Thiostrepton-Nanomedicine, a TLR9 Inhibitor, Attenuates Sepsis-Induced Inflammation in Mice.

Sepsis is a life-threatening clinical condition caused by infection and transposition of pathogens and pathogen-associated molecular patterns (PAMPs) into the host bloodstream. During sepsis, activation of toll-like receptors (TLRs) on immune cells triggers the release of pro-inflammatory cytokines and overstimulates the production of vasodilatory mediators such as nitric oxide (NO). These vascular changes lead to widespread inflammation, tissue damage, multiple organ failure, and often death. New therapeutic options are urgently needed. To this end, thiostrepton (TST) has emerged as a candidate for sepsis treatment due to its action as an antibiotic and anti-inflammatory molecule (TLR7-9 inhibitor). Reports in the literature suggest that TLR9 inhibition substantially suppresses the excessive host inflammatory response and attenuates sepsis-induced mortality in the cecal ligation and puncture (CLP) murine model of sepsis. However, to the best of our knowledge, TST has never been directly tested as a therapeutic option for the management of sepsis, possibly due to its low water solubility and drug delivery issues. These facts prompted us to test the central hypothesis that TST encapsulated in phospholipid sterically stabilized micelles (TST-SSM) could be developed into a novel treatment for sepsis. Thus, using our published method of encapsulating the hydrophobic antibiotic TST-SSM, we evaluated the in vivo efficacy of TST-SSM nanomedicine in the murine model of polymicrobial sepsis. We found that TST-SSM increased the median survival of CLP-induced septic mice from 31 to 44 hr by reducing the bacterial burden in the blood and peritoneal lavage. Moreover, plasma levels of pro-inflammatory cytokines (interleukin 6 and tumor necrosis factor-alpha) and NO derivatives were also reduced, whereas renal and hepatic function biomarkers creatinine and aspartate transferase were significantly improved. In conclusion, we identified that TST-SSM nanomedicine has significant potential as a therapeutic agent for sepsis management, primarily due to its anti-inflammatory and antibiotic properties.

TH-1 and TH-2 cytokines in stable chronic alcoholics.

UNLABELLED: In alcoholics, the activation of Kupffer cells by gram negative bacteriae leads to an inflammatory response and cytokine secretion, which in turn activate T-lymphocytes. Possibly, Th-1 lymphocytes are activated first, followed by a Th-2 response. Th-2 cytokines, especially interleukin (IL)-13 (scarcely studied in alcoholics), may be involved in the progression to chronic stages. AIMS: The aim of the study was to analyze the relationship of Th-1 and Th-2 cytokines with liver function, alcohol consumption, nutritional status and survival. METHODS: Serum Th-1 [interferon-γ (IFN-γ)] and Th-2 cytokines (IL-4, IL-13), IL-10, IL-6 and tumor necrosis factor (TNF-α), were determined for 18 controls and 47 stable alcoholics with variable liver function impairment, who were followed-up during a median time of 90 months, a period during which 14 patients died. RESULTS: IL-4 was lower among patients; no differences were observed regarding IL-6, but the remaining ILs were higher among alcoholics. IL-10 and IL-13 were even higher in cirrhotics (Z = 2.88, P = 0.004, and Z = 2.09, P = 0.037, respectively). A significant, direct, correlation was observed between IL-13 and IL-10 (ρ = 0.49, P = 0.001), and non-significant, inverse ones were observed between IFN-γ and IL-13 (ρ = -0.23), IL-4 (ρ = -0.14) and IL-10 (ρ = -0.09). IL-13 and IL-10 were inversely related with liver function and, directly with immunoglobulin A levels, but not with survival. CONCLUSION: Serum IFN-γ values were increased in alcoholics, who also showed raised IL-13 and IL-10, but lower IL-4 levels. Given the immunomodulatory roles of IL-10 and IL-13, this increase may be interpreted as a compensatory rise of anti-inflammatory cytokines. We failed to find any relation with mortality.

Alcoholic myopathy: vitamin D deficiency is related to muscle fibre atrophy in a murine model.

AIMS: Chronic myopathy has been described in alcoholics, characterized by atrophy of type II fibres, and vitamin D deficiency. Low serum vitamin D levels are frequent in alcoholics. The possibility exists that serum vitamin D levels are related to muscle changes in a murine experimental model. METHODS: Histological analysis of the right gastrocnemius muscle was performed in four groups of adult Sprague-Dawley rats, sacrificed after 5 weeks of treatment following the Lieber-DeCarli model. We studied the association between muscle histological changes and the activity of glutathione peroxidase (GPX), superoxide dismutase (SOD) and lipid peroxidation products (malondialdehyde); parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), free testosterone, 1,25 dihydroxyvitamin D3 (vitamin D) and corticosterone; and serum calcium and magnesium. RESULTS: Alcoholic animals showed type IIa and IIb fibre atrophy, especially the low-protein-fed ones, an effect dependent on protein deficiency. A significant relationship was observed between serum vitamin D levels and IIa fibre area (rho = 0.56, P = 0.002), and also, as a trend, between vitamin D and type IIb fibre area (rho = 0.39, p = 0.053); between vitamin D and muscle GPX (rho = 0.40, P = 0.025) and SOD activities (rho = 0.43, P = 0.012). Muscle GPX activity was significantly related with type I fibre area (rho = 0.49, P = 0.01) and muscle SOD, with type IIa fibre area (rho = 0.38, P = 0.045). Serum testosterone was also related with type IIa fibre area (rho = 0.61, P < 0.001). No relation was observed between serum PTH, corticosterone, or IGF-1 and fibre area PTH and antioxidant systems. Multiple regression analysis disclosed that the only parameter independently related with type IIa fibre area was serum vitamin D. CONCLUSION: Low vitamin D levels are related to muscle fibre atrophy, and altered levels of muscle antioxidant enzymes could play a role in alcoholic myopathy.

Osteopenia in alcoholics: effect of alcohol abstinence.

AIMS: The aims of this study were to assess bone mineral density (BMD) and content (BMC), osteocalcin, serum telopeptide, PTH and vitamin D in alcoholics, and to determine if a 6-month period of abstinence leads to changes in these parameters. METHODS: Serum osteocalcin, insulin-like growth factor 1 (IGF-1), telopeptide (40 patients) and 1,25 dihydroxyvitamin D, were measured in 28 controls and 77 alcoholic patients, 48 of whom were evaluated again 6 months later. All patients underwent whole-body assessment of BMD by a Hologic QDR-2000 (Waltham, MA, USA) bone densitometer, at the beginning of the study and 6 months later. RESULTS: Patients showed higher serum telopeptide levels (0.59 +/- 0.40 versus 0.19 +/- 0.10 nmol/100 ml, P < 0.001), lower IGF-1 [median = 49, interquartile range (IQR) = 31-121 ng/ml versus 135, IQR = 116-237 ng/ml, P < 0.001], vitamin D [26.5, IQR = 17.0-37.8 pg/ml versus 82.4 (IQR = 60.9-107.4 pg/ml, P < 0.001] and osteocalcin (2.1, IQR = 1.1-3.6 ng/ml versus 6.65, IQR = 4.9-8.8 ng/ml, P < 0.001) than those in controls. Patients also showed lower BMD values, Z- and T-scores at many levels of the skeleton and reduced total BMC. After 6 months, those who continued drinking showed a loss of bone mass, whereas those who abstained showed either no change or increase, differences being especially marked at pelvis, right arm and total BMD and BMC. Simultaneously, abstainers showed a significant increase in osteocalcin (versus a decrease among those who continued drinking). Serum telopeptide increased in both groups. CONCLUSION: Ethanol consumption leads to osteopenia, and decreased serum osteocalcin, which improve with abstinence, whereas those who continue drinking show a worsening of both parameters.

Alcoholic myopathy: lack of effect of zinc supplementation.

A chronic form of myopathy has been described in alcoholics, characterized by atrophy of type II fibers, due both to reduced protein synthesis and increased protein breakdown. Increased production of reactive oxygen species could probably play a role in increased protein breakdown. In addition, treatment with zinc might be beneficial, since it acts as a cofactor of several enzymes involved in the synthesis of proteins and antioxidants as copper-zinc-superoxidedismutase (SOD) and selenium dependent glutathione peroxidase (GPX). Based on these facts, we analyze the relative and combined effects of ethanol, protein malnutrition and treatment with zinc, 227 mg/l in form of zinc sulphate, on muscle changes in 8 groups of adult Sprague-Dawley rats fed following the Lieber-de Carli model during 5 weeks. We also study the association between muscle histological changes and the activity of GPX, SOD and lipid peroxidation products (MDA), with hormones such as IGF-1, and with trace elements involved in antioxidant systems and/or in lipid peroxidation, such as selenium, copper, zinc, and iron. We found type IIa and IIb fiber atrophy in the alcoholic animals, especially in the low-protein fed ones. This effect was mainly due to protein deficiency. Zinc played no role at all. Muscle iron increased in ethanol, low protein fed rats, either with or without zinc, and was directly related with muscle MDA levels, which in turn were related with muscle atrophy, as was also found for serum IGF-1 levels. Ethanol was the main responsible for all these changes, although protein undernutrition also played an independent role in MDA levels. A positive interaction between ethanol and protein deficiency on serum IGF-1 was also detected. These results suggest that both protein deficiency and ethanol contribute to muscle atrophy observed in alcoholized rats; this atrophy is associated with increased lipid peroxidation and muscle iron overload. Treatment with zinc sulphate confers no benefit.

Effect of zinc supplementation on ethanol-mediated bone alterations.

Ethanol consumption leads to bone alterations, mainly osteoporosis. Ethanol itself may directly alter bone synthesis, but other factors, such as accompanying protein malnutrition--frequently observed in alcoholics, chronic alcoholic myopathy with muscle atrophy, alcohol induced hypogonadism or hypercortisolism, or liver damage, may all contribute to altered bone metabolism. Some data suggest that zinc may exert beneficial effects on bone growth. Based on these facts, we analyzed the relative and combined effects of ethanol, protein malnutrition and treatment with zinc, 227 mg/l in the form of zinc sulphate, on bone histology, biochemical markers of bone formation (osteocalcin) and resorption (urinary hydroxyproline excretion), and hormones involved in bone homeostasis (insulin growth factor 1 (IGF-1), vitamin D, parathormone (PTH), free testosterone and corticosterone), as well as the association between these parameters and muscle fiber area and liver fibrosis, in eight groups of adult Sprague Dawley rats fed following the Lieber de Carli model during 5 weeks. Ethanol showed an independent effect on TBV (F=14.5, p<0.001), causing it to decrease, whereas a low protein diet caused a reduction in osteoid area (F=8.9, p<0.001). Treatment with zinc increased osteoid area (F=11.2, p<0.001) and serum vitamin D levels (F=3.74, p=0.057). Both ethanol (F=45, p<0.001) and low protein diet (F=46.8, p<0.01) decreased serum osteocalcin levels. Ethanol was the only factor independently related with serum IGF-1 (F=130.24, p<0.001), and also showed a synergistic interaction with protein deficiency (p=0.027). In contrast, no change was observed in hydroxyproline excretion and serum PTH levels. No correlation was found between TBM and muscle atrophy, liver fibrosis, corticosterone, or free testosterone levels, but a significant relationship was observed between type II-b muscle fiber area and osteoid area (rho=0.34, p<0.01). Osteoporosis is, therefore, present in alcohol treated rats. Both alcohol and protein deficiency lead to reduced bone formation. Muscle atrophy is related to osteoid area, suggesting a role for chronic alcoholic myopathy in decreased bone mass. Treatment with zinc increases osteoid area, but has no effect on TBV.

Relative and combined effects of ethanol and protein deficiency on some hair trace elements: lack of relationship with body stores.

This study was performed in order to analyze the relative and combined effects of ethanol and protein deficiency on hair copper, zinc, manganese, and iron content in four groups of seven animals each which were pair-fed during 8 wk with (1) a nutritionally adequate diet, (2) a 36% (as energy) ethanol-containing isocaloric diet, (3) a 2% protein, isocaloric diet, and (4) a 36% ethanol, 2% protein isocaloric diet, respectively, following the Lieber-DeCarli model, and to analyze the relationship between hair copper, zinc, manganese, and iron content, and the liver and muscle content of these elements. Although there was a trend to higher levels of all the elements analyzed in the the hair of the low-protein fed animals, differences were statistically significant regarding copper and manganese, effects being solely attributable to the low protein diet, not to ethanol. Moreover, hair copper was significantly, inversely related with final weight and weight loss. There were significant relationship between liver zinc and muscle zinc (r = 0.57, p = 0.002), but not between liver or muscle zinc and hair zinc; no correlations were observed between muscle copper and hair copper, nor between liver manganese and hair manganese. An inverse, statistically significant correlation was observed between liver copper and hair copper (r = -0.39, p < 0.05).

[Isotopic ventriculography in patients with obstructive hypertrophic cardiomyopathy treated with dual chamber stimulation]. / Ventriculografía isotópica en pacientes con miocardiopatía hipertrófica obstructiva tratados con estimulación bicameral.

BACKGROUND: Dual pacing stimulation improves symptoms and reduces intraventricular pressure gradient in obstructive hypertrophic cardiomyopathy. The mechanisms proposed for these effects are a change in ventricular contraction sequence, a paradoxical movement of the interventricular septum and a reduction in ventricular contractility. However, dual chambers pacing stimulation effects on systolic and diastolic function are unknown, and the study of this is the aim of this work. PATIENTS AND METHODS: We performed a radionuclide ventriculography in 10 patients with obstructive hypertrophic cardiomyopathy at 2 days and 1 and 6 months after placing a dual chamber pacemaker. The phase images, ventricular volumes, left ventricular ejection fraction and segmentary contractility were evaluated. Diastolic maximum peak-filling-rate and time to-peak-filling-rate were also studied. RESULTS: In DDD pacing stimulation, parametric phase images show a reversal up-down contraction sequence starting from the apical segment. At 6 months radionuclide ventriculography, a reduction in ejection fraction was found (60.9 +/- 12 vs 73.4 +/- 9.1; p < 0.05) which was attributed to apical, apical-septum and mainly basal-septum segmentary contraction decrease. A paradoxical mid-systolic septum movement was also found during DDD stimulation. No differences on diastolic parameters were noted. CONCLUSION: During dual chamber stimulation, a reversal ventricular contraction sequence, a paradoxical septum movement, hypokinesis of the septal ventricular segments and a reduction on left ventricular ejection fraction was observed.

[Alveolar recruitment under high-pressure transpulmonary inflation. An experimental morphometric study]. / Reclutamiento alveolar por insuflación transpulmonar a alta presión. Estudio morfométrico experimental.

We describe an experimental study in which morphometric techniques are used to question traditional conceptions about the behavior of the alveolus under changes in pressure. An increase in inflation pressure in the lung results in alveolar recruitment (an increase in distal air spaces) due to stretching of its walls. To test this hypothesis, the lungs of rats were filled to 25 to 35 cm water pressure. Lungs filled to a higher pressure were expected to present a decrease in alveolar size along with thinning of its walls, and an increase of internal perimeter of the alveolus with no change in amount of tissue. Morphometric data were processed by computer and results were analyzed by statistical tests. The lungs of 10 Wistar rats were examined under light microscope. The following variables were recorded: mean linear intersection (Lm), alveolar cord, wall thickness, internal alveolar perimeter and tissue percentage. Lower Lm in lungs filled to 35 cm water pressure allows us to consider that the number of alveoli increased; lower Lm and alveolar cord indicate that alveolar size is smaller; alveolar wall thickness decreased; internal alveolar perimeter increased. All these differences were statistically significant (p < 0.001), tissue percentage being the only variable that did not change significantly. All results lead us to consider that an increase in lung inflation pressure leads to alveolar recruitment and stretching of its walls.

[Morphometric assessment of rat lungs insufflated with liquid fixative at different pressures close to total lung capacity]. / Valoración morfométrica de pulmones de rata insuflados con líquido fijador a diferentes presiones próximas a la capacidad pulmonar total.

INTRODUCTION: Liquid lung fixing through the trachea to a pressure of 25 cmH2O is currently accepted to be ideal. However, some studies do not seem to confirm that assumption. MATERIAL AND METHODS: The lungs of Fischer rats were filled with fixing liquid to four different pressures: 20 cm, 25 cm, 30 cm and 35 cmH2O. The fixed lungs were processed for inspection under a light microscope for morphometric study. The following variables were recorded: lung volume, tissue volume, air volume, internal alveolar surface (IAS), alveolar chord to measure the size of the distal air space, and the number of alveoli. Statistical comparisons were performed. RESULTS: Lung volume increased with insufflation pressure, with significant differences related to pressure increases from 20 cm to 25 cm and from 30 cm to 35 cmH2O. Air volume did not change, although tissue volume changed when pressure increased from 20 cm to 30 cmH2O and from 30 cm to 35 cmH2O. The increase of tissue volume was related to extravasation of interstitial fixer. The number of alveoli increased with pressure from 20 to 30 cm and from 30 to 35 cmH2O. IAS increased with pressure from 20 cm and all the other pressures. Alveolar chord, which is related to size of alveoli, decreased significantly as pressure increased from 20 cm to 25 cm. CONCLUSION: A pressure of 25 cmH2O is ideal for liquid fixing of lung volumes. With lower pressures the lung is partially distended and with higher pressures the fluid can pass into the interstitial space.

[Scintigraphy with 99mTc-MIBI in the diagnosis of axillary lymph node invasion of breast cancer]. / Gammagrafía con 99mTc-MIBI en el diagnóstico de la invasión ganglionar axilar del cáncer de mama.

UNLABELLED: The presence of affected locoregional lymph nodes should be considered as one of the most important prognostic factors of breast cancer. At present, the clinician is conditioned by an absolute lack of an efficient methodology to evaluate the possible invasion of the axillary lymph nodes, which if negative, would make it possible to avoid surgical excision. In this study, we will evaluate the use of the 99mTc-MIBI scintigraphy in the pre-surgical diagnosis of axillary lymph node invasion and will analyze the relationship between the 99mTc-MIBI uptake and the number of lymph nodes affected. MATERIAL AND METHODS: 84 patients diagnosed of breast cancer were analyzed in this study. All of them underwent a 99mTc-MIBI scintigraphy, and the tumor/background ratio was determined semiquantitively for each image. The axillary lymph node invasion was determined following surgery. RESULTS: The sensitivity of the breast scintigraphy with 99mTc-MIBI for detection of lymph node invasion is 36% and the specificity is 100%. The positive predictive value is 100% and the negative one 48%. In the current study, we failed to detect correlation between the intensity of 99mTc-MIBI uptake in the primary tumor and the number of affected axillary lymph nodes. CONCLUSION: 99mTc-MIBI breast scintigraphy can provide complementary information for the presurgical diagnosis of breast cancer axillary lymph node invasion. 99mTc-MIBI breast scintigraphy shows high specificity and a high predictive value.

[Usefulness of the quantification of (99M)Tc-MIBI uptake in breast neoplasms in the preoperative assessment of tumor aggressiveness]. / Utilidad de la cuantificación de la captación de (99m)Tc-MIBI en neoplasias mamarias en la valoración preoperatoria de la agresividad tumoral.

UNLABELLED: (99m)Tc-MIBI has been proposed as an imaging diagnostic method in a large variety of human malignant tumors. At present, the mechanism by which (99m)Tc-MIBI is uptaken and concentrated by the malignant cells is not totally known. Some mammary neoplasms do not show any uptake of (99m)Tc-MIBI. This study aims to determine if there is any correlation between the uptake of (99m)Tc-MIBI by the tumor and the different histopathological parameters involved in tumoral aggressiveness. To do so, we have studied 100 patients with breast cancer. All of them underwent a breast scintimammography with (99m)Tc-MIBI with semiquantitative analysis by means of a tumor-to-background ratio calculated in every projection. After surgery, an experienced pathologist determined tumor size, axillary lymph node metastases, histological grade (Scarff Bloom Richardson) (SCBR), nuclear grade, mitotic index, presence of cellular atypia and estrogen and progesterone receptor expression. RESULTS: A statistically significant correlation (p < 0.005) has been found between tumor-to-background (T/B) ratios of (99m)Tc-MIBI uptake and tumor SCBR histological grade. A correlation between (99m)Tc-MIBI uptake and the mitotic index, cellular atypia and nuclear grade has also been found. No correlation was found in our study with tumor size, hormone receptor expression or axillary lymph node metastases. CONCLUSIONS: (99m)Tc-MIBI uptake in breast cancer is correlated with the tumoral differentiation grade: the smaller the tumoral cellular differentiation (greater aggressiveness), the greater the uptake. On the other hand, no correlation was found between the uptake of (99m)Tc-MIBI and the classical pathological parameters that define tumoral aggressiveness, such as size and axillary lymph node metastasis.

[99m Tc-MIBI scintigraphy compared to mammography in the diagnosis of breast cancer in dense, operated and young women breasts]. / Gammagrafía con 99mTc-MIBI frente a la mamografía en el diagnóstico de cáncer de mama en mamas densas, mamas intervenidas y mujeres jóvenes.

UNLABELLED: Breast scintimammography with 99mTc-MIBI has proven to be a useful complement to mammography in the diagnosis of breast cancer in the female population. Although the mammography, along with a physical examination, is the backbone of breast cancer diagnosis, there are groups of patients in whom the mammography has an even lower specificity. OBJECTIVE: Our study has aimed to assess the usefulness of breast 99mTc-MIBI scintimammography in those situations in which the mammography was indeterminate, such as, in dense breasts, young females or breasts with architectural distortion after surgery or radiation therapy. MATERIALS AND METHODS: We studied 109 females with mammographically dense breasts, 8 young females under 30 and 24 patients who had undergone previous surgery or radiation therapy. All cases were studied to rule out breast cancer. Final diagnosis was established with excisional biopsy. RESULTS: In dense breasts MIBI scintimammography sensitivity was 88% and the mammography one 81%. MIBI scintimammography specificity was 90% and the mammography 28%. In young females MIBI scintimammography sensitivity was 100% and the mammography 50%, MIBI scintimammography specificity 100% and the mammography 20%. In previous surgery, MIBI scintimammography sensitivity was 80% and the mammography 80%, MIBI scintimammography specificity 100% and the mammography 42%. CONCLUSION: Breast scintimammography with 99mTc-MIBI is an excellent diagnostic technique with high specificity. Undoubtedly it is complementary to mammography in those cases where mammography has major limitations such as dense breasts, young females and breasts with severe scarring after surgery or radiation therapy.

[Utility of 201Tl washout in syndrome X patients]. / Utilidad del washout del 201Tl en pacientes con síndrome X.

INTRODUCTION: The term Syndrome X defines a group of patients who present symptoms of typical angina pectoris, positive exercise test and normal coronaries in the arteriogram. This study aims to compare the results of 201Tl scan images and washout in Syndrome X patients. METHODS: 24 patients with typical angina and normal arteriogram and 30 normal controls were studied with 201Tl SPECT to evaluate uptake and washout. RESULTS: 201Tl perfusion defects were found in 3 out of 24 (12.5%) and washout defects in 22 out of 24 (91.6 %). There is a statistically significant reduction (0 < 0.05) in thallium uptake and washout in patients with syndrome X compared to the control group. In addition, patients with syndrome X had a higher thallium uptake at rest than during exercise compared to the healthy subjects. CONCLUSIONS: Analysis of the 201Tl washout and thallium uptake can provide addition information in the evaluation of patients with syndrome X since the visual assessment of the cardiac scintigraphy with 201Tl is of limited value. Alterations of the myocardial perfusion in patients with chest pain and normal coronaries can be detected non-invasively with a complete scintigraphic study.

Serum sclerostin in hepatitis C virus infected patients.

BACKGROUND: Sclerostin inhibits osteoblast functions, differentiations, and survival rates. As an endogenous inhibitor of the Wnt/ß-catenin pathway, the sclerostin should be related to decreased bone masses, although several studies indicate opposite results. In addition, it may be related to insulin resistances and carbohydrate metabolisms, a relation shared with other markers of bone metabolisms, such as osteocalcin. Hepatitis C virus (HCV) infected patients may present osteoporosis, and frequently show liver steatosis, which is a consequence of insulin resistance. The behaviour of sclerostin in these patients is yet unknown. The aim of this work is to analyse the relationships between serum sclerostin and osteocalcin levels and bone mineral density (BMD), liver functions, the intensity of liver steatosis and biochemical markers of bone homeostasis and insulin resistance in HCV-infected patients. METHODS: Forty HCV patients with 20 years of age and gender-matching controls were included in this study and underwent bone densitometry. Serum sclerostin, osteocalcin, collagen telopeptide, adiponectin, leptin, insulin, resistin, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were determined. Liver fat was histomorphometrically assessed. RESULTS: Sclerostin levels were slightly higher in patients than in controls, and were directly related to BMD at different parts of the skeleton, also to the serum telopeptide, and to the liver steatosis and TNF-α. On the contrary, osteocalcin showed a significant direct relationship with serum adiponectin, and an inverse one with IL-6. CONCLUSIONS: Serum sclerostin levels were within the normal range in HCV patients, and correlated directly with BMD and serum telopeptide. In addition, the relationships of sclerostin and osteocalcin with variables associated with insulin resistance suggested the role of bones for intermediary metabolisms.

Relative and combined effects of selenium, protein deficiency and ethanol on hepatocyte ballooning and liver steatosis.

Oxidative damage plays a key role in alcohol-mediated liver alterations. Selenium, a potent antioxidant, is decreased in alcoholics. This study was conducted to analyse if the supplementation with selenium may alter liver changes in a murine model fed ethanol and/or a 2 % protein-containing diet, following the Lieber-DeCarli design. Adult male Sprague Dawley rats were divided into eight groups which received the Lieber-DeCarli control diet; an isocaloric, 36 % ethanol-containing diet; an isocaloric, 2 % protein-containing diet; and an isocaloric diet containing 2 % protein and 36 % ethanol diet; and other similar four groups to which selenomethionine (1 mg/kg body weight) was added. After sacrifice (5 weeks later), liver fat amount and hepatocyte areas of pericentral and periportal cells were measured, and liver and serum selenium, activity of liver glutathione peroxidase (GPX), and liver malondialdehyde were determined. Ethanol-fed rats showed increased hepatocyte areas and fat accumulation especially when ethanol was added to a 2 % protein diet. Selenium caused a decrease in hepatocyte ballooning and liver fat amount, but an increase in GPX activity, and a marked increase in serum and liver selenium. The present study demonstrates that selenium, added to the diet of rats in the form of seleniomethionine, prevents the appearance of early signs of ethanol-mediated liver injury under the conditions of the Lieber-DeCarli experimental design.

Lipid peroxidation and serum cytokines in acute alcoholic hepatitis.

AIMS: Increased exposure of Kupffer cells to intestinal-borne Gram-negative bacteria enhances the metabolism and leads to cytokine production by these cells. Activation of Kupffer cells increases free radical release, which may, in turn, enhance cytokine secretion, creating a positive feedback loop, which contributes to liver inflammation. Cytokines act on T cells, inducing their proliferation and secretion of additional interleukins. Lipid peroxidation products (malondialdehyde; MDA) form adducts with proteins and acetaldehyde, triggering a T cell immune response. Controversy exists about the predominance of either Th-1 or Th-2 cellular responses. We performed the present study in order to analyse the cytokine pattern in patients with acute alcoholic hepatitis, its relation to MDA and the relation between all these parameters and liver function and prognosis. SUBJECTS AND METHODS: The study included 53 male alcoholics, 47 followed up for a median time of 32 months, during which 17 of them died. We measured serum MDA, tumour necrosis factor-alpha, interferon gamma (IFNG) and interleukins (IL) 4, 6, 8, and 10. RESULTS: MDA levels were raised in cirrhotics and non-cirrhotics with alcoholic hepatitis, maintaining a relationship with bilirubin and Maddrey index, and with mortality in the univariate analysis. Both IFNG and IL-4 were raised in our patients compared with controls, as well as IL-8, and IL-6, but IL-10 were below the detection limit in the majority of cases, especially in cirrhotics. Using a Cox regression model, Maddrey index displaced MDA in the survival analysis. CONCLUSIONS: Our data lend support to the hypothesis that activation of both Th-1 and Th-2 cell subsets take place. MDA levels are raised in alcoholics with alcoholic hepatitis and are closely related to liver function derangement and to survival, although this is displaced by Maddrey index using Cox regression model.

Receptors and inhibitory actions of gonadotropin-releasing hormone in the fetal Leydig cell.

The receptors and actions of gonadotropin-releasing hormone (GnRH) were analyzed in cultured testicular cells from 20.5-day fetal rats, in which treatment with luteinizing hormone (LH) maintained Leydig cell steroidogenesis and gonadotropic responses for up to 2 weeks. Testicular GnRH receptors were present on the 5th postnatal day, but were not demonstrable in fetal testes or 2-day cultures thereof. However, GnRH receptors were readily detectable in 4-day cultured fetal testes and were increased by exposure to GnRH agonists. In LH-treated cultures, GnRH sites were reduced by about 50% and did not increase during incubation with GnRH agonists. In such cultures, GnRH agonists inhibited LH-dependent steroid production and abolished the acute testosterone response to human chorionic gonadotropin. The half-maximal inhibitory concentration of [D-Ala6]des-Gly10-GnRH-N-ethylamide (3 X 10(-10)M) was commensurate with its binding affinity for testis receptors (Kd = 1.4 X 10(-10)M). In contrast, GnRH agonists had no inhibitory effects in 2-day cultures prior to the detection of GnRH receptors. The expression of functional GnRH receptors during culture in the absence of gonadotropin and their suppression in LH-treated cultures suggest that pituitary gonadotropins exert a tonic inhibitory effect upon testicular GnRH receptors. The demonstrated inhibitory actions of GnRH on steroidogenesis, with the expression of GnRH receptors in cultured fetal testes and 5-day postnatal testes, indicate that GnRH agonists could influence the actions of gonadotropins upon Leydig cell function in the neonatal testis.

Combined effects of steroids, ethanol and protein deficiency on tissue content and urinary and faecal excretion of zinc, copper and iron.

This study was performed in order to determine the relative and combined effects of ethanol, a low protein diet and steroid treatment on bone, muscle, liver, and urinary and faecal excretion of zinc, copper and iron in 64 rats divided into eight groups treated following the Lieber-DeCarli liquid diet technique, with and without dexamethasone, 1 mg/l. Steroids showed a lack of effect on liver zinc, but enhanced ethanol- and low protein-mediated liver iron overload when both factors were combined. Steroids also increased muscle copper, iron and zinc, and bone copper, especially in the low protein, ethanol-fed rats.