Biomedical subjects
Publications and source records attributed to D Thierry.
A 70 kDa protein of Halobacterium halobium cross-reacts with an antiserum directed against the v-myc gene product of the avian myelocytomatosis virus (MC29). This cross-reaction is in agreement with hybridization studies which indicate that H. halobium possesses DNA and RNA sequences homologous to the v-myc gene.
We have partially purified an archaebacterial protein of 84 kD which shares common epitopes with the human c-myc protein as shown by its cross-reactivity with a commercialized anti-human c-myc antiserum. An antiserum raised against the 84 kD protein recognizes a 60 kD protein from HL-60 nuclei. This protein is also recognized by the anti-human c-myc antiserum. Using this archaebacterial protein as antigen for Western blot analysis, we found that the human c-myc oncogene product could be immunogenic and that it is possible, in some spontaneously occurring human tumours, to detect antibodies against the c-myc gene product in the serum of cancer patients.
Antibodies raised in rabbits against pure rat T-kininogen did not cross-react with Ile-Ser-Bradykinin, bradykinin, nor with kininogens from other mammalian species. They presented a 1 to 15% cross-reaction with pure rat HMW kininogen, depending on the quantity of HMW kininogen. A direct radioimmunoassay for rat T-kininogen in plasma was developed and it enabled 89 fmol of the protein to be detected. A good correspondence was obtained between the direct RIA and the T-kinin generating assay. By the direct assay, it was found that T-kininogen is increased about ten fold in rats subcutaneously injected with turpentine. These data were confirmed by HPLC analysis of the plasma kinins released by trypsin which demonstrated that only T-kinins are increased, bradykinin being unchanged. It was possible according to the results obtained by the direct RIA and HPLC analysis to estimate that in the normal rat, HMW and LMW kininogen represent about 35% and T-kininogen 65%. In the turpentine-treated rat, T-kininogen reached 95%. This RIA will allow the study of the regulation of T-kininogen in the rat and the synthesis of this protein in cells in culture.
Fluorimetric analysis of DNA unwinding, which allows measurement of DNA strand breaks in human leukocytes, has been optimized by reducing the amount of cells required for the test and by modifying the DNA alkali unwinding conditions. This permitted measurement of DNA strand-break induction in cells irradiated with low (0.5-7 Gy) or high doses (5-20 Gy) of gamma rays. Linear dose-response curves were obtained for both dose ranges. Presence of cysteamine during irradiation caused a decrease in the extent of DNA strand breaks. The kinetics of the DNA strand-break rejoining process appeared to be biphasic over the dose range of 2-20 Gy when plotted on a linear vs linear axis (percentage of damage as a function of time). Since the rate of disappearance of damaged DNA was similar for any given dose and for all postirradiation incubation times tested, we have expressed the extent of repair after a given postirradiation incubation as the ratio of the slopes of the regression lines obtained from incubated and nonincubated cells. Leukocytes from 25 healthy donors were analyzed to determine an average value for controls. No difference in the level of DNA strand breaks and the rate of repair of these breaks was observed between leukocytes from three ataxia telangiectasia patients and those from normal donors.
In the present investigation, the immunoglobulins G of human sera were studied after isoelectrofocusing using polyacrylamide flat bed gel. The heterogeneity of immunoglobulins was demonstrated in normal serum as well as in pathological situations. Isoelectrofocusing study was performed using the total serum or the immunoglobulin's preparations obtained after absorption and elution on staphylococcus protein A. These methods respect the integrity of immunoglobulins and allow the study of native immunoglobulins. Furthermore it was demonstrated the micro-heterogeneity of immunoglobulins (antibody) induced by antitetanic vaccination.
Delayed hypersensitivity reactions to the carrier and antibody mediated reactions to the carrier and to the hapten have been studied in guinea pigs treated with carrageenan, a macrophage toxic agent, before or after immunization with a hapten-carrier complex. The results show that carrageenan which acted at the afferent limb of immunity neither depressed nor enhanced delayed hypersensitivity reactions but could affect antibody formation. Macrophage functions in antibody synthesis appeared to be more sensitive to carrageenan than those involved in the induction of cell-mediated immunity. Carrageenan could provide a useful tool for studying the functional heterogeneity of macrophages during the induction of the immune response.
In a previous study, we have showed a great heterogeneity of antitetanus antibodies using polyacrylamide thin layer isoelectrofocusing. In this study, we have improved the resolution of the focused antitetanus antibodies by electrofocusing in sucrose density gradients, which is a suitable procedure for preparative work. After isoelectrofocusing the elution is performed according to any pH variation. For each fraction, we have determined pH, absorbance at 280 nm, protein level by method of Lowry, IgG concentration using immunonephelometric method and antitetanus antibodies concentration by radioimmunoassay. Furthermore, the purity of each fraction is verified by polyacrylamide slab gel isoelectrofocusing. It is concluded that the isoelectric point of antitetanus antibodies is alkaline range between 7.5 and 8.5. This precise knowledge could afford an opportunity for the industrial preparation of antitetanus antibodies.
Sodium diethyl dithiocarbamate (DTC) is a low molecular weight sulphur compound which has been shown to be an adjuvant of immunity in vivo. In this report, DTC was shown to inhibit in vitro DNA-synthesis of human peripheral blood lymphocytes stimulated by phytohemagglutinin or concanavalin A. DTC could act directly at the cellular level and indirectly by interfering with culture medium or lectins.
Specific gene transfer into targeted tumor cells remains a critical issue for the development of systemic gene therapy protocols. With this end in view, we have tested the possibility of selectively directing genes to tumor cells through the recognition of tumor-associated antigens (TAA). This was approached in vitro on four human renal cell carcinoma (RCC) lines by means of the highly specific mouse G250 monoclonal antibody (mAb) chemically conjugated to a plasmid DNA conveying a reporter activity. This mAb directed to a TAA that is present on 95% of primary RCCs and on 60% of metastatic human RCCs was extensively characterized, including during clinical trials. Epifluorescence microscopy analysis indicated that upon specific binding to G250 TAA, G250 mAb alone or conjugated to plasmid DNA was internalized by an active endocytic process and colocalized with the transferrin concentrated in the late recycling perinuclear compartment. We also observed that both unconjugated G250 mAb or G250 mAb conjugated to plasmid DNA remained in the perinuclear region of the cells for > or = 20 hours and were not rapidly translocated to lysosomes or recycled to the plasma membrane. In contrast, unconjugated plasmid DNA was not internalized. After transfection of G250 TAA-positive RCC lines with G250 mAb conjugated to a plasmid cDNA encoding mouse interleukin-2, a significant and sustained production of mouse interleukin-2 protein was detected from days 5-15 and was abrogated by inhibiting the internalization process. Altogether, our data showed that endocytosis of G250 TAA should be the basis of gene transfer to RCC, suggesting that targeting of TAA capable of internalization may be the basis of new approaches for designing alternative cancer gene therapy procedures.
The rate of the clonogenicity of granulocyte macrophage forming cells (GM-CFU) in agar was analysed using mononuclear cells from the peripheral blood of cancer patients undergoing total body irradiation (TBI, 5 patients, 1.5 Gy in 15 days) or hemicorporal irradiation of the lower (LHBI, 5 patients, 15 Gy in 15 days) or upper (UHBI, 4 patients, 15 Gy in 15 days) part of the body. Although the GM-CFU level in the blood is low compared with that in the bone marrow, and shows considerable variability, the preliminary results demonstrated that in most cases the GM-CFU level was increased around the 10th day after the beginning of irradiation, followed by a return to base level within a few days, for LHBI and TBI but not for UHBI. The question of whether the observed peak for the clonogenicity was due to stem cells leaving a radiation-injured compartment of the bone marrow or to vicarious stem cells migrating from non-irradiated regions, or was the result of enhanced activity of colony-stimulating factors, remains to be investigated.
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