Stimulation of proteoglycan production by glucosamine sulfate in chondrocytes isolated from human osteoarthritic articular cartilage in vitro.

OBJECTIVE: This study investigated the in-vitro effects of a crystalline glucosamine sulfate (GS) preparation on DNA synthesis and on proteoglycan (PG) and type II collagen (coll II) production by human articular chondrocytes isolated from human osteoarthritic articular cartilage in a 3-dimensional culture system for 4, 8, and 12 days. MATERIALS AND METHODS: Human articular chondrocytes from osteoarthritic femoral heads were isolated from their matrix by collagenase digestion and then cultured in suspension. Under constant agitation, cells aggregated and formed a cluster within a few days. The effects of GS (1-100 micrograms/ml) on chondrocytes were determined by quantifying DNA synthesis (by measurement of [3H]-thymidine uptake) as well as PG and coll II production using radiommunoassays (RIAs) specific for coll II and to human human cartilage PG. Cross-reaction with GS in the RIAs was not detected. Moreover, PG size distribution was determined by exclusion chromatography under associative conditions to determine the association of PG monomers with hyaluronic acid (HA) to form large molecular weight PG aggregates. RESULTS: Under the above conditions, PG production in culture media and chondrocyte clusters was increased by GS (10-100 micrograms/ml). DNA synthesis and coll II production were not modified by GS. In addition, GS did not modify the physico-chemical form of PG produced by cells during culture. CONCLUSIONS: Glucosamine sulfate did not affect DNA synthesis nor coll II production but caused a statistically significant stimulation of PG production by chondrocytes from human osteoarthritic cartilage cultured for up to 12 days in 3-dimensional cultures.

Plasma estradiol concentrations and pharmacokinetics following transdermal application of Menorest 50 or Systen (Evorel) 50.

OBJECTIVES: In order to compare the pharmacokinetics of two transdermal estrogen replacement therapy (ERT) systems designed to release 50 micrograms 17 beta-estradiol/day, two studies were performed in healthy postmenopausal volunteers. METHODS: Both studies had a cross-over design and incorporated a 1-week wash-out period between treatments. In the first study, Menorest 50 and Systen 50 (Evorel 50) were compared over four days of application in 30 women. In the second, 13 women wore each of the two systems for a total of 12 days each (three patches each for 4 days), and comparison was made during the third patch period (steady state, between days 8 and 12). Plasma 17 beta-estradiol levels were assayed using specific direct radioimmunoassays, and pharmacokinetic parameters were calculated by standard methods. All the samples of the first study were re-analysed using a different radioimmunoassay and the results of both assays were compared. RESULTS: In both studies, plasma 17 beta-estradiol levels rose at a comparable rate and reached similar peak levels with each of the two formulations. Levels then remained relatively constant throughout both evaluation periods with Menorest 50, but began to decline after 12 hours in the first study and after 30 h under steady state conditions in the second study with Systen 50. The difference between the two products was statistically significant in both studies. Analysis of pharmacokinetic parameters confirmed the greater bioavailability of Menorest 50. In addition, 17 beta-estradiol levels remained within the suggested therapeutic ranges for relief of acute symptoms and protection against osteoporosis for longer periods of time with Menorest 50 than with Systen 50. CONCLUSION: Since the acute efficacy, long-term protective effects, side effects and risks associated with ERT may depend on critical threshold plasma levels, much attention should be paid to the pharmacokinetic profiles of different formulations. The comparison of these two different radioimmunoassays demonstrates the comparability of their results.

Duality of glutamatergic and GABAergic control of pulsatile GnRH secretion by rat hypothalamic explants: I. Effects of antisense oligodeoxynucleotides using explants including or excluding the preoptic area.

Using antisense oligodeoxynucleotides we aimed to study the role of N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) receptors in the mechanism of Gonadotrophin-releasing hormone (GnRH) secretion in vitro. Since GnRH cell bodies are located in the rat preoptic hypothalamus while most GnRH terminals are in the retrochiasmatic hypothalamus, we compared the effects of oligodeoxynucleotides on explants of the whole (preoptic area included) or retrochiasmatic hypothalamus. When GnRH secretion is evoked by muscimol and NMDA, a time-related reduction of GnRH secretion is caused by antisense oligodeoxynucleotides for the beta subunit of the GABAA receptor and the NR2A subunit of the NMDA receptor, respectively. After 6-7 h, binding studies of tritiated ligands show a decrease in GABA- and NMDA-receptor expression. While these antisense effects are observed using whole explants, no such effects are seen using retrochiasmatic explants, indicating that the facilitatory GABAA and NMDA receptors are encoded in the preoptic area. Using several missense oligodeoxynucleotides or antisense for the NR2B and NR2C subunits of the NMDA receptor, the muscimol- and NMDA-evoked release of GnRH is not affected. When spontaneous pulsatile GnRH secretion is studied, the NR2A antisense oligodeoxynucleotides cause an increase of the interpulse interval. This increase is seen using whole but not retrochiasmatic explants. In contrast, the GABAA and NR2C antisense oligodeoxynucleotides result in a reduction of GnRH interpulse interval. Such a reduction is seen using whole as well as retrochiasmatic explants, indicating that the GABAA and NMDA receptors which mediate inhibition of GnRH pulsatility are encoded in the retrochiasmatic hypothalamus. We conclude that NMDA receptors (NR2A subunit) encoded in the preoptic hypothalamus mediate a facilitatory effect on GnRH pulsatility while GABAA and NMDA (NR2C subunit) receptors encoded in the retrochiasmatic hypothalamus mediate an inhibition of GnRH pulsatility. Pulsatile GnRH secretion is affected differently than the agonist-evoked release of GnRH suggesting that the GnRH secretory neurons and the GnRH pulse generator consist of different cellular entities.

Duality of glutamatergic and GABAergic control of pulsatile GnRH secretion by rat hypothalamic explants: II. Reduced NR2C- and GABAA-receptor-mediated inhibition at initiation of sexual maturation.

N-methyl-D-aspartate (NMDA) receptors and gamma-aminobutyric acid (GABA) receptors are involved in the mechanism of pulsatile gonadotrophin-releasing hormone (GnRH) secretion. The aim of this study was to elucidate the role of those receptors in the acceleration of pulsatile GnRH secretion seen at onset of puberty. Using hypothalamic explants from prepubertal (15 days), early pubertal (25 days) and adult (50 days) male rats, we studied the effects of pharmacological antagonists and antisense oligodeoxynucleotides on GnRH release evoked by NMDA and GABA receptor agonists as well as the interval between spontaneous GnRH secretory pulses. At the three studied ages, the muscimol-evoked release of GnRh is similarly inhibited by the GABAA receptor antagonist bicuculline. In contrast, the frequency of pulsatility is stimulated by bicuculline as indicated by a reduction of the mean GnRh interpulse interval from 60 to 40 min and such an effect is seen at 15 days only. The GnRH interpulse interval is also reduced by GABAA receptor antisense oligodeoxynucleotides at 15 days while no effects are seen at 25 days. At the three studied ages, the NMDA-evoked release of GnRH and the GnRh interpulse interval are similarly inhibited by 100 or 500 microM of the NMDA receptor antagonist 7-chlorokynurenic acid (7CK). These effects are consistent with the increase of GnRH interpulse interval caused by NR2A antisense oligodeoxynucleotides at 15 days (86 vs 64 min in controls) as well as 25 days (44 vs 36 min). A low (5 microM) concentration of 7CK does not result in any effect except a reduction of GnRH interpulse interval which is seen at 15 days only. A similar reduction of GnRh interpulse interval is obtained using NR2C antisense oligodeoxynucleotides at 15 days (50 vs 64 min in controls) while no effects are seen at 25 days (35 vs 36 min). At 25 days, muscimol can prevent the developmental increase in frequency of pulsatile GnRH secretion. In summary, pulsatile GnRH secretion by the prepubertal hypothalamus characteristically involves an inhibition mediated through GABAA receptors and the NR2C subunit of NMDA receptors. Based on these data, we propose a model for the mechanism of the onset of puberty which involves the disappearance or inactivation of GABAergic neurons located in the retrochiasmatic hypothalamus and expressing the NR2C subtype of NMDA receptors.

Sepsis and serum cytokine concentrations.

OBJECTIVE: To look for relationships between the classification of sepsis and plasma cytokine concentrations. DESIGN: Prospective, consecutive entry study of patients meeting severe sepsis criteria and having bacteriologically documented infections. SETTING: University hospital, surgical intensive care unit. PATIENTS: Fifty consecutive patients developing severe sepsis or septic shock between December 1991 and December 1993. MEASUREMENTS AND MAIN RESULTS: Concentrations of tumor necrosis factor, interleukin (IL)-6, IL-8, and leukemia inhibitory factor were measured by immunoradiometric assay in the plasma of patients as soon as they developed severe sepsis or septic shock. Septic shock patients were divided into three groups in a blinded fashion (i.e., without knowing the results of the concentrations of cytokines), according to the presence of sustained hyperlactacidemia and to the rapidity of the onset of sepsis. Peak concentrations of all cytokines were statistically different between severe sepsis and septic shock patients. This finding was almost exclusively due to the data from patients with rapid onset of septic shock, who demonstrated very high but transient cytokine concentrations. Septic shock patients may thus have different profiles in the time course of their cytokine concentrations. The transient, high peak concentrations of cytokines were also related to transient leukopenia. Among the cytokines measured, IL-8 appeared to be the one that correlated best with lactacidemia, the presence of disseminated intravascular coagulation, severe hypoxemia, the Acute Physiology and Chronic Health Evaluation II score, and mortality rate. CONCLUSIONS: According to the profiles of the cytokines, septic shock patients do not represent a homogeneous population. These profiles should be described in order to distinguish between patients, and the profiles may be useful to identify those patients susceptible to new therapies.

Efficacy and safety of meloxicam in patients with rheumatoid arthritis.

OBJECTIVE: To evaluate the efficacy and safety of meloxicam, a new acidic enolic nonsteroidal anti-inflammatory drug, at doses of 7.5 and 15 mg once daily in patients with rheumatoid arthritis (RA). METHODS: Meloxicam 15 and 7.5 mg daily was administered for 21 days in this double blind, randomized, placebo controlled study. 159 patients received meloxicam 7.5 mg, 162 received meloxicam 15 mg, and 147 received placebo. RESULTS: Meloxicam 15 mg once daily was significantly superior (p < 0.05) to placebo in 3 of the 4 primary endpoints (disease activity assessed by the investigator, disease activity assessed by the patient, and reduction of the number of tender/painful joints). No difference was observed regarding number of swollen joints. The difference between meloxicam 7.5 mg once daily and placebo reached statistical significance in 2 of the 4 primary endpoints, disease activity assessed by the patient and number of tender/painful joints. A statistically significant difference between meloxicam 1.5 mg and 7.5 mg was not observed for any primary endpoint. The rating of global tolerance by investigators and patients at the end of the study was similar in the 3 treatment groups, indicating that meloxicam and placebo were generally similarly well tolerated. However, there was a slightly higher incidence of gastrointestinal (GI) disturbances reported by patients receiving meloxicam 15 mg. GI adverse events were reported by 11, 11, and 16% of patients in the placebo, meloxicam 7.5 mg, and meloxicam 15 mg groups, respectively. None were serious. CONCLUSION: Meloxicam in daily doses of 7.5 and 15 mg is effective in treating the signs and symptoms of RA.

Dimeric inhibin A and alpha-subunit immunoreactive material in maternal serum during spontaneous and in vitro fertilization pregnancies.

To increase our understanding of the nature and source of immunoreactive inhibin-related material during pregnancy, we studied inhibin secretion in women pregnant after spontaneous conception and after in vitro fertilization (IVF). Two solid phase enzyme-amplified immunoassays (EASIAs) were used to measure the inhibin A dimer and inhibin alpha-subunit immunoreactivities (alpha-inhibin). In spontaneous pregnancies, levels of both inhibin A and alpha-inhibin were low during the first two trimesters of pregnancy, but a significant increase was observed toward the end of gestation. Both assays confirmed that inhibin concentrations in IVF pregnancies exceeded those in spontaneous pregnancies during the entire first trimester. Moreover, the two assays displayed discordant profiles. The alpha alpha-EASIA, which detects all alpha-inhibin immunoactivity, displayed a major peak during the follicular phase and a second broader peak during the luteal phase and corpus luteum rescue. A progressive decline was observed during the subsequent weeks. EASIA measurements for inhibin A revealed distinct peaks during the follicular phase, the luteal phase, and the hCG peak. A marked fall, however, was seen at the time of corpus luteum rescue. In summary, these data indicate that the nature of the immunoreactive material changes considerably during the different phases of pregnancy. The available evidence further points to an ovarian source of dimeric inhibin in early pregnancy and a placental source toward the end of pregnancy.

Effects of ipriflavone and its metabolites on human articular chondrocytes cultivated in clusters.

Ipriflavone (IP) is an isoflavone derivative that was suggested to have bone-sparing effects in post-menopausal and senile osteoporosis. A moderate stimulatory effect of IP and its metabolites on proliferation of osteoblastic cells was reported in rat osteoblastic osteosarcoma cell line. We investigated the effects of different concentrations (0, 1, 10 and 100 micrograms/ml) of IP and its metabolites (MET I, II, III and V) on the incorporation of [3H] thymidine and production of proteoglycans (PG) and type II collagen (COL II) by human articular chondrocytes during a 12-day period, in a three-dimensional chondrocyte culture model. [3H]thymidine uptake was measured in chondrocyte clusters, and specific PG and COL II radioimmunoassays were performed every 4 days on the culture medium and cell clusters. Incubation with IP or its metabolites did not affect [3H]thymidine uptake regardless of the dose. PG released into the culture medium and PG cluster content rose significantly (P < 0.025) in presence of IP (1, 10 and 100 micrograms/ml). MET I increased PG release in culture medium (10 and 100 micrograms/ml) and PG cluster content (100 micrograms/ml). MET II has no effect on PG production. MET III increased PG in culture medium (100 microgram/ml) but did not influence PG cluster content while MET V (100 micrograms/ml) increased both PG release in culture medium and PG cluster content. COL II release in culture medium and COL II cluster content were significantly (P < 0.025) increased in presence of IP (10 and 100 micrograms/ml), MET III (1, 10 and 100 micrograms/ml) or MET V (100 micrograms/ml). MET I and II did not significantly affect COL II production.

UVB-irradiation of human bone marrow: potential for donor specific tolerance.

Bone marrow mononuclear cell (BMMC) transplant may serve to produce donor specific tolerance for a coincident solid organ graft, but with the risk of graft versus host disease (GVHD). We examined in vitro the immunomodulatory effect of UVB on human BMMCs as potential prophylaxis against GVHD for clinical transplantation. After 10-200 J/m2 UVB-irradiation, BMMCs were examined by proliferative response (in mixed lymphocyte reaction and following phytohemagglutinin stimulation) and by cytokine profile. We also evaluated CFU-GM, CFU-GEMM, and BFU-E progenitor viability by 2-week methyl cellulose cultures following UVB-irradiation. Parallel studies were applied to marrow that was T-cell depleted by soybean agglutination (SBA) or by SBA and sheep erythrocyte rosetting (SBA-E-). We found that (1) UVB produces a dose-dependent inhibition of the proliferative response to stimulators by human BMMCs; (2) increasing doses of UVB-irradiation and increasing levels of T-cell depletion (TCD) are both inversely related to production of lymphokines (IL2, IL3, LIF, IFN-gamma, and GMCSF) and (3) T-cell depletion, but not UVB-irradiation, decreases the production of monokines (IL1, TNF, IL6). Progenitor cell viability was decreased but preserved at 100 J/m2 of UVB. Our findings suggest that UVB compares favorably with TCD as a technique for inhibition of GVHD and therefore that UVB-modulation of bone marrow (BM) inoculum may be useful in the prevention of GVHD in clinical bone marrow transplantation accompanying a solid organ graft.

Biological and immunological characterization of inhibin forms in human plasma.

A number of immunoassay methods have been developed recently to detect specifically the bioactive alpha-beta A subunit inhibin dimer (inhibin A) in human plasma. However, the specificity of these assays in terms of their ability to detect the range of inhibin forms found in plasma and their relationship to bioactivity have not been investigated. Inhibin was fractionated from human follicular fluid (hFF) and serum/plasma from women stimulated with gonadotropins (IVF serum), and from postmenopausal and male plasma, using a combined immunoaffinity/preparative SDS-PAGE procedure. The molecular weight profile of inhibin was established by inhibin in vitro bioassay, three alpha-beta A subunit specific immunoassays, and three alpha subunit-directed immunoassays that detect the alpha subunit as well as inhibin A and B forms. In hFF inhibin forms of 33, 36, 55 and 66K were detected by in vitro bioassay and by most immunoassays except for 33 k inhibin, which was nondetectable by one alpha-beta A ELISA. The alpha subunit-directed assays also detected activity in the 29-31K region, in some assays in considerably high levels. In IVF serum in vitro bioactivity and immunoactivities were detected between 27 and 100K with the alpha-beta A assays failing to detect all bioactive forms. Alpha subunit-directed assays gave similar immunoactive profiles. Neither in vitro bioassay nor alpha-beta A assays detected activity in post-menopausal plasma or male plasma, while alpha subunit-directed assays showed peaks predominantly at 36 k, although at low levels. It is concluded that dimeric inhibin A specific assays detected bioactive inhibin forms in hFF and to a lesser extent in IVF serum. Alpha subunit-directed assays correlated poorly with in vitro bioassay in hFF because of the high alpha subunit levels in this sample. The higher correlation between these assays in IVF serum suggested that there was little free alpha subunit. The 36K form in male plasma may be free alpha subunit or inhibin B.

Plasma concentration of estradiol following transdermal administration of Systen 50 or Menorest 50.

Circulating levels of 17 beta estradiol (E2) following the administration of fixed doses of E2, show a great variability in kinetics depending upon the product administrated, the routes of administration, and the interindividual variations in absorption and metabolism. This might have important implications both in terms of tolerance and effectiveness. Two new forms of transdermal E2 (SYSTEN Cilag and MENOREST Rhone-Poulenc Rorer) have been recently accepted in Europe for the treatment of climacteric symptoms. The present study was undertaken to compare the pharmacokinetic characteristics of plasma E2 profile under these two drugs. It was carried out in 30 healthy postmenopausal volunteers according to good clinical practice after informed consent, as a single blind, randomised, cross-over study during the classical wearing period of 4 days. Plasma E2 concentration was determined 24 hours before, 1/2 hour before and then 2, 4, 8, 12, 24, 48, 72, 84, 96 hours after the first patch administration. E2 measurement was performed using a specific direct radioimmunoassay developed in the FRH laboratories. The main criteria for this method were an intraassay coefficient of variation (CV) less than 6%, an interassay CV less than 8% in a concentration range of 15-140 pg/ml and a quantitative detection limit (LOQ) of 2.7 pg/ml with a 20% CV. The following kinetic parameters were analysed: C(max), C(mean), C96 and MRT. The bioequivalence was assessed by analysis of variance of C(max), C(mean), C96 and AuC after logarithmic transformation, complemented by Westlake test (95%). Data show that these two products are identical in terms of C(max) but C(mean), C96 and AuC are statistically greater when MENOREST 50(R) is administered; furthermore, E2 levels decrease more rapidly and more deeply with SYSTEN 50 than MENOREST 50. The differences of pharmacokinetic profiles after administration of two different forms of the same dose of 50 micrograms transdermal 17 beta estradiol might have important medical consequences.

Prevention of postmenopausal bone loss by rectal calcitonin.

A group (150) of healthy women, who had been menopausal for less than 5 years and who had never received any form of treatment to prevent bone loss were entered into a randomized, controlled study comprising three arms. They were randomly allocated to the double-blind administration of five suppositories per week containing either 100 IU of salmon calcitonin or a placebo, or to a group receiving a suppository containing 200 IU of salmon calcitonin three times per week. All women received 500 mg/day of calcium supplementation. After 12 months, bone mineral density (BMD) of the spine, measured by dual energy X-ray absorptiometry, decreased significantly (P < 0.01) in the placebo group by 3.1% (SD: 3.6%) but did not change in the two calcitonin groups [+1.3% (3.5%) with 100 IU/day and +2.3% (4.0%) with 200 IU 3/week]. The differences in response between the placebo group and the two calcitonin groups were significant (P < 0.05), but the difference between the two regimens of calcitonin administration was not. No differences appeared among the three groups for the response at the level of the hip. Evolution of biochemical markers reflecting bone turnover did not differ significantly among groups. Nearly 40% of the women withdrew prematurely because of local (rectal or intestinal) intolerance to repetitive suppositories, with a nonsignificantly different frequency in the placebo or calcitonin groups. We conclude that rectal calcitonin might be an interesting preventive approach against trabecular postmenopausal bone loss but that long-term acceptability of suppositories should be evaluated in view of each patient's sensibility or cultural background.

A double-blind, placebo-controlled, dose-finding trial of intermittent nasal salmon calcitonin for prevention of postmenopausal lumbar spine bone loss.

PURPOSE: Nasal administration of salmon calcitonin (SCT) has been suggested for preventing trabecular bone loss during the first years following the menopause, but no conclusive evidence has appeared about the minimal effective dose. Since nasal calcitonin is highly expensive, it makes sense to define this dose. PATIENTS AND METHODS: We performed a double-blind, placebo-controlled, randomized, single-center study with a 3-arm parallel-group design. The subjects were 251 healthy women who had experienced natural menopause within the past 6 to 72 months and were not affected by any diseases or treatments that interfere with calcium metabolism. They were randomly allocated in groups of 6 to receive intranasal SCT 50 IU (n = 84), SCT 200 IU (n = 84), or placebo (n = 83). All treatments were given on 5 consecutive days per week. Statistical analysis was based on two populations: intention-to-treat (IT) and valid completers (VC). The main assessments performed were bone mineral density of the lumbar spine (LSBMD) and biochemical parameters reflecting bone turnover (serum alkaline phosphatase, urinary calcium/creatinine, and hydroxyproline/creatinine ratios). RESULTS: Changes over the treatment period were comparable in the IT and VC populations. In the group receiving the placebo, LSBMD decreased from baseline to end point by a mean of 6.28% (95% confidence interval [CI] -7.69 to -4.89) in the IT population and 6.98% (95% CI -8.86 to -5.11) in the VC population (P = 0.0001, end LSBMD versus baseline LSBMD). LSBMD increased slightly with the 50-IU/d dose of SCT, by 0.82% (95% CI -0.26 to 1.89) in the IT population, and 0.51% (95% CI -0.69 to 1.72) in the VC (P = NS, versus baseline). Subjects who received SCT 200 IU/d experienced significant increases of 2.03% (95% CI 0.92 to 3.15) in the IT population and 2.26% (95% CI 1.01 to 3.51) in the VC (both P = 0.001). The difference between the evolution of the combined groups receiving nasal SCT and the group treated with the placebo was highly significant (P = 0.0001). No significant changes were recorded in biochemical parameters reflecting bone turnover. CONCLUSIONS: SCT 50 IU/d administered nasally and intermittently appears to prevent lumbar bone loss in nonobese early postmenopausal women.

Changes in bronchial responsiveness, circulating leucocytes and ex vivo cytokine production by blood monocytes after PAF inhalation in allergic asthmatics.

We investigated the effects of inhaled platelet-activating factor (PAF) on methacholine bronchial responsiveness, circulating leucocyte counts, and ex vivo tumour necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) production from blood monocytes in eight allergic asthmatics. Bronchial responsiveness was defined as the provocative concentration of methacholine causing a 20% decrease in forced expiratory volume in one second (PC20). Circulating leucocytes were counted by means of an automatic haemocytometer, and cytokines were measured with specific immunoassays. The different variables were measured before and 4, 24, 48, 72 and 168 h after a PAF (225 micrograms), a lyso-PAF (225 micrograms) and a saline bronchial challenge. When compared with lyso-PAF and saline, inhalation of PAF resulted in a significant decrease in PC20 over a period of one week. Two falls in bronchial responsiveness were identified, the first by 4 h and the second beginning 48 h and reaching a maximum by 168 h. The increases in spontaneous TNF alpha and IL-1 production which occurred during the week after both PAF, lyso-PAF and saline, did not differ significantly. Likewise, the changes in circulating neutrophil counts, characterized by a transient rise by 4 h after PAF and lyso-PAF but not saline, followed by a fall by 24 h and a persistent decrease until 168 h, were not significantly different after PAF, lyso-PAF and saline. On the other hand, in comparison with lyso-PAF and saline, inhaled PAF caused a significant protracted augmentation in circulating eosinophil counts, which was maximal by 48 h but did not correlate with the delayed decline in PC20.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous glutamate involvement in pulsatile secretion of gonadotropin-releasing hormone: evidence from effect of glutamine and developmental changes.

The secretion of GnRH can be stimulated by glutamate (GLU) and GLU agonists, whereas GLU receptor antagonists inhibit GnRH. Using 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase, we aimed to study the involvement of endogenous GLU in GnRH secretion through the effects of impaired GLU biosynthesis from its precursor glutamine (GLN). GnRH secretion by hypothalamic explants of male rats, aged 15 and 50 days, was compared, because the frequency of spontaneous GnRH secretory pulses showed a 2-fold increase between those two ages. Using explants of 50-day-old rats, GLN elicited GnRH secretion in a similar dose-related manner as GLU. DON prevented GLN-evoked secretion of GnRH, whereas the effect of GLU was not altered. DON also markedly inhibited spontaneous pulsatile secretion of GnRH and the secretory response to veratridine, a Na+ channel opener. The inhibitory effect of DON on veratridine-evoked secretion of GnRH was directly related to the duration of exposure to DON and the frequency of GnRH secretory episodes. Using explants of 15-day-old rats, GLN could elicit GnRH release, although this response was lower than GLU-evoked secretion of GnRH. The DON concentrations required for inhibition of veratridine-evoked secretion of GnRH were lower at 15 days than at 50 days. These data indicate that 1) GLU biosynthesis from GLN is a prerequisite to the physiological mechanism of pulsatile GnRH secretion; and 2) inhibition of veratridine- or GLN-induced secretion of GnRH requires higher DON concentrations after the onset of puberty than before. This suggests that glutaminase, the enzyme controlling GLU biosynthesis from GLN, shows increased activity after the onset of puberty when the frequency of pulsatile GnRH secretion is increased as well.

Long-term performance in vitro and in vivo of dual-energy X-ray absorptiometry.

Dual-energy X-ray absorptiometry (DXA) is actually considered as one of the most appropriate techniques for measuring bone mineral content (BMC) and bone mineral density (BMD). An anthropomorphic phantom and a 25-year-old girl were repeatedly measured, 160 times and 50 times respectively, over an 18-month period to investigate performance in vitro and in vivo of a commercial DXA equipment (HOLOGIC QDR 1000). DXA is a highly accurate technique, the BMC and BMD determinations only overestimated the exact value of the phantom by 0.20% and 0.51% respectively. In vivo long-term (18 months) reproducibility of BMD of the spine is characterized by an interassay coefficient of variation (CVt) of 0.8% while, for the different regions of interest of the hip, BMD CVt varies from 1.1% (total zone) to 5.3% (Ward's triangle). In the subject tested, BMD sensitivity for changes of 2.2% at the lumbar spine and 3% at the hip were recorded.

Adverse effect of abdominal operations on production of interferon-gamma.

OBJECTIVE: To assess the effects of abdominal operations on the production of cytokines as one of the mechanisms of postoperative immunosuppression. DESIGN: Prospective study. SETTING: University hospital, Belgium. SUBJECTS: 19 Selected patients who underwent operations for benign (n = 10) or malignant (n = 9) diseases. INTERVENTIONS: Whole blood was collected in heparinised tubes before operation and on postoperative days 1, 2, 3, 5, 7, and 9. After 1/10 dilution in culture medium the whole blood cells were stimulated with 5 micrograms/ml phytohaemagglutinin and 25 micrograms/ml lipopolysaccharide, and incubated at 37 degrees C in 5% carbon dioxide. Concentrations of interleukin 1 (IL-1), tumour necrosis factor alpha (TNF alpha), and interleukin 6 (IL-6) were measured at 24 hours, and interferon-gamma and interleukin 2 (IL-2) were measured at 72 hours, with commercially available assays. OUTCOME MEASURES: Production of the monokines IL-1, TNF alpha, and IL-6, and of the lymphokines IL-2 and interferon-gamma, postoperatively. The monokines were expressed as a percentage of the preoperative values/monocyte, and the lymphokines as a percentage of preoperative values/lymphocyte. RESULTS: Production of IL-1 and TNF alpha, but not IL-6, decreased immediately after operation then returned to preoperative values. Production of IL-2 and interferon-gamma were significantly reduced immediately after operation, and that of interferon-gamma was still depressed on the ninth postoperative day. CONCLUSION: Cytokine production is altered after abdominal operations. The production of interferon-gamma may be a more sensitive indicator of altered immune response and vulnerability to infections and tumour growth than concentrations of other cytokines.

Investigation of the relationship between osteoporosis and the collagenase gene by means of polymorphism of the 5'upstream region of this gene.

Osteoporosis is a slowly progressing disease resulting from an imbalance between bone accretion and degradation. As interstitial collagenase is a key enzyme in the degradation of bone matrix, we investigated a possible relationship between the collagenase gene and osteoporosis. Analysis of an amplified genomic DNA fragment from -524 to +52 by denaturing gradient gel electrophoresis and sequencing allowed us to detect three dimorphic sites upstream of base -300, one of them leading to a BanI restriction site. None of the sites could be directly associated with osteoporosis. The allele frequencies of the three dimorphic sites were estimated. The interallelic ratios were high, thus providing new useful genetic markers for linkage analysis. When comparing these ratios in osteoporotic and nonosteoporotic subjects, no significant differences could be observed.

Defective cytokine expression but adult-type T-cell receptor, CD8, and p56lck modulation in CD3- or CD2-activated T cells from neonates.

Expression of IL-2, interferon-gamma, and IL-3 mRNA and proteins was investigated in peripheral blood mononuclear cells from cord blood after activation with phytohemagglutinin, CD2, or CD3 MAb. The results showed that interferon-gamma and IL-3 expression was decreased in cord peripheral blood mononuclear cells when compared with expression observed in adult peripheral blood mononuclear cells, irrespective of the stimulation used. In addition, in newborn cells a defect in IL-2 secretion and mRNA expression was observed in response to CD2 or CD3 MAb but not in response to phytohemagglutinin-mediated activation. We further analyzed the modulation of nonlymphokine genes under the same protocol of stimulations. The results indicate that in newborn cells, despite a reduced lymphokine expression observed after CD2 or CD3 MAb activation, the up-regulation of the T-cell receptor, CD8, and p56lck was similar to that found in adult cells, as was also found after phytohemagglutinin activation of both types of cells. These data are in favor of a deficient T-cell responsiveness to CD2 or CD3 MAb in newborn cells. This impairment of the T-cell response appears to selectively affect lymphokine gene expression because the modulation of other genes also implicated in T cell activation is not altered.