Tamara Griggi

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.

Colocalization and Complex Formation Between Prosaposin and Monosialoganglioside GM3 in Neural Cells

Abstract: Prosaposin, the precursor of saposins A, B, C, and D, was recently identified as a neurotrophic factor in vitro as well as in vivo. Its neurotrophic activity has been localized to a linear 12‐amino acid sequence located in the NH2‐terminal portion of the saposin C domain. In this study, we show the colocalization of prosaposin and ganglioside GM3 on NS20Y cell plasma membrane by scanning confocal microscopy. Also, TLC and western blot analyses showed that GM3 was specifically associated with prosaposin in immunoprecipitates; this binding was Ca2+‐independent and not disassociated during sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The association of prosaposin‐GM3 complexes on the cell surface appeared to be functionally important, as determined by differentiation assays. Neurite sprouting, induced by GM3, was inhibited by antibodies raised against a 22‐mer peptide, prosaptide 769, containing the neurotrophic sequence of prosaposin. In addition, pertussis toxin inhibited prosaptide‐induced neurite outgrowth, as well as prosaptide‐enhanced ganglioside concentrations in NS20Y cells, suggesting that prosaposin acted via a G protein‐mediated pathway, affecting both ganglioside content and neuronal differentiation. Our findings revealed a direct and right GM3‐prosaposin association on NS20Y plasma membranes. We suggest that ganglioside‐protein complexes are structural components of the prosaposin receptor involved in cell differentiation.

Role of autoimmunity in protein S deficiency during HIV-1 infection

SummaryThe objective of the study was to evaluate the role of autoimmune mechanisms in the pathophysiology of protein S deficiency during HIV-1 infection. In a prospective study the correlation between protein S activity and the presence of anti-protein S autoantibodies or anti-cardiolipin antibodies in HIV-1-positive patients and in a population of patients without HIV infection was investigated. Fifty-five HIV-1-infected patients and 15 hospitalized patients without HIV infection were analysed for protein S activity (functional assay), complement system activation, presence of autoantibodies against protein S (Dot Immunobinding) and levels of anti-cardiolipin IgG antibodies (ELISA). The presence of anti-protein S antibodies was detected in 31 (56.36%) out of the 55 HIV-1-positive patients and in three (20%) of the 15 control patients (Fishers exact test, p=0.012). The average value (± standard deviation) of protein S activity was 100.93 (14.73)% in the control group. For the HIV-1-infected patients it was 73.70 (20.67)% in those with anti-protein S antibodies compared to 88.08 (25.48)% in those without antibodies (Mann-Whitney U Test, p=0.01). In the HIV-1-positive group protein S activity was correlated with concentrations of circulating immune complexes (Spearman rank sum test, r=−0.41, p=0.018) and in the control group with concentrations of anti-cardiolipin antibodies (Spearman rank sum test, r=0.709, p=0.032). In conclusion, HIV-1 infection is associated with a high prevalence of antibodies against protein S. These antibodies are associated with a significantly low protein S activity. The exact correlation with plasma levels of these antibodies remains to be investigated.ZusammenfassungIn der vorliegenden Studie wurden Untersuchungen zur Bedeutung von Autoimmunmechanismen in der Pathophysiologie des Protein-S-Mangels bei HIV-1-Infektion durchgeführt. Die Beziehung zwischen Protein-S-Aktivität und Antikörpern gegen Protein S oder Kardiolipin wurde bei Patienten mit HIV-1-Infektion und ohne HIV-Infektion prospektiv untersucht. In einem funktionalen Assay wurde bei 55 HIV-1-infizierten Patienten und 15 stationären Patienten ohne HIV-Infektion Protein S bestimmt sowie die Aktivierung des Komplementsystems, Vorliegen von Autoantikörpern gegen Protein S (Dot Immunbindung) und IgG-Antikörperspiegel gegen Kardiolipin (ELISA) gemessen. Bei 31/55 HIV-1-positiven Patienten (56,36%) und bei 3/15 (20%) Kontrollpatienten waren Antikörper gegen Protein S nachzuweisen (Fishers exact test p=0,012). Die Protein-S-Aktivität betrug im Durchschnitt (± Standardabweichung) in der Kontrollgruppe 100,93 (14,73)%, bei HIV-Infizierten 73,70 (20,67)% bei Fällen mit Anti-Protein-S-Antikörpern und 88,08 (25,48)% bei Patienten ohne Anti-Protein-S-Antikörper (Mann-Whitney-U-Test p=0,01). In der HIV-1-positiven Gruppe korrelierte die Protein-S-Aktivität mit zirkulierenden Immunkomplexen (Spearman rank sum test r=− 0,41; p=0,018) und in der Kontrollgruppe mit Anti-Kardiolipin-Antikörpern (Spearman rank sum test r=0,709; p=0,032). Die HIV-Infektion ist folglich mit einer hohen Prävalenz von Antikörpern gegen Protein S und niedriger Protein-S-Aktivität assoziiert. Die genaue Korrelation dieser Antikörper mit Plasmaspiegeln muß noch bestimmt werden.

Autoantibodies Against Ganglioside GM3 Represent a Portion of Anti-Lymphocyte Antibodies in AIDS Patients

In this study we analysed the relationship between anti‐lymphocytic ganglioside antibodies and anti‐lymphocyte antibodies in AIDS patients. Anti‐lymphocytic ganglioside antibodies were detected by thin layer chromatography (TLC) immunostaining; three colour flow cytometry was used to analyse circulating antibodies against different lymphocyte subsets.

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.

Evidence for the existence of ganglioside molecules on Pneumocystis carinii from human lungs.

This study was undertaken to assess whether glycolipid antigens (particularly gangliosides) are associated with Pneumocystis carinii obtained from human lungs. Gangliosides were extracted, purified in high performance thin-layer chromatography and stained with resorcinol. Two resorcinol-positive bands, co-migrating with GM1 and GD1a were demonstrated, suggesting the existence of ganglioside molecules on P. carinii. No resorcinol-positive bands were revealed in the pulmonary control tissue. In addition, an antiserum obtained from rabbits immunized with P. carinii antigen reacted with gangliosides GM1 and GD1a, as revealed by a dot immunobinding assay. This reactivity was inhibited by first incubating the antiserum with ganglioside micelles. Furthermore, anti-glycosphingolipid antibodies (aGM1) reacted with the bands of 200 and 55 kDa of P. carinii antigen. These results suggest that ganglioside antigens expressed on P. carinii can trigger specific immune responses.

Prosaposin and prosaptide, a peptide from prosaposin, induce an increase in ganglioside content on NS20Y neuroblastoma cells.

Prosaposin has been recently identified as a neurotrophic factor eliciting differentiation in neuronal cultured cells (NS20Y). In this paper we investigate whether prosaposin and its active peptide (prosaptide) may modify the ganglioside pattern in neuroblastoma cells. The analysis by high performance thin layer chromatography did not reveal qualitative changes in the ganglioside pattern of NS20Y cells incubated in the presence of prosaposin, compared to control cells, but it did reveal an increase of the content of all three major resorcinol positive bands (GM3, GM2, GD1a). Cytofluorimetric and immunofluorescence microscopic analysis revealed that the increase of the ganglioside content was at the plasma membrane level. These findings suggest that the neurotrophic activity of prosaposin on NS20Y neuroblastoma cells might be mediated in part by the increase of cell surface gangliosides.

Overexpression of Lymphocytic GD3 Ganglioside and Presence of Anti-GD3 Antibodies in Patients with HIV Infection

This study was undertaken to analyze the role of disialoganglioside GD3 in HIV infection and disease progression. We report here the results obtained by both ex vivo and in vitro experiments on (1) surface and cytoplasmic expression and distribution of GD3 in HIV-infected cells, (2) the presence of anti-GD3 antibodies in sera of patients with HIV infection in various stages of the disease, and (3) the association of GD3 expression with HIV-related apoptotic events. GD3 expression was determined by high-performance thin-layer chromatography (HPTLC) and lipid-bound sialic acid and by static and flow cytometric analyses in peripheral blood lymphocytes from 22 AIDS patients, 20 anti-HIV Ab(+) asymptomatic subjects, and 25 healthy donors. Results obtained clearly indicated a significantly higher expression of plasma membrane GD3 content in lymphocytes from HIV-infected patients with respect to healthy controls. These HIV-induced perturbations of glycosphingolipid metabolism could be detected in all stages of the disease, including asymptomatic individuals. In addition, a significant percentage of patients showing disease progression displayed in serum samples an increased presence of anti-GD3 antibodies. Interestingly, ex vivo studies of lymphocytes from patients with HIV infection also indicated that GD3 expression is strictly associated with annexin V binding, an early marker of apoptosis. Moreover, cytofluorimetric analysis showed that virtually all anti-p24 Ab-positive cells were also immunolabeled with anti-GD3 antibodies. Accordingly, in vitro studies showed a significant redistribution and increase in GD3 expression in cultured U937 cells chronically infected with HIV-1 with respect to uninfected counterparts. In conclusion, our data clearly indicate that a significant increase in GD3 content in HIV-infected lymphocytes can occur and that this GD3 overexpression is paralleled by the presence of anti-GD3 antibodies in the plasma of patients. This is the first demonstration that disialoganglioside GD3, independent of the therapeutic schedule employed, can be considered as one of the early markers of HIV infection and can contribute to the early events leading to T cell depletion by apoptosis.

Undetectable phospho‐STAT1 in peripheral blood mononuclear cells from patients with chronic hepatitis C who do not respond to interferon‐α therapy

Abstract: Background: Recent studies have suggested that phosphorylated signal transducers and activators of transcription 1 (STAT1) plays an important role in interferon (IFN)‐mediated biological functions, including antiviral activity. Moreover, it has been demonstrated that suppressors of the cytokine signal 1 (SOCS1) negatively regulates IFN activities.

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.