Моlecular mechanisms of initiation of carcinogenesis in the testis.

In this review, literature data on the study of precancerous changes in testicular tissue and molecular changes, as well as the influence of environmental factors that can initiate carcinogenesis, were analyzed and summarized for the future determination of early diagnosis of germ cell tumors of the testis and the development of preventive measures. The review also discusses the significant new changes presented in the Fourth Edition of the World Health Organization Classification of Urogenital Tumors, published in 2016, and modern concepts of the etiology and pathogenesis of these diseases. Among the environmental factors that can initiate carcinogenesis, the most noteworthy are the biological effects of low doses of ionizing radiation, such as the effect of radiation-induced genome instability, which increases the risk of carcinogenesis, the "bystander effect", and chronic oxidative stress. Disruption of ubiquitin-proteasomal proteolysis, impaired molecular-level components of the blood-testis barrier, and impaired regulatory action of TGF-ß on the cell cycle can play a crucial role in the pathogenesis of male infertility and the initiation of carcinogenesis in the testis. The effect of low doses of ionizing radiation as an additional etiological factor leads to changes in the structural, as well as molecular, components of the testis, including epigenetic changes, which can be characterized as environmental pathomorphosis, which leads to impaired spermatogenesis and increased risk of malignancy. Summarizing the literature review data, we can state that patients with blocked spermatogenesis, in which atypical germ cell neoplasia in situ cells are detected in testicular tissue, constitute a group at increased risk of testicular carcinogenesis. The presence of additional etiological factors, such as chronic low doses of ionizing radiation, can initiate the progression of carcinogenesis in the testicle.

Rodent model systems for studies of HIV-1 associated dementia.

Understanding of HIV-1 neuropathogenesis and development of rationale therapeutic approaches requires relevant animal models. The putative mechanisms of neuroinflammatory and neurotoxic events triggered by HIV-1 brain infection are reflected by a number of rodent models. These include transgenic animals (either expressing viral proteins or pro-inflammatory factors), infection with murine retroviruses, and severe combined immunodeficient (SCID) mice reconstituted with human lymphocytes and injected intracerebrally with HIV-1-infected human monocyte-derived macrophages. The potential importance and limitations of the models in reflecting human disease are discussed with emphasis on their utility for development of therapies to combat HIV-1-associated neurologic impairment.

Alcohol-induced oxidative stress in brain endothelial cells causes blood-brain barrier dysfunction.

Brain microvascular endothelial cells (BMVEC) connected by tight junctions (TJ) form a tight monolayer at the blood-brain barrier (BBB). We investigated the idea that BBB dysfunction seen in alcohol abuse is associated with oxidative stress stemming from ethanol (EtOH) metabolism in BMVEC. Exposure to EtOH induced catalytic activity/expression of EtOH-metabolizing enzymes, which paralleled enhanced generation of reactive oxygen species (ROS). EtOH-mediated oxidative stress led to activation of myosin light chain (MLC) kinase, phosphorylation of MLC and TJ proteins, decreased BBB integrity, and enhanced monocyte migration across BBB. Acetaldehyde or ROS donors mimicked changes induced by EtOH in BMVEC. Thus, oxidative stress resulting from alcohol metabolism in BMVEC can lead to BBB breakdown in alcohol abuse, serving as an aggravating factor in neuroinflammatory disorders.

Effects of pluronic P85 on GLUT1 and MCT1 transporters in the blood-brain barrier.

PURPOSE: The amphiphilic block copolymer Pluronic P85 (P85) increases the permeability of the blood-brain barrier (BBB) with respect to a broad spectrum of drugs by inhibiting the drug efflux transporter, P-glycoprotein (Pgp). In this regard, P85 serves as a promising component for CNS drug delivery systems. To assess the possible effects of P85 on other transport systems located in the brain, we examined P85 interactions with the glucose (GLUT1) and monocarboxylate (MCT1) transporters. METHODS: Polarized monolayers of primary cultured bovine brain microvessel endothelial cells (BBMEC) were used as an in vitro model of the BBB. 3H-2-deoxy-glucose and 14C-lactate were selected as GLUT1 and MCT1 substrates, respectively. The accumulation and flux of these substrates added to the luminal side of the BBMEC monolayers were determined. RESULTS: P85 has little effect on 3H-2-deoxy-glucose transport. However, a significant decrease 14C-lactate transport across BBMEC monolayers is observed. Histology, immunohistochemistry, and enzyme histochemistry studies show no evidence of P85 toxicity in liver, kidney, and brain in mice. CONCLUSIONS: This study suggests that P85 formulations do not interfere with the transport of glucose. This is, probably, due to compensatory mechanisms in the BBB. Regarding the transport of monocarboxylates, P85 formulations might slightly affect their homeostasis in the brain, however, without any significant toxic effects.

Hippocampal synaptic dysfunction in a murine model of human immunodeficiency virus type 1 encephalitis.

Alterations in hippocampal physiology affect cognition in human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD). The mechanism for how this occurs is not well understood. To address this, we investigated how changes in synaptic transmission and plasticity are affected by viral infection and macrophage activation using a severe combined immunodeficiency mouse model of human HIV-1 encephalitis (HIVE). HIVE was induced in mice by stereotactic injection of HIV-1-infected human monocyte-derived macrophages (MDM) into the striatum. Animals were sacrificed after 3, 7 and 15 days. Hippocampal slices were prepared from HIV-1, MDM- and sham-injected animals. Electrically evoked field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus. Neuronal physiology was assessed by input-output and by long-term potentiation (LTP) assays. We observed that a higher stimulation intensity (mA) was required to induce a 1-mV response in the HIVE mice (0.32+/-0.06) compared with shams (0.17+/-0.01) at day 7. The stimulation intensities at day 15 were 0.44+/-0.07 and 0.23+/-0.05 in the HIVE and shams, respectively. An impairment of synaptic function was detected through measuring synaptic responses induced by stimuli with different intensities. Paired-pulse facilitation (PPF) showed deficits in HIVE mice at days 3, 7, and 15. At day 3, PPF ratios were 1.13+/-0.02 and 1.24+/-0.04 in HIVE and sham. The induction and maintenance of LTP was also impaired in HIVE mice. The average magnitude of LTP was 131.23+/-15.26% of basal in HIVE as compared with sham animals of 232.63+/-24.18%. MDM-injected mice showed an intermediate response. Taken together, the results show a range of neuronal synaptic transmission and plasticity changes in HIVE mice that may reflect the mechanisms of cognitive dysfunction in human HAD.

Model systems for assessing cognitive function: implications for HIV-1 infection and drugs of abuse.

Memory deficits are common among drug abusers and in those with chronic neurodegenerative disorders. Currently, the mechanisms through which diverse neurophysiologic processes alter memory are not known. This review describes the current systems and rationale for studying memory formation, consolidation, and recall. Special attention is given to physiologic (hippocampal long-term potentiation) and behavioral animal models. The principles and methods described can be applied to studies of diverse clinical disorders.

The regulation of alpha chemokines during HIV-1 infection and leukocyte activation: relevance for HIV-1-associated dementia.

Cellular immunity against human immunodeficiency virus type 1 (HIV-1)-infected brain macrophages serves to prevent productive viral replication in the nervous system. Inevitably, during advanced disease, this antiretroviral response breaks down. This could occur through virus-induced dysregulation of lymphocyte trafficking. Thus, we studied the production of non-ELR-containing alpha-chemokines and their receptor (CXCR3) expression in relevant virus target cells. Macrophages, lymphocytes, and astrocytes secreted alpha-chemokines after HIV-1 infection and/or immune activation. Lymphocyte CXCR3-mediated chemotactic responses were operative. In all, alpha-chemokine-mediated T cell migration continued after HIV-1 infection and the neuroinflammatory events operative during productive viral replication in brain.

Mononuclear phagocyte differentiation, activation, and viral infection regulate matrix metalloproteinase expression: implications for human immunodeficiency virus type 1-associated dementia.

The pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is mediated mainly by mononuclear phagocyte (MP) secretory products and their interactions with neural cells. Viral infection and MP immune activation may affect leukocyte entry into the brain. One factor that influences central nervous system (CNS) monocyte migration is matrix metalloproteinases (MMPs). In the CNS, MMPs are synthesized by resident glial cells and affect the integrity of the neuropil extracellular matrix (ECM). To ascertain how MMPs influence HAD pathogenesis, we studied their secretion following MP differentiation, viral infection, and cellular activation. HIV-1-infected and/or immune-activated monocyte-derived macrophages (MDM) and human fetal microglia were examined for production of MMP-1, -2, -3, and -9. MMP expression increased significantly with MP differentiation. Microglia secreted high levels of MMPs de novo that were further elevated following CD40 ligand-mediated cell activation. Surprisingly, HIV-1 infection of MDM led to the down-regulation of MMP-9. In encephalitic brain tissue, MMPs were expressed within perivascular and parenchymal MP, multinucleated giant cells, and microglial nodules. These data suggest that MMP production in MP is dependent on cell type, differentiation, activation, and/or viral infection. Regulation of MMP expression by these factors may contribute to neuropil ECM degradation and leukocyte migration during HAD.

Reduction in glial immunity and neuropathology by a PAF antagonist and an MMP and TNFalpha inhibitor in SCID mice with HIV-1 encephalitis.

The effects of anti-inflammatory drugs on glial immunity and neuropathology were determined in a severe combined immune deficiency (SCID) mouse model of HIV-1 encephalitis. HIV-1-infected human monocyte-derived macrophages (MDM) are stereotactically inoculated into basal ganglia resulting in a multinucleated giant cell encephalitis. A platelet activating factor antagonist and a matrix metalloproteinase inhibitor, which also inhibits tumor necrosis factor alpha release, were administered to animals at the time of the MDM inoculation. The drugs administered in combination markedly reduced brain inflammation, astrogliosis and microglia activation. These findings demonstrate that reduction of brain inflammatory responses, independent of viral replication, can affect HIVE pathology in an animal model system of disease.

The efficacy of potent anti-retroviral drug combinations tested in a murine model of HIV-1 encephalitis.

Development of anti-retroviral regimens with enhanced efficacy against brain HIV-1 is essential if viral eradication is to be achieved. To address this, a severe combined immune deficiency mouse model of HIV-1 encephalitis was used to assay the effect of protease-containing and protease-sparing drug regimens on viral replication in brain macrophages. Here, HIV-1-infected human monocyte-derived macrophages (MDM) are inoculated into basal ganglia, causing a multinucleated giant cell encephalitis reminiscent of human disease. Drugs were administered at the time of MDM inoculation and continued until sacrifice. Immunohistochemical tests evaluated ongoing viral replication, glial immunity, and neuronal survival. Treatment with ddI/d4T decreased the numbers of infected cells by 75%, while ddI/d4T/amprenavir or ZDV/3TC/ABC diminished infection by 98%. Triple drug regimens decreased astrogliosis by > or = 25%. This small-animal model may be used to screen drug regimens that affect ongoing HIV-1 replication within its brain sanctuary.

HIV-1 infected and immune competent mononuclear phagocytes induce quantitative alterations in neuronal dendritic arbor: relevance for HIV-1-associated dementia.

Neuronal loss, alterations in dendritic arbor, and decreased synaptic density, in infected brain tissue, are neuropathological signatures of HIV-1-associated dementia (HAD). Brain mononuclear phagocyte (MP) (macrophage and microglia) secretory products can effect neuronal compromise, although the underlying mechanism(s) remain incompletely defined. To these ends, we quantitatively assessed the effects of virus-infected and/or immune activated MP secretory products on multiple aspects of neuronal morphology. Rat cortical and hippocampal neurons were exposed to secretory products from HIV-1-infected and lipopolysaccharide (LPS)-activated human monocyte-derived macrophage (MDM). Our assays for alterations in neuronal dendritic arbor and cell loss included the quantification of neurofilament (NF), neuron-specific enolase (NSE), and MAP-2 by ELISA and cellular morphology. MDM conditioned media (MCM) enhanced neuronal survival. HIV-1 infection or activation by LPS had modest neurotoxic effects. In contrast, the combination of HIV-1 infection and activation of MDM produced significant neurotoxicity. Such MDM products altered dendritic arbor, decreased synaptic density, and increased LDH release. Comparable neurotrophic/toxic responses were observed when neurons were exposed to MCM collected from 12 separate human donors. Similar responses were observed with MCM from human fetal microglia, further supporting the role of HIV-1-infected and immune-activated brain MP in the overall neurotoxic responses. This work provides quantitative measures of neuronal damage by which virus infected and activated MP can elicit neuronal injury in HAD.

HIV-1 infected immune competent mononuclear phagocytes influence the pathways to neuronal demise.

Secretory products from HIV-1-infected immune-competent mononuclear phagocytes (MP) damage neuronal dendritic arbor (Zheng et al., 2001). The mechanism behind neuronal injury and whether it is species and/or viral strain dependent is not fully understood. To these ends, we investigated whether HIV-1-infected and lipopolysaccharide (LPS)-activated MDM elicit neuronal injury in primary human neurons. Neuronal damage was compared to that seen in rat neurons. Utilizing a spectrum of HIV-1 strains to infect human monocyte-derived macrophages (MDM), productive viral replication proved necessary, but not sufficient, for neuronal injury. Neuronal demise was induced by virion-free HIV-1-infected and immune-activated MDM culture supernatants. Maximal alterations in glutamate mediated neuronal signaling, resulted from exposure to secretory products from HIV-1-infected and immune-activated MDM. Apoptosis was the predominant mechanism of cell death induced by HIV-1-infected and LPS-treated MDM. Importantly, neuronal injury and increases in calcium influx mediated by HIV-1-infected and immune-activated MDM culture supernatants was partially blocked by the N-methyl D-aspartate (NMDA) receptor antagonist, MK 801. These data support a primary role for immune-activation in MP neurotoxic activities. The upregulation of NMDA receptor sensitive soluble factors and neuronal apoptosis by HIV-1-infected and immune-activated MDM provide unique insights into links between soluble factors, produced as a consequence of MP immunity, and neuronal demise in HAD.

Mononuclear phagocytes mediate blood-brain barrier compromise and neuronal injury during HIV-1-associated dementia.

The neuropathogenesis of HIV-1 infection revolves around the production of secretory factors from immune-activated brain mononuclear phagocytes (MP). MP-secreted chemokines may play several roles in HIV-1 encephalitis (HIVE). These can promote macrophage brain infiltration, blood-brain barrier (BBB) and neuronal dysfunction during HIV-1-associated dementia. We investigate how HIV-1-infected MP regulates the production of chemokines and how they influence HIV-1 neuropathogenesis. We demonstrate that HIV-1-infected and immune-activated MP (for example, microglia) and astrocytes produce beta-chemokines in abundance, as shown in both laboratory assays and within infected brain tissue. HIV-1-infected microglia significantly modulate monocyte migration in a BBB model system and in brains of SCID mice with HIVE. HIV-1-infected MP down-regulate tight junction protein and special polarized transport systems on brain microvascular endothelial cells as shown in human autopsy brain tissue and in SCID mice with HIVE. Chemokines can damage neurons directly. Toxicity caused by binding of stromal-derived factor-1alpha to its receptor on neurons exemplifies such mechanism. In toto, these works underscore the diverse roles of chemokines in HIV-1 neuropathogenesis and lay the foundation for future therapeutic interventions.

HIV-1 infected mononuclear phagocyte secretory products affect neuronal physiology leading to cellular demise: relevance for HIV-1-associated dementia.

Viral and cellular products from HIV-1-infected and/or immune competent mononuclear phagocytes (MP) (brain macrophages and microglia) affect neuronal function during HIV-1-associated dementia (HAD). Neurotoxic MP factors include, but are not limited to, pro-inflammatory cytokines, chemokines, platelet activating factor, arachidonic acid and its metabolites, nitric oxide, progeny virions and viral structural and regulatory proteins. The mechanisms for immune-mediated neural injury in HAD, only now, are being unraveled. In this regard, we reviewed the current knowledge of how postmitotic neurons, which can neither divide nor be replaced, are damaged by MP secretory activities. Linking neuronal function with brain MP activation was made possible by placing viral and/or immune products onto neurons and measuring cell signaling events or through ex vivo electrophysiological tests on MP-treated brain slices. Such linkages are shown, in this report, by select demonstrations of MP factors which cause neuronal dysfunction in HAD.

Evaluation of antiretroviral drug efficacy for HIV-1 encephalitis in SCID mice.

OBJECTIVES: To compare the efficacy of the nucleoside reverse transcriptase inhibitors (NRTIs) abacavir, zidovudine (AZT), lamivudine (3TC), didanosine (ddI), and stavudine (d4T) to inhibit viral replication in brain macrophages. A severe combined immunodeficiency (SCID) mouse model of HIV-1 encephalitis (HIVE) was used to monitor spreading viral infection in the CNS. BACKGROUND: The development of antiretroviral therapies with CNS efficacy against neuroinvasive virus is important if eradication of HIV-1 can be achieved within critical "hidden reservoirs." METHODS: HIV-1-infected human monocyte-derived macrophages (MDMs) (after a single round of viral replication) were inoculated into the caudate and putamen of SCID mice. This resulted in the spreading of viral infection with a concomitant multinucleated giant cell encephalitis (astrogliosis, microglial activation, and neuronal injury). NRTIs were administered to animals at the time of intracerebral MDM inoculations and continued until the time of sacrifice. Antiretroviral effects were assessed by viral load and percentages of infected MDMs. RESULTS: In brains of SCID mice with HIVE, abacavir and lamivudine reduced HIV-1 p24 antigen-positive cells by 80% and 95%, respectively, whereas both decreased viral load by approximately 1 log. Zidovudine, didanosine, and stavudine showed variable effects. CONCLUSION: Abacavir and lamivudine showed significant antiretroviral activity in SCID mice with HIVE when compared with other NRTIs. The extrapolation of these results to humans with HIV-1 dementia awaits future investigations.

Model systems for studies of leukocyte migration across the blood - brain barrier.

The blood - brain barrier (BBB) plays a crucial role in central nervous system (CNS) homeostasis. Serving as the brain's protective shield it regulates soluble factor and cellular exchanges from blood to brain. Critical to its function, the BBB is composed of brain microvascular endothelial cells (BMVEC), a collagen matrix, and astrocytes. Astrocytic endfeet surround the BMVEC abluminal surface and influence the 'tightness' and trafficking role of the barrier. In neurodegenerative disorders (for example stroke, multiple sclerosis and HIV encephalitis) the BBB becomes compromised. This is, in part, immune mediated. An accumulating body of evidence demonstrates that the cellular components of the BBB are themselves immunocompetent. Perivascular cells (astrocytes, macrophages and microglial cells) and BMVEC produce inflammatory factors that affect BBB permeability and expression of adhesion molecules. These affect cell trafficking into the CNS. Leukocyte BBB migration can be influenced by cytokines and chemokines produced by glia. Astrocytes and macrophages secrete a multitude of factors that affect brain immune responses. Interactions between BMVEC, leukocytes and/or glia, immunological activation and noxious (infectious, toxic and immune-mediated) brain insults all appear to play important roles in this BBB cell trafficking. New information gained into the mechanisms of leukocyte-brain penetration may provide novel insights in the pathogenesis and treatment strategies of neurodegenerative disorders.

Microglial and astrocyte chemokines regulate monocyte migration through the blood-brain barrier in human immunodeficiency virus-1 encephalitis.

The numbers of immune-activated brain mononuclear phagocytes (MPs) affect the progression of human immunodeficiency virus (HIV)-1-associated dementia (HAD). Such MPs originate, in measure, from a pool of circulating monocytes. To address the mechanism(s) for monocyte penetration across the blood-brain barrier (BBB), we performed cross-validating laboratory, animal model, and human brain tissue investigations into HAD pathogenesis. First, an artificial BBB was constructed in which human brain microvascular endothelial and glial cells-astrocytes, microglia, and/or monocyte-derived macrophages (MDM)-were placed on opposite sides of a matrix-coated porous membrane. Second, a SCID mouse model of HIV-1 encephalitis (HIVE) was used to determine in vivo monocyte blood-to-brain migration. Third, immunohistochemical analyses of human HIVE tissue defined the relationships between astrogliosis, activation of microglia, virus infection, monocyte brain infiltration, and beta-chemokine expression. The results, taken together, showed that HIV-1-infected microglia increased monocyte migration through an artificial BBB 2 to 3.5 times more than replicate numbers of MDM. In the HIVE SCID mice, a marked accumulation of murine MDM was found in areas surrounding virus-infected human microglia but not MDM. For human HIVE, microglial activation and virus infection correlated with astrogliosis, monocyte transendothelial migration, and beta-chemokine expression. Pure cultures of virus-infected and activated microglia or astrocytes exposed to microglial conditioned media produced significant quantities of beta-chemokines. We conclude that microglial activation alone and/or through its interactions with astrocytes induces beta-chemokine-mediated monocyte migration in HAD.

The neuropathogenesis of HIV-1 infection.

HIV encephalitis is the common pathologic correlate of HIV-dementia (HAD). HIV-infected brain mononuclear phagocytes (MP) (macrophages and microglia) are reservoirs for persistent viral infection. When activated, MP contribute to neuronal damage. Such activated and virus-infected macrophages secrete cellular and viral factors, triggering neural destructive immune responses. Our Center's laboratories have begun to decipher the molecular and biochemical pathways for MP-mediated neuronal damage in HAD. This review will discuss the salient clinical and pathological features of HAD and highlight the recent advances made, by our scientists and elsewhere, in unraveling disease mechanisms, including the role of chemokines and their receptors in the neuropathogenesis of HIV-1 encephalitis.

Intracellular CXCR4 signaling, neuronal apoptosis and neuropathogenic mechanisms of HIV-1-associated dementia.

The mechanism(s) by which HIV-1 affects neural injury in HIV-1-associated dementia (HAD) remains unknown. To ascertain the role that cellular and viral macrophage products play in HAD neurotoxicity, we explored one potential route for neuronal demise, CXCR4. CXCR4, expressed on lymphocytes and neurons, is both a part of neural development and a co-receptor for HIV-1. Its ligand, stromal cell-derived factor-1alpha (SDF-1alpha), affects neuronal viability. GTP binding protein (G-protein) linked signaling after neuronal exposure to SDF-1alpha, virus-infected monocyte-derived macrophage (MDM) secretory products, and virus was determined. In both human and rat neurons, CXCR4 was expressed at high levels. SDF-1alpha/beta was detected predominantly in astrocytes and at low levels in MDM. SDF-1beta/beta was expressed in HAD brain tissue and upregulated in astrocytes exposed to virus infected and/or immune activated MDM conditioned media (fluids). HIV-1-infected MDM secretions, virus and SDF-1beta induced a G inhibitory (Gi) protein-linked decrease in cyclic AMP (cAMP) and increase inositol 1,4, 5-trisphosphate (IP3) and intracellular calcium. Such effects were partially blocked by antibodies to CXCR4 or removal of virus from MDM fluids. Changes in G-protein-coupled signaling correlated, but were not directly linked, to increased neuronal synaptic transmission, Caspase 3 activation and apoptosis. These data, taken together, suggest that CXCR4-mediated signal transduction may be a potential mechanism for neuronal dysfunction during HAD.