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.

[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.

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.

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.

Liposomal mitoxantrone for the local treatment of peritoneal carcinomatosis induced by colon cancer cells in mice.

Since liposomes are slowly resorbed from serous cavities, they may constitute a valuable tool for the treatment of peritoneal carcinomatosis. We prepared mitoxantrone (MXN)-liposomes with various lipid compositions and checked their antitumoral activity on a peritoneal carcinomatosis induced by a colon cancer cell injection (1 x 10(5) C51 cells) in BALB/c mice. MXN entrapment in liposomes was rapid and stable due to its high lipophilicity. MXN carried in phosphatidylcholine: cholesterol (2:1; G-liposomes) displayed a reduced toxicity in mice compared to the free drug. When tested at a non-toxic dose (2 mg/kg), MXN entrapped in G-liposomes proved to be as efficient as the free drug. At a higher MXN dose (3 mg/kg), both G-liposomes and phosphatidylcholine:cholesterol:dipalmitoylphosphatidylethanolamine (7:2:1) liposomes, loaded with MXN, significantly increased the life span of mice compared to the free drug and six other liposome formulations. Increase in the MXN therapeutic index, when used in the liposomal form, could then merit further clinical investigations in regard to patients with malignancies confined to serous cavities.

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.

Human carcinoembryonic antigen cDNA expressed in rat carcinoma cells can function as target antigen for tumor localization of antibodies in nude rats and as rejection antigen in syngeneic rats.

We have tried to develop a new model consisting of rats transplanted with syngeneic colon carcinoma PROb cells transfected with cDNA coding for the carcinoembryonic antigen (CEA), the human tumor marker most commonly used as target for MAbs. The antigenic density of the 4 CEA-expressing clones selected for a precise characterization ranged from 5 x 10(4) to 1 x 10(6) CEA molecules per cell. In all clones the CEA was shown to be attached to the membrane by a phosphatidylinositol (PI) anchor. Using a panel of radiolabeled MAbs directed against the 5 major epitopes described on the CEA molecule, we showed that all these CEA epitopes were expressed by the 4 transfectants. Southern-blot analysis showed that the entire CEA cDNA was present in the transfectants. Western-blot analysis, however, showed that the size of the CEA expressed by the 4 transfectants was slightly smaller than that of CEA produced by 2 reference human colon-carcinoma cell lines. Two clones, expressing 1 x 10(5) and 1 x 10(6) CEA molecules per cell, respectively, were grafted s.c. in nude mice and rats. Injection of radiolabeled anti-CEA F(ab')2 fragments into these animals showed specific tumor localization with the highest percentages of injected doses for the transfectants expressing the highest CEA level. When grafted into immunocompetent syngeneic BDIX rats, the CEA-expressing clones induced a strong antibody response against CEA and tumor rejections in a majority of the animals. Although the analysis of the immune response against the CEA-cDNA-transfected carcinoma cells is under investigation, the present results demonstrate that human CEA could function as a rejection antigen when transfected into rat carcinoma cells.

Histological reactivity of a monoclonal antibody against rat colon cancer cells on human and rat normal gut and colonic tumours.

A monoclonal antibody, F11C, was raised against rat colon cancer cells. Its immunoreactivity on normal human and rat gut as well as human and rat colonic tumours was studied by the avidin-biotin-peroxidase complex technique. In both normal rat and human gastrointestinal tract, F11C stained surface epithelial cells from the fundus to distal colon, mainly as supranuclear vesicles. These vesicles appeared to be part of the Golgi apparatus on electron microscopy with immunogold labelling. Twenty primary rat colon tumours and 28 of 43 human colon tumours were also stained, with a heterogeneous pattern but much more strongly than the normal colonic mucosa. Biochemical purification suggested that in rat tumours F11C epitope was carried by a high molecular weight glycoprotein. Absorption experiments with synthetic oligosaccharides showed that F11C monoclonal antibody reacted with blood group A-related oligosaccharides. Nevertheless, F11C reactivity on human tissues was not related to the individual ABO or Lewis phenotype.

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.

F11C antigen: a membrane marker able to distinguish two regressive and progressive variants from a rat colon adenocarcinoma.

Cell variants that differ in their tumorigenicity and immunogenicity have been isolated from a BDIX rat colon adenocarcinoma cell line, DHD/K12. One variant, PRO, and the clones derived from it, are poorly immunogenic and induce progressive and metastatic tumors; the other one, REG, and its clones, are highly immunogenic and induce regressive tumors. When looking for a membrane marker distinguishing between PRO and REG lines, we obtained monoclonal hybridomas by immunizing BALB/c mice with PROb or REGb cell clones. Hybridoma F11C, producing an IgM monoclonal antibody (MAb) was able to distinguish between the cell variants on membrane immunofluorescence. All REGb cells strongly express F11C membrane antigen. On PROb cells, F11C antigen expression is weak, as demonstrated by cytofluorimetric analysis, and limited to a fraction of the cell population. The F11C membrane antigen is highly specific for the DHD/K12 cell line and the variants derived from it, but is not expressed on cells dissociated from the DHD transplanted tumor, from which DHD/K12 was established, suggesting that F11C antigen emerged during cell culture. Fluorescence absorption on synthetic oligosaccharides demonstrated that F11C antibody cross-reacts with A type 3, A type 4 and A type 5 chain blood group tetrasaccharides.

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.

Comparative effect of rat and fetal calf serum on measurement of the natural tumoricidal activity of rat lymphocytes, macrophages and polymorphonuclear cells.

The effect of rat serum versus fetal calf serum on the in vitro natural cytolytic activity of rat lymphocytes, macrophages and polymorphonuclear cells against syngeneic tumour cells was compared. The cytolysis level mediated by the three varieties of effector cells was lower when rat serum was used instead of fetal calf serum to supplement the culture medium. This could explain in part the discrepancies found between in vitro and in vivo studies.

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.

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.

Regression induced by lentinan, of peritoneal carcinomatoses in a model of colon cancer in rat.

Lentinan has been tested in a model of colon cancer in rats. Peritoneal carcinomatoses were induced in BDIX rats by i.p. injections of syngeneic cells isolated from a colon carcinoma, and established in a permanent cell line. The treatment consisted of five i.p. injections, 2 days apart, of 2 mg lentinan/kg at a concentration of 200 micrograms/ml. This was started on day 14 after tumor cell injection, when the rats bore numerous nodules of 1-5 mm. Lentinan significantly inhibited the growth of carcinomatoses. Eleven out of the 20 rats treated with the best lentinan therapy were tumor free at autopsy on day 42. Lentinan significantly increased the life span of carcinomatous rats. The half life following tumor cell injection was 42 days in the control and 70 days in the treated group. Four out of 10 treated rats were still alive on day 210. They were tumor free at autopsy, whereas all the controls died between the 40th and the 70th day. The effectiveness of lentinan was dependent on the number and frequency of the injections. A dose effect was obtained and a strong influence of the concentration was shown.

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.

Effects of a single injection of anti-asialo GM1 serum on natural cytotoxicity and the growth of a regressive colonic tumor in syngeneic rats.

The REGb tumor cell line is a cloned variant of the DHD-K12 cell line, established from a colon carcinoma chemically induced in the rat. Unlike the parent DHD-K12 cell line, or other clones, which give progressive tumors when inoculated to the syngeneic rat, REGb cells produce tumors which regress in 3 to 5 weeks and never cause metastasis. In order to explore the role of natural killer (NK) cells in REGb tumor regression, each rat was given one injection of anti-asialoGM1 (anti-asGM1) serum, a known inhibitor of NK activity. This injection was done 24 hr before REGb cell challenge. This injection significantly depressed the in vitro cytotoxicity of peripheral blood lymphocytes on REGb cells for 2 weeks. REGb tumors grew larger and regressed later in the treated animals than those in the controls. Furthermore, a progressive or recurrent tumor was observed in 4 out of 10 treated rats, giving lung and/or lymph-node metastases in 2 cases. Immuno-histological study of the cells infiltrating the REGb tumors in control and treated animals showed a decrease number of asGM1+ and OX8+ lymphocytes, presumably NK cells, after anti-asGM1 treatment. An increase in number of macrophages was demonstrated in the progressive tumors of treated animals. These results suggest that NK cells play an important role in the initial stage of the regression TSb tumors in untreated syngeneic rats.