Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration.

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, we show that Netrin-1 is expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. We demonstrate that alpha6beta4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of alpha6beta4 and alpha3beta1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells.

Novel expression of PINCH in the central nervous system and its potential as a biomarker for human immunodeficiency virus-associated neurodegeneration.

Particularly interesting cysteine histidine-rich (PINCH) protein functions as a shuttling protein in Schwann cells after peripheral nerve damage, during repair and remodeling, and in maintaining neuronal polarity. However, the presence of PINCH in the human CNS during disease has not been addressed. Because HIV-associated damage to cells of the CNS involves dysregulation of neuronal signaling and white matter damage, we hypothesized that PINCH may play a role in neuropathological processes during the course of HIV infection. To determine the expression of PINCH in the CNS, brain, and cerebrospinal fluid (CSF) obtained at autopsy from HIV patients with no CNS alterations, HIV encephalitic (HIVE) patients, and HIV-negative individuals with no CNS alterations were examined for PINCH immunoreactivity. Our results show that PINCH is expressed robustly in the brains and CSF of HIV patients, but is nearly undetectable in HIV-negative individuals. However, HIVE patients' CSF contained significantly less PINCH than HIV patients with no CNS alterations. PINCH immunolabeling was significantly more intense in the white matter than in the grey matter and was associated exclusively with neuronal cell bodies or processes, or with the extracellular matrix. Given the recently discovered importance of PINCH in maintaining neuronal fitness, our observations that PINCH is robustly expressed in the CNS of HIV patients suggests an important role for PINCH in HIV-associated neurodegenerative processes. Understanding mechanisms by which PINCH functions during HIV-associated CNS alterations will provide new insight into potential treatments to limit neurological alterations in HIV.

Alterations in the Notch4 pathway in cerebral endothelial cells by the HIV aspartyl protease inhibitor, nelfinavir.

BACKGROUND: Aspartyl protease inhibitors (PIs) used to treat HIV belong to an important group of drugs that influence significantly endothelial cell functioning and angiogenic capacity, although specific mechanisms are poorly understood. Recently, PIs, particularly Nelfinavir, were reported to disrupt Notch signaling in the HIV-related endothelial cell neoplasm, Kaposi's sarcoma. Given the importance of maintaining proper cerebral endothelial cell signaling at the blood brain barrier during HIV infection, we considered potential signaling pathways such as Notch, that may be vulnerable to dysregulation during exposure to PI-based anti-retroviral regimens. Notch processing by gamma-secretase results in cleavage of the notch intracellular domain that travels to the nucleus to regulate expression of genes such as vascular endothelial cell growth factor and NFkappaB that are critical in endothelial cell functioning. Since, the effects of HIV PIs on gamma-secretase substrate pathways in cerebral endothelial cell signaling have not been addressed, we sought to determine the effects of HIV PIs on Notch and amyloid precursor protein. RESULTS: Exposure to reported physiological levels of Saquinavir, Indinavir, Nelfinavir and Ritonavir, significantly increased reactive oxygen species in cerebral endothelial cells, but had no effect on cell survival. Likewise, PIs decreased Notch 4-protein expression, but had no effect on Notch 1 or amyloid precursor protein expression. On the other hand, only Nelfinavir increased significantly Notch 4 processing, Notch4 intracellular domain nuclear localization and the expression of notch intracellular domain targets NFkappaB and matrix metalloproteinase 2. Pre-treatment with the antioxidant Vitamin E prevented PI-induced reactive oxygen species generation and partially prevented Nelfinavir-induced changes in both Notch 4 processing, and cellular localization patterns. Moreover, in support of increased expression of pro-angiogenic genes after Nelfinavir treatment, Nelfinavir did not inhibit angiogenic capacity. CONCLUSION: Nelfinavir affects Notch 4 processing that results in induction of expression of the pro-angiogenic genes NFkappaB and matrix metalloproteinase 2 in cerebral endothelial cells.

Signalling crosstalk in FGF2-mediated protection of endothelial cells from HIV-gp120.

BACKGROUND: The blood brain barrier (BBB) is the first line of defence of the central nervous system (CNS) against circulating pathogens, such as HIV. The cytotoxic HIV protein, gp120, damages endothelial cells of the BBB, thereby compromising its integrity, which may lead to migration of HIV-infected cells into the brain. Fibroblast growth factor 2 (FGF2), produced primarily by astrocytes, promotes endothelial cell fitness and angiogenesis. We hypothesized that treatment of human umbilical vein endothelial cells (HUVEC) with FGF2 would protect the cells from gp120-mediated toxicity via endothelial cell survival signalling. RESULTS: Exposure of HUVEC to gp120 resulted in dose- and time-dependent cell death; whereas, pre-treatment of endothelial cells with FGF2 protected cells from gp120 angiotoxicity. Treatment of HUVEC with FGF2 resulted in dose- and time-dependent activation of the extracellular regulated kinase (ERK), with moderate effects on phosphoinositol 3 kinase (PI3K) and protein kinase B (PKB), also known as AKT, but no effects on glycogen synthase kinase 3 (GSK3beta) activity. Using pharmacological approaches, gene transfer and kinase activity assays, we show that FGF2-mediated angioprotection against gp120 toxicity is regulated by crosstalk among the ERK, PI3K-AKT and PKC signalling pathways. CONCLUSIONS: Taken together, these results suggest that FGF2 may play a significant role in maintaining the integrity of the BBB during the progress of HIV associated cerebral endothelial cell damage.

Altered P-glycoprotein expression in AIDS patients with HIV encephalitis.

Penetrance of anti-retroviral drugs into the CNS depends partly on the activity of P-glycoprotein (P-gp), an ATP-dependent efflux pump involved in restricting entry of lipophilic drugs into the brain. The present study characterizes the patterns of P-gp expression in the brains of AIDS patients and examines its relationship with clinical and neuropathological indicators of HIV encephalitis (HIVE). For this purpose, brain tissue collected at autopsy from 26 subjects with a history of HIV (9 without HIVE; 17 with HIVE) was analyzed. Immunocytochemical staining and Western blot analyses for regional P-gp expression were performed and levels were correlated with neuropathological indicators and with HIV RNA. Double labeling experiments were performed with antibodies against astroglial (GFAP), endothelial (CD31), microglial (CD45) and neuronal (MAP2) cell markers. In the HIVE-negative cases, P-gp immunoreactivity was associated primarily with endothelial cells. HIVE-positive cases showed extensive immunolabeling of astroglial and microglial cells, but relatively less endothelial cell immunolabeling. No neuronal P-gp immunostaining was detected in brain tissue from any cases in the study. In the HIVE-positive cases with extensive astroglial labeling, the most intense immunoreactivity was detected in white matter. A subset of HIVE-positive cases displayed intense P-gp immunostaining of astrocytes closely associated with blood vessels in the cortex. Both the immunocytochemical and Western blot analyses showed a significant correlation between P-gp expression and HIV RNA levels. In conclusion, P-gp immunoreactivity was detected largely in glial cells in tissue from HIVE-positive patients. Furthermore, in HIVE-positive patients, brain viral burden and P-gp levels were significantly higher than those in HIVE-negative patients. Taken together, our data suggest that P-gp may be part of a central pathway mediating viral compartmentalization in the brains of HIV-infected individuals and may play a significant part in HIV disease progression in the brain.

Most common causes of natural and injury-related deaths in Addis Ababa, Ethiopia.

In Ethiopia, like many developing countries, autopsy is rare unless conducted in the medico-legal arena, making vital statistics that include pathological diagnoses sparse. To determine the most common factors contributing to death among individuals who died from natural or injury-related events in Ethiopia 200 consecutive autopsies were conducted in 2006 at the Forensic Medico-legal Pathology Department, Menelik II Hospital, Addis Ababa, Ethiopia. The results describe significant pathological observations, putative cause of death, age distribution, and gender ratios. Eighty-one percent of the cases were male, and the mean age was 38.9 (+/-15.5 years). Fifty-two percent of the individuals died from natural causes, including infections, and 48% died from injury-related events. In the natural deaths group, as determined by gross examination at autopsy pulmonary complications were the most commonly reported cause of death, with suspected tuberculosis accounting for 12%. Tuberculosis (21, 8%) and liver disease (14, 5%) were the most common histopathological findings in the natural and injury-related causes groups, respectively. In the injury-related group, automobile accident was the most common cause of accidental death (80%), and homicide by beating was the most common cause of death in the intentional injury group (31%). These data provide valuable unbiased analyses of causes of death among individuals in Addis Ababa, Ethiopia.

The role of mitochondrial alterations in the combined toxic effects of human immunodeficiency virus Tat protein and methamphetamine on calbindin positive-neurons.

The use of methamphetamine (METH) continues to increase the risk of human immunodeficiency virus (HIV) transmission within both homosexual and heterosexual drug abuser groups. Neurological studies indicate that the progression of HIV encephalitis is also enhanced by illicit drug use. Recently, the authors' studies in the postmortem brains of HIV-positive METH users have shown that the combined effects of HIV and METH selectively damage calbindin (CB)-immunoreactive nonpyramidal neurons, which may contribute to the behavioral alterations observed in these patients. To better understand the mechanisms of toxicity associated with exposure to HIV and METH, neuronal survival, phenotypic markers, levels of oxidative stress, and mitochondrial potential were assessed in vitro in the hippocampal neuronal cell line, HT22, and in primary human neurons exposed to the HIV Tat protein and/or METH. Both Tat and METH were toxic to neurons in a time- and dose-dependent fashion. Neurons exposed to a combination of Tat and METH displayed early evidence of neuronal damage at 6 h, characterized by a decrease in CB and microtubule-associated protein 2 (MAP2) immunoreactivity followed by more extensive cell death at 24 h. Loss of CB immunoreactivity associated with the combined exposure to Tat and METH was accompanied by mitochondrial damage with increased levels of oxidative stress. The toxic effects of Tat and METH were inhibited by blocking mitochondrial uptake of intracellular calcium, whereas blocking calcium flux in the endoplasmic reticulum or from the extracellular environment had no effect on Tat and METH toxicity. These studies indicate that in vitro, when combined, the HIV protein Tat and METH damage CB-immunoreactive nonpyramidal neurons by dysregulating the mitochondrial calcium potential. In combination, Tat and METH may increase cell injury and death, thereby enhancing brain metabolic disturbances observed in HIV-positive METH users in clinical populations.