Immunochemical and molecular characterization of regulatory idiotopes expressed by monoclonal antibodies exhibiting or lacking beta 2-6 fructosan binding activity.

Hybridomas secreting antibodies bearing the ABPC48 (A48) regulatory idiotype (Id) were generated from BALB/c mice treated at birth or as adults with minute amounts of anti-A48-Id antibodies. The majority of these antibodies were recognized by the syngeneic monoclonal anti-A48-Id and anti-UPC-10-Id antibodies, IDA10 and 10-1, respectively. In Northern blotting experiments, most of these hybridomas were shown to use VH (heavy chain variable region) genes related to the 441-4 germline VH gene that encodes the A48 VH region. Hybridization was detected between polyadenylated H chain mRNA, isolated from the majority of the hybridomas, and the VH probe. Southern blots confirmed these results by showing a rearrangement of VH-related sequences to the JH (H chain joining segment) clusters on these same hybridomas. The antibodies from all of the hybridomas that derived from neonatal mice and half of those derived from adult mice showed specificity for fructosan determinants that, in most cases, was different from the beta 2-6 fructosan linkage specificity of A48. Surprisingly, several of the non-fructosan-binding hybridomas generated from the adult mice and the MOPC-173 myeloma demonstrated a clear specificity for the beta 1-6-D-galactan determinant. Of four galactan-binding myeloma proteins studied. XRPC 44 alone shared idiotypy with the UPC-10 myeloma. These findings suggest a possible clonal crossreactive regulation mediated by regulatory idiotopes. The crossreactive regulation concept is discussed.

High frequency of autoantibodies bearing cross-reactive idiotopes among hybridomas using VH7183 genes prepared from normal and autoimmune murine strains.

Hybridomas obtained by in vitro stimulation with lipopolysaccharides (LPS) of BALB/c, MRL/lpr, and NZB splenocytes were selected for expression of VH7183 by hybridization using slot blotting. Northern blot analysis showed that the majority of hybrids produce a full length message complementary to the VH7183 probe. The frequency of VH7183 hybridomas was significantly higher in NZB mice as compared with BALB/c mice. Using multiple binding assays, 60% of the total antibodies encoded by VH7183 were specific for self-epitopes. Finally, the vast majority express cross-reactive idiotypes borne by autoantibodies of various specificities.

Shared idiotypes and restricted immunoglobulin variable region heavy chain genes characterize murine autoantibodies of various specificities.

The study of the Ig variable region heavy chain (VH) genes used to encode antibodies specific for self-epitopes from murine hybridomas showed that three VH families are primarily utilized: VH J558, the largest family, and VH QPC52 and VH 7183, the families most proximal to the Ig joining region heavy chain genes. These monoclonal autoantibodies express cross-reactive idiotopes shared by rheumatoid factors and antibodies specific for Sm. The expression of these idiotypes is independent of major histocompatibility complex and Ig constant region heavy chain haplotypes, self-antigen specificity, and even the VH gene family utilized. Though the experiments described here are limited to murine autoantibodies, similarities exist between murine and human autoimmune diseases. Studies that aim to investigate the relationship between VH gene expression and the presence of cross-reactive idiotypes among human autoantibodies should enable us to better understand the mechanisms of autoimmunity and self-tolerance.

Inhibitory properties and antigenic specificity of monoclonal antibodies to pancreatic colipase.

To understand the mechanism by which colipase acts as a protein cofactor for anchoring pancreatic lipase at triacylglycerol/water interface, we have used an immunochemical approach. Ten monoclonal antibodies (Mabs) against porcine pancreatic procolipase were produced. Purified immunoglobulins and Fab fragments were studied for their capacity to inhibit colipase-dependent lipase activity. These studies were carried out by using procolipase, the secretory form of the cofactor, and its trypsin-treated form obtained by removal of the amino terminal pentapeptide by trypsin. Reactivities of Mabs with both forms of the cofactor were also studied by immunoenzymatic methods. Mabs 6.1, 49.20. 75.8, 270.13 and 419.1 were found to inhibit lipolysis by preventing the binding of procolipase or trypsin-treated colipase to the lipid substrate. Mab 72.11 inhibited procolipase binding but had no effect on trypsin-treated colipase. Mab 72.11 reacted with procolipase in ELISA but showed no reactivity with trypsin-treated colipase. Finally, preincubation of Mab 72.11 with porcine procolipase prevented specific cleavage at the Arg5-Gly6 bond by trypsin. It could be concluded, that the five first residues of procolipase are structural elements of the antigenic determinant recognized by Mab 72.11. Results of ELISA additivity tests (cotitrations) further indicated that epitopes for Mabs 6.1, 72.11, 270.13 and 419.1 and for Mabs 49.20 and 75.8 are located in two distinct antigenic regions of the procolipase molecule. It appears then that the lipid binding domain of the pancreatic lipase protein cofactor comprises two regions. The first region corresponds to the amino terminal fragment of the protein. The second region is likely identical with the peptide segment at position 51-59 as previously hypothesized from NMR and spectrophotometric studies. Studies carried out on procolipase chemically modified at tyrosine residues provided evidence that epitopes for Mabs 49.20 and 75.8 are in or close to the region which contains tyrosines at positions 55 and 59, and that the two peptide regions essential for interfacial binding are spatially adjacent in the procolipase and the trypsin-treated form of the cofactor. General conclusions are in accordance with the location of antigenic regions of procolipase determined by predictive methods.

The yemanuclein-alpha: a new Drosophila DNA binding protein specific for the oocyte nucleus.

The Drosophila yG 4.5 gene (now called yemanuclein-alpha gene), which maps at 98F, is a member of the yema gene cluster isolated in a search for differentially expressed maternal genes. The yemanuclein-alpha transcript (formerly yT 4.5) is specifically expressed in the female germ cells at early oogenic stages and displays a graded distribution along the antero-posterior axis of the oocyte. These provocative features are reminiscent of that of K10, bicoid and Bicaudal-D gene transcripts and lead us to hypothesize that the yemanuclein-alpha gene plays a key role in egg organization. We show in the present work that the yemanuclein-alpha is a nuclear protein highly specific for the oocyte nucleus. The sequence analysis of the 5696 bp EcoRI fragment containing the yemanuclein-alpha gene, and of 5 overlapping cDNAs, reveals a 3006 nucleotides long open reading frame (ORF) flanked by long untranslated 5' and 3' sequences. This ORF predicts a 109,215 kDa protein which is basic (pHi: 8.57), and serine rich (12.08%). It contains a 40 amino acid acidic domain in the first third of the protein with a potential alpha-helix organization; this domain has some similarity with the nucleolin acidic domain. Parts of the yemanuclein-alpha sequence are likely to form secondary structures known to interact with DNA. We demonstrate the DNA binding activity of the yemanuclein-alpha by affinity chromatography experiments. Our data indicate that the yemanuclein-alpha shares some of the features which are characteristic of genuine transcriptional activators.

Antigen specificity and cross-species reactivity of a monoclonal antibody (mAb 72.11) against porcine pancreatic procolipase.

We have studied the antigen specificity and cross-reactivity of a monoclonal antibody (mAb 72.11) of subclass IgG1, raised against the precursor form of porcine colipase (procolipase), whose epitope lies near the amino terminal region of the polypeptide. mAb 72.11 cross-reacts with native porcine, equine and human procolipase, as shown by immuno-inactivation and ELISA titration studies carried out on pure proteins, pancreatic tissue homogenate or pancreatic juice. The epitope site recognized by mAb 72.11 was further characterized by studying antibody binding to denatured procolipase. Reduced carboxymethylated procolipase reacted with mAb 72.11 in ELISA. Heat inactivated or reduced carboxymethylated porcine procolipase displaced antigen from the complex formed between antibody and native procolipase. The lack of sensitivity of epitope recognized by mAb 72.11 on procolipase to heat denaturation or reduction of the disulfide bridges is indicative that antigen specificity of mAb 72.11 is not dependent on the conformation of the antigenic site. Cross-reactivity of mAb 72.11 with procolipase from the three species demonstrates that substitution of amino acid at positions 1 and 3 causes no loss of antigenicity. Finally, mAb 72.11 was coupled to sepharose to isolate human procolipase from human pancreatic juice and to separate the precursor form from activated colipase non-adsorbed on the column.

Albumin, fibrinogen, prothrombin and antithrombin III variations in blood, urines and liver in rat nephrotic syndrome (Heymann nephritis).

Albumin, fibrinogen, prothrombin and antithrombin III (AT III) variations have been studied in blood, urines and liver during an experimental nephrotic syndrome in rats (Heymann nephritis). A quantitative morphometric study (light microscopy) has been performed in the liver using an immunocytochemical technique--(PAP) method--to evaluate the protein synthesis by the number of protein-containing hepatocytes. Some sections were also studied by electron microscopy. The nephrotic animals were compared with control rats. In the blood of nephrotic rats, fibrinogen and prothrombin concentrations were increased and albumin and AT III concentrations were decreased. In the urines of nephrotic rats, albumin, prothrombin and AT III were lost, but no fibrinogen. The morphometric study in the liver has shown a significantly higher number of fibrinogen and prothrombin-containing hepatocytes in nephrotic rats than in controls, suggesting an increased synthesis of these proteins; no change was observed concerning albumin and AT III between nephrotic and control animals. In electron microscopy, albumin was demonstrated in Golgi apparatus, proving that the peroxidase-positive cells are related to protein synthesis. These results show that the mechanisms of regulation of the protein synthesis during nephrotic syndrome are different from one protein to another and, particularly, that their blood level is not the only regulating factor for their synthesis.

Association of overt glomerulonephritis and liver disease: a study of 34 patients.

Thirty-four patients with overt glomerulonephritis and chronic liver disease were studied. Kidney specimens were examined by light, electron and immunofluorescence microscopy. Plasma C3 levels were measured and a search for cryoglobulinemia was carried out in all patients. Twenty-six out of the thirty-four patients had an immune complex type glomerulonephritis (membrano-proliferative glomerulonephritis or glomerulosclerosis with mesangial deposits) suggestive of hepatic glomerulonephritis. The glomerular deposits almost always contained IgA and very frequently other immunoglobulins as well as C3. The membrano-proliferative glomerulonephritis was characterized by severe renal symptoms, mixed cryoglobulinemia and the frequent finding of low C3 levels. These data suggest that there is a linkage between liver disease and glomerulonephritis. The immunomorphological type of glomerulonephritis and the cryoglobulinemia are both suggestive of an immune complex disease. The lowering of the C3 levels could be due to activation of complement components by immune complexes, to hepatic hyposynthesis, or to a combination of the two.

Adoptive transfer of experimental autoimmune uveoretinitis in HgCl2 injected rats.

We previously demonstrated that mercuric chloride (HgCl2) injected-(Lewis x Brown-Norway) F1 rats are protected against experimental autoimmune uveoretinitis (EAU) induced by active immunization with the retinal S-antigen (S-Ag). To better understand the mechanisms of the protection promoted by HgCl2, we studied the effect of HgCl2-induced autoimmune disease on transferred EAU. We demonstrate herein that HgCl2 has no effect on adoptively transferred EAU. Therefore, the HgCl2-induced autoimmune disease does not affect effector S-Ag specific T cells activated in vitro but acts at an earlier stage.

Inhibition of experimental autoimmune uveoretinitis by mercuric chloride injections in (Lewis x Brown Norway) F1 hybrid rats.

Experimental autoimmune uveoretinitis (EAU), induced in (LEW X BN) F1 rats by immunization with S antigen (S-Ag) is T cell and antibody (ab) mediated and anti-S-Ag IgE ab have been involved in the occurrence of ocular lesions. (LEW X BN) F1 rats repeatedly injected with HgCl2 develop an autoimmune disease characterized by numerous auto-ab and a high increase of serum IgE level. We hypothesize that large amounts of non anti-S-Ag IgE induced by HgCl2 would compete with anti-S-Ag induced by S-Ag immunization so as to prevent EAU to occur. Indeed (LEW X BN) F1 rats immunized with S-Ag 7 days after the first HgCl2 injection are strongly protected against EAU. The putative role of the different mercury-induced autoimmune phenomena in the protection against EAU are discussed.

Monoclonal antibodies to human pancreatic procolipase: production and characterization by competitive binding studies.

Hybridomas secreting monoclonal antibodies (MAbs) specific for human pancreatic colipase were established and 11 clones were selected by using a dot immunobinding assay. Characterization of the MAbs was carried out by using direct and competitive epitope mapping methods, including ELISA and inactivation of colipase-dependent pancreatic lipase. Monoclonal antibodies showed four distinct patterns of reactivity. Monoclonal antibody 5.30 (group I) inhibited colipase-dependent lipase activity. The dissociation constant of the inactive antibody-antigen complex was 10(-9) M. Monoclonal antibodies 48.30, 66.24, and 153.23 (group II) had no effect on activity although they bound competitively with MAb 5.30 to antigen as shown by their capacity to displace MAb 5.30 from the antibody-antigen complex and by ELISA additivity test. Dissociation constants calculated from the displacement curves were 0.9 10(-9) M, 0.6 10(-9) M, and 2 10(-9) M, respectively. Noninhibitory MAbs 13.29, 16.25, and 33.30 bound competitively with MAbs of group II but not with MAb 5.30 (group I). Monoclonal antibodies of group IV (MAbs 17.6, 18.1, 37.39, and 169.29) had no effect on activity and did not react with immobilized antigen. None of the MAbs reacted in ELISA with reduced and carboxymethylated human procolipase, indicating that epitopes involved conformationally dependent determinants on protein antigen. Anti-human colipase MAbs showed no cross-reactivity with porcine or equine procolipases. Monoclonal antibodies described here appear to be useful tools for studying surface hydrophobic domain of colipase and/or interaction between colipase and lipase in its active conformation (open lid).

[Treatment of a septic patient with acquired haemophilia]. / Traitement d'une hémophilie acquise chez un patient septique.

INTRODUCTION: Acquired haemophilia is a rare bleeding diathesis caused by auto-immune depletion of factor VIII. It is characterised by spontaneous haemorrhagic syndrome, which can be fatal sometimes. EXEGESIS: A 71 year-old man presents in a dysimmunitary context (rheumatoid arthritis complicates by an acquired haemophilia) a septicemia with a methicillin resistant staphylococcus aureus. At the time of the hospitalization, the patient is febrile (39 degrees C). The activated partial thromboplastin time is very much increased, the level of factor VIII is lowered by 7% and the title of the inhibitor to factor VIII amounts to 140 Bethesda unities. An haematoma of the right root thigh is also noted. In that case, the concomitant presence of septicemia makes difficult the use of immunosuppressive therapy usually recommended to decrease auto-antibody's level. For the management of the septicemia, an adapted antibiotherapy (vancomycin then teicoplanin) is organized to J1. To control haemorrhagic risk, immunoglobulins are prescribed from d12 to d16, without immediate results. Then prednisone is introduced. We observe a very fast decrease of the anticoagulant circulating title with a neat improvement of the clinical state, allowing so to realize a draining puncture of the psoas. This invasive investigation required the use of prothrombinic complex concentrates ((Feiba) in the dose of 80 UI/kg two to three times a day). Biopsy does not show infection source. CONCLUSION: The infection delayed the prescription of immunosuppressive therapy and the surgery. Use of corticoids, following 5 days of intravenous polyvalent immunoglobulin, was the good choice. After 7 weeks of hospitalization the patient has recovered a normal haemostasis results, and a good general state.

[Cytokines and immune response]. / Cytokines et réponse immune.

This review paper briefly describes the principal cytokines involved in immune response, and in particular their role in some of its key-steps: antigen presentation; activation, proliferation and differentiation of CD4+ T cell subsets; antibody response by B cells; manifestations of hypersensitivity. The role played by cytokines in cytotoxic T cell (CTL) differentiation and in T cell-mediated suppression phenomena is also discussed. It seems that the auxiliary CD4+ memory cells secrete either IL-2 and interferon gamma (Th1) or IL-4 and IL-5 (Th2). It has recently been suggested that cytotoxic/suppressor CD8+ cells can also be divided into cells that secrete interferon gamma and no IL-4 (CTL) and cells that secrete IL-4 and IL-5 (suppressor T cells). The reciprocal regulatory effects of these cell populations through these interleukins is also discussed.