Autoantibodies directed against ribosomal proteins in systemic lupus erythematosus and rheumatoid arthritis: a comparative study.

To assess the specificity of autoantibodies (aAbs) directed against the ribosomal P-proteins (RPPaAbs) in patients with systemic lupus erythematosus (SLE) and to investigate aAbs directed to other ribosomal proteins, 100 SLE, 100 rheumatoid arthritis (RA), 25 thyroiditis and 20 blood-donors were analyzed in a comparative study using an immunoblotting technique. Forty-eight percent of SLB sera contained aAbs directed against the ribosomal proteins of the 60 S subunit compared to 9% for RA, 5% for blood donors and 0% for thyroiditis. RPPaAbs were only found in SLE (25%) and aAbs directed to a 31 kDa and/or a 28 kDa protein of the 60 S subunit were found with a statistically higher frequency for SLE compared to RA (p < 0.0001). aAbs directed to proteins of the 40 S subunit were present in 63% of the SLE sera compared to 42% for RA, 4% for thyroiditis and 5% for blood donors. The number of positive sera was not statistically different between SLE and RA but a much more intense reactivity was observed for SLE sera. These data shows that the aAbs against the ribosomal proteins, especially the P-proteins along with the 28 and 31 kDa proteins of the 60 S subunit proteins, can be considered as useful biological markers for t he diagnosis of SLE inclinical practice.

Detection on immunoblot of new proteins from the soluble fraction of the cell recognized either by anti-liver-kidney microsome antibodies type 1 or by anti-liver cytosol antibodies type 1--relationship with hepatitis C virus infection.

Antibodies directed against liver cytosol protein, called anti-liver cytosol type 1 (LC1 Ab), have been described by both immunofluorescence (IF) and immunodiffusion techniques in sera from patients with autoimmune hepatitis (AIH). They have never been found in association with antibodies directed against the hepatitis C virus (HCV), unlike the anti-liver-kidney microsome antibodies type 1 (LKM1 Ab), the serological marker of AIH type 2. This suggests that there are two subgroups of AIH type 2, i.e., HCV-related and non-HCV-related. In this study, immunoblotting experiments were performed using proteins from the soluble phase of the rat liver cell; 141 sera which tested positive for LKM1 Ab by IF, 24 identified as having LC1 Ab by IF, and 50 from blood donors as controls were analyzed. Three bands were stained by LC1 Ab sera more often than by the control sera, and with a statistically significant frequency. These 3 proteins were located at apparent Mr 50,000, 55,000, and 60,000. The LKM1 Ab-positive sera as defined by IF stained six bands with a statistically significant frequency compared to the controls. Their apparent Mr were 35,000, 39,000, 47,000, 50,000, 55,000, and 60,000. LKM1 Ab-positive sera which were anti-HCV negative recognized a 60,000 protein belonging to the soluble phase of the cell, with a statistically significant frequency compared to LKM1 Ab-positive sera which were anti-HCV positive. This 60,000 protein was also recognized by LC1 Ab-positive sera, which were almost always anti-HCV negative. The presence of antibodies against a 60,000 protein from the soluble phase of the cell is discussed in terms of the anti-HCV serological markers found in the sera from patients with AIH.

Detection on immunoblot of new proteins from the microsomal fraction recognized by anti-liver-kidney microsome antibodies type 1.

Previous studies have demonstrated that sera from patients with autoimmune hepatitis type 1 contain antibodies which react with proteins other than the endoplasmic reticulum integral membrane protein of apparent Mr 50,000, now known to be a cytochrome P450 of the IID subfamily. Sera from 141 patients found by immunofluorescence to be positive for anti-liver-kidney microsome antibodies type 1, and sera from 50 blood donors used as controls, were analyzed by immunoblotting experiments on rat liver microsomes, microsomal subfractions, and also microsomes subjected to various treatments, as described in the text. These fractions were characterized morphologically by electronic microscopy and biochemically by different enzymatic activities. Five bands were found to be stained more often by the patients' sera than by the controls' and with a statistically significant difference in frequency. These antigenic proteins were located at apparent Mr 62,000, 58,000, 50,000, 40,000, and 35,000. The 50,000 protein was of course more often stained than the others. Antibodies against these antigens belonged essentially to the IgG1 subclass. For some of them, subcellular localization and membrane topography are discussed. Interestingly, the 58,000 protein is not an integral membrane protein.

Use of human sera containing autoantibodies for an immunochemical study of some ribosomal proteins in rat, trout, mussel and fly maggot.

Sera from human subjects affected by autoimmune connective tissue diseases and containing antiribosomal autoantibodies were used to analyze by immunoblotting ribosomal proteins from trout (Oncorhynchus mykiss) liver, mussel (Mytilus edulis) hepatopancreas and whole fly maggots (Calliphora vomitoria). As usual in medical analysis of autoantibodies, the reference antigen preparation was extracted from rat liver. With the used sera, six known ribosomal proteins from rat liver were characterized: P0, P1, P2, p30, p25 and p20. These six proteins were all targeted in trout; moreover an important 40 kDa fraction, undetectable in rat pattern, was seen. p30 and p20 were undetected in mussel and fly maggot; but p25, undetected in mussel, is clearly characterized in fly maggot. The interest of these data to infer phylogenic relationships is discussed.

In-vitro concentration of azithromycin in human phagocytic cells.

The in-vitro intraphagocytic uptake and retention of azithromycin in both human polymorphonuclear leucocytes (PMN) and alveolar macrophages was measured by an improved high-performance liquid chromatography (HPLC) method that was approximately three-fold more sensitive than previous methods. Azithromycin was accumulated in PMN and alveolar macrophages (about 300-fold), with maximum uptake being obtained after incubation for 60 min. Azithromycin was eliminated only partially from the cells during the washing process, and was released slowly during re-incubation of phagocytic cells in antibiotic-free medium. This intracellular retention distinguishes azithromycin from most of the macrolides and quinolones which, in spite of high I/E ratios, are released rapidly from cells.

Accumulation of pefloxacin in the lower respiratory tract demonstrated by bronchoalveolar lavage.

The in-vivo pulmonary disposition of pefloxacin in alveolar macrophages alveolar macrophages and in the alveolar epithelial lining fluid recovered by bronchoalveolar lavage was studied in 10 healthy volunteers. Bronchoalveolar lavage was performed either 2 or 4 h after oral intake of 800 mg of the drug. The recovered fluid was immediately centrifuged and processed for the assays. Pefloxacin was assayed by High Pressure Liquid Chromatography (HPLC) and by a microbiological method. The mean concentrations of pefloxacin assayed by HPLC were 106 +/- 11.1 mg/L in alveolar macrophages and 88.2 +/- 10 mg/L in the epithelial lining fluid, whereas the mean serum concentration was 6.67 +/- 0.47 mg/L. Therefore, pefloxacin accumulated rapidly in human alveolar macrophages. The high epithelial lining fluid concentrations may be attributed to lipophilicity of the drug and to rapid diffusion from blood, pulmonary cells and interstitium during the bronchoalveolar lavage procedure. The substantial accumulation of pefloxacin in alveolar components (alveolar macrophages and epithelial lining fluid) endorses its use in the treatment of intracellular bacterial infections such as legionellosis; for these diseases, pefloxacin represents an alternative to the macrolide antibiotics.

Impaired G-proteins and cyclic nucleotide phosphodiesterase activity in T-lymphocytes from patients with sarcoidosis.

Among the various immune abnormalities which characterize active sarcoidosis, a low proliferative response of peripheral blood lymphocytes to mitogenic lectins has long been observed. Since membrane-associated G-proteins are very likely to be crucial elements in lectin signal transduction, we investigated the binding of 5'-guanylylimidodiphosphate (GppNHp), a non hydrolyzable GTP analogue, to blood total lymphocyte membranes and to blood T-lymphocyte membranes from patients with active sarcoidosis, and from healthy control subjects. GppNHp binding was markedly decreased in peripheral cells from patients with sarcoidosis as compared to controls, suggesting the occurrence of a defect at the level of G-protein(s). A further characterization of G-proteins in these cells by means of ADP-ribose-labelling in the presence of bacterial toxins brought forward a significant decrease in the labelling of a 40 kDa protein, the major pertussis toxin substrate, in membranes from sarcoid patients, while the labelling of the major 44 kDa cholera toxin substrate proved to be unchanged with respect to control membranes. It is hypothesized that, in sarcoid lymphocytes, a defect in the negative control of adenylate cyclase mediated by the inhibitory G-protein Gi, prevents the lowering of cAMP necessary to normal mitogenic response of blood lymphocytes to stimulation. cAMP degradation by the specialized enzyme phosphodiesterase constitutes another critical step in the control of cAMP levels. Both cAMP and cGMP phosphodiesterase activities were found decreased in blood total lymphocyte preparations from sarcoid patients. With purified T-cells, although the mean cAMP and cGMP phosphodiesterase activities from sarcoid patients were found more markedly decreased with respect to healthy donors, only the decrease in cGMP phosphodiesterase was found statistically significant. The role these defects in cyclic nucleotide degradation potentially play in the disturbance of blood lymphocytes response associated with sarcoidosis is discussed.

Autoantibodies directed against the ribosomal P proteins are not only directed against a common epitope of the P0, P1 and P2 proteins.

The autoantibodies (aAbs) directed against the ribosomal P proteins (RPP aAbs) are known to react mainly against epitopes localized within the common C-terminal sequence of the three acidic ribosomal P proteins, P0, P1 and P2. In order to investigate the opportunity to select short recombinant peptides of this common C-terminal sequence to detect the RPP-aAbs, the location of the epitopes recognized by ribosomal proteins (RP) aAb(+)sera of systemic lupus erythematosus patients (SLE) was investigated. Immunoblotting and ELISA techniques using extracted or recombinant, entire or cleaved RPP showed that 55% of the RP aAbs were directed against the three ribosomal P0, P1, and P2 proteins. The epitopes recognized by the RPP aAbs are located not only within the C-terminal sequence common to the three proteins but also within the N-terminal sequence of the P2 or P1 protein. The other RP aAbs sera (45%) did not react with all three proteins but with some of them, and showed the following pattern: P0(+)P1(+); P1(+); P2(+); P0(+)and P1(+). They recognized epitopes located in the region of the C-terminal sequence of the protein but not common to the three proteins. In addition two out of the six monoclonal Abs produced by immunization of mice using the P1 protein did not react with the peptide N-65 or N-71 of the P2 protein or with the C-terminal sequence of the three proteins. In conclusion, this study showed that the RPP aAb in SLE patients are not only directed against epitopes within the C-terminal sequence shared by the three acidic ribosomal P proteins. In view of these data it seems necessary to be cautious in using only a C-terminal peptide of ribosomal P proteins in tests performed to detect RPP aAb in human sera.