Effect of high-density lipoproteins with varying ratios of apolipoprotein A-I to apolipoprotein A-II on steroidogenesis by cultured rat ovary granulosa cells.

Plasma high-density lipoproteins (HDL) can provide rat ovary steroidogenic tissue with cholesterol for steroid hormone production, but the mechanism of cholesterol transfer is unknown. To test the importance of apolipoprotein A-I (the major HDL apolipoprotein) in HDL-cell interactions, we examined the ability of canine-human HDL hybrids containing various proportions of canine apolipoprotein A-I and human apolipoprotein A-II to stimulate steroidogenesis by cultured rat ovary granulosa cells. We observed that as the apolipoprotein A-II to apolipoprotein A-II ratio decreased, the ability of the hybrid particles to stimulate granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, apolipoprotein A-I was not necessary for cholesterol transfer, since hybrids with less than 5% of their total apolipoprotein mass as apolipoprotein A-I stimulated progestin production 30% as effectively as canine HDL, which contained essentially only apolipoprotein A-I. These data indicate that the delivery of cholesterol from HDL into the rat ovary cell for steroidogenesis is not strictly dependent on the presence of a specific HDL apolipoprotein.

Estrogen regulation of steroidogenesis in rabbit luteal cells.

Estradiol is a potent modifier of gonadotropin-stimulated steroidogenesis. By the seventh day after ovulation, estradiol is the only agent required for the stimulation of progesterone synthesis by corpora lutea of superovulated pseudo-pregnant rabbits. To learn which control points in steroidogenesis are susceptible to regulation by estradiol alone, we have studied the production of pregnenolone, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one by corpora lutea of estradiol-stimulated and estradiol-deprived pseudopregnant rabbits. In previous investigations, we learned that estradiol deprivation in vivo, on day 9 of pseudopregnancy, causes an abrupt cessation of progesterone and 20 alpha-hydroxy-4-pregnen-3-one production, which is associated with accumulation of cholesterol and cholesteryl ester in the luteal tissue. We now report that production of pregnenolone, measured as its concentration in serum, also decreases abruptly by 84% within 48 h when the estradiol stimulus is removed on day 9 of pseudopregnancy. In addition, short term incubations of luteal tissue demonstrate that corpora lutea from estradiol-deprived rabbits do not use stores of luteal intracellular cholesterol for production of pregnenolone and progestin. These findings suggest that upon estradiol deprivation, rabbit luteal cells lose their capacity for using stored cholesteryl ester, or cholesterol synthesized de novo, for the production of pregnenolone and progestins. We, therefore, tested the hypothesis that a blockade of steroidogenesis caused by estrogen deprivation occurs at the point of cytochrome P-450 cholesterol side-chain cleavage (P-450scc), a principal rate-limiting step in the conversion of cholesterol to hormonal steroid products. To this end, we assayed the P-450scc activity in mitochondria-rich fractions of corpora lutea from rabbits that were deprived of estradiol for 24 and 48 h beginning on day 9 after induction of superovulation. Surprisingly, withdrawal of the estradiol stimulus did not cause loss of luteal P-450scc activity, measured as the amount of aminoglutethimide-inhibitable conversion of 25-hydroxycholesterol to pregnenolone by mitochondria-rich preparations. From these results, we infer that the luteotropic action of estradiol is probably not effected at P-450scc in the rabbit corpus luteum, but, presumably, occurs at control points that regulate the availability of stored cholesterol and/or its movement to or within the mitochondria for conversion to pregnenolone.

Further characterization of a rat ovarian testosterone receptor with evidence for nuclear translocation.

We have recently described receptor like testosterone (T)-binding protein in the cytosol of ovaries from estrogen-primed hypophysectomized immature female rats (HIFR). This binding protein is thermolabile, saturable, specific for T, and sediments at 7-8 S on sucrose gradients. We now report the further characterization of this protein. It is present in the cytosol of isolated granulosa cells, has a molecular weight of 240,000, a frictional ratio of 1.8, and a mean Stokes radius of 73 A. The binding protein is acidic and cysteine residues are necessary for binding. We have also demonstrated the selective nuclear accumulation of T by granulosa cells after the in vivo administration of (3H)T to estrogen primed HIFR. After the in vitro incubation of the ovaries with (3H)T, a thermolabile protein specific for T was extracted from nuclei under high-salt conditions. The cytosol and nuclear T-binding proteins described in this report have many characteristics of an androgen receptor, and may play a role in the regulation of granulosa cell proliferation during preantral follicular growth.

Progesterone "receptor" in rat ovary.

A soluble thermolabile protein with many characteristics of a progesterone receptor has been identified in ovaries of estrogen-stimulated, hypophysectomized, immature female rats. A potent synthetic progestin R5020 (17,21-dimethyl-19-nor-pregna-4, 9-diene-3, 20-dione) and a progestin-receptor complex stabilizer (glycerol) were employed. After the incubation of [3H]R5020 with ovarian cytosol, fractionation of a Sephadex G-200 column revealed a peak of radioactivity which eluted with the void volume. This peak, which represented saturable binding, disappeared after heating (37 C for 20 min) and trypsinization. In the absence of glycerol, binding decreased by 84%. Scatchard analysis of the binding curve showed the R5020 binding to be of moderately high affinity (Kd 4 nM), with 232 fmol binding sites/mg cytosol protein. Binding site number rose linearly with increasing cytosol protein concentration. The relative abilities of various steroids to inhibit [3H]R5020 Binding were: R5020 greater than progesterone greater than estradiol greater than testosterone greater than cortisol greater than diethylstilbestrol. [3H]R5020 was not metabolized and did not bind specifically to serum. In summary, we have identified a protein with characteristics of a progesterone receptor in the cytoplasmic fraction of ovarian tissue.

Localization and movement of newly synthesized cholesterol in rat ovarian granulosa cells.

The distribution and movement of cholesterol were studied in granulosa cells from the ovaries of estrogen-stimulated hypophysectomized immature rats cultured in serum-free medium. Plasma membrane cholesterol was distinguished from intracellular cholesterol with cholesterol oxidase, an enzyme that converts cell surface cholesterol to cholestenone, leaving intracellular cholesterol untouched. Using this approach we showed that 82% of unesterified cholesterol was associated with the plasma membrane in granulosa cells cultured for 48 h in serum-free medium in both the presence and absence of added androstenedione and FSH. FSH and androstenedione stimulated a marked increase in steroid hormone (progestin) production. The movement of newly synthesized cholesterol to the plasma membrane also was followed using cholesterol oxidase. Newly synthesized cholesterol reached the plasma membrane too rapidly to be measured in unstimulated cells (t1/2 less than 20 min); however, in cells stimulated by FSH and androstenedione, this rate was considerably slower (t1/2 approximately 2h). Therefore, cholesterol movement to the plasma membrane appears to be regulated by gonadotropins in these cells. We tested whether steroid biosynthesis used all cell cholesterol pools equally. To this end we administered [3H]acetate and [14C]acetate at different times and determined their relative specific contents in various steroids after defined intervals. The relative ages of the steroids (youngest to oldest) were: lanosterol, progestins, intracellular cholesterol, and plasma membrane cholesterol. This finding suggests that progestins use newly synthesized intracellular cholesterol in preference to preexisting intracellular or cell surface cholesterol. A measure of this effect is that the specific activity of secreted hormone was 15- to 30-fold greater than that of intracellular cholesterol. We conclude that the various cholesterol compartments in granulosa cells are discrete. While the major fraction of cholesterol in these steroidogenic cells resides in the plasma membrane, it is not in rapid equilibrium with intracellular cholesterol. Furthermore, steroidogenesis appears to use newly synthesized over preexisting cholesterol, suggesting a shunt pathway.

A receptor-like testosterone-binding protein in ovaries from estrogen-stimulated hypophysectomized immature female rats.

A soluble thermolabile protein with many characteristics of an androgen receptor has been demonstrated for the first time in the cytosol (100,000 X g supernatant) of estrogen-stimulated ovaries from hypophysectomized immature female rats (HIFR) treated with diethylstilbestrol in Silastic capsules (DESC). This binding protein is organ-specific and androgen-specific, has a high affinity with a Kd of 2.4 X 10(-9)M for testosterone, and is saturable with 2.1 X 10(-13) moles of binding sites per mg cytosol protein. The number of binding sites is linear with cytosol protein concentration and the binding protein sediments at 7-8 S on sucrose gradients. Estradiol is an effective inhibitor of testosterone binding. A role for this testosterone-binding protein as an effector of ovarian morphologic change is hypothesized.

Degradation of rat and human lipoproteins by cultured rat ovary granulosa cells.

We have investigated the degradation of 125I-labeled rat and human lipoproteins by rat ovary granulosa cells cultured in serum-free medium. The granulosa cells degrade rat [125I]iodo high density lipoprotein (HDL) to acid-soluble products, mainly monoiodotyrosine. The degradation of 125I-labeled rat HDL is a specific, saturable, high affinity (Km = 21 micrograms protein/ml) process. In studies of rat [125I]iodo-HDL degradation and progestin (progesterone plus 20 alpha-dihydroprogesterone) production by the same granulosa cell cultures, the cholesterol potentially made available to the cells by degradation can account for the majority of the substrate necessary for the increased progestin production. Granulosa cells degrade human [125I]iodo-HDL by a specific, saturable, high affinity (Km = 20 micrograms protein/ml) process. The degradation of human [125I]iodo-HDL can account for only 20% of the cholesterol substrate necessary for increased progestin production. The degradation of human [125I]iodo-low density lipoprotein (LDL) is saturable and a high affinity (Km = 8 micrograms protein/ml) process, but can be inhibited significantly by a 10-fold excess of unlabeled human HDL. In contrast to both rat [125I]iodo-HDL and human [125I]iodo-HDL, the degradation of human [125I]iodo-LDL can potentially provide twice the cholesterol necessary for increased progestin production. Pronase treatment of the granulosa cells inhibits human [125I]iodo-LDL degradation but stimulates rat [125I]iodo-HDL degradation, indicating that the mechanisms of degradation are separate. The data demonstrate that cultured rat ovary granulosa cells degrade rat HDL, human HDL, and human LDL, and this process has the potential for providing cholesterol for cellular steroid hormone synthesis.

Gonadotropin-releasing hormone and its agonist inhibit testicular luteinizing hormone receptor and steroidogenesis in immature and adult hypophysectomized rats.

The direct effect of gonadotropin-releasing hormone (GnRH) and its agonist on testicular LH receptor and steroidogenesis was studied in hypophysectomized immature and adult rats. Hypophysectomized rats were treated daily with varying doses of GnRH or [des-Gly10,D-Leu6(N alpha Me)Leu7, Pro9-NHEt]GnRH(a potent agonist). Some animals were also treated concomitantly with FSH, PRL, GH and/or LH to prevent the hypophysectomy-induced loss of testicular LH receptor and steroidogenic capacity. At the end of 5 days of treatment, testicular LH/hCG receptor concentration was measured by a [125I]-hCG-binding assay and steroidogenic responsiveness was determinded by in vitro incubations. GnRH and the GnRH agonist reduced testicular LH receptor in control and FSH-treated hypophysectomized immature rats. As little as 0.5 microgram agonist/day induced a greater than 40% decrease in the LH receptor content, whereas GnRH was less potent, with 50 micrograms/day inducing about a 50% decrease. The inhibitory effect of GnRH was shown to be the result of decreases in the concentration of LH receptor rather than changes in the receptor affinity (Kd = 1.1 X 10(-10)M). GnRH did not interfere with the [125I]hCG receptor assay. Treatment with PRL, GH, and FSH, alone or in various combinations, increased the testicular LH receptor content. The stimulatory effect of these pituitary hormones was depressed by concomitant treatment with the GnRH agonist. Similar inhibitory effects of GnRH and the agonist on testicular LH receptor were demonstrated in adult hypophysectomized rats. In vitro studies demonstrated that treatment with the GnRH agonist in vivo inhibited both basal and hCG-stimulated androgen production in FSH-primed immature hypophysectomized rats. Associated with decreases in androgens (testosterone and androstenedione) and reduced androgens (dihydrotestosterone, androstanediol, and androsterone), there was marked suppression of 17 alpha-hydroxylated precursors and C-21 steroid intermediates in animals treated with the GnRH agonist, thus suggesting that the inhibitory effect of the GnRH agonist was associated with possible defects in 17 alpha-hydroxylase and side-chain cleavage enzymes. Likewise, treatment with the GnRH agonist inhibited in vitro testicular steroidogenic responses in adult hypopysectomized rats. These results demonstrate the extrapituitary inhibitory effect of GnRH on testicular LH receptor content and Leydig cell steroidogenesis in immature and adult hypophysectomized rats.

Estrogen increases precursor for pregnenolone synthesis with temperature-sensitive occupancy of P-450scc in mitochondria of rabbit corpus luteum.

To examine the mechanism of estrogen's direct stimulation of steroidogenesis in the rabbit corpus luteum, we tested the hypothesis that the effect of estrogen on progestin production occurs at the site of processing of the precursor for pregnenolone (i.e. cholesterol) in the mitochondrion. For this purpose, we manipulated a model of estrogen stimulation by 1) removing sc estradiol-filled polydimethylsiloxane capsules from superovulated rabbits on day 9 of pseudopregnancy or 2) leaving the capsules in place to preserve a chronic estrogen stimulus. In the estrogen-deprived rabbits, the serum progesterone level fell precipitously in vivo within 24 h, but in rabbits with chronic estrogen stimulation, serum progesterone levels remained high. Our results show that the loss in progestin production caused by estrogen deprivation could not be attributed to loss of the mitochondrial cytochrome P-450 side-chain cleavage enzyme (P-450scc), a common rate-limiting step in progestin synthesis in many steroidogenic tissues. In addition, we confirmed that there was no loss in the catalytic activity of this enzyme. Treatment with aminoglutethimide in vivo followed by electron paramagnetic resonance spectroscopic analysis of mitochondria (prepared in aminoglutethimide-free buffers) showed that incubation of isolated mitochondria at 37 C and pH 6.2 caused an increased high spin state (g = 8.2 signal) and a concomitant decreased low spin state. This shift from low to high spin states, which is indicative of cholesterol-P-450scc complex formation, occurred in the luteal mitochondria from both estrogen-deprived and estrogen-stimulated rabbits. In further studies to localize estrogen's regulatory point, we determined that the initial (first minute) rate of production of pregnenolone (per mg protein or per U P-450scc) from endogenous precursor proceeded equally fast in mitochondria from estrogen-deprived and those from estrogen-stimulated rabbits. However, the rapid pregnenolone production in the estrogen-deprived group lasted for a shorter time and, after 30 min, yielded less pregnenolone per mg protein or per U P-450scc than did mitochondria from estrogen-stimulated rabbits. Addition of 25-hydroxycholesterol did not increase the initial rate of pregnenolone formation, indicating that precursor availability is not limiting during the initial period. In aggregate, these observations suggest that the effect of estrogen on progestin production in the rabbit corpus luteum is not regulation of the movement of cholesterol to the catalytic site on the inner mitochondrial membrane, even though this is a step in the regulation of protein hormone-stimulated steroidogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Progesterone production by cultured preantral rat granulosa cells? Stimulation by androgens.

Progesterone (P) production by isolated rat granulosa cells from preantral follicles was enhanced by addition of androgens to the tissue culture medium. Testosterone (T) at 10(-7), 10(-6), and 10(-4)M as well as 10(-6)M dihydrotestosterone (DHT) increased P production 400 to 700% over paired control cultures. Human chorionic gonadotropin (100 mIU/ml) and 17beta-estradiol (7.8 X 10(-10M) had no effect on P production. P was identified by both a specific radioimmunoassay and sephadex LH-20 column chromatography. The stimulatory influence of T and DHT on these preantral follicular cells is consistent with a direct role for androgens in granulosa cell differentiation.

Candida antigen detection in two premature neonates with disseminated candidiasis.

Two premature neonates with birth weight less than 1,200 g developed systemic candidiasis during treatment with multiple antibiotics and parenteral hyperalimentation. Clinical findings included signs of necrotizing enterocolitis in one patient and multiple fungal renal cortical abscesses in the other. The Candida antigen, mannan, was present in the sera of both patients at the time of clinical deterioration. Multiple blood cultures and urine and stool samples from both patients grew Candida albicans. Systemic antifungal therapy was given for a 6-week period and was associated with prolonged antigenemia despite negative findings on follow-up cultures. Antifungal therapy was stopped soon after antigen was no longer detected. Both patients recovered without evidence of further fungal infection. Candida antigen detection may be useful in the diagnosis and follow-up of premature infants with disseminated candidiasis.

Progestins inhibit FSH-stimulated steroidogenesis in cultured rat granulosa cells.

It has been hypothesized that progesterone (P) exerts a direct inhibitory effect on ovarian follicular development, an effect which could be mediated by P receptors located in granulosa cells. We tested this hypothesis by examining the effect of several progestins on FSH-stimulated estrogen (E), P, and 20 alpha-dihydroprogesterone (DHP) production by cultured rat granulosa cells, and correlated the results with the ability of the progestins to bind to the granulosa cell P receptor. Granulosa cells from immature hypophysectomized DES-treated rat produced 9 ng/ml E, 21 ng/ml P and 29 ng/ml DHP during a 2-day incubation in McCoy's 5a medium containing 10(-7) M androstenedione and 10 ng/ml of FSH. The FSH-induced increase in E production was inhibited by 50 and 95% following concomitant treatment with 3 x 10(-6) and 10(-5) M resp. of R5020, a potent synthetic progestin. Added R5020 at these concentrations also significantly inhibited P and DHP production. R5020 had no effect on granulosa cell viability or plating efficiency, and the inhibitory action of R50920 on E production was reversible. In studies of the specificity of the progestin inhibitory action, the relative abilities of various progestins to inhibit E production were R5020 > P > DHP > 17 alpha-hydroxyprogesterone (17OHP). The relative abilities of these progestins to bind to the ovary P receptor were also: R5020 > P > DHP > 17OHP. These results indicate that exogenous progestins directly inhibit the FSH-stimulation of granulosa cell steroidogenesis in vitro and suggest that the progestin effect may be mediated by the P receptor. Such results offer a possible mechanism whereby progesterone could exert a direct but reversible inhibitory action on ovarian follicular development.

Progestins inhibit FSH-stimulated granulosa estrogen production at a post-cAMP site.

The purpose of this investigation is to examine the mechanism by which progestins inhibit FSH-induced estrogen (E) production by cultured rat ovary granulosa cells. We have demonstrated that the highly potent synthetic progestin, R5020, is able to inhibit the induction of granulosa cell aromatase activity by cholera toxin, prostaglandin E2, dibutyryl cAMP or oFSH. Since the induction of E synthesis by these compounds is mediated through activation of adenylate cyclase and increased cellular cAMP production, these observations indicate that the progestin inhibitory effect is a post-cAMP event. In addition, we have demonstrated that R5020 does not inhibit FSH-stimulated granulosa cell cAMP production. The involvement of the granulosa cell progesterone (P) receptor as a mediator of this post-cAMP progestin effect is suggested by the relative abilities of various progestins to both bind the P receptor and to block the induction of granulosa cell aromatase activity by dibutyryl cAMP. While the precise mechanism of progestin action remains unclear, the kinetic analysis of aromatase enzyme activity demonstrates that progestins are not acting as competitive inhibitors of granulosa cell aromatase. Since E is necessary for follicular development, our in vitro data are consistent with the hypothesis that P is a factor which can inhibit FSH-induced follicular growth and development in the rat ovary.

Progestins inhibit FSH-induced functional LH receptors in cultured rat granulosa cells.

The role of intraovarian progesterone in the control of follicular growth and development remains unclear. The presence of a rat ovary granulosa cell progesterone receptor suggests that progesterone has a direct effect on the follicles. We have previously reported that progestins inhibit FSH-stimulated estrogen production by cultured granulosa cells by inhibiting the FSH induction of the aromatase enzyme. We now report that progestins can inhibit another FSH action on rat granulosa cells; the induction of LH/hCG receptors. The concomitant administration of 10(-5) M R5020, a potent synthetic progestin, with 10 ng/ml FSH during a 2-day culture period inhibits the FSH induction of LH/hCG receptors by 75 +/- 6% (mean +/- S.E.) The progestin inhibition of the induction of LH/hCG receptors is not mediated by its inhibitory action on the induction of aromatase. Scatchard analysis indicates that progestin decreases the number of LH/hCG receptors per cell but has no effect on receptor affinity. Both R5020 and progesterone have a dose-dependent inhibitory effect on the FSH induction of LH/hCG receptors, causing a 30 and 85% decrease in receptor number at concentrations of 10(-6) and 10(-5) M, respectively. The concomitant administration of R5020 with FSH also leads to a significant decrease in the ability of LH to stimulate cAMP production, indicating that progestin is inhibiting the induction of 'functional' LH/hCG receptors. R5020 (10(-5) M) also inhibits by 90% the induction of LH/hCG receptors by cholera toxin and dibutyryl cAmP, indicating that the progestin effect is at a post-cAMP site. Since the induction of LH/hCG receptors by FSH is a necessary event in follicular maturation, these results offer another mechanism by which progestins, at high concentrations, can inhibit follicular growth and development.

Inhibitory effect of gonadotropin releasing hormone upon cultured testicular cells.

The direct inhibitory of gonadotropin-releasing hormone (GnRH) upon testicular androgen production was studied in cultured testicular cells. Enzyme-dispersed testicular cells from immature hypophysectomized rats were cultured for 6 days in a serum-free medium with or without various hormones. Culture media were changed every 2 days and media concentration of a androgens was measured by radioimmunoassay. The testis cultures from immature rats secrete predominately 5 alpha-androstane-3 alpha, 17 beta-diol (A-diol) and androsterone but negligible amounts of androstenedione, testosterone and dihydrotestosterone. Incubation of testicular cells with hCG and FSH increased A-diol and androsterone production as compared to control cultures. In contrast, treatment with GnRH or a GnRH agonist (des-Gly10, D-Leu6 (N alpha Me)Leu7, Pro9NHEt-GnRH) inhibited the gonadotropin effect in a dose-dependent manner; 10(-8) M GnRH inhibited A-diol production by approximately 20% on day 4 and 6 of culture whereas 10(-6) M GnRH inhibited A-diol production by greater than 80%. The observed effect of GnRH upon testicular cells in primary culture demonstrates that the hypothalamic peptide directly inhibits testicular steroidogenesis.

Severe La Crosse encephalitis with significant neurologic sequelae.

La Crosse encephalitis, a member of the California arbovirus group, is the most common cause of reported mosquito-borne illness in the United States. Approximately 70 cases of La Crosse encephalitis are reported each year. The principal vector is the mosquito Aedes triseriatus. During the summer the virus is amplified horizontally in a cycle among small mammals such as chipmunks and squirrels. Infected female A. triseriatus deposit eggs in the basal holes of hardwood trees, although man-made containers and old tires containing water also supply a suitable breeding site. Some of these eggs infected with La Crosse virus hatch the next spring and give rise to infected adult A. triseriatus, and the host-vector cycle is renewed. Only a minority of children infected with the virus become ill. Clinical disease caused by La Crosse is usually mild, and neurologic sequelae are relatively uncommon. In this report we describe six patients with severe La Crosse meningoencephalitis diagnosed within a 4-week period. All patients required intensive care management, and there was a high rate of neurologic sequelae, suggesting that La Crosse is not necessarily a benign meningoencephalitis.

Low yield of bacterial stool culture in children with nosocomial diarrhea.

OBJECTIVE: To determine whether bacterial stool cultures (BSC) are useful in initial evaluation of children with symptoms of nosocomial diarrhea. To answer this question we performed a retrospective record review to determine the yield of BSC in children who developed diarrhea after the third hospital day (HD-3). METHODS: The hospital computer record keeping system was utilized to compile the result of BSC collected from children and adolescents ages 0 to 20 years between January 1, 1988, and October 31, 1996. All specimens were analyzed for Salmonella, Shigella, Yersinia and Campylobacter. We reviewed hospital charts of all children who developed a positive BSC beyond HD-3 to determine the time of onset of diarrhea and clinical circumstances. RESULTS: A total of 11 516 BSCs were submitted from 9262 children during the 8 1/2-year period. Five hundred sixty-eight (6.6%) of 9262 children had at least 1 positive BSC. Two thousand five hundred seventy-two children had the first BSC submitted after HD-3 and 13 (0.5%) of these children had a positive result. Chart review of these 13 children demonstrated that 6 had onset of diarrhea during the first 3 hospital days. Therefore only 7 children met our criteria for having nosocomially acquired diarrhea caused by a bacterial pathogen. Children whose first BSC was submitted after HD-3 accounted for 3767 (46%) of the total 8126 inpatient BSCs and in excess of $21000 annually in patient billing charges. CONCLUSION: In the absence of a known exposure the isolation of a bacterial pathogen from the stool of children with onset of diarrhea beyond HD-3 is a rare event. Under most circumstances BSC should not be part of the initial evaluation of children with symptoms of nosocomial diarrhea.

Transmission disequilibrium of maternally-inherited CTLA-4 microsatellite alleles in idiopathic recurrent miscarriage.

To elucidate the mechanisms that facilitate tolerance at the maternal-fetal interface, we are investigating the role of genes that are involved in peripheral self-tolerance in couples with idiopathic recurrent miscarriage. CTLA-4 is a negative regulator of T-cell proliferation and has been associated with human autoimmune disease. An AT(n) polymorphism in the 3'-untranslated region (UTR) of the human gene results in AT stretches that vary in length from 16 to 46 bp. We hypothesized that long stretches of AT repeats would result in mRNA instability, and reduced fetal survival in humans. We examined the transmission of AT(n) alleles in 60 couples with a history of > or = 3 unexplained spontaneous abortions to their 51liveborn children and 10 abortuses. The shorter allele was transmitted from heterozygous mothers to 26 of 35 liveborn children (chi2 = 8.3, P = 0.0040) and to three of nine aborted fetuses (chi2 = 1.0, P = 0.317). The shorter allele was transmitted from heterozygous fathers to 15 of 32 liveborn children (chi2 =0.12, P=0.726) and to five of eight aborted fetuses (chi2 = 0.5, P = 0.480). Furthermore, liveborn fetuses who inherited smaller alleles were more likely to represent the first successful pregnancy than liveborn fetuses who inherited larger maternal alleles (Pexact = 0.044) and fetuses of first pregnancies that inherited the smaller allele were significantly more likely to survive to term (Pexact = 0.0086). The preferential transmission of maternally-inherited shorter alleles to liveborn children, but random transmission of paternally-inherited alleles, suggests that CTLA-4 may be imprinted in humans and that this gene may play a role in inducing or maintaining tolerance at the maternal-fetal interface.

Treatment of infertility due to retrograde ejaculation: a simple, cost-effective method.

Our data indicate that an appropriate therapy for the infertility associated with retrograde ejaculation is isolation of sperm from voided urine after orgasm, plus IUI. This technique is simple and can be performed in the physician's office, in contrast to more complex techniques such as GIFT or in vitro fertilization.

Apolipoprotein E synthesis by cultured human ovarian granulosa cells: regulation by human chorionic gonadotropin and cholesterol.

OBJECTIVE: Apolipoprotein E (Apo E) is a surface component of several classes of plasma lipoproteins and functions as a receptor ligand for the low-density lipoprotein (LDL) (B/E) receptor, the hepatic E receptor, and the LDL receptor related protein. In continuing studies of Apo E synthesis by extrahepatic steroidogenic tissue, we have examined Apo E production and its control in cultured human granulosa cells (GC). DESIGN: Human GC obtained at the time of oocyte aspiration for in vitro fertilization were cultured, and the following parameters were measured: progesterone secretion into the medium, Apo E synthesis and secretion, and Apo E messenger ribonucleic acid (mRNA) content of the GC. RESULTS: Human chorionic gonadotropin induces a decrease in Apo E protein synthesis and Apo E mRNA content. The addition of aminoglutethimide (which blocks cholesterol conversion to pregnenolone) and/or LDL (which provides the cell with cholesterol), both result in an increase in Apo E protein synthesis and Apo E mRNA content. These latter findings are in agreement with studies in nonsteroidogenic cells, which show that Apo E production is positively correlated with cell cholesterol content.