Biomedical subjects
Publications and source records attributed to M S Hamilton.
Anticipating a change in the way Medicare pays for capital costs, healthcare organizations should evaluate their capital expenditure plans. To maximize payments under the current system, they should create an equipment acquisition plan that brings the greatest capital costs during the equipment's early life. They then should determine a cost allocation method that assigns the greatest allowable capital costs to Medicare.
The actions of the cytokine interleukin 1 (IL-1) in hematopoiesis involve induction of colony-stimulating factor (CSF) production on accessory cells in the hematopoietic microenvironment and synergy with CSF on early hematopoietic progenitor cells. We have used these properties to accelerate hematologic reconstitution in granulocytopenic mice. Mice with cyclophosphamide-induced granulocytopenia were injected i.p. at day 0 with a single dose (8-800 ng) of human recombinant IL-1 alpha. At daily intervals thereafter during a period of 7 days mice were sacrificed and blood granulocytes and bone marrow cellularity were assessed. Mice receiving a single dose of 80 or 800 ng IL-1 had significantly (p less than 0.05) higher blood granulocyte counts at days 4 and 5 than control animals receiving heat-activated IL-1. This activity of IL-1 was not affected by injection of the cyclooxygenase inhibitor ibuprofen. Bone marrow cellularity, as assessed in histological sections of femurs, was significantly greater (p less than 0.05) at day 2 in mice treated with 80 or 800 ng IL-1. These results show that a single low dose of IL-1 may be used to accelerate the reconstitution of granulocytes following granulocytopenia induced with chemotherapy.
A panel of sperm-reacted monoclonal auto-antibodies developed from spleen cells of vasectomized mice (BDF1) were characterized. Immunogenic antigens were mainly located on the acrosome, midpiece and principal piece. All of the monoclonals were IgM; three demonstrated a multispecific reaction with testis and/or epididymis antigen extracts by immunoblotting. Immunobead studies indicated that most of the antibodies were to surface molecules, a finding supported by the observation that five of the seven antibodies caused complement-mediated immobilization (although not sperm agglutination) of mouse sperm. In vitro fertilization was significantly impaired when antibodies (Vx5, 8 and 10) were added to the sperm prior to exposure to the eggs. Furthermore, passive immunization with Vx5 antibody reduced in vivo fertilization. Our findings indicate that vasectomy-generated antibodies can reduce sperm function in vitro and in vivo.
A well-characterized murine model of graft-vs-host disease (GVHD) that develops in response to minor histocompatibility antigens was used to study the mechanism of an immunodeficiency syndrome that is associated with GVHD. Lethally irradiated mice were transplanted with a combination of bone marrow and spleen cells from H-2 compatible donors that differed at multiple minor histocompatibility antigens, or from syngeneic donors. Four to 12 weeks later, the humoral responses of transplanted and control mice to the T dependent antigens bacteriophage phiX174 and TNP-sheep red blood cells (TNP-SRBC), and to the T independent antigen TNP-Brucella abortus (TNP-BA) were determined. The results demonstrate that mice with GVHD have relatively intact B Cell function and a profound defect in T helper cell function. The immune response to T dependent antigens normalized with repeated immunization. We conclude that immune dysfunction in mice with GVHD is due to a reversible defect in T helper cell function.
It has been postulated that an acquired immunodeficiency syndrome develops in neonates as the result of a maternally induced graft-vs-host disease (GVHD) that develops when sufficient numbers of maternal lymphocytes are transferred to the fetus across the placental barrier. The present study was done to determine whether major histocompatibility complex (MHC) antigens or non-MHC minor histocompatibility antigens (MiHA) were involved. Female C57BL/6 mice were bred to males of eight selected strains such that maternal-fetal disparity existed at MHC antigens and/or minor histocompatibility antigens. Offspring were tested for immune function at 6-7 weeks of age using a Jerne plaque assay to measure the humoral response to that T dependent antigen sheep red blood cells (SRBC). None of the offspring developed clinical signs of GVHD, but 3 of 124 mice tested made no immune response to SRBC. Immunodeficiency was associated with maternal-fetal disparity only at a small number of MiHA and not at the MHC. We postulate that immunodeficiency in this model is mediated by a subclinical maternally induced GVHD to paternally derived MiHA of the fetus.
Antisperm antibodies are implicated as one causative factor of infertility, but the target antigens have not been identified. Immune responses to sperm antigens are qualitatively variable even within a single mouse strain. We took advantage of this variability and immunized individual female mice to allogeneic sperm to reflect their natural exposure during mating. We determined the ability of the individual sera to inhibit in vitro fertilization and to bind to sperm antigens separated by electrophoresis. Compared to preimmune sera, four of five immune sera significantly inhibited in vitro fertilization. The serum from individual mice bound variable panels of sperm antigens. By comparing the panels, we identified two polypeptides with molecular weights of 40,000 and 44,000 that were bound by all sera. We propose that these molecules may be good candidates for further investigation of the immunoprophylaxis of pregnancy.
CBA/J female mice mated to DBA/2 male mice have a high level of fetal resorption. The rate of resorption can be influenced by the environment in which the animals are maintained.
Female mice from four congenic strains were bred to males of the same four strains to determine the effect of maternal-fetal disparity at major histocompatibility complex (MHC) and the effect of non-MHC minor histocompatibility antigens on the weight of the feto-placental unit. An increase in feto-placental weights was found in all three of the four strains that could be evaluated when mother and fetus differed at multiple minor histocompatibility loci, irrespective of whether disparity at the MHC was present. No increase in feto-placental weights was found when mother and fetus differed at the MHC alone. The fact that these results were found in all strains studied suggests that the effect of maternal-fetal disparity at multiple minor histocompatibility antigens (minor HA) on the weight of the feto-placental unit is biologically significant. We conclude that if the increase in weight of the feto-placental unit results from immunostimulation, then minor histocompatibility antigens are the primary target of the maternal immune response to the histocompatibility antigens of the fetus.
Inbred mice from selected unrelated and congenic strains were mated to determine the relative effects of maternal-fetal disparity at major histocompatibility complex (H-2) and non-H-2 minor histocompatibility antigens on the feto-placental unit at 14 days of gestation. A significant increase in weight of the feto-placental unit was observed only when mother and fetus differed at multiple minor histocompatibility loci. No increase in the weight of the feto-placental unit was observed when mother and fetus differed only at H-2. These results suggest that immunostimulation of the fetus results from a maternal response to minor histocompatibility antigens and not to H-2 antigens.
A monoclonal antibody designated 'EC-1' was derived from a fusion of myeloma cells with lymphoid tissue from a syngeneically multiparous, but otherwise unimmunized, mouse and was selected by screening for reactivity with teratocarcinoma cells. The IgM antibody binds to the cell surface of ova, zygotes, and 2-cell embryos. Binding is not detected on the 4- or 8-cell embryo but reappears on the morula and blastocyst. EC-1 binds to the trophoblast but not to the inner cell mass of in vitro attached blastocysts and the ectoplacental cone of the peri-implantation embryo. In adult tissues, EC-1 binds to the follicular cells of the ovary, the lining epithelium of the pregnant uterus, the interstitial region of the testes and to epididymal but not testicular sperm. In nongonadal tissues EC-1 binds to an epitope located in some, but not all, regions of connective tissues associated with basement membrane. The antigen detected by EC-1, as expressed on teratocarcinoma-derived cell line PYS-2, is a large glycoprotein which is sensitive to reduction. EC-1 inhibits in vitro fertilization and partially inhibits in vitro development of in vitro fertilized ova. The possible implications of EC-1 binding and activity are discussed.
Sperm maturation antigen 4 (SMA 4) is a glycoprotein secreted by the mouse epididymis that binds specifically to the sperm tail. Its fate has been examined on cauda epididymidal sperm in vivo and in vitro. SMA 4 was detected by indirect immunofluorescence (IIF) on sperm flushed from uteri of mice 5.5 h after natural or artificial insemination, but not on sperm attached to cumulus cells or zonae pellucidae of eggs recovered at that time. Detectable SMA 4 declines with time in vitro, as assayed by IIF on intact sperm or by enzyme immunoassay (EIA) of detergent extracts. After 3 h in vitro, 90% or more of sperm are not positive for SMA 4 by IIF. EIA of medium in which sperm have been incubated suggests that SMA 4 is being released from the cell surface. This time-dependent loss of SMA 4 is inhibited by mouse or rat cauda epididymidal fluid, low incubation temperature, or lack of protein in the incubation medium. However, the loss does not seem to be affected by the presence of eggs, cumulus cells, or oviduct fluids. SMA 4 is not removed from the sperm by selected treatments, suggesting that it is bound to the plasma membrane by strong, noncovalent interactions.
The effects of unilateral vasectomy upon fertility and antisperm antibody production were studied using DBA/1J mice. Thirty-six males underwent either unilateral vasectomy, unilateral orchiectomy, or sham surgery. In vivo effects upon fertility were monitored by matings. Antisperm antibody titers were measured monthly. In vitro fertilization was performed in the presence of serum obtained 4 months postoperatively, and serum testosterone levels were also determined. After 3 months, only 1 male in the vasectomy group induced a pregnancy (1 of 12), while all but 1 of the males in the two control groups induced a pregnancy (20 of 21). The geometric mean antisperm antibody titer was 1:169 in the vasectomy group, while the orchiectomy and sham surgery groups had titers of 1:4 and 1:14, respectively (P less than 0.0001). The percentage of eggs fertilized in vitro in the presence of serum from experimental mice fell from 80% in the two control groups to 40% in the unilateral vasectomy group. Unilateral vasectomy induced infertility in DBA/1J mice and an antisperm antibody response. Sera containing these antibodies inhibited in vitro fertilization. This suggests that infertility after unilateral vasectomy may be immunologically mediated by antisperm antibodies.
A monoclonal antibody to an antigen in the human germ cell membrane did not agglutinate or immobilize sperm but inhibited binding and penetration of zona-free hamster ova by human sperm and blocked murine fertilization in vitro. The antibody, of the 2a subclass of immunoglobulin G, was germ cell-specific but not species-specific. It recognized a single antigen of 23 kilodaltons that has been isolated from human germ cells. This fertilization antigen, located on the postacrosome , midpiece, and tail of human sperm, is a glycoprotein of testicular origin associated with some types of human involuntary immunoinfertility .
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The maternal host responds immunologically to antigens of the fetus. While the immune responses to paternally derived alloantigens and to placental antigens have been intensively studied, the immune responses to oncofetal antigens have been relatively unexplored. Oncofetal antigens are present on fetal and malignant tissues but absent from normal adult somatic tissues. These antigens elicit both cell-mediated and humoral immune responses in the parous female. Limited data suggest that these immune responses may influence reproductive processes. More investigation in this area is desirable.
Sera from vasectomized BDF1 mice were tested by immunofluorescence and radioimmunoassay for antibodies reacting with sperm and the mouse teratocarcinoma cell lines F9 and PYS. Antibody titers to both sperm and F9 cells increased with time after vasectomy and were highest in long-term (15 to 30 mo) vasectomized animals. By immunofluorescence assay, 35% of long-term vs 10% of age-matched control mice had antisperm antibodies (p less than or equal to 0.01), 54% of vasectomized vs 21% of controls had antibodies reacting with F9 teratocarcinoma (p less than or equal to 0.01), and 5% of vasectomized vs no controls reacted to the PYS teratocarcinoma cell line. No sera from either group reacted with thymocytes or cultured fibroblasts from male 129/Sv mice (negative somatic cell controls). Kendall's rank order statistical correlation test showed a significant association (p less than or equal to 0.01) between individual responses to sperm and F9 cells. Absorption of positive sera with sperm removed both sperm and F9 reactivity, indicating that the F9 cell-reactive antibodies in vasectomy sera are directed against identical or immunologically cross-reactive antigens present on the surface of sperm and F9 embryonal teratocarcinoma. A combination of electrophoresis and nitrocellulose blotting techniques (Western blot analysis) revealed that sera from some vasectomized mice contain IgG antibodies that react with 50,000 and 150,000 dalton (approximately) antigen bands present in sperm and F9 extracts, but not in PYS or thymocyte extracts. It remains to be determined whether the teratocarcinoma antigens detected in this study are true embryonic antigens or germinal antigens (the F9 cell line may have originated from primordial germ cells in transplanted embryonic tissue). In either case, teratocarcinoma cell lines provide an abundant source of material for the further characterization of these autoantigenic molecules.
Previous studies have demonstrated that the maternal host responds immunologically to antigens of the fetus, including oncofetal antigens (OFA). However, until now, the OFA that have been studied are those associated with the postimplantation embryo. In this paper we provide evidence that the maternal host also responds immunologically to antigens associated with the mouse teratocarcinoma (TC) cell lines F9 and PYS-2, which have been established as models for the preimplantation embryo. In a solid-phase radioimmunoassay using cell membrane extracts as antigen and 125I-protein A to detect binding antibodies, sera from parous C57BL/6 mice had a significantly greater reactivity than sera from age-matched virgin animals to F9 but not PYS-2, splenocyte or fibroblast antigen extracts. This finding was confirmed by surface immunofluorescence (IF) with fluorescein-labeled anti-mouse IgG. IF also detected a small number of multiparous mice with antibodies to PSY-2. Positive serum reactivity did not correlate directly with the number of previous litters. These TC cell lines express at least 2 OFA, F9 and Endo, which are found on preimplantation embryos and sperm, and preimmunization of female mice with F9 cells has been shown to reduce fertility. The parity-induced antibodies detected in this study could react with embryos at early stages of development and contribute to the natural decrease in litter size which occurs in mice after the second litter.
Oncofetal (OF) antigens have been isolated from mouse F9 teratocarcinoma cells, mouse testicular cells, and human molar tissue by detergent extraction followed by dialysis. The soluble antigens have been used in solid phase radioimmunoassay (SPRIA) and enzyme-linked immunosorbent (ELISA) assay. Specific antibodies have been raised to these antigens in mice. By using these antisera, extensive cross-reactivity was found between mouse and human OF antigens. A human trophoblastic tumor cell line BEWO absorbed out mouse anti-F9 reactivity. Patients with tumors of germinal origin were found to have antibodies which cross-react with mouse and human OF antigens. This new assay is a rapid and sensitive method for the screening of monoclonal antibodies against these antigens as well as for detecting antibodies to tumors bearing these antigens in patients.