Aminophylline increases parasternal intercostal muscle activity during hypoxia in humans.

To clarify the mechanism of action of aminophylline on the hypoxic ventilatory response in humans, we analyzed the effects of aminophylline on respiratory neural output. To evaluate the respiratory neural output, we analyzed the electromyogram (EMG) of the parasternal intercostal muscle, one of the major inspiratory muscles, in eight healthy subjects. Both before and during aminophylline administration, measurements of ventilatory parameters with EMG recordings were conducted in room air, mild hypoxia (F(I)(o)(2) 0.15), and severe hypoxia (F(I)(o)(2) 0.11). Before administering aminophylline, hypoxic stimulation elicited ventilatory augmentation in a hypoxia-intensity dependent manner. Administration of aminophylline caused significant increases in ventilation (V (I)), tidal volume (V(T)), respiratory frequency (f(R)), and the respiration-related phasic moving averaged EMG amplitude (tidal EMG), at corresponding levels of hypoxia compared to before aminophylline. Augmentation patterns of hypoxia-induced increases in V(T) and tidal EMG showed close similarity. These results indicate that augmentation of hypoxic ventilatory response by aminophylline is mainly mediated by an increase in the respiratory neural drive in healthy humans.

Serum dehydroepiandrosterone sulfate in Cushing's syndrome.

Serum concentrations of dehydroepiandrosterone sulfate (DHEA-S) were measured in patients with hyperadrenocorticism. When compared to normal subjects of corresponding age, serum DHEA-S levels were normal or elevated in 37 patients with Cushing's disease. In contrast, DHEA-S levels were significantly lower than those of normal subjects in all 28 patients with hyperadrenocorticism due to benign adrenocortical adenoma, suggesting that ACTH is the major determinant of DHEA-S secretion and that determination of serum DHEA-S concentrations is useful in the biochemical differential diagnosis of the etiology of Cushing's syndrome. In six patients with adrenocortical adenoma, the recovery of suppressed DHEA-S secretion after removal of the adrenal gland affected by a tumor was studied. Serum cortisol levels normalized by the end of the second year after unilateral adrenalectomy, while DHEA-S levels remained low for at least 2 succeeding yr. The results suggest that deficient ACTH secretion may result in a greater and longer lasting loss in the ability of the adrenal cortex to secrete androgens than in the ability to secrete cortisol.

Aromatization by skeletal muscle.

Because peripheral aromatization is the major source of circulating estrogens in men and postmenopausal women, we studied the aromatase activity in muscle tissue from both men and postmenopausal women. To do so, the in vitro conversion of tritiated androstenedione to estrogen in homogenates of skeletal muscles obtained at autopsy was studied. Samples from lower limb muscles of both men and postmenopausal women produced estrogen, ranging from 8.5-39.8 pg/g wet wt. The conversion was almost the same as that reported for human adipose tissue, suggesting that the contributions of muscle and fat to the extraglandular production of estrogens in these subjects might be similar. This is the first direct confirmation of muscle aromatase activity and indicates the possible importance of muscle as an extragonadal source of estrogen in both men and postmenopausal women.

Atrial natriuretic peptide in umbilical cord blood: evidence for a circulating hormone in human fetus.

To determine whether atrial natriuretic peptide (ANP) is a circulating hormone in the human fetus, ANP levels in umbilical cord plasma obtained at the time of delivery in 10 normal infants were measured by RIA. Plasma ANP levels were consistently higher in paired umbilical cord arterial than in cord venous samples. The mean umbilical cord arterial plasma ANP concentration [283 +/- 56 (+/- SEM) pg/ml] was significantly higher than that in cord venous plasma (165 +/- 27 pg/ml) or maternal peripheral venous plasma (155 +/- 25 pg/ml). Analyses by reverse phase high performance liquid chromatography revealed that the elution pattern of plasma ANP in cord arterial blood and that in maternal venous blood were nearly identical and that the retention time of the main ANP peak coincided with that of alpha-human ANP. These results suggest that alpha-human ANP is a circulating hormone in the human fetus.

Amniotic fluid lactoferrin in intrauterine infection.

Lactoferrin (LF) has been found in most biological fluids including amniotic fluid and cervical mucus in pregnant women and is released from neutrophils in response to inflammation. It is an important component of the host defence against microbial infections due to its antimicrobial properties. Premature labour is caused by amniotic infection and high concentrations of inflammatory cytokines in amniotic fluid with infection are well established. In the present study, LF levels of intrauterine infection in amniotic fluid were measured and the biological significance of LF was investigated. The effects of LF on IL-6 production in cultured amnion cells were also investigated. The concentrations of LF and IL-6 in amniotic fluid with chorioamnionitis (CAM) were 8.76+/-0.65 microg/ml and 6.92+/-4.88 ng/ml (n = 28), respectively, and both were significantly higher (P<0.01) than those without CAM (0.86+/-0.81 microg/ml and 0.34+/-0.25 ng/ml; n = 31). LF and IL-6 levels were significantly higher (P<0.01) with CAM. A significant positive correlation between LF and IL-6 levels in amniotic fluid was found (r = 0.91, P<0.01). To our knowledge, this was the first study of its kind, which shows that IL-6 production induced by lipopolysaccharide in cultured cells was significantly inhibited below physiological concentration of LF in the amnion. In addition, the immunohistochemical localization of LF in fetal membranes was investigated. In the fetal membranes with CAM, strong positive staining was observed in amniotic and chorionic membranes, with leucocyte migration, while weak staining was observed in membranes without CAM. These results show conclusively that LF suppresses amniotic IL-6 production under the conditions of intrauterine infection.

Serum levels of neurophysin in pregnancy and in the postpartum period: relation to 17beta-estradiol levels.

Changes in serum neurophysin levels were studied in women during normal pregnancy and after delivery. Neurophysin was elevated as early as the sixth week of gestation and gradually increased until, at the latest, the twentieth week. A significant positive correlation was obtained between serum concentrations of neurophysin and those of 17beta-estradiol. In serum samples with 17beta-estradiol levels exceeding 5 ng/ml, neurophysin was consistently elevated above the normal control range. After delivery, serum neurophysin concentrations declined quite rapidly. The levels returned to those of nonpregnant women by 6 days postpartum. These results support the view that a high rate of secretion of 17beta-estradiol may be one of the factors responsible for the elevated levels of serum neurophysin in pregnancy and that this effect disappears rapidly after the disposal of estrogen, independent of the duration of the elevated estrogen levels.

Prenatal diagnosis of the fetal RhD blood type using a single fetal nucleated erythrocyte from maternal blood.

OBJECTIVE: To develop a method that allows prenatal diagnosis of the fetal RhD blood type from maternal blood. METHODS: Maternal blood was obtained at 8-31 weeks' gestation, and nucleated erythrocytes were separated with Percoll using a discontinuous density gradient method, then collected individually by micromanipulation under microscopic observation. After whole genome amplification with primer extension pre-amplification, exon 7 of the RhD and RhCE as well as the ZFX/ZFY loci were further amplified by a nested polymerase chain reaction (PCR). RESULTS: Nucleated erythrocytes were detected in nine of ten maternal blood samples, and sex was determined in 13 of 21 nucleated erythrocytes. RhD genotype could be diagnosed in 12 of the 13 nucleated erythrocytes in which sex could be determined. The results of RhD blood type and sex in nucleated erythrocytes obtained from maternal blood were identical with those of newborns. Fetal RhD blood type could be determined in six of ten maternal blood samples. CONCLUSION: A new method for noninvasive prenatal diagnosis of the fetal RhD blood type using a single nucleated erythrocyte isolated from maternal blood was demonstrated. This diagnostic method offers extremely useful information for the management of Rh-negative pregnant women. Furthermore, this method of prenatal diagnosis can be applied to other genetic disorders and is expected to become the preferred method of noninvasive prenatal diagnosis of DNA.

Serum level of 19-hydroxyandrostenedione during pregnancy and at delivery determined by gas chromatography/mass spectrometry.

19-Hydroxyandrostenedione (19-OHA) is secreted from the adrenal glands in men and women and also from the placenta during pregnancy. It has been found to cause hypertension in animal models. We have synthesized [7,7-2H2]-19-OHA with high deuterium content and, together with [7,7-2H2]A and [9,11-2H2]estrone (E1), have developed a quantitative assay of serum level 19-OHA, A, and E1 using the gas chromatography/mass spectrometry-mass fragmentography method to monitor individual subjects throughout pregnancy. The labeled 19-OHA, used as internal standard, showed only 6.73% of unlabeled compound. Recovery of standard 19-OHA, A, and E1 (5,000 pg each) added to male plasma was 97.4 +/- 2.3%, 96.3 +/- 2.1%, and 100.1 +/- 4.1% (mean +/- SD), respectively; the intraassay coefficient of variation was 2.1%, 3.5%, and 3.8%, respectively. Ten pregnant subjects without complications and 10 pregnant subjects near term with hypertension were selected (with informed consent). The 19-OHA and E1 serum concentrations of maternal venous blood from uncomplicated pregnancies increased significantly as gestation progressed (19-OHA: first trimester, 225 +/- 72; second trimester, 656 +/- 325; third trimester, 1,518 +/- 544 pg/ml), reaching the highest level at delivery (19-OHA: 1,735 +/- 684 pg/ml). Whereas a positive correlation was found between the level of 19-OHA and E1, no apparent change of the A level was observed during pregnancy. Levels of the three steroid hormones in pregnancy complicated by hypertension in the second and third trimester were not found to be significantly different from those of normal pregnancy (19-OHA of hypertensive subjects: second trimester, 762 +/- 349; third trimester, 1,473 +/- 491 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Localization and expression of steroid sulfatase in human fallopian tubes.

Localization of steroid sulfatase, a membrane-bound microsomal enzyme, in human fallopian tubes was immunohistochemically investigated, and expression of RNA was confirmed by competitive RT-PCR. Human fallopian tubes were obtained from 10 patients in follicular and early luteal phases during gynecological laparotomy. An anti-human rabbit polyclonal antibody was prepared against sulfatase protein purified from human placenta. Total RNA was isolated from epithelium of fallopian tubes. A heterologous RNA competitor was designed, and competitive RT-PCR was carried out. Steroid sulfatase was localized to the cytoplasm of epithelial cells. With respect to the positive staining of cells, the number of positive secretory cells was higher than that of ciliated cells. A significantly higher number of positive cells was found in tissue obtained from the early luteal phase than that found in tissue from the follicular phase. An abundant expression of sulfatase mRNA in early luteal phase was also observed. This study demonstrates, for the first time, that steroid sulfatase is localized to human epithelial cells and that steroid sulfatase staining and mRNA expression changes with the menstrual cycle. These results suggest that sulfatase in the fallopian tube may be involved in controlling the local steroid environment, which appears to regulate aspects of the physiological reproductive function of the fallopian tube.

Noninvasive prenatal diagnosis using a single fetal nucleated erythrocyte isolated by micromanipulation from maternal blood.

Prenatal diagnosis of hereditary diseases is usually performed by collecting fetal samples using amniocentesis, chorionic villus sampling (CVS), or cordocentesis. However, these procedures are associated with some degree of risk. For example, abortion owing to hemorrhage or infection occurs in 0.2-0.4% of patients undergoing amniocentesis. Furthermore, CVS reportedly presents the potential risk of fetal limb malformation in 0.01-0.03% of cases (1). Each method has the risk of misdiagnosis because of contamination by maternal cells. Thus, the development of a suitable noninvasive method is important (Table 1). Table 1 Summary of Prenatal Diagnostic Methods Diagnostic methods Weeks of gestation Benefits Defects Fetal loss rate,% Amniocentesis 15-18 Relatively simple Necessary to culture 0 2-0.4 Contamination by maternal cells Chorionic 9-12 Early and direct Limb malformations 0 5-10 villus sampling diagnosis possible (0 01-0.03%) Contamination of maternal cells Placental mosaicism Cordocentesis 18- Early and direct Bleeding 0.5-2.0 diagnosis possible Fetal bradycardia Contamination of maternal cells Maternal 6- Noninvasive - 0 peripheral blood.

Behavioral and adrenocortical responses to stress in neonates and the stabilizing effects of maternal heartbeat on them.

Adrenocortical response to stress in neonates was assessed by estimating salivary concentrations of cortisol, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulfate (DHEA-S). Behavioral responses to stress were also scored clinically, according to the method of Lewis and Thomas [15]. The modulation of these measures by hearing maternal heartbeat was also evaluated. Levels of cortisol and DHEA in saliva in neonates correlated well with those of serum showing significant variation in painful stress. Hearing the maternal heartbeat significantly reduced the grade of variation, but the recorded sound of the Japanese drum with the same rhythm showed no significant effect. Scores of the behavioral response to stress also decreased significantly in neonates who were presented with the maternal heartbeat in comparison with those hearing no sound or the beat of a Japanese drum.

Lactoferrin and interleukin-6 interaction in amniotic infection.

Lactoferrin (Lf) has been found in most biological fluids including amniotic fluid and cervical mucoids in pregnant women, and released from neutrophils in response to the inflammation. As Lf possesses antimicrobial properties, it is widely considered to be an important component of the host defence against microbial infections. It is known that premature labor is caused by amniotic infection with the increase of prostaglandin production. High concentration of the inflammatory cytokines: interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) in the amniotic fluid has been known. However, changes of Lf in amniotic fluid with infection has not been reported. In the present study, Lf concentrations in amniotic fluid were measured under the intra-uterine infections state and the biological significance of Lf was investigated. The effects of Lf on the IL-6 and IL-6mRNA production in cultured amnion cells were also investigated. The concentrations of Lf and IL-6 in amniotic fluid with CAM were 8.76 +/- 0.65 micrograms/ml and 6.92 +/- 4.88 ng/ml (n = 28) respectively and both were significantly higher (p < 0.01) than those without CAM [0.86 +/- 0.81 microgram/ml and 0.34 +/- 0.25 ng/ml (n = 31)]. Significant positive correlation (r = 0.91, p < 0.01) between Lf and IL-6 levels in amniotic fluid was found. IL-6 production induced by lipopolysaccharide (LPS) (100 ng/ml) in cultured amnion cells was significantly inhibited (p < 0.05) under the physiological concentration of Lf in amnion. Total RNA was extracted from the amniotic cells by guianizine solution. RT-PCR procedure and product analysis were performed from one microgram aliquote of total RNA. beta-actin was used as an international standard and c-DNA samples were followed by 30 cycles of PCR. RT-PCR product of IL-6 mRNA was detected by Southern hybridization. Expression of IL-6 mRNA was inhibited by the addition of Lf. From the results, the possibility that Lf might suppress amniotic IL-6 production under the condition of amniotic infection is suggested. It is also suggested that Lf might act as self defence mechanism from intra-uterine infection.

Steroid formation in osteoblast-like cells.

For preventing the reduction of bone mass in postmenopausal women, oestrogen replacement is known to be useful and the importance of sex steroids in bone metabolism in both sexes is well established. The presence of steroid-converting-enzyme activities in various osteoblast and osteoblast-like cells has been demonstrated using in vitro culture systems. In the present study, we assessed the expression of messenger ribonucleic acid (mRNA) for aromatase, steroid sulphatase, 5 alpha-reductase, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and 3 beta-HSD by reverse transcription-polymerase chain reaction in the human osteoblast-like cell lines, MG 63 and HOS. Oestrogen, androgen and progesterone receptor mRNAs were also measured. Expression of mRNA for these enzymes and receptors was found in both cell lines without induction. From these and previous findings, we conclude that osteoblast-like cells have the capacity to form biologically potent oestrogens and androgens from peripheral circulating steroids. This may indicate an important role of bone in facilitating hormonal action.

Effect of dehydroepiandrosterone sulphate, oestrogens and prostaglandins on collagen metabolism in human cervical tissue in relation to cervical ripening.

To clarify the mechanism of cervical ripening at term, collagenase activity in human cervical tissue was studied. The effects of steroids and prostaglandins on collagenase activity were also examined. Collagenase activities in cervical tissues obtained from non-pregnant (n = 5) and pregnant women (early-pregnant, n = 3; late-pregnant, n = 14) were measured, with or without the addition of steroids and prostaglandins into the incubation medium prior to the measurement of enzyme activity. The enzyme activity was significantly (P < 0.01-0.05) higher in the cervical tissue obtained from late-pregnant women than that from non-pregnant and early-pregnant women. Collagenase activity was significantly (P < 0.05) elevated when steroid sulphates such as dehydroepiandrosterone sulphate and oestrone sulphate were added to the incubation medium, while the addition of free steroids, prostaglandin E2 and prostaglandin F2 alpha did not alter the activity. These data suggest that conjugated steroid produced in the foetoplacental unit during pregnancy may be involved in cervical ripening through the enhancement of collagenase activity.

[Effect of interleukin-1beta on aromatase activity in human osteoblast-like cells].

OBJECTIVE: To study the stimulation of interleukin (IL)-1beta and interleukin-1 receptor antagonist (IL-1ra) on aromatase activity in human osteoblast cells. METHODS: In the present study, the effect of IL-1beta and IL-1ra on aromatase (Arom) activity and mRNA expression in human osteoblast-like cells was investigated by [(3)H] water released upon the conversion of [1beta-(3)H] androstenedione to estrone and the reverse-transcription polymerase cain reaction (RT-PCR) method. The experimental group is divided into four group: G1 (IL-1beta 1 microgram/L), G2 (10 microgram/L), G3 (IL-1beta 10 microgram/L + IL-1ra 500 microgram/L) and G4 (IL-1ra 500 microgram,/L). RESULTS: The activity of G1 and G2 resulted in (4 211 +/- 348) pmol/10(6) cell.h(-1) and (4 958 +/- 231) pmol/10(6) cell.h(-1), respectively. IL-1ra neutralized the increased arom activity to control level in G3 (3 652 +/- 203) pmol/10(6) cell.h(-1). Arom mRNA expression was increased with the dose increasing of IL-1beta. CONCLUSION: These findings suggest that IL-1beta stimulates arom activity through the IL-1 receptor and addition of IL-1beta increased arom activity in a dose-dependent manner.

[Gonadal dysfunction].

Function of hypothalamic-pituitary-ovarian axis is an essential factor for the maintenance of regular cycles in mature women. The disturbance of function of those organs causes gonadal dysfunction such as anovulation, amenorrhea and menstrual disorders. Therefore, the correct diagnosis for the assessment of CNS and ovarian function is clinically important to treat the patients those who have an menstrual disorders. In this review, the mechanism of normal gonadal cycles and the diagnostic method and the treatment of gonadal dysfunction are described.

Phase II study of S-1 monotherapy in patients with previously treated, advanced non-small-cell lung cancer.

BACKGROUND: In this phase II clinical trial, we evaluated the efficacy and safety of S-1 monotherapy in patients with previously treated advanced non-small-cell lung cancer (NSCLC). We also measured plasma concentrations of 5-fluorouracil (5-FU) and 5-chloro-2,4-dihydroxypyridine components of S-1 and examined correlation with effectiveness and toxicity. METHODS: S-1 was given orally at a dose of 80 mg/m(2)/day for 14 consecutive days, followed by a 7-day rest period. This treatment course was repeated until disease progression or intolerable toxicity. RESULTS: We enrolled 30 patients. The response rate was 26.7% (8/30), and the disease control rate was 70% (21/30). Median progression-free survival (PFS) was 3.1 months, and median overall survival (OS) was 11.2 months. Mutations in the epidermal growth factor receptor (EGFR) gene were analyzed in 27 patients. The response rate was higher in patients with mutant EGFR (50.0%) than in those with wild-type EGFR (11.8%, P = 0.0288). Median PFS was 4.8 and 2.5 months (P = 0.038), and median OS was 22.4 and 8.4 months (P = 0.071). There was no grade 4 toxicity in this study. Five patients had grade 3 non-hematologic toxicity, and there was a trend toward higher plasma concentrations of 5-FU in those patients than in another patients. CONCLUSIONS: S-1 monotherapy is effective and well-tolerated treatment for previously treated advanced NSCLC.