Partial purification of uterine secretory protein capable of suppressing lymphocyte reactivity invitro

Porcine uterine flushings obtained on day 15 of the estrous cycle were fractionated using gel filtration, and preparative poly-acrylamide gel electrophoresis was employed to separate one molecular weight fraction into two groups of small uterine-specific proteins designated pAP and pLAP. The two groups were assayed for immuno-suppressive ability using phytohemagglutinin (PHA) stimulated porcine lymphocyte cultures and incorporated ³H-thymidine. It was found that the pAP preparation which was composed of two proteins inhibited lymphocyte reactivity to PHA (p<.001) while the pLAP preparation failed to exhibit a similar activity at levels as high as 1000 μg/ml. The immunosuppressive effect was determined to be independent of cytotoxicity, PHA inactivation by binding and other non-specific phenomena. The results of this study indicate that the immunosuppressive activity of porcine uterine flushings is caused, at least in part, by one or both of these proteins present in the pAP preparation.     

Characterization of immunosuppressive substances in the basic protein fraction of uterine secretions from pregnant ewes

The basic protein fraction of ovine uterine secretions collected late in pregnancy (Days 125-140) contains a substance capable of inhibiting in vitro blastogenic responses of lymphocytes to phytohemagglutinin (PHA) or mixed lymphocyte reactions. In this study, the immunosuppressive substance in the basic protein fraction of uterine secretions was further defined by gel filtration. The immunosuppressive activity resided in a group of high molecular weight proteins eluting at the void volume of Sephacryl S-200 and Sepharose CL-6B columns. For example, incorporation of thymidine by PHA-stimulated lymphocytes incubated with 20, 40, 80, and 120 micrograms/ml of protein from the void volume of Sepharose CL-6B was 65, 28, 2, and 0 percent of control lymphocytes, respectively. Based on polyacrylamide-gel electrophoresis in the presence of sodium dodeylsulfate (SDS), the immunosuppressive fraction from Sepharose CL-6B chromatography contained aggregates of uterine milk proteins (UTM-proteins) and a pair of proteins running at the top of a 5% (w/v) polyacrylamide gel. Other protein peaks resolved by Sephacryl S-200 and Sepharose CL-6B contained aggregates of UTM-proteins but were not immunosuppressive. The substance inhibiting in vitro lymphocyte function was not of conceptus origin, because it was found in fluid from the ligated uterine horn of unilaterally pregnant ewes and from the uterus of an ovariectomized ewe treated for 60 days with progesterone and estrone.     

Role of prostaglandins in development of porcine blastocysts

Rapid elongation of porcine blastocysts between Days 11 to 12 of pregnancy coincides with an increase in uterine luminal content of prostaglandins. The present study evaluated the effect of two prostaglandin synthesis inhibitors (indomethacin and flunixin meglumine) on elongation of porcine blastocysts from spherical to filamentous forms between Day 11 to 12 of pregnancy. Gilts were hemi-hysterectomized on Day 11 of prenancy. The excised uterine horn was flushed with 0.9% saline and diameter of blastocysts recovered were measured. Immediately following surgery, pregnant gilts were assigned to receive either: 1) vehicle every 4 h, 2) flunixin meglumine (banamine) every 4 h, or 3) indomethacin every 12 h. The remaining uterine horn was removed and flushed after the time of blastocyst elongation estimated for each gilt on basis of blastocyst development in the first horn. Uterine flushings were analyzed for total calcium, protein, acid phosphatase activity, estrone, estradiol-17β and prostaglandin F. Pretreatment blastocyst diameter was similar for all groups and ranged from 1 mm to 20 mm. Treatment of gilts with either banamine or indomethacin effectively inhibited (P<0.001) the increase in uterine luminal content of PGF. Total calcium, estrone and estradiol-17β were not influenced by treatment. Total uterine luminal protein and acid phosphatase activity were reduced (P<0.05) in banamine treated gilts compared to those receiving vehicle or indomethacin treatments. Although total PGF recovered in uterine flushings was reduced during the period of blastocyst elongation, treatment with PGF synthetase inhibitors failed to block rapid elongation of blastocysts from the spherical to filamentous forms.     

Immunosuppressive activity of uterine luminal protein from steroid-treated ovariectomized heifers

Uterine luminal protein (ULP) collected from ovariectomized steroid-treated crossbred heifers was tested for immunosuppressive activity in vitro. Heifers were allotted to treatment groups and for 16 d received daily injections of the following steroids or vehicle: Control (C, corn oil only, n=10); estradiol-17beta (E(2), 1.1 mug/kg body wt, n=10); progesterone (P(4), 2.2 mg/kg body wt, n=10); and E(2)+P(4) (1.1 mug + 2.2 mg/kg body wt, n=9). On Day 17, uterine flushings were collected, concentrated and quantitated for total ULP. ULP was tested for suppression of lymphocyte blastogenesis. For each experiment, 5 x 10(5) bovine lymphocytes were incubated with 0.4 mug of phytohemagglutinin (PHA) and ULP (25 to 400 mug ULP/ml) using standard culture conditions. At 48 h, 0.5 muCi of (3H) thymidine was added to cultures with cells harvested at 60 +/- 1 h by automation. Incorporated thymidine was measured by scintillation chromatography. Mean total ULP values for C-, E(2)-, P(4)- and E(2)+P(4)-treated groups were 4.7, 8.4, 13.6, and 25.5 mg, respectively (E(2)+P(4)>C and E(2), P<0.05). ULP from all treatment groups suppressed (P<0.0001) lymphocyte blastogenesis (thymidine incorporation) to PHA; however, suppression was greater (P<0.0001) for ULP from E(2)- and P(4)-than C-treated heifers at 100 and 200 mug ULP/ml. In conclusion, E(2) and P(4) injections enhanced immunosuppressive activity of ULP secretions.     

Immunosuppressive activity associated with early pregnancy in the bovine

Immunosuppressive activity was assessed in uterine flushings (UF) and uterine vein serum and plasma from nonpregnant and early-pregnant cows, and in media from the short-term culture of Day 18 bovine embryos. The preparations were tested for their ability to inhibit [3H] thymidine (3H-TdR) incorporation into phytohemagglutinin-stimulated bovine lymphocytes. On Days 2-3 (called Day 3), Days 9-10 (called Day 10), and Days 17-19 (called Day 18) of the estrous cycle (estrus = Day 0) and pregnancy, untreated and superovulated cows were anesthesized and jugular vein and uterine vein blood was collected. The uteri were removed and flushed to obtain UF and embryos. Uterine flushings were concentrated and tested for immunosuppressive activity at 400 micrograms uterine protein/ml culture fluid. Uterine flushings from both Day 18 pregnant and Day 18 nonpregnant cows were immunosuppressive (8/8), whereas Day 10 UF were usually not immunosuppressive (7/10). Day 3 UF were usually stimulatory or only marginally suppressive (8/8). Uterine vein serum and plasma from Day 18 cows were not suppressive when compared to jugular vein serum or plasma from the same cow; neither were Day 18 uterine vein serum or plasma suppressive when compared to those same samples taken from Day 3 cows. Embryo culture media obtained from the 48-h culture of Day 18 embryos was consistently suppressive. The activity was lost after dialysis in 1000-Mr cut-off tubing, removed by charcoal, and reduced by protease digestion. These results suggest two mechanisms whereby the embryo could escape immune rejection: 1) the progesterone-induced secretion of a uterine immunosuppressive substance(s) and 2) the production by the embryo of a low molecular weight immunosuppressive substance(s).     

The detection and purification of a cat uterine secretory protein that is estrogen dependent (CUPED)

An estrogen-dependent secretory protein (CUPED) was detected and purified from uterine flushings of ovariectomized cats treated with 17 beta-estradiol. The protein was not detected in uterine flushings obtained from untreated ovariectomized animals or estrogen-primed animals treated with progesterone for 4 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of uterine flushings showed the presence of 1 or 2 protein bands with relative mobility values less than reduced and denatured thyroglobulin (Mr = 330,000). The protein was purified by differential centrifugation and gel filtration chromatography. Antiserum was raised against this purified protein in rabbits. The specificity of the antiserum to uterine fluid proteins was assessed by immunoblotting of electrophoretically transferred proteins. The antiserum cross-reacted with electrophoretically separated CUPED protein bands in uterine flushings. This protein may represent the content of the estradiol-induced secretory granules present in endometrial epithelial cells.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis:

Summary Approximately 250 phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte polypeptides from three unrelated healthy males were compared by high-resolution two-dimensional gel electrophoresis and double-label autoradiography. Comparisons by all possible pairwise combinations of [¹⁴C]leucine-labeled proteins from an individual and [³H]leucine-labeled proteins from another revealed that only three polypeptides differed qualitatively among the three individuals. The degree of variation in lymphocyte polypeptides between different individuals was similar to that in fibroblast polypeptides reported previously. Among the three variant polypeptides, two polypeptides with mol.wt. 64,000 and mol. wt. 37,000 coexisted with a polypeptide with the same molecular weight, and they showed the behavior expected of two allelic gene products separated in the isoelectric focusing dimension by charge differences. Analysis of [¹⁴C]leucine labeled peripheral blood lymphocyte proteints, from the parents of each individual, by two-dimensional gel electrophoresis indicated that the variant polypeptides with mol. wt. 64,000 and mol. wt. 37,000 in the propositus were inherited from one of his parents. The data indicate that genetic analysis of PHA-stimulated peripheral blood lymphocyte proteins is feasible by high-resolution two-dimensional gel electrophoresis in combination with double-label autoradiography and pedigree analysis.     

Adenylate cyclase activity in porcine sperm in response to female reproductive tract secretions

Adenylate cyclase activities were studied in porcine sperm in the presence and absence of Mn⁺⁺ before and after incubation in vivo and in vitro. Incubation of sperm in vivo for 30 min increased the Mg⁺⁺-stimulated adenylate cyclase activity from 35.1 pmoles cyclic AMP formed per mg protein per 10 min to 50.4 pmoles. The activity stimulated by Mg⁺⁺ and Mn⁺⁺ increased from 392 to 729 pmoles after 30 min of in vivo incubation. Activity after incubation in vivo for 120 min was not different from activity after 30 min. In vitro incubation of porcine sperm in Ca⁺⁺-free Ringer-fructose resulted in no change, but incubation in oviductal and uterine flushings obtained from gilts soon after ovulation increased Mg⁺⁺-stimulated activity by 24% and Mg⁺⁺?+ Mn⁺⁺-stimulated activity by 49%. In vitro incubations in preovulatory flushings plus follicular fluid or in bovine serum albumin also increased adenylate cyclase activity.     

A simple technique for sampling the uterine cavity of the baboon

The uterine environment within which the primate conceptus develops is poorly understood. This study was undertaken a) to develop a technique by which the uterine lumen could be sampled simply and efficiently and b) to analyze the proteins present in these uterine flushings throughout the menstrual cycle. The instrument described in this communication consists primarily of a double lumen cannula which permits one to inject and aspirate the flushing medium simultaneously. Volume recoveries usually exceeded 75% and the concentration of protein did not change significantly throughout the menstrual cycle. Two-dimensional polyacrylamide gel electrophoresis of the uterine flushings revealed the presence of one polypeptide (M(r) congruent with 66,000; pI congruent with 5.7-6.0) that was not observed in serum and was presumed to be of uterine origin. The technique described here provides a rapid method by which baboon uterine secretions can be frequently collected in the lightly sedated animal.     

Role of prostaglandins in development of porcine blastocysts

Rapid elongation of porcine blastocysts between Days 11 to 12 of pregnancy coincides with an increase in uterine luminal content of prostaglandins. The present study evaluated the effect of two prostaglandin synthesis inhibitors (indomethacin and flunixin meglumine) on elongation of porcine blastocysts from spherical to filamentous forms between Day 11 to 12 of pregnancy. Gilts were hemi-hysterectomized on Day 11 of pregnancy. The excised uterine horn was flushed with 0.9% saline and diameter of blastocysts recovered were measured. Immediately following surgery, pregnant gilts were assigned to receive either: 1) vehicle every 4 h, 2) flunixin meglumine (banamine) every 4 h, or 3) indomethacin every 12 h. The remaining uterine horn was removed and flushed after the time of blastocyst elongation estimated for each gilt on basis of blastocyst development in the first horn. Uterine flushings were analyzed for total calcium, protein, acid phosphatase activity, estrone, estradiol-17 beta and prostaglandin F. Pretreatment blastocyst diameter was similar for all groups and ranged from 1 mm to 20 mm. Treatment of gilts with either banamine or indomethacin effectively inhibited (P less than 0.001) the increase in uterine luminal content of PGF. Total calcium, estrone and estradiol-17 beta were not influenced by treatment. Total uterine luminal protein and acid phosphatase activity were reduced (P less than 0.05) in banamine treated gilts compared to those receiving vehicle or indomethacin treatments. Although total PGF recovered in uterine flushings was reduced during the period of blastocyst elongation, treatment with PGF synthetase inhibitors failed to block rapid elongation of blastocysts from the spherical to filamentous forms.     

Metabolic compounds within the porcine uterine environment are unique to the type of conceptus present during the early stages of blastocyst elongation

The objective of this study was to identify metabolites within the porcine uterine milieu during the early stages of blastocyst elongation. At Days 9, 10, or 11 of gestation, reproductive tracts of White cross‐bred gilts (n = 38) were collected immediately following harvest and flushed with Roswell Park Memorial Institute‐1640 medium. Conceptus morphologies were assessed from each pregnancy and corresponding uterine flushings were assigned to one of five treatment groups based on these morphologies: (a) uniform spherical (n = 8); (b) heterogeneous spherical and ovoid (n = 8); (c) uniform ovoid (n = 8); (d) heterogeneous ovoid and tubular (n = 8); and (e) uniform tubular (n = 6). Uterine flushings from these pregnancies were submitted for nontargeted profiling by gas chromatography–mass spectrometry (GC–MS) and ultra performance liquid chromatography (UPLC)–MS techniques. Unsupervised multivariate principal component analysis (PCA) was performed using pcaMethods and univariate analysis of variance was performed in R with false discovery rate (FDR) adjustment. PCA analysis of the GC–MS and UPLC–MS data identified 153 and 104 metabolites, respectively. After FDR adjustment of the GC–MS and UPLC–MS data, 38 and 59 metabolites, respectively, differed (p < .05) in uterine flushings from pregnancies across the five conceptus stages. Some metabolites were greater (p < .05) in abundance for uterine flushings containing earlier stage conceptuses (i.e., spherical), such as uric acid, tryptophan, and tyrosine. In contrast, some metabolites were greater (p < .05) in abundance for uterine flushings containing later stage conceptuses (i.e., tubular), such as creatinine, serine, and urea. These data illustrate several putative metabolites that change within the uterine milieu during early porcine blastocyst elongation.     

Secretion of a progesterone-induced inhibitor of plasminogen activator by the porcine uterus

The indirect ¹²⁵I-fibrin plate assay has been used to measure the levels of plasminogen activator (PA) in uterine flushings from pigs through the estrous cycle and during early pregnancy, and to measure the production of PA by pig conceptuses cultured in vitro. Activity in the flushings was high at the beginning and end of the estrous cycle, but only low levels were detected in mid cycle (the luteal phase). In pregnant animals, uterine PA levels became low around day 12 and did not show any further increase. Cultured day 12 blastocysts, however, released large amounts of PA into the medium in a time-dependent fashion over a 48 hr period, suggesting that this activity was inhibited in vivo. The presence of a protease inhibitor in uterine flushings has been demonstrated in cycling gilts, and follows a hormone-directed trend, with flushings taken during the luteal phase showing inhibitory activity against PA secreted early or late in the cycle. By assaying flushings from ovariectomized gilts given daily injections of progesterone, estrogen, both hormones together, or corn coil, it has been verified that the inhibitor is progesterone-induced and is also active against both PA produced by day 12 conceptuses and urokinase. It also inhibits PA, as determined using a direct fluorometric assay with glutaryl-glycyl-L-arginine-4-methyl-coumarinyl-7-amide as substrate. The PA inhibitor is acid-stable, and of low molecular weight (15,000 ± 5000), as determined by Sephacryl S-200 gel filtration. Unlike most animals, the trophoblast of the pig is not invasive in the uterus, but is invasive if transplanted to some ectopic site. The progesterone-induced inhibitor may possibly play a role in preventing invasive implantation.     

A study of prostaglandin F2α as the luteolysin in swine: V comparison of prostaglandin F, progestins, estrone and estradiol in uterine flushings from pregnant and nonpregnant gilts

Uterine flushings were collected from 38 gilts representing Days 6,8,10,12,14,15,16 and 18 of the estrous cycle and pregnancy. The same group of gilts were represented within each of the respective days of the estrous cycle and pregnancy, i.e., three to six gilts per day per status. Uterine flushings (about 40ml) were assayed for prostaglandin F (PGF), estrone (E₁), estradiol (E₂), progestins (P) and protein. Nonpregnant gilts had higher (P<.01) concentrations of P in uterine flushings than pregnant gilts, but pregnant gilts had higher (P<.01) E₁ and E₂ concentrations. Significant day by status interactions were detected for E₁ (P<.05), but not for E₂ concentrations in uterine flushings. Total recoverable PGF and PGF concentrations in uterine flushings were greater (P<.01) in pregnant than nonpregnant gilts and significant (P<.01) day by status interactions were detected. In nonpregnant gilts, PGF increased between Days 12 and 16, i.e., during the period of corpora lutea (CL) regression. In pregnant gilts, PGF in uterine flushings increased markedly between Days 10 and 18. Total recoverable PGF on Day 18 of the estrous cycle was only 464.5 ± 37.6 ng as compared to 22,688.1 ± 1772.4 ng on Day 18 of pregnancy. Total recoverable protein was also higher (P<.01) in pregnant gilts. These data indicate that PGF synthesis and secretion by the uterine endometrium and/or conceptuses is not inhibited during pregnancy and suggest that PGF is sequestered within the uterine lumen of pregnant gilts, as is the total protein component of endometrial secretions referred to as histotroph.     

Suppression of phytohemagglutinin-stimulated lymphocyte blastogenesis by ovine uterine milk protein

Two basic glycoproteins (UTM-P) with molecular weights of 57,000 and 59,000 were purified from ovine uterine milk collected on Days 125 and 130 of pregnancy. The UTM-P were evaluated for immunosuppressive activity in phytohemagglutinin (PHA)-treated, mixed lymphocyte (MLC) and resting lymphocyte (RLC) cultures. For PHA and RLC cultures, UTM-P (2.5 to 800 micrograms UTM-P/ml) were added to 1 X 10(6) lymphocytes and 0.8 micrograms of PHA (for PHA cultures only), while for the MLC, UTM-P (50 to 1600 micrograms UTM-P/ml) were added to 5 X 10(5) lymphocytes combined from each of two ewes. Following [3H] thymidine addition, cells were later harvested for determination of thymidine incorporation. Lymphocyte blastogenesis was suppressed by UTM-P in PHA (R2 = 0.32 to 0.92, P less than 0.01 to 0.001), MLC (R2 = 0.8, P less than 0.001) and RLC (R2 = 0.65, P les than 0.01) experiments. To determine reversibility, PHA-treated lymphocytes were incubated with UTM-P for 6, 12 or 24 h, then washed to remove surface UTM-P. Incubation was continued in the presence of PHA as with other experiments. Exposure of lymphocytes to UTM-P for 6 or 12 h did not result in suppression of blastogenesis, whereas exposure for 24 h was sufficient for suppression (P less than 0.01). In an additional experiment, UTM-P were added to PHA-treated cultures at 0, 6, 12 or 24 h. Suppression (P less than 0.01) of blastogenesis was observed for each time period. Immunosuppressive activity was not mediated by overall cytotoxicity and was not affected by routine handling and storage of UTM-P. Data from these experiments provide one explanation for tolerance of the conceptus allograft during defined stages of ovine pregnancy.     

Detection of a progesterone-dependent secretory protein synthesized by cat endometrium

Uterine flushings and culture media from endometrial explants incubated in the presence of radiolabeled amino acids were analyzed using one-(1-D) and two-dimensional (2-D) gel electrophoresis to identify proteins synthesized by the endometrium and subsequently released into the uterine lumen. 1-D and 2-D analyses of uterine flushings and culture media of endometrial explants obtained from 7- to 11-day pregnant cats (pre-implantation) showed a Mr 30,000 protein that appeared on 2-D gels as a family of macromolecules with isoelectric points between 6.5 and 7.0. This family of macromolecules was also present in the culture media of implantation-site tissue obtained from 12- to 16-day pregnant cats and of nonimplantation-site endometrium obtained form 12- to 28-day pregnant cats. The Mr 30,000 protein was absent in uterine flushings and culture media from estrous and 3- to 5-day-pregnant cats. In ovariectomized, steroid-treated animals, the Mr 30,000 protein was only detected in flushings and media from those animals treated with progesterone, regardless of the presence or absence of estradiol-priming and/or simultaneous estradiol treatment. In daily flushings obtained from ovariectomized, steroid-treated cats equipped with an indwelling uterine catheter, the Mr 30,000 protein was absent during the 14 days of estradiol treatment and was first detected 3-4 days after the onset of estradiol plus progesterone treatment. This protein was not detected in serum from estrous, 9-day pregnant, ovariectomized, and ovariectomized, steroid-treated animals. This study shows that 1) a progesterone-dependent protein, with an approximate molecular weight of 30,000 and an isoelectric point of 6.5-7.0, first appears within the uterine lumen soon after the arrival of the blastocyst and continues to be present during implantation; 2) the synthesis and release of the Mr 30,000 protein is dependent on progesterone regardless of the presence or absence of estradiol; and 3) the onset of secretion of the Mr 30,000 protein requires 3-4 days of continuous progesterone treatment in the estradiol-primed cat.     

A study of prostaglandin F2α as the luteolysin in swine: IV an explanation for the luteotrophic effect of estradiol

This study evaluated effects of estradiol valerate on synthesis, secretion and direction of movement of immunoreactive prostaglandin F_2α (PGF) in swine. Gilts were randomly assigned to provide uterine flushings representing days 11, 13, 15, 17 and 19 of the estrous cycle (three gilts/day). The same gilts then were allowed one estrous cycle for recovery. During the second postoperative estrous cycle they were treated with estradiol valerate (EV) (5mg/day, SC) on days 11 through 15 and uterine flushings again were obtained on the same respective days with the same gilts represented within each day. Total recoverable PGF per uterine horn increased from day 11 (¯X = 1.98 ng) to day 17 (¯X = 210.20 ng) and then declined to day 19 (¯X = 66.20 ng) during the control period. Following EV treatment average total recoverable PGF was 1.9, 4,144.3 and 4,646.7 ng on the same respective days. EV treatment also resulted in maintenance of elevated levels of total protein and acid phosphatase activity in uterine flushings. These data suggest that estradiol may exert its luteotrophic effect by preventing the release of PGF from the uterine endometrium into the uterine venous system (endocrine secretion) while maintaining the movement of endometrial secretions into the uterine lumen (exocrine secretion).     

Antibacterial activity of mare uterine fluid

Luminal fluid from the mare uterus was used to investigate its relation to antibacterial defenses. Uterine flushings were collected at Day 3 of estrus, Day 8 postovulation and Day 15 postovulation. Uterine proteins were concentrated by ultrafiltration, dialyzed and examined for chemotactic activity to neutrophils and for antibacterial properties. Serum taken at the time of flushing was dialyzed and studied in a similar manner. Neutrophil migration in response to serum from Day 3 estrus and Day 8 postovulation was increased (P less than 0.05) above controls. Uterine protein from Day 8 postovulation and from Day 3 of estrus also stimulated neutrophil migration (P less than 0.05) above values of controls. Antibacterial activity was measured by incubation of S. zooepidemicus with concentrated uterine flushing or serum. Serum from all three estrous cycle intervals diluted 1:10 or used at a protein concentration equal to the protein concentration of uterine fluid did not inhibit growth. After 4 h of incubation, bacterial growth in estrous serum was significantly greater (P less than 0.01) than serum taken at Day 8 and Day 15 postovulation. Uterine flushings from Day 8 postovulation significantly decreased bacterial colony-forming units (P less than 0.01). Heating flushings at 56 degrees C for 30 min did not abolish the antimicrobial activity, while heating flushings for 30 min at 80 degrees C removed this activity. The antibacterial activity does not appear to be due to agglutinating antibody.     

Partial characterization of immunosuppressive proteins from bovine uterine luminal secretions

Unfractionated and fractionated uterine luminal protein (ULP) secretions collected from nonpregnant and pregnant beef cows on Day 17 post-breeding were tested in vitro for suppression of lymphocyte blastogenesis to the mitogen phytohemagglutinin (PHA). In replicated experiments, ULP from nonpregnant and pregnant cows was separated into five molecular weight (M_r) fractions with Sephacryl S-200. Unfractionated (25 to 400 μg/ml) and fractionated (25 to 100 μg/ml) ULP was added to cultures containing 5 × 10⁵ bovine lymphocytes and 0.4 μg of PHA in a complete culture medium. At 48 hr, 0.5 μCi of ³H-thymidine was added to cultures, and cells were harvested at 60 h by automation. Thymidine incorporation data were expressed as percentage of control (no ULP) values. Unfractionated and all S-200 ULP fractions from nonpregnant and pregnant cows suppressed (P<0.05 to 0.001) lymphocyte blastogenesis to PHA, but to varying degrees of suppression. Unfractionated ULP was more suppressive (P<0.05) for pregnant than nonpregnant cows, which was likely due to the greater (P<0.05) immunosuppressive activity of S-200 fractions I (>219,000 M_r) and V (∼14,000 M_r) from the pregnant cows. At 25 μg ULP/ml, mean (± SEM) % of control values for fraction I from pregnant and nonpregnant cows were 9.1 ± 3.3 and 36.6 ± 8.5%, respectively (P<0.05). Values for fraction V were 15.7 ± 6.5 and 46.6 ± 6.1%, respectively (P<0.01). Within each reproductive class, fractions I and V were more suppressive (P<0.05) than fractions II, III and IV. Immunosuppression was not mediated by lymphocyte cytotoxicity.     

Dose-dependent hyporreactivity to phytohemagglutinin in systemic lupus erythematosus

The response of peripheral blood lymphocytes to stimulation with optimal and suboptimal doses of PHA was measured in patients with active SLE before initiation of therapy. The [³H]thymidine uptake of SLE patient's lymphocytes was significantly lower than that of their matched controls when cells were stimulated with suboptimal PHA doses in the presence of autologous plasma. A moderate improvement in the PHA response was observed by culturing washed patient's lymphocytes in medium supplemented with pooled normal human plasma, but only in one case the response reverted to normal values. A significant inhibitory effect of SLE plasma on the response of normal donor's lymphocytes to stimulation with low PHA doses, which was independent from the presence of lymphocytotoxic antibodies and persisted after complement inactivation was observed in further experiments.The results indicate that depression of lymphocyte transformation could be demonstrated in patients with active SLE using suboptimal doses of PHA and suggest that this depression may be caused by both a defect in the responding lymphocyte populalation and the presence of inhibitory factor(s) in SLE plasma.