A new proline aminopeptidase assay for diagnosis of bacterial vaginosis.

Bacterial vaginosis is one of the most common occurring vaginal conditions among women of reproductive age. A rapid and reliable laboratory test for diagnosis of bacterial vaginosis would be helpful in the clinical detection of this disease. Elevated proline aminopeptidase activity has been identified as a reliable marker enzyme for bacterial vaginosis. A proline aminopeptidase assay has been shown to predict accurately women with a clinical diagnosis of bacterial vaginosis. However, this assay has significant practical disadvantages, the most notable of which is the production of a carcinogenic end product, alpha-naphthylamine. We have developed a modified assay for this bacterial vaginosis marker enzyme with L-proline p-nitroanilide, a substrate that does not yield a carcinogenic end-product. The new proline aminopeptidase assay is a one-step test that is analyzed colorimetrically with microsomal leucine aminopeptidase used as a standard enzyme (linear from 3 to 125 mU per well). We have determined the activity of proline aminopeptidase in vaginal wet preparations from 57 patients with both assay methods. In addition, vaginal smears were examined with Gram's stain and analyzed for bacterial vaginosis with the Spiegel method. When compared with the Spiegel method, the two proline aminopeptidase assay methods were similar with respect to assay sensitivity (93%), specificity (91% to 93%), and the predictive value of a positive result (78% to 82%) or a negative result (97% to 98%). Vaginal wash samples also were assessed for proline aminopeptidase activity. Values for samples identified as bacterial vaginosis positive were significantly different (p less than 0.0001) from those that were negative according to the Spiegel analysis of Gram's stain: negative results, 66 +/- 41 mU/ml; positive results, 704 +/- 145 mU/ml. These findings indicate that this improved proline aminopeptidase assay will offer a rapid, sensitive, and objective laboratory method for the diagnosis of bacterial vaginosis.

Antibiotic inhibition of bacterially induced fetal membrane weakening.

A fetal membrane model was used to evaluate in vitro the efficacy of two antibiotics, erythromycin and clindamycin, in preventing bacterial protease-induced weakening of amniochorion. Standardized inocula of protease-producing bacteria (10(9) colony-forming units [cfu]/mL Staphylococcus aureus, incubated at 37C for 20 hours) reliably reduced fetal membrane structural integrity as reflected by bursting tension and work to rupture. Supraminimal inhibitory concentrations (supra-MICs) (erythromycin 0.23 microgram/mL; clindamycin 0.56 microgram/mL) and subminimal inhibitory concentrations (sub-MICs) (erythromycin 0.13 microgram/mL; clindamycin 0.06 microgram/mL) of both antibiotics prevented fetal membrane impairment due to test bacteria. Supra-MICs of both antibiotics prevented bacterial cell growth and release of protease. Sub-MICs of both antibiotics allowed bacterial cell growth of test microorganisms but inhibited protease release and subsequent fetal membrane damage. These findings suggest that inhibitory and even subinhibitory doses of antibiotics such as erythromycin and clindamycin may be effective in reducing the occurrence of premature rupture of membranes and subsequent preterm birth mediated by susceptible microorganisms.

Bacteria and inflammatory cells reduce chorioamniotic membrane integrity and tensile strength.

Bacteria and the inflammatory response they engender are implicated in the pathophysiology of premature rupture of fetal membranes and preterm birth. We tested the hypothesis that bacteria and polymorphonuclear neutrophils may have both separate and combined effects in weakening the amniochorionic membrane, and thus predispose to premature rupture of membranes. We examined how three parameters of membrane integrity (bursting tension, work to rupture, and elasticity) were affected by standardized preparations (10(9) cfu/mL) of group B streptococci or Staphylococcus aureus in the presence or absence of purified human neutrophils (32 x 10(6)/mL). In addition, effects of purified human neutrophil elastase were evaluated. Exposure to either group B streptococcus or S aureus decreased membrane strength, elasticity, and work to rupture. Only activated neutrophils had significant effects on membrane strength. Membrane exposure to S aureus plus neutrophils led to an additive weakening of fetal membranes. On the other hand, group B streptococci did not interact with neutrophils to yield a significant further weakening of the membranes. Elastase (150 U/mL) also weakened the membrane. The results were correlated with measures of protease (Azocoll and ninhydrin assays) and peroxidase (dimethoxybenzidine) activity. Our findings support the concept that human neutrophils and their constituent enzymes may act in concert with bacteria and their protease(s) in weakening amniochorion, and may possibly predispose to premature rupture of membranes in some women. These observations require clinical correlations.

Evidence that serotonin is involved in the sexually dimorphic development of the preoptic area in the rat brain.

To investigate the possibility that serotonin plays a role in the sexually dimorphic development of a nucleus in the medial preoptic area of the rat brain, p-chlorophenylalanine, an inhibitor of serotonin biosynthesis, was administered to pregnant dams from day 8 of gestation until parturition. This treatment did not alter plasma steroid levels but increased the volume of the sexually dimorphic nucleus in female neonates to that of control males. Thus, serotonin is implicated as a neurochemical which may be involved in the sexually dimorphic development of the preoptic area.

Auditory and hormonal stimulation interact to produce neural growth in adult canaries.

Gonadal hormones can produce striking behavioral and neural plasticity in adult organisms. For example, systemic administration of testosterone to adult female canaries induces the development of male-typical song behavior and results in a striking increase in the size of brain nuclei that are known to be involved with song control. The mechanism whereby androgens produce such neural plasticity is not known, although it has seemed likely that growth-promoting effects of androgens are due to a direct induction of protein synthesis in cells containing hormone receptors (following activation of specific genes by the hormone-receptor complex). In this experiment we have examined the trophic effect of testosterone in the song-control nucleus HVc (caudal nucleus of the ventral hyperstriatum), which has been shown to contain androgen-concentrating cells as well as neurons that are especially responsive to conspecific song. We report here that testosterone administration increases the volume of HVc in hearing adult female canaries only; testosterone-induced growth of HVc is greatly attenuated in birds that are deprived of auditory stimulation via deafening. Thus, testosterone treatment alone is not a sufficient stimulus for neural growth in HVc. This result suggests that testosterone does not stimulate growth solely via a direct action on hormone receptors in HVc, but rather that testosterone and sensory stimulation can act synergistically to produce structural plasticity in the adult brain.

Differential effects of the perinatal steroid environment on three sexually dimorphic parameters of the rat brain.

Gonadectomy of male rats was performed at 0, 6-7 (6h), 12-13 (12h), or 24 h postnatally in order to examine the influence of testosterone exposure on sexual differentiation of the brain. The indices examined were: the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) titers following estradiol benzoate (EB) and progesterone (P) administration. Control animals were sham-operated at 0 h and gonadectomized at 29 days of age (sham). A decrease in the percentage of males with elevated plasma LH levels following P was found with increasing delay before gonadectomy. Significant (P less than 0.001) differences existed in the amplitude of plasma LH titers 5 h following P administration between sham, 0 h, and 6 h groups. Follicle-stimulating hormone was also elevated in all neonatally gonadectomized male groups following P administration, but there was no difference between the groups. Volume of the SDN-POA was significantly (P less than 0.001) smaller in all gonadectomized males when compared to that of sham-operated males, but no differences existed between males gonadectomized at the different hours postpartum. In female rats gonadectomized at 0 h (F0h), LH levels were elevated 5 h following P, but only to a magnitude of 36% of that of sham-operated controls (P less than 0.001). Volume of the SDN-POA of the F0h group was significantly reduced (P less than 0.05) when compared to that of sham females. Thus, in males, the presence of the tests prenatally may be responsible for the initiation of masculinization of LH release mechanisms and the SDN-POA, but both require further androgen exposure for their completion. In addition, the LH and FSH regulating systems show a differential sensitivity to the steroid hormone environment during development that shapes the animal's response to steroid as an adult.

A receptive period for estradiol-induced luteolysis in the rhesus monkey.

This study was performed to test the hypothesis that responsiveness to the luteolytic action of estradiol is acquired as the luteal phase of the menstrual cycle progresses. The luteolytic effect of fixed estradiol increment (270 +/- 12 pg/ml serum) was assessed at different stages of luteal function in rhesus monkeys. A 4-day elevation in estradiol early in the luteal phase (days 2--6 after the LH peak) caused a decrease in the concentration of serum progesterone but did not shorten luteal life span. In contrast, when provided during the midluteal phase (days 6--10), the same 4-day estradiol increment promptly induced premature luteolysis. Furthermore, during sustained exposure to the extradiol increment from days 2--10, signs of premature luteolysis were not evident until day 7 after the LH peak. Thus, the effects of estradiol early in the luteal phase do not alter luteal life span; it is the effects after day 6 that precipitate luteolysis. These observations support the existence of a receptive period for the luteolytic action of estradiol in the rhesus monkey.