A novel, spectrophotometric microneutralization assay for respiratory syncytial virus. We describe a simple and rapid microneutralization assay for respiratory syncytial virus (RSV) based on the colorimetric quantitation of the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to a formazan product by the mitochondria of viable cells. Conditions for RSV infectivity were first optimized for sensitivity and reproducibility based on cell density and on RSV concentration as a function of multiplicity of infection (MOI) and time post-infection and the resulting optical densities were shown to be inversely proportional to MOI. For RSV neutralization, dilutions of heat-inactivated human plasma were preincubated with RSV and complement prior to infection of cells in microtiter plates. Following MTT dye conversion, 50% RSV neutralization titers were determined by linear regression analysis of the optical density values and endpoints were markedly influenced by MOI. The MTT-based assay was shown to be comparably sensitive to the plaque reduction assay for quantitation of neutralizing antibody, but more readily adaptable to the screening of a large number of samples. Finally, we demonstrated that the MTT microneutralization assay for RSV was useful for quantitation assay of neutralization activity in sera of mice and cotton rats.


A statistical method for the detection of false positives and false negatives in microtitre format PCR assays. A method is described which can be used to determine whether a series of PCR reactions carried out in a microtitre plate are inherently unlikely to have occurred by chance, and hence to show 'false' results. The method is an extension of the familiar 'runs test' which can be used for tube-based PCRs. A Monte Carlo simulation program is discussed which can be used to generate expected probability distributions for either symmetric or asymmetric plate designs. In addition, systematic departures from a random pattern due to 'edge effects' can be detected.


Viral susceptibility of an immortalized human microvascular endothelial cell line. CDC/EU.HMEC-1 is the first immortalized human microvascular endothelial cell line that retains morphologic, phenotypic, and functional characteristics of a normal human microvascular endothelial cell. This study evaluates a variety of viruses and their effects on this human endothelial cell line. The data indicate that adenoviruses, some herpesviruses, reoviruses and most picornaviruses grow well in HMEC-1, with distinctive cytopathic effects. The paramyxoviruses, however, do not appear to propagate, nor does HIV. The findings indicate that microvascular endothelial cells may act as a reservoir of these viruses; it also suggests the possibility that microvascular endothelium could be involved in the processing and presentation of antigen to immune cells.


Highly sensitive digoxigenin-labelled DNA probe for the detection of potato spindle tuber viroid. A molecular probe pSPAv6.2(+), with concatameric insert representing 6.2-times repeated copy of potato spindle tuber viroid (PSTV) RNA, was labelled with digoxigenin and used to detect PSTV by dot-blot hybridization assay. The probe was highly sensitive and specific, detecting as little as 2.5 pg of PSTV RNA. Both severe and mild PSTV strains were detectable in 64-512-times diluted crude extracts from infected tomato leaves, and potato leaves, sprouts, and seeds. For extraction of plant tissue three buffers were compared to determine the lowest non-specific background and the highest sensitivity. The results showed that the digoxigenin-labelled probe is as sensitive as the 32P-labelled probe and can replace radioactive techniques in PSTV detection. With such high sensitivity, the probe is also potentially useful for detecting the viroid in composite samples of mass-indexing programs.


Recombinant parvovirus B19 capsids as a new substrate for detection of B19-specific IgG and IgM antibodies by an enzyme-linked immunosorbent assay. A new enzyme-linked immunosorbent assay for the detection of B19-specific IgG and IgM antibodies was established using B19 capsids synthesized in a baculovirus expression system. These B19 capsids, consisting of either coat protein VP2 alone or of both VP1 and VP2, have been shown to be similar to native virus in size and appearance. The results obtained for the detection of B19-specific antibodies showed good correlations with a radioimmunoassay which uses native B19 virus and an immunofluorescence assay based on insect cells expressing coat protein VP1. The course of the antibody response could be followed by determining the titers of sequential serum samples taken after a recent B19 infection. Both types of recombinant capsids form an excellent source of antigen for the detection of both B19 IgG and IgM antibodies and are a very promising substitute for native virus.


Competitive polymerase chain reaction using an internal standard: application to the quantitation of viral DNA. A general strategy for the construction of an internal standard for the polymerase chain reaction (PCR) is described together with its application in the evaluation of clinical samples. This internal standard is a plasmid containing a modified target sequence that is co-amplified with the native target using the same set of primers. The co-amplification reaction will generate two fragments of different size that are readily separated without the need for restriction enzyme digestion. Thereafter, they are detected and quantitated by hybridization to the same probe. Detection of HIV proviral DNA was chosen as a model for this competitive PCR. The assay proved to be a sensitive tool for the detection of PCR inhibitors and allowed quantitation of HIV with a 20-30% variation coefficient. Despite limitations that appear inherent to the amplification process, internal standards appear to be useful tools for quantitative analysis by PCR.


Comparison of eight different procedures for harvesting avian reoviruses grown in Vero cells. 14 avian reovirus isolates adapted to replicate in an African green monkey (Vero) cell line were studied for the nature of their replication. The growth curves of 5 viruses showed them to be highly cell-associated in Vero cells. Different procedures were examined for releasing the cell-associated virus following propagation in Vero cells, including several freeze-thaw cycles, treatment with sterile distilled deionized water (ddH2O), freon extraction, and trypsin treatment. Treatment of virus infected cultures with ddH2O was the most effective, and trypsin treatment was the least effective procedure for dissociation of virus from cells. Treatment of virus infected cultures with ddH2O is a simple and effective procedure which can be used where large amounts of virus are required for experimental purposes.


A rapid chemiluminescent detection method for barley yellow dwarf virus. Barley yellow dwarf virus (BYDV-PAV-IL) was detected with biotinylated in vitro transcript cDNA using a chemiluminescent substrate on nylon membranes. Signals were detected on X-ray film and quantified using either a densitometer or an ELISA plate reader. The time required for sample preparation was reduced so that the entire protocol could be completed in two days. The in vitro transcript probes could detect 1 ng of purified virus and as little as 1 microliter of sap extracts prepared from infected oat shoots.


The assessment of nucleotide sequence diversity by the polymerase chain reaction is highly reproducible. The polymerase chain reaction (PCR) is often used to assess the diversity of viral nucleotide sequences present in various biological samples (e.g., blood and tissues). However, it is not clear how reproducible this approach may be. DNA was extracted from the peripheral blood lymphocytes of a macaque that had been infected, experimentally, with SIVmac251-32H 6 months previously. The nef gene was then amplified by the PCR on three separate occasions from this same template preparation. A panel of clones was prepared from the product of each PCR, the entire nef gene was sequenced and the sequences obtained compared with each other. Phenogram analysis revealed that within each panel the same degree of sequence diversity was observed between clones. Furthermore, when the sequences obtained from all three panels were compared, the overall sequence diversity observed was no greater than that observed for each panel individually. These data indicate that the analysis of sequence diversity in SIV 'quasi-species' populations by the PCR is reliable and, more important, reproducible.


A nylon membrane enzyme immunoassay for rapid diagnosis of influenza A infection. A new membrane-enzyme immunofiltration assay (MIFA) was developed for rapid diagnosis of influenza A infection. The pretreated specimens were dispensed into a 1.2 micron Biodyne B nylon membrane-bottomed microplate and vacuum filtration was applied. Blocking solution, peroxidase-conjugated anti-influenza A nucleoprotein monoclonal antibody, washing buffer and substrate were added in that order. The assay was completed within 30 min. Out of 103 nasopharyngeal swabs collected in transport medium, 31 isolates of influenza A virus were obtained and 22 specimens were detected directly by the MIFA technique. The 9 isolation-positive MIFA-negative specimens required 6 days or more for viral detection in cell culture, and probably contained a very low quantity of virus. The 72 cell culture negative specimens were also negative by MIFA. Comparison with a classical immunocapture assay (ICA) gave a better sensitivity for MIFA, as only 15/103 specimens were positive by ICA. MIFA is a rapid test with 71% sensitivity and 100% specificity. It was also very useful to test the cell culture supernatants, as a sensitivity of 100% was obtained with MIFA when the immunofluorescence technique was positive. The same technique could be readily carried out on the same plate for other respiratory viruses since capture antibody is not used.


Effect of increasing doses of recombinant human insulin-like growth factor-I on glucose, lipid, and leucine metabolism in man. The metabolic effects of recombinant human insulin-like growth factor-I (IGF-I) were assessed in five groups of normal male overnight-fasted volunteers receiving infusions of either 0, 5, 7.5, 15, or 30 micrograms/kg.h IGF-I during 8 h, resulting in total plasma IGF-I concentrations 127 +/- 7, 247 +/- 30, 389 +/- 39, 573 +/- 62, 620 +/- 105 ng/ml, respectively. Glucose consumption (euglycemic glucose clamp) increased dose dependently during IGF-I infusion (P < 0.001) up to 6.7 +/- 1.3 mg/kg. min in the 30 micrograms/kg.h group. Plasma triglyceride concentrations decreased with increasing doses of IGF-I (P < 0.03); the fall was 43% in the 30 micrograms/kg.h group. Plasma free fatty acid concentrations decreased during 7.5, 15, and 30 micrograms/kg.h IGF-I by 23%, 34%, and 48%, respectively. IGF-I lowered plasma beta-hydroxybutyrate concentrations in a dose-dependent manner (P < 0.025). Plasma concentrations of leucine and alpha-ketoisocaproate decreased dose dependently (P < 0.001 and P < 0.015). Whole body leucine flux (1-13C-leucine infusion technique) decreased with increasing doses of IGF-I by 41% during 30 micrograms/kg.h, indicating decreased whole body protein breakdown. Leucine oxidation into 13CO2 decreased with increasing doses of IGF-I (P < 0.045) by 57% in the 30 micrograms/kg.h group, suggesting inhibition of irreversible loss of leucine. Plasma C-peptide and insulin concentrations decreased dose dependently (P < 0.005 and P < 0.02), indicating diminished insulin secretion. Thus, acute elevation of plasma IGF-I concentrations in man results in metabolic effects which are qualitatively similar to those described previously of insulin.


Low dose recombinant human insulin-like growth factor-I fails to affect protein anabolism but inhibits islet cell secretion in humans. The in vivo effects of recombinant human insulin-like growth factor-I (rhIGF-I) on whole body protein metabolism were studied to ascertain whether rhIGF-I has comparable effects as those reported with rhGH use in humans. The doses of rhIGF-I chosen achieved similar plasma IGF-I concentrations as those achieved after 7 days of rhGH injections. Eight normal volunteers were studied using [1-13C]- and [1-14C]leucine tracers, before, 4 h, and 28 h after a continuous infusion of rhIGF-I at 5 micrograms kg-1 h-1 (n = 6) and 10 micrograms kg-1 h-1 (n = 2). Two additional subjects were studied in a protein catabolic state after 7 days of high dose (0.8 mg kg-1 day-1) glucocorticosteroid administration. Plasma concentrations of rhIGF-I were similar using either 5 or 10 micrograms kg-1 h-1 and increased to values approximately 300% above baseline by 28 h of infusion. No decrease in the plasma glucose concentration was observed during the 28-h infusion; however, plasma insulin, C-peptide, and glucagon concentrations significantly decreased, whereas plasma free fatty acids were not affected. No changes were observed in the rate of proteolysis (as estimated by the rate of leucine appearance), the rate of leucine oxidation, or the rate of protein synthesis in the absence or presence of glucocorticosteroid treatment. Plasma concentrations of insulin-like growth factor binding protein-3 did not change during the rhIGF-I infusion whereas they increased 50% in subjects who received rhGH, and in whom rhGH caused a potent protein anabolic effect. These results suggest that rhIGF-I may have a somatostatin-like effect. In addition, we found that rhIGF-I infusion is insufficient to promote protein anabolism. This may be due to the failure of rhIGF-I alone to induce a pivotal GH-dependent cofactor(s) necessary for IGF-I to elicit an anabolic effect on protein metabolism in humans.


Temporal pattern of pancreatic insulin and C-peptide secretion and of plasma glucose levels after nutritional stimulation. The dependency of the secretory pattern of insulin and C-peptide on either oral ingestion of the energy substrates glucose and protein or gastric distension was determined in nine healthy male subjects. To analyze secretion dynamics, high frequency blood sampling, computed estimation of individual hormone half-lives, deconvolution of data, and pulse analysis of the deconvoluted data by the Cluster program were used. After stimulation with oral glucose and protein, baseline insulin, C-peptide, and glucose levels increased in parallel, forming two or three large increases (macropulses), with a mean duration of 63.8 min. The frequency of high frequency insulin and C-peptide pulses was unchanged, whereas a significantly increased amplitude formed the basis of insulin/C-peptide macropulses after both oral stimulations. No changes in baseline insulin/C-peptide concentrations or in amplitude or frequency were observed after a challenge with 400 mL H2O (n = 3). Gastric distension with an equal volume of H2O (400 mL) did not influence pancreatic hormone secretion. Insulin and C-peptide secretions were pulsatile, with a frequency of approximately one pulse per 12 min correlated to C-peptide pulses. When calculated by multiple regression analysis glucose, insulin and C-peptide plasma levels increased simultaneously after the challenge with either glucose or protein, suggesting a neuronal or humoral intestinal-pancreatic regulation of pancreatic hormone secretion. These findings suggest that high frequency insulin and C-peptide pulses form the basis of insulin and C-peptide plasma levels after meal stimulation.


Testosterone buciclate (20 Aet-1) in hypogonadal men: pharmacokinetics and pharmacodynamics of the new long-acting androgen ester. Due to unfavorable pharmacokinetics of the available androgen esters for substitution therapy of male hypogonadism, there is a demand for new testosterone (T) preparations producing constant serum levels in the physiological range. To assess the pharmacokinetics and pharmacodynamics of the new ester testosterone buciclate (TB) [20 Aet-1] in hypogonadal men a clinical phase I-study was performed. After two control examinations 8 male patients with primary hypogonadism were randomly assigned to 2 treatment groups (n = 2 x 4) given single doses of either 200 (group I) or 600 mg (group II) TB im. Blood samples were obtained 1, 2, 3, 5, and 7 days post injection and then weekly in the course of 4 months. In group I serum androgen levels did not rise to normal values. However, in group II androgens increased significantly and were maintained in the normal range up to 12 weeks with maximal serum levels of 13.1 +/- 0.9 nmol/L (mean +/- SE) in study week 6. No initial peak release of T was observed in either study group. Pharmacokinetic analysis revealed a terminal elimination t1/2 beta of 29.5 +/- 3.9 days and a mean residence time of 65.0 +/- 9.9 days in group II. In one patient in group II dihydrotestosterone levels slightly exceeded the upper normal limit during the study course. Sex hormone-binding globulin remained unchanged and estradiol serum levels never exceeded the normal range in any patient. In group II gonadotropins were significantly suppressed, whereas no change was seen in group I. A significant increase in body weight, hematological parameters, and libido/potency was observed after TB injection which was more pronounced in the higher dose group. Regardless of the dose administered, no significant change was seen in uroflow, prostate volume measured by transrectal ultrasonography, or prostate specific antigen. No adverse side-effects including changes in clinical chemistry were observed. In conclusion, single injections of 600 mg TB in hypogonadal patients show favorable pharmacokinetics and pharmacodynamics. This new long-acting T ester is a promising new agent for substitution therapy of male hypogonadism and for male contraception.


Acute effects of testosterone infusion and naloxone on luteinizing hormone secretion in normal men. To evaluate the role of endogenous opioid pathways in the acute suppression of LH secretion by testosterone (T) infusion in men, we studied eight normal healthy volunteers who received a saline infusion, followed 1 week later by a T infusion (960 nmol/h) starting at 1000 h and lasting for 33 h. After 2 h of infusion (both saline and T), four iv boluses of saline were given hourly, and after 26 h of infusion, four hourly iv boluses of naloxone were given. Blood was obtained every 15 min for LH and every 30 min for T. T infusion increased the mean plasma T concentration 2.1-fold (18.7 +/- 2.1 to 39.5 +/- 3.5 nmol/L, saline vs. T infusion, P < 0.01). The mean plasma LH concentration was 7.9 +/- 0.5 IU/L during the saline control study and was decreased to 6.9 +/- 0.6 IU/L by the infusion of T (P < 0.05). LH pulse frequency was similar during both saline and T infusions (0.48 +/- 0.02 vs. 0.43 +/- 0.04 pulses/man.h, saline vs. T infusion). The mean LH pulse amplitude decreased from 4.3 +/- 0.4 IU/L during saline infusion to 3.3 +/- 0.2 IU/L during T infusion (P < 0.05). The administration of naloxone increased the mean plasma LH concentration significantly during saline infusion (7.6 +/- 0.4 to 10.0 +/- 0.9 IU/L, saline vs. naloxone boluses, P < 0.01), but not during T infusion (6.9 +/- 0.6 vs. 7.3 +/- 0.6 IU/L). LH pulse frequency increased significantly after the administration of naloxone during both saline and T infusions (0.54 +/- 0.04 to 0.71 +/- 0.08 pulses/man.h, saline vs. naloxone boluses during saline infusion, and 0.46 +/- 0.08 to 0.60 +/- 0.07 pulses/man.h during T infusion; P < 0.05). LH pulse amplitude was suppressed by T infusion, but administration of naloxone did not reverse this suppression. The mean amplitude of the LH response to exogenous GnRH (250 ng/kg) was decreased by T infusion from 48 +/- 13.5 to 31.2 +/- 8.5 IU/L (P < 0.01). Therefore, in men, the administration of naloxone increases LH pulse frequency during both saline and T infusions, but the acute suppression of LH pulse amplitude seen with T infusion was not reversed by naloxone. This pattern contrasts sharply with the effects of T infusion in pubertal boys, as elucidated by our earlier studies. The negative feedback effects of T on LH secretion are primarily hypothalamic in early pubertal boys and change to pituitary suppression in men.


Autoantibody to human prolactin in patients with idiopathic hyperprolactinemia. We have demonstrated the presence of anti-PRL autoantibody in 5 patients with idiopathic hyperprolactinemia. The clinical features were suggestive of a weak biological activity of PRL, such as regular menses and no galactorrhea. Total PRL levels were markedly elevated (685 +/- 386 micrograms/L) (mean +/- SD) and the proportion of the bound form was 90.7 +/- 7.1%. Scatchard analysis revealed a low-affinity, high-capacity antibody: the association constant was 0.73 +/- 0.56 x 10(7) mol-1 and the maximal binding capacity was 2139 +/- 1792 micrograms/L. Gel filtration study showed that a substantial amount of PRL (64.6 +/- 19.5%) was eluted at the position of 150,000-170,000 mol wt PRL (big-big PRL). Immunoprecipitation study using the chain-specific antibodies showed that the anti-PRL autoantibody belonged to kappa-type immunoglobulin G. These results may indicate that there exists autoantibody-related hyperprolactinemia, especially in those with particularly high serum PRL levels, who had previously been diagnosed as "idiopathic" hyperprolactinemia.


Synthesis and release of endothelin-1 by human decidual cells. We have studied whether a novel vasoconstrictor, endothelin-1 (ET-1), is synthesized by and released from human decidual cells in early pregnancy, and whether ET-1 acts directly on their own cells. It was observed that ET-1-like immunoreactivity (ET-1-LI) was released from cultured decidual, but not villous, cells, as a function of time. Reverse-phase high-pressure liquid chromatography of the conditioned media from the decidual cells revealed a major peak of ET-1-LI coeluting with standard ET-1. Phorbol myristate acetate, a protein kinase C activator, dose-dependently increased the release of ET-1-LI from the decidual cells, while a protein kinase C inhibitor, H7, significantly attenuated the stimulatory effect of phorbol myristate acetate on ET-1-LI release. Northern blot analysis demonstrated the expression of messenger RNA for prepro-ET-1 in the decidual tissue, but no such messenger RNA was observed in the villous tissue. The human decidual tissue contained a noninteracting, single class of binding sites demonstrating higher affinity for ET-1 and ET-2 than ET-3. This would be most consistent with the ETA receptor subtype. An ET-1-induced, dose-dependent accumulation of total inositol phosphates was also observed in human decidual cells prelabeled with myo-[3H]inositol. The present results demonstrate for the first time that human decidual cells in early pregnancy can synthesize and release ET-1. These cells also possess specific functional receptors for ET-1 which are coupled to phosphoinositide hydrolysis. Thus our data suggest a possible role for ET-1 in autocrine and/or paracrine function in human decidual cells.


Growth hormone (GH)-binding protein regulation by estrogen, progesterone, and gonadotropins in human: the effect of ovulation induction with menopausal gonadotropins, GH, and gestation. The recently described serum GH-binding protein (GH-BP) may reflect the GH-receptor level. To assess the serum GH-BP levels under various physiological and supraphysiological levels of sex steroids, we have evaluated its concentration in 26 patients undergoing 34 cycles of ovulation induction with either human menopausal gonadotropins (hMG)/hCG or GH/hMG/hCG. The latter ovulation induction protocol was undertaken in "Clonidine negative" patients in a prospective, randomized, crossed-over manner, between GH/hMG/hCG or hMG/hCG. The increase in GH-BP levels in patients' sera undergoing ovulation induction directly correlated with peripheral estradiol (E2) (r = 0.577; P < 0.001), and with peripheral progesterone (P4) (r = 0.542; P < 0.001; n = 174) concentrations in hMG/hCG cycles, and also in GH/hMG/hCG cycles (r = 0.669, P < 0.001 for E2, and r = 0.722, P < 0.001 for P4, n = 84). GH-BP levels did not change significantly in response to 0.15 mg Clonidine ingestion. The baseline GH-BP levels significantly correlated with the body mass index of 47 patients (r = 0.547; P < 0.001). The insulin-like growth factor-I (IGF-I) concentrations increased in correlation with increasing E2 levels up to 5500 pmol/L but decreased thereafter, at very high E2 concentrations. In gestations generated by ovulation induction with hMG/hCG or GH/hMG/hCG, longitudinal measurements of GH-BP levels showed an initial sharp increase during early gestation, followed by a gradual decrease beginning around the end of the first trimester and continuing during the mid and late trimesters. In normal spontaneous pregnancies, GH-BP levels, measured from the fifth week until term, were negatively correlated with gestational age (r = -0.581; P < 0.001; n = 84). This pattern is highly suggestive of the possibility that GH-BP is coregulated not only with E2 and P4 but also with hCG, and possibly other gonadotropins as well. Indeed, in patients with "resistant ovaries", pharmacological amounts of hMG failed to increase E2 concentrations but moderately increased GH-BP levels. These data provide good indirect evidence for coregulation of the GH-BP with both sex steroids and gonadotropins. The mid and late trimesters decrease in GH-BP levels in spite of increasing sex steroids concentrations, may be attributed to the decreasing hCG concentrations, and/or to the increasing secretion of placental lactogen (PL) and placental GH with the advancing gestation.


Effects of unopposed conjugated equine estrogen on lipoprotein composition and apolipoprotein-E distribution. Administration of conjugated equine estrogen to 31 postmenopausal women for 3 months produced 14.6% and 9.4% decreases in low density lipoprotein cholesterol (LDL-C) and apolipoprotein-B (apoB), and 11.5%, 12.7%, and 9.6% increases in high density lipoprotein cholesterol (HDL-C), apoA-I and apoA-II, respectively. Phospholipids of HDL2 and HDL3 were increased 57.9% and 19.3%, respectively, while relatively small increases in cholesterol of the two subfractions were not significant. Compositions of LDL and HDL and its subfractions were altered substantially with estrogen treatment. The proportion of LDL triglyceride to LDL-C was increased. The phospholipid content in both the HDL2 and HDL3 subfractions (compared to cholesterol) was increased significantly (34.8% and 10.7%, respectively), while the triglyceride content was increased only in the HDL2 subfraction (43.6%). Estrogen use also caused a 9.1% reduction in total apoE levels and a redistribution of apoE to the very low density lipoprotein (VLDL) from the LDL plus HDL fraction, resulting in a significant 19.5% decrease in apoE in the LDL plus HDL fraction. Changes in apoE in the VLDL fraction were associated positively with changes in the cholesterol levels of the VLDL fraction and inversely with changes in LDL-C and apoB levels, while changes in apoE in the LDL plus HDL fraction were associated positively with changes in the levels of HDL-C. Thus, estrogen causes alterations in lipoproteins that could potentially affect their metabolism and/or function.


Sustained improvement in growth velocity and recovery from suboptimal growth hormone (GH) secretion after treatment with human pituitary GH-releasing hormone-(1-44)-NH2. The sustained effect of human pancreatic GH-releasing hormone [hpGHRH-(1-44)-NH2] on growth rate and GH secretory patterns was studied in 14 patients (10 males and 4 females; aged 10-16 yr; all Tanner stage I or II). Nine children had inadequate spontaneous GH secretion (ISGHS), while 5 had classic GH deficiency. Seven of 9 patients with ISGHS and 1 of 5 patients with GH deficiency were given 2 sc injections/day of 5 micrograms/kg GHRH for 2-3 months; the others received 5 pulses of GHRH (5 micrograms/kg BW.pulse) for 6 nights a week for 2-13 months, given every 3 h. Six of the nine ISGHS patients increased their growth velocity in response to GHRH therapy. These same six patients maintained an increased growth velocity for up to 24 months after GHRH was discontinued. The remaining three ISGHS patients did not show a significant growth response to GHRH administration. Neither a temporary nor a sustained growth response was correlated with spontaneous overnight GH secretion in these patients. In contrast, three of five classical GH deficiency patients exhibited increased growth velocity while undergoing GHRH therapy, but growth returned to preintervention rates upon discontinuation of treatment. The other two of the five classic GH deficiency patients failed to demonstrate any growth response to GHRH treatment. The increased growth velocity that was sustained for long intervals even after discontinuation of GHRH in ISGHS patients may indicate restoration of normal regulation of the hypothalamic-pituitary GH secretion axis.


Existence of multiple peaks in plasma ethinyl estradiol and norethindrone after oral administration of a contraceptive pill. Previous measurements of plasma ethinyl estradiol (EE2) and norethindrone (NE) over 24 h after oral administration of a contraceptive pill have demonstrated a single steroid peak occurring 1-2 h after pill ingestion, with a gradual decline over the next 22 h. In the present study plasma concentrations of EE2 and NE were measured 0, 0.5, 0.75, 1, 2, 4, 12, and 24 h after oral ingestion of a contraceptive pill containing 35 micrograms EE2 and 1 mg NE at 0, 3, 6, and 9 months of use in 58 normal healthy women. Contrary to previous reports, analysis of the 464 steroid curves (58 subjects x 4 time periods x 2 steroids) revealed the presence of multiple hormone peaks. Two peaks of EE2 were identified in 44.8% of women during the first pill cycle and in 75.9%, 55.2%, and 67.2% of women after 3, 6, and 9 months of pill use. Two hormone peaks of NE were observed in 29.3% of women during the first cycle and in 36.2%, 50%, and 44.8% at 3, 6, and 9 months, respectively. Existence of these multiple peaks at the frequency observed has not previously been reported. Further quantification of the frequency and magnitude of these peaks could be helpful in explaining differences in biological responses associated with pill use.


Frame shift by insertion of 2 basepairs in codon 394 of CYP11B1 causes congenital adrenal hyperplasia due to steroid 11 beta-hydroxylase deficiency. Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder of corticosteroid biosynthesis primarily caused by a deficiency in either of two heme-containing cytochrome P450-enzymes: steroid 21- or 11 beta-hydroxylase (causing approximately 90% and 5-8% of classical CAH cases, respectively). Depending on the patient's gender, the affected enzyme, and the extent of enzymatic dysfunction, symptoms include adrenal hyperplasia, androgen excess, virilization, growth disturbance, and electrolyte imbalance. To define the molecular basis of steroid 11 beta-hydroxylase-deficient CAH, we cloned and sequenced the CYP11B1 gene (encoding 11 beta-hydroxylase) of a female patient afflicted with this disorder. Exon 7 contained a 2-basepair insertion in codon 394, leading to a reading frame shift, multiple incorrect codons, and a premature stop in codon 469, resulting in complete destruction of the enzyme's heme-binding domain. Due to parental consanguinity, this defect was homozygous and, therefore, provides a full molecular explanation for this disease.


The effects of human proinsulin on glucose turnover and intermediary metabolism in insulin-dependent-diabetes mellitus. We have compared the action of human proinsulin and insulin on glucose turnover, intermediary carbohydrate, and lipid metabolism in insulin-dependent-diabetic (IDDM) subjects. Six, young, weight-matched (23 +/- 2 kg-2) IDDM subjects underwent separate hyperinsulinemic euglycemic clamps. Three, low dose, iv infusions of both insulin and proinsulin were used to construct dose response curves. The proinsulin infusions were chosen to give steady state levels approximately or equal to 20-fold higher on a molar basis than insulin, based on previous findings that proinsulin has only 5-10% the biological potency of insulin. Hepatic glucose production, measured using [6'6'2H2]glucose, was suppressed equally by proinsulin and insulin at the three dose levels; (I1) 2.8 +/- 0.7 (P1) 3.3 +/- 0.6, (I2) 2.3 +/- 0.9 (P2) 3.3 +/- 1.1, (I3) -2.0 +/- 1.7 (P3) -1.1 +/- 0.6 mumol/kg min-1. Percentage elevation of glucose disposal was significantly increased during the insulin infusions compared to proinsulin; (I1) 132 +/- 12 (P1) 78 +/- 4 p < 0.01; (I2) 157 +/- 18 (P2) 104 +/- 14; P < 0.05; (I3) 242 +/- 23 (P3) 159 +/- 24 p = 0.02. Dose response curve analysis demonstrated that proinsulin stimulated glucose disposal approximately or equal to 3.7% whereas suppression of HGP was congruent to 5.7% compared to insulin. Proinsulin had a significantly weaker effect than insulin, at the lowest infusion dose, in percent suppression of plasma nonesterified fatty acids (I1 34 +/- 4, P1 14 +/- 15%; P < 0.05), blood glycerol (I1 47 +/- 4, P1 30 +/- 3%; P < 0.01) and 3-hydroxybutyrate levels (I1 81 +/- 7, P1 42 +/- 17%; P < 0.05). Proinsulin caused significant net reductions in blood lactate levels compared to insulin at each infusion dose; (P1) -130 +/- 34, (I1) -32 +/- 30 mumol/L (P < 0.05) (P2) -139 +/- 76 (I2) +8 +/- 65 mumol/L (P < 0.05) (P3) 48 +/- 60 (I3) 230 +/- 64 mumol/L (P < 0.05). We conclude that in IDDM: 1) proinsulin has a preferential effect on the liver compared to muscle, in terms of glucose handling; 2) proinsulin may have a different effect on lactate metabolism compared to insulin; 3) proinsulin at the lowest dose resulted in an inability to suppress lipolysis and ketogenesis; 4) glucose turnover can be underestimated using [6'6'2H2]glucose.


Screening for Y-derived sex determining gene SRY in 40 patients with Turner syndrome. In Turner patients, the presence of a Y chromosome or derivative Y is correlated with the risk of gonadoblastoma induction. "Marker" chromosomes originating from Y, may not show characteristic fluorescence and then be very difficult to identify by conventional cytogenetic techniques, although they still predispose the patients to gonadal tumors. Using polymerase chain reaction of the gene from the sex-determining region of the Y chromosome, we screened 40 Turner patients (thirty seven 45X and three 45X,46XX) for the presence of Y chromosomal DNA. We were able to identify karyotypically unrecognized Y chromosome material in 1 patient out of the 40 studied. In this patient mild clinical and biological hyperandrogenism was observed. Reliability of our technique was ascertained by the detection of the expected 648 base pairs amplified DNA fragment in all normal male controls as well as in 3 Turner patients with confirmed 45X,46XY mosaicism. Despite the low frequency of unrecognized Y chromosome material (1 case over 40 in our experience), our data suggest that polymerase chain reaction of the gene from the sex-determining region of the Y chromosome is worthy of being performed in Turner patients considering the potential risk of the presence of a Y chromosome.


Effect of physiological hyperinsulinemia on counterregulatory hormone responses during hypoglycemia in humans. We evaluated the effect of continuous physiological hyperinsulinemia on counterregulatory hormone responses in seven healthy subjects, each studied on two occasions. Hormone responses were measured during identical 2-h periods of hypoglycemia (plasma glucose target 3.5 mmol/L) at insulin levels of 350 pmol/L or 640 pmol/L. During hypoglycemia, there were significant (50-1400%) increases in glucagon, epinephrine, norepinephrine, GH, and cortisol which were comparable in the two groups. We further evaluated the influence of the duration of mild hyperinsulinemia on the responses in an additional group of normal subjects (n = 7). Brief (30 min) exposure to insulin was compared to a prolonged (3.5 h) insulin infusion, each followed by identical hypoglycemia. Plasma insulin (approximately 350 pmol/L) and plasma glucose (target 3.3 mmol/L) were similar in both groups. The increases in epinephrine, norepinephrine, GH, and cortisol during hypoglycemia were virtually identical in the two groups. However, the secretion of glucagon was blunted following prolonged hyperinsulinemia, increasing to levels of 249 +/- 17 ng/L in the brief studies and to only 185 +/- 20 ng/L in the prolonged studies (P < 0.005). The insulin-induced decrement in plasma amino acids were similar in the two studies and could not account for the impaired glucagon secretory response. 1) Brief exposure to even high physiological levels of insulin do not alter the magnitude of counterregulatory hormone secretion during hypoglycemia; 2) prolonged hyperinsulinemia results in a selective blunting of the plasma glucagon response to hypoglycemia, perhaps due to a direct suppressive effect of insulin on alpha-cell secretion.


Serum growth hormone (GH)-binding protein and insulin-like growth factor-I levels in Turner's syndrome before and during treatment with recombinant human GH and ethinyl estradiol. Serum levels of GH-binding protein (GH-BP) and insulin-like growth factor-I (IGF-I) were measured in 14 adolescent girls with Turner's syndrome (TS) before and during treatment with recombinant human GH (rhGH) and oral ethinyl estradiol (EE2). Before treatment, the mean +/- SE GH-BP level in TS patients was 33.2 +/- 2.0%; this was higher (P < 0.05) than that in bone age-matched (27.9 +/- 1.1%; n = 13) or chronological age-matched (28.1 +/- 0.8%; n = 17) control girls. The mean +/- SE IGF-I level in TS girls (283 +/- 26 micrograms/L) was comparable to that in bone age-matched controls (255 +/- 17 micrograms/L), but lower (P < 0.005) than that in chronological age-matched pubertal controls (568 +/- 35 micrograms/L). In 7 TS girls treated with daily sc injections of rhGH in a dose of 0.8 U/kg.week for 18 months, serum levels of IGF-I increased from 330 +/- 39 to 707 +/- 48 micrograms/L after 3 months of treatment and remained elevated for the entire observation period (P < 0.002). In contrast, GH-BP levels did not change significantly. In 7 other TS girls, puberty was induced after 2 yr of daily sc injections with rhGH (1 U/kg.week) by adding 100 ng/kg.day EE2, orally, during ongoing rhGH therapy. During 18 months of pubertal induction, serum GH-BP levels increased gradually from 31.4 +/- 2.7% at the start of EE2 treatment to a maximum of 49.2 +/- 0.8% after 1 yr (P < 0.001). Serum IGF-I levels, in contrast, did not change significantly. These results show that in adolescent TS patients 1) pretreatment serum GH-BP levels are higher than in bone age-matched or chronological age-matched control girls, and IGF-I levels are similar to those in bone age-matched but lower than those in chronological age-matched controls; 2) during rhGH treatment GH-BP levels do not change, whereas IGF-I levels increase significantly; and 3) oral EE2 treatment during ongoing rhGH treatment increases GH-BP levels markedly, but does not modify IGF-I levels.


Clinically nonfunctioning pituitary adenoma and octreotide response to long term high dose treatment, and studies in vitro. We have studied seven patients with a clinically nonfunctioning or alpha-subunit-secreting pituitary macroadenoma, four of whom received long term, high dose octreotide treatment. We have attempted to correlate the presence of somatostatin receptors (SS-R) in the adenomas and the outcome of octreotide treatment, as measured by tumor size, improvements in visual field defects, and hormonal response. The presence of SS-R in the pituitary adenomas was demonstrated in vivo using [111indium]octreotide scintigraphy and in vitro by autoradiography of tissue fragments obtained after transsphenoidal surgery. Adenomas from six of the seven subjects were SS-R positive. High dose (1200 micrograms, sc, daily) octreotide treatment was given to four subjects, three of whom were SS-R positive. Improvement of the visual field defects was observed in three of four patients (including the SS-R-negative subject), although no computed tomographic scan-assessed tumor size reduction was found. Two of four patients showed small but significant reductions in serum FSH concentrations (to 83% and 93% of initial values) with treatment. These in vivo responses to high dose octreotide treatment could not be predicted by pretreatment responses to 200 micrograms TRH or 100 micrograms octreotide. Tissue fragments for cell culture were obtained from six patients, and in vitro release of gonadotropins and/or alpha-subunit could be demonstrated in five cultures. In vitro, octreotide (10 nmol/L) significantly decreased gonadotropiin or subunit release in three of five cultures, whereas bromocriptine (10 nmol/L) significantly reduced the release in four of five cultures and to a significantly greater extent than octreotide. In conclusion, in six of seven patients with a clinically nonfunctioning or alpha-subunit-secreting pituitary adenoma, SS-R were demonstrated in the tumor. In vitro incubation of adenoma cells with octreotide resulted in mild inhibition of gonadotropin or alpha-subunit release. Although in vivo long term treatment with high doses of octreotide did not result in substantial tumor size reduction, improvement of visual field defects was observed in three of four subjects.


Suppression of human spermatogenesis by testosterone implants. Hormonally induced azoospermia is an effective, reversible form of male contraception; however, some men treated with weekly im testosterone enanthate (TE) injections fail to become azoospermic. As weekly injections cause widely fluctuating and supraphysiological testosterone levels, we tested the hypothesis that more stable, physiological testosterone levels would consistently produce azoospermia. Using a depot testosterone formulation which provides stable, physiological range testosterone levels for up to 6 months, we studied nine men before and after insertion of six 200 mg testosterone implants under the abdominal wall skin and compared the results with 38 men treated in a previous study with weekly im injections of 200 mg TE. Testosterone implants suppressed sperm output to near-azoospermia between the second to fourth postimplant months returning to normal by the sixth postimplant month. The fall in sperm output at the first month was greater after testosterone implants than TE injections (58% vs. 17%, P = 0.011) but similar proportions of men became azoospermic (5/9 vs. 25/38) or severely oligozoospermic (< 1 million/ml; 9/9 vs. 37/38). Plasma testosterone and estradiol levels remained mostly within the eugonadal range after implants but were markedly supraphysiological during TE injections. Both treatments suppressed immunoreactive LH and FSH to undetectable levels by ultrasensitive fluoroimmunoassay. Sex hormone-binding globulin levels were decreased and PRL levels increased by TE injections but neither was changed by testosterone implants. Prostate-specific antigen demonstrated a small rise of marginal significance (P = 0.065) after testosterone implants. Fewer men experienced acne after implants (0/9 vs. 25/38, p = 0.0004). Therefore a depot testosterone preparation with quasi-zero-order release demonstrates higher dose efficiency with similar (but not uniform) efficacy at inducing azoospermia but may cause fewer androgenic side-effects than weekly TE injections.


The role of chorionic gonadotropin in transient hyperthyroidism of hyperemesis gravidarum. Biochemical evidence of hyperthyroidism is frequently encountered in hyperemesis gravidarum, but its relationship to the cause of hyperemesis is unknown. We studied the relationship of serum hCG, thyroid function, and severity of vomiting among 57 hyperemesis patients and 57 controls matched for gestational age. TSH was suppressed in 60% of hyperemesis patients and 9% of controls. hCG correlated directly with free T4(r = 0.45, P < 0.001) and inversely with TSH (r = -0.48, P < 0.001). Hyperemesis patients had significantly greater mean serum hCG, free T4, total T3, and estradiol, and lesser serum TSH compared to controls. Hyperemesis patients with suppressed TSH had significantly greater free T4 and hCG compared to those with TSH in the normal range. Control and hyperemesis subjects were divided into four groups based on the severity of vomiting. The degree of biochemical hyperthyroidism and hCG concentration varied directly with the severity of vomiting. Unextracted serum was tested for thyrotropic activity by measuring its effect on iodide uptake in cultured FRTL-5 rat thyroid cells. Thyrotropic activity correlated with serum hCG (r = 0.50, P < 0.001). These data show that biochemical hyperthyroidism is a common finding in patients with hyperemesis gravidarum and suggest that hCG is the thyroid stimulator in this state. The increased estradiol concentration in patients with hyperemesis gravidarum may be attributed to the effects of hCG on steroidogenesis.


Immunoradiometric analysis of circulating human glycosylated and nonglycosylated prolactin forms: spontaneous and stimulated secretions. The monoclonal antibodies (MAbs) obtained in mice immunized with human PRL coupled to an anti-PRL MAb were screened for their ability to distinguish the glycosylated (G-) and nonglycosylated (NG-) forms of PRL. The 431-29 MAb exhibited high affinity binding for NG-PRL but little or no cross-reactivity to G-PRL. Using this antibody in conjunction with other MAbs which equally recognized both forms, we developed 2 immunoradiometric assays which were used to determine the amount of G- and NG-PRL in plasma. In 85 normal subjects, NG-PRL baseline levels averaged 6.6 +/- 3 micrograms/L, and represented 76 +/- 8% of the total PRL immunoreactivity. In 74 pregnant women, this proportion was significantly higher during the last 2 trimesters (84 +/- 4% and 85 +/- 6%), as compared to the first trimester (76 +/- 7%). In 6 healthy volunteers studied over 24 h, 79% of the NG-PRL peaks detected using the cluster algorithm occurred concomitantly to a G-PRL peak. The mean NG-PRL/PRL ratio was significantly higher during NG-PRL pulses (81 +/- 9%) than during valleys (71 +/- 12%). Similarly, this ratio was significantly increased during TRH or metoclopramide stimulated PRL secretion (to 88 +/- 7% and 86 +/- 6%, respectively). We conclude that 1) NG-PRL is the predominant immunoassayable form of PRL in plasma; 2) both G- and NG-PRL are cosecreted but NG-PRL is the main PRL form released during spontaneous or pharmacologically induced PRL secretion.


Regulation of immunoreactive inhibin patterns in baboon pregnancy: maternal, placental, and fetal considerations. To better understand the sources and regulation of circulating inhibin during primate pregnancy, immunoreactive inhibin was measured in sera obtained from the maternal saphenous vein, uterine vein, and the fetus at varying times of baboon pregnancy. In both intact and fetectomized (fetus removed on day 100 of gestation; term = 184 days) animals, maternal serum inhibin concentrations were relatively constant between day 80 (first sampling day) and day 110 of gestation, after which they then steadily increased until days 155-165 (end of sampling). The increase in inhibin concentrations was significantly less in the fetectomized animals than in the intact baboons. Restoration of estrogen levels in the fetectomized animals did not significantly alter the circulating inhibin concentrations. Similarly, administration of the estrogen antagonist MER-25 to intact animals in the last trimester had no effect on maternal serum inhibin concentrations. Inhibin concentrations in uterine venous blood collected on day 100 of gestation were not significantly different from those in the maternal saphenous vein. However, the inhibin concentrations of uterine venous blood collected late in gestation (days 155-165) in either intact or fetectomized animals were significantly higher than the corresponding maternal venous concentrations, suggesting that the uteroplacental tissue becomes a source of circulating inhibin during the third trimester of pregnancy. Consistent with this suggestion was the detection of inhibin alpha-subunit mRNA in the placentae of intact or fetectomized animals obtained late in pregnancy, but its absence at midgestation. Immunoreactive inhibin concentrations were about 16 times higher (6500 +/- 831 mu Leq/mL) in fetal blood than in maternal blood (411 +/- 23 mu Leq/mL) at midgestation. The fetal blood concentrations significantly decreased to about 2800 mu Leq/mL by days 160-165 of gestation, but were still greater than those in the mother (approximately 1000 mu Leq/mL). The umbilical arterial and venous concentrations were the same as the fetal blood concentration of inhibin. The role of the baboon fetal adrenal in inhibin production was studied. Fetal adrenals collected from days 59, 135, and 167 of gestation contained the mRNA for the inhibin alpha-subunit in relatively high abundance. The in utero administration of ACTH for 30 min to five fetuses at midgestation (days 100-110) apparently did not alter the fetal concentration of immunoreactive inhibin. In summary, maternal serum inhibin levels increase during the last trimester of baboon pregnancy. This is suggested to be due to an increasing contribution of placental inhibin secretion, which is regulated not by placental estrogen production but, perhaps, by placental growth.(ABSTRACT TRUNCATED AT 400 WORDS)


Gestational age-dependent dual action of epidermal growth factor on human placenta early in gestation. In order to better understand the role of epidermal growth factor (EGF) in the regulation of placental growth and function, effects of EGF on proliferative activity and differentiated function of trophoblast were examined. Explants from very early (4-5 week) placentas and early (6-7 week, 8-9 week, 10-12 week) placentas were respectively cultured under a serum-free condition in the absence or presence of EGF (100 micrograms/L) for the first 48 h, and the cultures were continued for subsequent 72 h without EGF. The proliferative potential and differentiated function of trophoblast were assessed by immunohistochemical Ki-67 staining and by determining the ability to secrete human CG (hCG) and human placental lactogen (hPL), respectively. Quantitative estimates of proliferative activity based on mean percentage of Ki-67 positive nuclei showed that EGF stimulated proliferative potential of cytotrophoblast in very early (4-5 week) placental explants. The EGF stimulation of trophoblast proliferation was apparent at a 12-h EGF-treated period. By contrast, early (6-12 week) placental explants did not respond to EGF with increase in trophoblast proliferation. Instead, in early placental explant culture EGF stimulated hCG and hPL secretion with a lag period of 72 h, whereas very early placental explants did not respond to EGF with increase in hCG and hPL secretion. These results suggest that EGF exerts gestational age-dependent dual action on the first-trimester placenta: one is to stimulate trophoblast proliferation in 4-5 week placenta and the other is to stimulate differentiated trophoblast function in 6-12 week placenta.


Demonstration and localization of growth hormone receptor in human skin and skin fibroblasts. Clinical evidence suggests that skin is responsive to GH status in vivo. We sought to demonstrate the presence of GH receptors in human skin and in cultured skin fibroblasts using the techniques of immunohistochemistry and northern blotting. Human foreskin was obtained at surgery for preparation of sections and primary fibroblast cultures. Skin sections and fibroblast monolayers were immunostained using a monoclonal antibody which recognizes the hGH receptor (MAb 263). Positive immunoperoxidase staining was seen in all epidermal layers except the stratum corneum, in dermal sweat and sebaceous glands, and in dermal fibroblasts. In cultured fibroblasts capping of surface GH receptor was observed after aqueous formaldehyde fixation, whereas fixation in Carnoy's solution resulted in granular cytoplasmic staining. Fibroblast poly A+ RNA was prepared from cultured skin fibroblasts, separated by denaturing agarose gel electrophoresis, blotted onto nitrocellulose, and hybridized to a 32P-labeled, 847 base pair (bp) hGH receptor complementary DNA (cDNA) clone. Human liver and non-pregnant rabbit liver total RNA were used as controls. Fibroblast poly A+ RNA contained a single hybridizing species of approximately 5.2 kilobase. Human liver total RNA also contained a single hybridizing species of 4.9 kilobase. We have demonstrated the presence of GH receptor protein in human skin and growth hormone receptor mRNA and protein in cultured human skin fibroblasts. These observations suggest that GH may indeed have a direct role in modulating keratinocyte and fibroblast function.


Localization of renin gene expression to monkey ovarian theca cells by in situ hybridization. To investigate the sites of renin gene expression and localization of renin in primate ovaries, five cynomolgus (Macaca fascicularis) and one rhesus (Macaca mulatta) monkey were treated with gonadotropins to induce multiple follicle development. One ovary was removed before hCG injection (1200 IU) from three monkeys and one ovary was removed 36 h after hCG administration from three monkeys. In three monkeys, the remaining ovary was removed 3, 5, and 7 days after injection of hCG. To detect and localize renin messenger RNA, 35S-radiolabelled 1.1 kb length complementary DNA and RNA probes of human renin were used for in situ hybridization. To compare the synthesis with the presence and the storage of renin or prorenin, renin antigen was assessed by immunohistochemistry in the same tissues using a polyclonal antibody against human renin (R15). Renin mRNA was detected by in situ hybridization only in ovaries collected within 5 days of exposure to hCG. All such ovaries exhibited a positive signal. Renin mRNA was localized to the theca interna and theca lutein cells. Positive cells were observed in a few growing antral follicles, in occasional mature preovulatory follicles, in corpus luteum, and most strikingly in atretic follicles. No signal was detected in primordial, primary, or in small antral follicles of ovaries exposed to hCG. In contrast with the in situ hybridization data, no signal was detected by immunohistochemistry using antirenin antibodies which exhibited a positive signal in monkey kidney. These results indicate that hCG turns on renin gene expression. Renin is synthesized without significant intracellular storage in monkey ovarian theca interna cells and in corpus luteum. The absence of storage of renin is consistent with the high concentrations of prorenin found in ovarian follicular fluid of hCG stimulated primates and with our knowledge of cellular renin processing which indicate that prorenin is secreted constitutively as it is synthesized.


Polyvalent immunoglobulin for intravenous use interferes with cell proliferation in vitro. Intravenous immunoglobulin is used to an increasing extent in various immune-mediated diseases, but its mechanism(s) of action in vivo is incompletely understood. Previous studies have shown that intravenous immunoglobulin may interfere with autoantibodies and their production by B cells and also inhibit Fc-mediated antibody-dependent cytotoxicity. Here we describe a novel effect of intravenous immunoglobulin on proliferation of in vitro activated peripheral blood lymphocytes and autonomously growing cell lines of various origin. Independently of whether proliferation was autonomous or induced by antigen-specific or antigen-nonspecific reagents, proliferation was inhibited in a dose-dependent fashion, as measured by reduced 3H-thymidine and BrdU uptake and cell counting. The effect was not due to cytotoxic effects of intravenous immunoglobulin and was reversible after removing the intravenous immunoglobulin by washing. The IgG levels required for this inhibition of proliferation are supraphysiological but are reached in vivo during treatment with intravenous immunoglobulin.


An immunodominant haptenic epitope of carbamazepine detected in serum from patients given long-term treatment with carbamazepine without allergic reaction. An anticarbamazepine antibody was detected in the serum of a patient with severe carbamazepine-induced serum sickness. We found that the patient's T cells and IgG antibody recognized an epitope which appeared in subjects showing an allergic reaction, as well as that in subjects who showed no allergic reaction, after long-term carbamazepine therapy. These results show that an anti-carbamazepine immune response does not occur in the majority of subjects who undergo long-term carbamazepine therapy without developing allergic symptoms, although the immunodominant haptenic epitope of carbamazepine is present in their sera.


The influence of aging on the induction and manifestations of experimental systemic lupus erythematosus. The influence of age on autoimmunity was studied in a model in which experimental systemic lupus erythematosus (SLE) is induced in normal mice by the injection of a human monoclonal anti-DNA antibody expressing a common idiotype designated 16/6 Id. The resulting disease is expressed by the production of a variety of autoantibodies and clinical manifestations characteristic to human SLE. Female BALB/c mice, at ages of 2 and 12 months, were immunized with the 16/6 Id. Mice were tested periodically for the presence of autoantibodies. The production of all autoantibodies tested was significantly lower in the older mice as compared to the group of young mice. Clinical manifestations which included leukopenia, increased erythrocyte sedimentation rate and proteinuria were similar in both age groups. Kidney evaluations revealed differences among the two groups of mice. While in all kidney sections of young mice multiple immune complex deposits were detected, in the group of older mice half had similar pathology while the rest either were negative or had only segmental and partial glomerular immune complex depositions. Thus, aging is associated with a decrease in the capacity to respond to the pathogenic anti-DNA, 16/6 Id, by the production of antibodies and autoantibodies and in the expression of a milder disease.


Effects of alpha-interferon on gamma-interferon production of peripheral blood mononuclear cells in hepatitis B virus carriers. We studied gamma-interferon production of phytohemagglutinin-stimulated peripheral blood mononuclear cells in response to alpha-interferon in hepatitis B virus carriers and healthy individuals. The magnitude of gamma-interferon production was significantly higher in patients with anti-HBe antibody than in patients with HBe antigen and healthy individuals. Furthermore, alpha-interferon augmented the production of gamma-interferon of peripheral blood mononuclear cells from patients with active liver injury [serum alanine aminotransferase (ALT), greater than 40 U/L], but not that from patients with inactive liver injury (serum ALT, less than 40 U/L) or healthy individuals. These results suggested that alpha-interferon could enhance the cellular immune response against hepatitis B virus by augmenting the endogenous production of gamma-interferon in patients with active liver injury, implying that the responsiveness to alpha-interferon might be responsible for liver cell injury.


Disease severity in rheumatoid arthritis: relationships of plasma tumor necrosis factor-alpha, soluble interleukin 2-receptor, soluble CD4/CD8 ratio, neopterin, and fibrin D-dimer to traditional severity and functional measures. Rheumatoid arthritis is a complex inflammatory disease of unknown cause. Although various laboratory and clinical measurements are useful in managing these patients, there is a need for better tests to quantitatively assess disease activity. The purpose of this study was to investigate the association of certain immune and inflammation (I-I) parameters with four traditional disease severity measures and a functional measure in rheumatoid arthritis patients. A single set of patient blood samples was analyzed, and four traditional disease severity measures and patient functional statuses were determined from 64 consecutive outpatients with rheumatoid arthritis. Plasma tumor necrosis factor-alpha (TNF), soluble interleukin-2 receptor (sIL-2R), sCD4 and sCD8 (and the sCD4/sCD8 ratio), neopterin, and fibrin D-dimer were analyzed in relationship to Westergren erythrocyte sedimentation rate (ESR), physician assessment of disease activity, joint pain count, grip strength, and Arthritis Impact Measurement Scale (AIMS) scores. Rheumatoid arthritis patients had higher mean levels of all I-I measures (except sCD4) compared to healthy subjects. Initial significant correlations between TNF, sIL-2R, and D-dimer and several disease severity and functional measures were detected. When we controlled for the covariates age, gender, race, and medications, regression analyses indicated that, as a group, the I-I measures were significantly related to grip strength, physician disease severity rating, ESR, and total joint pain. When the predictive values of the I-I measures were tested controlling for the covariates and ESR, D-dimer was independently and significantly associated with variability in grip strength, physician disease severity, and AIMS physical disability, while TNF was associated with a significant amount of variability in total joint pain.(ABSTRACT TRUNCATED AT 250 WORDS)


Cold-dependent activation of complement: recognition, assessment, and mechanism. Cold-dependent activation of complement (CDAC) is a phenomenon characterized by low hemolytic complement activity in chilled serum. Complement component levels are normal when measured immunologically, and there is normal hemolytic activity in EDTA plasma or serum maintained at 37 degrees C. Little attention has been paid to CDAC except in Japan, and current unfamiliarity with it, even by clinical immunologists, can lead to confusion and unnecessary laboratory tests. A 66-year-old patient with a complex medical history is described whose complement tests showed abnormalities characteristic of CDAC. Evidence for classical complement pathway activation in the cold was obtained by CH50 measurements, by hemolytic C4 determinations, by C4a, C3a, and C4d generation, and by quantitating C1s-C1r-(C1 inhibitor)2 complexes. A good correlation was observed among these parameters. Cryoprecipitates were absent. CDAC activity has persisted for over 5 years and is greater at 13 than at 4 degrees C. Activation is ablated by heating at 56 degrees C and restored by the addition of C1 to the heated serum. Adsorption by streptococcal protein G-Sepharose and precipitation by 2.5% polyethylene glycol support the hypothesis that CDAC is caused by aggregated IgG. The CDAC factor(s) also induces complement activation in normal serum but has not interfered with Raji cell or C1q binding tests or with FACS analysis. More limited studies of a second individual experiencing CDAC yielded similar results.


Expression of a functional p75 interleukin-2 receptor on lung lymphocytes from patients with human immunodeficiency virus type 1 (HIV-1) infection. Lung involvement in patients affected by HIV-1 infection is characterized by an alveolitis sustained by the accumulation of CD8+ T lymphocytes. To investigate whether in situ T cell growth plays a relevant role in the pooling of CD8+ lymphocytes, we have analyzed the activity of two lymphokines involved in the mechanisms of T cell proliferation, i.e., interleukin-2 (IL-2) and interleukin-4. To this aim, following appropriate triggering and blocking, the expression and the functional role of IL-2 receptors (IL-2R) (both p55 and p75 chains) and IL-4 receptors have been analyzed on T lymphocytes obtained from the bronchoalveolar lavage (BAL) of 16 HIV-1+ patients. Molecular and phenotypic studies we performed demonstrated that CD8+ lymphocytes from the BAL of HIV-1 + patients strongly expressed the p75 chain of IL-2 receptor, while neither p55 mRNA nor its surface membrane product (Tac antigen) was detectable; in addition, there was no expression of IL-4 receptors. IL-2 stimulation was able to induce T cell growth in a dose-dependent manner, whereas IL-4 did not. Finally, using mAbs which specifically block the p55 or p75 IL-2R, we showed that both subunits of IL-2R were involved in the proliferative activity of lung lymphocytes. The results obtained in the present study directly demonstrate that BAL T lymphocytes of HIV-1 + patients express a fully functional IL-2 receptor apparatus, pointing to the role for this lymphokine in maintaining the alveolitis taking place in the lungs of AIDS patients.


[The clinical application of rotational stereo digital angiography to the field of neurosurgery]. A new system for 3-dimensional angiography, named "Rotational stereo digital angiography" (RSDA), was applied to 36 patients with neurosurgical lesions. This system consists of a new rotating X-ray apparatus and a digital processor, and accumulates 144 projectional images in 2.2 seconds during a 180 degree transverse rotation of the X-ray device. These images are digitally processed and displayed on two CRT's at 5 different degrees so that a rotating image of the angiogram is observed. Thirteen cerebral aneurysms, 16 brain and skull tumors, and 7 other neurosurgical lesions were examined with this system and compared with conventional angiography. The aneurysms were examined to 1) identify the aneurysm, 2) demonstrate the shape of the aneurysm, 3) demonstrate the aneurysmal neck, and 4) illustrate the relationship between the aneurysm and the mother vessels. The tumors were examined to 1) demonstrate the tumor stain, 2) identify the feeding arteries, 3) identify the draining veins, and 4) illustrate the relationship between the lesion and the major vessels. Similarly, 1) demonstration of the lesion, and 2) illustration of the relationship between the lesion and the major vessels were examined for other lesions. The findings in RSDA and conventional angiography were classified into -, +/-, +, or +2 with regard to the readiness to the aspects described above. They were then statistically compared. According to the statistic analysis, RSDA is significantly more illustrative for aneurysms than conventional angiography. For tumors and for other lesions, there was no significant difference between RSDA and conventional angiography. These results indicate the usefulness of RDSA in the field of neurosurgery.


Effect of exposure to benzene on natural killer (NK) cell activity and interleukin-2 (IL-2) production of C57BL/6 mice. To investigate the effect of benzene on murine splenic natural killer (NK) cell activity and interleukin-2 (IL-2) production, we exposed C57 BL/6 mice to benzene for 28 days. Benzene exposure via drinking water at doses of 27 and 154 mg/kg/day yielded the following results. 1) A decrease of spleen cell number was detected on the 14th day of benzene exposure at 154 mg/kg/day, and on the 21st and 28th day of exposure at both 27 mg/kg/day and 154 mg/kg/day. 2) NK activity per viable spleen cell was temporarily enhanced on the 21st day of exposure at doses of 27 and 154 mg/kg/day, thereafter returned to control levels. 3) IL-2 production was inhibited after 28 days of benzene exposure at 27 and 154 mg/kg/day. After ending exposure at doses of 27 mg/kg/day for 28 days, the decreased spleen cell number began to return to control levels from the 14th day on, and reduced IL-2 production returned to control levels. These results suggest that, NK cells are resistant to benzene toxicity, while IL-2 producing cells are sensitive. Thus, an imbalance in immune system was caused by toxic benzene action.


[Urodynamic abnormalities in patients with lumbar diseases. With special reference to autonomic dysfunction]. In order to investigate urinary dysfunction in lumbar disease from the point of view of autonomic dysfunction, urodynamic examinations and sympathetic skin responses (SSR) were evaluated in patients with lumbar diseases. Cystometry and uroflowmetry were performed as urodynamic examinations on 40 patients with lumbar diseases including intervertebral disc herniation (IDH), spinal canal stenosis (SCS), and spondylolisthesis with spondylolysis. SSR of the feet were recorded in 6 healthy men, as controls, and in 10 patients with IDH; The latencies and amplitudes of SSR were compared. Eighty percent of the patients showed some abnormality in the urodynamic examinations that indicated an occult neurogenic bladder. Also, the mean and maximal amplitudes were significantly lower in the patients with IDH than in the controls (p < 0.01). While the occurrence of a neurogenic bladder was considered to be a parasympathetic dysfunction, abnormalities in SSR were considered to be sympathetic dysfunctions. It was therefore considered that autonomic dysfunction was present in the bladder and lower extremities of patients with lumbar diseases.


[Spatial and quantitative analysis of the QRS and T waves by Frank-lead orthogonal electrocardiography in normal children. Comparative study in an early and late cord-clamping groups in newborns]. We studied a spatial and quantitative analysis of QRS and T waves by Frank-lead orthogonal electrocardiography (afterwards; ECG) in 50 cases of normal newborns, 30 cases of infants, 50 cases of adults, and also the variation in age was investigated. In the newborns, the comparative study was made serially, longitudinally in 21 infants with an early clamping of the cord and 29 infants with a late clamping of the cord. The results are mainly as follows. 1) The parameters reflecting the cardiac electromotive force of the right ventricle such as Sx, Qz and Sx+Qz decreased with aging. 2) Rx, i.e., the maximum leftward projection of QRS vector increased until the children, probably because the left ventricle was dominant in their growth and development and in the increased amount of work. A subsequent reduced value in the adults was considered to be mainly due to an increase of the distance between the heart and electrode by the increase in the thorax, particularly the transverse diameter and due to the growth in subcutaneous fat and muscle. 3) The values of Rx/Sx increased with aging. Rx and Sx in the adults were less than those in the children, but Rx/Sx was larger than that of Rx because Sx was less than Rx. 4) Age-related changes of Tz was most characteristic and was negative up to several days after birth. T vector was in forward direction and then turned positive (in backward). This was greatest at the period of the infants. Subsequently, it turned negative in the childhood, and extent was maximum in the adults.(ABSTRACT TRUNCATED AT 250 WORDS)


[A study to increase the therapeutic effects of adoptive immunotherapy in vivo. Influence on the generation of lymphokine activated killer (LAK) cells and therapeutic effects of LAK cells with anti-tumor drug (cyclophosphamide)]. To enhance the effect of adoptive immunotherapy (AIT), we investigated the induction and characteristics of lymphokine activated killer (LAK) cells and also analyzed the combined effects of AIT with an antitumor drug (cyclophosphamide: CPA) in mice models. LAK cells were generated from C57/BL/6 (B6) spleen cells. The spleen cells were passed through a nylon wool column and cultured in RPMI-1640 medium containing 10% FCS and 2 x 10(3) units of human recombinant IL-2 (hr IL-2) for up to 14 days. During this period, the time kinetic analyses of the LAK cells' cytotoxicity and motility were performed. The cytotoxicities against Lewis Lung Carcinoma (3LL), evaluated by standard 51Cr release assay, gradually increased during the cultured period, and the motilities, determined by a modified version of the Boyden chamber method, greatly increased within the first 7 days' incubation. Based on these in vitro findings, we examined the efficacy of AIT alone or in combination with chemotherapy (CPA) in in vivo studies. AIT was performed in the following way: LAK cells were intravenously infused and rIL-2 was intraperitoneally administered for 5 consecutive days following LAK cell administration. CPA was intraperitoneally administered. The therapy protocols were as follows. There were seven experimental groups. Group I; the mice were infused with 3-day cultured LAK cells (3DLAKs) on the second day after tumor inoculation (day 2). Group II; the mice were infused with 3DLAKs on day 5. Group III; 10-day cultured LAK cells (10DLAKs) on day 2. Group IV; 10DLAKs on day 5. Group V (AIT and CPA combination); AIT (10DLAKs) was started on day 5 followed by CPA on day 10. Group VI; CPA was performed on day 5 followed by AIT (10DLAKs) on day 10. Group VII; CPA was performed on day 5 without AIT. Each group consisted on 15 mice. The therapeutic efficacies were evaluated by calculating the median survival time of each group. The results of these experiments were as follows (mean +/- SD); Group I's median survival time was 16.8 +/- 3.2 days, Group II 15.1 +/- 2.1 days, Group III 19.2 +/- 5.4 days, Group IV 16.1 +/- 4.8 days, Group V 23 +/- 6.3 days, Group VI 32 +/- 8.4 days and Group VII 22 +/- 5.1 days. These results suggested that the efficacy of AIT is closely related to the LAK cells' cytotoxicity and motility. Although AIT alone in the advanced tumor bearing host had a limited effect, combination with CPA improved it's efficacy.(ABSTRACT TRUNCATED AT 400 WORDS)


The fertilization and development of mouse oocytes following cortical granule discharge in the presence of a protease inhibitor. The effect of leupeptin, a serine protease inhibitor, on the fertilization and development potential of oocytes stimulated to undergo cortical granule exocytosis has been investigated. An in-vitro bioassay system was used in which mouse oocytes were exposed to calcium ionophore, A23187, in the presence and absence of leupeptin, before their fertilization and development to the blastocyst stage was assessed. We have demonstrated that the presence of leupeptin in the incubation medium, at concentrations of 1 micrograms/ml and 10 micrograms/ml during the first 10 min of cortical granule exocytosis, reversed the ionophore-induced decrease in the capacity of oocytes to fertilize and develop to blastocysts. The induction of exocytosis of cortical granules by calcium ionophore was confirmed using fluorescence microscopy. Using this technique, we also confirmed that leupeptin did not inhibit ionophore-induced cortical granule exocytosis, thus supporting the contention that leupeptin acted upon released cortical exudate. It was concluded that leupeptin acted by inhibiting proteases released into the perivitelline space during the early stages of cortical granule exocytosis. Based on these results it was proposed that leupeptin could be used to prevent premature loss of fertility of human oocytes which are inadvertently activated under in-vitro conditions.


Effect of the number of inseminated spermatozoa on subsequent human and mouse embryonic development in vitro. It has been shown, in both human and mouse in-vitro fertilization (IVF), that an excess number of spermatozoa in the insemination medium leads to reduced fertilization rates. In this study, we evaluated human embryonic development after dividing the oocytes of each of 62 IVF attempts into two groups on the basis of insemination with two widely used concentrations (50,000 and 100,000 spermatozoa/ml). The embryonic growth was retarded in the group inseminated with 100,000 spermatozoa/ml: significantly fewer fast developing embryos (4-cell and 5- to 8-cell stages) were found (53.4% in the 100,000/ml group and 65.5% in the 50,000 group; P less than 0.05). In two experimental series, mouse embryonic development was evaluated in the presence of 0, 50,000, 100,000 and 500,000 spermatozoa per ml. In the first series, the spermatozoa were present during 5-20 h after insemination, while in the second series, the spermatozoa were present during the whole culture period of 120 h. The development of mouse embryos was impaired when 500,000/ml spermatozoa were present during the whole culture period. In contrast with human IVF results, the presence of up to 500,000 spermatozoa during the first 20 h after insemination did not have any significant detrimental effect on blastocyst formation in the mouse.


Gene activity and cleavage arrest in human pre-embryos. There is a high rate of spontaneous cleavage arrest around the four- to eight-cell stage of human development in vitro. Since this coincides with the time of activation of the embryonic genome it has been suggested that cleavage arrest may occur as a consequence of failure of gene activation. Gene expression in human pre-embryos is associated with an alpha-amanitin sensitive, qualitative change in protein synthesis. In order to ascertain the role of gene expression in cleavage arrest, we have examined the protein synthetic patterns of human pre-embryos which have undergone spontaneous cleavage arrest in vitro. Of 54 cleavage-arrested embryos, 27 demonstrated evidence of synthesis of proteins sensitive to alpha-amanitin, suggesting that cleavage arrest is not always accompanied by failure of activation of the genome. Our results would also suggest that activation of gene expression is simply related to neither cell number nor time spent in culture since fertilization, but may be related to continuing karyokinesis.


Factors controlling spontaneous human chorionic gonadotrophin in superfused first trimester placental explants. We have recently reported that secretion of human chorionic gonadotrophin (HCG) by placental explants in superfusion is pulsatile. In this study, the factors involved in regulation of spontaneous pulsatility were examined. In superfusion, observed secretion of HCG by isolated cells was continuous, without spontaneous episodic hormonal secretion. However, this was not due to diminished viability of the cells since these cells continued to secrete sex steroids. In addition, the stimulatory response of HCG to a highly effective dose of 10(-9) M gonadotrophin releasing hormone (GnRH) analogue was also maintained. Preincubation of explants with low concentrations of cycloheximide (10(-6)M) markedly reduced baseline HCG levels as well as pulse amplitude, suggesting that episodic hormone secretion is in part dependent on protein synthesis. Moreover, preincubation of explants with labelled leucine has shown that the secretion of placental proteins is also episodic in superfusion. The pattern was similar but not identical to that of HCG. Addition of 1 min pulses of CaCl2 caused a significant release of HCG by superfused explants, suggesting that HCG secretion occurs through the release of storage granules. It was concluded that for the expression of spontaneous pulsatile secretion of HCG cell to cell contact/communication is necessary. HCG secretion is likely to reflect exocytosis of storage granules. HCG pulsatility is partly dependent on protein synthesis and this intermittent type of secretion can be documented by overall protein secretion by the superfused explant.


'Chemical curettage' using intrauterine methotrexate injection. A 22-year-old woman with multiple urogenital anomalies presented for the second time with a blighted ovum situated in the rudimentary right horn of a uterus communicans bicornis. A dilatation and curettage was not possible owing to the inaccessibility of this horn. The first blighted ovum was aborted in 1989 (at age 21 years) following the use of intramuscular prostaglandin therapy. On this occasion, therapy was by the transvaginal, intrauterine (intra-amniotic) instillation of 100 mg methotrexate and a complete abortion of the products of conception occurred. Similar to the treatment of ectopic pregnancy, in certain cases, termination of intrauterine pregnancies using local methotrexate injection may be considered.


When genes determine motherhood: problems in gestational surrogacy. Gestational surrogacy in which a commissioning couple's egg and spermatozoon are united in vitro and the resulting embryo is implanted in a woman's uterus is, of all the new methods for overcoming infertility, the most genetically appealing. This is because the genes are often perceived as determining all aspects of human health, disease and even behaviour. Having a child with the genes of both parents has become far more attractive to most infertile couples than having one who is only genetically related to the father. However, gestational surrogacy has created a situation where one child has two mothers, each one claiming to be the 'true' mother having exclusive parental rights. Surrogacy arrangements also raise the question of the meaning of motherhood.


Annual and sub-annual rhythms in human conception rates: time-series analyses show annual and weekday but no monthly rhythms in daily counts for last normal menses. Methods of time-series analysis, which are widely used to good effect in physical sciences and econometrics, have found little use in much-needed analyses of cyclical biological phenomena. Here we apply those methods to analyses of rhythmic patterns in human conception rates. Our results confirm the annual periodicity of monthly counts of total viable conceptions, demonstrate a weekday rhythm reflecting interaction of conjugal coital rhythms with individual menstrual fertility cycles, and find no evidence of any other significant repeating pattern.


The endometrial capillaries during the normal menstrual cycle: a morphometric study. The areas of the capillary lumen, the entire capillary, the endothelial cells and the adventitia, as well as the thickness of the endothelial cell layer and the adventitia were studied using morphometric methods in endometrial samples from 34 fertile women who had a hormonal profile compatible with normal ovarian function. The biopsies were grouped around the luteinizing hormone surge. The results were calculated as mean values of 72-h periods and related to the mean levels of oestradiol and progesterone circulating in plasma 72 h prior to the biopsy. The results indicated that the sub-epithelial capillary plexus of the human endometrium undergoes dynamic changes during the normal menstrual cycle with a significant dilatation of the vessels during the post-ovulatory phase. A significant correlation was found between the area of the capillary lumen and the mean level of progesterone circulating in the plasma 72 h prior to the biopsy (P = 0.037). We conclude that the ovarian steroids produced during the normal menstrual cycle are likely to influence sub-epithelial vascularization causing dilatation in the post-ovulatory phase. This dilatation of the sub-epithelial capillaries may be related to the development of oedema appearing in the stroma at the time of the expected implantation. The possible functional significance of the capillary dilatation in terms of implantation, however, needs to be further investigated.


The aetiology of galactorrhoea in women with regular menstruation and normal prolactin levels. Fourteen primary infertile women with expressible galactorrhoea associated with regular ovulatory cycles and normal basal prolactin levels (group A) were matched for age and weight with 14 infertile women with regular menstruation but no galactorrhoea (group B). Both groups showed equivalent increases in prolactin levels after stimulation with 200 micrograms thyrotrophin-releasing hormone (TRH) during the follicular and luteal phases of the menstrual cycle. Patients in group A had a greater increase in luteinizing hormone levels after 100 micrograms i.v. injection of a luteinizing hormone-releasing hormone during the follicular phase (P less than 0.05). Following a 60 mg oral dose of buspirone hydrochloride on day 22 of the menstrual cycle, patients in group A had a greater increase in prolactin levels than patients in group B (P less than 0.01). This reflects hyper-responsive 5-hydroxytryptamine type 1A (5HT1A) receptors in group A patients and may explain the presence of galactorrhoea in these patients despite normal basal and post-TRH prolactin levels.


Biochemical analysis of cervical mucus by nuclear magnetic resonance spectroscopy. Biochemical evaluation of cervical mucus is difficult due to the characteristic rheological properties of this hydrogel. The application of high resolution nuclear magnetic resonance spectroscopy proved to be a valuable new method for differentiated biochemical analyses of human cervical mucus. A particular advantage is that it is non-destructive, that it can be applied to specimens of small volume and that no sample preparation, such as solubilization, is necessary.


Androstenedione as a predictor of ovarian hyperstimulation syndrome. Serum concentrations of testosterone, 4-androstene-3,17-dione (androstenedione), dehydroepiandrosterone (DHA) and its sulphate and sex hormone-binding globulin (SHBG) were measured in 30 ovulatory women before and after down-regulation with gonadotrophin releasing-hormone analogue, prior to ovarian stimulation in a programme of in-vitro fertilization and embryo transfer. Nine of the women developed ovarian hyperstimulation syndrome (OHSS) while the others did not. Pretreatment values of androstenedione and the androstenedione:DHA ratios were higher and the testosterone:androstenedione ratios lower in the OHSS women. The decrease in androstenedione levels during down-regulation was greater and the absolute levels following down-regulation were lower in the OHSS group. Higher levels of SHBG and lower testosterone:SHBG ratios, an index of biologically active testosterone, were observed in the OHSS group. The isolated elevation in pretreatment androstenedione levels and the high SHBG levels make the OHSS group different from patients with polycystic ovarian disease. Pretreatment values of testosterone:androstenedione or testosterone/SHBG:androstenedione ratios and/or the decrease in androstenedione during down-regulation may be used as markers, prior to stimulation, for identifying women at risk of developing OHSS.


Impaired glucose effectiveness in patients with polycystic ovary syndrome. Insulin resistance and glucose intolerance are common features of polycystic ovary syndrome (PCOS). We have investigated the effect of glucose on the fractional glucose disappearance in patients with PCOS and in age- and weight-matched control subjects. The minimal model method as applied to a frequently sampled intravenous glucose tolerance test was employed. The insulin sensitivity index (Si) and glucose effectiveness (SG) were calculated with the MINMOD program. Testosterone, androstenedione and free testosterone concentrations were significantly higher in PCOS subjects. Glucose-induced glucose clearance (SG) and insulin sensitivity were significantly lower in PCOS subjects than controls [SG: 2.7 +/- 0.3 versus 1.8 +/- 0.1 x 100/min; P less than 0.01; Si: 133.4 +/- 20.0 versus 65.6 +/- 6.4/min (nmol/ml)]. Six PCOS women had an SG value within the normal range (greater than 2.0 x 100/min) but had a similar Si to that found in PCOS women with abnormal SG. We suggest that independent alterations in both glucose- and insulin-mediated glucose uptake occur in patients with PCOS. The underlying disturbance in glucose effectiveness may be similar to that found in familial non-insulin dependent diabetes mellitus.


The correlation between interleukin 2 and soluble interleukin 2 receptors to oestradiol, progesterone and testosterone levels in periovulatory follicles of in-vitro fertilization patients. The present study was performed to evaluate the correlation between follicular fluid levels of interleukin 2 (IL-2) and IL-2 soluble receptor (sIL-2R), oestradiol, progesterone and testosterone levels, oocyte fertilization, embryo quality and pregnancy rates. Twenty-eight patients with a pure tubal factor and undergoing in-vitro fertilization and embryo transfer were randomly chosen and treated with gonadotrophin releasing hormone agonist (GnRHa) in the midluteal phase (long protocol) coupled with follicular phase administration of human menopausal gonadotrophin. Transvaginal follicular aspiration was performed 36 h after human chorionic gonadotrophin administration, followed 48 h later by embryo transfer. One hundred and twenty-three follicular fluids were sampled. The mean follicular fluid levels (+/- SD) were 2.30 +/- 0.80 fmol for IL-2, 458.2 +/- 236.0 units/ml for sIL-2R, 28.5 +/- 58.1 ng/ml for oestradiol, 2360.5 +/- 2846 ng/ml for progesterone and 7.22 +/- 7.08 ng/ml for testosterone. There was a significant (P less than 0.01) correlation between IL-2 and testosterone levels. No correlation was found between the lymphokines and serum oestradiol, follicular fluid progesterone, oocyte fertilization, embryo quality and pregnancy. It may be concluded that significant concentrations of IL-2 and sIL-2R exist in follicular fluid. Wide variations in follicular IL-2 and sIL-2R concentrations of different follicles were found in the same patients.


Spontaneous luteinizing hormone surges can be reliably prevented by the timely administration of a gonadotrophin releasing hormone antagonist (Nal-Glu) during the late follicular phase. A new gonadotrophin releasing hormone antagonist (Nal-Glu) was used during the late follicular phase of the natural cycle in order to prevent spontaneous surges of luteinizing hormone (LH). Eight regularly ovulating women (group 1) received two injections of Nal-Glu (5 mg) administered 48 h apart when plasma oestradiol levels exceeded 125 pg/ml. Human menopausal gonadotrophin (HMG, 225 IU) was administered simultaneously with Nal-Glu and repeated every 12 h thereafter until either a spontaneous LH surge occurred or human chorionic gonadotrophin (HCG, 5000 IU) was administered. HCG was arbitrarily administered 48 h after the second Nal-Glu injection. Six other women (group 2) receiving only HMG served as controls. In seven of the eight women in group 1, LH and progesterone remained low for 96 h following Nal-Glu, i.e. until HCG administration. In the remaining woman in this group, LH started to rise 12 h before HCG injection. In this group, Nal-Glu did not interfere with follicular development or the plasma profile of oestradiol. All women developed one single dominant follicle with the exception of one subject who had already spontaneously developed two dominant follicles prior to administration of Nal-Glu and HMG. In group 2, LH rose spontaneously in all women before the planned HCG injection. The luteal phase was apparently not altered by Nal-Glu. These results suggest that Nal-Glu administration during the late follicular phase of natural cycles supported by HMG, can prevent the spontaneous LH surge while not interfering with follicular growth.(ABSTRACT TRUNCATED AT 250 WORDS)


Early changes of affinity and binding sites of progesterone and oestrogen receptors in decidua exposed to RU 486. Sixty patients with 6-7 weeks of amenorrhoea were randomly allocated to three groups. The women in the first group (control) took a placebo 24 h before undergoing a vacuum aspiration. The patients in the second and third groups were given 200 mg of RU 486 orally, 12 or 24 h before surgical interruption of their pregnancy. Decidua were collected and frozen in liquid nitrogen. By Scatchard plot analysis, the number of cytosolic binding sites (1798 +/- 803 fmol/mg DNA) of progesterone in decidua in the control group was significantly reduced (P less than 0.01) to 696 +/- 408 or 626 +/- 179 fmol/mg DNA by RU 486 treatment for 12 or 24 h respectively. The dissociation constants of both cytosolic and nuclear progesterone receptors in RU 486-exposed decidua were increased (P less than 0.01). The number of nuclear binding sites of oestrogen receptor was significantly higher (P less than 0.05) in decidua with RU 486 treatment for 12 h (178 +/- 77 fmol/mg DNA) compared to the control (89 +/- 32 fmol/mg DNA). The results suggest that RU 486 might regulate progesterone and oestrogen receptors in the decidua of early human pregnancy, either directly or indirectly.


Consecutive versus alternating cycles of ovarian stimulation using human menopausal gonadotrophin. Ovarian stimulation is an effective treatment for patients with ovulatory dysfunction and unexplained infertility. An initial report has suggested that consecutive cycles of ovarian stimulation can be employed without causing a diminished response in the second cycle. However, this observation has neither been confirmed nor has a regimen of consecutive stimulation cycles been compared to one of alternating stimulation cycles. Accordingly, 44 consecutive and 54 alternating cycles of stimulation were evaluated in patients (n = 42) who were treated with human menopausal gonadotrophin (HMG) alone. Human chorionic gonadotrophin (HCG) 10,000 IU was administered i.m. when at least one follicle exceeded 16 mm in mean diameter, and this was followed by either intercourse or intrauterine insemination. Using each patient as her own control, we were unable to demonstrate any differences in mean HMG dose requirements, endocrine parameters or follicular development on the day of HCG administration, or ovulation rates in the second consecutive cycle compared to the second alternating cycle. Clinical pregnancies resulted significantly more often in a consecutive cycle (8/22) than in an alternating cycle (2/27, P = 0.029). We conclude that consecutive cycles of ovarian stimulation with HMG are not detrimental and may, in fact, result in increased cycle fecundity compared to alternating stimulation cycles.


The 24 h pattern of pulsatile luteinizing hormone, follicle stimulating hormone and prolactin release during the first 8 weeks of lactational amenorrhoea in breastfeeding women. In women, breastfeeding results in a variable period of ovarian inactivity which is apparently related to suppression of the normal pulsatile release of luteinizing hormone (LH). However, pulse profiles had only been studied during the daytime. Since resumption of pulsatile LH secretion during puberty is initiated at night, the present study determined the pattern of pulsatile LH secretion in relation to that of follicle stimulating hormone (FSH) and prolactin, and suckling and ovarian activity at 4 and 8 weeks postpartum in 20 fully breastfeeding women with lactational amenorrhoea. Blood samples were withdrawn at 10 min intervals for 24 h from 0900 h to 0900 h at either 4 weeks (n = 9) or 8 weeks (n = 11) postpartum, while the mothers and babies continued their normal pattern of suckling activity. At 4 weeks postpartum, no LH pulses occurred over 24 h in six of the nine women while one (n = 1) or two (n = 2) LH pulses occurred in three of the nine women. In contrast, LH pulses were present in nine of the 11 women at 8 weeks postpartum. The pulse frequency varied considerably from two to eight pulses over the 24 h and there was no influence of the time of day or sleep on the time of the pulse release. Lactational amenorrhoea was maintained for at least 10 weeks afterwards and there was no relationship between the time of resumption of ovarian activity and the presence or absence of pulsatile LH secretion at 4 or 8 weeks postpartum.(ABSTRACT TRUNCATED AT 250 WORDS)


Density differences between spermatozoa with antisperm autoantibodies and spermatozoa covered with antisperm antibodies from serum. Autoantibody-carrying spermatozoa from infertile men were processed using a discontinuous Percoll gradient. The treatment yielded a sperm fraction with significantly reduced antibody loading on the sperm head but did not affect antibody binding to the sperm tail. The immunodepleted sperm preparation demonstrated fertilizing ability when used in in-vitro fertilization. Normal, antibody-free semen samples were coated in vitro with antisperm antibodies from serum, followed by Percoll gradient centrifugation. No significant separation of antibody-free spermatozoa was obtained, regardless of the antibody binding site.


Outcome of treatment subsequent to the elective cryopreservation of all embryos from women at risk of the ovarian hyperstimulation syndrome. From 1st June 1989 to 31st May 1991, 78 women with a serum oestradiol level greater than 3500 pg/ml on the day of the ovulatory trigger, following pituitary suppression with buserelin and ovarian stimulation with human menopausal gonadotrophins (HMG), had all their embryos electively cryopreserved at the pronucleate stage to minimize the risk of developing ovarian hyperstimulation syndrome (OHS). Treatment with buserelin was continued in the luteal phase. A median of 19 oocytes (range 7-43) was obtained and 12 embryos (range 1-37) frozen per cycle. Twenty-one (27%) women developed OHS (six severe). Women developing OHS had higher (P less than 0.05) serum oestradiol concentrations on the 7th day after oocyte retrieval, compared to those who did not. No differences were found for any of the following criteria: aetiology of infertility, age, total dose of HMG, number of oocytes, fertilization rate or freeze-thaw survival of embryos. Subsequently, 125 frozen-thawed embryo replacements have been undertaken, using buserelin and hormone replacement therapy (HRT) (n = 93) or natural cycles (n = 32). The overall freeze-thaw survival and implantation rates per embryo were 71.8 and 11.7%, respectively. The pregnancy rates in natural cycles (19%) and buserelin/HRT cycles (29%) were not significantly different.


Ovarian torsion: a complication of GIFT. A report on two cases and literature review. Two cases of ovarian torsion after gamete intra-Fallopian transfer are described and the roles of ovarian manipulation during the procedure, ovarian hyperstimulation and pregnancy as predisposing factors are discussed. The relevant literature is reviewed.


Results of routine syphilitic and human immunodeficiency virus (HIV) serology in infertility. While it has been accepted practice to screen women undergoing infertility evaluation for syphilis, there are few data in the literature regarding the seroprevalence of human immunodeficiency virus (HIV) infection in infertile patients despite the increasing number of HIV-positive women. In the present study, six out of 2137 infertility patients were seropositive for syphilis (0.28%) and four out of 791 were HIV positive (0.5%). All four women with HIV antibodies had negative tests for syphilis and none of them related any risk factor for HIV infection on their initial visit. The 0.5% sero-positivity rate found in our study warrants routine HIV testing in infertile patients.