Human immunodeficiency virus infection of T cells and monocytes proceeds via receptor-mediated endocytosis.

The rates of internalization and uncoating of 32P-labelled human immunodeficiency virus (HIV) in the human T lymphoid cell line CEM are consonant with a receptor-mediated endocytosis mechanism of entry. This interpretation was affirmed by electron microscopic observation of virions within endosomes. Virus binding and infectivity were inhibited to the same extent by pretreatment with OKT4A antibody, therefore, the CD4 receptor-dependent pathway of internalization appears to be the infectious route of entry. The pattern of internalization by the human monoblastoid cell line U937 proved to be more complex, involving rapid and efficient CD4-independent internalization. Electron microscopy revealed the presence of large intracellular vesicles, each containing several virions. Antibody against the CD4 receptor for virus efficiently blocked infection, but did not reduce significantly HIV binding or internalization in the U937 cell line. Consequently, U937 cells have a CD4-independent pathway of virus internalization that does not coincide with the route of entry for infectious HIV.

Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.

Uterine leiomyoma in a 14-year-old girl.

Uterine leiomyomas are common benign tumors of the uterus in adult females but are rare in adolescents. This is a review of the literature and a case report of a 14-year-old female who presented with increasing, intermittent back pain and abdominal distention due to a large uterine leiomyoma treated by myomectomy. This is the 9th reported case of a uterine leiomyoma in an adolescent female under the age of 18 years in the English literature.

Determination of estrogen receptor messenger ribonucleic acid (mRNA) and cytochrome P450 aromatase mRNA levels in adipocytes and adipose stromal cells by competitive polymerase chain reaction amplification.

Clinical evidence suggests that sex hormones affect adipose tissue metabolism and deposition. To investigate the biosynthesis and possible action of estrogen in adipose tissue, we report the use of competitive, specific polymerase chain reaction amplifications to determine levels of estrogen receptor (ER) messenger RNA (mRNA) and cytochrome P450 aromatase mRNA in adipocytes and adipose stromal cells. This extremely sensitive technique uses coamplification of a homologous animal species complementary RNA to control for differences in amplification efficiencies. The DNA amplification products are identified by Southern hybridization with species-specific radiolabeled oligonucleotide probes. Abdominal adipose tissue obtained from female patients during elective abdominoplasty was separated by collagenase digestion and centrifugation into floating adipocytes and pelleted adipose stromal cells. Our results demonstrate higher ER mRNA levels in adipocytes compared to adipose stromal cells, whereas cytochrome P450 aromatase mRNA levels are higher in adipose stromal cells compared to adipocytes. The finding of ER mRNA in adipose tissue suggests the presence of the ER in adipose tissue. In addition the inverse correlation of ER mRNA and cytochrome P450 aromatase mRNA levels in adipocytes and adipose stromal cells suggests a paracrine relationship whereby estrogen produced by adipose stromal cells affects adjacent adipocytes.

A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription.

C19 steroids are converted to estrogens in a number of tissues by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom). Adipose tissue is the principal site of estrogen formation in postmenopausal women. Aromatase activity as well as P450arom transcripts primarily reside in the stromal cell component of the adipose tissue. Studies designed to investigate whether increased local aromatase activity in breast adipose tissue influences the growth of breast cancers have yielded discrepant results. In an attempt to clarify this controversy, adipose tissue was obtained from the four breast quadrants at the time of mastectomy (n = 13) performed for removal of a tumor. Breast fat P450arom messenger RNA levels were quantified and compared between the four quadrants within each specimen using competitive polymerase chain reaction after reverse transcription in which 10 micrograms human adipose total RNA together with 1 pg rat complementary RNA (internal standard) were reverse transcribed and coamplified. In 9 out of 13 patients (69%), highest P450arom transcript levels colocalized to the quadrants bearing tumors. This correlation was statistically significant (P < 0.001). The regional distribution of P450arom transcripts in breast adipose tissue of disease-free individuals, obtained during reduction mammoplasty (control group, n = 9), did not favor any particular region of the breast. We also quantified by morphometry the histological components of the adipose tissue samples from each quadrant in mastectomy specimens. The distribution of stromal cells significantly correlated with the distribution of P450arom transcript levels, in that quadrants containing highest proportions of stromal cells matched to highest transcript levels (P < 0.01). Although the quadrants bearing tumors contained the highest percentage of stromal cells, this correlation was not statistically significant. The adipose tissue surrounding a breast tumor displays increased estrogen biosynthesis, which may promote tumor growth. It is further suggested that the distribution of stromal cell components in breast adipose tissue gives rise to locally elevated P450arom expression, which in turn may favor neoplastic development and growth in these predisposed areas of the breast. The correlation between the presence of a tumor and elevated P450arom levels in the proximal adipose tissue is independent of tumor size, node involvement, histological type or grade, estrogen/progesterone receptor status, DNA index, or S-phase fraction.

Identification of progesterone receptor in human subcutaneous adipose tissue.

Sex steroids are postulated to play a role in adipose tissue regulation and distribution, because the amount and location of adipose tissue changes during puberty and menopause. Because of the nature of adipose tissue, receptors for the female sex steroids have been difficult to demonstrate. To date, estrogen receptor messenger RNA and protein have been identified in human subcutaneous adipose tissue, but the presence of progesterone receptor (PR) has not been reported. In this study, we demonstrate PR message by Northern blot analysis in RNA isolated from the abdominal subcutaneous adipose tissue of premenopausal women. These preliminary studies revealed that PR messenger RNA levels are higher in the stromal-vascular fraction as opposed to the adipocyte fraction. Western blot analysis demonstrates both PR protein isoforms (human PR-A and human PR-B) in human subcutaneous adipose tissue. Using an enzyme-linked immunosorbent assay, total PR could be quantitated. These studies substantiate that sex steroid receptors are present in human adipose tissue, thereby providing a direct route for regulation of adipose tissue by female sex steroids.

Effect of type 1 transforming growth factor-beta on the level of aromatase cytochrome P-450 in human fetal hepatocytes.

Aromatase cytochrome P-450 (P-450AROM) is the enzyme in the steroidogenic pathway controlling the formation of oestrogens from 19 carbon steroid precursors. Aromatase is present in various tissues of the human fetus. The liver is second only to the placenta in the level of P-450AROM activity in the fetus. In this study we examined the effects of type 1 transforming growth factor-beta (TGF beta) on P-450AROM expression in human fetal (HF) hepatocytes. The HF hepatocytes were dispersed into single cells which were placed into monolayer cell culture until confluent. Cells were then rinsed and treated in serum-free media with dibutyryl cyclic AMP (Bu2cAMP) for 72 h. Treatment with Bu2cAMP (2 mmol/l) caused a fivefold increase in aromatase activity in hepatocytes. The increase in aromatase activity apparently represented an increase in P-450AROM enzyme as determined by immunoblotting using an antibody directed against human placental aromatase. TGF beta blocked basal, as well as Bu2cAMP increases, in aromatase activity by over 50%. The effect of TGF beta was dose-dependent with maximal inhibition observed using 2-5 ng TGF beta/ml. Immunodetectable P-450AROM decreased in parallel with activity following TGF beta treatment. The mechanism of TGF beta action was not through increasing phosphodiesterase (PDE) breakdown of cAMP since inhibition of PDE had no effect on TGF beta action. Finally we examined the level of P-450AROM mRNA using competitive polymerase chain reaction amplification. Bu2cAMP increased mRNA levels of P-450AROM by 2.5-fold, while TGF beta inhibited this induction by 35%. The results of this investigation demonstrated that TGF beta is a potent regulator of P-450AROM expression in HF hepatocytes.

MCF-7 and T47D human breast cancer cells contain a functional peroxisomal response.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors that regulate transcription of target genes. Since attempts have been made to correlate the ingestion of high-fat diets, itself a peroxisome proliferator, with the occurrence of breast cancer, we set about to determine if human breast cancer cells contained a functional PPAR. In this report we demonstrate the presence of an mRNA in two breast cancer cell lines (MCF-7 and T47D) which is specifically recognized by a mouse PPARgamma2 probe. Furthermore, in gel shift assays a consensus PPAR response element (PPRE) was specifically bound by nuclear extracts from MCF-7 cells and was further retarded by antibodies raised to mouse PPARgamma. Finally, when transfected with a PPRE-luciferase transcriptional reporter construct, transcription was increased in response to activators of PPAR and its dimmeric partner the retinoic acid X receptor (RXR). These data indicate that peroxisomal proliferators are capable of mediating transcription in human breast cells and suggest the possibility of a physiological role in the breast.

Differential transcriptional activation of peroxisome proliferator-activated receptor gamma by omega-3 and omega-6 fatty acids in MCF-7 cells.

While the role of dietary fats in breast cancer remains controversial, the recent cloning of peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear hormone receptor, from human breast cancer cells lines provides a potential molecular link. Several fatty acids from four classes of dietary fats were tested for their ability to mediate the transcriptional activity of PPARgamma in MCF-7 and MDA-MB-231 cells using growth media with minimal serum. Whereas omega-3 fatty acids inhibit transactivation of PPARgamma to levels below control, omega-6, monounsaturated and saturated fatty acids stimulate the activity of the transcriptional reporter. These studies indicate that individual fatty acids differentially regulate the transcriptional activity of PPARgamma by selectively acting as agonists or antagonists. Furthermore, the transcriptional activation of PPARgamma correlates with cell proliferation in MCF-7 cells. Understanding the effects of individual fats on breast cancer cells and PPARgamma transactivation could provide important new insights into the epidemiology of breast cancer and the role of dietary fat.

Durability of confluent endothelial cell monolayers on small-caliber vascular prostheses in vitro.

Since implantation of small-caliber vascular grafts with preformed confluent endothelial cell monolayers (ECMs) may prevent acute platelet deposition and thrombus formation, we have evaluated the conditions necessary to produce durable ECMs. Cultured human umbilical-vein ECs in buffer were attached to vascular grafts--either expanded polytetrafluoroethylene (ePTFE) or knitted Dacron, both with inside diameters of 4 mm--precoated with collaten type I and perfused in vitro with serum-containing culture medium to achieve cell spreading into confluent monolayers. The number of cells attached was quantified by DNA measurements and indium-111 labeling. Morphology was evaluated by scanning electron microscopy. The maximum number of cells that attached acutely was 3.6 X 10(5) cells/cm2 graft, and the minimum number needed for confluence was 1.4 X 10(5) cells/cm2 graft. Confluence was morphologically complete after 2 hours of in vitro perfusion at 15 ml/min. When ECMs were exposed to varying flow rates, cell retention after 1 hour was 96.1% +/- 5.6% at 50 ml/min, 94.6% +/- 6.1% at 100 ml/min, 77.6% +/- 10.8% at 200 ml/min (p less than 0.001), and 40.1% +/- 10.4% at 400 ml/min (p less than 0.0001). Confluence was maintained for all grafts exposed to flows of 100 ml/min or less. Fewer cells were retained when acutely attached, unspread cells (71.5% +/- 11.5%) were compared with established ECMs (89.9% +/- 6.7%) at a flow rate of 100 ml/min (p less than 0.0001). ECMs on knitted Dacron were more durable than on ePTFE (82.7% +/- 5.3% versus 75.5% +/- 4.8% remained attached at 200 ml/min, P less than 0.05). 111Indium-labeled and unlabeled cells were equivalent with respect to saturation level attachment, spreading time to confluence, and durability under flow at 100 ml/min. We conclude that confluent and durable endothelial cell monolayers can be established on small-caliber vascular grafts within 2 hours.

In vitro endothelialization of small-caliber vascular grafts.

To establish the conditions for achieving immediate and complete endothelial cell coverage of the luminal surfaces of small-caliber (internal diameter:4 mm) vascular grafts in vitro, the attachment and spread of endothelial cells cultured from human umbilical veins to expanded polytetrafluoroethylene (PTFE) and knitted Dacron grafts was studied. Cell number was quantified by fluorescent measurements of deoxyribonucleic acid. The completeness of cell coverage and cell junction formation were assessed by means of both scanning and transmission electron microscopy. Cell attachment was compared after expanded PTFE or knitted Dacron grafts were precoated with gelatin, laminin, fibronectin, fibrin, or collagen, singly or in combination. Saturation cell attachment of 3.5 +/- 0.7 X 10(5) cm-2 was completed within 15 minutes when (1) type I collagen was used to form the substrate matrix, (2) human umbilical vein endothelial cells were suspended in phosphate-buffered saline solution supplemented with calcium and magnesium, and (3) the suspended cell number was greater than or equal to 5 X 10(5). In contrast, attachment to untreated or laminin-treated surfaces was 1.3 +/- 0.33 X 10(5) cells cm-2 and attachment to fibrin- or fibronectin-treated surfaces was 2.8 +/- 0.47 and 2.4 +/- 1.1 cells X 10(5) cm-2, respectively. However, to produce a confluent flow surface, the attached cells required several hours in culture medium to spread completely. Maintenance of confluent cell coverage on the graft surface for 3 days in vitro was achieved by means of continuous perfusion with oxygenated tissue culture medium RPMI-1640-HEPES supplemented with 20% fetal bovine serum. We conclude that optimum immediate confluent endothelial coverage of small-caliber vascular grafts requires a high concentration of cells, attachment to collagen-precoated grafts, and several hours of incubation in complete culture medium.

Fulminant hepatic failure due to herpes simplex after hysteroscopy.

BACKGROUND: Fulminant hepatic failure due to herpes simplex in healthy adults is a rare condition with a high mortality rate. The lack of specific symptoms and the absence of typical herpetic lesions in a majority of cases contribute to delayed diagnosis. CASE: We describe a fatal case of fulminant hepatic failure due to herpes simplex in a healthy woman presenting after laparoscopy and hysteroscopy for tubal infertility. The patient lacked evidence of mucocutaneous lesions or jaundice. The surgery likely contributed to viral dissemination. CONCLUSION: Although rare, disseminated herpes should be considered a possible cause of postsurgical pelvic infections, even in the absence of ulcerative lesions. Until a definitive diagnosis is made, antiviral therapy should be considered in patients with high fever, leukopenia, and abnormal liver function.

Differences in the estrogen content of breast adipose tissue in women by menopausal status and hormone use.

OBJECTIVE: To determine the levels of free estrone (E1) and estradiol (E2) in breast adipocytes of premenopausal women, premenopausal women using oral contraceptives (OCs), postmenopausal women, and postmenopausal women using estrogen replacement therapy (ERT). METHODS: Breast adipose tissue was obtained from 36 premenopausal and 29 postmenopausal women, and adipocytes were separated from stromal and epithelial cells through collagenase digestion and centrifugation. Oil was rendered from adipocytes, and E1 and E2 levels were measured by specific radioimmunoassays after extraction with methanol-water. RESULTS: Estrone and E2 levels were approximately 2.4- and 7.8-fold higher, respectively, in premenopausal women than in postmenopausal women. In premenopausal women, E1 and E2 correlated with the time since last menses (R2 = .55 and .62, respectively), whereas in postmenopausal women, E1 and E2 correlated with body mass index (BMI) (r = .48 and .52, respectively). Estrone levels were always greater than E2 levels in adipocytes, with the E1/E2 ratio being 2.7-fold higher in postmenopausal women than in premenopausal women. The use of OCs decreased E1 and E2 levels in premenopausal women, and ERT increased levels in postmenopausal women. CONCLUSION: Free estrogen in breast adipocytes is characterized by E1 dominance, with levels in premenopausal women correlating with the menstrual cycle and levels in postmenopausal women correlating with BMI.

Single-dose pharmacokinetics of sublingual versus oral administration of micronized 17 beta-estradiol.

OBJECTIVE: To investigate the pharmacokinetic profiles of different doses of micronized 17 beta-estradiol administered by oral or sublingual routes. METHODS: Single doses of micronized 17 beta-estradiol were administered orally (1 mg, 0.5 mg) or sublingually (1 mg, 0.5 mg, 0.25 mg) to six postmenopausal women in a randomized clinical trial. We calculated pharmacokinetic parameters for estradiol (E2) and estrone (E1) of maximum serum concentration, time to maximum serum concentration, terminal half-life, area under the concentration curve, and oral clearance. Serum levels of E1 sulfate also were compared at 4, 12, and 24 hours after dosing. RESULTS: Sublingual administration resulted in rapid absorption with significantly higher E2 levels than did comparable oral dosing. Estrone levels did not vary with route of administration but correlated with the dosage administered. Estrone sulfate levels correlated with the dosage administered and also tended to be higher with sublingual administration. Sublingual administration resulted in a significantly lower E1 to E2 ratio during the 24 hours than did oral administration. CONCLUSION: Sublingual administration of micronized 17 beta-estradiol results in a rapid, burst-like absorption into the systemic circulation, yielding high E2 levels that fall rapidly over the first 6 hours.

Control of air quality in an assisted reproductive technology laboratory.

OBJECTIVE: To investigate the effect of improved air quality on IVF and subsequent embryo development. DESIGN: Retrospective cohort study. SETTING: Hospital-based IVF facility composed of an anteroom, a cleanroom, and an adjacent operating room. PATIENT(S): Two-hundred seventy-five couples requesting IVF between 1993 and 1997. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Particle counts (sizes 0.3, 0.5, 1.0, and 5.0 microm); IVF rates; and embryo quality (stage and grade). RESULT(S): Clinical pregnancy rates decreased from 35% in 1993 to 16% in 1994 (numerous construction odors were detected during 1994) and increased steadily after the cleanroom was built (rates for 1995-1997 were 20%, 32%, and 59%, respectively). Fertilization rates decreased between 1993 (74%) and 1994 (60%) and then steadily increased after cleanroom installation (62% in 1995, 71% in 1996, and 69% in 1997). The proportion of embryos past the four-cell stage decreased from 66% in 1993 to 61% in 1994 but then increased steadily in the years after the cleanroom was built (78%, 77%, and 83% in 1995, 1996, and 1997, respectively). During the same 5-year period, there were no differences in embryo quality or number of embryos transferred. CONCLUSION(S): Construction of a Class 100 cleanroom improved air quality and IVF rate and increased the number of embryos past the four-cell stage available for transfer.

Validity and cost-effectiveness of antisperm antibody testing before in vitro fertilization.

OBJECTIVE: To determine the usefulness of and cost-effectiveness of antisperm antibody testing in the prediction of poor fertilization rates in couples undergoing IVF. DESIGN: Retrospective cohort study. SETTING: A hospital-based reproductive endocrinology and infertility practice. PATIENT(S): Male partners of 251 couples undergoing IVF between 1992 and 1997. MAIN OUTCOME MEASURE(S): Fertilization rates in couples undergoing conventional IVF. RESULT(S): One hundred nineteen couples were evaluated for antisperm antibodies; fertilization rates were similar in those couples whose husbands were and were not tested (64% versus 68%). Antisperm antibodies were detected in 16 men. Four (25%) of the 16 couples whose husbands had antisperm antibodies fertilized < or = 50% of oocytes, compared with 31 (30%) of the 103 couples whose husbands did not have these antibodies. Overall, 21 couples (8.4%) experienced complete fertilization failure. In a program that included antisperm antibody testing for selected couples and intracytoplasmic sperm injection (ICSI) for those who tested positive, it would cost $11,735 to prevent a fertilization failure (assuming ICSI were 100% effective), whereas it would cost $9,250 to perform ICSI in a second IVF cycle for those who initially failed. CONCLUSION(S): In this practice setting, antisperm antibody testing has low sensitivity in predicting low or no fertilization and does not appear to be cost-effective when selectively ordered as part of an IVF workup.

Amniotic fluid embolism. Three case reports with a review of the literature.

Amniotic fluid embolism is a catastrophic event of the intra- and early postpartum period which may also be seen with cesarean delivery and during abortions. Presenting symptomatology includes respiratory distress with cyanosis, shock, and possibly tonic-clonic seizures. DIC frequently occurs. The pathogenesis may include entry of amniotic fluid through lacerations or ruptures of the uterus or cervix, through endocervical veins and through abnormal uteroplacental sites, such as with placental abruption, placenta previa, or placenta accreta. Amniotic fluid probably causes cardiovascular-respiratory symptoms by pulmonary vascular obstruction and through a vasoactive substance causing pulmonary vascular constriction. The lethality of amniotic fluid may be enhanced by a high particulate content or meconium staining. The diagnosis of amniotic fluid embolism may be made ante mortem by demonstrating amniotic fluid debris in central blood samples or expectorated sputum. Postmortem diagnosis often requires meticulous examination of the pulmonary microvasculature with the utilization of special stains. Treatment is directed towards symptoms of shock, arterial hypoxemia, and DIC. Acute renal failure may complicate the picture after shock. If the patient survives the embolic and coagulative problems, recovery is usually complete without long-term sequelae.

Ablation of luteal phase symptoms of Menière's disease with leuprolide.

Symptoms of Menière's disease in women may be exacerbated during the luteal phase of the menstrual cycle. This suggests a role for progesterone production and subsequent fluid redistribution as a predisposing factor for Menière's symptoms. We report the use of leuprolide acetate, a gonadotropin-releasing hormone agonist, in a woman with cyclic Menière's symptoms. This drug, which abolishes gonadotropin-dependent ovarian sex steroid production, alleviated the patient's symptoms during therapy. This observation offers further support to the hypothesis of sex hormone-related exacerbations of Menière's symptoms and provides a possible future treatment option for this debilitating disease.

Expression of a mitochondrial progesterone receptor in human spermatozoa correlates with a progestin-dependent increase in mitochondrial membrane potential.

The hyperactivation of human spermatozoa necessary for fertilization requires a substantial increase in cellular energy production. The factors responsible for increasing cellular energy remain poorly defined. This article proposes a role for a novel mitochondrial progesterone receptor (PR-M) in modulation of mitochondrial activity. Basic science studies demonstrate a 38 kDa protein with western blot analysis, consistent with PR-M; whereas imaging studies with confocal and immunoelectron microscopy demonstrate a PR on the mitochondria. Treatment with a PR-specific progestin shows increased mitochondrial membrane potential, not related to induction of an acrosome reaction. The increase in mitochondrial membrane potential was inhibited by a specific PR antagonist, but not affected by an inhibitor to the progesterone-dependent Catsper voltage-activated channel. In conclusion, these studies suggest expression of a novel mitochondrial PR in human spermatozoa with a progestin-dependent increase in mitochondrial activity. This mechanism may serve to enhance cellular energy production as the spermatozoa traverse the female genital tract being exposed to increasing concentrations of progesterone.