A Tissue type Transglutaminase in Human Seminal Plasma

PROBLEM: Initial studies from this laboratory of human seminal plasma (SePI) permitted the presumptive identification of the participation of transglutaminase (TGase) and prostaglandins as principal molecules contributing to the immunoregulatory activity of SePI. As a step toward confirmation and extension of these studies, SePI TGase has been partially purified, characterized, and localized. METHOD OF STUDY: An enzyme-enriched and partially purified preparation of SePI TGase obtained by molecular sieve and ion-exchange chromatography and the polyclonal antibody to this preparation were characterized by enzymatic analysis and evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); immunoprecipitation and immunofluorescence (IF). RESULTS: A Ca²⁺ -dependent, thrombin-independent, TGase enzyme from SePI was identified. A pH of 7.5 and a concentration of 2 mM calcium chloride were determined to be optimal for ascertaining the SePI TGase activity in a filter paper assay. Immunoprecipitation using polyclonal antibody prepared with a semipurified TGase preparation, concurrently comparing increasing serum dilution and enzyme activity, revealed a predominant protein band on SDS-PAGE of 83 kDa. IF studies using the polyclonal antibody on human prostate tissue showed reactivity with a variety of epithelial and stromal components. A significant (P < 0.05) correlation between the biochemical, i.e., enzyme activity of the SePI TGase active preparation, and immunologic activity, i.e., indirect IF staining titre of antibody to acinar epithelial cells and luminal contents, was observed. CONCLUSIONS: The results confirm and extend initial studies of the presumptive identification of a tissue type TGase associated with the immunoregulatory activity of human SePl and permit the characterization and immunohistologic localization of this macromolecule to the prostate. These observations provide a basis for further investigation of the immunoregulatory role of SePl and prostate TGase in the biology of reproduction and associated pathoses.     

Complement-Inhibiting Activity of Seminal Plasma

ABSTRACT: Human seminal plasma has an anticomplement effect that can be measured by a standard immune hemolytic assay. We found that about 20% of samples lack complement-inhibiting activity. To determine the anatomical origin of a complement inhibitor, inhibition of hemolysis was measured in seminal plasma samples that were primarily of prostatic, vesicular, or epididymal/testicular origin, as well as in seminal plasma from vasectomized men. All samples contained complement-inhibiting activity, although epididymal fluid showed nearly twice the amount. Therefore, the factor (or factors) is ubiquitous in secretions of the male reproductive tract. Complement-inhibiting activity is eluted in two high-molecular-weight peaks upon gel filtration. We found evidence that the inhibitory factor is not lactoferrin, a proteinase, or one of the proteinase inhibitors known to be present in human seminal plasma. It seems likely that the complement inhibitor in seminal plasma protects gametes and reproductive tissues from complement-mediated damage.     

人精浆对抗原抗体反应免疫抑制作用的研究

本文采用体外实验的方法,研究人精浆对抗原抗体特异性反应的抑制作用。运用酶联免疫吸附试验原理检测人精浆对AsAb、A-dsDNA、A-TG、A-TM、RF五种自身抗体反应的抑制,抑制率分别为61.5±23.1(％)、57.1±19.4(％)、56.7±25.8(％)、42.0±28.2(％)、41.8±25.1(％),说明均有明显的抑制作用。经生育组与不孕组、流产组对比分析,发现不孕组精浆对RF反应的抑制率明显高于生育组,其它各组之间对比均无显著差异。本文还对精浆的免疫抑制作用进行了探讨。     

ORIGINAL ARTICLE: The Effect of Oxidative Environment on Immunosuppressive Properties of Human Seminal Plasma

Problem Human seminal plasma (SP) has an important immunosuppressive function that enables sperm survival in the female reproductive tract. The aim of this study was to evaluate how oxidized proteins, by oxidative stress, may influence seminal plasma immunosuppressive properties in male infertility. Method of study Human SP immunosuppressive ability was evaluated by a lymphocyte proliferation assay. We used phytohemagglutinin mitogen to induce lymphocyte proliferation in the presence of human SP from infertile and fertile men, and under in vitro oxidizing conditions. Human SP‐oxidized proteins (MDA‐protein) were determined by the thiobarbituric acid test. Results Significant high levels of oxidized proteins were found in SP from asthenozoospermic patients. There was a significant difference (P < 0.05) between lymphocyte proliferation in the presence of SP from normozoospermic and asthenozoospermic group compared to the fertile donor group. Oxidized human SP in vitro allows for higher lymphocyte proliferation than non‐oxidized SP. Conclusion Human SP proteins have an inhibitory ability on lymphocyte proliferation, but under oxidative stress conditions, these proteins lose their immunosuppressive function.     

Immunological Specificity of Beta-Glucuronidase From Human Seminal Plasma and its Immunolocalization in the Human Reproductive Tract*

ABSTRACT: Beta-glucuronidase (β-glucuronidase) in human seminal plasma consists of two forms of isozymes (major and minor forms). Antiserum prepared against the purified major form of β-glucuronidase in rabbits was used for studies of immunological specificity and immunohistochemical localization of β-glucuronidase in the human male reproductive tract. By ouchterlony immunodiffusion technique, anti-β-glucuronidase serum showed reaction of identity with human tissue extracts such as liver, spleen, lungs, testis, epididymis, prostate, and seminal vesicles. Though monkey testis and epididymis extracts completely reacted with human seminal β-glucuronidase antiserum, no cross-reaction was observed with mouse and rat tissue extracts. Immunofluorescent studies revealed that β-glucuronidase is localized in the early stages of spermatogenesis in testis, in columnar cells of the epididymis, and in glandular cells of the prostate and seminal vesicles. Consistent absence of fluorescence in the lumens of testis and epididymis as well as negative cross-reaction between antiserum and sperm extracts suggested that major form of β-glucuronidase in human seminal plasma is not immunologically identical with human spermatozoa. It is concluded that major form of β-glucuronidase in seminal plasma is not tissue-specific although it appears to have limited species specificity.     

Immunological Specificity of Hexosaminidases From Human Seminal Plasma

ABSTRACT: Two isozymes of hexosaminidases (Hex) were purified from human seminal plasma and found to be homogeneous as revealed by immunoelectropho-resis and immunodiffusion experiments. Anti-Hex A antibodies were precipitated with isozyme B and vice versa. Though the precipitin arcs were equally stained for protein in these two antigen and antibody systems, enzyme activity was weakly stained in the reverse systems (ie, Anti-Hex A with Hex B and Anti-Hex B with Hex A). Thus, some common sequential antigenic determinants were indicated in two isozymes of Hex. On the other hand, loss of enzyme activity in the precipitate of reverse antigen-antibody complexes as compared to direct system (Anti-Hex A with Hex A and Anti-Hex B with Hex B) revealed that the two isozymes are present in different sequential and conformational states near the active sites. Moreover, antibodies of both forms of Hex cross-reacted with human sperm and reproductive and other vital organs such as kidney, liver, lung, pancreas, and spleen. Though monkey tissues also cross-reacted with human seminal plasma Hex-antisera, mouse, rat, and goat testis and epididymis extracts failed to show any cross-reaction. It is concluded that human Hex(s) are tissue nonspecific but appear to be specific to the human and the subhuman primates.     

Characterization of the in vivo and in vitro Effects of Indomethacin on Human Natural Killer Cell Activity

The in vivo and in vitro effects of indomethacin on the natural killer (NK) cell activity against K 562 target cells were studied. In vivo administration of indomethacin, 3 X 50 mg for 7 days to normal donors did not influence baseline NK cell activity, which means that treatment with prostaglandin (PG) inhibitors can be allowed in studies on NK cell activity of persons with normal PG production. The NK cell activity of fresh mononuclear cells was boosted with pharmacological concentrations of indomethacin in vitro, while frozen cells were not. Our results indicate that indomethacin enhances the NK cell activity in vitro by blocking the prostaglandin production of monocytes, since monocyte depleted effector cells were not boosted by indomethacin.     

Anti-Seminal Plasma Antibodies Associated With Infertility: II. Comparative Investigation of Human Antibody Binding to Normo-, Astheno-, and Azoospermic Seminal Plasma Antigens

PROBLEM: Sera from infertile patients with elevated reactivity against normozoospermic seminal plasma (NSP) have been selected to investigate human antibody binding to seminal fluid antigens present in abnormal ejaculates. METHOD: Sera from 32 idiopathically infertile patients and 44 control sera from fertile individuals were examined by ELISA against: 1) pooled seminal plasma from asthenozoospermic ejaculates (AsthSP), 2) pooled seminal plasma from patients with aspermatogenic azoospermia (AzooSP), and 3) chromatographic fractions from NSP, AsthSP and AzooSP. RESULTS: Of 32 patients positive for anti-NSP antibodies, only four exhibited increased reactivity to whole AsthSP and/or AzooSP, while 14 recognized antigens of different Mr and various distributions in the corresponding chromatographic fractions. CONCLUSION: Targets of human anti-NSP antibodies might be lacking, less concentrated, and/or modified in AsthSP and AzooSP. These findings suggest their physiological importance and the possible relevance of the observed auto- and iso-immune responses to infertility.     

The Molecular Weights and Molecular Weight Heterodispersion of Some Antigens From the Trichloroacetic Acid Soluble Fraction of Human Seminal Plasma

ABSTRACT: The antigens isolated from the trichloroacetic acid soluble fraction of human seminal plasma show a polymodal molecular weight heterodispersion. The molecular weights corresponding to the modes of the distribution have limiting values of 2,500-57,000. Previous results obtained by polyacrylamide gel and cellogel electrophoresis, immunodiffusion and Immunoelectrophoresis, and ultracentrifugation analysis, which suggested homogeneity, were explained by the formation of molecular aggregates, which would also explain why the smaller fragments were not lost during the dialysis carried out in the preparation and purification of the samples. The average molecular weight of the antigens, determined by gel permeation chromatography and using the equation of Svedberg, is in the range of 10,000–40,000. It is suggested that the polymodal heterodispersion together with the associating-dissociating properties of these products is used to control their immunological behavior.     

Characterization of Latent Transforming Growth Factor-β From Human Seminal Plasma

PROBLEM : Human seminal plasma is known to exhibit immunosuppressive activity. Transforming growth factor β (TGF-β) has been identified as an immunosuppressive factor in human seminal plasma. Biologically active TGF-β represents a family of 25-kDa homodimeric proteins linked with disulfide bonds. TGF-β associates with high molecular weight proteins noncovalently to form a type of latency that is biologically inactive. Quantitative distribution of active form of TGF-β versus inactive latent form of TGF-β, and mechanism of the TGF-β activation in human seminal plasma remain to be elucidated. PURPOSE : To characterize seminal plasma latent form of TGF-β, including its concentration, and the mechanism underlying the activation of TGF-β. METHOD : Gel filtrations on ACA-34 and Biogel P-60 were used to fractionate seminal plasma. TGF-β was measured by enzyme immunoassay using antibodies specific for TGF-β₁ and TGF-β₂, respectively. Radioreceptor assay with recombinant human [¹²⁵I]-TGF-β₁ was applied to qualitatively identify TGF-β₁. Kinetic experiments with various pH, temperature and time, along with protease inhibitors, were performed to delineate the activation mechanism of latent TGF-β. RESULTS : Human seminal plasma contained both TGF-β₁ and TGF-β₂, predominantly in latent form. The total concentration of TGF-β₁ averaged 238 ng/ml versus an average of 18 ng/ml for TGF-β₂. The in vitro activation or release of TGF-β₁, from latent TGF-β₁ was achieved only at acidic pH of <4.0, and was time and temperature dependent. At pH 3.7 and 37°C, a significant activation of latent TGF-β₁ was achieved after an incubation of only 15 min, reached the maximum at 120 min, and the activated TGF-β₁ remained relatively stable for at least 24 h. The activation was not inhibitable by a series of protease inhibitors examined, alone or in combination (e.g., phenylmethylsulfonyl fluoride, E-64, pepstatin, leupeptin, ethylenediamine tetraacetic acid). Competitive radioreceptor assay established the functional identity of TGF-β₁ in human seminal plasma with recombinant human TGF-β₁. CONCLUSION : Human seminal plasma TGF-β is biologically activated from high molecular weight latent TGF-β by acid pH. The acidic environment of female lower genital tract could represent an in vivo physiological condition for activation of seminal plasma TGF-β that may immunologically protect the integrity of sperm.     

Detection of Monocyte Chemotactic and Activating Factor (MCAF) and Interleukin (IL)-6 in Human Seminal Plasma and Effect of Leukospermia on These Cytokine Levels

PROBLEM : To demonstrate whether monocyte chemotactic and activating factor (MCAF) and interleukin-6 (IL-6) are present in the seminal plasma, and whether these presence is modulated by leukospermia. METHODS : Semen samples from 53 men were obtained by masturbation and examined for the presence of MCAF and IL-6 by enzyme immunoassay (EIA). Semen samples were obtained from 28 infertile men without leukospermia, 16 infertile men with leukospermia, and nine proven-fertile men. The correlation between the amount of MCAF in the seminal plasma with some spermiogram parameters and other cytokines such as IL-6 and IL-8 was statistically evaluated. RESULTS : Immunoreactive MCAF was detected in the seminal plasmas of all 53 subjects. The MCAF titer in the seminal plasma of patients with leukospermia (11.19 ± 2.75 μg/1) was significantly higher than that in the seminal plasma of the patients without leukospermia (3.24 ± 0.53 μg/1) and the fertile men (2.78 ± 0.35 μg/1) (P < 0.001). The IL-6 titer in the seminal plasma of the patients with leukospermia (21.05 ± 4.49 ng/1) was also significantly higher than that in the seminal plasma of the patients without leukospermia (8.77 ± 1.92 ng/1) and the fertile men (6.94 ± 1.27 ng/1) (P < 0.01). There was a high degree of correlation among the levels of MCAF, IL-6 and IL-8 in the seminal plasma. CONCLUSIONS : These findings demonstrated the presence of MCAF and IL-6 in the seminal plasma, and that the levels of these cytokines were elevated in the seminal plasma of the infertile patients with leukospermia.     

Modulatory Effects of Autologous Plasma on Functional Activity of Human Immunocompetent Cells

Blast transformation of peripheral blood lymphocytes stimulated with phytohemagglutinin and concanavalin A was studied in children with pyelonephritis and glomerulonephritis. Activity of natural killer cells from children with pyelonephritis was estimated before and after treatment with 50% autologous plasma. The autologous plasma modulated blast transformation of lymphocytes and activity of natural killer cells, which depended on the stage of diseases.     

Umbilical-Cord-Blood-Derived Suppressor Cells of the Human Natural Killer Cell Activity Are Inhibited by Interferon

The natural killer (NK) activity of umbilical-cord-derived lymphocytes was studied. The general level of activity was lower than with adult lymphocytes against K-562 cells and fetal fibroblasts. The activity could be boosted by interferon pretreatment of effector cells, but not to the same extent as with adult lymphocytes. Umbilical cord lymphocytes were fractionated with Percoll density gradient centrifugation, and the suppressive activity of different fractions was tested on highly enriched adult buffy-coat-derived NK cells. Allogeneic adult NK cell activity could be inhibited in 9 of 20 cases tested with small and medium-sized T lymphocytes (Percoll fractions 4–5) from the umbilical cord. The suppressive capacity was further enriched in fractions forming rosettes (RFC) with antibody-coated human erythrocytes (EA). Such EA-RFC of Percoll fractions 4–5 from umbilical cord exerted a strong suppressive activity in each case tested. Pretreatment of EA-RFC with interferon regularly abolished the suppressive effect. We conclude that there are Fc-receptor-positive small/medium-sized T lymphocytes in the umbilical cord blood which can efficiently suppress the cytotoxic activity of NK cells and that the suppressive activity can be abolished by interferon pretreatment of the suppressor cells.     

Timed Sexual Intercourse Facilitates the Recruitment of Uterine CD56bright Natural Killer Cells in Women with Infertility

Problem  The aim of this study was to investigate the influence of sexual intercourse on uterine NK cell subsets.
Method of study  Mid-secretory endometrial samples obtained from 56 women were submitted for flow cytometric analysis. Basal body temperature was used to determine the day of ovulation. A total of 27 women had sexual intercourse before ovulation (pre-ovulation group) and eight women had only after ovulation (post-ovulation group) without any contraceptive devices. A total of 21 women did not have sexual intercourse during the experimental cycle (abstinence group). Endometrial NK cells were analyzed for the expression of CD16 and CD56 using 3-color flow cytometry.
Results  CD16⁻/CD56^bright cells were markedly increased in the pre-ovulation group as compared with that of the post-ovulation group ( P  < 0.01) and the abstinence group ( P  < 0.01). CD16⁺/CD56^dim cells were significantly decreased in the pre-ovulation group as compared with that of the post-ovulation group ( P  < 0.01) and the abstinence group ( P  < 0.05).
Conclusion  It is suggested that seminal plasma participates in the recruitment of CD56^bright NK cells into endometrium.     

T-Helper 2 Type Cytokine and Soluble Interleukin-2 Receptor Levels in Seminal Plasma of Infertile Men

PROBLEM: The role of cell-mediated immunity (CMI) in unexplained male infertility and impaired sperm function has been explored. METHOD OF STUDY: The presence of cytokines, namely, interleukin-4 (IL-4), interleukin-6 (IL-6), and the soluble interleukin-2 receptor (SIL-2R), was investigated in seminal plasma of 18 fertile and 20 infertile subjects, using specific enzyme-linked immunoadsorbent assays. RESULTS: IL-4 was not detected. SIL-2R was detected, but the concentration difference between the fertile and infertile group was not significant. IL-6 was detected with significantly higher levels in the infertile group compared to the fertile group. IL-6 levels in seminal plasma correlated positively with leukocyte count and negatively with sperm count, motility, and morphology. CONCLUSIONS: These findings show: a) a lack of IL-4 in seminal plasma; b) similar SIL-2R levels in fertile and infertile seminal plasma; c) increased IL-6 secretion in seminal plasma of infertile subjects; and d) specific correlations of IL-6 with the main semen parameters.     

干扰素对受照射机体NK细胞活性的调节作用

采用4h51Cr释放法,分别以小鼠脾脏淋巴细胞对YAC-1细胞和肿瘤患者外周血淋巴细胞对K562细胞的体外自然杀伤（NK）活性为指标,研究受电离辐射后小鼠脾脏NK细胞活性的变化以及干扰素（IFN－α）对NK细胞活性的影响;并观察肿瘤患者及其放疗后NK细胞活性的变化以及IFN－α的调节作用。结果表明NK细胞具有较强的辐射耐受性,经16Gy照射后24hNK细胞活性显著降低;肿瘤患者的NK细胞活性明显低于正常对照组（P＜0．01）,放疗后NK细胞活性进一步降低（P＜0．05）。IFN－α处理NK细胞可以显著增强NK细胞活性,这提示IFN-α有可能作为一种生物应答调节剂用于肿瘤患者、肿瘤放、化疗或手术患者。     

Ontogeny and Expansion of Human Natural Killer Cells: Clinical Implications

Our knowledge of NK cells and their critical role in the innate immune system has increased enormously since their discovery several decades ago. However, it is only within the last 10 years that rational cytokine therapies, such as those utilizing low doses of IL-2, have been successful in expanding NK cells in patients with cancer and/or immunodeficiency. Such experiences in vivo have highlighted the importance of basing immunotherapeutic strategies on the known cellular and molecular properties of the targeted cell population. Recent advances in our understanding of the physiologic factors and events that orchestrate NK cell ontogeny, including IL-15 and receptor tyrosine kinase ligands to c-kit and fit3, provide novel therapeutic possibilities for cytokine therapy. This review summarizes our current understanding of human NK cell ontogeny, and links this knowledge to ongoing and future clinical strategies for the endogenous expansion of NK cells in patients with cancer and/or immunodeficiency.     

Effect of 5''-Methylthioadenosine, 3-Deazaadenosine, and Related Compounds on Human Natural Killer Cell Activity

The effect of 5'-methylthioadenosine (MTA) on human natural killer (NK) cell activity was examined and compared with the effect of 3-deazaadenosine (c3-ado) and periodate-oxidized adenosine (ado-ox). MTA inhibited NK cell activity in concentrations above 30 microM, but in concentrations below 10 microM a slight enhancing effect was often observed. C3-ado and ado-ox were 10 and 3 times more potent, respectively, as inhibitory agents and did not increase NK cell activity in low concentrations. The inhibitory effect of c3-ado was unaffected by preincubation of the cells but was enhanced by the addition of L-homocysteine. In concentrations that caused inhibition of NK cell activity all three agents caused a fall in the methylation index (AdoMet/AdoHcy) but no or an inconsistent effect on the level of cyclic AMP. An increase in the level of AdoHcy was observed already after 1 h of incubation but was more pronounced after 4 h of preincubation with the adenosine derivatives. The inhibition of cytotoxicity was mainly on their initiation of lysis, with a smaller effect on target cell binding. Antibody-dependent cellular cytotoxicity and lectin-dependent cellular cytotoxicity appeared to be less sensitive to inhibition by c3-ado. Our results show that several adenosine analogues inhibit NK-cell-mediated cytotoxicity in parallel with a decreased methylation index. The results suggest that a methylation step is critical in lymphocyte-mediated cytotoxicity and that NK cell activity is more sensitive to inhibition of this step than antibody- or lectin-dependent cytoxicity.