ABSTRACT
Microscopy with direct analyte-probed nanoextraction coupled to nanospray ionization mass spectrometry (DAPNe-NSI-MS) is a direct extraction technique that extracts ultra-trace amounts of analyte. It has been proven to extract ink from documents with little to no physical or chemical footprint. In this study, DAPNe has been coupled to Raman spectroscopy, fluorescence microscopy, and NSI-MS to determine if an ink entry from a document was falsified. A handwritten number was altered using a different ink pen to test if the aforementioned techniques could discriminate the original number from the altered number, qualitatively and/or quantitatively. Chemical species from part of the original number, altered number, and a point at which both inks intersect were successfully differentiated by all techniques when using different pens. DAPNe coupled to fluorescence microscopy and Raman spectroscopy was not able to discriminate the forged ink entry when the exact same pen was used to modify the text (due to the same ink formula). However, DAPNe-NSI-MS successfully discerned that the pen was dispensed on different days by quantitating the oxidation process.
ABSTRACT
BACKGROUND: Simian immunodeficiency virus (SIV) infection and persistent CD8(+) lymphocyte depletion rapidly leads to encephalitis and neuronal injury. The objective of this study is to confirm that CD8 depletion alone does not induce brain lesions in the absence of SIV infection. METHODS: Four rhesus macaques were monitored by proton magnetic resonance spectroscopy ((1) H-MRS) before and biweekly after anti-CD8 antibody treatment for 8 weeks and compared with four SIV-infected animals. Post-mortem immunohistochemistry was performed on these eight animals and compared with six uninfected, non-CD8-depleted controls. RESULTS: CD8-depleted animals showed stable metabolite levels and revealed no neuronal injury, astrogliosis or microglial activation in contrast to SIV-infected animals. CONCLUSIONS: Alterations observed in MRS and lesions in this accelerated model of neuroAIDS result from unrestricted viral expansion in the setting of immunodeficiency rather than from CD8(+) lymphocyte depletion alone.
Subject(s)
Brain/pathology , CD8-Positive T-Lymphocytes/pathology , Lymphocyte Depletion/veterinary , Macaca mulatta , Simian Acquired Immunodeficiency Syndrome/pathology , Animals , Antibodies, Monoclonal/metabolism , Astrocytes/metabolism , Astrocytes/pathology , Astrocytes/virology , Brain/metabolism , Brain/virology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/virology , Disease Models, Animal , Encephalitis, Viral/immunology , Encephalitis, Viral/metabolism , Encephalitis, Viral/pathology , Encephalitis, Viral/veterinary , Flow Cytometry/veterinary , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry/veterinary , Magnetic Resonance Spectroscopy , Microfilament Proteins/metabolism , Microglia/metabolism , Microglia/pathology , Microglia/virology , Microtubule-Associated Proteins/metabolism , Monkey Diseases/immunology , Monkey Diseases/pathology , Monkey Diseases/virology , Neurons/metabolism , Neurons/pathology , Neurons/virology , Protons , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/physiology , Synaptophysin/metabolismABSTRACT
The macrophage is well established as a target of HIV and simian immunodeficiency virus (SIV) infection and a major contributor to the neuropathogenesis of AIDS. However, the identification of distinct subpopulations of monocyte/macrophages that carry virus to the brain and that sustain infection within the central nervous system (CNS) has not been examined. We demonstrate that the perivascular macrophage and not the parenchymal microglia is the primary cell productively infected by SIV. We further demonstrate that although productive viral infection of the CNS occurs early, thereafter it is not easily detectable until terminal AIDS. The biology of perivascular macrophages, including their rate of turnover and replacement by peripheral blood monocytes, may explain the timing of neuroinvasion, disappearance, and reappearance of virus in the CNS, and questions the ability of the brain to function as a reservoir for productive infection by HIV/SIV.
Subject(s)
Brain/virology , Macrophages/virology , Simian Acquired Immunodeficiency Syndrome/pathology , Simian Immunodeficiency Virus/physiology , AIDS Dementia Complex/pathology , AIDS Dementia Complex/virology , Animals , Antigens, Differentiation/analysis , Brain/pathology , Cerebrovascular Circulation , DNA, Viral/analysis , Humans , Immunophenotyping , Macaca mulatta , Macrophages/immunology , Macrophages/pathology , Microglia/pathology , Microglia/virology , Microscopy, Confocal , RNA, Viral/analysis , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/isolation & purification , Viral Proteins/analysisABSTRACT
RATIONALE: The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. EXPERIMENTAL APPROACH: LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). RESULTS: Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-ß,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPßS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,ß-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. CONCLUSIONS: Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.
Subject(s)
Neurons/drug effects , Neurons/physiology , Receptors, Purinergic/metabolism , Submucous Plexus/drug effects , Submucous Plexus/physiology , Adenosine Triphosphate/metabolism , Calcium/metabolism , Colectomy , Electric Stimulation , Excitatory Postsynaptic Potentials/physiology , Humans , Immunohistochemistry , Purinergic Agents/pharmacology , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Voltage-Sensitive Dye ImagingABSTRACT
We modified a combinatorial library display vector, pCOMB3, to provide a stable, easily manipulated, high-copy vector for the display of a random hexapeptide library. The propensity of the original phagemid to accumulate 800-1000-bp deletions in the region of the cloning site has been eliminated. Furthermore, the small 63-bp 'stuffer' at the cloning site was replaced with a 2114-bp DNA fragment from adenovirus 2. This produced a 5808-bp vector, that we have named pICD1LS, with the appropriate characteristics for single-peptide phage display. Libraries of greater than 10(6) molecules were produced with this vector.
Subject(s)
Bacteriophages/genetics , Genetic Vectors , Cloning, Molecular/methodsABSTRACT
Activation of endothelium is a critical step in leukocyte recruitment to the CNS and in development of neurological diseases, such as HIV-associated dementia. Due to limited availability of early disease course data, it is important to develop in vitro models of the blood-brain barrier (BBB) that can be used to address these early events. No such model of the BBB has been established for the macaque. Here, we characterize rhesus microvascular brain endothelial cells (MBEC), comparing them with rhesus umbilical vein endothelial cells (RUVEC), and discuss their suitability for future use in developing in vitro models of simian immunodeficiency virus (SIV) neuropathogenesis. We conclude that MBEC are distinct from RUVEC with respect to growth characteristics, culture requirements, morphology and expression of surface molecules important for leukocyte adhesion and immune activation.
Subject(s)
Brain/blood supply , Endothelium, Vascular/cytology , Umbilical Veins/cytology , Animals , Antigens, Differentiation/biosynthesis , Blood-Brain Barrier/immunology , Brain/cytology , Brain/immunology , Cell Adhesion Molecules/biosynthesis , Cell Division , Cell Separation , Cells, Cultured , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Histocompatibility Antigens Class II/biosynthesis , Immunohistochemistry , Immunophenotyping , Macaca mulatta , Microcirculation/cytology , Microcirculation/immunology , Microcirculation/metabolism , Receptors, Chemokine/biosynthesis , Umbilical Veins/immunology , Umbilical Veins/metabolismABSTRACT
Emerging data indicate that chemokine receptors on neurons and glia in the central nervous system (CNS) play a role in normal CNS development, intercellular communication, and the neuropathogenesis of AIDS. To further understand chemokine receptors in the brain and explore their potential role in HIV neuropathogenesis, particularly in pediatrics, we examined the regional and cellular distribution of CCR5 and CXCR4 in normal fetal, neonatal, and adult rhesus macaques. CCR5 and CXCR4 were detected by immunohistochemistry and immunofluorescence within the cytoplasm of subpopulations of neurons in the neocortex, hippocampus, basal nuclei, thalamus, brain stem, and cerebellum and by flow cytometry on the surface of neurons and glia. Interestingly, expression of CCR5 and CXCR4 increased significantly (p<0.05) from birth to 9 months of age. We further characterize this dynamic developmental pattern of CCR5 and CXCR4 expression in resident cells of the CNS.
Subject(s)
Brain Chemistry/immunology , Gene Expression Regulation, Developmental/immunology , Receptors, CCR5/genetics , Receptors, CXCR4/genetics , Acquired Immunodeficiency Syndrome/immunology , Age Factors , Animals , Animals, Newborn , Fetus , Flow Cytometry , Frontal Lobe/cytology , Frontal Lobe/embryology , Frontal Lobe/immunology , Immunohistochemistry , Macaca mulatta , Neuroglia/chemistry , Neuroglia/physiology , Neurons/chemistry , Neurons/physiology , RNA, Messenger/analysis , Receptors, CCR5/analysis , Receptors, CXCR4/analysis , Receptors, Cell Surface/genetics , Reverse Transcriptase Polymerase Chain Reaction , Simian Acquired Immunodeficiency Syndrome/immunologySubject(s)
Cell Movement , Central Nervous System/cytology , Leukocytes, Mononuclear/physiology , AIDS Dementia Complex/immunology , AIDS Dementia Complex/virology , Animals , Blood-Brain Barrier , Central Nervous System/immunology , Endothelium, Vascular/physiology , Endothelium, Vascular/virology , HIV/isolation & purification , HIV/physiology , HIV Infections/immunology , HIV Infections/virology , Humans , Lymphocyte Subsets/physiology , Lymphocyte Subsets/virology , Macrophages/physiology , Macrophages/virology , Microglia/physiology , Microglia/virology , Monocytes/physiology , Monocytes/virology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virologySubject(s)
Animal Feed , Drug Residues/analysis , Food Microbiology , Fungi/metabolism , Swine/metabolism , Animals , Anti-Bacterial Agents/analysis , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/pharmacology , Bacillus subtilis/drug effects , Drug Residues/pharmacology , Kidney/chemistry , Liver/chemistry , Muscles/chemistryABSTRACT
A simplified version of Piaget's sensorimotor levels was employed to teach a group of 25 extremely impaired individuals who live in a large residential facility. Throughout the facility, all available formal assessments using the Vineland Adaptive Behaviour Scales (VABS) were compared. Using this tool, an average of 13% of individuals from the common population pool increased their scores over a 6-year time period. All of the individuals who received training within the experimental group demonstrated increased scores. Scores increased such that an average gain of nearly 1 month in overall age equivalency per individual per year was realized. A matched group comparison, a prediction test for like sensorimotor skill attainment (the primary distinction of this curriculum methodology), and an historical review of subject skill training, all support the cognitively geared methodology as being primarily responsible for this accelerated progress.
Subject(s)
Education of Intellectually Disabled , Intellectual Disability/rehabilitation , Psychomotor Disorders/rehabilitation , Activities of Daily Living/psychology , Adult , Aged , Humans , Imitative Behavior , Intellectual Disability/diagnosis , Intellectual Disability/psychology , Male , Middle Aged , Neuropsychological Tests , Psychomotor Disorders/diagnosis , Psychomotor Disorders/psychology , Remedial Teaching , Residential Facilities , Treatment OutcomeABSTRACT
Hospitals recognize that attracting and retaining physicians to their medical staff is the single most important means of increasing utilization. Further, the competitive environment makes it imperative for most hospitals to seriously compete for physicians' allegiance. Hospitals often have marketable business expertise and also share many of the same business problems as the physicians who are on their staff. That means that hospitals might be able to diversify their product line and strengthen their physician ties at the same time. This twofold benefit is very attractive. Business software and hardware vendors are also aware of this potential. They have come forward with packages in the data processing area to help physician and hospital computers share information. Sharing information via computer can potentially increase billing accuracy and efficiency, among other things, for the participating physicians. There have been reports of success in this area with hints that this service is attracting physicians and retaining them as medical staff members. That is exciting to hospitals looking for another way to build and maintain their medical staff.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Attitude of Health Personnel , Commerce/statistics & numerical data , Hospitals , Marketing of Health Services , Medical Staff, Hospital/statistics & numerical data , Practice Management, Medical , Evaluation Studies as Topic , Humans , Interprofessional Relations , Medicine , Specialization , Surveys and Questionnaires , United StatesABSTRACT
Multiple sclerosis (MS), a putative autoimmune disease of unknown etiology, is characterized by CNS perivascular inflammation, foci of demyelination, and elevated intrathecal production of oligoclonal IgG's. T and B cells, macrophages, and microglia are all implicated in contributing to the initiation and perpetuation of the disease. In this brief review we discuss the possible role of T cells, B cells, macrophages, and microglia in contributing to the initiation and perpetuation of inflammation and demyelination in MS. Data from the rodent model of MS, experimental allergic encephalomyelitis (EAE) supporting a immunological basis for the pathology of MS is noted. This paper discusses recent data suggesting an interaction of the above-mentioned cells, as well as serum and CSF proteins including complement and anti-myelin/oligodendrocyte antibodies, in the pathogenesis of MS and EAE. Additionally, this review describes each cell type including the clinical and experimental evidence for their contribution to the immunologically mediated pathology of MS. Following the description of the role of individual cells, there is consideration of: the possible interaction of cells with the blood brain barrier (BBB) under normal and pathologic inflammatory conditions; the traffic of cells into the CNS in inflammation; and the role of antigen presentation within the CNS in the initiation, and perpetuation, of the CNS immune response. Finally, the review suggests a role for T cells in the initiation, amplification, and possibly the termination of CNS inflammatory events with particular attention paid to the pattern of T cell activation and T cell cytokine production.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Multiple Sclerosis/immunology , Animals , Cell Communication , Cytokines/physiology , Disease Models, Animal , Humans , Immunoglobulin G/cerebrospinal fluid , Microglia/immunology , T-Lymphocytes/immunologyABSTRACT
The role of T cell activation associated adhesion molecules on lymphocyte traffic and the initiation of inflammation has received considerable attention. This study, using a new monoclonal antibody (mAb) TLD-3A12, describes the distribution of PECAM-1 (CD31), an Ig supergene family adhesion molecule thought to be important in leukocyte transmigration during inflammation, in rat lymphoid organs and spinal cord. PECAM expression within the CNS is confined to endothelial cells of the blood brain barrier (BBB). Induction of inflammation within the CNS using the adoptive transfer of myelin reactive CD4+ T cells results in the de novo expression of immune adhesion and accessory molecules in the spinal cord, while the level of PECAM appeared only mildly increased. The distribution of PECAM on CNS endothelial cells became more diffuse during EAE induction, possibly the result of endothelial cell activation. In vitro studies demonstrate a partial inhibition of antigen-specific CD4+ T cell proliferation following anti-PECAM mAb treatment. Treatment of Lewis rats with TLD-3A12 antibody prior to T cell injection and throughout EAE induction does not result in a delay in the onset of clinical signs or weight loss, nor does it decrease the incidence and severity of disease. These data suggest that the expression of PECAM by CNS endothelial cells is not a requirement for the initiation of inflammation and clinical signs of EAE following the adoptive transfer of encephalitogenic lymphocytes. Thus, cells requiring PECAM-1 to migrate and perform their pathogenic functions are not critical to the development of rat EAE.
Subject(s)
Central Nervous System/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Lymphatic System/immunology , Nerve Tissue Proteins/immunology , Platelet Endothelial Cell Adhesion Molecule-1/immunology , Animals , Antibodies, Monoclonal , Cell Division/immunology , Epitopes , Female , Histocompatibility Antigens Class II/immunology , Immunohistochemistry , Rats , Rats, Inbred Lew , Receptors, Antigen, T-Cell/immunology , Reference Values , Staining and LabelingABSTRACT
Experimental allergic encephalomyelitis (EAE) is a frequently employed animal model of the human disease multiple sclerosis. EAE can be induced by adoptive transfer of CD4+ T cells that are specific for central nervous system (CNS) antigens, typically myelin proteins. Although the pathogenic mechanism or mechanisms responsible for the clinical signs and histological changes in EAE and multiple sclerosis are not fully defined, the entry of T lymphocytes and antigen recognition within the CNS are required. The present study describes the participation of a novel cell surface molecule with properties suggesting a role in cell-cell adhesion or co-stimulation, or both, in the development of EAE in the rat. The molecule is defined by the unique monoclonal antibody (mAb) TLD-4A2. The TLD-4A2 antigen is present on resting and activated T lymphocytes, activated CNS endothelial cells, and microglia. The antigen is normally distributed in many tissues including lymph node, thymus, and spleen, as well as in the inflamed CNS. Both its pattern of tissue distribution and immunoprecipitation and immunoblotting studies suggest that the TLD-4A2 antigen is a novel molecule. Treatment of rats with the purified 4A2 mAb resulted in the inhibition of the clinical signs of EAE and also decreased the number T cells and macrophages accumulating in the CNS parenchyma. TLD-4A2 antibody did not seem to directly interfere with T cell viability in vivo, as demonstrated by the ability to recover and stimulate CD4+ encephalitogenic T cells from cervical lymph nodes of 4A2-treated animals. In vitro, the antibody partially blocked T cell proliferation assays. These data suggest that the TLD-4A2 mAb recognizes a novel molecule expressed on lymphocytes, endothelial cells, and macrophages that may play a role in hematogenous cell traffic and the initiation of CNS inflammation.
Subject(s)
Antibodies, Viral/immunology , Antigens, Viral/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Endothelium/immunology , Lymphocytes/immunology , Microglia/immunology , Animals , Blotting, Western , Female , Immunohistochemistry , Rats , Rats, Inbred LewABSTRACT
A capillary gel electrophoresis (CGE) method is described for detection of the formation of circular DNA ligation products as an aid in the prediction of ligated DNA competent cell transformation efficiency. The separation is based upon the differences in the relative migrations of linear and circular DNA molecules of the same size. In CGE, circular ligation products are shifted significantly from linear DNA fragments of comparable size (to 40-42 min from 32-33 min migration time) in the presence of an intercalating dye. CGE separation and detection of circularized DNA can be correlated with transformation efficiencies of > 10(6) colony-forming units (CFU, colonies/micrograms/ml) or the high efficiency desired for phagemid display and cell expression libraries. CGE has several advantages over slab gel electrophoresis: (i) only a minute quantity (approximately 250 CFU or 0.02%) of the total library is sacrificed for analysis, (ii) verification of the circularized ligation products is easier by CGE, and (iii) CGE analysis of ligation success can be accomplished in less than 2 h, prior to transforming competent cells.
Subject(s)
DNA, Circular/analysis , Electrophoresis, Polyacrylamide Gel/methods , Bacteriophages/genetics , DNA/analysis , DNA, Superhelical/analysis , Electrophoresis, Agar Gel/methods , Genetic Vectors , Intercalating Agents , Ligases , Transformation, GeneticABSTRACT
The influence of host cytokine response on viral load, disease progression, and neurologic lesions was investigated in the simian immunodeficiency virus (SIV)-infected macaque model of AIDS. Cytokine gene expression (interleukin-1beta [IL-1beta], IL-2, IL-6, IL-10, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and viral loads were evaluated by semiquantitative reverse transcription-PCR in lymph nodes of 5 control animals and 28 animals infected with SIVmac251 at the terminal stages of AIDS. Infected animals showed higher expression of IFN-gamma, IL-6, and IL-10 mRNAs compared with controls. Levels of all cytokines were comparable between animals with rapid (survival, <200 days) or slow/normal (survival, >200 days) disease progression. However, among rapid progressors, the eight animals with SIV encephalitis had a unique cytokine profile (increased IL-2, IL-6, and IFN-gamma) that was associated with higher viral loads. These observations provide evidence that host cytokine responses may influence SIV neuropathogenesis independent of disease progression.
Subject(s)
Cytokines/genetics , Encephalitis, Viral/immunology , Gene Expression Profiling , Simian Acquired Immunodeficiency Syndrome/immunology , Animals , Central Nervous System/pathology , Disease Progression , Encephalitis, Viral/pathology , Encephalitis, Viral/physiopathology , Encephalitis, Viral/virology , Lymphoid Tissue/immunology , Macaca mulatta , Simian Acquired Immunodeficiency Syndrome/pathology , Simian Acquired Immunodeficiency Syndrome/physiopathology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/immunology , Time Factors , Viral LoadABSTRACT
Antigen presentation by endogenous glial cells is postulated to regulate reactivity of immune cells that gain entry into the CNS. We have previously observed, using a mixed lymphocyte reaction (MLR) system, that adult human-derived microglia can function as antigen-presenting cells (APC) for immediately ex vivo CD4+ T cells in a primary MLR (1 degree MLR) whereas astrocytes could not. We have now found that fetal human astrocytes can support CD4+ T cell proliferation in the presence of exogenous human recombinant (r) IL-2, and that astrocytes can support the continued proliferation of CD4+ T cells previously sensitized to sister astrocyte cultures in a secondary MLR. Additionally, adult human microglia, seeded into the nonpriming astrocyte: CD4+ T cell cocultures at non-T cell-stimulatory concentrations of 1000-5000 microglial cells per well, could reverse the inability of astrocytes to present antigen in the primary MLR. To examine the cellular basis for the inability of human astrocytes to function as APCs in the primary MLR, astrocyte- and microglial-enriched populations were established from human embryonic and adult brain, respectively, and analyzed for their ability to synthesize cytokines potentially relevant as accessory signals in the MLR. Microglia had transcript as determined by the reverse transcriptase-polymerase chain reaction (RT-PCR) and protein as determined by bioassay for IL-1 alpha, IL-6, and TNF alpha. Human fetal astrocytes had transcript for IL-6 but not for IL-1 alpha or TNF alpha under basal culture conditions and following IFN gamma stimulation. The addition of human rIL-1 from 1-50 U/ml could reverse the inability of astrocytes to present antigen in the primary MLR. These studies demonstrate that although in vitro highly enriched cultures of astrocytes absent of microglia cannot present antigen to immediately ex vivo blood-derived CD4+ T cells in the MLR, in situ, with the cooperative help of microglia-derived cytokines or accessory surface molecules, astrocytes may function as central nervous system APCs.
Subject(s)
Antigen-Presenting Cells/immunology , Astrocytes/immunology , CD4-Positive T-Lymphocytes/immunology , Interleukin-1/metabolism , Microglia/immunology , Adult , Astrocytes/cytology , Astrocytes/metabolism , Base Sequence , Cell Division , Fetus/immunology , Histocompatibility Antigens Class II/immunology , Humans , Interleukin-1/genetics , Interleukin-1/immunology , Interleukin-6/genetics , Interleukin-6/metabolism , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , Microglia/cytology , Microglia/metabolism , Middle Aged , Molecular Sequence Data , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Although the mechanisms of human immunodeficiency virus (HIV) neuroinvasion, neuronal injury, and subsequent development of HIV-1-associated AIDS dementia complex are not fully understood, a correlation between monocyte/macrophage infiltrates in the brain and neurological disease exists. In light of the many potential roles that chemokines and chemokine receptors may play in HIV neuropathogenesis, we sought to describe their pattern of expression in the SIV-infected rhesus macaque model of HIV encephalitis. We previously demonstrated elevated expression of the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, and interferon-inducible protein (IP)-10 in brain of macaque monkeys with SIV encephalitis. In this study, we demonstrate that the corresponding chemokine receptors CCR3, CCR5, CXCR3, and CXCR4 are expressed in perivascular infiltrates in these same tissues. In addition, we detected CCR3, CCR5, and CXCR4 on subpopulations of large hippocampal and neocortical pyramidal neurons and on glial cells in both normal and encephalitic brain. These findings suggest that multiple chemokines and their receptors contribute to monocyte and lymphocyte recruitment to the brain in SIV encephalitis. Furthermore, the expression of known HIV/SIV co-receptors on neurons suggests a possible mechanism whereby HIV or SIV can directly interact with these cells, disrupting their normal physiological function and contributing to the pathogenesis of AIDS dementia complex.
Subject(s)
Brain/metabolism , Encephalitis, Viral/metabolism , Receptors, Chemokine/metabolism , Simian Acquired Immunodeficiency Syndrome/metabolism , Simian Immunodeficiency Virus , Animals , Brain/pathology , Brain/virology , Chemokines/metabolism , Encephalitis, Viral/etiology , Encephalitis, Viral/pathology , Immunohistochemistry , Macaca mulatta , Simian Acquired Immunodeficiency Syndrome/etiology , Simian Acquired Immunodeficiency Syndrome/pathologyABSTRACT
Chemokines are believed to play a role in the neuropathogenesis of AIDS through their recruitment of neurotoxin-secreting, virally infected leukocytes into the CNS. Levels of chemokines are elevated in brains of patients and macaques with HIV/SIV-induced encephalitis. The chemokine receptors CCR3, CCR5, and CXCR4 are found on subpopulations of neurons in the cortex of human and macaque brain. We have developed an in vitro system using both macaque and human fetal neurons and astrocytes to further investigate the roles of these receptors in neuronal response to inflammation. Here we report the presence of functional HIV/SIV coreceptors CCR3, CCR5, and CXCR4 on fetal human and macaque neurons and CCR5 and CXCR4 on astrocytes immediately ex vivo and after several weeks in culture. Confocal imaging of immunostained neurons demonstrated different patterns of distribution for these receptors, which may have functional implications. Chemokine receptors were shown to respond to their appropriate chemokine ligands with increases in intracellular calcium that, in the case of neurons, required predepolarization with KCl. These responses were blocked by neutralizing chemokine receptor in mAbs. Pretreatment of neural cells with pertussis toxin abolished responses to stromal-derived factor-1alpha, macrophage inflammatory protein-1beta, and RANTES, indicating coupling of CCR5 and CXCR4 to a Gialpha protein, as in leukocytes. Cultured macaque neurons demonstrated calcium flux response to treatment with recombinant SIVmac239 envelope protein, suggesting a mechanism by which viral envelope could affect neuronal function in SIV infection. The presence of functional chemokine receptors on neurons and astrocytes suggests that chemokines could serve to link inflammatory and neuronal responses.