Role of thyrotropin in metabolism of thyroid hormones in nonthyroidal tissues

T(4) conversion into T(3) in peripheral tissues is the major source of circulating T(3). However, the exact mechanism of this process is ill defined. Several in vitro studies have demonstrated that thyrotropin facilitates deiodination of T(4) into T(3) in liver and kidneys. However, there is a paucity of in vitro studies confirming this activity of thyrotropin. Therefore, this study was conducted to examine the influence of thyrotropin on thyroid hormone metabolism in nonthyroidal tissues. We assessed T(4), T(3), reverse T(3) (rT(3)), and T(3) resin uptake (T(3)RU) responses up to 12 hours at intervals of 4 hours in 6 thyroidectomized female mongrel dogs rendered euthyroid with LT(4) replacement therapy before and after subcutaneous (SC) administration of bovine thyrotropin (5 U) on one day and normal saline (0.5 mL) on another in a randomized sequence between 08:00 and 09:00 am. Euthyroid state after LT(4) replacement was confirmed before thyrotropin administration. Serum T(4), T(3), rT(3), and T(3)RU all remained unaltered after SC administration of normal saline. No significant alteration was noted in serum T(3)RU values on SC administration of thyrotropin. However, serum T(3) rose progressively reaching a peak at 12 hours with simultaneous declines being noted in both serum T(4) and rT(3) concentrations (P < .05 vs prethyrotropin values for all determinations). The changes after SC administration were significantly different (P < .001) in comparison to those noted on SC administration of normal saline. Thyrotropin may promote both the conversion of T(4) to T(3) and metabolism of rT(3) into T(2) in nonthyroidal tissues via enhancement of the same monodeionase.     

Melatonin reduces phosphine-induced lipid and DNA oxidation in vitro and in vivo in rat brain

Phosphine (PH(3)), a widely used pesticide, was found in our recent study to induce oxidative damage in the brain, liver and lung of rats. We also observed that melatonin significantly blocked this action. The present study focused on brain and the magnitude and mechanism of protection of PH(3)-induced oxidative damage by melatonin in vitro and in vivo. PH(3) in whole brain homogenate (3 mg protein/mL Tris-HCl pH 7.4 buffer) induced increasing lipid peroxidation [as malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA)] dependent on concentration (0.25-2 mM) and time (30-150 min), reaching a maximum level of 2.9-fold at 90 min after PH(3) at 1 mM. Elevation of MDA + 4-HDA levels by PH(3) at 1 mM was also observed in homogenates of cerebral cortex, cerebellum, hippocampus and hypothalamus examined individually. Melatonin at 0.1-2 mM progressively inhibited PH(3)-induced lipid peroxidation in brain and regions thereof. Additionally, PH(3) induced brain DNA oxidation in vitro and in vivo determined as 8-hydroxyguanosine (8-OH-dG). Melatonin at 1 mM significantly suppressed PH(3)-induced brain DNA oxidation in vitro. PH(3) at 4 mg/kg i.p. significantly elevated 8-OH-dG in frontal cortex and melatonin prevented it. PH(3) in vivo marginally lowered brain glutathione peroxidase activity and melatonin restored it completely. In contrast, PH(3) and melatonin both stimulated superoxide dismutase production. Brain glutathione (GSH) levels in PH(3)-treated rats were significantly reduced at 30 min and recovered gradually. It is concluded that melatonin, probably because of its free radical scavenging ability, confers marked protection against PH(3)-induced oxidative toxicity in brain.     

Iodothyronine levels in the human developing brain: major regulatory roles of iodothyronine deiodinases in different areas

Thyroid hormones are required for human brain development, but data on local regulation are limited. We describe the ontogenic changes in T(4), T(3), and rT(3) and in the activities of the types I, II, and III iodothyronine deiodinases (D1, D2, and D3) in different brain regions in normal fetuses (13-20 wk postmenstrual age) and premature infants (24-42 wk postmenstrual age). D1 activity was undetectable.The developmental changes in the concentrations of the iodothyronines and D2 and D3 activities showed spatial and temporal specificity but with divergence in the cerebral cortex and cerebellum. T(3) increased in the cortex between 13 and 20 wk to levels higher than adults, unexpected given the low circulating T(3). Considerable D2 activity was found in the cortex, which correlated positively with T(4) (r = 0.65). Cortex D3 activity was very low, as was D3 activity in germinal eminence and choroid plexus. In contrast, cerebellar T(3) was very low and increased only after midgestation. Cerebellum D3 activities were the highest (64 fmol/min.mg) of the regions studied, decreasing after midgestation. Other regions with high D3 activities (midbrain, basal ganglia, brain stem, spinal cord, hippocampus) also had low T(3) until D3 started decreasing after midgestation. D3 was correlated with T(3) (r = -0.682) and rT(3)/T(3) (r = 0.812) and rT(3)/T(4) (r = 0.889).Our data support the hypothesis that T(3) is required by the human cerebral cortex before midgestation, when mother is the only source of T(4). D2 and D3 play important roles in the local bioavailability of T(3). T(3) is produced from T(4) by D2, and D3 protects brain regions from excessive T(3) until differentiation is required.     

Triiodothyronine production in Graves' hyperthyroidism.

OBJECTIVE: To quantify the relative contributions of thyroid secretion and peripheral generation to triiodothyronine (T(3)) production in untreated Graves' hyperthyroidism. PATIENTS, DESIGN, AND MEASUREMENTS: Thirty-one patients with hyperthyroidism, of whom 6 had T(3) toxicosis, and 21 surgically and radioiodine ablated patients with thyroid cancer on thyroid stimulating hormone-suppressive therapy in whom serum T(3) should reflect peripheral generation alone were compared with respect to serum free thyroxine (T(4)) and serum free T(3) concentrations. MAIN OUTCOMES: Serum free T(4)/free T(3) molar ratios were virtually identical in the patients with T(4)+T(3) toxicosis (2.7 +/- 0.4) and those with T(3) toxicosis (2.6 +/- 0.4) and were significantly lower than in the patients with thyroid cancer (4.0 +/- 0.4) (p < 0.001). In the hyperthyroid patients, peripherally generated T(3) was calculated as the quotient of the individual serum free T(4) concentration and the free T(4)/free T(3) molar ratio in thyroid cancer; this value was subtracted from the individual measured free T(3) concentration to derive the value for secreted T3. Secreted T(3) accounted for 33 +/- 6% of T(3) production in T(4)+T(3) toxicosis and 34 +/- 10% in T(3) toxicosis. CONCLUSIONS: This study indicates that about one third of T3 production in untreated Graves' hyperthyroidism, irrespective of whether presenting as T4+T3 toxicosis or T3 toxicosis, arises from thyroid secretion as compared to about 20% in normal individuals.     

Estriol replacement therapy in osteoporotic women

In cultured human osteoblast-like cells (HOS), combined treatment with estriol (E(3)) and vitamin D(3) (VD(3)) increases the cell viability relative to treatment with E(3) alone and that E(3) treatment remarkably increases VD(3) receptor mRNA expression in the cells. Our findings in the present study with osteoblast cells may explain at least in a part the molecular basis of the clinical efficacy of combined treatment with E(3) and VD(3) in osteoporotic women.     

Biological activity of fluorine-substituted 1,25-dihydroxyvitamin D3 in rats, in chicken and in Japanese quails

The biological activity of two fluorinated analogs of 1,25(OH)2D3 was compared with 1,25(OH)2D3 in various vitamin D assays. The effect of 24,24-F2-1,25(OH)2D3 on plasma calcium, bone weight and duodenal calcium binding protein in chicken, on calcium excretion via egg shell in Japanese quails and on mobilization of calcium from the bone in rats was twice as high as the effect of the most potent naturally occurring vitamin D3 metabolite 1,25(OH)2D3. In contrast, 24R-F-1,25(OH)2D3 has less than 50% of the potency of 1,25(OH)2D3. Due to the wider therapeutic dosage range, this compound might be of clinical value.     

Change in serum thyroid hormone levels in patients with acute myocardial infarction

It has been reported that there is a decrease in the serum concentration of thyroid hormones in non-thyroidal illness. In the present study we made serial measurements of serum concentration of thyroid hormones [triiodothyronine (T3), thyroxine (T4), free triiodothyronine (FT3), free thyroxine (FT4), reverse triiodothyronine (rT3)], thyroid stimulating hormone (TSH) and thyroxine binding globulin (TBG) in 10 patients with acute myocardial infarction (AMI, Grade I, according to the classification of Killip & Forrester) during 14 days after onset. In the early phase of AMI, serum T3, T4, FT3 and FT4 levels decreased while rT3 increased. TSH and TBG levels, however, were unchanged. In the patients with a high peak creatine phosphokinase activity (greater than or equal to 400 mU/ml), the decrease in thyroid hormone and increase in serum rT3 levels were greater than in patients with a low peak value (less than 400 mU/ml), suggesting a correlation between severity of AMI and changes in serum thyroid hormone levels. Especially, serum FT3 levels fell below the lower limit of controls within 14 days, with the lowest levels and the rT3 peak on the third day after onset. These data suggest that in AMI peripheral conversion of T4 favours rT3 production and that low levels of serum FT3 and T3 protect the infarcted heart muscle against thyroid hormone action.     

Mobilization studies in mice deficient in either C3 or C3a receptor (C3aR) reveal a novel role for complement in retention of hematopoietic stem/progenitor cells in bone marrow

The mechanisms regulating the homing/mobilization of hematopoietic stem/progenitor cells (HSPCs) are not fully understood. In our previous studies we showed that the complement C3 activation peptide, C3a, sensitizes responses of HSPCs to stromal-derived factor 1 (SDF-1). In this study, mobilization was induced with granulocyte colony-stimulating factor (G-CSF) in both C3-deficient (C3-/-) and C3a receptor-deficient (C3aR-/-) mice as well as in wild-type (wt) mice in the presence or absence of a C3aR antagonist, SB 290157. The data indicated (1) significantly increased G-CSF-induced mobilization in C3-/- and C3aR-/- mice compared with wt mice, (2) significantly accelerated and enhanced G-CSF-induced mobilization in wt, but not in C3-/- or C3aR-/-, mice treated with SB 290157, and (3) deposition of C3b/iC3b fragments onto the viable bone marrow (BM) cells of G-CSF-treated animals. Furthermore, mobilization studies performed in chimeric mice revealed that wt mice reconstituted with C3aR-/- BM cells, but not C3aR-/- mice reconstituted with wt BM cells, are more sensitive to G-CSF-induced mobilization, suggesting that C3aR deficiency on graft-derived cells is responsible for this increased mobilization. Hence we suggest that C3 is activated in mobilized BM into C3a and C3b, and that the C3a-C3aR axis plays an important and novel role in retention of HSPCs (by counteracting mobilization) by increasing their responsiveness to SDF-1, the concentration of which is reduced in BM during mobilization. The C3a-C3aR axis may prevent an uncontrolled release of HSPCs into peripheral blood. These data further suggest that the C3aR antagonist SB 290157 could be developed as a drug to mobilize HSPCs for transplantation.     

Stimulatory effect of nitric oxide on bicarbonate secretion in bullfrog duodenums in vitro.

The effect of nitric oxide (NO) on HCO-3 secretion was examined in vitro using an isolated preparation of bullfrog duodenum. The tissue was bathed in unbuffered Ringer's solution gassed with 100% O2 on the mucosal side and HCO-3 Ringer's solution gassed with 95% O2-5% CO2 on the serosal side. The HCO-3 secretion was measured by the pH-stat method using 2 mmol/l HCl as the titrant to keep the mucosal pH at 7.4. (+/-)-(E)-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamine (NOR3) was used as a NO donor and added to the serosal solution. To analyze the NOR3 action on HCO-3 secretion, the effects of dibutyryl adenosine-3',5'-cyclic monophosphate (dbcAMP), dibutyryl guanosine-3',5'-cyclic monophosphate (dbcGMP), methylene blue, and indomethacin on the HCO-3 response were also examined. NOR3 (1x10(-4) and 3x10(-4) mol/l) caused an increase in HCO-3 secretion in a dose-dependent manner, and this effect appeared with an about 30-min time lag, reaching the level of 1.5-2.5 times greater than basal values at 1-2 h later. Both dbcAMP (1x10(-3) mol/l) and dbcGMP (1x10(-3) mol/l) also caused a significant increase in HCO-3 secretion in bullfrog duodenums in vitro, although the onset of the HCO-3 response to dbcGMP was delayed as compared to the former. The stimulatory action of NOR3 on duodenal HCO-3 secretion was significantly attenuated by methylene blue (5x10(-5) mol/l) and indomethacin (1x10(-5) mol/l), the latter also inhibiting the HCO-3 response to dbcGMP. The release of prostaglandin E2 in the serosal solution was significantly increased after addition of NOR3 (3x10(-4) mol/l) and dbcGMP (1x10(-3) mol/l) in an indomethacin-sensitive manner. These results suggest that the NO donor increases duodenal HCO-3 secretion in vitro, and this action of NO donor is cGMP-dependent and mediated by endogenous prostaglandins. Duodenal HCO-3 secretion may be regulated locally by NO/cGMP in addition to prostaglandin/cAMP.     

Metabolism of androsterone and 5 alpha-androstane-3 alpha,17 beta-diol in human lung tissue and in pulmonary endothelial cells in culture

The metabolism of [3H]androsterone and [3H] 5 alpha-androstane-3 alpha,17 beta-diol ( [3H] 3 alpha-diol) was studied in slices of human lung tissue and cultures of human pulmonary artery endothelial cells. Lung tissue metabolized [3H]androsterone (0.25 microM) to 5 alpha-androstane-3,17-dione (30.3 pmol 100 mg-1 tissue h-1), isoandrosterone (0.7 pmol 100 mg-1 tissue h-1), 5 alpha-dihydrotestosterone (5 alpha-DHT; 0.1 pmol 100 mg-1 tissue h-1), 3 alpha-diol (0.1 pmol 100 mg-1 tissue h-1), and two polar metabolites. Pulmonary arterial endothelial cells produced the same metabolites of [3H]androsterone (0.083 microM), with the exception of the polar compounds [5 alpha-androstane-3,17-dione (1.3 pmol mg-1 protein h-1), isoandrosterone (0.1 pmol mg-1 protein h-1), 5 alpha-DHT (0.2 pmol mg-1 protein h-1), and 3 alpha-diol (0.2 pmol mg-1 protein h-1)]. Thus, the principal metabolite of [3H]androsterone in both lung tissue and endothelial cells was 5 alpha-androstane-3,17-dione. Human lung tissue metabolized [3H]3 alpha-diol (0.28 microM) to 5 alpha-DHT (8.8 pmol 100 mg-1 tissue h-1), androsterone (2.2 pmol 100 mg-1 tissue h-1), 5 alpha-androstane-3,17-dione (0.8 pmol 100 mg-1 tissue h-1), isoandrosterone (0.1 pmol 100 mg-1 tissue h-1), and four polar metabolites (0.2 pmol 100 mg-1 tissue h-1). 5 alpha-DHT was the principal metabolite of [3H]3 alpha-diol within the first hour of incubation, but the concentration of this androgen declined thereafter to 3.6 pmol 100 mg-1 tissue after 4 h of incubation. This decline was correlated with increased 5 alpha-androstane-3,17-dione synthesis (6.7 pmol 100 mg-1 tissue 4 h-1). Androsterone formation from [3H]3 alpha-diol, however, was linear with time of incubation for 4 h (8.9 pmol 100 mg-1 tissue 4 h-1). The formation of these products demonstrates that the principal 5 alpha-reduced-C19-steroid-metabolizing enzymes in human lung are 3 alpha-hydroxysteroid oxidoreductase.     

Inositol triphosphate participates in an oestradiol nongenomic signalling pathway involved in accelerated oviductal transport in cycling rats

Oestradiol (E(2)) accelerates oviductal transport of oocytes in cycling rats through a nongenomic pathway that involves the cAMP-PKA signalling cascade. Here we examined the role of the inositol triphosphate (IP3) and mitogen-activated protein kinase (MAPK) signalling cascades in this nongenomic pathway. Oestrous rats were injected with E(2) s.c. and intrabursally (i.b) with the selective inhibitors of phospholipase C (PLC) ET-18-OCH(3) or MAPK PD98059. The number of eggs in the oviduct assessed 24 h later showed that ET-18-OCH(3) blocked E(2)-induced egg transport acceleration, whereas PD98059 had no effect. Other oestrous rats were treated with E(2) s.c. and 1, 3 or 6 h later oviducts were excised and the levels of IP3 and phosphorylated MAPK p44/42 (activated) were determined by radioreceptor assay and western blot, respectively. Oestradiol administration increased IP3 level at 1 and 6 h after treatment, whereas activated MAPK p44/42 level was unchanged. Finally, we explored whether cAMP-PKA and PLC-IP3 signalling cascades are coupled. Inhibition of adenylyl cyclase by i.b. injection of SQ 22536 blocked the increase of IP3 levels induced by E(2), while inhibition of PLC by ET-18-OCH(3) had no effect on E(2)-induced PKA activity. Furthermore, activation of adenylyl cyclase by Forskolin increased oviductal IP3 levels. Thus, activation of PLC-IP3 by E(2) requires previous stimulation of cAMP-PKA. We conclude that the nongenomic pathway utilised by E(2) to accelerate oviductal transport of oocytes in cycling rats involves successive activation of the cAMP-PKA and PLC-IP3 signalling cascades and does not require activation of MAPK. These findings clearly illustrate a non-genomic pathway triggered by E(2) that regulates a complex physiologic process accomplished by an entire organ.     

Estriol conjugates in human breast cyst fluid and in serum of premenopausal women.

A sensitive method was developed for the assay of the four major estriol (E3) conjugates in human breast cyst fluid and in serum during the menstrual cycle. In the cyst fluid, estriol-3-sulfate (E3-3S) was found in each of ten samples, the concentrations ranging from 240-4310 pg/ml. Estriol-16-glucosiduronate (E3-16G) was detected in six samples at levels of 19-153 pg/ml; estriol-3-glucosiduronate (E3-3G) in five samples, 13-79 pg/ml and estriol-3-sulfate-16-glucosiduronate (E3-3S-16G) in four samples, 28-152 pg/ml. Unconjugated estriol was found in three of the ten cases (12-30 pg/ml). Serum samples obtained in the follicular and luteal phases of the cycle from eight different subjects were assayed in the same way. There was considerable variation between subjects and many values were indistinguishable from zero. But preliminary data suggest that E3-3G is the predominant E3 conjugate in the serum and E3-3S-16G is quantitatively least important. It appears that E3 conjugates in the cyst fluid are not derived from the blood directly, but are produced locally from precursors which have not been identified.     

Changes in liver and spleen volume in alcoholic liver fibrosis of man

Alcoholic liver fibrosis is a relatively common form of alcoholic liver disease in Japan. It is regarded by some investigators as a prodromal stage of alcoholic liver cirrhosis, but little is known about the volumes of the liver and spleen in this disease state. Therefore, liver and spleen volumes were measured by computed tomography in 32 patients with alcoholic liver fibrosis in comparison with 10 healthy volunteers. Patients with alcoholic liver fibrosis were divided into three subgroups (13 of Grade 1, 9 of Grade 2 and 10 of Grade 3) according to the severity of fibrosis. The volume was calculated from the sum of the area measurements of successive transverse sections of the two organs. The liver volume (mean +/- S.D.) in Grade 2 alcoholic liver fibrosis (1,281 +/- 112 cm3) was significantly (p less than 0.01) larger than in healthy volunteers (1,017 +/- 73 cm3) and in Grade 1 (1,090 +/- 157 cm3), and the liver volume in Grade 3 (1,490 +/- 132 cm3) was larger than in Grade 2 (p less than 0.01). The mean volume of hepatocytes estimated by a two-dimensional image analysis system was significantly (p less than 0.05) larger in Grade 3 than in Grade 2, and that in Grade 2 was larger than in Grade 1. The spleen volume in Grade 3 (151 +/- 40 cm3) was significantly (p less than 0.01) larger than in healthy volunteers (86 +/- 26 cm3), Grade 1 (89 +/- 38 cm3) and Grade 2 (68 +/- 19 cm3). The presumed reason for hepatic volume increase would be the ballooning of hepatocytes along with increased fibrotic component.     

Pharmacodynamics of 3-hydroxyquinidine alone and in combination with quinidine in healthy persons

The relation between serum concentration of 3-hydroxyquinidine (3-OHQ), a major metabolite of quinidine in humans, and the pharmacologic effect alone and in combination with the parent drug was studied. The heart rate-corrected, computer-averaged QT interval (QTc) was used as the pharmacologic endpoint. In a randomized, double-blind study, 5 healthy subjects received, on 3 separate days 1 week apart, either (1) 300 to 400 mg 3-OHQ orally or (2) 150 mg quinidine base intravenously or (3) a combination in the same doses. Blood samples and electrocardiographic recordings were obtained over the following 10 hours. Serum concentrations of 3-OHQ and quinidine were determined by high-pressure liquid chromatography and the free fraction by ultrafiltration. Peak concentrations of 3-OHQ varied between 1,362 and 3,480 ng/ml after oral 3-OHQ ingestion, but were negligible after intravenous quinidine infusion. The free fraction was 49% +/- 4.8 (mean +/- standard deviation) for 3-OHQ and 20% +/- 4.3 for quinidine. In all 5 subjects a statistically significant correlation was found between serum concentration and QTc prolongation for both quinidine and 3-OHQ (largest p value less than 0.025). The mean slope of the regression line was 0.0184 +/- 0.0128 for 3-OHQ and 0.0297 +/- 0.0111 for quinidine. Multiple linear regression revealed in each subject a significant additive effect of 3-OHQ when administered together with quinidine (largest p value less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of HCV Genotypes in the Populations of Inmates in Polish Prison Potulice and Patients Hospitalised in Bydgoszcz

Background:

According to many studies, one of the social groups with high rate of HCV infections are prisoners.

Objectives:

The aim of the study was to determine and compare the genotypes distribution among prisoners and patients of hospital.

Patients and Methods:

HCV genotypes among prisoners (281 inmates) and patients of hospital (1415 patients) were determined in years 2002-2012. HCV genotypes were determined in 2002-2005 with INNO-LiPA HCV II test (Innogenetics, Gent, Belgium) and since 2006 with LINEAR ARRAY assay (Roche, Mannheim, Germany), after isolation and amplification of the material with COBAS AMPLICOR v 2.0 (Roche, Mannheim, Germany).

Results:

The most frequent HCV genotype among inmates was genotype 3, which was detected in169 of 281 patients (60.1%). Most frequent genotype among hospitalized was genotype 1, which was found in 1127 cases (79.6%). Comparing the results of prisoners with a group of patients with HIV/HCV co-infection gave similar results. In both groups most frequent was genotype 3 (respectively 60.1 and 45.5%). However, most prisoners in this study (96%) were HIV-negative.

Conclusions:

The current study shows that the predominant HCV genotype among inmates from prison in Potulice is genotype 3.     

Point mutations in the juxtamembrane domain of FLT3 define a new class of activating mutations in AML

In acute myeloid leukemia (AML), two clusters of activating mutations are known in the FMS-like tyrosine kinase-3 (FLT3) gene: FLT3-internal tandem duplications (FLT3-ITDs) in the juxtamembrane (JM) domain in 20% to 25% of patients, and FLT3 point mutations in the tyrosine-kinase domain (FLT3-TKD) in 7% to 10% of patients, respectively. Here, we have characterized a new class of activating point mutations (PMs) that cluster in a 16-amino acid stretch of the juxtamembrane domain of FLT3 (FLT3-JM-PMs). Expression of 4 FLT3-JM-PMs in interleukin-3 (IL-3)-dependent Ba/F3 cells led to factor-independent growth, hyperresponsiveness to FLT3 ligand, and resistance to apoptotic cell death. FLT3-JM-PM receptors were autophosphorylated and showed a higher constitutive dimerization rate compared with the FLT3-wild-type (WT) receptor. As a molecular mechanism, we could show activation of STAT5 and up-regulation of Bcl-x(L) by all FLT3-JM-PMs. The FLT3 inhibitor PKC412 abrogated the factor-independent growth of FLT3-JM-PM-expressing cells. Compared with FLT3-ITD and FLT3-TKD mutants, the FLT3-JM-PMs showed a weaker transforming potential related to lower autophosphorylation of the receptor and its downstream target STAT5. Mapping of the FLT3-JM-PMs on the crystal structure of FLT3 showed that these mutations reduce the stability of the autoinhibitory JM domain, and provides a structural basis for the transforming capacity of this new class of gain-of-function mutations of FLT3.