Cristian L. Achim

Human macrophages convert l-tryptophan into the neurotoxin quinolinic acid

Substantial increases in the concentrations of the excitotoxin and N-methyl-D-aspartate-receptor agonist quinolinic acid (QUIN) occur in human patients and non-human primates with inflammatory diseases. Such increases were postulated to be secondary to induction of indoleamine 2,3-dioxygenase in inflammatory cells, particularly macrophages, by interferon-gamma. To test this hypothesis, human peripheral-blood macrophages were incubated with L-[13C6]tryptophan in the absence or presence of interferon-gamma. [13C6]QUIN was quantified by gas chromatography and electron-capture negative-chemical-ionization mass spectrometry. [13C6]QUIN was detected in the incubation medium of both unstimulated and stimulated cultures. Exposure to interferon-gamma substantially increased the accumulation of [13C6]QUIN in a dose- and time-dependent manner. The QUIN concentrations achieved exceeded those reported in both cerebrospinal fluid and blood of patients and of non-human primates with inflammatory diseases. Macrophages stimulated with interferon-gamma may be an important source of accelerated L-tryptophan conversion into QUIN in inflammatory diseases.

Blood-Brain Barrier Tight Junction Disruption in Human Immunodeficiency Virus-1 Encephalitis

The blood-brain barrier (BBB) plays a critical role in regulating cell trafficking through the central nervous system (CNS) due to several unique anatomical features, including the presence of interendothelial tight junctions that form impermeable seals between the cells. Previous studies have demonstrated BBB perturbations during human immunodeficiency virus encephalitis (HIVE); however, the basis of these permeability changes and its relationship to infiltration of human immunodeficiency virus type 1 (HIV-1)-infected monocytes, a critical event in the pathogenesis of the disease, remains unclear. In this study, we examined CNS tissue from HIV-1-seronegative patients and HIV-1-infected patients, both with and without encephalitis, for alterations in BBB integrity via immunohistochemical analysis of the tight junction membrane proteins, occludin and zonula occludens-1 (ZO-1). Significant tight junction disruption (P < 0.001), as demonstrated by fragmentation or absence of immunoreactivity for occludin and ZO-1, was observed within vessels from subcortical white matter, basal ganglia, and, to a lesser extent, cortical gray matter in patients who died with HIVE. These alterations were also associated with accumulation of activated, HIV-1-infected brain macrophages, fibrinogen leakage, and marked astrocytosis. In contrast, no significant changes (P > 0.05) were observed in cerebellar tissue from patients with HIVE compared to HIV-seronegative patients or HIV-1-infected patients without encephalitis. Our findings demonstrate that tight junction disruption is a key feature of HIVE that occurs in regions of histopathological alterations in association with perivascular accumulation of activated HIV-1-infected macrophages, serum protein extravasation, and marked astrocytosis. We propose that disruption of this key BBB structure serves as the main route of HIV-1-infected monocyte entry into the CNS.

Microglial activation and increased microglial density observed in the dorsolateral prefrontal cortex in autism

BACKGROUND In the neurodevelopmental disorder autism, several neuroimmune abnormalities have been reported. However, it is unknown whether microglial somal volume or density are altered in the cortex and whether any alteration is associated with age or other potential covariates. METHODS Microglia in sections from the dorsolateral prefrontal cortex of nonmacrencephalic male cases with autism (n = 13) and control cases (n = 9) were visualized via ionized calcium binding adapter molecule 1 immunohistochemistry. In addition to a neuropathological assessment, microglial cell density was stereologically estimated via optical fractionator and average somal volume was quantified via isotropic nucleator. RESULTS Microglia appeared markedly activated in 5 of 13 cases with autism, including 2 of 3 under age 6, and marginally activated in an additional 4 of 13 cases. Morphological alterations included somal enlargement, process retraction and thickening, and extension of filopodia from processes. Average microglial somal volume was significantly increased in white matter (p = .013), with a trend in gray matter (p = .098). Microglial cell density was increased in gray matter (p = .002). Seizure history did not influence any activation measure. CONCLUSIONS The activation profile described represents a neuropathological alteration in a sizeable fraction of cases with autism. Given its early presence, microglial activation may play a central role in the pathogenesis of autism in a substantial proportion of patients. Alternatively, activation may represent a response of the innate neuroimmune system to synaptic, neuronal, or neuronal network disturbances, or reflect genetic and/or environmental abnormalities impacting multiple cellular populations.

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 β‐chemokines monocyte chemotactic protein‐1, macrophage inflammatory protein‐1α, and RANTES were detected on brain macrophages. Their presence was associated with the histopathological signs of HIV encephalitis. The α‐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.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 en

DISTRIBUTION OF BRAIN HIV LOAD IN AIDS

Approximately one quarter of patients with AIDS develop severe cognitive deficits called HIV‐associated dementia complex (ADC). There is some controversy regarding the importance of viral load in mediating neurologic disease. With the advent of sensitive, quantitative and reproducible RNA assays for HIV load in plasma and CSF, we quantified viral load in brains from 10 autopsied HIV‐infected subjects and 2 non‐infected controls. The new quantitative HIV RNA assays showed general agreement with previously used semi‐quantitative immunocytochemical assessments of HIV envelope protein, and were performed without professional subjective interpretation. All cases with very high levels of HIV in the CSF, had high overall levels in the brain, suggesting that CSF viral loads exceeding 106 copies per mL may be a surrogate marker of high viral load in the brain. Levels of virus in the spleen showed no clear association with those found in the brain. HIV RNA was not uniformly distributed throughout the brain. Selective regions, including basal ganglia and hippocampus, showed higher levels of virus than the cerebellar cortex and mid‐frontal cortical gray matter. Assessment of overall brain viral load requires careful attention to regional quantitation.

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 HI V encephalitis. Double-label immunocytochemical analysis confirmed neuritic damage to intemeurons. 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.

Brain deposition of beta-amyloid is a common pathologic feature in HIV positive patients.

Background:We planned to analyze the prevalence and distribution of β-amyloid deposition in the HIV+ brain in the HAART era. Our working hypothesis is that long term survival, aging and the secondary effects of HAART may contribute to increased beta-amyloid accumulation in this patient population. Methods:Paraffin embedded archival brain autopsy tissues were assessed by immunocytochemistry for β-amyloid. Detailed in-vivo neuro-behavioral assessments and ApoE genotyping were available for a subset of the studied population. Results:Immunoreactivity with the antibodies 4G8 and 6E10 was found predominantly in neuronal soma and dystrophic axonal processes. Extracellular, often perivascular plaques were also identified in many cases. Conclusions:We propose that prolonged HAART and aging may contribute to an overall increase in amyloid deposition, potentially mediated by inhibition of insulin degradation enzyme (IDE) or disruption of the axonal transport of the amyloid precursor protein.

Cortical and subcortical neurodegeneration is associated with HIV neurocognitive impairment.

Objective:To determine the association of markers of regional neurodegeneration (ND) at autopsy to degree of neurocognitive impairment in persons with HIV. Design:In a prospectively followed cohort of HIV-infected individuals we examined the relationship between antemortem neuropsychological (NP) abilities and postmortem neuropathological data. Methods:Twenty-seven HIV-infected individuals with both neuropsychological and neuropathological data were identified. Laser confocal scanning microscopy was used to determine the degree of ND based on: (1) microtubule-associated protein (MAP2; reflecting neuronal cell bodies and dendrites) and (2) synaptophysin (SYN; a measure of presynaptic terminals). A regional combined score, based on the distribution of percentage neuropil occupied by MAP2 and SYN and emphasizing severity of ND, was created for each brain region: midfrontal cortex, hippocampus, and putamen. Results:The regional combined scores from each brain region studied were better correlated with level of global NP impairment than measures of SYN and MAP2 individually. In a regression, hippocampal and putamen regional combined scores were independent predictors of degree of antemortem NP impairment (F3,23 = 6.17; P < 0.01; R2 = 0.45). The correlations among regional ND measures demonstrated that ND is unevenly distributed across multiple brain regions. Conclusions:As the anatomic distribution and temporal progression of neuropathologic changes appears to differ across individuals, it is important to consider both cortical and subcortical brain regions in studies of neuropathogenesis and treatment of HIV-related brain disease. Furthermore, combining information from several markers of neural injury provided the strongest association with degree of neurocognitive impairment during life.

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.

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.