The hypothalamic-pituitary-adrenal and -thyroid axes activation lasting one year after an earthquake swarm: results from a big data analysis.

PURPOSE: To cope physical and/or psychological threats, the human body activates multiple processes, mediated by a close interconnection among brain, endocrine and inflammatory systems. The aim of the study was to assess the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-thyroid (HPT) axes involvement after an acute stressful event (Emilia Romagna earthquake swarm) with a big data approach. METHODS: A retrospective, observational trial was performed, collecting all biochemical examinations regarding HPA and HPT axes performed in the same laboratory the year before and the year after the earthquake swarm (20-29 May 2012). RESULTS: Comparing 2576 pre-earthquake to 3021 post-earthquake measurements, a cortisol serum level increase was observed (p < 0.001). Similar increase was evident for urinary free cortisol (p = 0.016), but not for adrenocorticotropic hormone (p = 0.222). The biochemical hypercortisolism incidence increased from 7.6 to 10.3% after earthquakes (p = 0.001). Comparing 68,456 pre-earthquake to 116,521 post-earthquake measurements, a reduction in thyroid-stimulating hormone (TSH) levels was evident (p = 0.018), together with an increase in free triiodothyronine and free thyroxine levels (p < 0.001 and p < 0.001). Moreover, a significant increase in altered TSH after earthquakes was registered considering the epicenter-nearest measurements (p < 0.001). No clinically relevant alterations were observed considering thyroid-specific autoantibodies. CONCLUSION: A long-term HPA axis activation in the inhabitants of the earthquake-affected areas was highlighted for the first time. Moreover, an increased incidence of biochemical hypercortisolism emerged after earthquakes. We confirmed a recruitment of HPT axis after stressful events, together with increased incidence of altered TSH in the. Our big data study allowed to increase knowledge about the connection between external stressors and endocrine regulation.

The steroid response to human chorionic gonadotropin (hCG) stimulation in men with Klinefelter syndrome does not change using immunoassay or mass spectrometry.

PURPOSE: Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) was developed in parallel to Immunoassays (IAs) and today is proposed as the "gold standard" for steroid assays. Leydig cells of men with Klinefelter syndrome (KS) are able to respond to human chorionic gonadotropin (hCG) stimulation, even if testosterone (T) production was impaired. The aim was to evaluate how results obtained by IAs and LC-MS/MS can differently impact on the outcome of a clinical research on gonadal steroidogenesis after hCG stimulation. METHODS: A longitudinal, prospective, case-control clinical trial. (clinicaltrial.gov NCT02788136) was carried out, enrolling KS men and healthy age-matched controls, stimulated by hCG administration. Serum steroids were evaluated at baseline and for 5 days after intramuscular injection of 5000 IU hCG using both IAs and LC-MS/MS. RESULTS: 13 KS patients (36 ± 9 years) not receiving T replacement therapy and 14 controls (32 ± 8 years) were enrolled. T, progesterone, cortisol, 17-hydroxy-progesterone (17OHP) and androstenedione, were significantly higher using IAs than LC-MS/MS. IAs and LC-MS/MS showed direct correlation for all five steroids, although the constant overestimation detected by IAs. Either methodology found the same 17OHP and T increasing profile after hCG stimulation, with equal areas under the curves (AUCs). CONCLUSIONS: Although a linearity between IA and LC-MS/MS is demonstrated, LC-MS/MS is more sensitive and accurate, whereas IA shows a constant overestimation of sex steroid levels. This result suggests the need of reference intervals built on the specific assay. This fundamental difference between these two methodologies opens a deep reconsideration of what is needed to improve the accuracy of steroid hormone assays.

Human chorionic gonadotropin stimulation gives evidence of differences in testicular steroidogenesis in Klinefelter syndrome, as assessed by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Men with Klinefelter syndrome (KS) show hypergonadotropic hypogonadism, but the pathogenesis of hypotestosteronemia remains unclear. Testicular steroidogenesis in KS men was evaluated over three decades ago after human chorionic gonadotropin (hCG) stimulation, but inconclusive results were obtained. Intriguingly, some recent studies show increased intratesticular testosterone concentrations in men with KS. OBJECTIVE: To analyze serum steroid profile, as a proxy of testicular steroidogenesis, after hCG stimulation in KS compared with control men. DESIGN: A prospective, longitudinal, case-control, clinical trial. METHODS: Thirteen KS patients (36±9 years) not receiving testosterone (TS) replacement therapy and 12 eugonadic controls (32±8 years) were enrolled. Serum steroids were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at baseline and for five consecutive days after intramuscular injection of 5000IU hCG. RESULTS: Progesterone (P), 17-hydroxyprogesterone (17OHP), TS, and estradiol (E2) showed a significant increase (P<0.001) after hCG stimulation in both groups. On the contrary, androstenedione (AS) and dehydroepiandrosterone did not increase after hCG stimulation. The 17OHP/P ratio increased in both groups (P<0.001), the TS/AS ratio (17ß-hydroxysteroid dehydrogenase type 3 (17ßHSD3) activity) did not increase after hCG in any group, and the E2/TS ratio (aromatase activity) increased significantly in both groups (P=0.009 in KS and P<0.001 in controls). Luteinizing hormone decreased after hCG in both groups (P=0.014 in KS and P<0.001 in controls), whereas follicle-stimulating hormone decreased only in control men (P<0.001). CONCLUSION: This study demonstrates for the first time using LC-MS/MS that Leydig cells of KS men are able to respond to hCG stimulation and that the first steps of steroidogenesis are fully functional. However, the TS production in KS men is impaired, possibly related to reduced hydroxysteroid deydrogenase activity due to an unfavorable intratesticular metabolic state.

Polysiloxane-poly(propylene oxide) hybrid discs as solid phase in anti-HCV detection using a recombinant core protein.

In this work, siloxane-poly(propylene oxide) discs (PPO disc) prepared using the sol-gel process were used as solid phase in enzyme-linked immunosorbent assays (ELISA) for the detection of anti-hepatitis C virus (HCV) antibodies. The HCV RNA from serum (genotype 1b) was submitted to the RT-PCR technique and subsequent amplification of the HCV core 408 pb. This fragment was cloned into expression vector pET42a and expressed in Escherichia coli as recombinant protein with glutathione S-transferase (GST). Cell cultures were grown and induced having a final concentration of 0.4 x 10(-3) mol L(-1) of IPTG. After induction, the cells were harvested and the soluble fraction was analyzed using polyacrilamide gel 15% showing a band with an approximate molecular weight of 44 kDa, the expected size for this GST-fused recombinant protein. The recombinant protein was purified and confirmed by immunological detection using HCV-positive serum and showed no cross-reactivity with positive samples for other infectious diseases. An ELISA was established using 1.25 ng of recombinant protein per PPO disc, a dilution of 1:10,000 and 1:40 for a peroxidase conjugate and serum, respectively, and solutions of hydrogen peroxide and 3,3',5,5'-tetra-methylbenzidine in a ratio of 1:1. The proposed methodology was compared with the ELISA conventional polystyrene-plate procedure and the performance of the PPO discs as a matrix for immunodetection gave an easy synthesis, good performance and reproducibility for commercial application.

CCR5 genotype and plasma beta-chemokine concentration of Brazilian HIV-infected individuals.

The 32-bp deletion in the HIV-1 co-receptor CCR5 confers a high degree of resistance to HIV-1 infection in homozygous individuals for the deleted allele and partial protection against HIV-1 during disease progression in heterozygotes. Natural ligands for CCR5, MIP-1alpha, MIP-1beta and RANTES, have been shown to inhibit HIV replication in CD4+ T cells. In the present study, we examined the CCR5 genotype by PCR and the plasma levels of RANTES and MIP-1alpha by ELISA among blood donors (N = 26) and among HIV-1-infected individuals (N = 129). The control group consisted of healthy adult volunteers and HIV-1-infected subjects were an asymptomatic and heterogeneous group of individuals with regard to immunologic and virologic markers of HIV-1 disease. The frequency of the CCR5 mutant allele (Delta32ccr5) in this population was 0.032; however, no Delta32ccr5 homozygote was detected. These results could be related to the intense ethnic admixture of the Brazilian population. There was no correlation between circulating beta-chemokines (MIP-1alpha, RANTES) and viral load in HIV-infected individuals. RANTES concentrations in plasma samples from HIV+ patients carrying the homozygous CCR5 allele (CCR5/CCR5) (28.23 ng/ml) were higher than in the control samples (16.07 ng/ml; P<0.05); however, this HIV+ patient group (mean 26.23 pg/ml) had significantly lower concentrations of MIP-1alpha than those observed in control samples (mean 31.20 pg/ml; P<0.05). Both HIV-1-infected and uninfected individuals heterozygous for the Delta32ccr5 allele had significantly lower concentrations of circulating RANTES (mean 16.07 and 6.11 ng/ml, respectively) than CCR5/CCR5 individuals (mean 28.23 and 16.07 ng/ml, respectively; P<0.05). These findings suggest that the CCR5 allele and beta-chemokine production may affect the immunopathogenesis of HIV-1.

CCR5 genotype and plasma ß-chemokine concentration of Brazilian HIV-infected individuals

The 32-bp deletion in the HIV-1 co-receptor CCR5 confers a high degree of resistance to HIV-1 infection in homozygous individuals for the deleted allele and partial protection against HIV-1 during disease progression in heterozygotes. Natural ligands for CCR5, MIP-1alpha, MIP-1ß and RANTES, have been shown to inhibit HIV replication in CD4+ T cells. In the present study, we examined the CCR5 genotype by PCR and the plasma levels of RANTES and MIP-1alpha by ELISA among blood donors (N = 26) and among HIV-1-infected individuals (N = 129). The control group consisted of healthy adult volunteers and HIV-1-infected subjects were an asymptomatic and heterogeneous group of individuals with regard to immunologic and virologic markers of HIV-1 disease. The frequency of the CCR5 mutant allele (delta32ccr5) in this population was 0.032; however, no delta32ccr5 homozygote was detected. These results could be related to the intense ethnic admixture of the Brazilian population. There was no correlation between circulating ß-chemokines (MIP-1alpha, RANTES) and viral load in HIV-infected individuals. RANTES concentrations in plasma samples from HIV+ patients carrying the homozygous CCR5 allele (CCR5/CCR5) (28.23 ng/ml) were higher than in the control samples (16.07 ng/ml; P<0.05); however, this HIV+ patient group (mean 26.23 pg/ml) had significantly lower concentrations of MIP-1alpha than those observed in control samples (mean 31.20 pg/ml; P<0.05). Both HIV-1-infected and uninfected individuals heterozygous for the delta32ccr5 allele had significantly lower concentrations of circulating RANTES (mean 16.07 and 6.11 ng/ml, respectively) than CCR5/CCR5 individuals (mean 28.23 and 16.07 ng/ml, respectively; P<0.05). These findings suggest that the CCR5 allele and ß-chemokine production may affect the immunopathogenesis of HIV-1

Thallium 201 for detection of viable myocardium: comparison of different reinjection protocols.

BACKGROUND: The finding fo false fixed 201Tl defects by the conventional stress-redistribution protocol is a well-known phenomenon. The aim of this study was to compare two different 201Tl reinjection protocols to identify viable myocardium in the same group of patients. METHODS AND RESULTS: Twenty-seven patients with ischemic heart disease and at least one persistent defect on 201Tl uptake redistribution images 3 hours after stress were investigated. In the same-day protocol (R1) patients were reinjected with 1 mCi 201Tl immediately after redistribution images, with imaging starting 15 minutes later; in the different-day protocol the patients were reinjected with 2 mCi 48 to 96 hours later. Two sets of images were obtained, 30 (R2) and 180 (R3) minutes after reinjection. The comparison of redistribution and reinjection versus stress images showed a significant (p < 0.01) frequency distribution. The uptake of 201Tl of the 111 irreversible segments at redistribution was enhanced in 35.1% with R1, 43.2% with R2, and 49.5% with R3. The agreement among the three procedures in classifying the segmental defects was high between R2 and R3 (r = 0.81) and lower between the same- and different-day protocols. Of the 19 patients with a dominant scar pattern demonstrated by the conventional stress-redistribution study, 37%, 47%, and 53% were judged mainly ischemic after R1, R2, and R3, respectively. All but three of the 55 segments-showing an increased 201Tl uptake by R3 had an echocardiographic score of 2 or greater. CONCLUSION: The best technique to differentiate scarred from viable myocardium seems to be the reinjection of a second dose of 201Tl on a different day followed by imaging 3 hours later.

Effect of sulpiride on ischemia- and reperfusion-induced heart damage, in rats.

In an experimental model of heart ischemia, obtained in anesthetized rats with the permanent ligature of the left anterior descending coronary artery, the intravenous (iv) injection of I-sulpiride (6-25 micrograms/kg) dose-dependently reduced the lethality rate, the incidence and severity of ventricular dysrhythmias and infarct size during the early phase of ischemia (first 30 min after coronary ligation). Lethality and there incidence and duration of ventricular dysrhythmias were also significantly reduced by the same IV doses of I-sulpiride in a model of coronary reperfusion. These results show that a specific dopamine antagonist is able to limit ischemia- and reperfusion-induced myocardial damage and suggest that endogenous dopamine may exert a deleterious effect in such conditions.

Capsaicin prevents the adrenocorticotropin-induced improvement of cardiovascular function and survival in hemorrhage-shocked rats.

A volume-controlled hemorrhagic shock was produced in anesthetized rats by intermittent bleeding from an iliac vein over a period of 20-30 min, until the carotid mean arterial pressure (MAP) stabilized around 20-24 mmHg. In this condition, which caused the death of all saline-treated animals within 25-30 min, the intravenous (i.v.) bolus injection of the adrenocorticotropin fragment 1-24 (ACTH(1-24)) at a dose of 160 micrograms/kg promptly restored MAP, as well as pulse pressure, heart rate and respiratory function, and greatly prolonged the survival time. Capsaicin (125 mg/kg cumulatively, s.c., 1 week before) completely prevented the anti-shock effect of ACTH(1-24), which, on the other hand, was shared by i.v. [Nle11]-substance P (SP) (200-300 micrograms/kg). Finally the SP-antagonist [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP prevented the effect of ACTH(1-24). These results suggest that SP-containing nerve fibers are required for the effect of ACTH in hemorrhagic shock.

Reversal of experimental hemorrhagic shock by dimethylphenylpiperazinium (DMPP).

In a rat model of hemorrhagic shock which caused the death of all control rats within 30 min, i.v. injection of the ganglion-stimulating drug dimethylphenylpiperazinium (DMPP) caused a dose-dependent reversal of the shock condition--without the need for reinfusion of the shed blood--starting from the dose of 4 ng/kg i.v. Shock reversal was associated with the mobilization of residual blood and improvement in blood flow, particularly at the carotid level. These results could influence our thinking on pathophysiology and first-aid management of shock.

Influence of ACTH-(1-24) on metabolic acidosis and hypoxemia induced by massive hemorrhage in rats.

In anesthetized rats, step-wise bleeding to a severe condition of hemorrhagic shock causes a decrease in arterial and venous pH and in venous PO2 and SO2 and an increase in arterial PO2 and in venous PCO2 and lactic acid. The intravenous bolus injection of ACTH-(1-24) (160 micrograms/kg)--which causes a rapid and sustained reversal of the shock condition--produces a gradual and almost complete recovery (within 60 min) of venous PO2, PCO2 and SO2; on the other hand, the normalization of blood pH and lactate is preceded by a further worsening during the first minutes after treatment. On the whole, these data are compatible with the ACTH-(1-24)-induced mobilization of the residual blood--which is pooled in poorly oxygenated tissues--and with the improved circulatory and respiratory functions.

Influence of TRH on regional blood flow and metabolic acidosis in a model of volume-controlled hemorrhagic shock in rats.

In anesthetized rats, massive bleeding to a severe condition of hemorrhagic shock (invariably leading to death within 30 min) was obviously associated with a dramatic decrease in tissue blood flow and with profound modifications of several blood parameters leading to metabolic acidosis: decrease in arterial and venous pH, bicarbonate and BE, decrease in arterial pCO2 and in venous pO2 and SO2, increase in arterial pO2, venous pCO2 and venous lactate. The i.v. bolus injection of protirelin tartrate (TRH-T, 4 mg/kg), which produces a prompt and sustained reversal of the shock condition, caused a rapid increase in venous pO2, pCO2 and SO2; on the other hand, arterial and venous pH, bicarbonate and BE continued to decrease--and venous lactate to increase during the first few minutes after treatment. However venous pCO2 and lactate, as well as arterial and venous pH, returned to the pre-bleeding values within 60 min after treatment. The data are in keeping with the TRH-T-induced improvement of circulatory and respiratory functions, with mobilization of the residual blood from its capillary pooling and consequent immission of acid metabolites into the blood stream.

Afferent vagal fibres and central cholinergic mechanisms are involved in the TRH-induced reversal of haemorrhagic shock.

In a model of haemorrhagic shock causing the death of all saline-treated rats within 25.8 +/- 2.7 min after treatment, the intravenous injection of thyrotropin-releasing hormone tartrate (TRH-T) at the dose of 4 mg/kg induces a prompt and sustained increase of arterial pressure and pulse amplitude, with survival of all rats. Bilateral vagotomy, atropine sulphate (2 mg/kg intraperitoneally) and hemicholinium-3 (20 micrograms/rat intracerebroventricularly) partially prevent the TRH-T-induced shock reversal, whereas atropine methylbromide has no effect. These data indicate that afferent vagal fibres, brain cholinergic neurons and central muscarinic receptors play a role in the mechanism of the anti-shock effect of TRH-T.

Nicotine reverses hemorrhagic shock in rats.

Cholinergic mechanisms are currently thought to play an essential role in blood pressure homeostasis. Here we show that, in urethane-anaesthetized rats bled to severe hemorrhagic shock, the i.v. administration of nicotine 0.2-50 micrograms/kg causes a prompt, sustained and dose-dependent improvement in cardiovascular and respiratory functions, the animals' survival rate being significantly higher than that of animals treated with saline. These effects are prevented by bilateral cervical vagotomy and by concurrent local anaesthesia of the carotid bodies, which suggests that stimulation of visceral afferents is the main mechanism of action of nicotine in hemorrhagic shock.

Ischemia- and reperfusion-induced arrhythmias are prevented by putrescine.

The influence of putrescine on cardiac arrhythmias induced by either permanent ligature of the left anterior coronary artery or heart reperfusion following a 5-min coronary occlusion was studied in anesthetized rats. Reperfusion-induced arrhythmias were significantly prevented by the i.v. injection of 150-200 mg/kg of putrescine, the survival rate being 100% in treated animals and 40% in controls. At a dose level of 200-300 mg/kg i.v., putrescine also significantly reduced the duration of ventricular tachycardia induced by permanent coronary occlusion. These findings show that putrescine significantly reduces the consequences of cardiac ischemia and reperfusion, probably as a consequence of its multiple stabilizing effects at the membrane level.

TRH reverses the ECG and EEG ischemic changes induced by massive hemorrhage in rats.

In a rat model of volume-controlled hemorrhagic shock causing the death of all saline-treated animals within 30 min of treatment, the intravenous bolus injection of thyrotropin- releasing hormone tartrate (TRH-T) at the dose of 4 mg/kg induced the prompt and sustained disappearance of the ECG and EEG signs of heart and brain ischemia, along with the reversal of hypotension and respiratory depression and with 100% survival rate at the end of the 2 h observation period. These data confirm that, in a pre-terminal condition induced by massive hemorrhage, timely treatment with TRH-T will restore heart and brain perfusion to levels compatible with survival and with functional recovery from ischemia and maintain it at those levels for some hours.

Antidiuretic and nephrotoxic effects of putrescine in rats.

Putrescine, intraperitoneally injected either into intact or into hypophysectomized rats, caused a reduction in urine volume at doses of 200-300 mg/kg. At doses of 100 mg/kg or more, there was also a significant loss of potassium. The highest dose (300 mg/kg) caused haemoglobinuria, proteinuria, increased natriuresis, increased urinary osmolarity, reduced aldosteronaemia, ectasis of glomerular capillaries and tubular damage. The underlying mechanism(s) are probably mostly linked to the strong cationic charge of putrescine and to its binding to fixed anions of tubular-cell membrane.

Influence of gonadotropin-releasing hormone on castration-induced 'depression' in mice: a behavioral and binding study.

Long-term (33-35 days) castration caused a significant increase in the duration of immobility of male and female mice in the tail suspension test (an animal model of depression), and a significant decrease in the maximum number (Bmax) of [3H]imipramine binding sites in the cerebral cortex of male mice. In the tail suspension test, gonadotropin-releasing hormone (GnRH), s.c. injected 3 times at 3-h intervals at doses of 0.2, 2 or 20 micrograms/kg, did not significantly modify the duration of immobility of castrated animals and did not reduce that of sham-operated ones, while desipramine (20 mg/kg s.c. 1 h before testing) restored immobility to normal in castrated animals and reduced it significantly in sham-operated ones. The same treatment schedule with GnRH produced an increase in the number of [3H]imipramine Bmax in cortical membranes that was statistically significant at the dose of 2 micrograms/kg. It is concluded that the castration-induced depression-like behavior in mice seems not to be due to the decreased levels and release of GnRH, and that GnRH has no antidepressant-like effect in mice, at least at our dose levels; however, GnRH seems to increase the number of cortical [3H]imipramine binding sites.