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J Charlemagne

Publications and source records attributed to J Charlemagne.

At least 55 records · Page 3Linked to original sources
PubMed1988

Monoclonal antibodies to axolotl immunoglobulins specific for different heavy chains isotypes expressed by independent lymphocyte subpopulations.

An immunoblotting analysis of purified axolotl immunoglobulins (Ig) separated by SDS-PAGE reveals two heavy (H) chains isotypes: a 76 kDA chain recognized by the monoclonal antibody (mAb) 33.45.1 and a 66-68 kDa doublet recognized by the mAb 33.39.2. The 76 kDa chain is associated to high molecular weight (HMW) Ig molecules and the 66-68 kDa H chains are associated to low molecular weight (LMW) Ig of 172 kDa. Both H chains isotypes are linked to identical light (L) chains, labelled in immunoblotting by the mAb 33.101.2. Two different axolotl lymphocyte subpopulations are characterized by these two distinct H chains isotypes. One population of splenic lymphocytes (approximately 40%) is labeled by indirect immunofluorescence with mAb 33.45.1, specific for the 76 kDa H chain isotype. Another population (approximately 20%) is labeled by mAb 33.39.2 specific for the 66-68 kDa H chain isotype. Both populations of splenic lymphocytes are stained by mAb 33.101.2 specific for the axolotl L chains. Therefore, the presence of at least two independent Ig classes is now confirmed in a urodele amphibian species at the humoral and cellular levels.

Ambystoma↗
PubMed1988

Light and electron microscopic immunolocalization of two nuclear antigens in the liver of Pleurodeles waltl using monoclonal antibodies.

The distribution of 2 nuclear antigens in the interphase nuclei of liver of Pleurodeles waltl was determined with the help of monoclonal antibodies, using immunofluorescence for light microscopy and indirect immunoperoxidase and immunogold labeling procedures for electron microscopic localization. The antibodies C36/1 and A33/22 label antigens with relative molecular masses of 270 kDa and 80 kDa, and isoelectric points of 7.0 and 6.4, respectively. The liver of urodels is characterized by the presence of a peripheral layer of hematopoietic cells around the parenchymatous tissue formed by typical hepatocytes. The antibody C36/1 labels the nuclei of both types of cells, whereas the antibody A33/22 labels the nuclei of hepatocytes but not those of the peripheral hematopoietic cells. With both these antibodies, labeling, whenever observed, is restricted to fibrillar structures in the interchromatin space, i.e., to peri- and inter-chromatin fibrils; condensed chromatin, nucleoli, and nuclear envelope are not labeled.

Animals↗
PubMed1988

Surface markers of axolotl lymphocytes as defined by monoclonal antibodies.

In an attempt to identify urodele amphibian lymphocyte subpopulations by their surface markers, we prepared hybridomas from BALB/c mice spleen immunized with axolotl (Ambystoma mexicanum) blood and splenic leucocytes and purified immunoglobulins. Sixty-five hybridomas were selected and subsequently subcloned. Among numerous monoclonal antibodies (mAbs) thus obtained, four mAbs were extensively characterized by immunoblotting, single and double fluorescence and immunohistology. MAb 34.38.6 recognizes polypeptides between 65,000 and 72,000 MW and labels in immunofluorescence nearly all thymocytes, 60-63% splenic lymphocytes of normal animals but only 9% splenic lymphocytes in thymectomized animals. MAb 19.14.2 reacts with a 98,000 MW protein and labels a restricted lymphocyte population in thymus (52-77%) and spleen (20-25%). The immunohistological study demonstrates that 34.38.6 and 19.14.2 label most thymocytes and a large proportion of spleen leucocytes including lymphocytes, granulocytes and macrophages. In addition, 19.14.2 labels some large interdigitating cells in thymic epithelial areas and splenic cords. MAbs 33.45.1 and 33.101.2, respectively, recognize heavy (72,000-88,000 MW) and light (20,000-27,000 MW) axolotl immunoglobulin chains. They do not react with thymocytes but label a splenic lymphocyte population not labelled by mAb 34.38.6. The proportion of surface immunoglobulin-positive (sIg+) lymphocytes in spleen is not altered by thymectomy. MAb 33.101.2 labels 40-48% of splenic lymphocytes, 33.45.1 stains only 14% of these same cells. This suggests some interesting heavy-chain isotypic differences in axolotl. For the first time in urodele amphibians, mAbs differentiate T-like and B-like lymphocyte populations by their membrane markers. This will allow further analysis of the axolotl immune system.

Ambystoma mexicanum↗
PubMed1988

Specificity of natural serum antibodies present in phylogenetically distinct fish species.

The following fish orders, at different stages of phylogenetic development, were tested for serum antibody activity against actin, myosin, tubulin, thyroglobulin, haemocyanin, single-stranded DNA and trinitrophenyl (TNP): Elasmobranches (sharks), Chondrosteans (sturgeons), and Teleosts (bony fish). Significant antibody activity was found against all these self and non-self antigens but, in particular, high titres of natural anti-TNP antibodies were noted. Anti-TNP antibodies were then isolated by affinity chromatography from pooled sera of several representative fish species, and studied by a non-competitive and a competitive enzyme immunoassay. When the isolated antibodies were tested by the non-competitive assay, Teleost (tench) anti-TNP antibodies bound almost exclusively to TNP, whereas Chondrostean and Elasmobranche antibodies bound mainly to TNP but also to one or more other antigens. When the isolated antibodies were examined by the competitive assay, they could be separated into three groups: (i) anti-TNP antibodies whose binding could be inhibited by TNP only (Teleosts); (ii) anti-TNP antibodies mainly inhibited by TNP but also significantly inhibited by two or three other antigens (Chondrosteans); and (iii) anti-TNP antibodies binding to different antigens but only slightly inhibited by them (Elasmobranches). These antibodies were found by immunocytochemical staining to bind to normal cell constituents, and especially to cytoskeletal proteins. The results strongly suggest that natural antibodies against the seven antigens examined are present in normal fish serum, and that their specificities differ with the stage of phylogenetic development.

Animals↗
PubMed1987

Antibody diversity in amphibians. Noninbred axolotls used the same unique heavy chain and a limited number of light chains for their anti-2,4-dinitrophenyl antibody responses.

Noninbred axolotls (Ambystoma mexicanum, amphibia, urodela) were immunized with trinitrophenylated sheep red blood cells (TNP-SRBC) and anti-2,4-dinitrophenyl (DNP)/TNP antibodies were individually purified by affinity chromatography. The isolated IgM-like antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) under reducing conditions. The SDS-PAGE and IEF-separated heavy (H) and light (L) chains were electroblotted onto nitrocellulose, probed with mouse monoclonal antibodies specific for H or L axolotl Ig chains and stained by a rabbit anti-mouse Ig horseradish peroxidase conjugate. The specific detection of axolotl anti-DNP/TNP H chain spectrotypes shows for each of the 14 individually analyzed samples a very similar pattern of 4-5 ordered spaced bands. This suggests that all animals express the same VH chain segment representing the germinal expression of a unique VH gene. When the same analysis was performed starting from a pool of nonimmunized axolotl sera, a low background of natural anti-DNP antibodies was detected. When analyzed by IEF, the H chains of the pooled anti-DNP natural antibodies display the same pattern of restricted heterogeneity when compared to the H chain spectrotypes of the individual immune anti-DNP/TNP antibodies. The specific detection of the axolotl anti-DNP/TNP L chain spectrotypes indicates at the individual level more heterogeneous and polymorphic patterns compared with H chains, although most animals share the majority of their bands. Our experiments indicate that in axolotl, the production of antibodies to DNP results from the germinal expression of a very limited set of V genes, already expressed as naturally occurring anti-DNP antibodies before immunization. This seriously restricts the possible extension of the antibody repertoire and perhaps even the nature of antibody "specificity" in this primitive vertebrate.

Ambystoma↗
PubMed1987

Antibody diversity in trouts obtained by gynogenesis or self-fertilization. Comparative analysis of the heavy chain spectrotypes.

Conventionnal (C) trouts and trouts obtained by gynogenesis (G) or self-fertilization (SF) were immunized with DNP-KLH and anti-DNP antibodies were individually purified by affinity chromatography. The isolated IgM-like antibodies were separated by an iso-electrofocusing technique in reducing conditions and electroblotted onto nitrocellulose. The transfers were probed with a mouse monoclonal antibody specific for trout heavy (H) antibody chain and revealed with a rabbit anti-mouse IgG horse-radish peroxydase conjugate. When comparing the IEF H chain spectrotypes of C, G and SF trouts, it was observed that individual C spectrotypes are more different between themselves than G and SF spectrotypes, and that individual SF spectrotypes were less heterogeneous than C or G ones. These results suggest that in trout, inbreeding induces a reduction of antibody diversity and heterogeneity. The inheritance of antibody repertoire might be taken in account in the inbreeding selection schedules for fish of economical interest.

Animals↗
PubMed1987

Characterization of axolotl heavy and light immunoglobulin chains by monoclonal antibodies.

Axolotl specific antibodies to 2,4-dinitrophenyl (DNP) were purified by affinity chromatography from the sera of animals immunized with 2,4,6-trinitrophenylated sheep red blood cells (TNP-SRBC). The purified anti-TNP/DNP antibodies, when analyzed by SDS-PAGE, were constituted of high molecular weight molecules, which in reducing conditions, were separated into heavy 72-88 kD and light 27-30 kD polypeptides. The axolotl heavy antibody chains strongly bound Concanavalin-A and migrate faster in SDS-PAGE after endoglycosidase-F (Endo-F) treatment. Using the same techniques, no carbohydrate components were detected onto light chains. Monoclonal antibodies (MAbs) were obtained against these purified axolotl immunoglobulins (Ig) and their specificities were studied by immunoblotting. MAbs 33.45.1 and 33.101.2 respectively recognized heavy and light chains determinants of the Ig molecule. These determinants were resistant to Endo-F digestion, suggesting that the two MAbs were not directed to polypeptide-associated N-linked high mannose or complex oligosaccharides. MAbs 33.45.1 and 33.101.2 were compared to 11.5.2, an anti-axolotl thymocytes MAb which was reactive for both axolotl leucocytes and soluble Ig. MAb 11.5.2 reacted in immunoblotting against several high molecular weight axolotl serum proteins, including heavy Ig chains. Light chains were not recognized. However, 11.5.2 did not further recognize Endo-F treated Ig, suggesting its specificity for a carbohydrate determinant of the heavy chain, and link to a large diversity of soluble or membrane glycoproteins.

Ambystoma↗
PubMed1985

Monoclonal antibodies to lampbrush chromosome antigens of Pleurodeles waltlii.

Germinal vesicles of oocytes from Pleurodeles waltlii were used for immunization of BALB/c mice to obtain hybridomas secreting monoclonal antibodies. The hybridomas were screened for reactivity of their antibodies against lampbrush chromosomes of oocytes, as revealed by indirect immunostaining. Antibodies labelling the lampbrush chromosomes were also tested on histological sections of oocytes, embryos, and larvae of Pleurodeles. Characterization of the antigens was accomplished through immunoblotting of two-dimensional electrophoretic gels of germinal vesicle proteins. The ten monoclonal antibodies giving a positive reaction were classed into five groups. Group 1, exemplified by antibody A33, recognizes all the lampbrush chromosome transcribing sites (loops). Moreover, it differentially labels the cell nuclei during embryonic and larval development. Group 2, antibody B71, also stains all the loops of the lampbrush chromosomes, but does not react with cell nuclei of embryos and larvae. Group 3, antibody A1, labels specific loops, some of which are heterozygous in the strain of P. waltlii used. These heterozygosities have allowed us to localize and to characterize a chromosomal segment on bivalent IV which is heteromorphic in the two partners of the bivalent. We suggest that this heteromorphism represents a morphological distinction between Z and W heterochromosomes. Moreover, this antibody reacts with only one transcription unit along a loop that contains several units. Group 4, antibody B24, stains the only two structures in the lampbrush chromosomes of P. waltlii that do not have a loop organization, the mass "M" and the spheres. Group 5, antibody A35, reacts with the chromomeres. The antigens corresponding to antibodies A33 and B24 have been identified as proteins, which have apparent molecular weights of 80 and 104 kilodaltons, respectively. They correspond to proteins abundant in the germinal vesicles. All the antibodies described here cross-react with the lampbrush chromosomes of five other species of Urodeles.

Animals↗
PubMed1985

Antibody diversity in fish. Isoelectrofocalisation study of individually-purified specific antibodies in three teleost fish species: tench, carp and goldfish.

Natural anti-DNP antibodies were isolated by affinity chromatography from individual sera of three Cyprinid fish species (carp, goldfish and tench) and their electrofocusing (IEF) spectra were analysed in reducing conditions. In addition, immune anti-penicillin and anti-BSA antibodies were isolated from individual and pooled tench sera, and studied by IEF techniques on reduced samples. Diversity rates appeared to be rather low in the three fish species, and striking similarities arose between individuals of a same species. These results can be interpreted by the existence of particular selective pressures operating in poikilothermic species as it was already suggested by Du Pasquier. No enhancement of antibody heterogeneity could be detected in the tetraploid (carp and goldfish) species. This result is also in accordance with the selection of a restricted germ-line determined antibody repertoire in lower vertebrates.

Animals↗
PubMed1984

Structural and functional analysis of spontaneous anti-nitrophenyl antibodies in three cyprinid fish species: carp (Cyrinus carpio), goldfish (Carassius auratus) and tench (Tinca tinca).

High spontaneous anti-trinitrophenyl (TNP) activities were found in three Cyprinid fish species: Carp (Cyprinus carpio), Goldfish (Carassius auratus) and Tench (Tinca tinca). The molecules involved, isolated by affinity chromatography on dinitrophenyl-lysine Sepharose (DNP-lysine-Sepharose), had the main characteristics of a high molecular weight immunoglobulin (IgM-like). Affinity measurements were performed on natural anti-DNP/TNP antibodies isolated from nine individual tench sera, using the inhibition of DNP-T4 bacteriophage inactivation technique. The antibodies analysed were more specific for TNP than for DNP. No activity was found against paranitrophenyl hapten. Affinities were all very low, even for TNP. In the three species, natural anti-DNP/TNP antibodies constitute as much as 11 to 16% of the total immunoglobulin concentration. This high level of nitrophenyl-binding serum immunoglobulins either suggests the existence of a particular regulatory mechanism in fish or reflects a generally low antibody diversity in these species.

Animals↗
PubMed1981

Regulation of antibody synthesis in the X-irradiated Mexican axolotl.

The effects of X-irradiation were studied on the Mexican axolotl antibody synthesis. To reduce the anti-horse red blood cell (HRBC) antibody titers, 150 rd and smaller doses are ineffective, 200-450 rd are increasingly effective, and 700 rd are maximally effective (and lethal). A significant enhancement of the anti-HRBC titers was observed in low doses (50-150 rd X-irradiated animals). This enhancement was also observed when a low X-ray dose was applied only on the thymic areas. In whole body, but thymus area-shielded, 100 rd X-irradiated animals, the anti-HRBC titers were similar to those of the nonirradiated, HRBC-immunized control group. To explain these phenomena, it is suggested that a radiosensitive, adult thymectomy-sensitive and hydrocortisone-sensitive suppressor T cell subpopulation regulates the antibody synthesis in the axolotl.

Ambystoma↗
PubMed1981

The goldfish immune response. I. Characterization of the humoral response to particulate antigens.

Anti-red blood cells (RBC) and anti-hapten antibody synthesis were studied in the goldfish, Carassius auratus. Spontaneous haemagglutination titres were found against all the antigens tested. A weak secondary response was observed in RBC-primed fish boosted during the end-phase of the primary antibody production. However, when the second antigenic challenge was performed during the early exponential phase of a primary stimulation, an important amplified response was obtained. The antibody production and immunological memory can be dissociated: no antibody synthesis occurred in glutaraldehyde-fixed RBC (F-RBC) primed was obtained when untreated or F-RBC were given to F-RBC primed animals. The amplified response to sheep red blood cells (SRBC) was significantly inhibited when fish were primed with a mixture of SRBC and Xenopus red blood cells (XRBC), demonstrating an antigenic competition phenomenon. Studies on anti-trinitrobenzene responses confirm the efficiency of E. coli lipopolysaccharide as a carrier for fish anti-hapten immunization. The kinetics and regulation of antibody synthesis in fish are discussed in relation to the described results.

Animals↗
PubMed1980

Development of amphibian thymus. I. Morphological differentiation, multiplication, migration and lysis of thymocytes in the urodele Pleurodeles waltlii.

Different stages of thymus morphogenesis and thymocyte differentiation have been studied at the ultrastructural level in the urodele amphibian Pleurodeles waltlii Michah. From stage 38 to 42 the undifferentiated lymphoid stem-cells colonize the epithelial thymic buds. From stage 43 to 45 the lymphoid stem-cells differentiate into lymphoblasts and then transform into typical lymphocytes. A clasmatosis phenomenon seems to be involved in this transformation. From stage 46 to 52, a phase of intense proliferation occurs and relations between dense reticular epithelial cells and lymphocytes are described. At stage 53, numerous lymphocytes die in the thymic tissue and are phagocytosed by macrophages. At the same time, lymphocytes undergo migrations through the intra- and peri-thymic blood vessels. These lymphocytes should populate the peripheral lymphoid organs, according to the previous finding that stage 52 was the last developmental step for an efficient abrogation of cell-mediated immunity by thymectomy in Pleurodeles.

Animals↗