Platelet decline commonly occurs in human immunodeficiency virus (HIV)-infected individuals and has been linked to adverse sequelae of infection, including central nervous system (CNS) disease. A ...role for platelets in the pathogenesis of HIV infection has yet to be demonstrated, and existing literature on both causes and consequences of platelet decline has yet to illuminate a clear consensus. As platelets may play different roles in acute, asymptomatic and terminal stages of HIV infection, we used the simian immunodeficiency virus (SIV)/macaque model to examine whether causes of platelet decline differed throughout infection. We found that platelet decline was biphasic, with an acute transient drop in numbers of circulating platelets preceding a decline that persisted from mid-asymptomatic to terminal infection. We therefore examined decreased production, increased destruction, and increased platelet activation and use as possible causes of platelet decline during acute and asymptomatic infection. We demonstrated that platelet activation and sequestration of platelets in CD16+ plateletmonocyte aggregates drove platelet decline during acute infection, and herein discuss the implications of the association of platelets with greater than 90% of pro-migratory CD16+ monocytes for the development of CNS disease. During asymptomatic infection, we identified decreased production, increased destruction, and increased activation, with decreased platelet production dominating. Thrombopoietin (THPO) stimulates platelet production by promoting maturation of platelet producing megakaryocytes within bone marrow, and THPO is transcriptionally downregulated by cytokines such as transforming growth factor β (TGFβ). We found that elevated plasma TGFβ during asymptomatic infection correlated inversely with a 10,000-fold downregulation of THPO transcription, and that THPO mRNA correlated positively with megakaryocyte numbers. As platelet decline during asymptomatic infection does not generally occur in patients using combined antiretroviral therapy (cART), we evaluated whether cART increased platelet production, and demonstrated that cART corrected plasma TGFβ levels and platelet count. Finally, we identified an association between frontal cortex transcription of THPO, which is pro-apoptotic to neurons in culture, and the presence of CNS disease during asymptomatic and terminal SIV infection. In summary, this dissertation establishes that the cause of platelet decline differs depending upon the stage of lentiviral infection, and presents candidate mechanisms that may link platelet decline to the pathogenesis of HIV-associated CNS disease.
An 8-year old, male Rhesus macaque (
Macaca mulatta
) previously used for dengue virus vaccine research with viral challenge, presented with adult-onset, chronic, cyclic thrombocytopenia. Platelet ...number, morphology and function were evaluated by automated hematology, peripheral blood smears, electron microscopy, flow cytometry, and impedance aggregometry. Bone marrow was evaluated by cytology. Both, serum anti-dengue non-structural protein 1 (NS1) antibodies and anti-platelet antibodies were detected by ELISA. Platelet characterization showed a lack of aggregation to all agonists (ADP, ASP, and collagen), increased activation with increased expression of surface marker (HLA-ABC), and an absence of surface receptor GPIX during clinical episodes of petechiae and ecchymoses, even in the presence of normal platelet counts. Bone marrow aspirates identified potential mild megakaryocytic hypoplasia. All platelet functions and morphologic attributes were within normal limits during clinically normal phases. Presence of anti-dengue NS1 serum antibodies confirmed a positive dengue virus titer 8 years post-vaccination. Based on the history and clinical findings, a primary differential diagnosis for this chronic, cyclic platelet pathology was autoimmune platelet destruction with potential bone marrow involvement.
It is unknown if extremely early initiation of antiretroviral therapy (ART) may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure ...prophylaxis (PrEP) program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male) and 12 days (Participant B; 31-year-old male) after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI) following 32 weeks of continuous ART.
Colorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF), plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs) were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA). Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative) followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse), but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input cells/mouse) experienced very low level viremia (201 copies/mL); sequence confirmation was unsuccessful. PrEP Participant A stopped ART and remained aviremic for 7.4 months, rebounding with HIV RNA of 36 copies/mL that rose to 59,805 copies/mL 6 days later. ART was restarted promptly. Rebound plasma HIV sequences were identical to those obtained during acute infection by single-genome sequencing. Mathematical modeling predicted that the latent reservoir size was approximately 200 cells prior to ATI and that only around 1% of individuals with a similar HIV burden may achieve lifelong ART-free remission. Furthermore, we observed that lymphocytes expressing the tumor marker CD30 increased in frequency weeks to months prior to detectable HIV-1 RNA in plasma. This study was limited by the small sample size, which was a result of the rarity of individuals presenting during hyperacute infection.
We report HIV relapse despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete or complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission. However, the small numbers of latently infected cells in individuals treated during hyperacute infection may be associated with prolonged ART-free remission.
Macaques with self-injurious behavior (SIB) have been used as a model of human SIB and have previously been shown to respond to treatments targeting enhancement of central serotonin signaling, ...whether by supplementation with tryptophan, or by inhibiting synaptic reuptake. Decreased serotonin signaling in the brain has also been implicated in many human psychopathologies including major depression disorder. A disturbance in tryptophan metabolism that moves away from the production of serotonin and toward the production of kynurenine has been proposed as a major etiological factor of depression. We hypothesized that in macaques with SIB, central tryptophan metabolism would be shifted toward kynurenine production, leading to lower central serotonin (5-hydroxytryptamine). We analyzed tryptophan metabolites in the cerebral spinal fluid (CSF) of macaques with and without SIB to determine whether and where tryptophan metabolism is altered in affected animals as compared with behaviorally normal controls. We found that macaques with SIB had lower CSF concentrations of serotonin than did behaviorally normal macaques, and that these deficits were inversely correlated with the severity of abnormal behavior. However, our results suggest that this decrease is not due to shifting of the tryptophan metabolic pathway toward kynurenine, as concentrations of kynurenine were also low. Concentrations of IL6 were elevated, suggesting central inflammation. Determining the mechanism by which serotonin function is altered in self-injurious macaques could shed light on novel therapies for SIB and other disorders of serotonin signaling.
EcoHIV is a model of HIV infection that recapitulates aspects of HIV-1 pathology in mice. However, there are limited published protocols to guide EcoHIV virion production. Here, we present a protocol ...for producing infective EcoHIV virions and essential quality controls. We describe steps for viral purification, titering, and multiple techniques to analyze infection efficacy. This protocol produces high infectivity in C57BL/6 mice which will aid investigators in generating preclinical data.
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•Improved EcoHIV preparation for optimal acute and chronic infection•Enhanced infectivity rate with ∼100% of mice infected and actively producing EcoHIV•Multiple quality controls to calculate the virus titer and check viral infectivity
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
EcoHIV is a model of HIV infection that recapitulates aspects of HIV-1 pathology in mice. However, there are limited published protocols to guide EcoHIV virion production. Here, we present a protocol for producing infective EcoHIV virions and essential quality controls. We describe steps for viral purification, titering, and multiple techniques to analyze infection efficacy. This protocol produces high infectivity in C57BL/6 mice which will aid investigators in generating preclinical data.
Macaques with self-injurious behavior (SIB) have been used as a model of human SIB and have previously been shown to respond to treatments targeting enhancement of central serotonin signaling, ...whether by supplementation with tryptophan, or by inhibiting synaptic reuptake. Decreased
serotonin signaling in the brain has also been implicated in many human psychopathologies including major depression disorder. A disturbance in tryptophan metabolism that moves away from the production of serotonin and toward the production of kynurenine has been proposed as a major etiological
factor of depression. We hypothesized that in macaques with SIB, central tryptophan metabolism would be shifted toward kynurenine production, leading to lower central serotonin (5-hydroxytryptamine). We analyzed tryptophan metabolites in the cerebral spinal fluid (CSF) of macaques with and
without SIB to determine whether and where tryptophan metabolism is altered in affected animals as compared with behaviorally normal controls. We found that macaques with SIB had lower CSF concentrations of serotonin than did behaviorally normal macaques, and that these deficits were inversely
correlated with the severity of abnormal behavior. However, our results suggest that this decrease is not due to shifting of the tryptophan metabolic pathway toward kynurenine, as concentrations of kynurenine were also low. Concentrations of IL6 were elevated, suggesting central inflammation.
Determining the mechanism by which serotonin function is altered in self-injurious macaques could shed light on novel therapies for SIB and other disorders of serotonin signaling.
An 8-year-old, male Rhesus macaque (Macaca mulatta), previously used for dengue virus (DENV) vaccine research with viral challenge, was presented with adult-onset, chronic, cyclic thrombocytopenia. ...Platelet number, morphology, and function were evaluated by automated hematology, peripheral blood smears, electron microscopy, flow cytometry, and impedance aggregometry. Bone marrow was evaluated by cytology. Both serum anti-dengue nonstructural protein 1 (NS1) antibodies and anti-platelet antibodies were detected by ELISA. Platelet characterization showed a lack of aggregation to all agonists (ADP, ASP, and collagen), increased activation with increased expression of surface marker (HLA-ABC), and an absence of surface receptor GPIX during clinical episodes of petechiae and ecchymoses, even in the presence of normal platelet counts. Bone marrow aspirates identified potential mild megakaryocytic hypoplasia. All platelet functions and morphologic attributes were within normal limits during clinically normal phases. Presence of anti-dengue NS1 serum antibodies confirmed a positive DENV titer 8 years postvaccination. Based on the history and clinical findings, a primary differential diagnosis for this chronic, cyclic platelet pathology was autoimmune platelet destruction with potential bone marrow involvement.
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