Glucocorticoid regulation of the humoral immune system. I. In vivo effects of dexamethasone on IgA and IgG in serum and at mucosal surfaces

The present studies were undertaken to determine whether glucocorticoids influence the levels of Ig in serum, saliva, and vaginal secretions. When measured by RIA, IgA levels in serum were elevated when increasing doses of dexamethasone, a potent synthetic glucocorticoid, were administered to intact- and adrenalectomized-ovariectomized rats. In contrast, IgA levels decreased in saliva and vaginal secretions over the same dose range. Time course studies indicated that the decline in salivary IgA, observed at 24 h after a single injection of dexamethasone, preceded a rise in serum IgA detected at 24 h after the second hormone treatment. Both responses were maximal at day 2 and did not change with further hormone exposure. After immunization and boosting with SRBC at two mucosal sites (intestinal Peyer's patch and uterine lumen), dexamethasone increased anti-SRBC IgA antibody levels in serum and reduced their presence in vaginal secretions. In contrast, anti-SRBC IgG-antibody levels in serum and vaginal secretions were reduced with hormone treatment. In the absence of hormone treatment, pooled sera from nonimmunized animals, when analyzed by HPLC, contained polymeric and dimeric IgA that was present in roughly equal proportion. In response to dexamethasone, polymeric IgA increased to a greater extent than did monomeric IgA. In summary, these studies demonstrate that dexamethasone alters the levels of IgA as well as specifically directed IgA and IgG antibodies in secretions and serum. Further, it suggests that glucocorticoid controlled IgA increases in serum and decreases in vaginal and salivary secretions may be due, in part, to a redistribution of polymeric IgA from mucosal surfaces to serum.     

Specific IgA and IgG antibodies in the secretions of the female reproductive tract: effects of immunization and estradiol on expression of this response in vivo

Uterine and vaginal secretions collected from intact adult female rats were analyzed to determine whether immunization at sites distal to the reproductive tract had any effect on the presence of specific IgA and IgG antibodies in genital tract secretions. Peyer's patch and i.p. immunization and boost with sheep red blood cells (SRBC) stimulated the appearance of specific IgA antibodies in uterine and vaginal secretions of uterine-ligated animals. IgG antibodies were also induced in uterine but not in vaginal secretions. In contrast, subcutaneous immunization and boost elicited a weak IgA uterine and IgG vaginal response. To establish the role of estradiol in regulating the presence of specific antibodies in the female genital tract, ovariectomized rats received primary and/or secondary Peyer's patch immunizations with hormone treatment. Administration of estradiol daily for 3 days before sacrifice resulted in a significant accumulation of IgA and IgG antibodies to SRBC in uterine secretions. In the absence of estradiol, antibody content was negligible. Vaginal antibody levels were also clearly influenced by estradiol. In contrast to the uterus, however, specific IgA and IgG antibodies were present in the vaginal secretions of saline-injected immunized animals and were markedly inhibited in animals treated with estradiol. These results indicate that antibodies in genital tract secretions can be induced by immunization of the Peyer's patches and that their presence in uterine secretions is clearly dependent on estradiol. Further, they indicate that gut-derived specific antibodies enter the vagina in the absence of hormone stimulation and that estradiol exerts an inhibitory effect on their presence in vaginal secretions.     

Prevalence of Trichomonas vaginalis in Women Visiting 2 Obstetrics and Gynecology Clinics in Daegu,South Korea

This study explored epidemiological trends in trichomoniasis in Daegu, South Korea. Wet mount microscopy, PCR, and multiplex PCR were used to test for Trichomonas vaginalis in vaginal swab samples obtained from 621 women visiting 2 clinics in Daegu. Of the 621 women tested, microscopy detected T. vaginalis in 4 (0.6%) patients, PCR detected T. vaginalis in 19 (3.0%) patients, and multiplex PCR detected T. vaginalis in 12 (1.9%) patients. Testing via PCR demonstrated high sensitivity and high negative predictive value for T. vaginalis. Among the 19 women who tested positive for T. vaginalis according to PCR, 94.7% (18/19) reported vaginal signs and symptoms. Notably, more than 50% of T. vaginalis infections occurred in females younger than 30 years old, and 58% were unmarried. Multiplex PCR, which simultaneously detects pathogens from various sexually transmitted infections, revealed that 91.7% (11/12) of patients were infected with 2 or more pathogens. Mycoplasma hominis was the most prevalent co-infection pathogen with T. vaginalis, followed by Ureaplasma urealyticum and Chlamydia trachomatis. Our results indicate that PCR and multiplex PCR are the most sensitive tools for T. vaginalis diagnosis, rather than microscopy which has been routinely used to detect T. vaginalis infections in South Korea. Therefore, clinicians should take note of the high prevalence of T. vaginalis infections among adolescent and young women in order to prevent persistent infection and transmission of this disease.     

Antigenic Heterogeneity in the 115,000 Mr Major Surface Antigen of Trichomonas vaginalis1

ABSTRACT The 115,000-molecular-weight antigen of Trichomonas vaginalis was characterized using monoclonal antibodies developed to three different strains of T. vaginalis and one strain of Tritrichomonas foetus. The antigen was found to be present on all strains or isolates of T. vaginalis examined and was demonstrated to be located on the external surface plasma membrane by agglutination assays and complement-mediated lysis assays. Characteristics of the antigen were assessed with a proteolytic enzyme and periodate oxidation. Periodate treatment of whole T. vaginalis abrogated binding for eight antibodies while use of pronase-treated antigen resulted in loss of antibody binding for two different antibodies. Screening of 19 axenized clinical isolates of T. vaginalis and one strain each of T. foetus and Giardia lamblia with type-specific antibodies delineated three major groups of T. vaginalis based on antigenic specificities (epitope distributions) within the 115,000-molecular-weight antigen. In addition, one epitope of the 115,000-molecular-weight antigen was found only on the immunizing strain. Two epitopes were present on all T. vaginalis isolates as well as T. foetus and G. lamblia. One epitope was common to all T. vaginalis except one. A minimum of six different epitopes of the 115,000-molecular-weight antigen were identified. Antigens purified with type-specific or “common” monoclonal antibodies shared the same partial peptide maps demonstrating relatedness.     

Analysis of the humoral responses of Toxoplasma gondii-infected cats using immunofluorescent assays with tachyzoite, bradyzoite, and gametogenic stages

We investigated levels of Toxoplasma gondii specific antibodies present in sera, intestinal secretions, and fecal extracts obtained from cats following primary and challenge infections. Antibodies specific to T. gondii tachyzoites, bradyzoites, sporozoites, and enteroepithelial stages were detected by indirect immunofluorescence assay. Enteroepithelial stage-specific antibodies were detected in serum as early as 2 wk after infection, whereas antibodies from intestinal secretions did not appear until 3 wk following infection. The T. gondii-specific IgG and IgA antibodies were present in serum, but only specific IgA antibodies were detected in the intestinal secretions. Serum IgG bound to tachyzoites, bradyzoites, sporozoites, and enteroepithelial stages of T. gondii, whereas serum IgA bound strongly to enteroepithelial stages but only weakly to tachyzoites and bradyzoites. IgA from intestinal secretions bound to antigens on all enteroepithelial stages and the distal tips of sporozoites and bradyzoites but did not bind to tachyzoites. IgA present in fecal extracts also bound to enteroepithelial stages of T. gondii. Toxoplasma gondii infection in cats induces the production of antibodies that bind with all forms of the parasite, including the enteroepithelial stages. Comparison of the staining patterns of T. gondii stages for serum and intestinal secretion IgA indicated differences. Thus, the intestinal antibody immune response may be uniquely focused on the intestinal stages relative to the circulating antibodies, resulting in a compartmentalization of the humoral response.     

Estradiol regulation of the secretory immune system in the female reproductive tract: IgA in uterine and vaginal secretions of rats following portacaval anastomosis

The uterine immune system is under the control of estradiol which acts to increase the levels of both IgA and secretory component (SC) in uterine secretions. The objective of the present study was to determine whether serum is the primary source of the IgA which enters uterine secretions in response to estradiol. To examine this, serum IgA levels in rats were surgically elevated by portacaval anastomosis which prevents hepatic clearance of IgA. Under these conditions, IgA levels in serum were 2- to 4-fold higher than those of intact or sham-operated animals. Levels of IgA in uterine secretions of portacaval animals, however, were significantly lower than those measured in controls when animals were ovariectomized and treated with estradiol. IgA in vaginal secretions of portacaval animals was greater than that in sham-operated or intact rats. To determine whether IgA had leaked from the uterus into vaginal secretions, a second group of animals had their uteri ligated at the utero-cervical junction prior to hormone treatment. Following estradiol stimulation, uterine IgA levels in portacaval animals were the same as those measured in intact and sham-operated animals. When free SC was measured in uterine secretions of ligated rats, levels were the same in all three groups. These studies indicate that elevated levels of serum IgA did not lead to a rise in uterine IgA. Further, since SC, which is thought to be a receptor for transporting IgA into mucosal secretions, remained unchanged, it appears unlikely that IgA movement into the uterine lumen was transport limited. These studies suggest that the presence of IgA in uterine and vaginal secretions is not due exclusively to serum contributions but may involve local synthesis of IgA.     

HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities

Background

Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.

Methods and Findings

We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.

Conclusion

Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.     

Immunoglobulin G is the main protective antibody in mouse vaginal secretions after vaginal immunization with attenuated herpes simplex virus type 2.

We investigated the protective role of antibodies in vaginal secretions of mice that were immune to vaginal challenge with herpes simplex virus type 2 (HSV-2). Unfractionated vaginal immunoglobulins from immune and nonimmune mice and affinity-purified immunoglobulin G (IgG) and secretory IgA (S-IgA) from immune secretions were adjusted to their concentrations in vivo. Wild-type HSV-2 was incubated in the immunoglobulin preparations for 15 min in vitro, followed by inoculation into vaginae of nonimmune mice. HSV-2 was neutralized by unfractionated antibody and purified IgG from immune secretions but not by unfractionated nonimmune antibody or by purified immune S-IgA. The protective effect of IgG in vivo was investigated by passively transferring purified serum IgG from immune and nonimmune donors to nonimmune recipients before vaginal challenge infection. Immune IgG significantly reduced the percentage of vaginal epithelium infected, concentrations of shed virus protein in the vaginal lumen, and illness scores, even though the viral antibody titers in serum and vaginal secretions of recipient mice at the time of challenge were only 29 and 8%, respectively, of those in actively immunized mice. Additionally, removal of vaginal secretions from immune mice 10 min before vaginal challenge with HSV-2 significantly increased the concentration of shed virus protein in the vaginal lumen after challenge. Collectively, the data indicate that IgG antibody in vaginal secretions of immune mice provides early protection against vaginal challenge infection, probably by neutralizing virus in the vaginal lumen. In contrast, S-IgA antibody contributed relatively little to immune protection of the vagina.     

IgA and IgG antibodies to Candida albicans in the genital tract secretions of women with or without vaginal candidosis

Levels of anti-Candida albicans immunoglobulin A (IgA) and IgG were measured by enzyme-linked immunosorbent assay in serum and cervicovaginal secretions from 64 non-pregnant women with vaginal candidosis and 158 uninfected non-pregnant women. Specific IgA and IgG were detected in the serum and secretions of all 222 women. There was no significant difference between the mean levels of specific IgA or IgG in secretions from women with candidosis and those of uninfected women. Neither was there a significant difference between mean levels of specific IgA or IgG when women using oral contraception were compared with others who were not. There was a significant correlation between the levels of IgA and IgG in serum and secretions from women with candidosis and from uninfected women. Blastospore and hyphal forms of C. albicans were seen in vaginal smears from 29 of the 64 women with culture-proven candidosis: in nine, both IgA- and IgG-coated C. albicans cells were recovered from the genital tract; in a tenth, IgG-coated cells were found.     

Clinical and immunological effect of intravesical interleukin-2 on superficial bladder cancer

TheBamHI Z EBV replication activator (ZEBRA) protein is involved in the switch from latency to productive cycle of Epstein-Barr virus. A recombinant ZEBRA protein was synthesized and assessed in enzymelinked immunosorbent assay (ELISA) for serum IgG response in nasopharyngeal carcinoma (NPC) patients. In 100 NPC serum samples that were positive for IgA to the EBV viral capsid antigen (VCA), 75% had IgG anti-ZEBRA antibodies. In contrast, only 3/83 (3.6%) serum samples from healthy donors and 2/50 (4%) from other cancers were positive for IgG to ZEBRA. Interestingly, in a selected group of 100 NPC sera negative for IgA to VCA, 25% contained IgG anti-ZEBRA antibodies. This suggests that the ELISA for IgG anti-ZEBRA may also identify earlier cases of NPC not detected by the conventional immunofluorescence test for IgA to VCA.     

Trichomonas vaginalis: Investigation of a novel diagnostic method in urine samples using cysteine proteinase 4 gene and PCR technique

Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted disease that leads to vaginitis, urethritis, ectocervicitis and has been associated with human immunodeficiency virus (HIV). Detection of T. vaginalis based on wet-mount microscopy and culture methods is insensitive and time consuming, respectively. Thus the quest for reliable PCR techniques of T. vaginalis in vaginal discharge and urine sample is more importance. In this study, 500 urine and vaginal-discharge samples were collected from women referred to Sexual Transmitted Disease Clinic of Mirzakuchakkhan Hospital in Tehran, Iran between May 2008 and March 2009. Wet-mount and culture methods were done on the vaginal discharges, and PCR assay targeting cysteine proteinase 4 (CP4) was performed on the urine samples. The present study demonstrated 16 (3.2%) of patients were infected with T. vaginalis using culture and wet-mount, whereas PCR assay using CP4 could detect 12 (2.4%) positivity. Sensitivity and specificity of urine PCR assay compared to culture were 80% (95% CI, 54-96) and 99.6% (95% CI, 98.96-100), respectively. These results indicate that using urine-based detection method for T. vaginalis may not be appropriate in women.     

测定Epstein-Barr病毒IgA/EA抗体的改进方法

用免疫酶法(IE)检查鼻咽癌(NPC)病人血清中IgA/EA抗体,阳性率为73％,几何平均滴度为25,将血清用马抗人IgG血清或葡萄球菌菌体A蛋白(SPA)处理后,以除去竞争性IgG类抗体后,阳性率可增高至92％,几何平均滴度提高到89,有15例NPC病人血清,经马抗人IgG血清处理前IgA/EA抗体为阴性,处理后均呈阳性反应。     

Increased intracellular Cl− concentration mediates Trichomonas vaginalis-induced inflammation in the human vaginal epithelium

Trichomonas vaginalis is a primary urogenital parasite that causes trichomoniasis, a common sexually transmitted disease. As the first line of host defense, vaginal epithelial cells play critical roles in orchestrating vaginal innate immunity and modulate intracellular Cl⁻ homeostasis via the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that plays positive roles in regulating nuclear factor-κB (NF-κB) signalling. However, the association between T. vaginalis infection and intracellular Cl⁻ disequilibrium remains elusive. This study showed that after T. vaginalis infection, CFTR was markedly down-regulated by cysteine proteases in vaginal epithelial cells. The intracellular Cl⁻ concentration ([Cl⁻]_i) was consequently elevated, leading to NF-κB signalling activation via serum- and glucocorticoid-inducible kinase-1. Moreover, heightened [Cl⁻]_i and activated NF-κB signalling could be sustained in a positive feedback regulatory manner resulting from decreased intracellular cAMP through NF-κB-mediated up-regulation of phosphodiesterase 4. The results conclusively revealed that the intracellular Cl⁻ of the human vaginal epithelium could be dynamically modulated by T. vaginalis, which contributed to mediation of epithelial inflammation in the human vagina.     

A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

Background  

Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.     

Superoxide Anion Production by Human Neutrophils Activated by Trichomonas vaginalis

Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O₂^.-) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.     

Further studies on the surface charge of various strains ofTrichomonas vaginalis andTritrichomonas foetus

The surface charge of three strains ofTrichomonas vaginalis and five strains ofTritrichomonas foetus was determined by direct measurement of the mean cellular electrophoretic mobility (EPM) of cells suspended in solutions of different ionic strength and pH. No differences were observed in the mean EPM among the two species, although significant differences among the strains exist. Strains that are more pathogenic to mouse, as measured using the subcutaneous assay, had a surface more negative. Treatment of the parasites with trypsin or neuraminidase reduced significantly their mean EPM and increased their isoelectric point.Tritrichomonas foetus was more sensitive to the enzyme treatment thanT. vaginalis. Enzyme-treated cells recovered their normal EPM if, after enzyme treatment, they were incubated in fresh culture medium. The recovery process of trypsintreated cells was inhibited 10–20% by addition of inhibitors of either protein synthesis (puromycin) orN-glycosylation of proteins (tunicamycin) to the incubation medium, suggesting that a cytoplasmic pool of sialoglycoproteins may exist. The recovering of the EPM ofT. foetus andT. vaginalis previously treated with neuraminidase was inhibited by puromycin or tunicamycin about 40–50% and 17–30%, respectively. These observations suggest that sialoglycolipids exist on the surface of both parasite species, and that they contribute more to the surface charge ofT. vaginalis than to that ofT. foetus.     

Adenosine,but not guanosine,protects vaginal epithelial cells from Trichomonas vaginalis cytotoxicity

Trichomonas vaginalis causes the most common non-viral sexually transmitted disease worldwide. The cytoadherence and cytotoxicity upon the vaginal epithelial cells are crucial for the infection. Extracellular nucleotides are released during cell damage and, along with their nucleosides, can activate purinoceptors. The opposing effects of nucleotides versus nucleosides are regulated by ectonucleotidases. Herein we evaluated the hemolysis and cytolysis induced by T. vaginalis, as well as the extracellular nucleotide hydrolysis along with the effects mediated by nucleotides and nucleosides on cytotoxicity. In addition, the gene expression of purinoceptors in host cells was determined. The hemolysis and cytolysis exerted by all T. vaginalis isolates presented positive Pearson correlation. All T. vaginalis isolates were able to hydrolyze nucleotides, showing higher NTPDase than ecto-5′-nucleotidase activity. The most cytotoxic isolate, TV-LACM6, hydrolyzes ATP, GTP with more efficiency than AMP and GMP. The vaginal epithelial cell line (HMVII) expressed the genes for all subtypes of P1, P2X and P2Y receptors. Finally, when nucleotides and nucleosides were tested, the cytotoxic effect elicited by TV-LACM6 was increased with nucleotides. In contrast, the cytotoxicity was reversed by adenosine in presence of EHNA, but not by guanosine, contributing to the understanding of the purinergic signaling role on T. vaginalis cytotoxicity.     

Secretory IgA and secretory component in women affected by recidivant vaginal candidiasis

Local immunity was evaluated in 47 patients affected by recidivant vaginal candidiasis and 33 control women. IgG, IgA, IgM and secretory component (SC) were determined by single radial immunodiffusion in samples of cervicovaginal secretion. IgG in dosable levels was detected in 17/47 samples (36.2%) and IgA in 15/47 patients (31.9%) whereas in the controls, the incidence was 31/33 (93.9%) for IgG and 24/33 (72.7%) for IgA. The difference was significative (P< 0.001) for both immunoglobulins. Significant differences were not obtained for IgM. The SC was detected in 4/47 cervicovaginal secretions of patients affected by candidiasis (8.5%) whereas in the control samples the incidence was 21/33 (63.6%) (P<0.001). In only 2/15 patients with dosable levels of IgA (13%) the secretory nature of this immunoglobulin could be shown by its reaction with anti-SC serum. In the control group, secretory IgA was detected in 19/24 cases (79%) (P< 0.001). Serum immunoglobulins levels were normal. The lack of secretory IgA and SC in the secretion could be related to the adherence capacity of the Candida albicans to epithelial cells.     

Detection of Microtubules of the Flagellate Trichomonas vaginalis by Monoclonal Antibodies Specific for β-Tubulin1

ABSTRACT. Monoclonal antibodies specific for mammalian β-tubulin recognized the microtubule cytoskeleton of the flagellated protozoon Trichomonas vaginalis. Of seven antibodies, two demonstrated the axostyle, costa, recurrent flagellum, and anterior flagella by indirect immunofluorescence microscopy. The remaining five stained a hazy reticular pattern in the cytoplasm of formaldehyde-fixed, detergent-extracted organisms. Western immunoblots of whole T. vaginalis extracts treated with protease inhibitors and electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate showed a major band at molecular weight 50,000 when probed with only one of the antibodies which stained the axial cytoskeleton. The antibodies which stained only the cytoplasm showed a different western blot pattern with a major doublet band at MW 58,000–60,000. Another antibody, which stained both the axial cytoskeleton and the reticular cytoplasmic pattern showed major bands at MW 58,000–60,000 and also at MW 40,000–42,000. The recognition of microtubule populations in T. vaginalis by these monoclonal antibodies was different than we found earlier with Leishmania donovani and Toxoplasma gondii, where all seven antibodies recognize cytoskeletal microtubules and produce western blots characteristic of tubulin. Only one of these seven antibodies recognizes tubulin in T. vaginalis by immunoblot. The microtubules of T. vaginalis do not demonstrate all epitopes recognized by monoclonal antibodies specific for mammalian β-tubulin; one of the antibodies appears to recognize an epitope which is morphologically associated with microtubules but does not have the characteristic MW of tubulin.