Diagnostic value of anti-F-actin antibodies in a French multicenter study.

According to international criteria, autoimmune hepatitis (AIH) type 1 is characterized by the presence of antinuclear or anti-smooth muscle antibodies (SMA) with F-actin specificity. SMA have been found in 85% of AIH patients, but are not specific to this disease, and anti-F-actin specificity is not always verified when SMA are detected. The objective of this study was to determine the diagnostic value of anti-F-actin antibodies in a large population. A multicenter study involving 12 clinical centers was performed. Patients were selected on the basis of the presence of F-actin SMA detected by indirect immunofluorescence (IIF) on rat liver-kidney-stomach sections and was confirmed by IIF on Hep2 cells treated with colchicine, or F-actin dot-blot. The clinical status of patients was determined from their medical records. One hundred sixty-eight patients were included: 76% women, 24% men; mean age of 45 years (range, 2-88 years), with a bimodal age distribution. Sixty percent had AIH type 1, and 40% had another disease. In the group of women younger than 25 years, 90% had AIH type 1. Other pathologies associated with antiactin were other liver diseases (19%), including viral hepatitis C (7%), and non-liver diseases (21%), including connective tissue diseases (12%). Antibody titers were higher in AIH than in other diseases. Antiactin antibodies are of major diagnostic value in AIH, especially in young women; they may be found in other disease settings, but mostly at low levels.

[Analysis of nine autoantibodies associated with systemic autoimmune diseases using the Luminex technology. Results of a multicenter study]. / Apport de la technologie Luminex pour la recherche simultanée de neuf autoanticorps associés aux connectivites. Résultats d'une étude multicentrique.

UNLABELLED: Luminex technology allows simultaneous detection of several analytes in a single well. Applications have been recently developed for the detection of autoantibodies. PURPOSE: To evaluate the performances and convenience of the Fidis analytical system (BioMedical Diagnostics, Marnes-la-Vallee, France) for the detection of nine autoantibodies associated with connective diseases: SS-A, SS-B, Sm, RNP, Scl-70, Jo-1, CENP-B, P ribosomal and double stranded DNA antibodies. MATERIALS AND METHODS: Three hospital laboratories analysed 366 samples taken from their serum banks and corresponding to the main systemic autoimmune diseases. Control samples included 120 sera from blood donors and 42 sera from patients with dysglobulinemia. RESULTS: In each laboratory, handling of this new analytical system was easy. Results are readily obtained: nine autoantibodies are quantitated in fourty-four sera in less than two hours. A clear-cut discrimination between negative and positive results was observed, due to very low backgrounds. Intra-assay and inter-assay variations were low: coefficients of variation were under 10% in 80 and 64% of the cases, respectively. Results obtained with Fidis correlated satisfactorily with those obtained with the numerous routine techniques used in each laboratory. The overall concordance exceeded 93%. CONCLUSION: Fidis is a reliable and time-saving analytical system for the detection of autoantibodies associated with systemic autoimmune diseases.

Cytotoxic activity of lymphocytes infiltrating progressive and regressive tumor variants from a rat colonic cancer.

Two tumor cell variants (PROb and REGb) isolated from a single chemically-induced rat colon adenocarcinoma were previously shown to differ in their tumorigenicity. When injected into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells give rise to tumors which always regress. PROb and REGb variants also differ in their capacity to induce an immune response in the syngeneic host. Regression of REGb tumors could have been mediated by cytotoxic effector cells acting at the tumor site. To test this hypothesis, the cytolytic activity of non-adherent lymphoid cells isolated from PROb and REGb tumors and from the spleen of the same animals were compared. The same number of infiltrating lymphocytes was recovered per gram of PROb or REGb tumor. The cytolytic activity of tumor infiltrating lymphocytes, as that of spleen lymphocytes, was predominantly non specific, as demonstrated by their ability to kill YAC-1 cells, an NK-sensitive cell line. PROb cells were relatively resistant to the cytotoxic activity of spleen or tumor infiltrating lymphocytes. In the regressing REGb tumors, the cytotoxic activity of tumor infiltrating lymphocytes to homologous cells or to YAC-1 cells was low and significantly inferior to that of the corresponding spleen lymphocytes. These results suggest that the cytotoxic activity of lymphocytes was impaired at the local, intratumoral level, even in spontaneously regressing tumors.

In vitro natural killer activity against progressive and regressive variants of a rat colon adenocarcinoma. Effect of treatments with anti-asialo GM1 plus complement.

In a previous work, a cell line (DHD/K12) was established from a colon adenocarcinoma induced in a BDIX rat by 1,2-dimethylhydrazine. From this line, two cloned sublines, PROb and REGb, were then isolated. When subcutaneously inoculated into syngeneic rats, PROb cells yield progressive tumors, whereas REGb cells yield tumors which regress. In this study, in a 16-h 51Cr release assay, natural cytotoxicity mediated by BDIX splenic nonadherent lymphoid cells (NK cells) was shown to be much higher against REGb cells than against PROb cells. Whatever the target cells, NK cytotoxicity was always higher when the effector cells were obtained from males rather than from females. Treatment of BDIX splenic lymphocytes by anti-asGM1 serum plus complement revealed that both anti-asGM1 sensitive and non-sensitive NK cells exist. The activity of anti-asGM1 non-sensitive NK cells appeared to be minor and to be detected only when the level of cytotoxicity before treatment was sufficiently high. The difference between PROb and REGb tumor growth appears to be linked, at least in part, to a higher sensitivity of REGb cells to NK cells and especially to anti-asGM1-sensitive NK cells.

Synergistic effect of liposomes and endotoxins on the activation of rat macrophage tumoricidal activity.

Macrophage biological responses to endotoxins have been extensively studied; nevertheless, the mechanisms by which endotoxins activate macrophage tumoricidal activity are not currently understood. We used liposomes to investigate the interaction of endotoxins with macrophages. In a medium containing 10 micrograms endotoxin/ml, macrophage-mediated cytolysis ranged from -7 to 36%. In all the experiments, 1mM dipalmitoyl phosphatidyl choline (DPPC) small unilamellar liposomes significantly induced or enhanced cytolysis, ranging from 30-90%. Liposomes and endotoxins had a synergistic effect on the macrophage cytolytic activity. This effect was dose-dependent on liposome concentration, ranging from 0.25-1 mM or 2 mM. Liposomes decreased the endotoxin concentration threshold necessary to induce cytolysis. They did not modify the kinetics of macrophage activation. Liposomes did not modify the binding of tumor cells to macrophages. The optimum synergistic effect was obtained when liposomes were present during the first 18 h of the mixed culture of macrophages and target cells, before adding endotoxins for the next 18 h. When cholesterol was added to DPPC (M/M), liposomes did not enhance but rather inhibited macrophage activation by endotoxins.

Prevalence of ANCA in a hospitalized elderly French population.

OBJECTIVE: The prevalence of some autoantibodies in the elderly population has been reported to be greater than in younger controls. The prevalence of ANCA has been shown to be low in a generally healthy population, but has not been established in the elderly. Thus, the presence of ANCA in elderly patients might not have the same clinical significance as in younger people. The aim of this study was to evaluate the prevalence of ANCA in elderly subjects. PATIENTS AND METHODS: Serum samples from 137 subjects (96 females, 41 males; mean age = 82.2 years +/- 6.97 SD) were evaluated. Criteria for exclusion included suspected or established systemic vasculitis, connective tissue or neoplastic diseases, acute infection, HIV infection, current therapy with corticosteroids or cytotoxic drugs, and recent blood transfusion. ANCA were detected by indirect immunofluorescence on ethanol-fixed normal human neutrophils. Fluorescence patterns were classified as c-ANCA, p-ANCA or nuclear. Sera exhibiting p-ANCA or nuclear fluorescence were further tested by IIF on formalin-acetone fixed normal human neutrophils. Sera whose reaction pattern was cytoplasmic were considered as positive for "true" pANCA. Additionally, sera were tested for the presence of antinuclear antibodies (IIF), anti-double-stranded DNA (enzyme immunoassay) and IgM rheumatoid factors (enzyme immunoassay). RESULTS: The prevalence of c-ANCA was 0% (95% CI = 0-2.66), the prevalence of p-ANCA was 2.2% (95% CI = 0.45-6.3), and the prevalence of "true" p-ANCA was 0.73% (95% CI = 0.02-4). The prevalence of ANA, anti ds-DNA and RF were respectively 38%, 3.6%, and 11.7%. CONCLUSION: The prevalence of ANCA is not increased in elderly people. Thus, the presence of ANCA in elderly subjects may have the same clinical significance as in younger people.

In vivo and in vitro effects of fish-containing diets on colon tumour cells in rats.

Polyunsaturated n-3 fatty acids, abundant in sea fish, can inhibit the growth of chemoinduced or transplanted mammary tumours in the rat. Since mammary and colonic cancers have both been linked to a high fat consumption, we studied the effect of 2 diets moderately (7% fish meal) or strongly (9% fish oil) enriched in fish fatty acids on the growth of colon cancer cells subcutaneously inoculated into syngeneic rats. The diets had no effect on the in vivo tumor growth and on the in vitro tumouricidal activity of peritoneal macrophages or splenic lymphocytes.

Involvement of effector cells in the treatment with endotoxins of peritoneal carcinomatosis induced by colon tumour cells.

Peritoneal carcinomatoses, an ordinary way for human colon carcinoma to spread, are incurable. When rat peritoneal carcinomatoses of colon origin were treated with endotoxins i.p. administered 3 days after the tumour cell injection, 70% of the BDIX rats were alive 30 weeks after the PROb tumour cell injection whereas all the untreated rats died of their tumour within 14 weeks. The study of the effector cells involved in the antitumour effect of endotoxins suggests that T lymphocytes are required for this effect. The endotoxin effect is local and is not reflected by the cytolytic activity of peritoneal cells.

Differential retention of doxorubicin in the organs of two strains of rats.

Fluorescence microscopy and high pressure liquid chromatography were used to study the decrease of doxorubicin (DXR) concentrations in the liver, spleen, heart, lung, kidney and skeletal muscle of two strains of rats at various times after a single intravenous injection of the drug (8 mg kg-1). DXR was located within the cell nucleus and was mostly undegraded, it persisted, especially in heart, lungs and spleen where it was detectable 10 days after injection. The DXR/DNA ratio, was used as an index of nuclear fixation of the drug. A major difference in the DXR/DNA ratio between the two strains were observed in heart and spleen results; the DXR/DNA ratio was significantly higher in heart and spleen compared with lung, liver and kidney in both strains.

[Diagnostic value of a new serum marker of human digestive cancer: the carbohydrate antigen 19.9; comparison with the carcinoembryonic antigen]. / Valeur diagnostique d'un nouveau marqueur sérique des cancers digestifs humains: l'antigène carbohydrate 19.9 (CA 19.9); comparaison avec l'antigène carcino-embryonnaire.

In order to evaluate its diagnostic value for digestive cancers, the carbohydrate antigen 19.9 (CA 19.9) was quantitated by radioimmunoassay in the serum of 102 patients suffering from malignant or non-malignant diseases of the digestive or pulmonary tract and of 20 healthy subjects. The carcinoembryonic antigen (CEA) was quantitated by enzyme-immunoassay in the same sera. No correlation was found between the CA 19.9 and the CEA levels. The sensitivity of the CA 19.9 test for the detection of digestive cancers (0.51) was not significantly higher than neither that of the CEA assay (0.46), nor that of the combined tests (0.59). Numerous false positive results were encountered in the CA 19.9 assay in pulmonary diseases and in non-cancerous liver diseases, especially cirrhosis. Its specificity (0.84) did not prove to be superior to that of CEA (0.90). Thus, in our experience, the clinical interest of serum CA 19.9 determination for the diagnosis of digestive cancers seems doubtful.

[Hepatic mesenchymal hamartoma in children. Immunohistochemical, ultrastructural and flow cytometric case study]. / L'hamartome mésenchymateux du foie de l'enfant. Etude immunohistochimique, ultrastructurale et en cytométrie en flux d'un cas.

Mesenchymal hamartoma is a rare liver lesion. This lesion was found in a 7-month-old girl with high serum alphafaetoprotein serum levels and was composed of loose connective tissue containing a certain number of epithelial cells of biliary or hepatic origin. Immunohistochemical studies showed that cytokeratins 7 and 19 were localized in bile duct epithelium. The ultrastructural study showed that the hamartoma was composed of well differentiated ductal structures surrounded by a myxoid mesenchyma with cysts formed either from degenerative mesenchymal areas or from dilated ducts. Flow cytometric analysis of nuclei from frozen tissue revealed that the lesion was DNA aneuploid, with a DNA index of 1.28.

Elevated serum CA19-9 levels in poorly controlled diabetic patients. Relationship with Lewis blood group.

Many non-malignant diseases may be associated with elevated serum CA19-9 levels. Recent reports suggest that diabetes mellitus may also be responsible for some elevations. In this study we investigated the influence of the glycaemic level and Lewis phenotype on the serum CA19-9 levels in diabetic patients. In 15 out of 84 patients (17.8%) serum CA19-9 exceeded 100 kU/L (highest value: 208 kU/L). CA19-9 concentrations were significantly correlated with glycosylated haemoglobin levels. On the other hand, no correlation was found between CA19-9 levels and the type of diabetes, lipase levels, or the presence of anti-islet cell antibodies. Le(a-b-) patients had significantly lower serum CA19-9 levels. This study emphasizes the frequency of CA19-9 elevations in diabetic patients without cancer.

In vivo and in vitro reactivity of rat spleen cells against regressor and progressor colon-cancer cell variants.

Previous reports demonstrated that progressor and regressor tumor-cell variants isolated from the same colon carcinoma chemically induced in a BD-IX rat differed in their capacity to induce an immune response. The present study was aimed at analyzing the characteristics of the responses to the regressor REGb and progressor PROb clones. Spleen cells from rats bearing early REGb tumors neutralized PROb cell tumorigenicity in a Winn-type local transfer assay, but responded occasionally to REGb and PROb cells in vitro. However, spleen cells from rats immunized by several injections of REGb and PROb cells strongly proliferated when cultured with PROb or REGb cells. This response was selective for the cell lines generated from the individual tumor at the origin of PROb and REGb lines, was dependent on CD4+ spleen cells, and was partially inhibited by an antibody against major histocompatibility complex class-II molecules. Although PROb cells shared tumor-rejection antigen(s) with REGb cells, splenocytes from PROb tumor-bearing rats did not neutralize PROb-cell tumorigenicity in vivo, nor did they proliferate when cultured with PROb or REGb cells in vitro. The unresponsiveness of spleen cells from PROb tumor-bearing rats was not due to the presence of immune suppressive cells, and a defect of antigen-presenting cells was shown to be unlikely. This unresponsiveness was limited to a lymphocyte subpopulation, since spleen cells from tumor-bearing rats responded normally to stimulation by PHA or allogeneic lymphocytes. These results strongly suggest that unresponsiveness of spleen cells from tumor-bearing rats is due to a tumor-specific anergy of the lymphocyte clones able to respond to tumor-associated antigens.

Liposome induction or enhancement of macrophage-mediated cancer cell lysis.

Liposomes of different composition have been used to modify macrophage-mediated destruction of syngeneic cancer cells through a modulation of membrane lipid content of macrophages and/or tumor cells. Dipalmitoylphosphatidylcholine (DPPC)1 liposomes induce cancer cell lysis by normal, non-tumoricidal, peritoneal macrophages and enhance tumor cell destruction by BCG-activated macrophages. This effect was produced by large and small unilamellar liposomes, which are in the 25,000 g supernatant of sonicated preparations. Addition of cholesterol or negative charges carried by dicetylphosphate supressed the effect of DPPC liposomes on macrophage-mediated cytolysis. Enhancement of macrophage-mediated tumor cell lysis was observed when both cancer cells and macrophages were incubated with DPPC liposomes, but not when macrophages and/or tumor cells were preincubated with liposomes prior to their coincubation. Liposomes did not promote the binding of the cancer cells to the macrophages. Liposomes could promote formation of phospholipid domains within the plasma membrane of both tumor cells and macrophages leading to the destruction of cancer cells through a temporary fusion with the macrophages.

[Modulation of macrophage-mediated toxicity to cancer cells in vitro by liposomes and endotoxins]. / Modulation par des liposomes et des endotoxines de la cytotoxicité anti-tumorale des macrophages in vitro.

The cytotoxic effect of rat peritoneal macrophages on syngeneic colon cancer cells is modified by the addition of liposomes. The direction and intensity of this modification depends on the concentration of endotoxin in the culture medium.

High expression of NKR-P1 is not an absolute requirement for natural killer activity in BDIX rats.

NKR-P1 has been identified as a triggering structure selectively expressed on rat natural killer (NK) cells and adherent lymphokine-activated killer (A-LAK) cells. In vivo treatment with anti-NKR-P1 monoclonal antibody (mAb 3.2.3) was shown to induce complete inhibition of NK cytotoxicity and elimination of LAK cell precursors in Lewis and Fisher rat strains. We investigated the effects of mAb 3.2.3 in a colon tumor model in BDIX rats. Inoculation of animals with mAb 3.2.3 even at very high doses induced a strong but incomplete inhibition of NK cytotoxicity in nylon-wool-non-adherent spleen and peripheral blood cells. Generation of adherent A-LAK cells from their spleen precursors was also strongly by not fully inhibited. We also investigated the effect of treatment with mAb 3.2.3 on the tumorigenicity of the NK-sensitive REGb cell line. When subcutaneously inoculated in syngeneic animals, REGb cells induce tumors that first grow for 2 weeks, then spontaneously regress and disappear. In contrast with previous results using anti-asialoGM1, no significant difference in tumor growth was observed between rats treated with mAB 3.2.3 and control animals, even with a long-term treatment. In vitro, mAb 3.2.3 exhibited the same incomplete efficiency. Nylon-wool-non-adherent spleen cells treated with mAb 3.2.3 plus complement were completely free of 3.2.3(bright) cells, but retained a substantial NK activity and generated LAK cells after culture with IL-2. After an overnight incubation in standard medium of 3.2.3-depleted spleen cells, 3.2.3(bright) cells were partially recovered and the NK cytotoxic activity, as well as the generation of LAK cells, was significantly enhanced. These results suggest that a strong expression of NKR-P1 is not required for BDIX mononuclear cells to exhibit NK function and generate LAK cells under IL-2 activation.

Differential sensitivity to natural cell-mediated cytotoxicity of two rat colon adenocarcinoma variants differing in their tumorigenicity: identification of the effector cells as natural killer cells.

DHD/K12 TRb (PROb) and DHD/K12 TSb (REGb) are two cancer cell variants originating from the same rat colon adenocarcinoma. They differ in their tumorigenicity: when inoculated into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells induce tumors which always regress. As previously described, there is an inverse relation between their tumorigenicity and their susceptibility to NCMC mediated by syngeneic spleen or peripheral blood lymphocytes: PROb cells are significantly less sensitive to NCMC than REGb cells. This suggests a role for NCMC in the regression of REGb tumors. In this work the BDIX NCMC effector cells active in vitro against REGb cells were identified as NK cells according to four criteria: (1) efficacy in a 4-h 51Cr release assay, (2) sensitivity to anti-asGM1 antibody plus complement, (3) LGL morphology, and (4) ability to bind with the same affinity REGb and YAC-1 cells. In spleen, these NK cells were heterogeneous with respect to their asGM1 surface density and their morphology. PROb cells were not lysed by these NK cells in a short-term cytotoxicity assay, but only in a 16-h assay. It was shown that PROb and REGb cells were bound with the same affinity by NK cells, thus they certainly differ in their ability to resist to NK lytic mechanisms. This difference could play a role in the different tumorigenicity of the two variants.

Lack of effect of a high polyunsaturated fat diet on the growth of transplantable colon tumors and on the cytolytic activity of macrophages in rats.

The effect of a high dietary level of polyunsaturated fat was tested on the growth of three different colon cancer cell lines injected subcutaneously into syngeneic rats. This effect was also tested on the in vitro cytolytic activity of resident peritoneal macrophages, natural or endotoxin and/or indomethacin-modulated. A 12% corn oil dietary supplement had no effect in any of the cases tested.