Dhirendra N. Misra

Maternal plasma leptin is increased in preeclampsia and positively correlates with fetal cord concentration

OBJECTIVE We tested the hypothesis that the maternal leptin concentration would be increased in preeclampsia, independent of maternal obesity. STUDY DESIGN Maternal and cord plasma leptin concentrations were compared in 2 groups of women with either preeclampsia (n = 24) or normal pregnancy (n = 24), matched 1:1 for prepregnancy body mass index and fetal gestational age at sampling. RESULTS Median leptin concentrations were significantly higher (P <. 03) in women with preeclampsia (45.6 ng/mL) than in normal pregnant women (27.0 ng/mL) and fell rapidly shortly after delivery (26.7 ng/mL and 25.4 ng/mL, respectively). Cord leptin was not significantly different between groups (5.4 ng/mL and 5.8 ng/mL, respectively). Maternal and cord leptin correlated significantly (rho = 0.76, P <.01) only in preeclampsia. CONCLUSION Preeclampsia is associated with an increase in maternal plasma leptin concentrations that strongly correlates with the fetal cord concentration at delivery.

Lymphocyte plasma membranes I. Thymic and splenic membranes from inbred rats

Abstract Thymic and splenic lymphocyte plasma membranes were isolated from inbred ACI and F344 rats. Chemical and enzymic analyses indicated a highly purified plasma membrane preparation with a high cholesterol to phospholipid ratio and over 12-fold enrichment of the membrane enzyme 5′-nucleotidase. Polyacrylamide-gel electrophoresis of externally radioiodinated membranes showed a major 117 000 dalton glycoprotein in both thymic and splenic lymphocytes. Thymocytes differ from splenic lymphocytes by having an unique external glycoprotein of 27 000 daltons and by the relative lack of membrane components above 200 000 daltons.

Lymphocyte plasma membranes. III. Composition of lymphocyte plasma membranes from normal and immunized rats.

Isolated plasma membranes of thymic and splenic lymphocytes from unimmunized and immunized rats of the inbred ACI and F344 strains were analyzed for chemical and enzymatic composition, for membrane protein patterns by polyacrylamide gel electrophoresis and for membrane-associated immunoglobulins. After immunization, the thymic and splenic lymphocyte membranes from F344 rat contained less carbohydrate and higher phospholipid contents than control animals. In both ACI and F344 inbred rat strains the membrane phospholipid to cholesterol weight ratio increased significantly after immunization. The electrophoretic patterns of solubilized membrane proteins and of iodinated external membrane proteins were similar in unimmunized and immunized animals. When thymic and splenic lymphocytes of normal or immunized animals were surface radioidinated, solubilized in Triton X-100, NP-40 or 10 M urea in 1.5 M acetic acid and analyzed by immunoprecipitation, labeled IgM immunoglobulin was recovered from thymic lymphocytes but both labeled IgG and IgM were recovered from splenic lymphocytes. However, when unlabeled isolated plasma membranes were solubilized in 1 percent Triton X-100 and analyzed by immunodiffusion in agarose gels both IgG and IgM were identified in thymic and splenic cells.

Elicitation of the maternal antibody response to the fetus by a broadly shared MHC class I antigenic determinant

The antibody response to the semiallogeneic fetus in the rat was examined in eight mating combinations that were chosen specifically to across different major histocompatibility complex (MHC)2 and nonMHC loci. The BN×DA and BN×BN.IA(DA) matings (n anti-a) were the only ones to show an antibody response to the fetus, and the response occurred in 25–64% of the matings. All the other six mating combinations studied were negative by hemagglutination, cytotoxicity, and cellular radioimmunoassay. The response appeared in the postpartum period after the first litter and reached its maximal frequency during the postpartum periods of the second and third matings. There was no correlation between the litter size and the presence or absence of an antibody response to the fetal antigens. The antibodies elicited by the fetus were hemagglutinating but not cytotoxic, and consisted of only one isotype, IgG2a, whereas in the alloantiserum raised by skin grafting and the injection of lymphocytes, they were distributed among the IgG2a > IgG1 > IgM isotypes and were both hemagglutinating and cytotoxic. Using reciprocal congenic strains, the pregnancy-induced antibody response was shown to be against MHC antigens only. This finding was confirmed by testing the sera against rats from a population that was segregating for the haplotypes a, a/n, and n/n. Comparison of the pregnancy-induced antibody with the BN anti-DA alloantibodies by absorption studies showed that the former was less crossreactive, did not react with the RT1.Aa haplotype-specific antigenic determinant, and did react with an antigenic determinant shared among the a, d, o, i, e, f, r10, b, and m haplotypes—but not with the n, c, g, k, l, h, or u haplotypes. Using the DA.II and r10 strains in cellular radioimmunoassay, absorption, and immunoprecipitation experiments, it was shown that the pregnancy-induced antibody was elicited by a class I antigenic determinant, provisionally designated Pa, which is encoded by a locus in the vicinity of RT1.A.

Lymphocyte plasma membranes II. Cytochemical localization of 5′-nucleotidase in rat lymphocytes

Abstract Cytochemical studies of thymic and splenic lymphocytes from rats showed that 5′-nucleotidase was restricted to the plasma membranes. Isolated plasma membranes contained the highest specific activity of 5′-nucleotidase of any cellular fractions. The results indicate that this enzyme can be used as a plasma membrane marker for lymphocytes.

Growth of rat-mouse hybridomas in nude mice and nude rats.

Athymic (nude) mice and rats were inoculated intraperitoneally with rat-mouse hybridoma cells secreting monoclonal antibodies to rat MHC class I antigens in order to improve the yield of antibodies. Pristane priming and subsequent intraperitoneal injection of the hybridoma cells in to nude mice yielded ascites which contained antibody in high concentration (10-15 mg/ml). Complete Freunds adjuvant, mineral oil, pristane or antibody-antigen complexes were used to induce ascites in nude rats, but only pristane priming did so consistently. The hybridoma cells in the ascitic fluid failed to secrete antibody, although they contained intracellular antibody. However, when the pristane-primed nude rats received 250-500 rads of total body radiation prior to injection with the hybridoma cells, they produced large amounts of antibody. When the nude rats were splenectomized and injected with the hybridoma cells, they also produced antibody in high titers. There was no in vitro inhibition of antibody formation by the hybridoma cells cultured in medium containing 15% serum from nude rats, but co-culture of the hybridoma cells with splenic lymphocytes from normal or nude rats markedly inhibited antibody production. These results indicate that the defect in antibody secretion by the hybridoma cells in the ascites of nude rats is due to the presence of radiation-sensitive suppressor cells in the spleen.

The RT1.G locus in the rat encodes a Qa/TL-like antigen.

A new antigenic system in the rat homologous to theQa/TL antigen system in the mouse has been characterized. It was detected by antibodies raised in donor-recipient combinations that were matched for theRT1. A, B, D, E loci in the major histocompatibility complex (MHC): (R11×BN)F1 anti-BN.1L(LEW), (R18×BN)F1 anti-BN.1L, and BN.1LV1(F344) anti-BN.1L. Absorption analyses using these antisera and a variety of inbred, congenic and recombinant strains identified three alleles,RT1.Ga,Gb,Gc, of whichGc is a null allele. The strain distribution of these alleles was determined, using 37 strains of rats representative of all of the prototypic haplotypes and a number of congenic and recombinant strains. The use of the congenic and recombinant strains showed that theRT1.G locus was linked to the MHC and that the most probable gene order wasA-E-G. Testcross analysis showed that the map distance betweenA andG was 1.4 cM(4/285 recombinants). The RT1.G antigen has a heavy chain ofMr 46 000 and is present on both T and B cells.

Lymphocyte plasma membranes—V. Immunoglobulins on isolated plasma membranes of the thymic and splenic lymphocytes of the rat

Abstract The plasma membranes from thymic and splenic lymphocytes of inbred ACI rats were isolated and compared for immunoglobulin content before and after immunization of the animals with poly(Glu 52 Lys 33 Tyr 15 ). The highly purified plasma membranes were solubilized in the nonionic detergent Triton X-100 and analyzed by double immunodiffusion with antisera specific for heavy chains of rat IgM and IgG immunoglobulins. Both the thymic and splenic lymphocyte plasma membranes from unimmunized and immunized animals had IgM and IgG immunoglobulins. The IgM content was lowest in thymocyte plasma membranes from unimmunized animals. Immunization resulted in an increase in the IgM content of the thymocyte plasma membranes only. Both thymic and splenic cell membranes appeared to have similar amounts of IgG before and after immunization. Semiquantitative estimates indicated that the content of IgG in thymocyte membranes was about 1% of the total membrane protein. Since our previous studies of rat thymocytes by surface radioiodination demonstrated IgM but no IgG immunoglobulin, the present findings suggest that IgG in rat thymocyte membranes may lie within the plasma membrane matrix.

STRUCTURE AND FUNCTION OF THE MAJOR HISTOCOPATIBILITY COMPLEX OF THE RAT

The rat is a major experimental animal in biochemistry, physiology and transplantation, but the lack of systematic definition of its genetics has been detrimental to its use, particularly in transplantation studies. Following the pioneering immunogenetic work of Palm (Palm & Black, 1971) and of Stark (Stark et al., 1967), a steadily growing group of investigators has addressed itself to the study of rat genetics (International Workshops, 1977, 1979, 1981, 1983; Altman & Katz, 1979). These efforts have led to a fairly detailed view of the structure and function of the major histocompatibility complex (MHC), and this article will present a concise review of the current state of knowledge in this field.

Immunochemical evidence for multiple class I antigens coded by the MHC of the rat (RT1) and their differential expression on red blood cells and lymphocytes.

Five monoclonal antibodies reacting with class I MHC antigens were produced by fusing lymphocytes from WF (RT1u) rats immunized against DA (RT1a) rats with P3‐X63‐Ag8‐653 myeloma cells. Sequential immunoprecipitation studies with the mAb and the WF anti‐DA alloantiserum demonstrated the presence of four different class I molecules: all four molecules were reactive with the alloantiserum; three of them contained the determinant for mAb 155; two of the latter three molecules shared the determinants for mAb 3, 56 and 60, and one of these two molecules also contained the determinant for mAb 118. The four molecules could be isolated from the antigen preparation by sequential immunodepletion first with 118, next with 3, then with 155 and finally with the alloantiserum or by sequential absorption with affinity columns of Sepharose 4B coupled to the antibodies. The three antigens which were sequentially isolated with the mAb 118, 3, and 155, respectively, were analysed by SDS‐PAGE after digestion with Staphylococcus aureus V8 protease, and they showed differences in peptide patterns. The relative amounts of the antigens expressed on red blood cells and on lymphocytes were different based on the results of sequential isolation and indirect cellular radioimmunoassay: the antigen which reacted with both mAb 3 (and 56 or 60) and 155 was the major class I antigen on red blood cells, and the antigen which reacted with mAb 118, 3 (and 56 or 60) and 155 was the major class I antigen on lymphocytes.