Lisa M. Mangus

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.

Neuroinflammation and Virus Replication in the Spinal Cord of Simian Immunodeficiency Virus-infected Macaques

Abstract Studies of neurologic diseases induced by simian immunodeficiency virus (SIV) in Asian macaques have contributed greatly to the current understanding of human immunodeficiency virus pathogenesis in the brain and peripheral nervous system. Detailed investigations into SIV-induced alterations in the spinal cord, a critical sensorimotor relay point between the brain and the peripheral nervous system, have yet to be reported. In this study, lumbar spinal cords from SIV-infected pigtailed macaques were examined to quantify SIV replication and associated neuroinflammation. In untreated SIV-infected animals, there was a strong correlation between amount of SIV RNA in the spinal cord and expression of the macrophage marker CD68 and the key proinflammatory mediators tumor necrosis factor and CCL2. We also found a significant correlation between SIV-induced alterations in the spinal cord and the degree of distal epidermal nerve fiber loss among untreated animals. Spinal cord changes (including elevated glial fibrillary acidic protein immunostaining and enhanced CCL2 gene expression) also were present in SIV-infected antiretroviral drug–treated animals despite SIV suppression. A fuller understanding of the complex virus and host factor dynamics in the spinal cord during human immunodeficiency virus infection will be critical in the development of new treatments for human immunodeficiency virus–associated sensory neuropathies and studies aimed at eradicating the virus from the central nervous system.

Unraveling the Pathogenesis of HIV Peripheral Neuropathy: Insights from a Simian Immunodeficiency Virus Macaque Model

Peripheral neuropathy (PN) is the most frequent neurologic complication in individuals infected with human immunodeficiency virus (HIV). It affects over one third of infected patients, including those receiving effective combination antiretroviral therapy. The pathogenesis of HIV-associated peripheral neuropathy (HIV-PN) remains poorly understood. Clinical studies are complicated because both HIV and antiretroviral treatment cause damage to the peripheral nervous system. To study HIV-induced peripheral nervous system (PNS) damage, a unique simian immunodeficiency virus (SIV)/pigtailed macaque model of HIV-PN that enabled detailed morphologic and functional evaluation of the somatosensory pathway throughout disease progression was developed. Studies in this model have demonstrated that SIV induces key pathologic features that closely resemble HIV-induced alterations, including inflammation and damage to the neuronal cell bodies in somatosensory ganglia and decreased epidermal nerve fiber density. Insights generated in the model include: finding that SIV alters the conduction properties of small, unmyelinated peripheral nerves; and that SIV impairs peripheral nerve regeneration. This review will highlight the major findings in the SIV-infected pigtailed macaque model of HIV-PN, and will illustrate the great value of a reliable large animal model to show the pathogenesis of this complex, HIV-induced disorder of the PNS.

Persistent peripheral nervous system damage in simian immunodeficiency virus-infected macaques receiving antiretroviral therapy

Abstract Human immunodeficiency virus (HIV)–induced peripheral neuropathy is the most common neurologic complication associated with HIV infection. In addition to virus-mediated injury of the peripheral nervous system (PNS), treatment of HIV infection with combination antiretroviral therapy (cART) may induce toxic neuropathy as a side effect. Antiretroviral toxic neuropathy is clinically indistinguishable from the sensory neuropathy induced by HIV; in some patients, these 2 processes are likely superimposed. To study these intercurrent PNS disease processes, we first established a simian immunodeficiency virus (SIV)/pigtailed macaque model in which more than 90% of animals developed PNS changes closely resembling those seen in HIV-infected individuals with distal sensory neuropathy. To determine whether cART alters the progression of SIV-induced PNS damage, dorsal root ganglia and epidermal nerve fibers were evaluated in SIV-infected macaques after long-term suppressive cART. Although cART effectively suppressed SIV replication and reduced macrophage activation in the dorsal root ganglia, PGP 9.5 immunostaining and measurements of epidermal nerve fibers in the plantar surface of the feet of treated SIV-infected macaques clearly showed that cART did not normalize epidermal nerve fiber density. These findings illustrate that significant PNS damage persists in SIV-infected macaques on suppressive cART.

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.

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.

Tracking Epidermal Nerve Fiber Changes in Asian Macaques Tools and Techniques for Quantitative Assessment

Quantitative assessment of epidermal nerve fibers (ENFs) has become a widely used clinical tool for the diagnosis of small fiber neuropathies such as diabetic neuropathy and human immunodeficiency virus–associated sensory neuropathy (HIV-SN). To model and investigate the pathogenesis of HIV-SN using simian immunodeficiency virus (SIV)-infected Asian macaques, we adapted the skin biopsy and immunostaining techniques currently employed in human patients and then developed two unbiased image analysis techniques for quantifying ENF in macaque footpad skin. This report provides detailed descriptions of these tools and techniques for ENF assessment in macaques and outlines important experimental considerations that we have identified in the course of our long-term studies. Although initially developed for studies of HIV-SN in the SIV-infected macaque model, these methods could be readily translated to a range of studies involving peripheral nerve degeneration and neurotoxicity in nonhuman primates as well as preclinical investigations of agents aimed at neuroprotection and regeneration.

CAUSES OF MORTALITY IN CAPTIVE PANAMANIAN GOLDEN FROGS (ATELOPUS ZETEKI) AT THE MARYLAND ZOO IN BALTIMORE, 2001–2013

Abstract:  The Maryland Zoo in Baltimore is home to the largest captive assurance population of the critically endangered Panamanian golden frog (Atelopus zeteki). With the ongoing extinction that is occurring worldwide in amphibians, the need for amphibian captive assurance populations is growing, and few mortality reviews on amphibian species exist. Necropsy and histopathologic examination of animals that die in captivity can help identify population-level disease problems, direct research needs in amphibian medicine and husbandry, and improve the success of captive breeding programs. This study reviews postmortem findings from 406 frogs, greater than 1 yr of age, which died in this population from 2001 to 2013. Frogs were categorized by age and sex, and the cause of mortality was determined. Dermatitis associated with filamentous-type fungal organisms was the most common cause of mortality in both age and sex categories and accounted for one-third of frog deaths in this study (36.0%; n = 146 out of 406 frogs). Other major causes of mortality included renal disease, gastrointestinal disease, septicemia, and a previously undescribed myopathy condition associated with a tetany syndrome. Increased mortality of frogs occurred during the breeding season, highlighting the need for further research into methods to minimize mortality during this time.