Joseph L. Mankowski

Heterozygous disruption of Hic1 predisposes mice to a gender-dependent spectrum of malignant tumors

The gene hypermethylated in cancer-1 (HIC1) encodes a zinc-finger transcription factor that belongs to a group of proteins known as the POZ family. HIC1 is hypermethylated and transcriptionally silent in several types of human cancer. Homozygous disruption of Hic1 impairs development and results in embryonic and perinatal lethality in mice. Here we show that mice disrupted in the germ line for only one allele of Hic1 develop many different spontaneous malignant tumors, including a predominance of epithelial cancers in males and lymphomas and sarcomas in females. The complete loss of Hic1 function in the heterozygous mice seems to involve dense methylation of the promoter of the remaining wild-type allele. We conclude that HIC1 is a candidate tumor-suppressor gene for which loss of function in both mouse and human cancers is associated only with epigenetic modifications.

Pivotal role of cardiomyocyte TGF-β signaling in the murine pathological response to sustained pressure overload

The cardiac pathological response to sustained pressure overload involves myocyte hypertrophy and dysfunction along with interstitial changes such as fibrosis and reduced capillary density. These changes are orchestrated by mechanical forces and factors secreted between cells. One such secreted factor is TGF-β, which is generated by and interacts with multiple cell types. Here we have shown that TGF-β suppression in cardiomyocytes was required to protect against maladaptive remodeling and involved noncanonical (non-Smad-related) signaling. Mouse hearts subjected to pressure overload and treated with a TGF-β-neutralizing Ab had suppressed Smad activation in the interstitium but not in myocytes, and noncanonical (TGF-β-activated kinase 1 [TAK1]) activation remained. Although fibrosis was greatly reduced, chamber dysfunction and dilation persisted. Induced myocyte knockdown of TGF-β type 2 receptor (TβR2) blocked all maladaptive responses, inhibiting myocyte and interstitial Smad and TAK1. Myocyte knockdown of TβR1 suppressed myocyte but not interstitial Smad, nor TAK1, modestly reducing fibrosis without improving chamber function or hypertrophy. Only TβR2 knockdown preserved capillary density after pressure overload, enhancing BMP7, a regulator of the endothelial-mesenchymal transition. BMP7 enhancement also was coupled to TAK1 suppression. Thus, myocyte targeting is required to modulate TGF-β in hearts subjected to pressure overload, with noncanonical pathways predominantly affecting the maladaptive hypertrophy/dysfunction.

Increased Macrophage Chemoattractant Protein-1 in Cerebrospinal Fluid Precedes and Predicts Simian Immunodeficiency Virus Encephalitis

Macrophage chemoattractant protein-1 (MCP-1) may be a key trigger for the influx of macrophages into the brain in human immunodeficiency virus (HIV) encephalitis. In this study, simian immunodeficiency virus-infected macaques that developed moderate-to-severe encephalitis had significantly higher MCP-1 levels in cerebrospinal fluid (CSF) than in plasma as early as 28 days after inoculation, which was before the development of brain lesions. In contrast, CSF:plasma MCP-1 ratios remained constant at preinoculation levels in macaques that developed minimal or no encephalitis. Abundant MCP-1 protein and mRNA were detected in both macrophages and astrocytes in the brain. Macaques with increased MCP-1 in CSF had significantly greater expression of markers of macrophage and microglia activation and infiltration (CD68; P= .003) and astrocyte activation (glial fibrillary acidic protein; P= .019 and P= .031 in white and gray matter, respectively). The results suggest that the CSF:plasma MCP-1 ratio may be a valuable prognostic marker for the development of HIV-induced central nervous system disease.

The Central Nervous System as a Reservoir for Simian Immunodeficiency Virus (SIV): Steady-State Levels of SIV DNA in Brain from Acute through Asymptomatic Infection

Latent reservoirs of human immunodeficiency virus (HIV) present significant challenges for eradicating HIV from infected persons, particularly reservoirs in the brain established during acute infection. A simian immunodeficiency virus (SIV)/macaque model of HIV dementia was used to show that viral DNA levels in the brain remained at constant levels from acute through asymptomatic infection, despite significant down-regulation of viral RNA in the brain after the acute phase of infection. Viral replication in the brain coincided with activation of macrophages and microglia in the central nervous system; down-regulation of viral replication coincided with increased infiltration of cytotoxic lymphocytes and reduced activation of macrophages and microglia in the brain. Comparison of viral genotypes in the central nervous system and peripheral blood mononuclear cells suggests that recrudescence of viral replication in brain occurs by reactivation of latent viral DNA. Latent virus in the brain must be considered in therapeutic strategies to eliminate HIV from infected persons.

HIV-associated neurocognitive disorder — pathogenesis and prospects for treatment

In the past two decades, several advancements have improved the care of HIV-infected individuals. Most importantly, the development and deployment of combination antiretroviral therapy (CART) has resulted in a dramatic decline in the rate of deaths from AIDS, so that people living with HIV today have nearly normal life expectancies if treated with CART. The term HIV-associated neurocognitive disorder (HAND) has been used to describe the spectrum of neurocognitive dysfunction associated with HIV infection. HIV can enter the CNS during early stages of infection, and persistent CNS HIV infection and inflammation probably contribute to the development of HAND. The brain can subsequently serve as a sanctuary for ongoing HIV replication, even when systemic viral suppression has been achieved. HAND can remain in patients treated with CART, and its effects on survival, quality of life and everyday functioning make it an important unresolved issue. In this Review, we describe the epidemiology of HAND, the evolving concepts of its neuropathogenesis, novel insights from animal models, and new approaches to treatment. We also discuss how inflammation is sustained in chronic HIV infection. Moreover, we suggest that adjunctive therapies — treatments targeting CNS inflammation and other metabolic processes, including glutamate homeostasis, lipid and energy metabolism — are needed to reverse or improve HAND-related neurological dysfunction.

A Simian Immunodeficiency Virus-Infected Macaque Model To Study Viral Reservoirs That Persist during Highly Active Antiretroviral Therapy

ABSTRACT The treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART), a combination of three or more antiretroviral drugs, suppresses viremia below the clinical limit of detection (50 HIV-1 RNA copies/ml), but latently infected resting CD4+ T cells serve as lifelong reservoirs, and low-level viremia can be detected with special assays. Recent studies have provided evidence for additional reservoirs that contribute to residual viremia but are not present in circulating cells. Identification of all the sources of residual viremia in humans may be difficult. These discoveries highlight the need for a tractable model system to identify additional viral reservoirs that could represent barriers to eradication. In this study, simian immunodeficiency virus (SIV)-infected pig-tailed macaques (Macaca nemestrina) were treated with four antiretroviral drugs to develop an animal model for viral suppression during effective HAART. Treatment led to a biphasic decay in viremia and a significant rise in levels of circulating CD4+ T cells. At terminal infection time points, the frequency of circulating resting CD4+ T cells harboring replication-competent virus was reduced to a low steady-state level similar to that observed for HIV-infected patients on HAART. The frequencies of resting CD4+ T cells harboring replication-competent virus in the pooled head lymph nodes, gut lymph nodes, spleen, and peripheral blood were reduced relative to those for untreated SIV-infected animals. These observations closely parallel findings for HIV-infected humans on suppressive HAART and demonstrate the value of this animal model to identify and characterize viral reservoirs persisting in the setting of suppressive antiretroviral drugs.

Resting CD4+ T Lymphocytes but Not Thymocytes Provide a Latent Viral Reservoir in a Simian Immunodeficiency Virus-Macaca nemestrina Model of Human Immunodeficiency Virus Type 1-Infected Patients on Highly Active Antiretroviral Therapy

ABSTRACT Despite suppression of viremia in patients on highly active antiretroviral therapy (HAART), human immunodeficiency virus type 1 persists in a latent reservoir in the resting memory CD4+ T lymphocytes and possibly in other reservoirs. To better understand the mechanisms of viral persistence, we established a simian immunodeficiency virus (SIV)-macaque model to mimic the clinical situation of patients on suppressive HAART and developed assays to detect latently infected cells in the SIV-macaque system. In this model, treatment of SIV-infected pig-tailed macaques (Macaca nemestrina) with the combination of 9-R-(2-phosphonomethoxypropyl)adenine (PMPA; tenofovir) and beta-2′,3′-dideoxy-3′-thia-5-fluorocytidine (FTC) suppressed the levels of plasma virus to below the limit of detection (100 copies of viral RNA per ml). In treated animals, levels of viremia remained close to or below the limit of detection for up to 6 months except for an isolated “blip” of detectable viremia in each animal. Latent virus was measured in blood, spleen, lymph nodes, and thymus by several different methods. Replication-competent virus was recovered after activation of a 99.5% pure population of resting CD4+ T lymphocytes from a lymph node of a treated animal. Integrated SIV DNA was detected in resting CD4+ T cells from spleen, peripheral blood, and various lymph nodes including those draining the gut, the head, and the limbs. In contrast to the wide distribution of latently infected cells in peripheral lymphoid tissues, neither replication-competent virus nor integrated SIV DNA was detected in thymocytes, suggesting that thymocytes are not a major reservoir for virus in pig-tailed macaques. The results provide the first evidence for a latent viral reservoir for SIV in macaques and the most extensive survey of the distribution of latently infected cells in the host.

Simian Immunodeficiency Virus-Infected Macaques Treated with Highly Active Antiretroviral Therapy Have Reduced Central Nervous System Viral Replication and Inflammation but Persistence of Viral DNA

BACKGROUND During the era of highly active antiretroviral therapy (HAART), the prevalence of HIV-associated central nervous system (CNS) disease has increased despite suppression of plasma viremia. METHODS In a simian immunodeficiency virus (SIV) model system in which all animals develop AIDS and 90% develop CNS disease by 3 months after inoculation, pigtailed macaques were treated with a regimen of tenofovir disoproxil fumarate, saquinavir, atazanavir, and an integrase inhibitor starting at 12 days after inoculation and were euthanized at approximately 175 days after inoculation. RESULTS Plasma and cerebrospinal fluid (CSF) viral loads declined rapidly after the initiation of HAART. Brain viral RNA was undetectable at necropsy, but viral DNA levels were not different from those in untreated SIV-infected macaques. CNS inflammation was significantly reduced, with decreased brain expression of major histocompatibility complex class II and glial fibrillary acidic protein and reduced levels of CSF CCL2 and interleukin 6. Brain from treated macaques had significantly lower levels of interferon beta, type 1 interferon-inducible gene myxovirus (influenza) resistance A, and indolamine 2,3-dioxygenase messenger RNA, suggesting that immune hyperactivation was suppressed, and fewer CD4(+) and CD8(+) T cells, suggesting that trafficking of T cells from peripheral blood was reduced. Brain levels of CD68 protein and tumor necrosis factor alpha and interferon gamma RNA were reduced but were not significantly lower, indicating continued CNS inflammation. CONCLUSIONS These data, generated in a rigorous, high-viral-load SIV-infected macaque model, showed that HAART provided benefits with respect to CNS viral replication and inflammation but that no change in the level of viral DNA and continued CNS inflammation occurred in some macaques.

Searching for Clues: Tracking the Pathogenesis of Human Immunodeficiency Virus Central Nervous System Disease by Use of an Accelerated, Consistent Simian Immunodeficiency Virus Macaque Model

An accelerated model of human immunodeficiency virus central nervous system disease was developed in which more than 90% of infected macaques develop typical simian immunodeficiency virus (SIV) encephalitis with neuronal dysfunction by postinoculation (pi) day 84. Infected macaques had replicating virus and microglial activation in the brain 10 days after inoculation; viral replication and microglial activation were suppressed at pi day 21. By pi day 56, viral recrudescence in the brain was detected in 2 of 6 infected macaques. CD4 cells were the predominant lymphocytes in the brain during acute and asymptomatic infection; cytotoxic T lymphocytes and NK cells predominated in macaques with encephalitis. Low levels of peripheral blood NK lytic activity at pi day 10, elevated cerebrospinal fluid (CSF) monocyte chemoattractant protein-1 after 28 days, and high CSF viral RNA after 42 days predicted SIV encephalitis. This model is ideal to track the viral, cellular, and immunologic changes in the brain during acute and asymptomatic infection and during viral recrudescence and SIV encephalitis.

The central nervous system is a viral reservoir in simian immunodeficiency virus-infected macaques on combined antiretroviral therapy: A model for human immunodeficiency virus patients on highly active antiretroviral therapy

This study used a simian immunodeficiency virus (SIV)-macaque model to determine whether virus persists in the central nervous system (CNS) of human immunodeficiency virus (HIV)-infected individuals in which plasma viral load has been suppressed by highly active antiretroviral therapy. SIV-infected macaques were treated with two reverse transcriptase inhibitors: PMPA (9-R-(2-phosphonomethoxypropyl)adenine), which does not cross the blood-brain barrier, and FTC (beta-2′,3′-dideoxy-3′-thia-5-fluorocytidine), which does. Viral DNA and RNA were quantitated in the brain after 6 months of suppression of virus replication in blood and cerebrospinal fluid (CSF). Viral DNA was detected in brain from all macaques, including those in which peripheral viral replication had been suppressed either by antiretroviral therapy or host immune responses. Significant neurological lesions were observed only in one untreated macaque that had active virus replication in the CNS. Expression of the inflammatory markers, major histocompatibility complex (MHC) II and CD68 was significantly lower in macaques treated with PMPA/FTC. Thus, although antiretroviral treatment may suppress virus replication in the periphery and the brain and reduce CNS inflammation, viral DNA persists in the brain despite treatment. This suggests that the brain may serve as a long-term viral reservoir in HIV-infected individuals treated with antiretroviral drugs that suppress virus replication.