GM-CSF expression by tumor cells correlates with aggressivity and with stroma reaction formation.

Granulation tissue involved in tissue repair and in the stroma reaction to epithelial tumors is characterized by the presence of myofibroblastic cells. It has been previously reported that granulocyte macrophage-colony stimulating factor (GM-CSF) induces a fibrotic reaction containing numerous myofibroblasts. This reaction results from a cascade of events, including stimulation of transforming growth factor-beta1 (TGF-beta1) production by macrophages which, in turn, promotes alpha-smooth muscle actin and collagen synthesis by fibroblasts. Moreover, GM-CSF is known to be expressed by many tumor cell types. In this study we have analyzed, by means of reverse transcription-polymerase chain reaction, GM-CSF mRNA expression in a progressive and a regressive rat colon carcinomas and in the corresponding cell lines, eliciting different degrees of desmoplastic reaction. We have also evaluated the expression of GM-CSF protein in selected cases. The expression of GM-CSF mRNA and, when tested, protein were higher in progressive compared to regressive cancer cells both in vivo and in vitro. We then investigated GM-CSF mRNA and protein expression in different human colon cancer cell lines known to exhibit different degrees of aggressivity in vivo. We found high levels of GM-CSF mRNA and protein in the most aggressive cell lines. Similar results were also obtained on human breast and cervical cancer cell lines. Our results are in agreement with the assumption that GM-CSF expression is correlated to tumor aggressivity. Conceivably, one of the GM-CSF actions affecting tumor progression is exerted through its influence on stroma reaction development.

Cellular retinol-binding protein-1 is expressed by distinct subsets of rat arterial smooth muscle cells in vitro and in vivo.

Previous work (M.-L. Bochaton-Piallat, P. Ropraz, F. Gabbiani, G. Gabbiani, Arterioscler Thromb Vasc Biol 1996, 16:815-820) has shown that a subset of smooth muscle cell (SMC) clones derived from the normal rat aortic media displays an epithelioid phenotype similar to that of the whole SMC population cultured from the intimal thickening 15 days after endothelial injury (IT-15). We show here that the whole IT-15 SMC population and the epithelioid clones, derived either from the normal media or from the IT-15, express cellular retinol-binding protein-1 (CRBP-1), a protein involved in retinoid metabolism. The expression of CRBP-1 is accompanied by the expression of cytokeratin 8. In both whole SMC population cultured from IT-15 and epithelioid clones, retinoic acid modulates the transition from the epithelioid phenotype to the spindle phenotype, typical of whole SMC populations cultured from the rat normal aortic media. Moreover, after endothelial injury in vivo, a CRBP-1 expressing SMC subset appears transiently in the IT and disappears, allegedly by apoptosis, when re-endothelialization takes place. Our results suggest that the expression of CRBP-1 is a marker of arterial SMC activation after endothelial injury in vivo and that CRBP-1 and probably retinoids participate in this process.

Autoimmune syndrome after neonatal induction of tolerance to alloantigens: analysis of the specificity and of the cellular and genetic origin of autoantibodies.

BALB/c mice neonatally injected with 10(8) semiallogeneic (C57BL/6 x BALB/c)F1 spleen cells become tolerant to the H-2b alloantigens, but also develop a wide range of autoimmune manifestations characteristic of systemic lupus erythematosus (SLE). Indeed, in these mice, the presence of a hypergammaglobulinaemia, autoantibodies--including anti-ssDNA, anti-platelet, thymocytotoxic and rheumatoid factor antibodies--circulating immune complexes, cryoglobulins as well as renal glomerular deposition of immunoglobulins have been observed. In this study, we have shown that the allogenic effect and B cell chimaerism which characterize these F1 cell-injected mice is associated with the expression of a large spectrum of autoantibodies, including anti-ssDNA and anti-cytoskeleton antibodies, and that these autoantibodies are not multispecific. We took advantage of the fact that, in this model, autoantibodies are exclusively produced by F1 donor B cells to inject newborn BALB/c mice with F1 Xid spleen cells lacking the CD5+ B cell subset. Injection of 2 x 10(8) F1 Xid spleen cells triggers the production of anti-ssDNA as well as anti-BrMRBC antibodies, and these mice developed tissue lesions. Finally, analysis of the VH gene family expressed by monoclonal autoantibodies derived from F1 cell-injected mice showed that they used the 2 largest families J558 and 7183. These results suggest that the allogenic effect and B cell chimerism which characterize the neonatal induction of tolerance to MHC alloantigens is associated with the selective triggering of autoreactive B cells producing monospecific IgG autoantibodies. They also imply that upon stimulation by persisting alloreactive CD4+ T cells, either CD5- B cells are able to produce autoantibodies or autoantibody-producing CD5+ B cells can differentiate from Xid spleen cells.

Correlation between the distribution of smooth muscle or non muscle myosins and alpha-smooth muscle actin in normal and pathological soft tissues.

The distribution of smooth muscle (SM) and non muscle myosins was compared with that of alpha-SM actin in various normal and pathological tissues and in cultured cells by means of indirect immunofluorescence using a monoclonal antibody specific for alpha-SM actin [anti-alpha sm-1, Skalli et al., 1986b] and two polyclonal antibodies raised against bovine aortic myosin (ABAM) and human platelet myosin (AHPM), respectively. In normal tissues ABAM stained vascular and parenchymal smooth muscle cells (SMC), myoepithelial cells and myoid cells of the testis in a pattern similar to that reported by other authors with antisera raised against non vascular SM myosin. Cells stained with ABAM were always positive for anti-alpha sm-1. In human and experimental atheromatous plaques, most cells were positive for AHPM; a variable proportion was also stained for ABAM plus anti-alpha sm-1. Myofibroblasts from rat granulation tissue, Dupuytren's nodule and stroma from breast carcinoma were constantly positive for AHPM and negative for ABAM; however, myofibroblasts from Dupuytren's nodule and breast carcinoma were anti-alpha sm-1 positive. Early primary cultures of rat aortic SMC were positive for ABAM and anti-alpha sm-1 and became negative for ABAM and positive for AHPM after a few days in culture. They remained positive for AHPM and anti-alpha sm-1 after passages; the staining of AHPM and anti-alpha sm-1 appeared to be colocalized along the same stress fibers. These results may be relevant for the understanding of SMC function and adaptation, and show that in non malignant SMC proliferation, alpha-SM actin represents a more general marker of SM origin than SM myosin.

A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.

A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.

Circulating immune complexes in regularly dialyzed patients with chronic renal failure.

The prevalence of circulating immune complexes (CIC) was investigated using the C1q binding assay (C1q BA) and the conglutinin binding assay (Kg BA) in 200 patients undergoing maintenance hemodialysis. Increased C1q binding was found in 45% (87 of 194) of the patients, and the modified Kg BA gave elevated values in 31% (20 of 65). The prevalence of CIC was similar in American and Swiss patients, and in patients undergoing hemodialysis, self-dialysis or peritoneal dialysis. In patients with 'nonimmunological' renal diseases, CIC were detected with similar frequency. No change in CIC was noted during hemodialysis in 6 additional patients tested. The abnormality was not related to age, sex, duration of dialysis, hepatitis B antigenemia, bacterial infections, or transfusions. Anti-DNA antibodies were absent in all subjects tested and the results of the C1q BA were not changed by DNase digestion of eight sera with high C1q binding. Rheumatoid factor activity (RF) was detected in approximately one-fifth of the patients, and there was a direct correlation between positive C1q binding and RF. There was no correlation between CIC and lymphocytotoxic antibodies. This study demonstrated a high prevalence of CIC in dialyzed uremic patients and established its relationship to other immunological abnormalities.

HLA antigens in Bali (Indonesia) with a special reference to an isolated community.

One hundred eighty-two Balinese were typed for HLA-A and -B locus antigens. From these, 103 were also typed for HLA-C, 51 for HLA-DR, 172 for Bf and 173 for GLO. These results and the significant phenotypic associations are situated with respect to other South-East Asian populations. In addition to this first study, 175 individuals from an isolated Balinese village typed for HLA-A, -B, -DR, Bf and GLO are presented. The effect of isolation on haplotype (HLA-A/-B/Bf/-DR) variability is discussed.

Association of a better kidney graft survival with the presence of circulating immune complexes before transplantation.

The presence of circulating immune complexes was investigated using the C1q-binding assay before and after kidney transplantation in 48 patients with renal failure. Circulating immune complexes were found in 54% of the patients. The presence of circulating immune complexes prior to grafting was associated with a better renal graft survival. Median survival time of grafts in patients with circulating immune complexes was more than 18 months as compared with 21/2 months in patients without such complexes. The incidence of circulating immune complexes in patients before transplant could not be related to the renal disease, viral infections, blood transfusions, or serum levels of lymphocytotoxic antibodies, IgG, or IgM.

Donor-specific B and T lymphocyte antibodies and kidney graft survival.

The prognostic significance of a positive B and/or T cell crossmatch test before transplantation was analysed in 174 cadaver kidney transplants. Thirty-one B cell-positive crossmatches were observed. In 15 of these cases, T cell crossmatches were also found to be positive retrospectively (long incubation assays). In 16 patients with a positive B cell crossmatch, an anti-HLA-DR antibody was present, as judged by platelet absorption studies, whereas in the other patients the B cell activity of the sera was attributable to weak anti-HLA-A,B,C antibodies or to cold autoantibodies. The success rate at 3 months of positive B cell crossmatch transplants because of anti-HLA-A,B,C or anti-HLA-DR antibodies was 84% whereas that of grafts with negative B and T cells crossmatches was 79%. Patients with B cell autoantibodies had excellent graft survival. Serial serum samples from patients receiving transplants were tested against specific donor B and T cells kept frozen. B cell antibodies appeared or persisted in 38 of 44 patients tested. Many patients also had donor T cell antibodies. Platelet absorption studies showed that about one-half of the B cell antibodies were presumably anti-HLA-DR and one-half anti-HLA-A,B,C. Donor-specific anti-B cell antibodies were detected in 86% of the patients at the time of graft rejection but they were also detected in approximately 80% of patients with excellent graft survival. In these cases they were usually also reactive with the patient's own B cells. These results indicate that at least two types of donor-specific B and/or T cell antibodies must be distinguished, those directed against HLA (A,B,C, or DR) antigens possibly deleterious, and those against autologous B lymphocytes, possibly enhancing.

Increased frequency of HLA-DRw3 in systemic lupus erythematosus.

The distribution of HLA antigens was studied in 40 patients suffering from systemic lupus erythematosus. For loci A and B, increased frequencies of A1, B8 and the presence of only one B antigen were found. Nevertheless, in relation to the number of antigens tested, this increase was not significant. For the DRw locus, DRw3 was significantly increased (P < 0.001, after correction for the 39 antigens tested). However, the patients with DRw3 did not show any correlation with a specific clinical picture or the presence in their serum of lymphocytotoxic antibodies or autoantibodies against T and B lymphocytes.

Actin is unevenly distributed in the pituitary gland.

In view of the suggestion that actin-like proteins might be involved in the final steps leading to hormone secretion, the actin content of pituitary glands of adult rats was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (for total actin), by the DNAse method (which measures predominantly monomeric actin) and by immunocytochemistry. The amount of actin present in the neural lobe, expressed per mg total protein, was found to be comparable to that of other neural tissues. In contrast, in the anterior lobe, the ratio was significantly lower. The intensity of immunofluorescent staining with anti-actin antibodies was higher in the neural lobe than in either anterior or intermediate lobes. The intensity and distribution of tubulin immunofluorescent staining with anti-tubulin antibodies resembled that of anti-actin antibodies. Thus, three independent methods point to an uneven distribution of actin in the subdivisions of the pituitary gland, although all these subdivisions are believed to secrete their hormones by exocytosis. These data suggest that the bulk of actin present in pituitary cells is unlikely to be involved only in exocytosis, but may be implicated also in the intracellular translocation of secretory products.

Supercontraction in crayfish muscle: correlation with a peculiar actin localization.

Crayfish muscle, like muscles from some other invertebrates, can supercontract. This muscle shortening is characterized by an overlap of thin filaments with crossing of thick filaments through the Z discs. In intact muscle cells, supercontraction does not seem to induce irreversible structural modifications in the tissue. Isolated crayfish myofibrils in the relaxed state cannot be distinguished from vertebrate myofibrils under light microscope, either by phase contrast or by immunofluorescence, with antiactin antibodies, actin being localized in the I bands. However, when isolated crayfish myofibrils are supercontracted, irreversible dammage occurs, most thin filaments being lost. Actin becomes then hardly detectable, being visible, by immunofluorescence, either in the Z discs or evenly distributed in the whole myofibril. During myofibril supercontraction, high amounts of denatured actin, become soluble as shown by SDS-PAGE, by double immunodiffusion, and by DNAse inhibition.

Immunofluorescent subcellular localization of some muscle proteins: a comparison between tissue sections and isolated myofibrils.

The localization of parvalbumin in fish white muscle and of the calcium binding protein, of arginine kinase and of glycogen phosphorylase in crayfish tail muscle have been investigated by immunofluorescence using isolated myofibrils and muscle sections as starting materials. It is shown that the four proteins appear to be localized on the thin filaments when myofibrils are used as starting material. This result contrasts with previous observations where it appeared that parvalbumin in fish muscle and arginine kinase in crayfish muscle were distributed uniformly within the cell. This discrepancy is discussed in relation to the high solubility of these proteins. In the light of the present knowledge about striated muscles from these two organisms, it seems that the roles of parvalbumin in fish and of the calcium binding protein in crayfish are probably different.

Cellular distribution of sarcoplasmic calcium-binding proteins by immunofluorescence.

Specific antibodies against carp paravalbumin, crayfish calcium binding protein and crayfish arginine kinase were used for indirect immunofluorescence localization of the respective proteins. Simultaneous staining of the same muscle sections with human serum containing anti-actin autoantibodies served as a probe to identify the isotropic band. Parvalbumin appears to be evenly distributed in carp white muscle. The crayfish calcium binding protein however shows a distinct localization, in the isotropic band, coincident with the actin staining. Arginine kinase, which has the same molecular weight and is extractible in the same way as the calcium binding protein does not show this distinct localization, but is evenly present in crayfish tail muscle, similarly to parvalbumin. The possible meaning of the different distribution of the two calcium binding proteins is discussed.