Anti-phospholipid antibodies in serum from patients with Guillain-Barré syndrome.

OBJECTIVE: The Guillain-Barré syndrome (GBS) is an acute inflammatory polyneuropathy related to autoimmunity. However, no conclusive etiological concept has yet been found. We examined the variation in autoantibodies to lipids in serum of GBS patients in response to the course of the disease but investigated titer modifications during treatment with gamma-globulin. DESIGN AND SETTING: Prospective clinical study in a 14-bed general ICU. PATIENTS: Nine patients with GBS and nine controls were included in the study. MEASUREMENTS AND RESULTS: Four blood samples were obtained before and after treatment. Serum samples, diluted 1:60, were tested by enzyme-linked immunosorbent assay for IgM, IgA, and IgG antibodies to phosphatidylcholine, phosphatidylinositol, cardiolipin, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine, sphingomyelin, and gangliosides. Anti-phospholipid antibodies of the IgM, IgA, and IgG families were detected in all GBS patients but in none of the controls. Phosphatidylinositol, cardiolipin, phosphatidylcholine, and phosphatidic acid were the main antigens. All patients developed anti-phosphatidylinositol antibodies of the IgM family and anti-cardiolipin antibodies of the IgA and IgG families. A decrease in the level of anti-phospholipid autoantibodies was observed after 1 day of treatment with gamma-globulin. Two days after ending gamma-globulin administration the IgG antibodies increased again. CONCLUSIONS: Our findings suggest that in GBS there is an extensive immune reaction, which is altered after gamma-globulin treatment. Anti-cardiolipin and anti-phosphatidylinositol antibodies could be useful markers for the response to treatment.

Autoantibodies to lipids in bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome.

OBJECTIVE: To investigate the presence of autoantibodies to lipids in the bronchoalveolar lavage (BAL) fluid from adult patients with acute respiratory distress syndrome (ARDS). DESIGN: Analysis of immunoglobulin G (IgG) in BAL fluid by electrophoresis followed by immunoblotting and characterization of immunoglobulins as antilipid autoantibodies. SETTING: Intensive care unit of a university hospital and two research university laboratories. SUBJECTS: Twenty-seven mechanically ventilated patients in total, including nine patients with ARDS and two control groups. INTERVENTIONS: Patients were ventilated with a mechanical ventilation mode. Six aliquots of 20-mL sterile normal saline at 37 degrees C were infused through the working channel of the bronchoscope. MEASUREMENTS: Total protein, detection of IgG by electrophoresis followed by immunoblotting, and characterization of IgG by enzyme-linked immunosorbent assay using different lipids as target antigens. MAIN RESULTS: Antiphospholipid autoantibodies are present in BAL fluid of ARDS patients. Among the phospholipids tested, phosphatidic acid and phosphatidylserine gave the most significant activity. The IgG fraction, purified from BAL fluids by affinity chromatography, gave the same pattern of binding as that of the BAL fluid. CONCLUSION: The presence of antiphospholipid autoantibodies in BAL fluid suggests involvement of autoimmune mechanisms in the pathogenesis of ARDS.

Phosphatidylserine delivery to endoplasmic reticulum-derived vesicles of plant cells depends on two biosynthetic pathways.

Vesicles formed from endoplasmic reticulum (ER) by a cell-free system of leek cells (Allium porrum) are enriched in phosphatidylserine (PS), especially species containing very long chain fatty acids (VLCFA, at least 20 carbon atoms). In plant cells, PS is formed either by PS synthase or the serine exchange enzyme, although it is not known which pathway(s) contribute(s) to PS delivery in the ER-derived vesicles (EV), nor to what extent this occurs. Taking advantage of a cell-free system, we have shown that PS enrichment originates mainly from the serine exchange enzyme which is the only pathway that synthesizes the VLCFA-PS species. On the other hand, both enzymes synthesize PS with long chain fatty acids (up to 18 carbon atoms), but these species are given to the EV by PS synthase.

The characteristics of bronchoalveolar lavage from a patient with antiphospholipid syndrome who developed acute respiratory distress syndrome.

The purpose of this study was to investigate the biochemical characteristics as well as the occurrence and specificity of antiphospholipid antibodies in the bronchoalveolar lavage (BAL) fluid from a patient with both antiphospholipid antibodies syndrome (APS) and acute respiratory distress syndrome (ARDS). Proteins, lipids, cells and autoantibodies were determined. Immunoglobulins were purified with affinity chromatography. Autoantibody identification was assessed with enzyme-linked immunosorbent assay (ELISA) and with electrophoresis, followed by immunoblotting and revelation with antihuman IgG-peroxidase conjugate. Antiphospholipid antibodies were found to be present in the BAL fluid as well as in the serum from a patient with APS. Specifically, antiphosphatidylserine and antiphosphatidic acid IgG antibodies in the BAL fluid and antiphosphatidylcholine and anticardiolipin IgG antibodies in the serum were detected at high levels. BAL fluid protein and the percentage of neutrophils were found to be increased. A quantitative as well as qualitative deficiency of surfactant phospholipids was also observed. Antibodies directed against surfactant phospholipids could cause surfactant abnormalities and an inflammatory reaction. These disorders may be one of the causes of the ARDS or a factor in the perpetuation of the inflammation.

One of the origins of plasma membrane phosphatidylserine in plant cells is a local synthesis by a serine exchange activity.

In plant cells, as in animal cells, the endoplasmic reticulum (ER) is considered to be the major site of phospholipid synthesis, and it has been shown that phosphatidylserine (PS) reaches the plasma membrane via the vesicular ER-Golgi-plasma membrane pathway in leek cells. However, it has never been determined whether the plasma membrane of leek cells is able to synthesize PS. We have analyzed the distribution of PS synthesizing enzymes along the vesicular pathway. In ER, Golgi and plasma membrane fractions isolated from leek cells, we have measured the activity of the two biosynthetic pathways leading to the synthesis of PS, i.e. serine exchange and CTP cytidylyltransferase plus PS synthase. We have found a high serine exchange activity in the plasma membrane fraction, and then determined that this membrane is able to synthesize both long chain fatty acid- and very long chain fatty acid-containing PS. Therefore, the PS in the plasma membrane of leek cells has two different origins: the intracellular vesicular pathway from the ER and a local synthesis in the plasma membrane.

Immunocytochemistry of lipids: chemical fixatives have dramatic effects on the preservation of tissue lipids.

We report here the effects of chemical fixatives on lipids studied under conditions simulating the immunogold labelling of phosphatidylserine. Using anti-phosphatidylserine antibodies, it is shown that the labelling intensity of a phosphatidylserine/phosphatidylcholine coating depends largely on the conditions of fixation. In fact, the usual aldehydic fixatives washed out most of the phostphatidylserine, thus preventing the binding of anti-phosphatidylserine antibodies. This was confirmed on biological samples such as rat liver and brain by measuring the loss of radiolabelled lipids during the fixation procedure. Furthermore, the complete procedure of tissue preparation for electron microscopical observation was investigated. The loss of (radiolabelled) lipids was studied in tissue samples during fixation and resin embedding. The results showed that the classical procedure (glutaraldehyde fixation followed by epoxy resin embedding) results in the loss of 73-91% of the tissue lipids whereas in unfixed, freeze-substituted samples, more than 76% of the tissue lipids are preserved.

ATP-Dependent formation of phosphatidylserine-rich vesicles from the endoplasmic reticulum of leek cells

Leek (Allium porrum) plasma membrane is enriched in phosphatidylserine (PS) by the vesicular pathway, in a way similar to that already observed in animal cells (B. Sturbois-Balcerzak, D.J. Morre, O. Loreau, J.P. Noel, P. Moreau, C. Cassagne [1995] Plant Physiol Biochem 33: 625-637). In this paper we document the formation of PS-rich small vesicles from leek endoplasmic reticulum (ER) membranes upon addition of ATP and other factors. The omission of ATP or its replacement by ATPgamma-S prevents vesicle formation. These vesicles correspond to small structures (70-80 nm) and their phospholipid composition, characterized by a PS enrichment, is compatible with a role in PS transport. Moreover, the PS enrichment over phosphatidylinositol in the ER-derived vesicles is the first example, to our knowledge, of phospholipid sorting from the ER to ER-derived vesicles in plant cells.

Antibodies to long-chain acyl-CoAs. A new tool for lipid biochemistry.

Antibodies directed against long chain acyl-CoAs (having 16 and 18 carbon atoms) have been prepared and are reported for the first time. A modified ELISA procedure adapted to these amphiphilic molecules has been developed: it is a rapid, simple and sensitive test permitting to detect as little as 3 pmol of acyl-CoA. These antibodies represent a new tool for studying long-chain acyl-CoAs. Their use in an immunochemical approach for the study of protein-acyl-CoA interactions is presented.

Immunogold labelling of fatty acyl chains.

For the first time, antibodies against a hydrophobic hapten have been used for immunogold labelling of a lipid antigen (BSA-C18:1 conjugate) coated on polystyrene. The labelling was visualised either directly in transmission electron microscopy or in light microscopy after silver enhancement. Good recognition of the fatty acyl chain was obtained even after treatment of the antigen coat with various cross-linking fixatives used for electron microscopy, i.e. formaldehyde, glutaraldehyde and osmium tetroxide.

Cell-free transfer of phospholipids between the endoplasmic reticulum and the Golgi apparatus of leek seedlings.

The transfer of lipids between the endoplasmic reticulum and the Golgi apparatus was investigated in vitro using a cell-free system from leek seedlings. Lipids of the donor membranes (endoplasmic reticulum) were radiolabeled either by incubating leek seedlings with [1-14C]acetate or [3H]acetate. Acceptor membranes (Golgi apparatus) were unlabeled and immobilized on nitrocellulose strips. The assay measured the lipid transfer resulting from both an ATP-independent process and an ATP- and cytosol-dependent process. A significant ATP- and cytosol-dependent lipid transfer was observed only in the case of the endoplasmic reticulum as donor and the Golgi apparatus as acceptor. Lipids transferred in an ATP-dependent manner were chiefly phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The stimulation of lipid transfer by ATP as compared to the ATP-independent process was +79% (PC), +123% (PS) and +69% (PE). On the other hand, PI was not transferred in an ATP-dependent manner (the stimulation by ATP was only 20%). This supports the theory that a sorting of phospholipids takes place in the donor membrane. Moreover, a formation of lysoPC was observed only in the presence of ATP (+330%). The ATP-dependent lipid transfer was inhibited by N-ethylmaleimide, indicating the involvement of cytosolic (but no phospholipid transfer proteins) or membrane proteins in the transfer process. The ATP-dependent transfer of lipids was also diminished at 12 degrees C showing the sensitivity to low temperatures of the transfer of lipids between the endoplasmic reticulum and the Golgi apparatus.

Demonstration that anti-phospholipid auto-antibodies react with both anionic and zwitterionic phospholipids.

The specificity of anti-phospholipid auto-antibodies present in the serum of systemic lupus erythematosus patients towards the phospholipids has been studied by two different methods. The antibodies have been characterized either after affinity purification or by inhibition experiments. Our results clearly demonstrate that anti-phospholipid auto-antibodies recognize all phospholipids, whatever their polar head.

Fatty acids rendered immunogenic.

A method allowing the covalent binding of fatty acids to a carrier was developed. The absence of absorbed fatty acids was controlled by HPTLC. Rabbits were immunized with these immunogens. The antibodies obtained were studied by ELISA and were shown to be directed against the fatty acyl chain, whatever its length.

Synthesis of a monoclonal antibody-indium-111-porphyrin conjugate.

Antibodies were labelled with indium-111 with a view to their use in the radio-immunodetection of cancers. The covalent coupling between indium-111 porphyrin and monoclonal antibodies (IgG and F(ab')2 fragment) was achieved using the ester activated method [N-hydroxy-succinimide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide]. After purification, this provided conjugated with specific activities of 6 muCi/micrograms Mab (9.3 molecules per Mab) or 1 muCi/microgram (F(ab')2 fragment (1.5 molecule per F(ab')2). ELISA procedures suggested the full retention of immunoreactivity by the radiolabelled antibodies.

Autoantibodies against all the phospholipids: a comparative systematic study with systemic lupus erythematosus and healthy sera.

The sera of systemic lupus erythematosus patients were tested by ELISA for the presence of autoantibodies against all the phospholipids: cardiolipin, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylserine. The quantity of phospholipid coated (in comparison with that initially deposited) on the microtitration plates was precisely evaluated in order to determine if the results (absorbance values) obtained for each phospholipid could be compared directly. The systemic lupus erythematosus sera tested gave positive results for all the phospholipids. The highest level of autoantibodies was observed with phosphatidic acid followed by phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine. The sera seemed to contain antibodies directed either against all the 8 phospholipids tested or more specifically against one or two phospholipids. The results were compared with those obtained with 17 healthy sera. Much lower values were obtained for the sera of healthy subjects, the majority of which showed a weak binding, similar for all the phospholipids. These results suggest that the anti-phospholipid autoantibodies present in systemic lupus erythematosus sera are significantly higher than those of healthy subjects. It is concluded that in the investigation of anti-phospholipid antibodies, tests should be carried out for all the phospholipids.

Specific immunocytochemical visualization of phosphatidylserine.

In order to determine whether anti-phosphatidylserine antibodies are able to detect phosphatidylserine in situ, an immunocytochemical approach has been developed using human platelets. A strong positive reaction was obtained when anti-phosphatidylserine serum was applied to platelets whereas no reaction was observed with preimmune serum, or with immune serum which had been preincubated with 10(-5) M phosphatidylserine, suggesting that phosphatidylserine was indeed specifically detected by the antibodies.

Demonstration of high specificity antibodies against phosphatidylserine.

Anti-phosphatidylserine antibodies were raised in rabbits immunized with phosphatidylserine-polyacrylamide gels and with phosphatidylserine-cytochrome c vesicles. A solid-phase immunoenzymatic assay (ELISA) was developed to study the immune serum. The optimal conditions were defined and the technique used to obtain quantitative results. The anti-phosphatidylserine serum diluted to 1/1000 was tested against several phospholipids and was found to be highly specific to phosphatidylserine. This represents the first experimental demonstration of the specificity of antibodies raised against phosphatidylserine.

Natural seric anti-fatty acid antibodies in multiple sclerosis.

Naturally occurring anti-oleic acid conjugate antibodies were detected in human sera using an adapted direct immunoenzymatic assay. They were present to a higher level in the sera of patients with multiple sclerosis in acute relapse compared to patients with other neurological diseases or healthy subjects and even patients with multiple sclerosis in progressive form.

Membrane labeling of the cortex of 'aging' rats by anti-fatty acid antibodies.

The cortex of 'aging' rat brain exhibits no significant difference in the lipid and fatty acid composition in comparison with that of control rat brain, except for a lowered value of the cholesterol/phospholipids ratio. However, an antiserum raised against oleic acid, specifically labels neurones of the cortex of the 'aging' rat mainly within layers IV and V. An electron microscopical study revealed that immunoreactivity was associated with cytoplasmic vesicular inclusions (lipofuscin) and with membranes. Thus, these anti-fatty acid antibodies may help in the evaluation of local modifications of membranes which are not predictable on the basis of biochemical analysis of lipids.

Antibodies to histamine. Specificity studies and radioimmunological assay.

Antibodies against conjugated histamine were raised in rabbits. This amine was coupled to different protein carriers by a bifunctional agent, hexamethylene diisocyanate. The specificity of the antibodies was determined with radioimmunological tests in equilibrium dialysis using an iodinated ligand: 125I-labelled histamine-hexamethylene diisocyanate-glycyl-tyrosine. The latter mimicked the antigenic determinant present in immunogens. Competition experiments were established between the radiolabelled ligand and conjugated histamine, conjugated analogs or unconjugated histamine. Cross-reactivity ratios and affinity constants were calculated from displacement curves, thereby allowing the antibody site to be characterized. The antibodies were found to be highly specific and were used for the assay of histamine in biological samples. For this, polystyrene beads coated with purified antiserum were used to establish a simple and reproducible test.