Detection of antiphospholipid antibodies by immunostaining on thin layer chromatography plates.

There is increasing interest in the role of antiphospholipid antibodies in the so-called 'antiphospholipid antibody syndrome' (APS). The two major methods currently employed for detecting the autoantibodies are the solid phase ELISA and the LAI test (inhibition of phospholipid dependent coagulation assay). In our study we have tested the possibility of detecting antiphospholipid antibodies by immunostaining on thin layer chromatography (TLC) plates, since this technique permits the use of pure phospholipid molecules as antigen. Sera were collected from 20 patients with SLE without APS, 20 patients with APS, 20 anti-HIV positive subjects, ten patients with signs of APS but antiphospholipid negative (ELISA), 20 patients with syphilis and 40 matched blood donors. Results showed that only 72.3% of sera containing detectable levels of aCL antibodies in solid phase ELISA were also positive for aCL in TLC immunostaining; these discrepancies may be due to the presence of antibodies reacting with a protein complexed with phospholipid (beta 2-glycoprotein-I) or, alternatively, to the different antigenic presentation of phospholipids on chromatograms compared to the surface of microtitre wells. Furthermore, aCL monoclonal antibody CAL-3, as well as nine sera positive for aCL, also reacted with PS and PE. Previous absorption of these sera with CL micelles completely abolished the reactivity with PS and PE, demonstrating cross-reactivity among these three phospholipids. In conclusion, our findings reveal that TLC immunostaining is more specific, but less sensitive, than ELISA for the detection of antiphospholipid antibodies in human sera.

Anticardiolipin and anti-beta 2-GPI are two distinct populations of autoantibodies.

This study has been undertaken to assess whether anticardiolipin and anti-beta 2-GPI are two distinct populations of (auto)antibodies, and to clarify whether the beta 2-GPI region critical for phospholipid binding is also crucial for anti-beta 2-GPI reactivity. Fourteen of the 62 anticardiolipin (aCL) ELISA positive sera (22.6%) were positive for anti-beta 2-GPI by immunoblotting, 42 (67.7%) for aCL using TLC immunostaining. IgG fractions from 5 sera gave the same anticardiolipin reactivity detected by TLC immunostaining in the corresponding sera. All anti-beta 2-GPI-positive sera were reactive with the phenylthiocarbamyl derivative of the protein, indicating that binding of phenylisothiocyanate with lysine residues does not modify the molecule antigenicity. In addition, incubation of IgG fractions with the phospholipid binding site did not modify reactivity with beta 2-GPI. These findings demonstrate that: a) "true" antiphospholipid antibodies are detectable in patients' sera; b) aCL and anti-beta 2-GPI have a different immunological profile; c) the beta 2-GPI phospholipid-binding site is not the region recognized by the antibodies.

Inhibition of protein S by autoantibodies in patients with acquired protein S deficiency.

This study was undertaken to analyze antibodies to protein S (PS) in patients with an acquired PS deficiency. Plasma from symptomatic patients with acquired (n = 14) or congenital (n = 10) PS deficiency and 10 healthy donors was screened for PS antibodies by immunoblotting and for anti-phospholipid antibodies. PS antibodies (IgG) were detected in five of the patients with acquired PS deficiency. These antibodies belonged to the G1 and G4 immunoglobulin subclasses. IgG fractions from the same 5 patients were shown to inhibit PS activity. The inhibition of PS activity by the 5 IgG fractions was shown to be time- and dose-dependent and was abolished following incubation with purified PS, while no effect was found after absorption with cardiolipin micelles. In addition, anticardiolipin monoclonal or human purified antibodies, failed to exert significant PS inhibition. These findings demonstrate that anti-PS antibodies are able to inhibit PS activity and that this is independent of anti-phospholipid antibodies. Given the clinical features of the patients, these antibodies should be regarded as an expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins.

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.

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.

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

The 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 (Fisher's 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 (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.(ABSTRACT TRUNCATED AT 250 WORDS)

GM3 as a target of anti-lymphocytic ganglioside antibodies in AIDS patients.

IgG antibodies reacting with the GM3-comigrating band extracted from pooled AIDS lymphocytes were detected in 33.3% of AIDS patients sera, in 8% of asymptomatic anti-HIV-positive subjects, in none of the sera obtained from asymptomatic anti-HIV-negative drug abusers, from patients with acute B and chronic C hepatitis, and from healthy donors. All positive sera reacted selectively with the GM3-comigrating band obtained from AIDS lymphocytes but not with the corresponding band from normal lymphocytes. The lymphocytic ganglioside autoantigen was revealed as GM3. In addition, two main data were shown: (a) AIDS lymphocytes have an increased concentration of GM3 and (b) the ceramide of AIDS lymphocytic GM3 has a different percentual composition of fatty acids in contrast to control cells. It is suggested that these quantitative and qualitative changes might be responsible for the appearance of circulating anti-lymphocytic GM3 antibodies.

Characterization of autoantibodies to natural killer cells in HIV-infected patients.

Natural killer (NK) cells in HIV-infected patients have a reduced ability to generate non-MHC restricted cytotoxicity to a variety of target cells. The authors investigated antibodies to NK cells in HIV-infected patients and evaluated effects of these antibodies to NK cell numbers and function. Antibodies to NK cells were determined in 160 HIV-infected patients and 35 healthy controls. Flow cytometric whole blood methods were developed to detect antibodies to NK cells. Antibodies to asialo-GM1 were detected by TLC immunostaining. The presence of antibodies to NK cells was demonstrated in plasma of about one-third (54/160) of HIV-infected patients but rarely in controls (2/35). Auto-antibodies bound to NK cells in vivo and were detected by a strong increase of surface immunoglobulin (Ig) on NK cells of HIV-infected patients. Anti-NK cell antibodies were warmreactive antibodies rather of IgG than of IgM phenotype. The prevalence of specific antibodies to asialo-GM1 was low (12.5%). Numbers of circulating NK cells did not differ significantly between antibody positive (99.5/microliters) and antibody negative (141/microliters) patients (P = 0.3). However, pre-incubation of healthy donors' NK cells with autoantibody positive plasma significantly inhibited cytotoxicity to K562 leukaemic cells (P = 0.002). Autoantibodies to NK cells in HIV-infected patients are present in the plasma of one-third of HIV-infected patients and are bound to NK cells in vivo. There is evidence that these autoantibodies can induce NK cell defects similar to those seen in vivo.

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.

Cerebrospinal fluid antiganglioside antibodies in patients with AIDS.

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.

Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies.

The antigen specificity of anti-phospholipid antibodies in infectious mononucleosis (IM) was studied using ELISA for the detection of anti-beta2-glycoprotein I (beta2-GPI), anti-annexin V, anti-protein S and anti-prothrombin antibodies and TLC immunostaining for the detection of anti-phospholipid antibodies. This technique enabled us to look at antibodies reacting to 'pure' phospholipid antigens in the absence of protein contamination. Sera from 46 patients with IM, 18 with systemic lupus erythematosus (SLE), 21 with primary anti-phospholipid antibody syndrome (PAPS), 50 with Helicobacter pylori infection and 30 healthy blood donors were tested. This study highlights anti-phospholipid antibodies in patients with IM as specific 'pure' anti-cardiolipin antibodies, while in PAPS and SLE patients anti-phosphatidylserine and anti-phosphatidylethanolamine antibodies were also found. This investigation also shows that the anti-cardiolipin antibodies found in IM can be present with anti-cofactor protein antibodies. The higher prevalence of anti-cofactor antibodies found in IM sera than in Helicobacter pylori sera may be due to the immunostimulatory effect and/or the polyclonal activation often observed in course of Epstein-Barr virus infection. However, anti-beta2-GPI and, to a lesser extent, anti-prothrombin antibodies occur with a significantly lower prevalence in IM than in PAPS patients. This finding suggests that these antibodies should be regarded as the expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins.

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.

Evidence for the existence of ganglioside molecules in the antigen of Entamoeba histolytica.

Gangliosides were found to be present in Entamoeba histolytica. They were extracted from lyophilized trophozoites of the pathogenic strain HM-1:IMSS and purified by high performance thin-layer chromatography. Two resorcinol-positive bands, comigrating with GM2 and GD1a were demonstrated, revealing the existence of ganglioside molecules in Entamoeba histolytica. The GM2 content, determined as lipid-bound sialic acid, was 1.5 micrograms/10(8) amoebae, the content of the GD1a comigrating band was 0.32 microgram/10(8) amoebae. The identity of the GM2 comigrating band was confirmed by TLC immunostaining, using the monoclonal anti-GM2 antibody GMB28. Furthermore, six out of ten anti-amoeba positive sera selectively reacted with the GM2 comigrating band, as revealed by immunostaining on TLC plates. Absorption tests revealed that preincubation of anti-amoeba positive sera with standard GM2 was followed by a significant decrease in the reaction with amoeba trophozoites by indirect immunofluorescence. These results demonstrate that a GM2 comigrating component of Entamoeba histolytica may be one of the antigens responsible for the appearance of circulating antibodies in patients with amoebiasis.

Beta-2-glycoprotein I expression on monocytes is increased in anti-phospholipid antibody syndrome and correlates with tissue factor expression.

It is well known that monocytes may play an active role in thrombogenesis, since they may express on their surface tissue factor, the major initiator of the clotting cascade. The results of this investigation demonstrate beta-2-glycoprotein I (beta2-GPI) mRNA expression by human peripheral blood monocytes, indicating that these cells synthesize beta2-GPI. In addition, we show beta2-GPI expression on cell surface of these cells by flow cytometric analysis, and the presence of this protein in cell lysate by Western blot. Interestingly, beta2-GPI expression on monocytes is significantly increased in patients with anti-phospholipid syndrome (APS) or systemic lupus erythematosus (SLE) as against healthy blood donors and correlates with tissue factor expression on monocytes. These findings support the view that monocytes are able to synthesize beta2-GPI and suggest that patients with APS may have increased beta2-GPI exposure on cell surface, which leads to persistently high monocyte tissue factor expression and consequently to a prothrombotic diathesis.

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. Anti-lymphocytic ganglioside antibodies were detected in 23 out of 49 AIDS patients sera (46.9%). All positive sera reacted selectively with the GM3 comigrating band from AIDS lymphocytes. Twenty two out of the 23 anti-lymphocytic GM3 positive sera also had antibodies against CD4+T cells, versus 17/26 anti-GM3 negative. Furthermore, patients with lymphocytic GM3 antibodies showed a significantly higher antibody reactivity against CD4+ T cells than patients in which these antibodies were not detected. The absorption tests revealed that preincubation of positive sera with GM3 was followed by a decrease in the reaction with target lymphocytes. These findings suggest that anti-GM3 antibodies are a portion, but not the majority, of antibodies reacting with CD4+ T cells.

Colocalization and complex formation between prosaposin and monosialoganglioside GM3 in neural cells.

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 tight GM3-prosaposin association on NS20Y plasma membranes. We suggest that ganglioside-protein complexes are structural components of the prosaposin receptor involved in cell differentiation.