Hepatitis B virus Dane particles bind to human plasma apolipoprotein H.

Human apolipoprotein H (apo H) was found to bind specifically to hepatitis B surface antigen (HBsAg) from hepatitis B virus (HBV)-infected individuals. We used recombinant HBsAg proteins to analyze HBV domains recognized by apo H. We showed that the myristylated pre-S1 domain of HBsAg strongly interacted with apo H. This binding involved phospholipid components of the HBV envelope because their removal by detergent prevented apo H-HBsAg interaction. The opposite effects of iron and zinc metal ions on binding suggest that the oxidation of phospholipids also affects apo H-HBsAg interaction. After fractionation of viral particles on a sucrose gradient, and their addition to microtiter plates coated with apo H or anti-HBsAg, we observed that the maximal anti-HBsAg capture activity corresponded to a sucrose concentration of 36%, whereas the maximal apo H capture activity corresponded to a concentration of 39%. Electron microscopy and polymerase chain reaction (PCR) Southern blot studies of these fractions showed that the fraction with maximal apo H binding predominantly contained full Dane particles. Finally, we studied apo H-HBsAg binding relative to the presence of hepatitis B virus markers and observed that apo H binding activity for HBsAg was higher in sera from patients in the active virus replication phase.

Human plasmatic apolipoprotein H binds human immunodeficiency virus type 1 and type 2 proteins.

Apolipoprotein H (apo H), isolated from human plasma albumin solution, was shown to capture HIV-1-related antigens from antigen-positive sera (HIV-1 AG+) of AIDS patients, by using HIV-1-specific polyclonal antibodies. In an enzyme-linked immunosorbent assay and ligand blot and dot assays, apo H was able to bind recombinant retroviral HIV antigens, especially Gag proteins p18 of HIV-1, p26 of HIV-2, and Env gp160 of HIV-1. Binding was shown to be pH and NaCl dependent, with an optimum at acidic pH and low ionic strength. Specificity was demonstrated by saturation of this binding and inhibition either by homologous competition or by specific antisera. Binding was also observed in cell line-harvested viral proteins. The mechanism of this apo H-polyspecific binding is discussed in relation to conformational changes due to the influence of lipids or detergents.

[IgG autoantibodies against cellular p72 antigen crossing with (MLV) p15-gag antigen: presence in early HIV 1 infection, in HBV infection and in primary Gougerot-Sjögren]. / Autoanticorps IgG contre un antigène cellulaire p72 croisant avec l'antigène (MLV) p15-gag: présence dans l'infection HIV 1 précoce, dans l'infection HBV et le Gougerot-Sjögren primitif.

IgG antibodies of autoimmune SLE (Systemic Lupus Erythematosus) serum S detected a HeLa hnRNP 72 kDa protein, cross-reacting with the retroviral (MLV) p15-gag polypeptide. Since serum S disclosed a ubiquitous 72 kDa antigen in HeLa cell fractions, was prepared the so-called cytoplasmic "X fraction", enriched for the 72 kDa protein, defined here as p72. This autoantigen was detected by antibodies of HIV 1+ patients, recently of seroconverted (RSC) asymptomatic subjects, of HBV+ sera, and of primary Gougerot-Sjögren (prGS) sera. The presence of these autoantibodies in different autoimmune and infectious pathologies raises the question of the involvement of p72 in the immune processes and in the early HIV1 infection.

The B cell repertoire in rheumatoid arthritis. I. Frequency of EBV-inducible circulating precursors producing autoantibodies.

The frequency of B cell precursors producing antibodies against various autoantigens (Fc fragment of IgG, F(ab')2 fragment of IgG, type II collagen, cytoskeleton filaments and insulin) was determined in patients with rheumatoid arthritis (RA) using immortalization of peripheral blood B cells by the Epstein-Barr virus (EBV) and limiting dilution analysis. Equally large numbers of B cell precursors producing IgM-rheumatoid factors (RFs) were present in the peripheral blood of seronegative and seropositive RA patients and of controls. On average, 1 out of 15,000 B cells could be induced by EBV to secrete IgM-RFs, which represents 0.5-1% of the EBV-induced proliferating clones. By cloning or somatic hetero-hybridization of EBV cell lines derived from patients and controls, we obtained two types of monoclonal RFs: one polyreactive, reacting with Fc but also with the other autoantigens tested, and the other monoreactive, reacting with Fc only and that previously had only been found in the RA B cell repertoire. Moreover, patients and controls had similar numbers of circulating B cell precursors secreting IgM antibodies against other autoantigens that might be regarded as specific targets of RA (F(ab')2 fragment of IgG and type II collagen), and against cytoskeleton filaments that are targets of natural autoantibodies, increased in RA. The frequencies of EBV-induced B cells producing antibodies against all these autoantigens were of the same order of magnitude as the frequency of EBV-induced B cells producing RFs. The patients also possessed a similar number of precursors producing antibodies against insulin, an autoantigen irrelevant to the pathogenesis of the disease, taken as control. These data tend to demonstrate no abnormality in the autoantibody repertoire of B cells activable by EBV in RA, especially those secreting RFs. In vitro spontaneous RF secretion by circulating B cells was observed in seropositive RA patients but not in seronegative patients and in the controls tested. We enumerated the number of B cells spontaneously secreting RFs in seropositive RA patients and found that it correlated with the serum RF titer, but not with the number of RF-secreting B cells activated by EBV. The mean frequency values of B cells secreting RFs either spontaneously or after EBV infection were of the same order of magnitude, showing that the expanded population of in vivo-activated B cells was not (at least partially) infectable by EBV. This raised the possibility that EBV triggers a repertoire which may not reflect the status of B cells secreting autoantibodies in autoimmune diseases.

B-B' proteins from small nuclear ribonucleoproteins have an endoribonuclease catalytic domain inactive in native particles.

Native small nuclear ribonucleoproteins (snRNPs) purified by several conventional procedures or reconstituted in vitro have no ribonuclease activity. However, when these same snRNPs are centrifuged in cesium chloride gradients at low [Mg2+] and in the presence of sarkosyl, an endoribonuclease is unmasked at the density of core particles (i.e. containing only the set of low molecular weight proteins common to all snRNPs), while an inhibitory component is released in soluble form. The nature of this inhibitor was not further investigated and the molecular events underlying this inhibition/activation process remained only a matter of speculation. On the other hand, evidence was obtained that the nuclease activity is carried by B-B' on the basis of its comigration with B-B' as well as with two of their cleavage products after SDS/polyacrylamide gel electrophoresis of snRNP proteins. One was identified by a B-B'-specific monoclonal antibody. Another one, especially prominent and migrating between D and E core proteins, was identified as the N-terminal half of B-B' by microsequence analysis. Although tightly associated with core snRNPs, the activity is not dependent upon the presence of an snRNA. For the time being, the functional significance of this nuclease remains entirely elusive.

[Detection of visna-maedi virus antibodies and antigens by immunoblotting]. / Détection d'anticorps et d'antigènes du virus de Visna-Maedi par immunotransfert.

The visna-maedi virus was immunologically diagnosed using an immunoblotting technique from an antigenic preparation of the visna-maedi virus K796 purified by sucrose density gradient centrifugation. After SDS-PAGE electrophoresis and transfer onto a nitrocellulose sheet, the immunoblotting procedure was adapted to the search for specific antibodies in ovine serum samples. The results obtained showed that, in the natural visna-maedi disease, antibodies are not systematically detectable against every viral protein of the virus core. We have demonstrated the existence of antibodies directed against the proteins coded by the gag and the pol genes.

Clinical significance of anti-RNP and anti-Sm autoantibodies as determined by immunoblotting and immunoprecipitation in sera from patients with connective tissue diseases.

Antibodies to Sm and RNP antigens have been detected by immunoblotting and immunoprecipitation of small nuclear ribonucleoproteins in 168 sera from patients with connective tissue diseases previously characterized by immunodiffusion. Anti-RNP and anti-Sm antibodies immunoprecipitated U1 and U1-U6 snRNA respectively. By immunoblotting anti-Sm reacted with B-B' and D polypeptides and we have distinguished two types of anti-RNP sera: 1) 'full spectrum' anti-RNP sera reacted with the 68 kD, A, C and B-B' polypeptides; 2) 'partially reactive' anti-RNP sera reacted with various combinations of these polypeptides but not the four of them. A strong specificity of anti-Sm antibodies for systemic lupus erythematosus (SLE) was found with all three methods but immunoblotting was more sensitive and detected anti-Sm in 76% of SLE sera. Sera containing a high titer of 'full spectrum' anti-RNP without anti-Sm activity were only detected in mixed connective tissue disease (MCTD) whereas anti-68 kD antibodies alone seemed to be less specific. This strong association between 'full spectrum' anti-RNP antibodies and MCTD supports the hypothesis that MCTD is a distinct clinical entity associated with a specific serologic marker.

Human autoimmune serum antibodies against gag gene p30 retroviral protein also react with a U1-SnRNP 68K comigrant protein.

Using Immunoblotting procedure, we showed that autoimmune human antibodies reacting with mouse retrovirus gag-p30 also reacted with a HnRNP 68 Kd protein. Since U1-SnRNP 68 K and p30-gag proteins show 40% homology, the detected 68 Kd protein is likely to be the U1-RNA 68 K.

Presence of circulating antibodies against gag-gene MuLV proteins in patients with autoimmune connective tissue disorders.

An immunoblotting procedure using viral proteins from purified murine sarcoma virus or MSV-(MLV) has been developed to characterize antiviral antibodies in sera from patients with autoimmune connective tissue disorders. Fifty-eight sera with anti-Sm, anti-RNP, anti-SS-B (La), and other undefined specificities were found to react with several major viral polypeptide bands. Most of them corresponded to gag-gene-encoded products: pr65gag, p40gag, p30, p15, p12 and p10. Other bands with molecular weights averaging 90K, 60K, 45K, and 28K were recognized by a few sera. Immunological specificity of the reaction was assessed by reproducing the tests with IgG purified from sera and from corresponding F(ab')2 fragments. Moreover, the specificity of the reaction with gag proteins was confirmed by repeating the tests with p30 and p15 prior purified by immunoprecipitation with anti-p30 and anti-p15 goat sera. Furthermore, the gag polypeptides were recognized by human sera by replacing MSV-(MLV) by three other murine retroviruses of different origin. An indirect confirmation of these results was obtained by applying this method to sera of MRL lpr/lpr mice which develop an autoimmune syndrome comparable to that of human systemic lupus erythematosus. In agreement with previously published results (C. Rordorf, C. Gambke, and J. Gordon (1983), J. Immunol. Methods 59, 105-112), we found that anti-gag-gene antibodies were present in the sera of individual mice. Patterns of reactivity were found to vary with the age of the animals. No retroviral polypeptide was significantly detected in the great majority (80%) of sera from normal donors. However, 5 out of 25 sera showed faint bands although to a lesser extent than pathological sera. These five sera also reacted with HeLa cell purified HnRNPs, suggesting that their normal status should be reconsidered.

Solubilization of a human lymphocyte factor for autorosette.

With incubation at 45 degrees C, human peripheral blood lymphocytes (PBL) loose 80% of their capacity to form 1-hr autorosettes (AR). However, the addition of supernatant from heated lymphocytes (SHL) restores 93% of their rosette-forming capacity, while producing an inhibitory effect on nonincubated lymphocytes. A soluble factor present in SHL is active to a 1/5000 dilution; is absorbable on autologous red blood cells but not on sheep red blood cells; is RNase and DNase resistant and sensitive to trypsin and pronase; and acts variably on allogenic cells.

Human autologous rosettes. IV. Their relation with interleukin 2 activity production and natural killer cells in cancer patients.

Peripheral blood lymphocytes (PBL) of solid-tumor-bearing cancer patients produced a lower interleukin 2 (IL-2) activity after lectin stimulation than did those from normal subjects. Moreover natural killer (NK) cell activity and autologous rosette forming (ARF) cell rate are found significantly correlated with IL-2 production in these patients. No direct relation is observed between ARF cell ratio and NK cell activity in a given patient. A central role for IL-2 in cancer patient immune dysfunctions is suggested. Two lines of pathogenetic mechanisms are documented. First, PBL exhibited cellular function defects, namely, autologous receptor expression, IL-2 production, and NK activity. Second, these dysfunctions involved, at least partly, plasma factors. The possibility of specific deficiency, (e.g., thymic factors) is not documented. Conversely it is demonstrated that patient plasma contain immunosuppressive factor(s) that block(s) IL-2 production and ARF cell expression. Involvement of ARF cell receptor in T-cell activation is discussed.

Prognostic evaluation of human tumor cell in vitro cloning assay.

In vitro cloning of human tumor cells were carried out for 19 patients: 13 breast cancers (4 metastatic effusions and 9 primary tumors), 3 ovarian cancers, 2 glioblastomas and 1 meningioma. Cloning efficiency varied from 1 X 10(-5) to 2.8 X 10(-3). Tumor cells with the highest cloning rates were provided by patients with rapidly evolving tumors. 7 drug assays were performed: in vitro-in vivo correlation was observed in all cases (drug resistance in 5 cases: drug sensitivity in 2).

Use of trypsin for rapid and efficient purification of murine sarcoma and leukemia virus.

A method is described for purification of MSV-MuLV from culture supernatant of chronically infected 78A,1 rat embryo cell line. This method involves direct polyethylene glycol-NaCl precipitation of the low speed supernatant of culture fluid followed by digestion of the pellet with trypsin. This procedure efficiently disrupts large aggregates which normally entrap most of the virus. Highly purified virus can be obtained in very good yield by a combination of sedimentation velocity and isopycnic centrifugation : yields up to 100 A280 units (17 mg of protein) of purified virus per liter of culture fluid can be observed. This procedure appears well suited for large scale isolation of virion associated enzymatic activities.

[Evidence of protein kinase activity in 2 murine oncornaviruses]. / Mise en évidence d'une activité protéine kinase dans 2 oncornavirus murins.

A protein kinase activity has been detected in two strains of murine Oncornaviruses, MSV/MLV and EFV. This activity phosphorylates not only endogenous viral proteins but also exogenous substrates (histones and phosvitin). The stimulation of enzyme activity by detergents along with the increase of specific activity in viruses treated with trypsin during purification suggest that the enzyme is located in the viral particle.

Studies on amikhellin. II.-Inhibition of dna-polymerase from murine sarcoma leukemia virus.

We have previously reported that amikhellin binds to double-stranded DNA by an intercalation process (1). We now report that this drug inhibits the DNA-polymerase from murine sarcoma leukemia virus. The extent of inhibition was found to vary with the nature of the primer-template used : maximum with poly(rA)n-oligo(dT)10 (nucleotide ratio 20:1), minimum with poly(rA)n-poly(dT)n and intermediate with native calf thymus DNA. Experiments performed with synthetic templates of the (rA)-(dT) type have led to the following conclusions as to the mechanism of inhibition: 1) Amikhellin acts at an early stage of the synthesis reaction because the drug is no longer inhibitory when a limited extension of the oligo(dT) primers has been allowed to occur. However, mere incubation of the enzyme with the template in the absence of dTTP is not sufficient to confer resistance to the drug. 2) Progression of enzyme molecules actively engaged in polymerization is stopped when they reach downstream duplex regions to which amikhellin is bound.