Unique alterations in infection-induced immune activation during pregnancy.

BACKGROUND: Immune responses to infection are uniquely regulated during gestation to allow for antimicrobial defence and tissue repair, whilst preventing damage to developing fetal organs or the triggering of preterm labour. OBJECTIVE: A review and analysis of studies delineating gestation-specific immune modulation and intra-amniotic regulation of pro-inflammatory immunity. SEARCH STRATEGY: Identification of the alterations between the fetus/neonate and adult with regard to the endogenous and infection-induced expression of molecules with immune regulatory properties, and the characterisation of intra-amniotic immune mediators that inhibit bacterial-induced pro-inflammatory cytokine production. SELECTION CRITERIA: English and non-English publications from 1985 to the present. DATA COLLECTION AND ANALYSIS: An electronic literature search using MEDLINE, PubMed, articles cited in the primary sources, as well as pregnancy-related immunology research from our laboratory at Weill Medical College of Cornell University. MAIN RESULTS: During fetal development, interleukin (IL)-23, IL-10 and IL-6, as well as T-helper-17 (Th17)-mediated immune responses, are upregulated, whereas tumour necrosis factor-α (TNF-α) and IL-1ß- and Th1-mediated immune responses are downregulated in the intrauterine environment (both the fetal compartment and the amniotic compartment). Infection-related immunity during gestation is preferentially directed towards combating extracellular microbial pathogens. Amniotic fluid and the neonatal circulation contain multiple components that improve the ability of the developing neonate to tolerate microbial-induced immune activation. CONCLUSIONS: The repertoire of immune mechanisms to control infection and inflammation differ between fetal and adult life. The dual mechanisms of resistance to infection and tolerance to infection-induced immune activation prevent damage to the developing fetus and the triggering of premature labour.

Endocervical hyaluronan and ultrasound-indicated cerclage.

OBJECTIVE: To investigate whether, at the time of ultrasound-indicated cerclage, the endocervical concentration of hyaluronan (HA), 27-kDa heat shock protein (HSP-27) and/or interleukin-8 (IL-8) would predict pregnancy outcome. METHODS: Endocervical samples, obtained from 40 women undergoing ultrasound-indicated cerclage at 15 + 3 to 25 + 0 weeks' gestation, were assayed by enzyme-linked immunosorbent assay for HA, HSP-27 and IL-8. All subjects had a cervical length of < 1.5 cm or dramatic cervical length change on serial endovaginal ultrasound, no uterine contractions or tenderness, no fever and intact membranes and underwent a modified Shirodkar cerclage. RESULTS: The median HA level was 10.0 ng/mL in the 12 women who delivered at < 37 weeks' gestation as opposed to 39.7 ng/mL in the 28 women delivering at 37-41 weeks (P = 0.017). Median HSP-27 and IL-8 concentrations were not significantly different in these groups. CONCLUSION: A higher endocervical HA level at the time of ultrasound-indicated cerclage is associated with a longer interval before birth.

Serum antibodies to the 27-kd heat shock protein in women with gynecologic cancers.

OBJECTIVES: Among women the association between heat shock protein immunity and cancer has been examined primarily for breast cancer. Autoantibodies to the 27-kd heat shock protein were detected in some patients with breast cancer but not in control subjects, and the presence of these antibodies was correlated with improved survival. We examined the relationship between autoimmunity to heat shock proteins and the diagnosis of malignancies of the female genital tract. STUDY DESIGN: Serum samples from women seen for possible gynecologic malignancies or returning for evaluation after surgery, radiation, chemotherapy, or a combination for gynecologic cancers were tested for immunoglobulin G antibodies to the 27-kd, 60-kd, 70-kd, and 90-kd heat shock proteins by enzyme-linked immunosorbent assay with the purified recombinant proteins bound to wells of a microtiter plate. Serum samples from women with no history of malignancies served as control preparations. RESULTS: Antibodies to the 27-kd heat shock protein were detected in only 1 of 29 healthy control subjects (3.4%) and 1 of 23 women whose lesions were benign (4.3%). In marked contrast, 39 of 96 women with gynecologic cancers (40.6%) had positive antibody detection (P =.0004 vs benign). The percentages of positive results seen for ovarian (17/34, 50%), endometrial (13/34, 38.2%), cervical and uterine (3/10, 30%), vaginal and vulvar (3/5, 60%), and other (3/13, 23.1%) cancers were not significantly different from each other. Similar prevalences of antibodies to the 27-kd heat shock protein were seen among patients with cancer who had untreated active disease and after treatment. Unlike the results with antibodies to the 27-kd heat shock protein there was no relationship between antibodies to the other heat shock proteins and any gynecologic cancer. CONCLUSION: Circulating autoantibodies to the 27-kd heat shock protein were found to be associated with malignancies of the female genital tract.

IgA antibodies to the 27-kDa heat-shock protein in the genital tracts of women with gynecologic cancers.

Heat-shock proteins promote cell survival under adverse environmental conditions. Synthesis of the 27-kDa (HSP27), 70-kDa (HSP70), and 90-kDa (HSP90) heat-shock proteins is increased in malignantly transformed cells and has been associated with tumor proliferation, metastasis, and resistance to chemotherapeutic agents. The increased expression of heat-shock proteins and their association with tumor-specific antigens may result in local immunity to the heat-shock proteins. We examined the occurrence of IgA antibodies to HSP27, HSP70, and HSP90 in the lower genital tracts of women with possible gynecologic cancers. Cervical samples were obtained from 119 consecutive women being evaluated for a gynecologic malignancy or returning for a follow-up examination following cancer treatment. Aliquots were tested for IgA anti-heat-shock protein antibodies by ELISA. Aliquots were also tested for IgG antibodies to HSP27 as well as for human papillomavirus. Anti-HSP27 IgA was detected in 85.7% of 21 women with endometrial cancer tested prior to diagnosis and in 41.1% of 17 women tested after treatment. In women with ovarian cancer, 77.8% of 9 women tested prior to diagnosis and 75.0% of 24 women evaluated after treatment were anti-HSP27 IgA-positive. Of 6 women with cervical cancer tested prior to diagnosis, 5 were positive for this antibody. None of 25 women with benign diagnoses or 46 healthy women were cervical IgA anti-HSP27-positive (P < 0.0001). In contrast, anti-HSP27 IgG was not associated with a gynecologic malignancy. HSP27 cervical antibodies were not associated with the presence of human papillomavirus. Cervical IgA antibodies to HSP90 were associated with ovarian cancer; antibodies to HSP70 were not cancer-associated. We conclude that cervical IgA antibodies to HSP27 may be indicators of a gynecologic malignancy.

Induction of heat shock protein expression in cervical epithelial cells by human semen.

OBJECTIVE: The 70kD heat shock protein (Hsp70), induced when cells are subjected to environmental stress, prevents the denaturation and incorrect folding of polypeptides and may expedite replication and transmission of DNA and RNA viruses. We analyzed whether messenger RNA (mRNA) for Hsp70 was expressed following exposure of a cultured human cervical cell line (HeLa cells) to human semen or in cervical cells from sexually active women. STUDY DESIGN: HeLa cells were co-cultured with a 1:50 dilution of semen from four men or with purified spermatozoa or cell-free seminal fluid. Endocervical swabs were acquired at mid-cycle from 53 women. Heat shock protein 70 mRNA was detected by a reverse transcriptase-polymerase chain reaction utilizing specific primer pairs and analysis on agarose gels. In cervical cells Hsp70 mRNA was measured identically followed by hybridization with an Hsp70-specific internal probe and detection by enzyme-linked immunosorbent assay (ELISA). Cervical immunoglobulin A (IgA) antibodies to the human Hsp70 were determined by ELISA. RESULTS: HeLa cell-semen co-culture resulted in the induction of Hsp70 mRNA. In addition, cell-free seminal plasma and motile sperm incubated individually with HeLa cells also induced this mRNA. Heat shock protein 70 mRNA was detected in 28 (52.8%) of 53 endocervical samples obtained from women at various time points following intercourse. The percentage of samples expressing this mRNA was 37.5% at less than 10 hours, 64.3% at 10 hours, 70% at 11 hours, and between 36% and 50% at later times after semen exposure. The detection of cervical IgA antibodies to the Hsp70 was highly associated with Hsp70 gene transcription. CONCLUSION: Human semen induces transcription of Hsp70 in cervical epithelial cells.

Detection of endocervical anti-Chlamydia trachomatis immunoglobulin A in pregnant women by a rapid, 6-minute enzyme-linked immunosorbent assay: comparison with PCR and chlamydial antigen detection methods.

There is a need for a rapid, uncomplicated, and inexpensive test for Chlamydia trachomatis infection in women. We evaluated the ability of a 6-min enzyme-linked immunosorbent assay (ELISA) that requires no laboratory equipment (IgA Rapid SeroTest; Savyon Diagnostics) to detect C. trachomatis immunoglobulin A (IgA) in the endocervices of 167 inner-city pregnant women and compared the results with DNA amplification (Amplicor PCR; Roche Diagnostics) and antigen detection (Chlamydiazyme; Abbott Laboratories) performed on the same women. Anti-C. trachomatis IgA was detected in the cervices of 32 women (19.2%). Samples from 23 women (13.8%) were PCR positive, while chlamydial antigen was present in 20 women (12.0%). There was only 1 sample (4.3%) that was positive by PCR but negative by ELISA; 10 samples were ELISA positive and PCR negative. In contrast, seven samples (30.4%) were PCR positive but Chlamydiazyme negative and four were Chlamydiazyme positive and PCR negative. Compared to PCR, the IgA ELISA had a sensitivity of 95.7%, a specificity of 93.1%, a positive predictive value of 68.8%, and a negative predictive value of 99.3%. The antigen assay had a sensitivity of only 69.6%, a specificity of 97.2%, a positive predictive value of 80.0%, and a negative predictive value of 95.2%. In high-risk groups where laboratory testing is not available, or where the patient might not return to obtain her testing result and be treated, the Rapid IgA SeroTest is a viable alternative for detection of cervical C. trachomatis in pregnant women.

Chlamydia trachomatis Infection, Immunity, and Pregnancy Outcome.

Chlamydia trachomatis can ascend from the cervix to the fallopian tubes and survive for long periods of time without causing symptoms. The immune response to infection clears the extracellular organisms but leads to development of a persistent intracellular infection. Repeated cycles of productive infection and persistence eventually induce tubal occlusion and infertility. Persistently infected cells continue to synthesize the chlamydial 60 kD heat shock protein (hsp60). Immunity to conserved regions of hsp60 may result in autoimmunity to human hsp60. Expression of hsp60 by the embryo and decidua during early pregnancy may reactivate hsp60-sensitized lymphocytes, disturb pregnancy-induced immune regulatory mechanisms, and lead to immune rejection of the embryo. Due to this mechanism women with tubal infertility who are sensitized to the human hsp60 may have a decreased probability of successful outcome after undergoing in vitro fertilization and embryo transfer.

Immune regulation in the male genital tract.

Spermatozoa are not produced until puberty, long after the establishment of tolerance to self-antigens. Therefore, sperm-specific antigens are immunogenic in men. Most men, however, do not produce antibodies to their own gametes. Development of mechanisms to prevent or limit autoimmune responses to spermatozoa were essential for preservation of reproductive capacity. Tight junctions between adjacent Sertoli cells, as part of the blood-testis barrier, prevent sperm-immune cell contact. In some portions of the genital tract this barrier is thin or incomplete. Immune mechanisms have evolved to actively suppress the autoimmune response to spermatozoa within the genital tract. Unlike in the circulation where CD(4+) helper T lymphocytes predominate, CD(8+) suppressor/cytotoxic T lymphocytes are the most prominant T cells in the epididymis and vas deferens. In addition, spermatozoa suppress pro-inflammatory lymphocyte immune responses, possibly by inducing production of anti-inflammatory cytokines. Antisperm antibody production is induced in the male genital tract when a local infection or disruption in the genital tract physical barrier leads to an influx of CD(4+) T cells. In response to induction of a productive immune response, two additional mechanisms downregulate humoral immunity within the genital tract. T lymphocytes possessing the gammasigma form of the antigen receptor (gammasigma T cells) are concentrated in the male genital tract and in semen. These cells become activated and proliferate in men with evidence of sperm autoimmunity. Activated gammasigma T cells inhibit production of antibodies by activated B lymphocytes, thereby limiting antisperm antibody production. Heat shock proteins (hsps) are also present in semen in association with infection and antisperm antibody formation. Hsp gene transcription leads to inhibition of transcription of the genes coding for pro-inflammatory cytokines and, conversely, to activation of gammasigma T cells. Activated gammasigma T cells also promote hsp synthesis. The mechanisms to inhibit immunity to sperm may hinder effective immune elimination of microoganisms in the male genital tract.

Relationship between an asymptomatic male genital tract exposure to Chlamydia trachomatis and an autoimmune response to spermatozoa.

The relationship between a localized genital tract humoral immune response to Chlamydia trachomatis and the presence of antisperm antibodies on the surface of motile spermatozoa in the ejaculate was examined in 227 asymptomatic male partners of infertile couples with no history of exposure to C.trachomatis. Semen and serum samples were assayed for immunoglobulin (Ig) A and IgG antibodies to C.trachomatis by enzyme-linked immunosorbent assay employing a recombinant Chlamydia-specific lipopolysaccharide fragment (Medac, Hamburg, Germany), while motile spermatozoa were tested for bound autoantibodies by immunobead binding. Semen samples from 24.7 and 10.9% of the men were positive for IgA and IgG antibodies to C.trachomatis respectively. In comparison, antichlamydial IgA was less prevalent in sera (14.5%) than in semen (P = 0.01), while antichlamydial IgG was most prevalent (21.5%) in sera (P = 0.003). In 75.0% of the men with antichlamydial IgA in their semen, this antibody was undetectable in sera obtained at the time of semen collection. Conversely, 84.0% of the men with seminal antichlamydial IgG were also IgG seropositive. Antisperm IgG and/or IgA were detected on motile spermatozoa from 16.3% of the men; their occurrence was strongly correlated with the presence of antichlamydial IgA in semen (P < 0.0001). Weaker associations between antisperm antibodies and either seminal IgG antibodies to C.trachomatis (P = 0.01) or circulating IgA and IgG antichlamydial antibodies (P = 0.03) were also observed. Men with antichlamydial IgA in their semen had a lower median sperm count (82 versus 144 x 10(6)/ml) than those men without (P = 0.003); sperm morphology and motility were comparable in both groups. These data suggest that asymptomatic male genital tract exposure to C.trachomatis is a frequent event among this population and that the presence of a humoral immune response to this organism is correlated with the development of an autoimmune response to spermatozoa.

Characterization of cytosolic glucocorticoid receptor of fetal rat epiphyseal chondrocytes.

The cytosolic glucocorticoid receptor of 21st gestational day rat epiphyseal chondrocytes has been evaluated. The receptor, a single class of glucocorticoid binding component approached saturation, utilizing [3H]triamcinolone acetonide ([3H]TA) as the radiolabeled ligand, at approximately 1.8-2.0 x 10(-8) M. The dissociation constant (Kd) reflected high-affinity binding, equaling 4.0 +/- 1.43 x 10(-9) M (n = 7) for [3H]TA. The concentration of receptor estimated from Scatchard analysis was approximately 250 fmol/mg cytosolic protein and when calculated on a sites/cell basis equalled 5800 sites/cell. The relative binding affinities of steroid for receptor were found to be triamcinolone acetonide greater than corticosterone greater than hydrocortisone greater than progesterone greater than medroxyprogesterone acetate much greater than 17 alpha-hydroxyprogesterone much greater than testosterone greater than 17 beta-estradiol. Cytosolic preparations activated in vitro by warming (25 degrees C for 20 min) were shown to exhibit an increased affinity for DNA-cellulose. 46% of the total specifically bound activated ligand-receptor complex was bound to DNA-cellulose. Cytosol maintained at 0-4 degrees C in the presence of 10 mM molybdate or activated in vitro in the presence of molybdate, bound to DNA-cellulose at 8 and 10% respectively. DEAE-Sephadex elution profiles of the nonactivated receptor were indicative of a single binding moiety which eluted from the columns at 0.4 M KCl. Elution profiles of activated receptor were suggestive of an activation induced receptor lability. The 0.4 M KCl peak was diminished, while a concomitant increase in the 0.2 M KCl peak was only modestly discernible. Evaluation of endogenous proteolytic activity in chondrocyte cytosol using [methyl-14C]casein as substrate show a temperature-dependent proteolytic activity with a pH optimum of 5.9-6.65. The proteolytic activity was susceptible to heat inactivation and was inhibitable, by 20 mM EDTA. The sedimentation coefficient of the nonactivated receptor was 9.3s (n = 6) on sucrose density gradients and exhibited steroid specificity and a resistance to activation induced molecular alterations when incubated in the presence of 10 mM molybdate. Receptor activation in vitro, in the absence of molybdate induced an increased receptor susceptibility to proteolytic attack and/or enhanced ligand receptor dissociation as evidenced by a diminution of the 9.3s binding form without a concomitant increase in 5s or 3s receptor fragments.

Are ovarian steroids required for ovum maturation and fertilization? Effects of cyanoketone on the in vitro perfused rabbit ovary.

An isolated perfused rabbit ovary preparation was used to determine the effects of cyanoketone, a potent inhibitor of 3 beta-hydroxysteroid dehydrogenase, on ovulation, ovum maturation and fertilizability, and steroid production. In the first experiment, cyanoketone (10(-4) M) was added to the perfusate of one ovary. The contralateral control ovary was perfused with medium alone. Thirty minutes after the onset of perfusion, hCG (50 IU) was added to the perfusate of both ovaries. The ovulatory efficiency of ovaries treated with cyanoketone plus hCG (82.3 +/- 4.6%) was similar to that of ovaries treated with hCG alone (84.8 +/- 4.4%). No difference was observed in the degree of ovum maturity or degeneration between control and cyanoketone-treated ovaries. Progesterone and estradiol production were significantly reduced by cyanoketone treatment; concentrations in the perfusate of ovaries treated with cyanoketone were 9.7% and 8.0% of the control values, respectively, 2 h after exposure to hCG. The concentration of 17-hydroxypregnenolone was not affected by cyanoketone treatment. Exposure to cyanoketone resulted in a significant (P less than 0.005) reduction in the fertilizability of ova ovulated and fertilized in vitro. In the second experiment, the percentage of ova that showed evidence of normal fertilization was significantly (P less than 0.025) increased in ovaries perfused with cyanoketone plus estradiol (64.5%) compared to that in ovaries perfused with cyanoketone alone (32.4%). In the third experiment, the addition of progesterone to the perfusate did not affect fertilizability of ovulated ova in ovaries perfused with cyanoketone plus estradiol. These results suggest that the presence of estradiol in the ovarian steroid environment may be essential for fertilizability of ova, but not for the processes of ovulation or meiotic maturation.

The effect of ovarian steroidogenesis on ovulation and fertilizability in the in vitro perfused rabbit ovary.

The effects of aminoglutethimide phosphate (AGP) on ovulation, ovum maturation, fertilizability, and steroid production were studied with the use of an isolated perfused rabbit ovary preparation. AGP (10(-3) or 10(-4) M) was added to the perfusate of one ovary. The contralateral control ovary was perfused in medium alone. Thirty minutes later human chorionic gonadotropin (hCG) (50 IU) was added to the perfusate of all ovaries. No difference was observed in time of ovulation or ovulatory efficiency between controls and AGP-treated ovaries. The degree of ovum maturity and degeneration was also comparable in the two groups. Progesterone and estradiol production were significantly reduced by AGP treatment. A second experiment examined fertilizability of ova ovulated in vitro after perfusion with 10(-3) M AGP. AGP significantly reduced the rate of normal fertilization as observed 12 h after insemination. The percentage of inseminated ova with evidence of degeneration was greater in ova from AGP-treated ovaries than in those from controls, however, this difference was not significant. The study indicates that AGP affects neither hCG-induced ovulation nor meiotic resumption; however, fertilizability of ova from ovaries treated with AGP is impaired. These data suggest that the intrafollicular steroid environment may participate in cytoplasmic maturation of ovulated ova.

An epidemic of precocious development in Puerto Rican children.

An alarming incidence of premature sexual development has been reported in Puerto Rico during the last 7 years. A significant increment of premature thelarche, premature pubarche, prepubertal breast enlargement in boys, and precocious pseudopuberty in girls has been observed throughout the island. Several food specimens analyzed by chromatography and cytosol receptor assay revealed significant levels of estradiol equivalent in some meat samples. We suspect that the early sexual development is caused by exogenous estrogen contamination in the food ingested by the children and by their mothers.

Antibodies to the neutral glycolipid asialo ganglio-N-tetraosylceramide: association with gynecologic cancers.

As part of our efforts to define subpopulations at increased risk for gynecologic malignancies, sera from 145 women were obtained prior to diagnosis and analyzed for antibody to asialo ganglio-N-tetraosylceramide. This neutral glycolipid is present on the surface of thymocytes and natural killer cells, and asialo ganglio-N-tetraosylceramide antibody has been shown in animals to block natural killer cell activity and promote tumor cell proliferation. With the use of an enzyme-linked immunosorbent assay and with a value of 2 SD above the mean for healthy women designated as the boundary for a positive response, antibody to asialo ganglio-N-tetraosylceramide was detected in only one of 30 (3%) healthy women, none of 16 pregnant women, none of 18 women with benign masses, and two of 24 (8%) women with microbial infections. All of the above samples that contained antibodies were barely over the 2 SD limit. In marked contrast, 19 of 35 (54%) women with gynecologic malignancies had asialo ganglio-N-tetraosylceramide antibodies, with positive values ranging to greater than 10 SD above the control mean. Asialo ganglio-N-tetraosylceramide antibody was found in six of eight (75%) patients with cervical cancer, five of eight (63%) with endometrial cancer, and seven of 15 (47%) with ovarian cancer. Of the eight patients with Stage I gynecologic cancer at any site, five (62%) had asialo ganglio-N-tetraosylceramide antibodies. Four of 22 (18%) women with Hodgkin's disease also had antibodies, with values just exceeding 2 SD above control levels. The presence of these antibodies may contribute to an impaired immune surveillance system in these women and so increase their susceptibility to malignancy.

Detection and characterization of immune complexes in the circulation of infertile women.

The incidence of circulating immune complexes (CICs) was evaluated in sera from 39 female partners of infertile marriages and from 38 fertile women. Fifteen (38%) of the infertile women had CICs, as determined by the Raji cell assay, in levels ranging from 300 to 8000 micrograms/ml; whereas only 1 (3%) of the fertile women displayed CICs (P less than 0.001). Analysis of the CICs from nine of the women following polyethylene glycol precipitation and acid dissociation revealed that four contained C1q and three contained an antigen reactive with rabbit antibody to human spermatozoa. These latter three women all lacked free sperm antibody, as determined by enzyme-linked immunosorbent assay and agglutination. Thus, CICs are not uncommon as a manifestation of infertility in females. Their presence may lead to an underestimation of sperm antibody levels and may be indicative of underlying infection or autoimmunity.

Analysis of sera from ovarian cancer patients for immune complexes.

In contrast to other malignancies, circulating immune complexes (CICS) are usually not detected by conventional assays in the sera of ovarian cancer patients. However, a polyethylene glycol (PEG) precipitation assay has been reported to detect putative CICS in ovarian cancer. To determine if CICS were indeed present, we analyzed sera from 12 women with ovarian cancer. All were negative for CICS by the Raji cell assay; 5 (42%) were positive by the PEG assay. However, the PEG precipitate did not possess characteristics of immune complexes. IgG in sera or in the precipitate sedimented in sucrose gradients solely at the same rate as 7S monomeric IgG. In addition, the precipitates were not able to activate the complement system and the four IgG subclasses were present in the same relative concentration as that found in normal serum. The results suggest that it is probably a misnomer to label the material detected in ovarian cancer sera by the PEG precipitation assay as CICS. Instead a non-immune interaction of IgG with other components, possibly membrane fragments, is probably being measured.