infections may be serious complications of immunosuppressive therapy in chronic antibody mediated diseases, such as neuromyelitis optica spectrum disorders (NMOSD) associated with aquaporin-4 antibodies (AQP4-Abs). However, no studies have yet investigated the frequency and risk factors for immunosuppression-associated severe infections in NMOSD patients.
Abstracts
resident cells and recruitment of immune cells from the periphery. We were interested to analyze the phenotype, function and dynamics of myeloid cell-subsets. Here we show, that depletion of CCR2+ Ly6Chi inflammatory monocytes, a subset of recruited myeloid cells, results in decreased survival, suggesting their critical importance in host defense against T. gondii infection. Moreover, CCR2+ Ly6Chi monocytes produce pro-inflammatory mediators, such as IL-1a, IL-1b, iNOS, TNF and ROS, confirming their anti-parasitic role. Furthermore, we demonstrate by adoptive transfer that inflammatory monocytes develop into two distinct subpopulations: CCR2+ Ly6Cint F4/80int and CCR2+ Ly6Cneg F4/80hi. The CCR2+ Ly6Cint F4/80int cells perform dendritic cell like functions such as interacting with T cells via MHC I and MHC II molecules. The CCR2+ Ly6Cneg F4/80hi cell subset displays elevated phagocytic capacity and upregulates TREM2. Finally, we show that recruitment of Ly6Chi monocytes to the CNS is mediated by selectins. These results indicate the crucial role of recruited Ly6Chi monocytes upon cerebral toxoplasmosis, unraveling the dynamics and function of further differentiated mononuclear cell subsets in parasite control and immune regulation in the CNS. doi:10.1016/j.jneuroim.2014.08.199
361 Peripheral sympathectomy affects the course and severity of Friend retrovirus infection in mice Dominique Bloemkera, Kathrin Gibbertb, Adriana Del Reyc, Ulf Dittmerb, Manfred Schedlowskia, Harald Englera a Institute of Medical Psychology and Behavioral Immunobiology, University Hospital Essen, Essen, Germany; bInstitute of Virology, University Hospital Essen, Essen, Germany; cDepartment of Immunophysiology, Institute of Physiology and Pathophysiology, Philipps University of Marburg, Marburg, Germany
The regulation of immune responses is not exclusively mediated by factors intrinsic to the immune system. In fact, a variety of important immune processes such as leukocyte trafficking, antigen presentation, cell activation, and clonal expansion were shown to be affected by noradrenaline (NA), the main neurotransmitter of the sympathetic nervous system. Here we demonstrate that also a lack in sympathetic output can have profound effects on immune responses and can influence the severity and course of an acute retroviral infection. Adult C57BL/6 mice were chemically sympathectomized with two injections (150 mg/kg) of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) and subsequently infected with Friend retrovirus (FV), a murine erythroleukemia virus that induces a strong immune response and causes a transient enlargement of the spleen during the acute infection. Neurotoxin treatment effectively destroyed peripheral noradrenergic nerve terminals and led to a substantial reduction (N85%) in splenic NA levels compared to vehicle-treated controls. More importantly, sympathectomized mice developed a significantly more pronounced splenomegaly and exhibited higher splenic viral loads on days 5, 10 and 15 post-infection compared to vehicle-treated mice, indicating a more severe disease course in animals lacking peripheral NA. This suggests that NA and/or certain sympathetic tone are required for normal immune functioning during acute FV infection. The underlying mechanisms are currently under investigation and will provide new insights into the relevance of SNS–immune interactions during viral infections.
doi:10.1016/j.jneuroim.2014.08.200
422 Droplet digital PCR for the precise quantification of HTLV-1 Breanna Caruso, Giovanna Brunetto, Raya Massoud, Emily Leibovitch, Steven Jacobson NINDS, NIH, Bethesda, United States Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that is the etiologic agent in a clinical neuromyelopathy known as HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/ TSP). Quantification of the amount of virus in patient samples is critical for monitoring infection, evaluating the efficacy of therapeutic agents, and assisting with clinical diagnosis. Real-time PCR (qPCR) is the standard method for determining HTLV-1 proviral load (PVL). However, qPCR is dependent upon well-defined standards and calibration to a standard curve, precluding precise and accurate quantification particularly at low cell concentrations. In this study, we use a third generation PCR technique, droplet digital PCR (ddPCR), which allows for direct quantification of PVL. DNA samples are partitioned into thousands of nanoliter-sized droplets, amplified on a thermocycler, analyzed for fluorescent signal, and normalized to a housekeeping gene. Poisson distribution statistics are used to determine absolute copy numbers independently of a standard curve. We found that the intra-assay and inter-assay reliability of ddPCR are robust and more consistent than those of qPCR. Moreover, ddPCR yields similar patient PVL by quantifying different viral genes. Our preliminary data suggests ddPCR can also be used to examine viral gene expression, a key player in the pathogenesis of HAM/TSP and an important parameter to monitor during therapeutic interventions in this condition. The reliability and precision of ddPCR for HTLV-1 quantification should be widely acknowledged as it might be an important tool to further understand the clinical correlates of the viral load in the context of HAM/TSP and other HTLV-1 related conditions; however, further investigation is required to determine the efficacy of ddPCR for mRNA analysis. doi:10.1016/j.jneuroim.2014.08.201
582 Risk factors for immunosuppression-associated infections in aquaporin-4 antibody mediated neuromyelitis optica spectrum disorders Fan Chenga, Jithin Georgea, J. Rochab, George Tackleya, John Revisa, Paddy Watersa, John Elstonc, Jacqueline Palacea, Nicola Jonesd, Maria Isabel Leitea a Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, United Kingdom; bDepartment of Clinical Neurology, Hospital S. Marcos, Braga, Portugal; cThe Oxford Eye Hospital, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom; dDepartment of Infectious Diseases, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom
Introduction: Opportunistic, atypical and severe infections may be serious complications of immunosuppressive therapy in chronic antibody mediated diseases, such as neuromyelitis optica spectrum disorders (NMOSD) associated with aquaporin-4 antibodies (AQP4-Abs). However, no studies have yet investigated the frequency and risk factors for immunosuppression-associated severe infections in NMOSD patients.