The factors associated with natural disease progression from HIV to AIDS in the absence of ART, a propensity score matching analysis.

This study aimed at comparing the factors associated with the natural progression between typical progressors (TPs) and rapid progressors (RPs) in HIV-infected individuals. A retrospective study was conducted on 2095 eligible HIV-infected individuals from 1995 to 2016 in a high-risk area of Henan Province, China. Propensity score matching was used to balance covariates, and the conditional logistic regression analyses were performed to explore the factors of natural disease progression among HIV infectors. A total of 379 pairs of RPs and TPs were matched. The standardised difference values of all covariates were less than 10%. HIV-infected individuals transmitted through sexual transmission (odds ratio (OR) 0.56, 95% confidence interval (CI) 0.36-0.85) were more likely to progress to AIDS compared with those infected through contaminated blood. Older age at diagnosis of HIV-infected individuals (OR 0.72, 95% CI 0.58-0.89) exhibited a faster progression to AIDS. HIV-infected individuals identified through a unique survey (OR 7.01, 95% CI 2.99-16.44) were less likely to progress to AIDS compared with those identified through medical institutions. HIV-infected individuals who had higher baseline CD4+T cell counts (OR 3.37, 95% CI 2.59-4.38) had a slower progression to AIDS. These findings provide evidence for natural disease progression from HIV to AIDS between TPs and RPs.

[Job stress in locomotive attendants in a locomotive depot and related influencing factors].

Objective: To investigate the current status of job stress in locomotive attendants in a locomotive depot and related influencing factors. Methods: From 2012 to 2013, cluster sampling was used to select 1500 locomotive attendants in a locomotive depot in Zhengzhou Railway Bureau as respondents.The contents of the investigation included general data and occupational information.A job satisfaction questionnaire was used to investigate the degree of satisfaction, a depression scale was used to investigate the frequency of symptoms, and a daily stress scale was used to investigate the frequency of fatigue and stress. Results: There was a significant difference in depression score between locomotive attendants with different ages, working years, degrees of education, working situations of spouse, total monthly family incomes, numbers of times of attendanceat night, monthly numbers of times of attendance,ormonthly attendance times(P<0.05). There was a significant difference in job satisfaction score between locomotive attendants with different ages,working years, degrees of education, working situations of spouse, total monthly family incomes, numbers of times of attendance at night, monthly attendance times,or ways to work(P<0.05). There was a significant difference in daily stress score between locomotive attendants with different ages, working years, marital status,working situations of spouse, total monthly family incomes, types of work,numbers of times of attendance at night,monthly attendance times,attendance times at night,or ways to work(P<0.05). The multiple stepwise regression analysis showed that the type of locomotive was positively correlated with job satisfaction(ß=1.546)and monthly number of times of attendance,working years,attendance time at night,and degree of education were negatively correlated with job satisfaction(ß=-0.185,-0.097,-0.020,and -1.106); monthly number of times of attendance andcommute time were positively correlated with depression(ß=0.243 and 0.029); attendance time at night,working situation of spouse,commute time,monthly number of times of attendance,degree of education,and working years were positively correlated with daily stress(ß=0.006,0.473,0.010,0.043,0.585, and 0.028). Conclusion: Number of times of attendance, attendance time,working years,and spouse are influencing factors for job stress in locomotive attendants. Improvement in work process and care for their personal life help to reduce the level of job stress.

Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice.

We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (kappa) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 V kappa genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.

[Effects of regional citrate anticoagulation in continuous veno-venous hemofiltration of severe burn patients].

Objective: To investigate the effects of regional citrate anticoagulation in continuous veno-venous hemofiltration (CVVH) of severe burn patients. Methods: A retrospective non-randomized controlled study was conducted. From January 2017 to August 2020, sixty-eight severe burn patients who met the inclusion criteria were treated with CVVH in Affiliated Hospital of Nankai University. According to the different methods of blood anticoagulation in CVVH treatment, patients were divided into citrate group (n=40) and heparin group (n=28). In the citrate group, 32 males and 8 females were (40±18) years old with total burn area of (62±14)% total body surface area (TBSA); in the heparin group, 22 males and 6 females were (38±16) years old with total burn area of (57±20)%TBSA. Creatinine level, C-reactive protein (CRP) value, and urea nitrogen level in serum of patients were recorded at 0 (immediately), 48, and 96 h after CVVH treatment in 2 groups, urea clearance index was calculated based on urea nitrogen level at 0, 48, and 96 h after CVVH treatment in 2 groups, platelet count (PLT), prothrombin time (PT), and activated partial thromboplastin time (APTT) in total coagulation of patients were recorded. The frequency of forced hemofiltration termination caused by adverse reactions such as severe hypocalcemia, aggravated wound bleeding, and new bleeding on non-wound surface of patients was recorded within 96 h of CVVH treatment. The duration of daily CVVH use from the beginning to the end was recorded. Data were statistically analyzed with chi-square test, analysis of variance for repeated measurement, independent samples t test, and Bonferroni correction. Results: There were no significant differences in urea nitrogen level, creatinine level, and CRP value in serum of patients between 2 groups at 0 h after treatment (P>0.05). At 48 and 96 h after treatment, urea nitrogen level, creatinine level, and CRP value in serum of patients in citrate group were significantly lower than those in heparin group (t=3.366, -2.315, 2.942, -2.657, 2.011, -2.441, P<0.05), and urea clearance index of patients in citrate group was significantly higher than that in heparin group (t=1.017, 2.233, P<0.05). There were no statistically significant differences in PLT, PT, and APTT of patients between 2 groups at 0 h after treatment (P>0.05). At 48 and 96 h, PLT of patients in citrate group was significantly higher than that in heparin group (t=-3.417, -4.143, P<0.05 or P<0.01), PT of patients in citrate group was significantly shorter than that in heparin group (t=2.760, -3.655, P<0.01), APTT of patients in citrate group was significantly shorter than that in heparin group (t=3.719, 5.146, P<0.05 or P<0.01). Within 96 h of treatment, there was 1 case of hypocalcemia and 1 case of aggravated wound bleeding resulting in forced hemofiltration termination in citrate group, but there was no new bleeding on non-wound surface; in heparin group, there was no hypocalcemia, but 7 cases of aggravated wound bleeding and 2 cases of new bleeding on non-wound surface (both at the tracheotomy site) resulting in forced hemofiltration termination. The use time of blood purification filter of patients in citrate group was (11.7±4.8) h, obviously longer than (6.6±2.5) h in heparin group (t=3.310, P<0.01). Conclusions: The use of regional citrate anticoagulation in CVVH treatment of severe burn patients has the advantages including little effect on coagulation function and high safety, can effectively prolong the use time of filter and improve the therapeutic effect, but this conclusion still needs to be further verified in clinical application.

[Influence of directed restrictive fluid management strategy on patients with serious burns complicated by severe inhalation injury].

Objective: To explore the influence of directed restrictive fluid management strategy (RFMS) on patients with serious burns complicated by severe inhalation injury. Methods: Sixteen patients with serious burns complicated by severe inhalation injury hospitalized in our department from December 2014 to December 2017, meeting the inclusion criteria and treated with RFMS, were enrolled in directed treatment group. Thirty-four patients with serious burns complicated by severe inhalation injury hospitalized in our department from December 2012 to December 2017, meeting the inclusion criteria and without RFMS, were enrolled in routine treatment group. Medical records of patients in 2 groups were retrospectively analyzed. Within post injury day 2, mean arterial pressure (MAP), central venous pressure (CVP), extravascular lung water index (ELWI), global end-diastolic volume index, and pulmonary vascular permeability index of patients in directed treatment group were monitored by pulse contour cardiac output monitoring technology, while MAP and CVP of patients in routine treatment group were monitored by routine method. On post injury day 3 to 7, patients in 2 groups were treated with routine fluid supplement therapy of our Department to maintain hemodynamic stability, and patients in directed treatment group were treated according to RFMS directed with goal of ELWI≤7 mL·kg(-1)·m(-2). On post injury day 3 to 7, total fluid intake, total fluid output, and total fluid difference between fluid intake and output within 24 h, value of blood lactic acid, and oxygenation index of patients in 2 groups were recorded. Occurrence of acute respiratory distress syndrome (ARDS) on post injury day 3 to 7 and 8 to 28, mechanical ventilation time within post injury day 28, and occurrence of death of patients in 2 groups were counted. Data were processed with chi-square test, t test, and analysis of variance for repeated measurement. Results: The total fluid intakes within 24 h of patients in directed treatment group were close to those in routine treatment group on post injury day 3, 4, 5, 6, 7 (t=-0.835, -1.618, -2.463, -1.244, -2.552, P>0.05). The total fluid outputs and total fluid differences between fluid intake and output within 24 h of patients in 2 groups on post injury day 3 were close (t=0.931, -2.274, P>0.05). The total fluid outputs within 24 h of patients in directed treatment group were significantly higher than those in routine treatment group on post injury day 4, 5, 6, 7 (t=2.645, 2.352, 1.847, 1.152, P<0.05). The total fluid differences between fluid intake and output within 24 h of patients in directed treatment group were (2 928±768), (2 028±1 001), (2 186±815), and (2 071±963) mL, significantly lower than (4 455±960), (3 434±819), (3 233±1 022), and (3 453±829) mL in routine treatment group (t=-4.331, -3.882, -3.211, -4.024, P<0.05). The values of blood lactic acid of patients in directed treatment group and routine treatment group on post injury day 3, 4, 5, 6, 7 were close (t=0.847, 1.221, 0.994, 1.873, 1.948, P>0.05). The oxygenation indexes of patients in directed treatment group on post injury day 3 and 4 were (298±78) and (324±85) mmHg (1 mmHg=0.133 kPa ), which were close to (270±110) and (291±90) mmHg in routine treatment group (t=-1.574, 2.011, P>0.05). The oxygenation indexes of patients in directed treatment group on post injury day 5, 6, 7 were (372±88), (369±65), and (377±39) mmHg, significantly higher than (302±103), (313±89), and (336±78) mmHg in routine treatment group (t=3.657, 3.223, 2.441, P<0.05). On post injury day 3, 4, 5, 6, 7, patients with ARDS in directed treatment group were less than those in routine treatment group, but with no significantly statistical difference between the 2 groups (χ(2)=0.105, P>0.05). On post injury day 8 to 28, patients with ARDS in directed treatment group were significantly less than those in routine treatment group (χ(2)=0.827, P<0.05). The mechanical ventilation time within post injury day 28 of patients in directed treatment group was apparently shorter than that in routine treatment group (t=-2.895, P<0.05). Death of patients in directed treatment group within post injury day 28 was less than that in routine treatment group, but with no significantly statistical difference between the 2 groups (χ(2)=0.002, P>0.05). Conclusions: Under the circumstance of hemodynamics stability, RFMS directed with goal of ELWI≤7 mL·kg(-1)·m(-2) on post injury day 3 to 7 is an useful strategy, which can reduce occurrence rate of ADRS and shorten mechanical ventilation time of patients with serious burns complicated by severe inhalation injury at late stage of burns.

Transcriptional regulation of the rat tissue type plasminogen activator gene: localization of DNA elements and nuclear factors mediating constitutive and cyclic AMP-induced expression.

We have characterized tissue type plasminogen activator (tPA) promoter elements and nuclear factors required for follicle-stimulating hormone (FSH)-induced transcription of the rat tPA gene in granulosa cells and constitutive expression of the gene in the rat neuroblastoma cell line B103. Run-on transcription analysis of isolated nuclei revealed that B103 cells transcribe the tPA gene at a high and constitutive level, while FSH was found to induce tPA gene transcription in a rapid and transient manner in granulosa cells. The maximal FSH-induced transcription rate was obtained after 20 min and was similar in the absence or presence of the protein synthesis inhibitor cycloheximide. However, in the presence of cycloheximide, tPA transcription was not turned off but continued at a high rate for several hours. This phenomenon may at least partly explain the earlier finding that tPA mRNA is superinduced by FSH in the presence of cycloheximide. DNase I footprinting analysis of the first 621 bp of the tPA promoter revealed a total of six regions that interact with nuclear factors from B103 and granulosa cells. Deletion of the promoter region from positions -269 to -621, a region that includes the two most-upstream footprints, had no effect on constitutive or FSH-induced transcription in transient expression experiments. Nuclear extracts from both granulosa cells and B103 cells showed strong binding to a consensus cyclic AMP-responsive element (CRE) at positions -178 to -185 and a neighboring binding site for nuclear factor 1 (NF1) at positions -145 to -158. The factors binding to these two regions were identified as members of the CRE-binding protein and NF1 families of transcription factors, respectively. Footprints were also obtained over two GC boxes at positions -64 to -71 and -41 to -49. These footprints were more pronounced with nuclear extracts from B103 cells than with extracts from untreated or FSH-treated granulosa cells, but gel shift assays indicate that similar amounts of two distinct factors bind to the two GC boxes in both cell types. Transfection experiments using promoter constructs with inactivated promoter elements indicate that both the CRE and NF1 sites contribute to the FSH responsiveness of the rat tPA gene in granulosa cells, while only the NF1 site is important for constitutive expression in B103 cells. The two GC boxes were found to be necessary both for constitutive expression in B103 cells and for FSH-induced expression in granulosa cells, and inactivation of both GC boxes essentially eliminated the tPA promoter activity in both cell types.

Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy.

A fully human IgG2kappa monoclonal antibody (MAb), E7.6.3, specific to the human epidermal growth factor (EGF) receptor (EGFr) was generated from human antibody-producing XenoMouse strains engineered to be deficient in mouse antibody production and to contain the majority of the human antibody gene repertoire on megabase-sized fragments from the human heavy and kappa light chain loci. The E7.6.3 MAb exhibits high affinity (KD = 5 x 10(-11) M) to the receptor, blocks completely the binding of both EGF and transforming growth factor alpha (TGF-a) to various EGFr-expressing human carcinoma cell lines, and abolishes EGF-dependent cell activation, including EGFr tyrosine phosphorylation, increased extracellular acidification rate, and cell proliferation. The antibody (0.2 mg i.p. twice a week for 3 weeks) prevents completely the formation of human epidermoid carcinoma A431 xenografts in athymic mice. More importantly, the administration of E7.6.3 without concomitant chemotherapy results in complete eradication of established tumors as large as 1.2 cm3. Tumor eradication of A431 xenografts was achieved in nearly all of the mice treated with total E7.6.3 doses as low as 3 mg, administered over the course of 3 weeks, and a total dose of 0.6 mg led to tumor elimination in 65% of the mice. No tumor recurrence was observed for more than 8 months after the last antibody injection, which further indicated complete tumor cell elimination by the antibody. The potency of E7.6.3 in eradicating well-established tumors without concomitant chemotherapy indicates its potential as a monotherapeutic agent for the treatment of multiple EGFr-expressing human solid tumors, including those for which no effective chemotherapy is available. Being a fully human antibody, E7.6.3 is expected to exhibit minimal immunogenicity and a longer half-life as compared with mouse or mouse-derivatized MAbs, thus allowing repeated antibody administration, including in immunocompetent patients. These results suggest E7.6.3 as a good candidate for assessing the full therapeutic potential of anti-EGFr antibody in the therapy of multiple patient populations with EGFr-expressing solid tumors.

Ligand-induced down-regulation of testicular and ovarian luteinizing hormone (LH) receptors is preceded by tissue-specific inhibition of alternatively processed LH receptor transcripts.

Down-regulation of plasma membrane receptors by homologous hormones has been found in diverse cell types. In testicular Leydig and ovarian luteal cells, treatment with LH/hCG decreases LH receptor content. Although suppression of LH-binding sites may result from ligand-induced receptor internalization, sequestration, and/or phosphorylation, the gonadotropins may also regulate receptor mRNA levels. We examined the regulation of testis LH receptor mRNAs in adult rats that received 10 or 200 IU hCG, using cRNA probes derived from the 5' extracellular domain (EC) or the 3' transmembrane domain (TM) of the rat receptor cDNA. Probe EC hybridized to predominant signals of 7 and 1.8 kilobases (kb) and weaker signals of 4.2 and 2.5 kb. However, probe TM hybridized to the three larger forms of the LH receptor mRNA, but not to the 1.8-kb species, suggesting that the latter form lacks the transmembrane domain. After 6 and 12 h of treatment with 200 or 10 IU hCG, respectively, hybridization to the larger mRNA species decreased by more than 60%, preceding decreases in testicular [125I]hCG binding. These transcripts were further inhibited (greater than 93%) between 24-72 h after hCG treatment and returned to 40% and 100% of control levels by days 6 and 9, respectively. In contrast, the truncated 1.8-kb LH receptor transcript was not affected by hCG treatment, indicating a differential suppressive effect of the ligand on its receptor mRNA levels. In the ovary, hybridization to probe EC revealed four transcripts with similar sizes as those found in the testes, with a predominant 7-kb species.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of human luteinizing hormone (LH) receptor: interaction with LH and chorionic gonadotropin from human but not equine, rat, and ovine species.

Studies on human LH receptors are difficult due to the limited availability of clinical samples. Recent cloning of rat and porcine LH receptor cDNAs indicated that these binding sites are single polypeptides of the G-protein-coupled receptor family with seven transmembrane domains. Based on the conserved sequences of rat and porcine receptors, we performed reverse transcription polymerase chain reaction, using human ovarian mRNA as template and obtained partial human LH receptor cDNA clones. Further screening of a human ovary cDNA library and subsequent ligation of individual cDNA clones generated a human LH receptor cDNA containing the entire amino acid-coding region. Sequence analysis indicated that the human receptor cDNA displays 89% and 82% homology at the nucleotide level with its porcine and rat counterparts, respectively. A region spanning the second extracellular and third transmembrane domains is highly conserved among the human LH, FSH, and TSH receptors. The ovarian LH receptor clone is, however, significantly different from an incompletely spliced LH receptor cDNA recently obtained from a human thyroid library. Unlike the thyroid clone, the ovarian LH receptor cDNA could be expressed in the human fetal kidney cell line (293), and radioligand receptor assay identified high affinity (Kd, 1.2 x 10(-10) M) LH/hCG-binding sites on the plasma membrane. Binding specificity of the human LH receptor was studied using recombinant human CG, LH, and FSH secreted by CHO cells transfected with the respective genes. Human CG and LH displaced [125I]hCG binding with an ED50 of 4.3 and 4.8 ng/ml, respectively. In contrast, recombinant FSH was not effective. Treatment of transfected cells with recombinant gonadotropins also induced dose-dependent increases in extracellular cAMP production (hCG = LH much greater than FSH; ED50 25, 10, and greater than 3000 ng/ml). Although equine, rat, and ovine LH as well as equine CG competed effectively for rat testicular LH receptor binding, these hormones were unable to displace [125I]hCG binding to the human receptor, suggesting evolutionary changes in receptor binding specificity and the importance of using human receptors for clinical studies. Thus, the cloning and expression of the human LH receptor cDNA allowed analysis of interactions between human LH receptor and gonadotropins from diverse species. The present work should provide the basis for future design of therapeutic agents capable of interacting with the human receptor and for understanding the structural basis for LH receptor binding to different gonadotropins.

Granulosa cell aromatase bioassay for follicle-stimulating hormone: validation and application of the method.

Stimulation of rat granulosa cell aromatase activity by FSH has recently been used as a sensitive biological end point to develop an in vitro FSH bioassay. The present report provides a detailed validation and application of this assay. In the presence of androstenedione and diethylstilbestrol, FSH stimulated estrogen production in a dose-dependent manner. Although addition of high doses of a phosphodiesterase inhibitor [1-methyl-3-isobutyl xanthine (MIX)] decreased maximal estrogen production, treatment with 0.125 mM MIX increased the sensitivity of granulosa cells to FSH, presumably by minimizing endogenous cAMP breakdown. Addition of insulin and human CG (hCG) further synergistically enhanced granulosa cell sensitivity to FSH. Although inclusion of gonadotropin-free serum obtained from hypophysectomized male rats decreased the assay sensitivity, pretreatment of serum with polyethylene glycol [(PEG) 10-14%] resulted in a dose-dependent decrease in the serum-interfering effect. Studies using exogenous [125I]iodo-rat FSH or RIA measurement indicated recovery of 94-98% FSH after pretreatment of serum with 12% PEG. In the presence of the PEG-pretreated gonadotropin-free serum (4%), ovine, rat, and human FSH preparations induced parallel dose-response curves for estrogen production with minimal detectable doses of 0.12 ng, 0.12 ng, and 0.12 mIU/culture, respectively. In contrast, treatment with GH, PRL, TSH, and ACTH did not affect estrogen production. The apparent stimulatory effect of high doses (greater than 60 ng/culture) of LH and hCG could be attributed to FSH contamination or intrinsic FSH activity in these preparations. Changes in serum bioactive FSH levels were studied in adult male rats after GnRH administration. GnRH (5 micrograms/rat) treatment significantly elevated FSH levels within 30 min after injection. Maximal increases (approximately 2.8-fold) in serum bioactive FSH were observed between 60-120 min. At 8 h after treatment, FSH levels decreased to control levels. Comparison between granulosa cell aromatase bioassay and RIA results indicated no apparent changes in the bio- to immuno- ratio of FSH after GnRH treatment. In conclusion, extreme sensitivity of the bioassay allows the measurement of circulating levels of bioactive FSH. Since rat granulosa cells respond to FSH preparations from different species, the in vitro assay should also provide valuable information on FSH levels in many animal species including those lacking a specific RIA. Measurement of serum levels of bioactive FSH should provide insight regarding the role of FSH in various physiological and pathological conditions.

Homologous regulation of hormone receptors: luteinizing hormone increases its own receptors in cultured rat granulosa cells.

LH receptors in granulosa cells are essential for ovulation and luteinization of ovarian follicles. We have studied the possible role of LH to regulate its own receptors in vitro. Granulosa cells obtained from immature hypophysectomized estrogen-treated rats were primed with FSH for 2 days to induce LH receptors. The cells were then challenged with or without increasing doses of LH or hCG for an additional 2 days, and the concentration of LH receptors was measured by [125I]iodo-hCG binding. FSH-induced LH receptors in granulosa cells decreased to negligible levels in cultures without gonadotropins, while LH receptor numbers were further increased by LH or hCG in a biphasic manner. Maximal stimulation of LH receptor content was obtained with gonadotropin doses of 6, 10, and 2.5 ng/ml for rat LH, ovine LH, and hCG, respectively. In contrast, higher doses of the gonadotropins were less effective. LH stimulation of [125I]iodo-hCG-binding sites was associated with increases in the number of LH receptors, without changes in the Kd value (control, 1.22 +/- 0.22 X 10(-10) M; LH-treated, 2.55 +/- 0.55 X 10(-10) M). Also, the changes in LH receptor numbers were correlated with the responsiveness of granulosa cells to LH stimulation of cAMP production. LH and hCG did not affect overall granulosa cell protein content. However, treatment with cycloheximide, a protein synthesis inhibitor, decreased LH-induced receptors by 46%, suggesting the involvement of new protein synthesis. Thus, these studies have demonstrated that LH, like FSH, is capable of stimulating granulosa cell differentiation by inducing its own receptors. This serves as an interesting model for studies on the positive autoregulation of hormone receptors and explains the important role of LH during advanced stages of follicular maturation.

Growth hormone enhances follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells.

Suppression of serum GH levels in immature rats is associated with delayed onset of puberty and decreased ovarian steroidogenic responsiveness to FSH. To investigate possible direct effects of GH on the differentiation of ovarian cells, granulosa cells from hypophysectomized estrogen-treated rats were cultured with FSH in the presence or absence of GH for 3 days. FSH stimulated granulosa cell LH receptor formation and steroid production in a dose-dependent manner. Concomitant treatment with GH increased LH receptor content by enhancing the action of low doses of FSH without substantial increases in the maximal response. This increase was due to an elevation in the receptor number rather than changes in their affinity for hCG. At 3 ng/ml FSH, concomitant treatment with ovine or bovine GH increased LH/hCG binding in a dose-dependent manner, with 300 ng/ml GH increasing the FSH action by about 3-fold. LH receptors in the GH-treated cells were functional, as indicated by the enhanced cAMP production of these cells in response to LH treatment. The cellular protein content in the FSH-treated cultures was slightly increased by GH (18%), but cell number and viability were unaffected. The change in cell protein content could not account for the increases in the amount of LH receptors. In addition to its effects on LH/hCG receptor content, GH also augmented FSH-stimulated progesterone and 20 alpha-hydroxy-4-pregnen-3-one production in a dose-dependent manner, with 100 ng/ml GH causing significant increases in FSH-induced progesterone production. In contrast, GH treatment did not significantly affect FSH-stimulated estrogen production. The augmentating effects of GH on LH receptor formation and progestin biosynthesis were associated with an enhancement of FSH-stimulated cAMP production. In addition, GH increased forskolin- and 8-bromo-cAMP-induced LH receptor formation and progestin production. Thus, GH-augmented LH receptor induction and progestin biosynthesis may be due to both increased cAMP production and enhanced action of cAMP. The present data have demonstrated that GH augments gonadotropin-stimulated differentiation of ovarian granulosa cells, suggesting an important regulatory role of GH in follicular growth and pubertal development.

Androgen inhibition of follicle-stimulating hormone-stimulated luteinizing hormone receptor formation in cultured rat granulosa cells.

Since LH receptors are decreased in atretic follicles known to contain high androgen levels, we have studied the androgen modulation of LH receptor formation in vitro. Granulosa cells from hypophysectomized, diethylstilbestrol-treated rats were cultured for 3 days with FSH in the presence or absence of nonaromatizable androgens, dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol, or a synthetic androgen, R1881 (17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one). FSH increased LH receptor content in granulosa cells, while concomitant androgen treatment decreased LH receptor content in a dose- and time-dependent manner, without changing the equilibrium dissociation constant (Kd) for human CG. R1881 (10(-7) M), dihydrotestosterone (10(-6) M), and 5 alpha-androstane-3 alpha, 17 beta-diol (10(-6) M) inhibited LH receptor content by 68%, 65%, and 65%, respectively. Similar to earlier findings, these androgens enhanced FSH-stimulated progesterone biosynthesis and aromatase activity in the same cells. To study their LH responsiveness, androgen-treated cells were washed and reincubated for 2 more days with or without LH. Although basal progesterone production was elevated by R1881 pretreatment, the androgen-pretreated cells were less responsive to LH. Treatment with cyanoketone, an inhibitor of 3 beta-hydroxysteroid dehydrogenase, did not alter the inhibitory effects of R1881 on LH receptors, indicating that the androgen action is not mediated by endogenous progestins. Furthermore, R1881 inhibited the stimulation of LH receptor formation by forskolin, cholera toxin, and 8-bromo-cAMP, suggesting that androgens may inhibit LH receptor induction by affecting post-cAMP events. Estrogen treatment enhanced the FSH induction of LH receptor content, while concomitant addition of R1881 also suppressed the estrogen action. Thus, androgens inhibit FSH-induced functional LH receptors in cultured rat granulosa cells. The androgen effect is exerted, at least partially, at post-cAMP sites and is independent of changes in progestin biosynthesis.

Deglycosylated human chorionic gonadotropin (hCG) antagonizes hCG stimulation of 3',5'-cyclic adenosine monophosphate accumulation through a noncompetitive interaction with recombinant human luteinizing hormone receptors.

In the rat, the antagonistic properties of deglycosylated (dg) gonadotropins in vitro are characterized by high affinity receptor binding but impaired ability to stimulate cAMP accumulation. In human, the functional role of N-linked sugars in human CG (hCG) action is unclear because of the unavailability of totally deglycosylated hCG and because of the difficulty involved in obtaining human gonadal tissues. We have recently prepared completely deglycosylated hCG using site-directed mutagenesis and expressed functional human LH (hLH) receptors using cloned complementary DNA. Since hLH receptor shows distinct ligand specificity from that of rat LH receptor, we examined binding kinetics and signal transduction of recombinant dg-hCG using recombinant hLH receptors. In embryonic human kidney cells (293) transfected with hLH receptor complementary DNA, 125I-hCG binding to its receptor was studied in the presence of varying amounts of unlabeled dg-hCG or wild type (WT)-hCG. Lineweaver-Burk analysis of the binding kinetics showed that the displacement of 125I-hCG by dg-hCG was noncompetitive whereas that seen for WT-hCG was competitive. The noncompetitive nature of dg-hCG binding was further confirmed using rat LH receptors present in testis membrane preparations. After preincubation of LH receptor-expressing 293 cells with WT-hCG, inclusion of 125I-hCG competitively displaced WT-hCG. In contrast, preincubation with dg-hCG prevented subsequent 125I-hCG binding to human LH receptor for at least 46 h. WT-hCG caused a dose-dependent increase in cAMP accumulation in the 293 cells with an ED50 of 10 ng/ml. However, dg-hCG was ineffective in inducing cAMP production with a maximal effect of only 12% of that stimulated by WT-hCG. In the presence of increasing doses of dg-hCG, stimulation of cAMP by WT-hCG was antagonized in a dose-dependent manner. In contrast, forskolin stimulation of cAMP was not antagonized by dg-hCG, indicating receptor-mediation of dg-hCG action. Similar to binding studies, preincubation with dg-hCG also dose-dependently blocked the subsequent stimulatory effect of WT-hCG on cAMP production. Thus, the noncompetitive binding of dg-hCG to hLH receptors and its antagonism of hCG stimulation of cAMP accumulation suggest that dg-hCG is an irreversible receptor blocker with unique antagonistic properties.

Gonadotropin-induced up- and down-regulation of rat ovarian LH receptor message levels during follicular growth, ovulation and luteinization.

Induction of follicular growth by PMSG is associated with increased ovarian LH receptor content, whereas the preovulatory surge of LH decreases LH binding sites, followed by a secondary increase in receptor numbers coincident with corpora lutea formation. Based on the recently reported LH receptor cDNA sequence, we have performed a reverse transcription-polymerase chain reaction to obtain LH receptor cDNA clones and generated a 32P-labeled cRNA probe to examine the dynamic changes in ovarian LH receptor mRNA levels during gonadotropin induction of follicular growth, ovulation and luteinization. Northern blot analysis of ovary RNA revealed hybridization signals of about 7.0, 4.2, 2.5 and 1.8 kb, with no hybridization to nongonadal tissues. PMSG increased the intensity of all four LH receptor messages at 24 h, preceding an increase in LH receptor number, with peak LH receptor mRNA and receptor content observed at 52 h. Treatment with hCG resulted in decreased LH receptor binding and mRNA levels by 6 h after injection, with maximal inhibition (greater than 85%) of message at 12 to 24 h after hCG treatment. Subsequently, LH receptor message levels increased again at 3 days after hCG, concomitant with increased [125I]hCG binding. A further increase in LH receptor content, but not message levels, was observed 5 days after hCG. These results demonstrate that the induction of LH receptors by PMSG is preceded by increased LH receptor mRNA levels. Furthermore, ligand-induced down-regulation of the LH receptor following an ovulatory dose of hCG is associated with decreased LH receptor message content, followed by increases in LH receptor message levels and binding sites during subsequent luteinization. Thus, the up- and down-regulation of ovarian LH receptors during follicle growth, ovulation and luteinization is probably due, at least in part, to changes in receptor message modulation.

Disparate effects of triphenylethylene antiestrogens on estrogen and progestin biosyntheses by cultured rat granulosa cells.

The influence of triphenylethylene antiestrogens on FSH-stimulated production of estrogen, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) was examined in cultured rat granulosa cells. The cells were cultured with increasing concentrations of FSH, with or without diethylstilbestrol (DES) or various antiestrogens. After 3 days, medium progesterone and 20 alpha-OH-P contents were determined. Cells were reincubated for an additional 8 h with delta 4-androstene-3,17-dione, and estrogen formation was measured. Steroid production was negligible by cultures treated with DES or antiestrogens alone. FSH treatment increased estrogen and progestin production, while the addition of DES (10(-7) M) further enhanced FSH-stimulated steroidogenesis. Likewise, treatment with 10(-6) M of the antiestrogens tamoxifen (Tam), nafoxidine (Naf), or clomiphene citrate (CC) also enhanced FSH-stimulated aromatase activity. In contrast, the antiestrogens each inhibited FSH stimulation of progesterone and 20 alpha-OH-P production (greater than 73% inhibition at 300 ng/ml FSH). Increasing concentrations (3 X 10(-8)-10(-6) M) of the antiestrogens augmented the stimulatory effect of FSH (10 ng/ml) on estrogen production in a dose-related manner (CC greater than Tam greater than Naf). Similar doses of these antiestrogens inhibited the stimulatory effect of FSH (300 ng/ml) on progesterone and 20 alpha-OH-P production (Tam greater than CC greater than Naf). The observed inhibition of progestin production is associated with decreases in FSH-stimulated pregnenolone biosynthesis in antiestrogen-treated cells incubated with 10(-6) M cyanoketone. Furthermore, the antiestrogens inhibited the binding of [3H]estradiol to ovarian estrogen receptors with binding affinity constants of 364, 437, and 2144 nM for CC, Tam, and Naf, respectively. Thus, antiestrogens exert disparate actions on granulosa cell estrogen and progestin biosyntheses. Like estrogens, CC, Tam, and Naf enhance FSH-stimulated aromatase activity with potencies comparable with their abilities to interact with ovarian estrogen receptors. However, unlike estrogens, the antiestrogens inhibit FSH-stimulated progestin biosynthesis, partially via suppression of pregnenolone biosynthesis. The present granulosa cell culture system provides a valuable model for elucidating the disparate actions of estrogens and antiestrogens on ovarian steroidogenesis.

Acute stimulation of human chorionic gonadotropin secretion by recombinant human activin-A in first trimester human trophoblast.

Inhibin and activin are synthesized by the placenta, raising questions as to their functions in this tissue. The possibility of local regulation of other placental hormones critical during pregnancy was investigated by examining the effects of recombinant human inhibin-A and activin-A on hCG secretion. Potential interactions with GnRH-stimulated hCG secretion were also explored. First trimester human placental tissue was physically dissociated to isolate trophoblast cells. Cells were cultured on microcarrier beads for 7-10 days and loaded into 1.5-ml chambers in a perifusion system. After a 1-h control period, hormone treatments were administered in the perifusion medium for 5 min, and perifusate was collected for an additional 55 min. Perifusate fractions were analyzed for hCG concentration by RIA. Both activin and GnRH stimulated a rapid and transient hCG secretory response in the perifusion system. Unlike the effect of GnRH, the activin-stimulated hCG response was not blocked by concomitant treatment with a GnRH antagonist. The hCG RIA results were confirmed by preliminary experiments using a novel hCG bioassay. This suggested that activin did not facilitate hCG secretion by stimulation of endogenous GnRH release. Inhibin alone did not affect hCG secretion or GnRH-stimulated hCG secretion. However, treatment with inhibin and activin in combination resulted in a transient increase in hCG, followed by a decrease in hCG secretion to below pretreatment levels. The results suggested that in addition to GnRH, activin may play a role in the regulation of hCG secretion in first trimester placenta.

Expression of recombinant human follicle-stimulating hormone receptor: species-specific ligand binding, signal transduction, and identification of multiple ovarian messenger ribonucleic acid transcripts.

The ligand specificity and biochemical properties of the human (h) FSH receptor are poorly characterized due to the low abundance of these receptors and the limited availability of human tissues. Using a fragment of rat FSH receptor cDNA, we screened a human testicular cDNA library and obtained a FSH receptor cDNA covering the entire amino acid-coding region. After transfection of a human fetal kidney cell line (293) with the hFSH receptor cDNA, radioligand receptor analysis revealed the presence of high affinity (Kd, 1.7 x 10(-9) M) FSH-binding sites on the plasma membrane. Both recombinant and wild-type hFSH displaced [125I]hFSH binding, with ED50 values of 25 and 70 ng/ml, respectively, whereas hLH, hCG, and hTSH were ineffective. Although human, rat(r), and ovine FSH as well as equine CG competed for rat testicular FSH receptor binding, only hFSH and rFSH interacted effectively with the recombinant hFSH receptor, suggesting that species-specific ligand recognition exists between human and rodent receptors. After incubation of transfected cells with hFSH, but not recombinant hLH or hCG, a dose-dependent increase (ED50, 10 ng/ml) in extracellular cAMP accumulation was observed, indicating a functional coupling of the expressed human receptor with the endogenous adenyl cyclase. In cells cotransfected with the FSH receptor expression plasmid and a luciferase reporter gene driven by the promoter of a cAMP-responsive gene, treatment with hFSH, but not hCG, resulted in a dose-dependent increase in luciferase activity. Northern blot analysis using a cRNA probe derived from the human receptor cDNA indicated the presence of multiple FSH receptor mRNA transcripts (7.0, 4.2, and 2.5 kilobases) in RNA prepared from human follicular phase ovary, but not from human corpus luteum or placenta. Additionally, two FSH-binding sites of 76 and 112 kilodaltons were detected in transfected 293 cells after ligand/receptor cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These results demonstrate the expression of functional hFSH receptor with unique ligand specificity and provide new data on the biochemical properties of the human receptor at the mRNA and protein levels.

Serum bioactive follicle-stimulating hormone during the human menstrual cycle and in hyper- and hypogonadotropic states: application of a sensitive granulosa cell aromatase bioassay.

A sensitive in vitro assay based on the stimulation of estrogen production by cultured rat granulosa cells was recently developed for the measurement of biologically active FSH. This bioassay system is specific for FSH, highly sensitive, and capable of measuring basal FSH levels in rat serum. The granulosa cell aromatase bioassay was improved by the use of additives known to enhance FSH activity and by pretreatment of serum with 12% polyethylene glycol to remove inhibitory substances. We applied this method to the measurement of bioactive FSH levels in serum samples from human subjects. As determined in daily blood samples during ovulatory menstrual cycles in seven women, bioactive FSH levels exhibited a pattern closely resembling that of immunoreactive FSH. The mean bioactive serum FSH levels were 29.9, 20.5, 39.2, and 14.8 mIU/ml for the early follicular phase, late follicular phase, preovulatory surge, and luteal phase, respectively. The bio- to immunoratio (B:I) throughout the menstrual cycle ranged from 1.4-3.4, with a mean of 2.5. The ratios for early follicular phase, late follicular phase, preovulatory surge, and luteal phase were 2.7, 2.3, 1.4, and 2.6, respectively. The correlation coefficient (r) of the serum FSH values obtained by bioassay and RIA was 0.91. FSH bioactivity was also measured in patients in each of the following categories with the following mean values: oral contraceptive pill users (undetectable), hypothalamic amenorrhea (18.7 mIU/ml; B:I, 2.6), premature ovarian failure (163 mIU/ml; B:I, 1.7), and postmenopausal women (191 mIU/ml; B:I, 1.6). These findings suggest that measurement of immunoreactive FSH levels correctly reflects the biological activity of FSH in serum of cycling women and patients in certain hyper- and hypogonadotropic states. The granulosa cell aromatase bioassay represents a new tool for future assessments of biologically active FSH in physiological and pathophysiological conditions.