Genotoxic and functional consequences of transplacental zidovudine exposure in fetal monkey brain mitochondria.

Mitochondrial toxicity was assessed in the brains of developing Erythrocebus patas monkey fetuses exposed in utero to the nucleoside analogue drug zidovudine (3'-azido-3'deoxythymidine or AZT). Pregnant E. patas monkeys were given 0 (n = 5), 10 (n = 3), and 40 (n = 3) mg of AZT/day, equivalent to 21 and 86% of the human daily dose, for the last half (about 10 weeks) of gestation. Mitochondria were isolated from fetal cerebrum and cerebellum at birth and mitochondrial morphology was examined in these tissues by transmission electron microscopy (TEM). Oxidative phosphorylation (OXPHOS) enzyme specific activities were measured spectrophotometrically. Mitochondrial DNA (mtDNA) integrity and quantity were determined by Southern blot and slot blot analysis. In the cerebral mitochondria, reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (complex I) specific activity decreased by 25% in monkeys treated with 40 mg of AZT/day compared with unexposed monkeys (p > or = .05). At the same AZT dose in the cerebral mitochondria, succinate dehydrogenase (complex II) and cytochrome c reductase (complex IV)-specific activities showed dose-dependent increases (p > or = .05), compared with those in controls. In the cerebellum, no difference was seen in mitochondrial OXPHOS enzyme activities between unexposed and exposed fetuses. Furthermore, TEM demonstrated no difference in mitochondrial morphology in frontal cerebrum or cerebellum from unexposed and exposed fetuses, and all fetuses had similar amounts of mtDNA in both tissues. Cerebral mtDNA degradation was noted in the highest AZT dosage group, whereas mtDNA from cerebellum was uneffected. Thus, in fetal patas monkeys given a human equivalent daily dose of AZT during the last half of pregnancy, mitochondria in the fetal cerebrum appear to sustain moderate damage, while the fetal cerebellum mitochondria were not effected.

Fetal mitochondrial heart and skeletal muscle damage in Erythrocebus patas monkeys exposed in utero to 3'-azido-3'-deoxythymidine.

3'-azido-3'-deoxythymidine (AZT) is given to pregnant women positive for the human immunodeficiency virus type 1 (HIV-1) to reduce maternal-fetal viral transmission. To explore fetal mitochondrial consequences of this exposure, pregnant Erythrocebus patas monkeys were given daily doses of 1.5 mg (21% of the human daily dose) and 6.0 mg (86% of the human daily dose) of AZT/kg body weight (bw), for the second half of gestation. At term, electron microscopy of fetal cardiac and skeletal muscle showed abnormal and disrupted sarcomeres with myofibrillar loss. Some abnormally shaped mitochondria with disrupted cristae were observed in skeletal muscle myocytes. Oxidative phosphorylation (OXPHOS) enzyme assays showed dose-dependent alterations. At the human-equivalent dose of AZT (6 mg of AZT/kg bw), there was an approximately 85% decrease in the specific activity of NADH dehydrogenase (complex I) and three- to sixfold increases in specific activities of succinate dehydrogenase (complex II) and cytochrome-c oxidase (complex IV). Furthermore, a dose-dependent depletion of mitochondrial DNA levels was observed in both tissues. The data demonstrate that transplacental AZT exposure causes cardiac and skeletal muscle mitochondrial myopathy in the patas monkey fetus.

Effects of endometrial serpin-like proteins on immune responses in sheep.

PROBLEM: The uterine milk proteins (UTMP) are a pair of related glycoproteins that are the major secretory products of the endometrium of the pregnant ewe. UTMP are members of the serpin superfamily of serine protease inhibitors but have no known antiprotease activity. One possible role for UTMP is to inhibit uterine immune responses--UTMP inhibit mitogen and mixed lymphocyte-induced proliferation of peripheral blood lymphocytes, natural killer (NK) cell activity and abortion caused by NK cell activation. Present objectives were to further evaluate the lymphocyte-inhibitory activity of UTMP and test whether UTMP modify immune responses in vivo. METHOD: One experiment demonstrated that UTMP inhibited antigen-induced lymphocyte proliferation induced by Candida albicans extract. In another experiment, ewes were immunized with OVA mixed with 3.75 mg/ml of UTMP or ovine serum albumin (OSA control). Injections of 1 mg OVA+UTMP or OSA in incomplete adjuvant were administered 6 wk later. Titers of antibody to OVA were lower (P < 0.001) for ewes administered UTMP than for ewes administered OSA. Effects of UTMP on delayed hypersensitivity reactions were evaluated in three experiments using skin-fold thickness assays. RESULTS: UTMP did not inhibit the increase in skin-fold thickness caused by PHA and Mycobacterium tuberculosis but rather tended to increase the response to PHA. CONCLUSION: Results strengthen the thesis that UTMP are physiologically relevant immunoregulatory molecules. Nonetheless, effects on skin-fold responses indicate that actions of UTMP can be more complex than would be predicted based on the proteins only having a single biological effect.

Identification of the predominant proteins in uterine fluids of unilaterally pregnant ewes that inhibit lymphocyte proliferation.

Uterine fluid from unilaterally pregnant ewes contains activity inhibitory to lymphocyte proliferation. The molecules responsible for this activity may thereby regulate uterine immune responses during pregnancy. The purpose of the experiment described here was to identify the major protein in uterine fluid responsible for this activity. When uterine fluid was fractionated by a combination of cation exchange chromatography, gel filtration, and lectin affinity chromatography, the majority of the lymphocyte activity co-migrated with a pair of proteins previously identified as related, serpin-like glycoproteins. Together, this pair of proteins, called the uterine milk proteins (UTM-proteins), are the predominant endometrial secretory protein produced under the influence of progesterone. Preparations of uterine protein highly enriched for the UTM-proteins inhibited lymphocyte proliferation induced by phytohemagglutinin, concanavalin A, and mixed lymphocyte reactions but did not inhibit proliferation induced by pokeweed mitogen. In some experiments, UTM-proteins also reduced viability of cultured lymphocytes. Another previously described lymphocyte-inhibitory factor, megasuppressin, was also observed. Megasuppressin, which eluted at an apparent molecular weight of greater than 4 x 10(6) even after treatment with urea, guanidine-HCl, and beta-mercaptoethanol, was a more potent inhibitor of lymphocyte proliferation than UTM-proteins. Megasuppressin is not very abundant, however, and probably is responsible for only a small fraction of the lymphocyte inhibitory activity in uterine fluid. The majority of lymphocyte-inhibitory activity is caused by the UTM-proteins or by a molecule that co-purifies in trace amounts with UTM-proteins.

Inhibition of lymphocyte proliferation by bovine trophoblast protein-1 (type I trophoblast interferon) and bovine interferon-alpha I1.

Bovine trophoblast protein-1 (bTP-1) is a Type I interferon secreted by the bovine trophoblast from about Day 15 of pregnancy. It is not known whether bTP-1 has functional properties in common with other interferons. The aim of the present study was to determine whether bTP-1 inhibits proliferation of lymphocytes induced by mitogens, mixed lymphocyte cultures (MLC) and interleukin-2 (IL-2) and, if so, whether this activity is similar to that of a related interferon, bovine interferon-alpha I1 (bIFN-alpha I1). Stimulation of lymphocyte proliferation caused by phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) was inhibited by bTP-1 and bIFN-alpha I1 without any reduction in cell viability. Maximum or near-maximum inhibition (less than 50%) was achieved at concentrations of 0.5-5.0 nM of bTP-1 and bIFN-alpha I1. Cells stimulated with PWM were less inhibited than cells stimulated with PHA and Con A. Both bTP-1 and bIFN-alpha I1 inhibited MLC to a greater degree than lectin-stimulated cells (maximum inhibition was 78% or greater). Also, bTP-1 and bIFN-alpha I1 slightly inhibited incorporation of [3H]thymidine ([3H]TdR) induced by the combination of phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), and calcium ionophore A23187. Finally, bTP-1 and bIFN-alpha I1 had bimodal effects on incorporation of [3H]TdR by IL-2-induced lymphocytes. Incorporation of [3H]TdR was increased at 0.005 nM and 0.05 nM concentrations while higher concentrations caused a slight decrease in [3H]TdR incorporation. Results confirm that bTP-1 inhibits lymphocyte proliferation in a manner similar to that caused by the leukocyte-derived interferon, bIFN-alpha I1. Incomplete inhibition of mitogen-induced proliferation and differences in degree of inhibition between various stimulators suggest that bTP-1 and bIFN-alpha I1 preferentially inhibit certain lymphocyte subpopulations. Local inhibition of lymphocyte proliferation caused by bTP-1 may help protect the allogeneic conceptus from immune responses to fetal antigens or regulate the release of cytokines from endometrial lymphocytes.