Spontaneous monoclonal immunoglobulin-secreting peripheral blood mononuclear cells as a marker of disease severity in multiple myeloma.

Peripheral blood from patients with multiple myeloma (MM) contains a small number of plasma cells related to the bone marrow tumour cells by their cytoplasmic immunoglobulin (Ig), their cell membrane antigen expression and/or their gene rearrangements, but hitherto the monoclonal Ig (M-Ig) production by circulating cells has not been reported. Using a two-colour ELISPOT assay, Ig-secreting cells (Ig-SCs) were detected in the blood of 28 MM and five Waldenstrom's macroglobulinaemia (WM) patients. The number of cells that spontaneously produced an Ig isotype similar to that of the M-Ig in serum was greater than that of the other Ig-SCs. MM patients presented an excess of circulating heavy-chain (alpha or gamma) Ig-SCs (0.38% of the PBMC) with kappa or lambda light chains (0.48%) compared with the number of cells secreting the other heavy- (0.02%) and light-chain isotypes (0.03%). WM patients also presented high numbers of cells secreting the mu-heavy-chain isotype (0.66%). The Ig synthesized in vitro was characterized as monoclonal, and the M-Ig secretory capacity of the peripheral blood cells was similar to that observed for Ig-SCs from polyclonal activated B cells in vivo. The number of these monoclonal cells was significantly increased in patients in an advanced stage of MM (I/II vs. III, P < 0.001) and correlated with the serum beta-2 microglobulin concentration (r = 0. 69; P < 0.0003). The number of M-Ig-SCs in MM patients could be a useful marker for evaluating the progression of multiple myeloma.

Impairment of B-lymphocyte differentiation induced by dual triggering of the B-cell antigen receptor and CD40 in advanced HIV-1-disease.

OBJECTIVE: This study was performed to investigate the hyporeactivity of purified B lymphocytes from HIV-1-infected patients. DESIGN: Given the importance of the B-cell Ag receptor (BCR) and CD40 in B-lymphocyte activation, we assessed the capacity of purified peripheral blood B lymphocytes from HIV-1-infected patients to differentiate into Ig-secreting cells in a T-cell- and accessory-cell-independent system of BCR and CD40 costimulation. METHODS: B lymphocytes from 21 HIV-1-infected patients were purified by immunomagnetic cell separation and costimulated with immobilized anti-CD40 monoclonal antibodies and Staphylococcus aureus Cowan I particles in the presence of interleukin (IL)-2 and IL-10. Homotypic aggregate formation, apoptosis, cell cycle entrance, proliferation and Ig secretion of B cells were analysed. RESULTS: Costimulation by the BCR and CD40 induced proliferation and differentiation of B lymphocytes into Ig-secreting cells in 13 patients (group I) but not in eight patients (group II). For three patients in group II, the dual triggering induced apoptosis of B cells. The unexpected inability of these cells to differentiate was associated with a high CD38 expression and a weak spontaneous production of Ig or anti-HIV-1 antibodies in patients with a high viral load and a low CD4+ lymphocyte count. Despite this anomaly, the B cells from group II were able to progress through the cell cycle after stimulation with a combination of phorbol ester and ionomycin in complete medium, suggesting an impairment in BCR and CD40 early signal transduction. CONCLUSION: Intrinsic in vitro hyporeactivity of B lymphocytes to dual triggering of BCR and CD40 was observed in advanced HIV-1 disease and appeared to be related to in vivo hyperactivation of B cells.

Oligomerization of recombinant penton base of adenovirus type 2 and its assembly with fiber in baculovirus-infected cells.

Full-length Ad2 penton base (PbFL571) and amino-terminal (PbAT69) and carboxy-terminal deletion mutants (PbCT550 and PbCT531) were expressed at high levels in recombinant baculovirus-infected insect cells. PbFL571, and with a lesser efficiency PbAT69, assembled penton base capsomers (9S pentamers), whereas pentamer assembly was abolished with the deletion of the 21 C-terminal amino acids (as in PbCT550). A discrete population of penton base capsomers in PbFL571 and PbAT69 was found to occur as 12S decamers. PbFL571 and PbAT69 penton base were able to bind to full-length (but not to N-terminal deletion mutants of) fiber trimer to form authentic penton capsomer when coexpressed in trans in double-infected cells. Penton base monomers accumulated by PbCT550 and PbCT531 were capable of interacting with both fiber trimers and monomers. This suggested that mutual binding sites exist in the fiber and penton base subunits, and that the fiber-binding domain is located between residues 70 and 531 in the penton base, with its complementary domain situated at the N-terminus of the fiber. Intranuclear inclusions of recombinant protein were observed with the three deletion mutants PbAT69, PbCT550, and PbCT531, implying the existence of karyophilic signal(s) in the central domain of penton base.

An antisense interferon-beta RNA abolishes repression of c-fos gene expression.

To determine the cellular functions which are modified when interferon-beta (IFN-beta) gene expression is inhibited, a plasmid allowing the constitutive expression of RNA complementary to IFN-beta mRNA was constructed and stably introduced into L929 cells. Some of the selected clones expressing this antisense IFN-beta mRNA, named L-ASI, were unable to produce IFN-beta and lost the ability to arrest in the G0 phase of the cell cycle. Indeed, the usual transrepression of the c-fos gene observed in quiescent cells was blocked in IFN-beta antisense L-ASI clones and the c-fos gene was permanently stimulated. This overexpression of c-fos was not modified in response to protein kinase C agonists such as phorbol esters, but increased in response to the adenylate cyclase activator forskolin. In addition, the ability to induce major histocompatibility class I genes following recombinant IFN-beta treatment was impaired in antisense IFN-beta L-ASI clones, suggesting an important alteration of this cell with regard to the interferon system. Unexpectedly, the tumorigenicity of the clones was significantly diminished. We postulate that IFN-beta antisense RNA blocks the repression of the c-fos gene and thus prevents the arrest of cells in the G0 phase of the cycle.

Reversion of thermosensitive splicing defect of Moloney murine sarcoma virus ts110 by oversplicing of viral RNA.

Moloney murine sarcoma virus ts110 possesses a thermosensitive splicing defect. By continuously growing nonproducer cells at the nonpermissive temperature, a new class of revertant cells, termed 6m3, that had lost the thermosensitive splicing defect was produced, and six distinct clones were selected. These cell clones were transformed at either permissive or restrictive temperatures. Unlike parental 6m2 cells, which contain two virus-specific RNA species of 4.0 and 3.5 kilobases (kb) at temperatures permissive for transformation, the 3.5-kb RNA was the only virus-specific RNA species detected in 6m3 clones. No new v-mos-containing DNA fragment was observed in Southern blot analysis of these cell clones compared with parental 6m2 cells, indicating that the 3.5-kb RNA was a splicing product rather than a direct transcript. Moreover, these cells expressed P85gag-mos but not P58gag at any temperature. The reversion of the phenotype in 6m3 cell clones appears to be the result of a selective loss of the temperature sensitivity of the splicing reaction, without affecting the thermosensitivity of the protein kinase activity. This change also appears to alter the mechanism regulating the efficiency of the genomic RNA-splicing reaction.

A common cellular pathway for v-mos and v-Ki-ras is not required for v-Ki-ras-induced tumorigenicity in a nonmalignant, v-mos-expressing revertant cell.

A revertant cell line was selected from Moloney sarcoma virus-transformed BALB/c cells after long-term treatment with type I interferon. Despite an actively transcribed and transfectable v-mos gene, these revertant cells were nontumorigenic in nude mice. The functionality of the mos protein was investigated, focusing on the alpha 2(1) collagen promoter regulation, which is known to be affected by mos-induced trans-acting factors. Both in transient expression assays and after stable integration into the cellular genome, the transfected alpha 2(1) collagen promoter fused to the cat reporter gene was activated in the revertant while being downregulated in the original transformed cells. In retransformation assays of the revertant by Moloney sarcoma virus strains homologous to the original transforming virus, no detectable change was noted in the in vitro phenotype or in tumorigenicity. These results reveal that the mos-directed factors were no longer effective on their specific targets. Thus, the R.MSVIF cell could be either an oncoprotein-deficient or a target-related revertant. Attempts at retransformation with unrelated sarcoma viruses bearing v-sis, v-fms, or v-fos oncogenes were also negative. In contrast, tumorigenicity was obtained with the unrelated Kirsten sarcoma virus without any change in the revertant morphology or collagen expression. These findings showed that the common pathway blocked by the reversion and shared by v-mos and v-ras was not required for ras-induced tumorigenicity.

Increased proto-oncogene expression in peripheral blood T lymphocytes from patients with systemic sclerosis.

The expression of c-myc, c-myb, and c-ras proto-oncogenes, determined using RNA hybridization techniques (slot-blot), was significantly increased in peripheral blood T lymphocytes, but not in B cells, from 17 patients with systemic sclerosis with diffuse scleroderma, compared with the expression in normal control subjects. The magnitude of expression of c-myc and c-myb tended to be higher in patients with early, active disease. These results demonstrate an in vivo activation of T cells from systemic sclerosis patients, which may play an important role in the pathogenesis of the disease.

Presence of a constitutive paracrine beta-interferon in v-mos-bearing nonmalignant reverted cells.

A stable nonmalignant revertant cell line was derived from Moloney murine sarcoma virus-transformed BALB/c cells after long-term cultivation in the presence of murine type I interferon (IFN). These cells gradually established resistance to exogenous IFN and were also seen to contain IFN-dependent proteins. The presence of an endogenous IFN was confirmed by the results of Northern blot analysis and in situ hybridization with an IFN-beta probe, showing that only mRNA specific for IFN-beta- could be found in the uninduced reverted cells. The latter synthesized only a small amount of IFN-beta protein and exhibited few IFN-specific membrane receptors, which bound recombinant IFN-beta with a high affinity. After treatment with IFN antibody, the overexpression of H-2 major histocompatibility antigen genes was significantly down-regulated. These findings strongly suggest the existence in this reverted cell line of a constitutive IFN which, acting through an autocrine and/or paracrine mechanism, might play a role in maintaining the reverted state.

Persistent expression of v-mos oncogene in transformed cells that revert to nonmalignancy after prolonged treatment with interferon.

BALB/c embryonic fibroblasts productively transformed by Moloney sarcoma virus and cultivated for over 600 generations in the presence of mouse alpha/beta interferon reverted to an apparently normal phenotype and were unable to produce tumors in nude mice. Nevertheless, the presence of an integrated Moloney sarcoma virus genome in the nonmalignant Moloney sarcoma virus-transformed interferon-treated cell DNA could be shown by focus formation upon transfection and by hybridization with a v-mos probe. After digestion with various restriction endonucleases, similar hybridization patterns of v-mos sequences were obtained with DNAs from both reverted and transformed cells. However, additional integration sites and at least twice as many copies of the oncogene were found in the nonmalignant Moloney sarcoma virus-transformed interferon-treated cell DNA. Polyadenylylated RNA extracted from reverted and control cells contained two mos-specific transcripts. Interestingly, the nonmalignant Moloney sarcoma virus-transformed interferon-treated cells produced helper virus, but no detectable mos-containing virions, suggesting that a posttranscriptional block in the v-mos gene expression had occurred in these cells. It should be stressed that, after up to 100 additional passages, cells cultured in the absence of interferon maintained their nontumorigenic character in spite of the persistent transcription of the mos oncogene.

Early nuclear structural changes of transformed human amniotic cells (WISH) in presence of type IV collagen detected by the nuclear refringency assay.

An early nuclear activation of transformed human amniotic epithelial (WISH) cells triggered by type IV collagen is visualized by a modification of the nuclear refringency obtained by mercury binding on condensed chromatin. This phenomenon is quantified by the nuclear refringency test. The nuclear activation of WISH cells by basement membrane collagen is also shown by the DNase I sensitivity of chromatin and by the measurement of mRNA synthesis. These nuclear phenomena are concomitant with WISH cell attachment and laminin synthesis. Reversible effects on nuclear refringency, cell attachment, and laminin synthesis are tested by the addition and removal of different metabolic inhibitors.

Interferon effect on collagen and fibronectin distribution in the extracellular matrix of murine sarcoma virus-transformed cells.

The transformation of murine BALB/c embryonic fibroblasts by murine sarcoma virus, Moloney strain, followed by prolonged treatment with murine interferon, resulted in the appearance of a new cell population (MSV-IF+). These MSV-IF+ cells are characterized by the recovery of a normal phenotype, contact inhibition, and lack of colony formation in agar. This phenotypic change of the MSV-IF+ cells is associated to the neosynthesis of a dense fibrous matrix beyond the cell periphery. Ultrastructural studies using peroxidase-labeled antibodies enabled us to localize the extracellular distribution of fibronectin and collagen in the MSV-IF+ cell line, compared to normal BALB/c and murine sarcoma virus-transformed cells. In parallel, a significant increase of collagen and fibronectin deposit in the intercellular space of interferon-treated murine sarcoma virus-transformed cells was observed.

Chemical characterization and functional role of human glomerular basement membrane components.

Subfractions of human glomerular basement membrane isolated by enzymatic procedures were analyzed for their chemical structure and biological activity. Pepsin or collagenase digestion of purified basement membranes released three groups of components: collagenous and noncollagenous fractions and also a mixed material consisting of a proteolysis-resistant association between collagen sequences and heteropolysaccharidic glycopeptides. These different fractions were explored in vitro for their ability to interact with cell membranes. In fact, only fractions containing the heterogenous material agglutinated human transformed or embryonic cells within 2 h. The cell agglutination was specifically inhibited by N-acetylosamines and N-acetylneuraminic acid. An additional incubation of 20 h at 37 degrees C of the agglutinated cells, maintained in a minimal medium devoid of serum, led to a morphological change of the transformed cells due to an increased cell adhesion and cell spreading. These experimental data suggest that the basement membrane possesses active sites - consisting of an association between collagen and structural glycoproteins - which could play a primordial role in cell matrix interactions.

Lectin-like activity of components extracted from human glomerular basement membrane.

Different fractions of human glomerular basement membranes have been isolated by enzymatic and chemical methods. These fractions were analyzed for their chemical structure and biological activity. The hypothesis that human glomerular basement membrane glycopeptide fractions related to collagenous sequences could act as a lectin-like substance was explored; in fact, extracellular glycoprotein factors play a role in cell-cell interactions and cell adhesion. These collagenous glycopeptides agglutinate human transformed or embryonic cells within 2 hr. The cell agglutination is inhibited by the following sugars: N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid. In addition, the rapid cytoagglutination is followed by a cell spreading effect after a further 20-hr incubation. These data led to the postulate that the maintenance of tissue differentiation is governed by the interaction of cells with peculiar sites of the basement membrane consisting of a proteolysis-resistant association between collagen and matrix glycoprotein.

[Presence of lectin-like substances in the basement membrane of the human kidney glomerulus]. / Présence de substances de type lectine dans la membrane basale du glomérule de rein humain.

Crude extracts from the human glomerular basement membranes solubilized by pepsin or bacterial collagenase agglutinate normal or transformed human cells. Cytoagglutination is inhibited by N-acetyl-osamines. These properties are reminiscent of lectins. When agglutinated cells are incubated for an additional 20 hrs. period in minimal serum free medium but in presence of these basement membrane extracts, they attach to the glass and spread out.

[Study of the nephrotoxicity of antibodies directed against the glomerular basement membrane glycoproteins of the Wistar rat]. / Etude de la néphrotoxicité d'anticorps anti-glycoprotéines de membrane basale glomérulaire du rat Wistar

The nephrotoxic activity of prepared antibodies against previously isolated glycoproteic fractions of the Rat glomerular basement membrane was studied using Masugi's model of Nephritis. This activity was determined by following the daily evolution of the proteinuria and the value of the seric complement. It seems linked to numerous factors.