Comparison of antibody- and cell-mediated immune responses after intramuscular hepatitis C immunizations of BALB/c mice.

Current treatments for hepatitis C infection have limited efficacy, and there is no vaccine available. The goal of this study was to compare the immune response to several immunization combinations against hepatitis C virus (HCV). Six groups of mice were immunized at weeks 0, 4, and 8 with different combinations of a candidate HCV vaccine consisting of 100 microg recombinant HCV core/E1/E2 (rHCV) DNA plasmid and/or 25 microg rHCV polyprotein and 50 microL Montanide ISA- 51. Four weeks after the last injection, all groups of mice were sacrificed and blood samples and spleens were collected for measuring the levels of specific HCV antibodies (total IgG, IgG1, and IgG2a). Cell proliferation and intracellular interferon-gamma were also measured. Among the groups of immunized mice, only the mice immunized with rHCV DNA plasmid, rHCV polyprotein, and montanide (group D) and mice immunized with rHCV polyprotein and montanide (group F) demonstrated a significant increase in the total IgG titer after immunization. IgG1 was the predominant antibody detected in both groups D and F. No IgG2a was detected in any of the groups. Proliferation assays demonstrated that splenocytes from group D and group C (rHCV DNA primed/rHCV polyprotein boost) developed significant anti-HCV proliferative responses. The combination of an rHCV DNA plasmid, rHCV polyprotein, and montanide induced a high antibody titer with a predominance of IgG1 antibodies and recognized the major neutralization epitopes in HVR1. In contrast, group C did not show an increase in anti-HCV antibodies, but did show a proliferative response.

Migration of the fungal pathogen Candida albicans across endothelial monolayers.

Migration of the fungal pathogen Candida albicans across the endothelial cell layer is considered a prerequisite for the invasion of multiple organs occurring in systemic candidiasis. We developed an experimental system in which C. albicans migrates from a luminal compartment across a monolayer of bovine aortic endothelial cells on a porous filter support to an abluminal compartment. In this system, a C. albicans wild-type strain (ATCC 10261) traverses the endothelial monolayer in a time-, glucose-, and cell concentration-dependent manner. A mutant derivative unable to grow and form hyphae (SGY-243) migrates at a reduced rate. Concomitant to transendothelial migration, the permeability of the endothelial monolayer for dextran diffusion markers is significantly increased. This increase in transendothelial exchange occurs before fungal cells are detectable in the abluminal compartment and is time, glucose, and cell concentration dependent. A mutant strain (hOG301) unable to interact with endothelial cells does not alter endothelial permeability. Thus, transendothelial migration of C. albicans is able to damage the barrier function of an endothelial monolayer. Our experimental system, which reflects key stages of transendothelial migration of C. albicans including adherence and passage across endothelial cells and the extracellular matrix, may be a useful model for comparisons of transendothelial migration characteristics of Candida strains.

The effect of pituitary suppression and the women's age on embryo viability and uterine receptivity.

OBJECTIVE: To examine the effect of pituitary suppression and the women's age on embryo viability and uterine receptivity. DESIGN: Retrospective analysis of 394 embryo transfers (ET) after in vitro fertilization (IVF). SETTING: Community hospital IVF program from 1986 to 1990. PATIENTS: Three groups were studied: women less than 40 years with pituitary suppression (group 1) and without pituitary suppression (group 2); women 40 years of age and older with pituitary suppression (group 3). INTERVENTIONS: Pituitary suppression was achieved in groups 1 and 3 with daily leuprolide acetate starting in the luteal phase; human menopausal gonadotropin and progesterone were given intramuscularly. MAIN OUTCOME MEASURES: Ongoing and multiple ongoing pregnancy rates (PRs) were compared in the three groups. A mathematical model of implantation was used to estimate embryo viability and uterine receptivity. RESULTS: Ongoing and multiple ongoing PRs per ET in group 1 (28.6% and 12.3%) were significantly higher than the corresponding rates in group 2 (16.9% and 2.4%) and in group 3 (16.9% and 3.4%). Implantation analysis revealed higher embryo viability without change in uterine receptivity with pituitary suppression (group 1 versus 2). Decrease in both embryo viability and uterine receptivity was noted in women greater than 40 (group 1 versus 3). CONCLUSIONS: (1) Pituitary suppression improved implantation outcome by increasing embryo viability with no change in uterine receptivity and was associated with a high multiple PR in women less than 40; (2) in women greater than 40 both embryo viability and, to a lesser extent, uterine receptivity were decreased; (3) transfer of a larger number of embryos in older patients may improve IVF outcome without excessive risk of multiple pregnancy.

Improved pregnancy outcome with the addition of leuprolide acetate to gonadotropins for in vitro fertilization.

In vitro fertilization treatment outcomes were compared prospectively in unselected patients with and without the addition of leuprolide acetate to gonadotropins for ovarian hyperstimulation. While the leuprolide patients required greater quantities of exogenous gonadotropins to achieve ovarian stimulation, significant improvements in the fertilization, implantation, and spontaneous abortion rates were observed compared with patients not receiving leuprolide. The ongoing pregnancy ("take home baby") rates per aspiration with and without leuprolide were 29% and 12%, respectively. Furthermore, the multiple pregnancy rate was markedly increased in the leuprolide group (44% versus 8%), suggesting that the improved pregnancy outcome with pituitary suppression was due primarily to higher oocyte and embryo quality.

Isofluorane inhibits early mouse embryo development in vitro.

In vitro development of 2-cell mouse embryos to the blastocyst stage was used to assess the toxicity of isofluorane, nitrous oxide, fentanyl, and meperidine. Isofluorane at concentrations similar to those employed during human oocyte recovery for IVF significantly inhibited mouse embryo development. Since the other agents were without effect, balanced anesthesia with nitrous oxide and narcotics may be preferable to isofluorane for IVF and GIFT.

Optimization of hydrogen-ion concentration during aspiration of oocytes and culture and transfer of embryos.

Rapid and marked shifts in pH can have deleterious effects on oocytes and embryos. Such shifts are most likely to occur during laparoscopy and examination of gametes and embryos. We studied the rate of pH change of follicular fluid and bicarbonate- and phosphate-based media during exposure to 100% CO2 with a constant surface area to volume ratio. All solutions tested developed a pH below the physiological range of 7.30-7.50 within 2 min of exposure to 100% CO2. We also examined the rate of increase in pH due to CO2 loss from media in room air within standard organ culture dishes and tubes. After exposure to ambient conditions for 2 min, the pH of modified Ham's F-10 medium in organ culture dishes rose above the physiological range. We have used a needle laparoscope to confirm intraperitoneal placement prior to the insufflation of a 5% CO2 gas mixture for laparoscopic oocyte retrieval. We have carried out all examinations of oocytes and embryos within a pediatric isolette equipped with an automatic CO2 controller to maintain a 5% CO2 environment. We have transferred embryos in 15% fetal cord serum to avoid the alkaline pH associated with a high concentration of equilibrated heat-inactivated preovulatory serum. These simple techniques can optimize the hydrogen-ion concentration of media during the entire process of in vitro fertilization and embryo transfer.

Relation of circulating estradiol and progesterone to gonadotropin secretion and estrous cyclicity in aging female rats.

The progressive cessation of regular ovulatory function in aging female rats is preceded by a significant decrease in the magnitude of the proestrous LH surge during regular estrous cycles. However, our recent study has demonstrated that normal LH secretion and regular estrous cycles can be maintained for an extended period of time in aging females housed with fertile males and allowed to undergo repeated pregnancies. Since progesterone (P) secretion is persistently increased in pregnant rats, the present study examined whether repeated increases in circulating progesterone accounted for these results. Starting at 8 months of age and continuing to 13 months, multiparous rats were grouped and treated as follows: controls: females were housed five per cage; mated: five females were housed with one fertile male and allowed to undergo repeated pregnancies; and P-implanted: females were housed five per cage and implanted sc with Silastic capsules containing P for 3 of every 4 weeks. During the 4.5 months of study, serum concentrations of estradiol (E2) in the P-implanted rats remained between 13 and 27 pg/ml, similar to levels in pregnant females (8-26 pg/ml) of the mated group. These E2 values were less than the preovulatory increase in serum E2 on proestrus (mean +/- SE, 56 +/- 10 pg/ml) in cyclic control females. In contrast, serum P values were persistently elevated in both the pregnant and the P-implanted rats, although the values in the latter (27-55 ng/ml) were about one third to one half of those in the former group (117-125 ng/ ml). All treatments were stopped at 13 months of age, and estrous cycle patterns were determined thereafter. Between 13 and 17 months of age, the percentages of regularly cycling rats were significantly (P less than 0.01) greater in the mated group (50%, 36%, and 15% at 13, 15, and 17 months, respectively) than in the control group (23%, 20%, and 9%, respectively). During this same period, 50% of the females from the P-implanted group continued to display regular cycles. By the age of 11 months, 8 of 21 untreated retired breeder females exhibited attenuated LH surges on proestrus and subsequently ceased to display regular estrous cycles within 2 months, whereas the other 13 rats showed normal LH and FSH surges and continued to maintain regular cycles. In contrast to these, aged female rats from the previously mated (15-month-old) and the P-implanted (19-month-old) groups exhibited normal profiles of proestrous LH and FSH surges during regular estrous cycles.(ABSTRACT TRUNCATED AT 400 WORDS)

Proestrous hormonal changes preceding the onset of ovulatory failure in lightly androgenized female rats.

Proestrous hormonal profiles were characterized in lightly androgenized female rats prior to the onset of the delayed anovulatory syndrome (DAS). In these females, ovulatory failure and persistent vaginal estrus (PVE) occur at a very early age. Female Sprague-Dawley rats were injected with 10 micrograms testosterone propionate (TP) on postnatal Day 5. Control rats were untreated. All animals were weaned at 21 days of age, and following the onset of puberty, estrous cyclicity was monitored by vaginal smear. Rats showing regular 4-day cycles were used. Between 50-70 days of age, intra-atrial cannulae were implanted on a morning of proestrus (0700-0900 h) and blood was sampled at 2-h intervals from 1000 to 2000 h. Additional samples were taken at 0.5-h intervals from 1600 to 1800 h. Plasma was assayed for luteinizing hormone (LH), follicle-stimulating hormone (FSH) and progesterone (P) by radioimmunoassay (RIA). All animals were monitored for the onset of PVE or other alterations in estrous cyclicity. Females treated neonatally with TP that subsequently showed PVE by 150 days of age (PRE DAS) displayed a reduced peak amplitude (P less than 0.01) and delay in onset (1600 vs. 1400 h) of LH but not FSH secretion, when compared to controls. Females treated neonatally with TP that did not enter PVE by 150 days of age (No DAS) also showed a delayed rise in LH when compared to controls. However, the amplitude of LH secretion was not different from controls or PRE DAS females.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in the luteinizing hormone secretory pattern in peripubertal female rhesus monkeys: comparisons between gonadally intact and ovariectomized animals.

Developmental changes in LH release patterns were observed longitudinally in female rhesus monkeys at 10-65 months of age. The average ages of menarche and first ovulation in this experiment (n = 14) were 31.1 +/- 2.6 and 47.0 +/- 2.6 months (mean +/- SE), respectively. To assess the ovarian influence on developmental changes in LH, data were simultaneously obtained from neonatally ovariectomized animals at similar ages. The estimation of circulating LH was made with RIA as well as biological assay. During the prepubertal period (10-20 months of age), basal LH was very low, and there was no circadian fluctuation of LH in gonadally intact monkeys. During the early pubertal stage (20-30 months of age), before menarche, basal LH levels started to increase, and a circadian LH rhythm (nocturnal increases) appeared. At the midpubertal stage (30-50 months of age), a period between menarche and first ovulation, basal LH levels further increased, and the circadian LH rhythm was maximal. At the late pubertal stage (50-60 months of age), a period after the first ovulation during which the animals were not able to reproduce fully as adults, basal LH declined, and the circadian rhythm diminished. Similar but more exaggerated developmental changes in basal LH and the circadian fluctuation of LH were observed in females ovariectomized neonatally. Basal LH levels at 10-20 months were as low as those in intact animals with no circadian rhythm present. During the early pubertal period, a circadian fluctuation appeared at the time when a slight increase in the basal LH level occurred. Furthermore, the amplitude of circadian fluctuation (the difference between morning and evening LH values) increased linearly with the increase in basal LH during the midpubertal stage. These LH parameters in ovariectomized animals reached their peaks at 40-44 months, an age before the first ovulation in intact animals. As basal LH levels declined during the late pubertal stage to postpubertal stage, circadian fluctuation disappeared. The results suggest that the increase in LH output and concomitant circadian fluctuations occur in close association with the pubertal process, and this change in LH release is not dependent on the presence of the ovary. Therefore, we suggest that alteration of the LHRH release pattern during maturation, as reflected by LH release, rather than resetting of the gonadostat, is the key factor involved in the mechanism of the onset of puberty.

Posterior hypothalamic lesions advance the onset of puberty in the female rhesus monkey.

The effects of experimental lesions in the posterior hypothalamus and the anterior hypothalamus on menarche and first ovulation were examined in nonhuman primates. With the aid of x-ray ventriculography, bilateral lesions were made by passing a radiofrequency current through a thermister electrode in the posterior hypothalamus (n = 7) or the anterior hypothalamus (n = 6) of female rhesus monkeys at 18 months of age. Four animals that received sham lesions as well as four normal females of a similar age served as controls. All animals were caged individually and examined daily for vaginal bleeding and sex skin color change. Developmental changes in gonadotropins, ovarian steroids, body weight, and nipple size were monitored throughout the experiments. The time of first ovulation was determined by laparoscopic observation of the newly formed corpus luteum and by the level of circulating progesterone. Histological examination confirmed that the bilateral lesions in the hypothalamus were approximately 2-3 mm in diameter and overlapped midline. Primary sites of posterior hypothalamic lesions included the premamillary area and the posterior nucleus, while the infundibular nucleus and the median eminence were entirely spared. The posterior lesions encroached upon the mamillary nuclei caudally in most cases and upon the ventromedial nucleus rostrally in some cases. Primary sites of anterior hypothalamic lesions included the medial preoptic area, the periventricular preoptic nucleus, and the anterior hypothalamic nucleus. Partial lesions of the diagonal bundle of Broca, the medial preoptic nucleus, and the paraventricular nucleus were also detected. Posterior hypothalamic lesions advanced the ages at menarche (22.2 +/- 1.3 months; P less than 0.001) and first ovulation (40.7 +/- 2.7 months; P less than 0.05) compared to those of control animals (menarche, 30.3 +/- 3.1; first ovulation, 51.2 +/- 3.3 months). The body weight at menarche of these lesioned animals (2.62 +/- 0.11 kg) was smaller (P less than 0.05) than that of controls (3.14 +/- 0.20 kg), but the body weight at first ovulation of lesioned animals (4.36 +/- 0.28 kg) was not different from that of controls (4.57 +/- 0.13 kg). Hormonal and physical changes during maturation, i.e. an increase in circulating estradiol and growth in nipple size before menarche and first ovulation, occurred earlier in the lesioned animals and the growth spurt before first ovulation not only began earlier but also attained mature levels several months earlier than that in control animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Prepubertal treatment with estrogen or testosterone precipitates the loss of regular estrous cyclicity and normal gonadotropin secretion in adult female rats.

To examine the effects of prepubertal steroid environment on subsequent estrous cyclicity and gonadotropin secretion, Silastic implants containing 25, 50 or 100% 17 beta-estradiol (E2;n=34), 50% diethylstilbestrol (DES; n=16) or 50% testosterone (T; n=17) were placed into female rats at 12 days of age and removed on the day of vaginal opening (18-24 days of age). At 80 days of age, the percentages of regularly cycling females in the E2-(three groups combined), DES- and T-implanted groups were 59%, 0% and 59%, respectively. By 110 days of age, the percentages were reduced to 24%, 0% and 0%, and at 140 days of age 6%, 0% and 0%, respectively. Many of these females displayed irregular estrous cycles followed by a persistent estrous (PE) state. By contrast, 89% of the control females (blank implants or no implant) maintained regular cycles up to 140 days of age. At 150 days of age, an i.p. injection of gonadotropin-releasing hormone (GnRH; 100 ng/100 g BW) markedly increased serum luteinizing hormone (LH), but not follicle-stimulating hormone (FSH), in intact PE females treated prepubertally with E2 implants. After the test with GnRH, PE rats were ovariectomized (OVX). Thirty days after OVX, similar GnRH administration significantly increased serum levels of both LH and FSH, but these responses were significantly (P less than 0.01) reduced when compared with those in OVX controls. Progesterone administration to estradiol benzoate-primed, acutely (3 days) OVX, or long-term (43 days) OVX-PE females did not increase LH or FSH release. These results indicate that exposure to exogenous estrogen or T prior to puberty precipitates the decline in estrous cyclicity associated with the loss of gonadotropin surge response, presumably due to an alteration in hypothalamic GnRH release.

Developmental changes in luteinizing hormone secretion in the female guinea pig. I. Effects of ovariectomy, estrogen, and luteinizing hormone-releasing hormone.

Negative feedback regulation of LH release was studied by ovariectomy and estrogen treatment in female guinea pigs of four different ages (days 10, 30, 50, and 150). The average age at first ovulation in guinea pigs in our colony was 60.1 +/- 2.2 days (n = 33). Bilateral ovariectomy (OVX) on day 30, 50, or 150 of age resulted in a sharp increase in serum LH concentrations within 3 days, and this rise continued for 9 days, when LH values reached a plateau. In contrast, when OVX was performed on day 10 of age, only a slight elevation in LH levels was observed during the first 16 days after surgery. Twenty days after OVX (day 30 of age), LH concentrations started to increase rapidly and reached the maximum value on day 38 of age; this latter response was similar to that observed immediately after OVX in animals in the three older age groups. Serum LH concentrations after LHRH administration (2 micrograms/kg BW) were similar in ovariectomized females on days 23 and 43 of age, suggesting that the postcastration increase in LH secretion after day 26 of age was not due to a change in pituitary sensitivity. Daily injections of estradiol benzoate at doses of 0.5 and 2.0 micrograms/kg BW from 15-20 days after OVX suppressed LH levels in all four age groups. The present findings indicate that an increase in the capacity for LH release occurs around day 30 in the female guinea pig. Since this increased LH release is not due to an apparent change in pituitary sensitivity to LHRH, the endogenous release of LHRH may be inhibited before day 30 of age. These results suggest that an alteration in the regulation of LH release by the brain may occur before the onset of puberty in female guinea pigs.

Developmental changes in luteinizing hormone secretion in the female guinea pig. II. Positive feedback effects of ovarian steroids.

Developmental changes in the positive feedback effects of ovarian steroids on LH release in female guinea pigs were studied. Administration of estradiol benzoate (EB; 10 micrograms) to intact females on day 30 of age (prepubertal) elicited a LH surge in only 1 of 14 instances. However, by day 46 of age (peripubertal), EB induced a sharp rise in serum LH concentrations in 9 of 12 females. The magnitude and timing of the LH surge in these peripubertal animals were similar to those in cycling adults given EB on day 13 of the estrous cycle. Progesterone (P; 1.0 mg) administration to intact females 30 h after a priming dose (1.5 microgram) of EB was also effective in eliciting a LH surge on day 46, but not on day 30, of age. The magnitude of the P-induced LH surge was significantly greater (P less than 0.001) than that of the EB-induced LH surge on day 46 of age. Pentobarbital anesthesia delayed, but did not prevent, the EB-induced LH surge in peripubertal females, while it completely abolished the P-induced LH surge. These results suggest that ovarian steroids can stimulate LH release in the immature female guinea pig, but not until an age approaching the normal time of the first ovulation. Those observations together with results presented in a preceding paper suggest that establishment of the positive feedback system between days 30 and 46 of age is associated with an increased capacity of the hypothalamic-pituitary axis to stimulate LH release; such developmental changes may represent the major events leading to the onset of puberty in the female guinea pig.

The effects of naloxone on hot flashes and gonadotropin secretion in postmenopausal women.

Hot flashes have a close temporal relationship with the initiation of LH pulses, suggesting that factors stimulating gonadotropin release are involved in the mechanism of this disturbance. It has been reported that the opiate antagonist naloxone acutely blocked subjective hot flashes, a seemingly paradoxical effect, since the use of this agent in premenopausal women increases the magnitude and frequency of LH pulses. We, therefore, studied the effects of naloxone in 16 postmenopausal women with frequent hot flashes using continuous recordings of finger temperature and skin resistance as objective indices of flushing and perspiration, respectively. After baseline recordings, the subjects were randomized into equal groups, and the recordings were repeated during 8-h infusion of either saline or naloxone (22 micrograms/min). Serum gonadotropin levels were measured at 15-min intervals before and during the last 4 h of the infusion. Naloxone did not change the rate of objectively measured hot flashes, mean serum LH or FSH levels, or the frequencies or amplitudes of gonadotropin pulses. These data suggest that there is a very low input of endogenous opiates on gonadotropin secretion in postmenopausal women and that opioid peptides do not play a role in the initiation of the postmenopausal hot flash.

Alterations in ovarian steroid and gonadotrophin secretion preceding the cessation of regular oestrous cycles in ageing female rats.

To determine whether discernible alterations in neuroendocrine and/or ovarian function precede the loss of regular oestrous cycles in ageing female rats, the present study examined the pattern of gonadotrophin secretion near the time of ovulation and the pattern of ovarian steroid secretion in the early morning of pro-oestrus in middle-aged (10-12 months old) females displaying regular oestrous cycles and compared these with young (4 months old) animals. In addition, the subsequent reproductive patterns in these animals were observed and correlations between the changes in hormonal profiles and the decline in regular reproductive cyclicity were established. In middle-aged females which subsequently ceased to display regular oestrous cycles (middle-aged non-regular; M-NR) within 1-2 months, the pro-oestrous surge of LH was significantly reduced in magnitude. There was no difference in the LH surge between young females and middle-aged animals which maintained regular oestrous cycles (middle-aged regular; M-R) for at least 2 months. There also was no difference in the magnitude of the pro-oestrous FSH surge or in the secondary rise in FSH in the early morning of oestrus among young, M-R and M-NR females. In a separate group of middle-aged females which subsequently became M-NR, serum concentrations of both oestradiol and testosterone in the early morning of pro-oestrus were markedly raised over those in the young and M-R groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotropin secretion during prolonged hyperprolactinemia: basal secretion and the stimulatory feedback effect of estrogen.

Inoculation of cyclic female rats with the prolactin (Prl)/growth hormone-secreting pituitary tumor, MtT.W15, resulted in a cessation of estrous cyclicity within 5--10 days. Associated with this acyclicity was a persistently low serum concentration of estradiol and marked increases in both circulating Prl and progesterone. At Day 26 of acyclicity, basal serum luteinizing hormone (LH) values measured in samples taken every 20 min from 0900--1100 h were significantly reduced when compared to cyclic, nontumor animals on diestrus Day 2. There was no difference in basal follicle-stimulating hormone (FSH) concentrations. In a separate group of acyclic, tumor-bearing females 42--56 days after transplantation, a single s.c. injection of 20 micrograms estradiol benzoate (EB) at 1030 h elicited significant increases in both serum LH and FSH values between 1700 and 1830 h on the next day. The magnitude of the LH surge was reduced and that of FSH was increased in tumor-bearing animals when compared to cyclic, nontumor females given a similar EB injection on diestrus Day 1. These results demonstrate that chronic hyperprolactinemia is associated with inhibition of basal LH secretion and ovarian estrogen production and an increase in circulating progesterone concentrations. Nevertheless, the stimulatory feedback effects of estrogen on LH and FSH release are still present and functioning in acyclic female rats under chronically hyperprolactinemic conditions. These data suggest that the cessation of regular ovulatory cycles associated with hyperprolactinemia may be due to a deficiency of LH and/or estrogen secretion, but not to a lack of central nervous system response to the stimulatory feedback action of estrogen.

Luteal phase deficiencies in peripubertal rhesus monkeys: mechanistic considerations.

Short luteal phases in peripubertal monkeys were identified by abbreviated progesterone secretion. Evaluation of the hormonal profiles for LH, FSH, and estradiol revealed that depressed serum concentrations of FSH during the follicular phase and LH and estradiol during the luteal phase were associated with luteal defects. In addition, morphological differences in the corpora lutea of short and normal luteal phases were identified. A mechanism is presented whereby inadequate follicular phase FSH secretion results in abbreviated luteal function.