Rows of dimeric-particles within the axolemma and juxtaposed particles within glia, incorporated into a new model for the paranodal glial-axonal junction at the node of Ranvier.

Using freeze-fracture techniques, we have analyzed the glial-axonal junction (GAJ) between Schwann cells and axons in the peripheral nervous system, and between oligodendrocytes and axons in the central nervous system of the rat. We have identified a new set of dimeric-particles arranged in circumferential rows within the protoplasmic fracture faces (P-faces) of the paranodal axolemma in the region of glial-axonal juxtaposition. These particles, 260 A in length, composed of two 115-A subunits, are observed in both aldehyde-fixed and nonfixed preparations. The rows of dimeric-particles within the axonal P-face are associated with complementary rows of pits within the external fracture face (E-face) of the paranodal axolemma. These axonal particles are positioned between rows of 160-A particles that occur in both fracture faces of the glial loops in the same region. We observed, in addition to these previously described 160-A particles, a new set of 75-A glial particles within the glial P-faces of the GAJ. These 75-A particles form rows that are centered between the rows of 160-A particles and are therefore superimposed over the rows of dimeric-particles within the paranodal axolemma. Our new findings are interpreted with respect to methods of specimen preparation as well as to a potential role for the paranodal organ in saltatory conduction. We conclude that this particle-rich junction between axon and glia could potentially provide an intricate mechanism for ion exchange between these two cell types.

Improved Detection of Subtle Mesial Temporal Sclerosis: Validation of a Commercially Available Software for Automated Segmentation of Hippocampal Volume.

BACKGROUND AND PURPOSE: Identification of mesial temporal sclerosis is critical in the evaluation of individuals with temporal lobe epilepsy. Our aim was to assess the performance of FDA-approved software measures of hippocampal volume to identify mesial temporal sclerosis in patients with medically refractory temporal lobe epilepsy compared with the initial clinical interpretation of a neuroradiologist. MATERIALS AND METHODS: Preoperative MRIs of 75 consecutive patients who underwent a temporal resection for temporal lobe epilepsy from 2011 to 2016 were retrospectively reviewed, and 71 were analyzed using Neuroreader, a commercially available automated segmentation and volumetric analysis package. Volume measures, including hippocampal volume as a percentage of total intracranial volume and the Neuroreader Index, were calculated. Radiologic interpretations of the MR imaging and pathology from subsequent resections were classified as either mesial temporal sclerosis or other, including normal findings. These measures of hippocampal volume were evaluated by receiver operating characteristic curves on the basis of pathologic confirmation of mesial temporal sclerosis in the resected temporal lobe. Sensitivity and specificity were calculated for each method and compared by means of the McNemar test using the optimal threshold as determined by the Youden J point. RESULTS: Optimized thresholds of hippocampal percentage of a structural volume relative to total intracranial volume (<0.19%) and the Neuroreader Index (≤-3.8) were selected to optimize sensitivity and specificity (89%/71% and 89%/78%, respectively) for the identification of mesial temporal sclerosis in temporal lobe epilepsy compared with the initial clinical interpretation of the neuroradiologist (50% and 87%). Automated measures of hippocampal volume predicted mesial temporal sclerosis more accurately than radiologic interpretation (McNemar test, P < .0001). CONCLUSIONS: Commercially available automated segmentation and volume analysis of the hippocampus accurately identifies mesial temporal sclerosis and performs significantly better than the interpretation of the radiologist.

Quantitation of human immunodeficiency virus, immune activation factors, and quinolinic acid in AIDS brains.

HIV encephalitis is unusual in that neurologic damage occurs in the absence of significant infection of neuronal or glial cells. Because the predominant infected cell in the brain is the macrophage, it has been proposed that release of viral or immune activation factors from macrophages may mediate neurologic damage. Numerous studies have examined the concentration of immune activation factors in the cerebrospinal fluid (CSF), however, there has been no correlation between these CSF measurements and severity of HIV encephalitis (Wiley, C.A., C.L. Achim, R.D. Schrier, M.P. Heyes, J.A. McCutchen, and I. Grant. 1992. AIDS (Phila.). 6:1299-1307. Because CSF measurements may not represent tissue concentrations of these factors, we examined the concentrations of HIV p24, quinolinic acid (QUIN), IL-1, IL-3, IL-6, TNF-alpha, and GMCSF within the brains of 10 AIDS autopsies. Homogenization and extraction of cortical gray, cortical white and deep gray matter showed a good correlation between the amount of HIV gp41 immunostaining and extracted HIV gag protein p24. The concentrations of cytokines were low in the tissue extracts and showed no correlation with severity of HIV encephalitis. Brain extracts from mild cases of HIV encephalitis showed elevated levels of TNF-alpha in deep gray matter, while in more severe cases, elevated TNF-alpha levels were also found within cortical white and cortical gray matter. Brain tissue and CSF QUIN concentrations were substantially increased compared to control values. QUIN concentrations were not correlated with the severity of HIV encephalitis. We conclude that increased tissue levels of TNF-alpha and QUIN may have a role in the etiology of HIV-related neurologic dysfunction.

Pathogenic and protective correlates of T cell proliferation in AIDS. HNRC Group. HIV Neurobehavioral Research Center.

To investigate the association of antigen specific CD4 T cell activation with HIV disease progression and AIDS-related central nervous system damage, T cell proliferation responses to HIV, CMV, and HSV were evaluated in infected individuals. CD4 T cell loss and neurocognitive impairment were assessed at 6-mo intervals. Individuals with known times of seroconversion who responded to more HIV peptides were at greater risk of progressing to < 200 CD4 T cells (P = 0.04) and dying (P = 0.03) than those with responses to fewer peptides. A positive correlation (0.52) was seen between the breadth of the HIV proliferation response and HIV plasma RNA levels. Higher proliferation responses to CMV and HSV were also associated with more rapid CD4 loss (P = 0.05). HLA phenotyped individuals (n = 150) with two HLA-DR alleles associated with response to more HIV peptides and CMV (DR-2,5,w6,10) were less likely to develop neurocognitive (P = 0.002) and neurologic impairment (P = 0.04), but were not protected from CD4 loss and death. Thus, the ability to generate a greater T cell proliferation response to HIV and opportunistic herpes viruses may lead to resistance to central nervous system damage, but also risk of more rapid HIV disease progression.

Immune predispositions for cytomegalovirus retinitis in AIDS. The HNRC Group.

CMV retinitis develops in approximately 28-35% of all AIDS patients at later stages of disease, often leading to blindness. To determine whether the subset of AIDS patients who developed CMV retinitis (CMV-R) were immunologically predisposed, T cell proliferation responses to CMV were examined prospectively in an HIV infected, HLA typed, longitudinal study population. Individuals who developed CMV-R had significantly lower T cell proliferation responses to CMV, both early and late in disease, compared to CD4 matched controls who have not developed CMV-R. Since HLA proteins influence T-cell recognition, phenotypes of 21 CMV-R patients were examined to determine whether certain HLA alleles were associated with low immune response and predisposed AIDS patients to CMV-R. HLA DR7 and B44 were at increased (nearly twice the expected) frequency in those with CMV-R. The combined association of either B44, 51 or DR7 with CMV-R was highly significant (P = .008, relative risk of CMV-R = 15) with correction for multiple comparisons. Low immune responses were twice as frequent in those with (61%) compared to those without (30%) predisposing alleles. Thus, AIDS patients with immunogenetically related hyporesponsiveness to CMV antigens may be at increased risk of retinitis.

Human immunodeficiency virus: infection of the nervous system.

Cynics would say it has taken the scientific community a long time to achieve very little progress in our understanding of HIV-mediated CNS damage. We cannot yet say with surity how neuronal function is affected. However, when viewed through the perspective that retroviral diseases of the human nervous system are newly recognized diseases, significant progress has been made in the 3 years since HIV infection was noted within the CNS. We have a lot to learn about how retroviruses damage the CNS, but at least the questions are better defined.

Expression of HIV regulatory and structural mRNA in the central nervous system.

OBJECTIVE: HIV encephalitis is observed in approximately one-half of AIDS autopsies. Although most investigators would agree that central nervous system (CNS) macrophages are the predominant infected cell in HIV encephalitis, there remains some controversy regarding whether other CNS cells can be infected by HIV and thus show the molecular characteristics of such an infection. DESIGN AND METHODS: Using reverse transcriptase polymerase chain reaction (PCR) and immunocytochemistry (ICC), we examined CNS tissues from AIDS and control autopsies for the presence of non-productive HIV infection. RESULTS: Single-spliced mRNA for structural envelope proteins were detected in the basal ganglia of only one of nine HIV-seropositive autopsies without HIV encephalitis and none of five seronegative autopsy controls. Double-spliced mRNA for regulatory proteins (e.g., Nef and Tat) were not detected in either the seropositive non-HIV encephalitis or seronegative controls. Both single and double-spliced viral RNA could be detected in basal ganglia of 10 out of 13 autopsies with HIV encephalitis. Similar findings were obtained when cerebral white matter was examined. Using PCR primers that distinguish single from double-spliced mRNA, we found no evidence for selective expression of the Nef regulatory gene. CONCLUSIONS: These data suggest that expression of HIV mRNA in the CNS is limited to those patients with HIV encephalitis. Further HIV encephalitis appears to be a chronic permissive infection of the CNS, without evidence of restricted Nef transcript expression.

Expression of major histocompatibility complex and HIV antigens within the brains of AIDS patients.

HIV establishes a chronic infection in the central nervous system (CNS) of AIDS patients. The immunopathogenesis of this chronic encephalitis is unknown. Because of the importance of major histocompatibility (MHC) class I and class II antigens in modulating the immune response, we examined the tissue expression of MHC molecules in relation to CNS damage and expression of viral antigens. By immunocytochemical staining we found that beta 2-microglobulin (beta 2M) expression is elevated in all cases with signs of viral encephalitis. beta 2M was expressed at high levels on endothelial cells, macrophages and possible oligodendroglia within regions of histopathology. In histologically normal regions elevated expression of beta 2M was noted only on endothelial cells. MHC class II expression was elevated only in the HIV encephalitis cases, and was restricted to macrophages/microglia and occasional endothelial cells. When compared with other viral encephalitides these findings suggest that the intra-CNS immune response to HIV is appropriate for viral presentation; however, the absence of responsive systemic T cells may lead to chronic viral infection.

Relationship of cerebrospinal fluid immune activation associated factors to HIV encephalitis.

OBJECTIVES AND DESIGN: Because macrophages are the predominant immune cell and the predominant infected cell in the brains of patients with HIV encephalitis, we studied macrophage and immune activation-associated factors in the cerebrospinal fluid (CSF) from 39 autopsied AIDS cases for whom complete neuropathologic evaluation of the brain was available. RESULTS: CSF HIV p24 antigen was present in less than one-third of cases (11 out of 39). Less than half of the autopsies with moderate to severe parenchymal infection by HIV had high levels of CSF p24, although all autopsies with elevated levels of HIV p24 had moderate to severe HIV encephalitis. Elevated levels of cytokines, beta 2-microglobulin, neopterin, and quinolinic acid were observed. CONCLUSIONS: Although many of the CSF findings showed a strong correlation with each other, none showed a strong correlation with the severity of HIV infection of the brain itself. The absence of a close association between CSF abnormalities and HIV encephalitis could reflect the abundance of complicating opportunistic infections in these terminally ill patients or the inadequacy of CSF as a marker of basal ganglia involvement in HIV encephalitis. These findings complicate interpretation of clinical studies of CSF in patients with AIDS where neuropathologic evaluation is unavailable.

HIV mediates a productive infection of the brain.

BACKGROUND: Approximately one quarter of patients with AIDS develop severe cognitive deficits called HIV-associated dementia complex. There is some controversy regarding the importance of viral load and distribution in mediating this neurologic disease. OBJECTIVE: Brain HIV proviral and RNA loads were compared to define the molecular nature of HIV infection of the brain. METHOD: Neuropathologic examination was performed on brains from 10 autopsies of patients with AIDS that had short post-mortem intervals and no evidence of opportunistic infection. Viral DNA and RNA were extracted and quantified from multiple brain regions. These findings were compared with triple-label immunofluorescence for viral and cell markers. RESULTS: Brains with histopathologic evidence of HIV encephalitis contained abundant HIV RNA and DNA. Regions without productive HIV infection showed minimal proviral load. By immunocytochemistry, only brain macrophages/microglia double labeled for viral proteins. CONCLUSIONS: HIV mediates a productive infection of brain macrophages/microglia. There was no evidence supporting the hypothesis of substantial neuronal or macroglial infection, or evidence of substantial proviral burden prior to the development of productive infection.

Chemokines and receptors in HIV encephalitis.

BACKGROUND: Chemokines are involved in the migration of leukocytes and have been implicated in several inflammatory diseases of the central nervous system. Some of their receptors have been proposed to mediate HIV infection. OBJECTIVE: To determine changes in chemokine and receptor expression in HIV encephalitis, and to determine whether upregulation leads to recruitment of infected monocytes across the blood-brain barrier and participates in HIV neuropathology. METHODS: Immunocytochemistry and double-label immunofluorescent laser confocal microscopy was performed with antibodies to chemokines and their receptors on brain tissues from patients who died with or without HIV encephalitis. In vivo distribution was compared with in vitro cultures of human neuroglial cells. RESULTS: The beta-chemokines monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, and RANTES were detected on brain macrophages. Their presence was associated with the histopathological signs of HIV encephalitis. The alpha-chemokines IP-10 (10 kDa inflammatory protein) and interleukin-8 were expressed by astrocytes in all tissues, including controls. Presence of the CXC-chemokine receptor (CXCR)-4 was seen on brain macrophages/microglia, neurons, and astrocytes. CC-Chemokine receptor (CCR)-5 was detected only on macrophages/microglia. CCR-3 and CCR-1 were expressed by macrophages and endothelial cells. In vitro studies examining the presence of CCR-3, CCR-5, and CXCR-4 on human brain cell cultures demonstrated abundant neuronal and microglial expression. CONCLUSIONS: Expression of a variety of chemokines and receptors was shown to be increased in HIV encephalitis brain tissues particularly in areas of neuroglial reaction. The expression pattern supported their involvement in the recruitment of inflammatory infiltrates and formation of microglial nodules. Presence of chemokine receptors on neurons may be involved in the pathogenesis of neurologic damage in AIDS patients.

Expression of major histocompatibility complex antigens in the brains of patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is caused by JC virus (JCV) infection of the central nervous system (CNS) in immunosuppressed patients. The immunopathogenesis of this chronic encephalitis is unknown. Because major histocompatibility (MHC) class I and class II antigens are important in modulating the immune response and viral clearance, we examined the tissue expression of MHC molecules in relation to CNS damage and presence of virus. By immunocytochemical staining, both MHC class I and class II antigens were expressed at high levels within PML lesions. Beta-2 microglobulin (beta-2m) was present on endothelial cells and JCV-infected oligodendroglia within the lesions. Also, many astrocytes with bizarre morphology expressed MHC class I antigens. In histologically normal regions of PML brains expression of beta-2m was noted only on endothelial cells. Expression of MHC class II also was focused within demyelinating lesions and was restricted to macrophages/microglia and occasional endothelial cells. When compared to other viral encephalitides (e.g. human immunodeficiency virus) these findings suggest that intra-CNS immune response to JCV is appropriate for antigenic presentation; however, the absence of responsive systemic T-cells may lead to chronic viral infection with progressive neuropathology.

Expression of major histocompatibility complex antigens and serum neutralizing antibody in murine retroviral encephalitis.

Murine leukemia virus infection serves as a model for noninflammatory degeneration of the central nervous system (CNS). During the course of infection with either of the molecularly cloned viruses pNE-8 or ts-1, we observed that ts-1 spread twice as rapidly as pNE-8, and ascended higher in the neuraxis. Endothelial cells were infected first, followed by oligodendrocytes and neurons, while astrocytes containing glial fibrillary acidic protein were not infected. Additionally, ts-1 also infected macrophages/microglia. Major histocompatibility complex (MHC) class I beta 2-microglobulin expression was minimal in pNE-8 infected mice, while it was elevated in endothelial cells of early ts-1 lesions, and in macrophages/microglia during later stages. Occasional infected cells expressed beta 2-microglobulin while rare endothelial and parenchymal cells expressed MHC class II in both viral infections. Limited intra-CNS MHC expression may be one of the mechanisms of viral persistence and will present a barrier to developing immunotherapy for CNS retroviral infections. The few mice that escaped lethal infection had higher serum titers of neutralizing antibodies and showed no neuropathologic changes or detectable virus in the CNS. Higher titers of neutralizing antibodies may protect the CNS from infection.

Neocortical pathology in chronic murine retroviral encephalitis.

Retroviral infection of the central nervous system (CNS) causes chronic functional and morphological damage in a wide variety of mammals. Neuropathological studies have focused on subcortical pathology, however, the neocortex is also affected. Because studies of human CNS pathology have been limited to the use of material from terminal stages of disease, we used two neuropathogenic murine leukemia virus (MuLV) models to study the development of neocortical damage. MuLV infection caused spongiform change in the spinal cord, brainstem and cerebellum but not in the cerebrum. However, over the course of disease, we observed a reduction of neocortical thickness, accompanied by diminished neuronal and dendritic spine density. Electron microscopic studies showed minimal to no ultrastructural alterations of dendritic spines. Since there was no evidence of extensive direct viral infection of the neocortical neurons or glia at the ultrastructural level, we hypothesize that neocortical damage may be an indirect effect of subcortical retroviral infection.

Brain viral burden in HIV infection.

We quantitated the brain viral burden in autopsy material from AIDS patients with and without HIV encephalitis. Central nervous system (CNS) samples from 45 AIDS autopsies with less than 48 hours postmortem autolysis and without significant non-viral opportunistic infections were analyzed using immunocytochemistry (ICC), antigen capture assay (ACA) and polymerase chain reaction (PCR). Approximately three-quarters of the cases contained HIV DNA by PCR. The majority of these had abundant gp41 detected by ICC, but approximately one-third had no HIV p24 detected by ACA. With all assays, HIV proteins and DNA were most abundant in deep gray matter. Approximately one-quarter of the cases contained HIV p24 by ACA in both CNS tissue and cerebrospinal fluid. In more than half of the cases cytomegalovirus was detectable in the brain by PCR, however, only in the basal ganglia of one case was human herpes virus-6 detectable by PCR. In conclusion, HIV infection of the CNS was observed in the majority of AIDS autopsies, however, the quantity of virus was variable between cases and within different neuroanatomical regions. Differences between the quantitation methods could be either technical or biological, however, any of them could be used to compare assessment of HIV burden by different laboratories.

Differential vulnerability of calbindin-immunoreactive neurons in HIV encephalitis.

Recent studies have suggested that the neuronal damage during human immunodeficiency virus encephalitis (HIVE) might be mediated by increased intracellular calcium. Since in vitro studies have shown that calcium-binding proteins protect neurons from calcium-mediated toxicity, we hypothesized that calbindin-expressing neurons might be resistant to HIV1-mediated damage. We compared patterns of calbindin immunoreactivity in the cortex and subcortex of autopsied AIDS cases with and without HIVE. Calbindin-immunoreactive neurons in the neocortex were significantly reduced in HIVE (one-way ANOVA, p < 0.001), while these neurons in the basal ganglia and hippocampus were unaffected. The loss of calbindin-immunolabeled neurons in the neocortex was correlated with viral burden (r = -0.45, p < 0.001). Differential loss of calbindin-immunoreactive neurons in HIVE suggests that neuronal damage in different regions of the CNS may be mediated by different pathogenic mechanisms.

Selective neuronal vulnerability in HIV encephalitis.

Recent studies of human immunodeficiency virus type 1 (HIV-1) encephalitis have shown that in addition to well established white matter damage, the neocortex shows thinning, loss of large neurons and dendritic damage. In order to identify neuronal populations affected in HIV encephalitis and to determine how neuronal damage relates to the severity of HIV infection within the nervous system, we quantified parvalbumin (PV+) and neurofilament (NF+) immunoreactive neurons in the frontal cortex and hippocampus. We found that in the neocortex, the density of NF+ and PV+ neurons was independent of severity of HIV encephalitis, and therefore changes in these neuronal subsets did not account for previously reported neuronal loss. However, neuritic processes of PV+ neurons were fragmented, atrophic and in some cases distended. In contrast to the frontal cortex, there was a trend toward decreased density of PV+ neurons in the hippocampus which only reached significance in the CA3 layer where there was a 50-90% decrease in PV+ neurons. This decrease was closely correlated with the severity of HIV encephalitis. Double-label immunocytochemical analysis confirmed neuritic damage to interneurons. These results suggest that HIV encephalitis differentially involves specific subpopulations of neurons. Since direct HIV infection of neuronal cells was not detected, damage to PV+ cells and fibers may be indirectly mediated by cytokines released by HIV-infected microglia.

Damage and repair of DNA in HIV encephalitis.

Neuronal damage and dementia are common sequelae of HIV encephalitis. The mechanism by which HIV infection of CNS macrophages results in neuronal damage is not known. We examined the brains from 15 AIDS autopsies (8 with HIV encephalitis and 7 without) and 4 non-infected control autopsies for the presence of DNA strand breaks, for associated changes in the expression of the DNA repair enzymes KU80 and Poly (ADP-ribose) polymerase (PARP), and for accumulation of amyloid precursor protein (APP). Abundant DNA damage was observed with terminal transferase-mediated dUTP nick end-labeling (TUNEL), however, there was no morphologic evidence of significant neuroglial apoptosis. The DNA repair enzyme KU80 was immunocytochemically detectable in neuronal and glial cells in autopsy brains from patients with and without HIV encephalitis; however, in cases with HIV encephalitis the staining was more prominent than in the infected or non-infected controls without encephalitis. There was no difference in KU80 immunostaining in oligodendroglia from autopsies with and without encephalitis. Immunostaining for PARP was more intense in gray and white matter of cases with HIV encephalitis. No clear spatial relationship existed between expression of DNA repair enzymes and the spatial proximity of microglial nodules or HIV-infected macrophages. The cytoplasm of cortical and subcortical neurons immunostained for APP Stronger cortical neuronal APP staining was observed in cases without HIV encephalitis. Staining of deep gray matter neurons was similar, irrespective of the presence or absence of encephalitis. While foci of intense APP staining were noted in white matter not related to HIV infection, they were associated with foci of opportunistic infections (e.g. due to CMV or PML). We conclude that damaged DNA and altered patterns of expression of DNA repair proteins and APP are common findings in the brains of AIDS patients at autopsy, but do not have a spatial relationship to HIV-infected macrophages.

Localization of cytomegalovirus proteins and genome during fulminant central nervous system infection in an AIDS patient.

Approximately one-half of autopsied acquired immune deficiency syndrome (AIDS) patients demonstrate probable human cytomegalovirus (CMV) infection of the central nervous system (CNS). Because CMV in brain tissue or cerebrospinal fluid is difficult to culture, we used antisera, and radioactive probes to diagnose CMV infection in the brain of an autopsied AIDS patient, who died of a fulminant CNS and systemic infection with CMV, suggesting a complete seeding of the ependymal regions possibly followed by a uniform ventriculofugal spread of the virus deep into the parenchyma. Cytomegalic cells were observed in optic nerve, retina, ependymal and subependymal regions of the brain and in the motor (but not sensory) root-CNS junctions. Immunocytochemistry demonstrated viral antigen predominantly in cytomegalic cells, which also stained positively for glial fibrillary acidic protein, S-100, or neuron-specific enolase, but not a common leukocyte antigen. Virions were visible in these cells examined by electron microscopy. No viral replication was observed in pineocytes, pituicytes or the choroid plexus. Morphologically normal cells that were CMV antigen-negative proved to be infected after in situ hybridization with well-defined human CMV DNA fragments. Hence, morphologically normal glia and neurons show restricted replication of CMV, indicating that such cells may be latently infected.

Neurological and neuropsychological complications of HIV infection, Monterey, California, June 16-19, 1990.

In summary this important conference covered the entire gamut of issues regarding retroviral damage of the central nervous system. Neuropathologists interested in this rapidly moving field should strongly consider attending the next satellite conference to be held in June 1991 in Florence, Italy. Given the almost carnival atmosphere that the parent International AIDS Conference has fallen into, this is an important satellite conference that lends itself well to the close sharing of data from a wide spectrum of neuroscience fields.