A. Circella

Cardiolipin and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis

AbstactWe previously reported that during death receptor-mediated apoptosis, cardiolipin (CL) relocates to the cell surface, where it reacts with autoantibodies from antiphospholipid syndrome sera. Here, we analysed the intracellular distribution of CL and its metabolites during the early phase of cell death signalling triggered by Fas stimulation in U937 cells and mouse liver. We found a redistribution of mitochondrial CL to the cell surface by using confocal microscopy and flow cytometry. Mass spectrometry revealed that CL and its metabolites relocated from mitochondria to other intracellular organelles during apoptosis, with a conversion into non-mitochondrial lipids. Concomitantly, cytosolic Bid relocated to the light membranes comprised in fraction P100, including the plasma membrane and associated vesicular systems. A direct Bid–CL interaction was demonstrated by the observation that CL and monolysoCL coimmunoprecipitated with Bid especially after Fas stimulation, suggesting a dynamic interaction of the protein with CL and its metabolites.

Prion protein is a component of the multimolecular signaling complex involved in T cell activation.

In this study we analyzed the interaction of prion protein PrPC with components of glycosphingolipid‐enriched microdomains in lymphoblastoid T cells. PrPC was distributed in small clusters on the plasma membrane, as revealed by immunoelectron microscopy. PrPC is present in microdomains, since it coimmunoprecipitates with GM3 and the raft marker GM1. A strict association between PrPC and Fyn was revealed by scanning confocal microscopy and coimmunoprecipitation experiments. The phosphorylation protein ZAP‐70 was immunoprecipitated by anti‐PrP after T cell activation. These results demonstrate that PrPC interacts with ZAP‐70, suggesting that PrPC is a component of the multimolecular signaling complex within microdomains involved in T cell activation.

Cardiolipin on the surface of apoptotic cells as a possible trigger for antiphospholipid antibodies

This study provides evidence that cardiolipin (CL) molecules are expressed on the surface of apoptotic cells and are recognized by antiphospholipid antibodies, purified from patients with the antiphospholipid antibody syndrome (APS). CL expression on cell surface was demonstrated by high performance thin layer chromatography analysis of phospholipids from plasma membrane purified fractions and by the positive staining with the CL‐specific dye nonyl‐acridine orange. This finding was complemented with the observation that aCL IgG purified from patients with APS bind to the surface of apoptotic cells. This staining shows a clustered distribution mostly localized on surface blebs. Interestingly, CL exposure on the cell surface preceded the DNA fragmentation, as shown by cytofluorimetric analysis. These findings demonstrate that exposure of CL molecules on the cell plasma membrane is an early event of the apoptotic cellular program that may represent an in vivo trigger for the generation of aCL.

Protein S and HIV infection the role of anticardiolipin and anti-protein S antibodies

It has recently been reported that a large proportion of patients with HIV infection have low free protein S levels. In this study we show that protein S (PS) activity levels, as well as PS antigen (Ag), were significantly lower in 35 HIV-1 infected patients than in the control population (p < 0.001). When we divided HIV infected patients into three groups according to their CD4+ counts, we found that PS levels were significantly lower in patients with < 100 CD4+ cells/ul. In order to investigate the possible role of (auto)immune response in the pathogenesis of PS deficiency, the presence of anticardiolipin antibodies (aCL) and/or of the specific antibodies to protein S was evaluated. A high prevalence (77.1%) of aCL in both symptomatic and asymptomatic subjects was observed. The screening for specific anti-PS antibodies, performed by immunoblotting, showed an overall positivity of 28.6% in anti-HIV+ patients, with a higher prevalence in symptomatic than in asymptomatic patients. Interestingly, the prevalence of the positivity for anti-PS antibodies was found to be higher in anti-HIV+ patients with PS levels < 50%. Taken collectively, our findings suggest that at least one of the mechanisms through which PS levels are decreased in HIV infection, is due to the presence of specific autoantibodies.

Dopamine transporter immunoreactivity in peripheral blood lymphocytes discriminates Parkinson’s disease from essential tremor

SummaryPeripheral blood lymphocytes (PBL) provide a model to study the changes of neurotransmitter-receptor systems in neurodegenerative disorders, including Parkinson’s disease (PD). In this study, densitometric analysis was applied to measure dopamine transporter (DAT) immunoreactivity in PBL from dopaminergic drug-free patients suffering PD or essential tremor (ET) with respect to healthy subjects. The results showed a significant reduction of DAT immunoreactivity in PBL in PD but not in ET. These finding suggests that DAT immunoreactivity in PBL may discriminate between PD and ET in the early clinical stages.

Dopamine transporter immunoreactivity in peripheral blood mononuclear cells in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a chronic progressive neuromuscular disorder of unknown etiology, characterized by weakness, muscle wasting, fasciculations, and increased reflexes, with conserved intellect and higher functions. The neuropathology of ALS is mostly confined to damage of the motor neurons in the cerebral cortex, some motor nuclei of the brainstem, and anterior horns of the spinal cord. However, there is evidence for the involvement of other neuronal systems in the disease. In particular, damage of the dopamine neurons has been shown by neurochemical and imaging studies in the brain and spinal cord of ALS patients. Recent reports suggest that peripheral blood mononuclear cells (PBMC) may represent a useful in vivo model to study neurochemical alterations that occur in neurodegenerative disorders. Here we demonstrate the significant reduction of dopamine transporter immunoreactivity in PBMC of patients affected by ALS with respect to healthy subjects. These results extend our knowledge of damage of the dopamine system in ALS to peripheral cells. Thus, the original concept of ALS as an isolated degeneration of motor neurons seems to extend to a more widespread understanding of the disease with involvement of other neuronal systems in the central as well as peripheral nervous system.

Overexpression of monosialoganglioside GM3 on lymphocyte plasma membrane in patients with HIV infection

SUMMARY This study was undertaken to analyze both the GM3 expression on peripheral blood lymphocytes of HIV-infected patients and the relationship between ganglioside content and anti-GM3 reactivity. GM3 expression was determined as a percentage of lipid-bound sialic acid and by cytofluorimetric analysis in 25 AIDS patients, 20 anti-HIV+ asymptomatic subjects, 25 patients with different viral disease, and 25 healthy donors. GM3 distribution was analyzed by immunofluorescence and immunoelectron microscopy. A follow-up study to detect anti-lymphocytic GM3 antibodies was performed in progressive and nonprogressive anti-HIV+ subjects. Lymphocytes from HIV-infected patients showed a significant increase of plasma membrane GM3 content; no difference was found between CD4+ and CD8+ cells. Immunofluorescence and immunoelectron microscopic analysis showed that GM3 was distributed in large clusters over the cell plasma membrane. The follow-up study revealed that the occurrence of anti-lymphocytic GM3 antibodies was significantly higher in patients with progressive disease, compared with asymptomatic non-progressive subjects. These findings revealed that (1) the increased GM3 content in HIV-infected patients is detected at the plasma membrane level, (2) GM3 overexpression is able to induce an increased reactivity with anti-GM3 antibodies, and (3) the appearance of anti-lymphocytic GM3 antibodies in asymptomatic anti-HIV+ subjects could have prognostic relevance for the risk of developing AIDS.

Central and peripheral dopamine transporter reduction in Parkinson's disease

Abstract Objective: Previous reports showed the reduction of dopamine transporter immunoreactivity in peripheral blood lymphocytes in Parkinsons disease. In this work, we sought to investigate the possible correlation between central and peripheral dopamine transporter immunoreactivity values in a group of 11 drug-naive patients with Parkinsons disease. Methods: Densitometric measurements of dopamine transporter immunoreactivity in peripheral blood lymphocytes was accomplished as described recently, using a monoclonal antidopamine transporter antibody. Dopamine transporter binding in the caudate and putamen nuclei was measured by means of 123I-fluopane single-photon emission computed tomography in the same patients. Results: The results failed to show any significant correlation between dopamine transporter immunoreactivity in peripheral blood lymphocytes and the caudate or putamen dopamine transporter binding. Moreover, dopamine transporter immunoreactivity in peripheral blood lymphocytes was reduced also in the single patient with normal striatal dopamine transporter binding. Discussion: These results indicate the lack of correlation between central and peripheral dopamine transporter reduction in Parkinsons disease, using the methodologies applied herein. They therefore suggest that the two phenomena are unlikely to share a common pathogenetic mechanism.

Dopamine transporter immunoreactivity in peripheral blood lymphocytes in multiple system atrophy.

Previous studies showed the reduction of dopamine transporter immunoreactivity (DAT-IR) in peripheral blood lymphocytes (PBL) in Parkinson’s disease. Here we report the reduction of DAT-IR in PBL in the extrapyramidal variant of multiple system atrophy. These results suggest the reduction of DAT-IR in PBL in a variety of neurodegenerative disorders, provided the presence of damage of the central dopaminergic systems. The reduction of DAT-IR in PBL in these disorders may represent a compensatory phenomenon aimed at reducing intracellular dopamine influx and, consequently, dopamine-mediated aggravation of oxidative stress in these cells.

Cerebrospinal Fluid Antiganglioside Antibodies in Patients with AIDS

SummaryIn this study the presence of brain antiganglioside antibodies in the cerebrospinal fluid (CSF) of patients with HIV infection was analysed. CSF samples were collected from 45 patients with AIDS and from 45 anti-HIV negative subjects, 15 of whom presented aseptic meningitis. Nineteen AIDS patients had clinically well-documented encephalopathy. Thirteen of these patients had white matter lesions shown by magnetic resonance imaging (MRI). Both IgG and IgM antiganglioside antibodies were detected by immunostaining on thin layer chromatography plates in three CSF samples from AIDS patients with progressive encephalopathy with signs of a diffuse demyelination, as revealed by MRI. Two of these CSF samples reacted specifically with GM3, GM1 and GD1a and one with GD1a. In none of the HIV infected patients without demyelinating encephalopathy, but with opportunistic infections or cerebral lymphoma, nor in the anti-HIV negative control subjects were antiganglioside antibodies detected. No association with JCV DNA, CMV DNA, EBV DNA, detected by nested PCR, nor HIV antigen p24 was found. These findings show the presence of brain antiganglioside antibodies in the CSF of AIDS patients for the first time. However, the findings do not suggest relating the presence of these antibodies to HIV encephalopathy or particular viral agents, but indicate that the antibodies are detectable in subjects with progressive encephalopathy with a diffuse demyelination.ZusammenfassungDas Vorkommen von Antigangliosid-Antikörpern im Liquor cerebrospinalis wurde bei Patienten mit HIV-Infektion untersucht. Liquorproben wurden von 45 Patienten mit AIDS und von 45 anti-HIV-negativen Personen (davon 15 mit aseptischer Meningitis) entnommen. 19 der AIDS-Patienten hatten eine klinisch eindeutig dokumentierte Encephalopathie. Bei 13 dieser Patienten waren mit Kernspintomographie (MRI) Läsionen in der weißen Substanz nachzuweisen. Antigangliosid-Antikörper der IgG und IgM-Klassen wurden mit Immunstaining auf Dünnschichtchromatographie-Platten in drei Liquorproben von AIDS Patienten mit progressiver Encephalopathie mit den Zeichen einer diffusen Entmarkung, wie sie sich im MRI dargestellt hatten, entdeckt. Zwei dieser Liquorproben reagierten spezifisch mit GM3, GM1 und GD1a und eine mit GD1a. Anti-Gangliosidantikörper waren bei keinem der HIV-infizierten Patienten ohne demyelinisierende Encephalopathie aber mit opportunistischen Infektionen oder mit zerebralem Lymphom und auch bei keinem der anti-HIV negativen Kontrollpersonen nachzuweisen. Es fand sich keine Beziehung mit JCV DNA, CMV DNA oder EBV DNA, die mit Schachtel-PCR nachgewiesen wurden und auch nicht mit dem HIV-Antigen p24. Mit diesen Untersuchungen wird erstmal der Nachweis eines Vorkommens von Antigangliosid-Antikörpern im Liquor von AIDS-Patienten erbracht. Doch ist aus den Befunden keine Beziehung des Antikörpernachweises zur HIV-Encephalopathie oder definierten viralen Agentien abzuleiten. Vielmehr ist festzustellen, daß die Antikörper bei Personen mit progressiver Encephalopathie mit diffuser Demyelinisierung zu finden sind.In this study the presence of brain antiganglioside antibodies in the cerebrospinal fluid (CSF) of patients with HIV infection was analysed. CSF samples were collected from 45 patients with AIDS and from 45 anti-HIV negative subjects, 15 of whom presented aseptic meningitis. Nineteen AIDS patients had clinically well-documented encephalopathy. Thirteen of these patients had white matter lesions shown by magnetic resonance imaging (MRI). Both IgG and IgM antiganglioside antibodies were detected by immunostaining on thin layer chromatography plates in three CSF samples from AIDS patients with progressive encephalopathy with signs of a diffuse demyelination, as revealed by MRI. Two of these CSF samples reacted specifically with GM3, GM1 and GD1a and one with GD1a. In none of the HIV infected patients without demyelinating encephalopathy, but with opportunistic infections or cerebral lymphoma, nor in the anti-HIV negative control subjects were antiganglioside antibodies detected. No association with JCV DNA, CMV DNA, EBV DNA, detected by nested PCR, nor HIV antigen p24 was found. These findings show the presence of brain antiganglioside antibodies in the CSF of AIDS patients for the first time. However, the findings do not suggest relating the presence of these antibodies to HIV encephalopathy or particular viral agents, but indicate that the antibodies are detectable in subjects with progressive encephalopathy with a diffuse demyelination. Das Vorkommen von Antigangliosid-Antikörpern im Liquor cerebrospinalis wurde bei Patienten mit HIV-Infektion untersucht. Liquorproben wurden von 45 Patienten mit AIDS und von 45 anti-HIV-negativen Personen (davon 15 mit aseptischer Meningitis) entnommen. 19 der AIDS-Patienten hatten eine klinisch eindeutig dokumentierte Encephalopathie. Bei 13 dieser Patienten waren mit Kernspintomographie (MRI) Läsionen in der weißen Substanz nachzuweisen. Antigangliosid-Antikörper der IgG und IgM-Klassen wurden mit Immunstaining auf Dünnschichtchromatographie-Platten in drei Liquorproben von AIDS Patienten mit progressiver Encephalopathie mit den Zeichen einer diffusen Entmarkung, wie sie sich im MRI dargestellt hatten, entdeckt. Zwei dieser Liquorproben reagierten spezifisch mit GM3, GM1 und GD1a und eine mit GD1a. Anti-Gangliosidantikörper waren bei keinem der HIV-infizierten Patienten ohne demyelinisierende Encephalopathie aber mit opportunistischen Infektionen oder mit zerebralem Lymphom und auch bei keinem der anti-HIV negativen Kontrollpersonen nachzuweisen. Es fand sich keine Beziehung mit JCV DNA, CMV DNA oder EBV DNA, die mit Schachtel-PCR nachgewiesen wurden und auch nicht mit dem HIV-Antigen p24. Mit diesen Untersuchungen wird erstmal der Nachweis eines Vorkommens von Antigangliosid-Antikörpern im Liquor von AIDS-Patienten erbracht. Doch ist aus den Befunden keine Beziehung des Antikörpernachweises zur HIV-Encephalopathie oder definierten viralen Agentien abzuleiten. Vielmehr ist festzustellen, daß die Antikörper bei Personen mit progressiver Encephalopathie mit diffuser Demyelinisierung zu finden sind.