HIV-1 gp41 ectodomain enhances Cryptococcus neoformans binding to human brain microvascular endothelial cells via gp41 core-induced membrane activities.

Cryptococcus neoformans causes life-threatening meningoencephalitis, particularly prevalent in AIDS patients. The interrelationship between C. neoformans and HIV-1 is intriguing, as both pathogens elicit severe neuropathological complications. We have previously demonstrated that the HIV-1 gp41 ectodomain fragments gp41-I33 (amino acids 579-611) and gp41-I90 (amino acids 550-639) can enhance C. neoformans binding to HBMECs (human brain microvascular endothelial cells). Both peptides contain the loop region of gp41. In the present study, we used immunofluorescence microscopy and transmission and scanning electron microscopy to explore the underlying mechanisms. Our findings indicated that both C. neoformans and gp41-I90 up-regulated ICAM-1 (intercellular adhesion molecule 1) on the HBMECs and elicited membrane ruffling on the surface of HBMECs. The HIV-1 gp41 ectodomain could also induce CD44 and ß-actin redistribution to the membrane lipid rafts, but it could not enhance PKCα (protein kinase Cα) phosphorylation like C. neoformans. Instead, gp41-I90 was able to induce syncytium formation on HBMECs. The results of the present study suggest HIV-1 gp41-enhanced C. neoformans binding to HBMECs via gp41 core domain-induced membrane activities, revealing a potential mechanism of invasion for this pathogenic fungus into the brain tissues of HIV-1-infected patients.

Polarized response of endothelial cells to invasion by Aspergillus fumigatus.

Hyphal invasion of blood vessels is a prominent feature of invasive aspergillosis. During invasive pulmonary aspergillosis, Aspergillus fumigatus hyphae invade the abluminal endothelial cell surface, whereas they invade the luminal endothelial cell surface during haematogenous dissemination. We investigated the endothelial cell response to abluminal and luminal infection with A. fumigatus hyphae in vitro. We found that these hyphae invaded the abluminal endothelial cell surface without inducing the formation of endothelial cell pseudopods. Also, the internalized hyphae were surrounded by a loose network of microfilaments. In contrast, A. fumigatus hyphae invaded the luminal endothelial cell surface by inducing by the formation of endothelial cell pseudopods. These endocytosed hyphae were surrounded by a tight network of microfilaments. Abluminal infection induced greater E-selectin, IL-8, tissue factor and TNF-alpha gene expression, but less endothelial cell damage than did luminal infection. Endothelial cell stimulation by infection of either surface was mediated by endothelial cell-derived TNF-alpha, and was not influenced by gliotoxin secreted by A. fumigatus. These differences in the endothelial cell response to abluminal versus luminal infection may contribute to differences in the pathogenesis of invasive versus haematogenously disseminated aspergillosis.

Alzheimer disease macrophages shuttle amyloid-beta from neurons to vessels, contributing to amyloid angiopathy.

Neuronal accumulation of oligomeric amyloid-beta (Alphabeta) is considered the proximal cause of neuronal demise in Alzheimer disease (AD) patients. Blood-borne macrophages might reduce Abeta stress to neurons by immigration into the brain and phagocytosis of Alphabeta. We tested migration and export across a blood-brain barrier model, and phagocytosis and clearance of Alphabeta by AD and normal subjects' macrophages. Both AD and normal macrophages were inhibited in Alphabeta export across the blood-brain barrier due to adherence of Abeta-engorged macrophages to the endothelial layer. In comparison to normal subjects' macrophages, AD macrophages ingested and cleared less Alphabeta, and underwent apoptosis upon exposure to soluble, protofibrillar, or fibrillar Alphabeta. Confocal microscopy of stained AD brain sections revealed oligomeric Abeta in neurons and apoptotic macrophages, which surrounded and infiltrated congophilic microvessels, and fibrillar Abeta in plaques and microvessel walls. After incubation with AD brain sections, normal subjects' monocytes intruded into neurons and uploaded oligomeric Abeta. In conclusion, in patients with AD, macrophages appear to shuttle Abeta from neurons to vessels where their apoptosis may release fibrillar Abeta, contributing to cerebral amyloid angiopathy.

Performance of multisite silicon microprobes implanted chronically in the ventral cochlear nucleus of the cat.

A central auditory prosthesis based on microstimulation within the ventral cochlear nucleus (VCN) offers a means of restoring hearing to persons whose auditory nerve has been destroyed bilaterally and cannot benefit from cochlear implants. Arrays of silicon probes with 16 stimulating sites were implanted into the VCN of adult cats, for up to 314 days. Compound neuronal responses evoked from the sites in the VCN were recorded periodically in the central nucleus of the contralateral inferior colliculus (ICC). The threshold and growth of most of the responses were stable for at least 250 days after implantation of the arrays. The responses evoked from the deepest and shallowest electrode sites did exhibit some changes over time but none of the thresholds exceeded 10 microA. The thresholds and growth of the compound responses from most of the stimulating sites were very stable over time, and comparable to those of chronically implanted single-site iridium microelectrodes. Multiunit neuronal activity evoked from the stimulating sites in the VCN was recorded along the dorsolateral-ventromedial (DLVM) axis of the ICC. The distribution, span and degree of overlap of the multiunit activity demonstrated the utility of the multisite, multishank array configuration as a means of accessing the neuronal populations in the VCN that encode various acoustic frequencies. These findings are encouraging for the prospects of developing an auditory prosthesis employing multi-site silicon microprobes.

Microelectrode array for chronic deep-brain microstimulation and recording.

We have developed an array of microelectrodes that is suitable for long-term implantation into the subthalamic nucleus (STN) or the globus pallidus and is able to record from single neurons, as well as deliver localized microstimulation. This device can be used to investigate the mechanisms by which deep brain stimulation can ameliorate the symptoms of Parkinson's disease and other movement disorders, and also may be the basis for a new clinical tool for the treatment of Parkinson's disease, by capitalizing on the high spatial specificity of intranuclear microstimulation. The array includes 16 activated iridium microelectrodes, 5-6 mm in length, within a cluster approximately 1.8 mm in diameter. We have fabricated the array using materials carrying the USP Category VI classification, and we have developed an apparatus and a procedure for implanting the microelectrode arrays into the deep brain. Ten arrays have been implanted into the STN of domestic cats, and one into the internal segment of the globus pallidus, for 140-415 days. During that time, we were able to record action potentials from individual neurons, on 4 to 8 of the 16 channels. The microelectrode' active surface areas ranged from 500 to 2000 microm2. Controlled-current pulses, 26.5 microA in amplitude and 150 micros/phase in duration (4 nC/phase) were used to excite neurons in the cat's STN. In addition to direct activation, the stimulus modulated the neuronal activity over a distance of at least 1.2 mm from the site of stimulation. These parameters did not induce histologically detectable changes around the tip sites after 35 hours of stimulation at 100 Hz (7 hours of stimulation per day, on 5 successive days), if the electrode' active surface area was 1000 microm2 or greater.

Ineffective phagocytosis of amyloid-beta by macrophages of Alzheimer's disease patients.

The defective clearance of amyloid-beta (Abeta) in the brain of Alzheimer's disease (AD) patients is unexplained. The immunohistochemical studies of the frontal lobe and hippocampus show perivascular and intraplaque infiltration by blood-borne macrophages containing intracellular Abeta but only inefficient clearance of beta deposits. Neurons and neuronal nuclei, respectively, express interleukin-1beta and the chemokine RANTES, which could induce the inflammatory cell infiltration. To clarify the pathophysiology ofbeta clearance, we examined Abeta phagocytosis by monocytes and macrophages isolated from the blood of age-matched patients and controls. Control monocytes display excellent differentiation into macrophages and intracellular phagocytosis of Abeta followed by beta degradation or export. AD monocytes show poor differentiation and only surface uptake of Abeta and suffer apoptosis. HLA DR and cyclooxygenase-2 are abnormally expressed on neutrophils and monocytes of AD patients. AD patients have higher levels of intracellular cytokines compared to controls. Thus Abeta clearance is not restricted to brain microglia and involves systemic innate immune responses. In AD, however, macrophage phagocytosis is defective, which may elicit compensatory response by the adaptive immune system.

HIV-1 induces cardiomyopathyby cardiomyocyte invasion and gp120, Tat, and cytokine apoptotic signaling.

We examined heart tissues of AIDS patients with or without HIV cardiomyopathy (HIVCM) by immunohistochemistry, in situ polymerase chain reaction, in situ riboprobe hybridization, and the TUNEL technique for apoptosis. In HIVCM tissues, only inflammatory cells, but not endothelial cells or cardiomyocytes, displayed HIV-1 DNA and RNA. However, macrophages, lymphocytes, and--in a patchy fashion--cardiomyocytes and endothelial cells exhibited virus envelope protein gp120. Macrophages infiltrated the myocardium in a perivascular fashion and expressed tumor necrosis factor family ligands; adjacent cardiomyocytes suffered apoptosis. In vitro HIV-1 strongly invaded neonatal rat ventricular myocytes (NRVMs) and coronary artery endothelial cells (CAECs) and induced microvilli but did not replicate. HIV-1, gp120, or Tat induced Erk 1/2 phosphorylation, activation of caspase-3, and apoptosis of NRVMs and CAECs; all of these were inhibited by a MAPK/ERK-kinase (MEK) inhibitor U0126. The pathogenesis of HIVCM involves HIV-1 replication in inflammatory cells and induction of cardiomyocyte apoptosis by (1) the extrinsic pathway through apoptotic ligands and (2) the intrinsic pathway through direct virus entry and gp120- and Tat-proapoptotic signaling.

Arrays for chronic functional microstimulation of the lumbosacral spinal cord.

Our objective is to develop neural prostheses based on an array of microelectrodes implanted into the sacral spinal cord, that will allow persons with spinal cord injuries to regain control of their bladder and bowels. For our chronic cat model, we have developed two microelectrode arrays, one type containing nine discrete activated iridium microelectrodes and the second utilizing silicon substrate probes with multiple electrode sites on each probe. Both types can elicit an increase in the pressure within the urinary bladder of more than 40-mm Hg and/or relaxation of the urethral sphincter. A stimulus of 100 microA and 400 micros/ph at 20 Hz (charge-balanced pulses) was required to induce a large increase in bladder pressure or relaxation of the urethral sphincter. We found that 24 h of continuous stimulation with these parameters induced tissue injury (disrupted neuropil, infiltration of inflammatory cells, and loss of neurons close to the tip sites). However, a neural prosthesis that is intended to restore bladder control after spinal cord injury would not operate continuously. Thus, when this stimulus was applied for 24 h, at a 10% duty cycle (1 min of stimulation, then 9 min without stimulation) only minimal histologic changes were observed.

HIV-1 Antigens in Neurons of Cocaine-Abusing Patients.

Cocaine opens the blood-brain barrier by deregulating transcription of target genes. Here we show that cocaine at blood concentrations in drug abusers disrupts endothelial cell junctions in parallel with signaling by phosphorylation of extracellular signal-regulated kinase, myristoylated alanine-rich C kinase and myosin light chain. Cocaine effects may be important in vivo since the neurons of drug abusing patients with HIV-1 associated dementia displayed gp120, p24 and Nef.

Cholesterol-depleting statin drugs protect postmitotically differentiated human neurons against ethanol- and human immunodeficiency virus type 1-induced oxidative stress in vitro.

The majority of human immunodeficiency virus type 1 (HIV-1)-infected individuals are either alcoholics or prone to alcoholism. Upon ingestion, alcohol is easily distributed into the various compartments of the body, particularly the brain, by crossing through the blood-brain barrier. Both HIV-1 and alcohol induce oxidative stress, which is considered a precursor for cytotoxic responses. Several reports have suggested that statins exert antioxidant as well as anti-inflammatory pleiotropic effects, besides their inherent cholesterol-depleting potentials. In our studies, postmitotically differentiated neurons were cocultured with HIV-1-infected monocytes, T cells, or their cellular supernatants in the presence of physiological concentrations of alcohol for 72 h. Parallel cultures were pretreated with statins (atorvastatin and simvastatin) with the appropriate controls, i.e., postmitotically differentiated neurons cocultured with uninfected cells and similar cultures treated with alcohol. The oxidative stress responses in the presence/absence of alcohol in these cultures were determined by the production of the well-characterized oxidative stress markers, 8-isoprostane-F2-alpha, total nitrates as an indicator for various isoforms of nitric oxide synthase activity, and heat shock protein 70 (Hsp70). An in vitro culture of postmitotically differentiated neurons with HIV-1-infected monocytes or T cells as well as supernatants from these cells enhanced the release of 8-isoprostane-F2-alpha in the conditioned medium six- to sevenfold (monocytes) and four- to fivefold (T cells). It was also observed that coculturing of HIV-1-infected primary monocytes over a time period of 72 h significantly elevated the release of Hsp70 compared with that of uninfected controls. Cellular supernatants of HIV-1-infected monocytes or T cells slightly increased Hsp70 levels compared to neurons cultured with uninfected monocytes or T-cell supernatants (controls). Ethanol (EtOH) presence further elevated Hsp70 in both infected and uninfected cultures. The amount of total nitrates was significantly elevated in the coculture system when both infected cells and EtOH were present. Surprisingly, pretreatment of postmitotic neurons with clinically available inhibitors of HMG-coenzyme A reductase (statins) inhibited HIV-1-induced release of stress/toxicity-associated parameters, i.e., Hsp70, isoprostanes, and total nitrates from HIV-1-infected cells. The results of this study provide new insights into HIV-1 neuropathogenesis aimed at the development of future HIV-1 therapeutics to eradicate viral reservoirs from the brain.

Cocaine increases human immunodeficiency virus type 1 neuroinvasion through remodeling brain microvascular endothelial cells.

Cocaine is a suspected cofactor in human immunodeficiency virus (HIV)-associated dementia but cocaine's effects are not clear. Herein the authors describe investigations of the mechanisms by which cocaine increases HIV-1 invasion through brain microvascular endothelial cells (BMVECs). Cocaine binds to a site on BMVECs, which is not a biogenic amine transporter, a binding site for estrogen, or a muscarinic receptor and for which benztropine and tamoxifen have the highest affinity. Cocaine treatment of BMVECs disrupts intercellular junctions and induces cell ruffling, which could account for their increased permeability and decreased electrical resistance. HIV-1 enters BMVECs by macropinocytosis and is transported to lysosomes and inactivated. In cocaine-treated BMVECs, the virus enters and persists in large cytoplasmic "lakes." Cocaine exposure of BMVECs up-regulates transcription of genes important in cytoskeleton organization, signal transduction, cell swelling, vesicular trafficking, and cell adhesion. The toxicity of cocaine for the blood-brain barrier may lead to increased virus neuroinvasion and neurovascular complications of cocaine abuse.

Human immunodeficiency virus type 1 enters brain microvascular endothelia by macropinocytosis dependent on lipid rafts and the mitogen-activated protein kinase signaling pathway.

Brain microvascular endothelial cells (BMVECs) present an incomplete barrier to human immunodeficiency virus type 1 (HIV-1) neuroinvasion. In order to clarify the mechanisms of HIV-1 invasion, we have examined HIV-1 uptake and transcellular penetration in an in vitro BMVEC model. No evidence of productive infection was observed by luciferase, PCR, and reverse transcriptase assays. Approximately 1% of viral RNA and 1% of infectious virus penetrated the BMVEC barrier without disruption of tight junctions. The virus upregulated ICAM-1 on plasma membranes and in cytoplasmic vesiculotubular structures. HIV-1 virions were entangled by microvilli and were taken into cytoplasmic vesicles through surface invaginations without fusion of the virus envelope with the plasma membrane. Subsequently, the cytoplasmic vesicles fused with lysosomes, the virions were lysed, and the vesicles diminished in size. Upon cell entry, HIV-1 colocalized with cholera toxin B, which targets lipid raft-associated GM1 ganglioside. Cholesterol-extracting agents, cyclodextrin and nystatin, and polyanion heparin significantly inhibited virus entry. Anti-CD4 had no effect and the chemokine AOP-RANTES had only a slight inhibitory effect on virus entry. HIV-1 activated the mitogen-activated protein kinase (MAPK) pathway, and inhibition of MAPK/Erk kinase inhibited virus entry. Entry was also blocked by dimethylamiloride, indicating that HIV-1 enters endothelial cells by macropinocytosis. Therefore, HIV-1 penetrates BMVECs in ICAM-1-lined macropinosomes by a mechanism involving lipid rafts, MAPK signaling, and glycosylaminoglycans, while CD4 and chemokine receptors play limited roles in this process.