Françoise Gray

Neuronal apoptosis in HIV infection in adults

Productive infection of the central nervous system by HIV predominantly involves the white matter and basal ganglia. Involvement of the cerebral cortex with neuronal loss is also described in AIDS patients but not in asymptomatic HIV‐positive patients. The mechanism of neuronal damage is unknown. To enquire whether neuronal loss in AIDS may be due to an apoptotic process, we examined the cerebral cortex from 12 patients who died from AIDS using two different methods: in situ end labelling and gel electrophoresis of DNA to demonstrate DNA fragmentation. None of the patients had cerebral opportunistic infection or tumour. Four patients had no significant neuropathological changes, eight patients had variable cerebral atrophy and four of them also had productive HIV infection of the brain. These patients were compared with four HIV‐positive asymptomatic patients, five seronegative asymptomatic controls, and two seronegative patients with Alzheimers disease. We demonstrated neuronal apoptosis in the cortex in all AIDS patients, as well as in the Alzheimers patients. Apoptosis was not observed in the asymptomatic cases whether seropositive or seronegative. Neuronal apoptosis was more severe in atrophic brains, and did not directly correlate with productive HIV infection, suggesting an indirect mechanism of neuronal damage is most likely.

Neuropathology of early HIV-1 infection

Early HIV‐1 invasion of the central nervous system has been demonstrated by many cerebrospinal fluid studies; however, most HIV‐1 carriers remain neurologically unimpaired during the so called “asymptomatic” period lasting from seroconversion to symptomatic AIDS. Therefore, neuropathological studies in the early pre‐AIDS stages are very few, and the natural history of central nervous system changes in HIV‐1 infection remains poorly understood. Examination of brains of asymptomatic HIV‐1 positive individuals who died accidentally and of rare cases with acute fatal encephalopathy revealing HIV infection, and comparison with experimental simian immunodeficiency virus and feline immunodeficiency virus infections suggest that, invasion of the CNS by HIV‐1 occurs at the time of primary infection and induces an immunological process in the central nervous system. This includes an inflammatory T‐cell reaction with vasculitis and leptomeningitis, and immune activation of brain parenchyma with increased number of microglial cells, upregulation of major histocompatibility complex class II antigens and local production of cytokines. Myelin pallor and gliosis of the white matter are usually found and are likely to be the consequence of opening of the blood brain barrier due to vasculitis; direct damage to oligodendrocytes by cytokines may also interfere. These white matter changes may explain, at least partly, the early cerebral atrophy observed, by magnetic resonance imaging, in asymptomatic HIV‐1 carriers. In contrast, cortical damage seems to be a late event in the course of HIV‐1 infection. There is no significant neuronal loss at the early stages of the disease, no accompanying increase in glial fibrillary acid protein staining in the cortex, and only exceptional neuronal apoptosis. Although HIV‐1 proviral DNA may be demonstrated in a number of brains, viral replication remains very low during the asymptomatic stage of HIV‐1 infection. This makes it likely that, although opening of the blood brain barrier may facilitate viral entry into the brain, specific immune responses including both neutralising antibodies and cytotoxic T‐lymphocytes, continuously inhibits viral replication at that stage.

Apoptosis of neurons in cardiovascular autonomic centres triggered by inducible nitric oxide synthase after death from septic shock

BACKGROUND Results of experimental and clinical studies have shown that septic shock is associated with cardiovascular autonomic failure. Thus, we aimed to investigate the existence of ischaemia and apoptosis within the cerebral autonomic centres that control the cardiovascular system in patients with septic shock. METHODS In a prospective cohort study, we did post-mortem examinations of supraoptic and paraventricular nuclei, cerebral amygdala, locus coeruleus, and medullary autonomic nuclei in 19 patients with septic shock, seven with non-septic shock and five who died suddenly from extracranial injury. Ischaemic and apoptotic neurons and microglial cells, and expression of tumour necrosis factor alpha (TNFalpha) and inducible nitric oxide synthase (iNOS) were scored. FINDINGS Ischaemic, neuronal, and microglial apoptosis scores differed between groups (p=0.0007, p<0.0001, and p=0.0037, respectively) and were higher in patients with septic shock than in those with non-septic shock (p=0.0033, p=0.0005, and p=0.0235, respectively), and extra-cranial injury related deaths (p=0.0027, p=0.0007, and p=0.0045, respectively). There was little microglial activation and glial expression of TNFalpha. The scores for endothelial iNOS expression were different between the three groups (p<0.0001), and were higher in septic shock than in non-septic shock (p=0.0009) and than in extracranial injury related deaths (p=0.0007). Vascular expression of iNOS also correlated (Spearman tau=0.57) with autonomic-centre neuronal apoptosis in the combined septic and non-septic shock group. INTERPRETATION Septic shock is associated with neuronal and glial apoptosis within the autonomic centres, which is strongly associated with endothelial iNOS expression.

Neuronal apoptosis does not correlate with dementia in HIV infection but is related to microglial activation and axonal damage

To characterize the distribution of apoptotic neurons and their relationships with the stage of disease, a history of HIV‐dementia, and the degree of productive HIV infection, microglial activation and axonal damage, we examined the brains of 40 patients. Samples of frontal and temporal cortex, basal ganglia and brain stem were taken post‐mortem from 20 patients with AIDS (including three with HIV‐dementia, and eight with cognitive disorders that did not fulfil the criteria for HIV‐dementia), 10 HIV‐positive asymptomatic cases and 10 seronegative controls. Neuronal apoptosis was demonstrated by in situ end labelling in 18 AIDS cases and two pre‐AIDS cases; a single apoptotic neuron was present in the temporal cortex of a control. Semiquantitative evaluation showed that the severity of neuronal apoptosis in the cerebral cortex correlated with the presence of cerebral atrophy, but not with a history of HIV dementia. There was no global quantitative correlation between neuronal apoptosis and HIV encephalitis or microglial activation. However, there was some topographical correlation between these changes. In the basal ganglia, apoptotic neurons were much more abundant in the vicinity of multinucleated giant cells and/or p24 expressing cells. Microglial activation was constantly present in these areas. Axonal damage was identified using β‐amyloid‐precursor protein (βAPP) immunostaining in 17 AIDS and eight pre‐AIDS brains. Although no global quantitative correlation could be established between axonal damage and neuronal apoptosis there was an obvious topographic correlation supporting the view that axonal damage, either secondary to local microglial activation or due to the intervention of systemic factors, may also contribute to neuronal apoptosis.

The neuropathology of septic shock.

The neuropathological correlates of encephalopathy and autonomic dysfuction in septic shock are unclear. We performed post mortem analysis of 5 brain areas susceptible to ischemia and 5 autonomic nuclei (AN) in 23 pqtients who had died in our intensive care unit (ICU) from septic shock and 8 dying from non‐septic shock as well as 5 controls who had died suddenly from extracranial injury. Proinflammatory cytokine (IL1‐β and TNF‐α) and inducible nitric oxide synthase (iNOS) expressin was assessed by immunocytochemistry. Abnormalities in septic shock were: hemorrhages (26%), hypercoagulability syndrome (9%), micro‐abscessed (9%), multifocal necrotizing leukoencephalopathy (9%) and ischemia (100%). The incidence of cerebral hemorrhage or hypercoagulability syndrome was not related to clotting distrurbances. The intensity of ischemia within susceptible areas was the same on both ICU groups, but more pronounced in the autonomic centers of septic patients (P<0.0001). Neuronal apoptosis assessed using anti‐caspase 3 immunocytochemistry and in situ end labeling was more pronounced in the autonomic nuclei septic patients. (P<0.0001). INF‐alpha expression did not differ between groups but vascular iNOS expression assessed by immunocytochemistry was higher in sepsis (p<0.0001) and correlated with autonomic center neuronal apoptosis (P<0.02). We conclude that septic shock is associated with diffuse cerebral damage and specific autonomic neuronal apoptosis which may be due to circulating factors particularly iNOS.

Depletion of neurohypophyseal content of vasopressin in septic shock.

OBJECTIVES To assess the mechanisms underlying the inappropriately low plasma vasopressin levels reported in septic shock. DESIGN Prospective case series. SETTING A 26-bed general medical intensive care unit at a university hospital. PATIENTS Septic shock patients. MEASUREMENTS AND MAIN RESULTS In three consecutive patients with septic shock, plasma vasopressin levels, circulating vasopressinase activity, baroreflex sensitivity, and neurohypophyseal vasopressin content were assessed. Plasma vasopressin concentration was unexpectedly within normal range in two patients (1.6 pg/mL and 1.8 pg/mL) and increased in one (16 pg/mL). In all cases, vasopressinase activity was undetectable, baroreflex sensitivity was decreased, and the high signal intensity of the posterior lobe of the pituitary gland on T1-weighted magnetic resonance images was absent. Magnetic resonance imaging and plasma vasopressin levels normalized after recovery from shock in the patient who survived. CONCLUSION These data suggest that in septic shock, inappropriately low plasma levels of vasopressin are at least partly related to a depletion of vasopressin stores in the neurohypophysis.

Neuronal apoptosis in Creutzfeldt-Jakob disease.

Neuronal loss is a salient feature of prion diseases; however, its causes and mechanisms are unclear The possibility that it could occur through an apoptotic process has been postulated and is consistent with the lack of inflammation in prion disorders as supported by experimental studies. In order to test this hypothesis in humans, we examined samples of frontal and temporal cerebral cortex, striatum, thalamus, and cerebellum from 16 patients who died from Creutzfeldt-Jakob disease. They included 5 sporadic cases, 5 familial, 3 iatrogenic, and 3 cases with the new variant. These were compared with age and sex matched controls. Using in situ end labelling, we identified apoptotic neurons in all the cases of Creutzfeldt-Jakob disease. A single labelled neuron was found in the eldest control. Apoptotic neurons were mostly found in damaged regions and their presence and abundance seemed to correlate closely with neuronal loss. This supports the view that apoptosis of neurons is a feature of prion diseases and may contribute to the neuronal loss which is one of the main characteristics of these conditions. Neuronal apoptosis also correlated well with microglial activation, as demonstrated by the expression of major histocompatibility complex class II antigens, and axonal damage, as identified by beta-amyloid protein precursor immunostaining. In contrast, we found no obvious relationship between the topography and severity of neuronal apoptosis and the type, topography, and abundance of prion protein deposits as demonstrated by immunocytochemistry.

Pathogenesis of Cerebral Cryptococcus neoformans Infection after Fungemia

The pathogenesis of cerebral infection after Cryptococcus neoformans fungemia in outbred mice was investigated. Confocal microscopy and cultures on ficoll-hypaque gradient-separated blood cells were used to detect yeasts in the cytoplasms of monocytes. In semithin brain sections, poorly capsulated yeasts were seen in macrophages in the leptomeningeal space, in monocytes circulating in leptomeningeal capillaries, or in the endothelial cells themselves, strengthening the hypothesis that monocytes and endothelial cells play key roles in the pathogenesis of cryptococcal meningitis. Similar fungal loads and cellular reactions were seen in mice and in 1 patient with acquired immune deficiency syndrome (AIDS), all with acute cryptococcal meningoencephalitis, and in mice and in 1 patient with AIDS, all with cured cryptococcal infection. Immunostaining revealed both the presence of cryptococcal polysaccharide in various brain cells and antigenic variability both from yeast cell to yeast cell and over time. Thus, our data established the relevance and interest that this experimental model has for investigation of the pathogenesis of human cryptococcal meningitis.

Early Entry and Widespread Cellular Involvement of HIV-1 DNA in Brains of HIV-1 Positive Asymptomatic Individuals

There is overwhelming evidence that invasion of the central nervous system (CNS) by HIV-1 takes place at an early stage of the infection. It has been demonstrated that HIV-1 DNA is present in brains of asymptomatic individuals. Evidence of immune activation and increased expression of cytokines suggested that neuropathological changes and neuronal and axonal damage could be the effect of the presence of the virus. The purpose of the study is to ascertain whether target cells for HIV-1 in brain of patients at early stage of the infection are the same as those found in AIDS sufferers or if the distribution seen in AIDS patients results from the late spreading of the infection from cells considered traditionally the reservoir of the virus, i.e. microglial cells. Eighteen brains, all HIV-1 DNA positive, as shown by nested polymerase chain reaction (PCR), were selected among the group of HIV-1 positive asymptomatic cases. In 6 of them, HIV-1 DNA was detected by PCR in situ. Positive cells included astrocytes and endothelial cells, in addition to microglial cells. We conclude that astrocytes and endothelial cells are already infected at an early (asymptomatic) stage of the infection and suggest that they might contribute to the damage of the CNS.

Early Brain Changes in HIV Infection: Neuropathological Study of 11 HIV Seropositive, Non-AIDS Cases

Abstract. We examined 11 brains of human immunodeficiency virus (HIV) seropositive cases who died from unnatural causes (10 intravenous drug abusers who died from heroin overdose and 1 homosexual dead from a gunshot injury); 10 brains of HIV seronegative heroin addicts who died from overdose and 1 seronegative drug abuser who died from gunshot injury served as controls. Complete postmortem examination did not show evidence of acquired immune deficiency syndrome (AIDS) or AIDS related complex. Terminal changes including nerve cell ischemia, edema and diffuse vascular congestion were observed in all cases. Perivascular pigment deposition with macrophages was a constant finding in drug addicts and was probably related to chronic intravenous injection. In contrast, cerebral vasculitis was significantly more frequent and marked in HIV seropositive cases and was often associated with lymphocytic meningitis. Granular ependymitis, myelin pallor with reactive astrocytosis and microglial proliferation were also more frequent and more severe in HIV seropositive cases. Immunocytochemistry was negative for HIV antigens. Our study further supports the view that early central nervous system changes occur in HIV infection.