Sarah E. Beck

Constitutive BDNF/TrkB signaling is required for normal cardiac contraction and relaxation

Significance BDNF plays a key role in neuron development, survival, and function, with actions occurring through the stimulation of the tropomyosin-related kinase receptor B (TrkB) receptor. Whether BDNF/TrkB signaling has any physiologic role in governing myocardial function is unknown. Here we report that intact BDNF/TrkB signaling is required for the heart to fully contract and relax. These actions occur independently from and in addition to β-adrenergic influence. BDNF-induced enhancement of myocardial performance occurs via direct modulation of Ca2+ cycling in a calmodulin-dependent protein kinase II-dependent manner. Thus, BDNF/TrkB signaling represents a previously unidentified way by which the peripheral nervous system controls cardiac muscle physiology. Our study suggests that loss or alterations in BDNF/TrkB stimulation may contribute to the pathogenesis of myocardial dysfunction in acute or chronic disease conditions. BDNF and its associated tropomyosin-related kinase receptor B (TrkB) nurture vessels and nerves serving the heart. However, the direct effect of BDNF/TrkB signaling on the myocardium is poorly understood. Here we report that cardiac-specific TrkB knockout mice (TrkB−/−) display impaired cardiac contraction and relaxation, showing that BDNF/TrkB signaling acts constitutively to sustain in vivo myocardial performance. BDNF enhances normal cardiomyocyte Ca2+ cycling, contractility, and relaxation via Ca2+/calmodulin-dependent protein kinase II (CaMKII). Conversely, failing myocytes, which have increased truncated TrkB lacking tyrosine kinase activity and chronically activated CaMKII, are insensitive to BDNF. Thus, BDNF/TrkB signaling represents a previously unidentified pathway by which the peripheral nervous system directly and tonically influences myocardial function in parallel with β-adrenergic control. Deficits in this system are likely additional contributors to acute and chronic cardiac dysfunction.

Neuroprotective maraviroc monotherapy in simian immunodeficiency virus-infected macaques: Reduced replicating and latent SIV in the brain

Objective:HIV-associated neurocognitive deficits remain a challenge despite suppressive combined antiretroviral therapy. Given the association between HIV-induced central nervous system (CNS) disease and replication of HIV in immune-activated macrophages, CCR5 antagonists may attenuate CNS disease by modulating inflammatory signaling and by limiting viral replication. Design:To establish whether initiating CCR5 inhibition during early infection altered CNS disease progression, outcomes were compared between simian immunodeficiency virus (SIV)-infected macaques treated with maraviroc (MVC) versus untreated SIV-infected macaques. Methods:Six SIV-infected rhesus macaques were treated with MVC monotherapy for 5 months beginning 24 days postinoculation; 22 SIV-infected animals served as untreated controls. SIV RNA levels in plasma, cerobrospinal fluid, and brain, and CNS expression of TNF&agr; and CCL2 were measured by qRT-PCR. Immunostaining for CD68 and amyloid precursor protein in the brain was measured by image analysis. Plasma sCD163 was measured by ELISA. Results:SIV RNA and proviral DNA levels in brain were markedly lower with MVC treatment, demonstrating CCR5 inhibition reduces CNS replication of SIV and may reduce the CNS latent viral reservoir. MVC treatment also lowered monocyte and macrophage activation, represented by CNS CD68 immunostaining and plasma sCD163 levels, and reduced both TNF&agr; and CCL2 RNA expression in brain. Treatment also reduced axonal amyloid precursor protein immunostaining to levels present in uninfected animals, consistent with neuroprotection. Conclusion:CCR5 inhibitors may prevent neurologic disorders in HIV-infected individuals by reducing inflammation and by limiting viral replication in the brain. Furthermore, CCR5 inhibitors may reduce the latent viral reservoir in the CNS. Adding CCR5 inhibitors to combined antiretroviral regimens may offer multiple neuroprotective benefits.

Paving the path to HIV neurotherapy: Predicting SIV CNS disease.

HIV-induced damage to the CNS remains a major challenge for over 30 million people in the world despite the successes of combined antiretroviral therapy in limiting viral replication. Predicting development and progression of HIV-associated CNS disease is crucial because prevention and early intervention could be more effective than attempts to promote repair. The SIV/macaque model is the premier platform to study HIV neuropathogenesis, including discovery of predictive factors such as neuroprotective host genes and both blood and CSF biomarkers that precede and predict development of SIV CNS disease. This report details the role of macaque MHC class I genes, longitudinal alterations in biomarkers in the circulation, and expression of inflammatory and neuronal damage markers in CSF using samples from SIV-inoculated pigtailed macaques collected during acute, asymptomatic, and terminal stages of infection.

CCR5 Inhibition Prevents Cardiac Dysfunction in the SIV/Macaque Model of HIV

Background Diastolic dysfunction is a highly prevalent cardiac abnormality in asymptomatic as well as ART‐treated human immunodeficiency virus (HIV) patients. Although the mechanisms underlying depressed cardiac function remain obscure, diastolic dysfunction in SIV‐infected rhesus macaques is highly correlated with myocardial viral load. As cardiomyocytes are not productively infected, damage may be an indirect process attributable to a combination of pro‐inflammatory mediators and viral proteins. Methods and Results Given the diverse roles of CCR5 in mediating recruitment of leukocytes to inflammatory sites and serving as a receptor for HIV entry into cells, we investigated the role of CCR5 in the SIV/macaque model of diastolic dysfunction. We found that in SIV‐infected macaques, CCR5 inhibition dramatically impacted myocardial viral load measured by qRT‐PCR and prevented diastolic dysfunction measured by echocardiography. Complementary in vitro experiments using fluorescence microscopy showed that CCR5 ligands impaired contractile function of isolated cardiomyocytes, thus identifying CCR5 signaling as a novel mediator of impaired cardiac mechanical function. Conclusions Together, these findings incriminate SIV/HIV gp120‐CCR5 as well as chemokine‐CCR5 interactions in HIV‐associated cardiac dysfunction. These findings also have important implications for the treatment of HIV‐infected individuals: in addition to antiviral properties and reduced chemokine‐mediated recruitment and activation of inflammatory cells, CCR5 inhibition may provide a cardioprotective benefit by preventing cardiomyocyte CCR5 signaling.

Macaque species susceptibility to simian immunodeficiency virus: increased incidence of SIV central nervous system disease in pigtailed macaques versus rhesus macaques

Immune pressure exerted by MHC class I-restricted cytotoxic T cells drives the development of viral escape mutations, thereby regulating HIV disease progression. Nonetheless, the relationship between host immunity and HIV central nervous system (CNS) disease remains poorly understood. The simian immunodeficiency virus (SIV) macaque model recapitulates key features of HIV infection including development of AIDS and CNS disease. To investigate cell-mediated immunity regulating SIV CNS disease progression, we compared the incidence of SIV encephalitis and the influence of MHC class I allele expression on the development of CNS disease in rhesus macaques (Macaca mulatta) versus pigtailed macaques (Macaca nemestrina). After inoculation with the immunosuppressive swarm SIV/DeltaB670 and the neurovirulent molecular clone SIV/17E-Fr, pigtailed macaques progressed more rapidly to AIDS, had higher plasma and cerebrospinal fluid (CSF) viral loads, and were more likely to progress to SIV-associated encephalitis (SIVE) compared to rhesus macaques. In addition, MHC class I alleles were neuroprotective in both species (Mamu-A*001 in rhesus macaques and Mane-A1*084:01:01 in pigtailed macaques); animals expressing these alleles were less likely to develop SIV encephalitis and correspondingly had lower viral replication in the brain. Species-specific differences in susceptibility to SIV disease demonstrated that cell mediated immune responses are critical to SIV CNS disease progression.

Loss of Corneal Sensory Nerve Fibers in SIV-Infected Macaques: An Alternate Approach to Investigate HIV-Induced PNS Damage

Peripheral neuropathy is the most frequent neurological complication of HIV infection, affecting more than one-third of infected patients, including patients treated with antiretroviral therapy. Although emerging noninvasive techniques for corneal nerve assessments are increasingly being used to diagnose and monitor peripheral neuropathies, corneal nerve alterations have not been characterized in HIV. Here, to determine whether SIV infection leads to corneal nerve fiber loss, we immunostained corneas for the nerve fiber marker βIII tubulin. We developed and applied both manual and automated methods to measure nerves in the corneal subbasal plexus. These counting methods independently indicated significantly lower subbasal corneal nerve fiber density among SIV-infected animals that rapidly progressed to AIDS compared with slow progressors. Concomitant with decreased corneal nerve fiber density, rapid progressors had increased levels of SIV RNA and CD68-positive macrophages and expression of glial fibrillary acidic protein by glial satellite cells in the trigeminal ganglia, the location of the neuronal cell bodies of corneal sensory nerve fibers. In addition, corneal nerve fiber density was directly correlated with epidermal nerve fiber length. These findings indicate that corneal nerve assessment has great potential to diagnose and monitor HIV-induced peripheral neuropathy and to set the stage for introducing noninvasive techniques to measure corneal nerve fiber density in HIV clinical settings.

Proliferative Thyroid Lesions in Three Diplodactylid Geckos: Nephrurus amyae, Nephrurus levis, and Oedura marmorata

Abstract:  Over a 5-mo period, three diplodactylid geckos housed at the National Aquarium were diagnosed with proliferative thyroid lesions: a rough knob-tail gecko (Nephrurus amyae), a smooth knob-tail gecko (Nephrurus levis), and a marbled velvet gecko (Oedura marmorata). Clinical signs included an intraoral mass or ventral throat swelling (or both), oral bleeding, and weight loss. Both of the knob-tail geckos died. The histologic diagnosis for the rough knob-tail gecko was thyroid carcinoma with metastases to the liver and lungs, and thyroid carcinoma with no metastases was reported in the smooth knob-tail gecko. A thyroidectomy was performed on the marbled velvet gecko with a histologic diagnosis of adenomatous hyperplasia. Postoperative weight loss and bradycardia resolved following oral supplementation with levothyroxine. The animal is in normal health 10 mo post-surgery. Five other diplodactylid geckos in the collection remain unaffected, giving a 38% prevalence of proliferative thyroid lesions (3/8). The etiology remains undetermined. This is the first report of a cluster of proliferative thyroid lesions in geckos.

An SIV/macaque model targeted to study HIV-associated neurocognitive disorders

Simian immunodeficiency virus (SIV) infection of pigtailed macaques is a highly representative and well-characterized animal model for HIV neuropathogenesis studies that provides an excellent opportunity to study and develop prognostic markers of HIV-associated neurocognitive disorders (HAND) for HIV-infected individuals. SIV studies can be performed in a controlled setting that enhances reproducibility and offers high-translational value. Similar to observations in HIV-infected patients receiving antiretroviral therapy (ART), ongoing neurodegeneration and inflammation are present in SIV-infected pigtailed macaques treated with suppressive ART. By developing quantitative viral outgrowth assays that measure both CD4+ T cells and macrophages harboring replication competent SIV as well as a highly sensitive mouse-based viral outgrowth assay, we have positioned the SIV/pigtailed macaque model to advance our understanding of latent cellular reservoirs, including potential CNS reservoirs, to promote HIV cure. In addition to contributing to our understanding of the pathogenesis of HAND, the SIV/pigtailed macaque model also provides an excellent opportunity to test innovative approaches to eliminate the latent HIV reservoir in the brain.

Central nervous system-specific consequences of simian immunodeficiency virus Gag escape from major histocompatibility complex class I-mediated control

In the fourth decade of the HIV epidemic, the relationship between host immunity and HIV central nervous system (CNS) disease remains incompletely understood. Using a simian immunodeficiency virus (SIV)/macaque model, we examined CNS outcomes in pigtailed macaques expressing the MHC class I allele Mane-A1*084:01 which confers resistance to SIV-induced CNS disease and induces the prototypic viral escape mutation Gag K165R. Insertion of gag K165R into the neurovirulent clone SIV/17E-Fr reduced viral replication in vitro compared to SIV/17E-Fr. We also found lower cerebrospinal fluid (CSF), but not plasma, viral loads in macaques inoculated with SIV/17E-Fr K165R versus those inoculated with wildtype. Although escape mutation K165R was genotypically stable in plasma, it rapidly reverted to wildtype Gag KP9 in both CSF and in microglia cultures. We induced robust Gag KP9-specific CTL tetramer responses by vaccinating Mane-A*084:01-positive pigtailed macaques with a Gag KP9 virus-like particle (VLP) vaccine. Upon SIV/17E-Fr challenge, vaccinated animals had lower SIV RNA in CSF compared to unvaccinated controls, but showed no difference in plasma viral loads. These data clearly demonstrate that viral fitness in the CNS is distinct from the periphery and underscores the necessity of understanding the consequences of viral escape in CNS disease with the advent of new therapeutic vaccination strategies.

Lymphocyte-Dominant Encephalitis and Meningitis in Simian Immunodeficiency Virus–Infected Macaques Receiving Antiretroviral Therapy

A retrospective neuropathologic review of 30 SIV-infected pigtailed macaques receiving combination antiretroviral therapy (cART) was conducted. Seventeen animals with lymphocyte-dominant inflammation in the brain and/or meninges that clearly was morphologically distinct from prototypic SIV encephalitis and human immunodeficiency virus encephalitis were identified. Central nervous system (CNS) infiltrates in cART-treated macaques primarily comprised CD20+ B cells and CD3+ T cells with fewer CD68+ macrophages. Inflammation was associated with low levels of SIV RNA in the brain as shown by in situ hybridization, and generally was observed in animals with episodes of cerebrospinal fluid (CSF) viral rebound or sustained plasma and CSF viremia during treatment. Although the lymphocytic CNS inflammation in these macaques shared morphologic characteristics with uncommon immune-mediated neurologic disorders reported in treated HIV patients, including CNS immune reconstitution inflammatory syndrome and neurosymptomatic CSF escape, the high prevalence of CNS lesions in macaques suggests that persistent adaptive immune responses in the CNS also may develop in neuroasymptomatic or mildly impaired HIV patients yet remain unrecognized given the lack of access to CNS tissue for histopathologic evaluation. Continued investigation into the mechanisms and outcomes of CNS inflammation in cART-treated, SIV-infected macaques will advance our understanding of the consequences of residual CNS HIV replication in patients on cART, including the possible contribution of adaptive immune responses to HIV-associated neurocognitive disorders.