Immunoglobulin heavy chain associated protein in multiple sclerosis cerebrospinal fluid.

Immunoglobulins G, A, and M (IgG, IgA and IgM) were isolated from multiple sclerosis (MS) cerebrospinal fluid (CSF) and sera by Protein A-Sepharose (PAS) affinity chromatography or using solid-phase immunoabsorbents. An isoelectric point heterogeneous protein with apparent mol. wt of 74,000-80,000 was seen consistently when CSF Igs were immunoaffinity purified but was never seen when PAS was used for Ig purification. Control experiments exclude the binding of this protein non-specifically either to Sepharose or to Igs or to other CSF proteins. Analysis of purified Igs under non-reducing conditions leads to some reduction in staining pattern indicating that a portion of the protein may be disulfide linked to itself or to other CSF proteins. Immunoblot analysis of CSF Igs separated on 2-DE gels is consistent with a portion of the total CSF Ig protein existing as "free" heavy (H)-chains. PAS requires dimeric Fc fragment of Ig for binding; the differences in 2-DE gels of PAS and immunoaffinity purified Igs may be due to interaction of the 74,000-80,000 protein with "free" H-chain, which nevertheless, still leaves this complex available for binding by anti-H chain antisera. It has been previously suggested that the cytotoxicity of "free" H-chains is abrogated in the absence of the complementary L-chains by H-chain binding proteins; the protein reported here has features similar to these proteins reported previously and may be involved in some way in regulating Ig production in the MS central nervous system.

Cerebrospinal fluid p24 antigen levels and intrathecal immunoglobulin G synthesis are associated with cognitive disease severity in HIV-1.

OBJECTIVE: To test the hypothesis that selected cerebrospinal fluid (CSF) markers [intrathecal immunoglobulin G (IgG) synthesis rate, oligoclonal IgG bands, and p24 antigen levels] are associated with the presence and severity of clinical HIV-1 neurologic disease. DESIGN AND METHODS: CSF and blood parameters from 142 HIV-seropositive subjects from the baseline examination of a longitudinal study were measured and analyzed in relationship with clinically derived cognitive impairment groups (none, mild, moderate) and with other neurologic and clinical classification groups. Subjects with opportunistic infections, lymphomas or neurosyphilis were excluded. RESULTS: The mean intrathecal IgG synthesis rate and mean CSF p24 antigen levels both differed significantly among cognitive impairment groups; more impairment was associated with a higher rate or level. Mean CSF p24 antigen levels were significantly higher in HIV-1-seropositive subjects with any HIV-1 neurologic disease than in subjects without neurologic disease. In contrast, there were no significant differences among seropositive groups in any CSF parameter when stratified by systemic disease classification (asymptomatic HIV-seropositives, AIDS-related complex, or AIDS), independent of neurologic status. CONCLUSION: We conclude that there may be a relationship between the severity of HIV cognitive disease and increasing levels of intrathecal IgG synthesis and CSF p24 antigen levels.

HIV-1 in postmortem brain tissue from patients with AIDS: a comparison of different detection techniques.

OBJECTIVE: The presence of HIV-1 in postmortem brain tissue from 31 patients with AIDS and 12 HIV-1-negative controls was investigated. DESIGN: Most laboratories have access to the methods used. We readily applied in situ hybridization and immunohistochemistry to archival formalin-fixed paraffin-embedded (FFPE) brain specimens. METHODS: The techniques used to detect HIV-1 were explant culture, in situ hybridization with 35S-labeled polymerase (pol) gene riboprobes and immunohistochemistry with monoclonal antibody to gp41. RESULTS: HIV-1 was isolated from explant cultures in 13 out of 20 (65%) patients, whereas HIV-1-infected cells were detected in FFPE brain tissue from nine out of 26 (35%) patients examined by in situ hybridization and in seven out of 26 (27%) patients examined by immunohistochemistry. CONCLUSIONS: Although the isolation technique was the most sensitive of the three techniques tested, infected cells may be identified with in situ hybridization in conjunction with immunohistochemistry.

Intra-blood-brain barrier IgG synthesis in cerebral cysticercosis.

Patients with central nervous system cysticercosis show elevated binding of cerebrospinal fluid (CSF) IgG to homogenized cysticercus. To determine whether any of the CSF IgG was the result of de novo intra-blood-brain barrier (BBB) synthesis, CSF and serum samples from six patients were examined for elevated rate of synthesis and oligoclonal bands. Five of the six patients had increased intra-BBB IgG synthesis rate and four of these patients also had oligoclonal IgG bands present in the CSF that were absent in the serum. These results demonstrate intra-BBB IgG synthesis similar to that observed in other infectious and inflammatory diseases of the central nervous system.

Quantitative multiple sclerosis plaque assessment with magnetic resonance imaging. Its correlation with clinical parameters, evoked potentials, and intra-blood-brain barrier IgG synthesis.

Magnetic resonance imaging (MRI) of the cerebrum, cerebellum, brain stem, and upper cervical cord was performed in 62 individuals with clinically definite chronic, progressive multiple sclerosis (MS). The total area of MRI-demonstrated lesions was measured from film enlargements for each region using an interactive image analysis system. While the MRI was abnormal in 60 (97%) of 62 patients, the visual-evoked potentials in 51 (85%) of 60 patients, the brain stem auditory-evoked potentials (BAEPs) in 24 (46%) of 52 patients, and the somatosensory-evoked potentials (SSEPs) in 45 (89%) of 54 patients, an abnormal intra-blood-brain barrier (BBB) IgG synthesis rate, IgG oligoclonal bands, or both were found in all 62 patients. The total area of MRI abnormality in the cerebrum was significantly correlated only with the intra-BBB IgG synthesis rate, abnormal visual-evoked potentials, impaired performance on the Symbol Digit Modalities Test (SDMT), and one test of standing duration in the quantitative examination of neurologic function (QENF). The brain stem lesion area correlated with the Kurtzke expanded disability status scale and brain stem functional systems score, the ambulation index, abnormal BAEPs, and impaired performance on the SDMT as well as multiple tests of upper and lower extremity function in the QENF. The cerebellar lesion area correlated with impaired performance on the SDMT and primarily upper extremity testing in the QENF.(ABSTRACT TRUNCATED AT 250 WORDS)

HIV-1 propagates in human neuroblastoma cells.

A major question in the pathogenesis of AIDS encephalopathy and dementia is whether HIV-1 directly infects cells of the central nervous system (CNS). The propagation of HIV was attempted in six cell lines: three related and three unrelated to the nervous system. HIV was able to propagate in two human neuroblastoma cell lines and a lymphocytic cell line control but did not result in infections of African green monkey kidney cells, human cervix carcinoma cells, and one human brain astrocytoma cell line. Neuroblastoma cell lines infected with HIV showed peaks of reverse transcriptase activity at 10-14 days postinfection. After prolonged growth in cell cultures, one of the neuroblastoma cell lines showed multiphasic virus production, additional high peaks of reverse transcriptase activity, 20-fold greater than the first, lasting from 36 to 74 days and 110 to 140 days postinfection. The presence of HIV was confirmed by p24 antigen capture. The neuroblastoma cell lines had weak but detectable levels of CD4 immunoreactivity by immunoperoxidase and flow immunocytometric analysis. Although no T4-specific RNA sequences were detected by hybridization of Northern blots of total and poly A-selected RNA extracted from the two neuroblastoma cell lines by using a T4 specific complimentary DNA probe, monoclonal antibodies to the CD4 receptor blocked HIV infection in both neuroblastoma cell lines. Thus, the infection of neuroblastoma cells by HIV occurs in part by a CD4-dependent mechanism. Passaging the neuroblastoma cell lines weekly and bimonthly resulted in similar cell cycle-DNA content patterns for the more permissive cell line and with significant numbers of cells in the S phase. HIV-infected neuroblastoma cell lines provide an in vitro model for the evaluation of virus-host cell interactions and may be useful in addressing the issue of the persistence of HIV in the human CNS.

Preliminary laboratory studies of inactivation of HIV-1 in needles and syringes containing infected blood using undiluted household bleach.

To evaluate the effectiveness of bleach disinfection of injection equipment, we tested HIV-1 inactivation by household bleach in needles and syringes. We obtained blood from HIV-1 infected injecting drug users (IDUs), placed small aliquots in needles and syringes. Blood with and without anticoagulant was incubated at room temperature for 3, 6, 18, and 24 h, and some needles and syringes from each condition were exposed to undiluted bleach for 15 and 30 s. The needles and syringes were then rinsed and the rinses were used to inoculate peripheral blood mononuclear cells (PBMNCs). HIV-1 replication was monitored using p24 enzyme linked immunosorbent assay (ELISA). We describe results that HIV-1 is inactivated in clotted and unclotted blood allowed to stand at room temperature for 3, 6, 18, and 24 h in needles and syringes using undiluted household bleach at 30 s of exposure time. These results are consistent with earlier findings that micropellets of HIV-1 were inactivated by bleach under similar conditions of exposure to bleach; 10% bleach was not effective at an exposure time of 30 s and undiluted bleach was not effective at an exposure time of 15 s to inactivate HIV-1 in clotted blood. Bleach concentration and exposure time are critical and HIV disinfection may not occur with inadequate exposure to bleach HIV.

Compliance to bleach disinfection protocols among injecting drug users in Miami.

Bleach cleansing of injection equipment has been recommended to reduce the risk of human immunodeficiency virus (HIV) transmission associated with the reuse of injection equipment by injecting drug users (IDUs). We evaluated the recall and performance of the most commonly recommended bleach cleansing procedure of two complete fillings of the syringe with bleach, followed by two complete fillings with rinse water, and not putting used bleach and water back into source containers. IDUs were taught this procedure on enrollment in an HIV prevention demonstration project in Dade County, Florida. During follow-up session 6-12 months after initial training, the knowledge and ability of IDUs to perform bleach cleansing were assessed by trained observers using a standardized method. In 1988-90, we assessed the knowledge and ability of 450 IDUs to perform the bleach cleansing procedure taught at enrollment. More than 90% of IDUs assessed performed the basic steps. However, only 43.1% completely filled the syringe with bleach and only 35.8% completely filled the syringe with bleach at least twice. Substantial proportions of IDUs did not perform all the steps of the previously taught bleach cleansing procedure. Compliance decreased as the number of steps required was increased. This limited compliance may make bleach cleansing less effective and suggests that some IDUs may fail to adequately disinfect injection equipment and therefore sterile needles and syringes are safer than bleach-cleansed ones. Compliance testing can help assess the effectiveness of HIV prevention programs.

Early penetration of the blood-brain-barrier by HIV.

CNS dysfunction occurs frequently in patients with HIV infection. To better define the role of HIV in the pathogenesis of neurologic dysfunction, HIV isolation and antibody studies were investigated from the CSF in 52 seropositive patients, 29 with and 23 without neurologic signs and symptoms, in various stages of disease development ranging from asymptomatic to ARC to AIDS. HIV was recovered from the CSF of 5 of 29 (17%) patients with neurologic signs and symptoms and 5 of 23 (22%) neurologically asymptomatic patients. All patients with positive CSF HIV cultures had antibodies directed against HIV p24 and gp41 in serum and CSF by Western blot analysis and elevated intra-blood-brain-barrier total IgG and HIV-specific IgG synthesis rates. The frequency of CSF HIV isolation from the group of seropositive patients without AIDS, 9 of 32 (28%), exceeded that of patients with AIDS, 1 of 20 (5%) (p less than 0.05). These findings indicate that HIV infects the CNS early in the course of viral infection and prior to the development of HIV-associated neurologic abnormalities.

Copolymer 1 as therapy for multiple sclerosis: the cons.

One of the hallmarks of multiple sclerosis (MS) is intra-blood-brain-barrier (BBB) IgG synthesis, a byproduct of plasma cells located in and around active inflammatory demyelinating plaques. The rate of IgG synthesis can be measured by plugging CSF and blood IgG and albumin concentrations into our equation. When done in conjunction with CSF and serum analyses for IgG oligoclonal bands, 99% of definite MS patients demonstrate intra-BBB IgG synthesis. At autopsy the pathologic criterion of an inactive plaque of demyelination is absence of inflammatory cells. Hence, we propose that modulation downward or eradication of intra-BBB IgG synthesis (ie, a manifestation of reduced white matter inflammation) in a living patient is a reasonable therapeutic criterion and goal of MS therapy. In a preliminary trial of five severely disabled MS patients, we evaluated the effects of copolymer 1 (COP-1) in daily intramuscular doses of 20 mg (2 patients) and twice daily subcutaneous doses of 15 mg (3 patients) on clinical parameters and on intra-BBB IgG synthesis over a 2-month study period. The results of this trial showed no beneficial effect on neurologic function or on inflammatory demyelination, as assessed by monitoring of intra-BBB IgG synthesis.

Subacute sclerosing panencephalitis: measles virus matrix protein nucleic acid sequences detected by in situ hybridization.

Subacute sclerosing panencephalitis (SSPE) is characterized by a hyperimmune state toward the polypeptides of measles virus except the matrix (M) protein. Using cloned (3H)-labeled complementary DNA probes for in situ hybridization, we found the M protein and nucleocapsid (NP) protein nucleotide sequences in glial cells and neurons of cryostat sections from two SSPE brains. In one SSPE brain, M protein was lacking, but the other measles polypeptides were present. IgG and IgM antibodies eluted from that brain lacked antibodies to M protein, but antibodies to other measles polypeptides were present. In SSPE brain, the viral M-protein defect is not a deletion of the M gene, but rather a block in gene expression.

Expression of HIV-1 and interleukin-6 in lumbosacral dorsal root ganglia of patients with AIDS.

We examined the immunopathology and the expression of human immunodeficiency virus type 1 (HIV-1) in lumbosacral dorsal root ganglia (DRGs) from 16 patients with acquired immunodeficiency syndrome (AIDS) and 10 HIV-1-seronegative controls. Using in situ hybridization, we detected HIV-1 RNA in a few perivascular cells in DRGs from five of 16 AIDS patients (31%). In addition, using polymerase chain reaction, we detected HIV-1 DNA more frequently in DRGs from four of five AIDS patients (80%) examined. We detected interleukin-6 (IL-6) immunoreactivity in endothelial cells in DRGs from seven of 16 AIDS patients (44%) but from none of 10 HIV-1-seronegative controls (0%). We found more nodules of Nageotte, CD8+ T lymphocytes, and intercellular adhesion molecule-1 (ICAM-1)-positive endothelial cells and mononuclear cells in DRGs from AIDS patients than in DRGs from controls. Increased numbers of nodules of Nageotte in DRGs of AIDS patients were associated with detection of HIV-1 RNA by in situ hybridization and detection of IL-6 by immunohistochemistry. We conclude that low levels of replication of HIV-1, through cytotoxic T lymphocytes or expression of cytokines, may play a role in the subclinical degeneration of sensory neurons frequently observed in DRGs of AIDS patients.

Analytic approaches to differential gene expression in AIDS versus control brains.

We previously showed that specific strains of human immunodeficiency virus (HIV)-1 infect the brain and contribute to Neuropathology, Cognitive Distress, and Neuropsychiatric Disease. To study further brain disease that results from HIV-1 infection, we commenced analysis of changes in gene expression in brain. We analyzed RNA purified from Frontal Cortex of 5 HIV-1 infected and 4 HIV-1 negative control subjects RNA was amplified and Affymetrix technology was used to analyze gene expression using the 12,585 gene Affymetrix Human Genome U95A chip. The expressed genes showed highly significant Pearsons correlations with each other within the two groups. Expression intensities were transferred to Microsoft Excel and Spotfire was used to analyze the results. Twenty-group K-means cluster analysis was done for HIV+ and HIV- subjects. Genes that were expressed in the same cluster numbers in the two groups were removed from further analysis. Analysis of Gene expression in the top 13 HIV+ clusters showed expression in the 40 gene categories designated in our prior studies. Genes from several categories occurred in more than one K-means cluster. Genes identified in these lists included several genes that have been previously studied: MBP, Myelin-PLP, NMDA receptor, MAG, astrocytic protein, Notch 3, APP, Senescence, proteasome, Ferritin, signaling, cell cycle, iNOS, Chemokine, splicing, synapse, protein tags, and ribosomal proteins. The first (primary significant) axis of both Principal Component Analyses ordered the genes in the same patient groups as the K-means cluster analysis for the respective patient groups. PCA was thus not more informative than K-Means cluster analysis. Ratios of HIV+ to HIV- intensities were calculated for all the averaged gene expression intensities. The ratio range was 0.14 to 9.26. The genes at the extremes (ad extrema) did not correspond to the gene order by K-means clustering (or PCA). The genes in the top 13 K-means clusters showed low-level changes by expression ratio. Genes ad extrema by ratio were in clusters with very large memberships. Mann-Whitney analysis confirmed expression ratio results. Several inferences result from our preliminary study. First, study design will be different in future studies involving additional replicates. Second, ratios inform us of the extent of changes in gene expression quantitatively. Third, Cluster methodology provides us with more subtle information, how bunches (clusters) of genes behave in terms of their centroids (attractors). Fourth, genes that change extensively by ratio tend to be in the larger k-Means clusters. We conclude that ranking gene expression with the use of expression ratio or by K-means clustering, yield different representations of the data.

Isoelectric point restriction of cerebrospinal fluid and serum IgG antibodies to measles virus polypeptides in multiple sclerosis.

Thirty consecutive isoelectric point (pI)-discrete IgG fractions were isolated from multiple sclerosis (MS) cerebrospinal fluid (CSF) and used to immune precipitate measles virus (MV) polypeptides. Most basic fractions were enriched in activity against nucleocapsid protein (NP), and to a lesser extent against hemagglutinin (H) protein; intermediate fractions were enriched in activity against H and fusion (F) proteins; and more anodic pI fractions were almost exclusively enriched in activity against the large (L) protein of MV. In MS there are marked differences between CSF and autologous serum in regard to antibody activity to MV. In contrast, there were similar profiles of antibody response to MV proteins in SSPE CSF and serum.

The long march of the cerebrospinal fluid profile indicative of clinical definite multiple sclerosis; and still marching.

Much progress has been made, especially in the last two decades, in laboratory aids to diagnosis and to follow the course of patients with multiple sclerosis (MS). The cerebrospinal fluid (CSF) profile indicative of MS, though not pathognomonic of MS, is present in almost every case of clinical definite MS in a chronic progressive phase (probably also true for early MS). The cardinal aspect of the profile is intra-blood-brain barrier (BBB) IgG synthesis which can be qualitatively detected by determining unique CSF oligoclonal IgG bands and quantitated by rate formula, mg/day. We believe that intra-BBB IgG synthesis is caused by a persistent antigen, most likely a virus, possibly measles. A number of issues about the profile are proposed and opportunities are presented to resolve them.

Cocaine abuse and HIV-1 infection: epidemiology and neuropathogenesis.

The epidemiology of cocaine abuse and potential relationships of cocaine withdrawal to human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are discussed. Neuroendocrinological changes in HIV-1 infection of the central nervous system (CNS) are discussed with the relevant impact of cocaine abuse. HIV-1 load in the brain tissue of infected substance users is described along with possible associations with neuropathology and HAD. Finally, the molecular epidemiology and sequence heterogeneity of HIV-1 and their implications for neuropathogenesis are summarized. The complex context of addressing cocaine abuse in the setting of HIV-1 infection appears more tractable when decomposed into its components.

Autologous and allogeneic antibody responses to canine distemper virus isolates from dogs with chronic neurological diseases.

We describe the antibody responses to three strains of canine distemper virus (CDV) isolated from dogs with chronic neurological disease in the Los Angeles area using the naturally occurring sera and cerebrospinal fluids (CSFs) of these animals as probes for comparison. CDV/CDE-2 was derived from a dog with chronic distemper encephalitis, and CDV/ODE-8 and CDV/ODE-10 were derived from dogs with old dog encephalitis. Sera and CSFs were used in autologous (same dog) and allogeneic (different dog) combinations to immune precipitate the [35S]-methionine-labelled H, P, NP, F1, and M polypeptides of the virus-infected cell cultures. The polypeptides were separated by SDS-PAGE and detected by fluorography. There was decreased recognition by the CSF and sera of the polypeptides of the viral isolates in several autologous as well as allogeneic combinations. It is concluded that the immune responses to the CDV strains are not identical, and it is likely that viral mutations occurred after the animals were infected. Some mutations may have contributed to the pathogenesis of distemper encephalitis in these animals and some may have occurred during subsequent passage of the viruses in cell culture. This may explain the decreased recognition of the polypeptides of the viral isolates by the CSF and sera.

Independent evolution of HIV type 1 in different brain regions.

HIV-1-associated brain pathology exhibits regional variability and we therefore studied the genetic differences in the V1-V5 domains of the HIV env gene in up to four regions of brain (frontal lobe, basal ganglia, medial temporal lobe, and nonmedial temporal lobe) from three patients. We found that in each separate brain region HIV-1 forms different quasispecies and that there is little gene flow among these regions. In further support of brain region-specific evolution of HIV-1, we analyzed amino acid signatures in these clones. In addition to known amino acid signatures associated with macrophage tropism and the lack of syncytium formation, we found 15 majority amino acid signature patterns from the V1-V5 env sequences associated with the neuroanatomical regions analyzed from the three individuals. Furthermore, on average, intrabrain genetic distances for the HIV-1 env were estimated to be much smaller than genetic distances between brain regions. Specific strains of HIV-1 may be neurotropic or neuroinvasive (replication preference in brain tissue) and may contribute to pathology, cognitive loss, and neuropsychiatric disease.

Aging and neuro-AIDS conditions and the changing spectrum of HIV-1-associated morbidity and mortality.

Older individuals (>50 years of age) now comprise over 11% of patients with AIDS in the United States. This percentage is expected to continue to grow, due both to the improved longevity of patients prescribed highly active antiretroviral therapy (HAART) and to new infections among older individuals. This review focuses on the neuropsychiatric and neurological conditions that are most likely to be affected by advancing age-HIV-1-associated cognitive-motor disorder, peripheral neuropathy, progressive multifocal leukoencephalopathy, primary CNS lymphoma, and risk for cerebrovascular accident. Age associations with incidence of these disorders and with treatment foci are specified. Implications for future changes in management are discussed.

Bone marrow examination in patients with AIDS and AIDS-related complex (ARC). Morphologic and in situ hybridization studies.

Bone marrow examinations were performed on 20 patients with acquired immune deficiency syndrome (AIDS) and 39 with AIDS-related complex (ARC). Fever of unknown origin and thrombocytopenia were common in ARC, but anemia and leukopenia were most frequent in AIDS. Changes in stromal cells and perivascular cuffing of plasma cells were found significantly more often in patients with AIDS than in those with ARC. Malignancies were common in both groups. Human immunodeficiency virus (HIV) nucleic acids were detected with the use of a 3H-labeled cDNA probe with an in situ hybridization method in 11 bone marrow samples (three ARC and eight AIDS). Most commonly positive cells were mononucleated, resembling lymphocytes and histiocytes. Endothelial cells, interdigitating reticulum cells, nucleated red blood cells, and immature myeloid cells also had positive results in some instances. The number of HIV-positive cells was not related to the size of the bone biopsies or the clinical diagnoses. The authors postulate that changes in the peripheral blood and bone marrow of these patients may be related to latent persistent infection with HIV.