Sequence heterogeneity and polymorphic gene arrangements of the Leishmania guyanensis gp63 genes.

The Leishmania GP63 major surface protein gene family encodes multiple isoforms which differ predominantly in the carboxyterminal region. We have isolated 4 full-length gp63 cDNA clones derived from stationary-phase promastigote RNA of a cloned isolate of Leishmania guyanensis, a member of the braziliensis complex. These genes, along with the previously published L. guyanensis gp63 gene sequence [15], appeared to be mosaics of different combinations of 5' and 3' untranslated regions and sequences encoding the propeptide, internal, and C-terminal regions of GP63. The predicted L. guyanensis GP63 isoforms shared as little as 55% sequence identity, comparable to the inter-species diversity of GP63. The genomic organization of gp63 genes in L. guyanensis is highly complex: there are at least 4 distinct polymorphic forms of tandemly linked gene clusters, with intra-gene cluster variation in gene sequence and in the number of gene repeats. Southern blot analysis suggested that the arrangement of gp63 genes in this L. guyanensis isolate did not differ from that in the parental lines.

Inhibition of antigen- and mitogen-induced activation of mouse splenocytes by uterine secretions from pregnant and progesterone-treated ewes.

Pregnant and progesterone-treated, ovariectomized ewes accumulate secretory products in the uterus that are immunosuppressive in mitogen-stimulated and mixed lymphocyte sheep cell cultures. In this study, uterine secretions from pregnant (Preg-UTM) and progesterone-treated, ovariectomized (P-UTM) ewes were equally effective in suppressing 3H-Tdr incorporation in mouse spleen cells stimulated with PHA. P-UTM inhibited PHA-stimulated, purified T-cells and separated L3T4+ and Lyt2+ T-cell subpopulations more than Preg-UTM, however. Both fluids were slightly inhibitory to conA-stimulated mouse spleen cells, enriched T-cells, and Lyt2+ T-cells but neither inhibited L3T4+ T-cells. For LPS-stimulated cells, P-UTM caused more suppression than Preg-UTM of enriched B-cells; however, suppression was similar for the two fluids on unseparated splenic cells. In antigen-stimulated mouse spleen cell cultures, both fluids inhibited antibody-forming cell responses to sheep erythrocytes, a thymus-dependent antigen, but neither suppressed antibody-forming cell responses to TNP-Ficoll, a thymus-independent antigen. These data indicate that uterine secretions in the ewe produced under the influence of progesterone or pregnancy contain immunoregulatory molecule(s) which modulate the activity of both homologous sheep and unrelated mouse lymphocytes. These studies establish the mouse as a useful in vivo model for studying the biological actions of these molecules.

Induction by progesterone of immunosuppressive activity in uterine secretions of ovariectomized ewes.

Immune responses in the uterus are depressed when concentrations of progesterone in the blood are elevated. We tested whether this action of progesterone involves induction of secretion of immunosuppressive molecules into the uterine lumen. Uterine fluid from ovariectomized ewes treated with progesterone for 60 days inhibited [3H]thymidine (TdR) incorporation by phytohemagglutinin-stimulated lymphocytes more than uterine flushings from vehicle-treated ewes. Uterine fluid from progesterone-treated ewes also inhibited TdR uptake by other mitogen-stimulated lymphocytes and mixed lymphocyte cultures, but had no effect on the growth of three nonlymphocyte cell lines. A group of ewes immunized with ovalbumin mixed with uterine fluid from progesterone-treated ewes produced an antibody titer that was lower than the titer for ewes immunized with ovalbumin without uterine fluid. Analysis of the physical properties of the active molecules in uterine fluid from progesterone-treated ewes indicated that activity could be separated into two fractions: a large mol wt (Mr; greater than 5000) fraction that was sensitive to pronase and a low Mr (less than 1000) fraction that was more resistant to pronase. There was more inhibitory activity in the high mol wt fraction. Further analysis of this fraction indicated that most activity was of basic isoelectric point (pI greater than 8.2). The most active fraction of basic material eluted at the void volume of a Sepharose CL-6B column (Mr, 4 x 10(6)). In conclusion, progesterone caused accumulation of factors in the uterine lumen that inhibited lymphocyte function in vitro and antibody formation in vivo. These molecules may play an important role in regulating immune responses in the uterus during pregnancy.

Inhibition of lymphocyte proliferation by uterine fluid from the pregnant ewe.

Immunosuppressive molecules in uterine fluid from the nonpregnant uterine horn of unilaterally pregnant ewes at Days 60, 100, and 140 of gestation were examined. Uterine fluid from all days inhibited [3H]thymidine incorporation into phytohemagglutinin-stimulated lymphocytes. Inhibitory activity increased with advancing gestation. Uterine fluid also inhibited lymphocyte proliferation caused by other mitogens or by mixed lymphocyte reactions. Inhibitory activity was found in both salt volume (Mr less than 1000) and void volume (Mr greater than 5000) fractions of uterine fluid resolved by Sephadex G-25 desalting columns. Only activity in the void volume was sensitive to pronase. Several fractions containing inhibitory activity were resolved when dialyzed uterine fluid was fractionated into acidic and basic components by cation-exchange chromatography and further resolved by gel filtration using Sepharose CL-6B. The most active fractions (inhibition/micrograms protein) for both acidic and basic components eluted at the void volume of Sepharose CL-6B (Mr greater than 4 x 10(6). The inhibitory factor in the basic component that eluted at the void volume of Sepharose CL-6B was rich in carbohydrate, slightly cytotoxic, and partially sensitive to digestion with trypsin or oxidation with periodate. In conclusion, uterine fluid of unilaterally pregnant ewes is enriched in molecules that inhibit lymphocyte proliferation in vitro. Among these are low molecular weight, non proteinaceous factors and a very high molecular weight (Mr greater than 4 x 10(6) fraction that contains protein and carbohydrate.

Secretion of the major progesterone-induced proteins of the sheep uterus by caruncular and intercaruncular endometrium of the pregnant ewe from days 20-140 of gestation.

An experiment was performed to evaluate the types and quantity of proteins secreted by intercaruncular endometrium at days 20, 60, 100 and 140 of gestation and caruncular endometrium from days 100 and 140 of gestation. Tissues were obtained from ewes made unilaterally pregnant. Based on SDS polyacrylamide gel electrophoresis (SDS-PAGE), the major proteins present in uterine fluid at days 60-140 of gestation were the uterine milk proteins (UTM-proteins), a pair of structurally-related, progesterone-induced polypeptides with molecular weights of 55,000 and 57,000. These proteins were also present in uterine fluid at day 20, but the major protein at this time migrated coincident with albumin. Cultured explants of endometrium at all days of pregnancy produced UTM-proteins as their major radiolabelled product for both caruncular and intercaruncular endometrium. The amount of protein secretion in vitro was greater (P less than .04) for intercaruncular endometrium than for caruncular endometrium but was not significantly affected by stage of gestation or local presence of the conceptus. Immunohistochemical analysis revealed that UTM-proteins were present in glandular and luminal epithelium of intercaruncular endometrium and in both epithelial and stromal elements of caruncular endometrium. It was concluded that the UTM-proteins are produced earlier than previously described (i.e., day 20). In addition, caruncles contribute to the uterine secretory protein milieu through the secretion of proteins that are similar to that produced by the glandular intercaruncular epithelium.

Presence of the major progesterone-induced proteins of the sheep endometrium in fetal fluids.

Allantoic and amniotic fluids were collected on Days 60 (n = 3), 100 (n = 4), and 140 (n = 3) of pregnancy. The presence of uterine milk proteins (UTM-proteins) in these samples was evaluated by Ouchterlony immunodiffusion and enzyme-linked immunoabsorbant assay (ELISA). Eight of ten samples of allantoic fluid and three of ten samples of amniotic fluid produced one or two immunoprecipitin bands against antiserum to UTM-proteins. Each band fused with immunoprecipitin bands from UTM-proteins purified from uterine fluid. Data from a semi-quantitative ELISA indicated that allantoic fluid from all ewes and amniotic fluid from six of ten ewes contained immunoreactive UTM-proteins. Concentrations of UTM-proteins in these fluids were not statistically affected by day of gestation (p greater than 0.10), but tended to decline as gestation advanced. Greater concentrations of UTM-proteins were detected in allantoic fluid than in amniotic fluid (p less than 0.05). The physical characteristics of the immunoreactive material in allantoic and amniotic fluids were examined by polyacrylamide gel electrophoresis and Western blotting. The immunoreactive material was found to possess pIs and molecular weights identical to UTM-proteins. These results indicate that fetal fluids contain material that reacts with antiserum to UTM-proteins and has physical properties similar to UTM-proteins. It is likely, therefore, that the UTM-proteins are transported across the placenta during gestation, perhaps to serve some function in the fetal compartment.

Modification of immune function during pregnancy by products of the sheep uterus and conceptus.

The preimplantation sheep conceptus produces several molecules that inhibit lymphocyte responses in vitro to mitogens or allogeneic cells, including prostaglandin E-2 and a lactosaminoglycan-containing glycoprotein of Mr 800,000-900,000. Additionally, the definitive placenta releases immunosuppressive factors when placed in culture. One of these is a heat- and protease-stable, carbohydrate-containing molecule released by explants of cultured fetal cotyledons. The pregnant uterus also produces molecules that alter lymphocyte responsiveness in vitro, including a dialysable, heat- and trypsin-stable factor and a high-molecular weight factor (Mr greater than 4 x 10(6]. An important regulator of uterine immune function is progesterone. This hormone can induce the production of uterine immunosuppressants such as seen during pregnancy and prolong the life of skin allografts placed in utero. Taken together, results suggest that the conceptus resides in an immunosuppressive environment throughout most of gestation, a situation that may be critical to maintenance of pregnancy.

Calcium kinetics in male white-tailed deer.

Calcium kinetic analysis was combined with calcium balance data on two 2-year-old and two 4-year-old male white-tailed deer. There were three 10-day trials. Trial 1, February 3-13, was conducted after velvet shedding. Trial 2, May 26-June 5, was conducted after about a month of slow antler growth. Trial 3, August 18-28, was conducted shortly before velvet shedding, after the peak of antler growth. Significant differences (P less than 0.05) between trials 1 and 2 included increased calcium absorption and fractional calcium absorption from the intestine. Skeletal resorption decreased, resulting in an increased calcium balance. Significant differences (P less than 0.05) between trials 1 and 3 included increased skeletal accretion and total calcium flow into the pool. Nonsignificant differences (P greater than 0.05) between trials 2 and 3 included decreased average intestinal calcium absorption and increased skeletal resorption resulting in a decreased calcium balance. The younger deer had nonsignificantly lower (P greater than 0.1) endogenous fecal and significantly lower (P less than 0.1) urinary calcium excretions. The younger deer also maintained higher fractional absorption rates from the intestine than the older deer. The percent decrease in skeletal resorption and increase in calcium balance was greater in the younger deer between trials 1 and 2 and between trials 1 and 3.