Biomedical subjects

G Squadrito

Publications and source records attributed to G Squadrito.

At least 19 recordsLinked to original sources

PubMed2003

TT virus has a ubiquitous diffusion in human body tissues: analyses of paired serum and tissue samples.

The tissue tropism and possible correlation with liver disease of the TT virus (TTV) as well as its prevalence and genotype distribution remain undefined. TTV-DNA was investigated in paired sera and tissue samples from 144 patients, and sera and cerebrospinal fluids (CSF) from additional six subjects. Of the 144 tissue samples, 128 were liver biopsy specimens from subjects with hepatic disease while 16 were surgically obtained nonliver specimens from patients with extrahepatic disease. TTV cloning, sequencing and genotype analyses were performed on isolates from sera, tissue specimens and peripheral blood mononuclear cells of two patients with hepatic and four patients with extrahepatic pathologies, as well as from sera and CSFs of two subjects. TTV was found in 100% of the examined tissues and in 60.1 and 50% of sera from patients with hepatic and extrahepatic pathologies, respectively. Moreover, TTV was detected in four of the six CSFs analysed but only in two correspondent sera. Genotyping revealed the coexistence of multiple TTV genotypes and genetic variants in each infected individual, and the analysis of TTV mRNA showed the presence of transcripts in all the six different tissues studied. These results indicate that the entire adult population in our area is more likely infected by TTV, although several subjects are not viraemic and that TTV infects many different human tissues and is able to invade the central nervous system.

Adult↗

PubMed2002

Is investigation of hepatitis C virus NS5A gene heterogeneity a tool for predicting long-lasting response to interferon therapy in patients with HCV-1b chronic hepatitis?

Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) may repress the interferon (IFN)-induced protein kinase R (PKR). High variability of different regions in the carboxy-terminal half of NS5A implicated in the interaction with PKR (particularly the interferon sensitivity determining region (ISDR)) may be a predictor of response to IFN in patients infected with genotype 1b of HCV. We examined pretreatment serum samples from 17 HCV-1b infected patients included in the same schedule of IFN therapy. Seven patients were a rare series of sustained responders (SR) with a post-treatment follow-up of 5-7 years, while ten were nonresponders (NR). The carboxy-terminal half of the NS5A gene was amplified and directly sequenced in all 17 cases. In addition, the entire NS5A gene and the part of the HCV E2 gene coding for the hypervariable region 1 (HVR1) were amplified, cloned and sequenced in six cases (three NR and three SR). No difference in number and distribution of amino acid mutations was observed between isolates from SR and NR in any portion of the protein, including the ISDR region. Analysis of full length NS5A confirmed no difference between the two groups. The NS5A gene sequence was different among the six cases cloned although it appeared to be conserved in each individual patient independently of the quasispecies complexity evaluated through HVR1 examination. These data indicate that pretreatment analysis of theNS5A genomic variability has no value in predicting long-lasting response to IFN therapy in HCV-1b-infected patients, and that the HCV NS5A gene has high quasispecies homology.

Adult↗

PubMed2002

Immunohistochemical evidence of lactoferrin in hepatic biopsies of patients with viral or cryptogenetic chronic liver disease.

Lactoferrin (Lf) expression has been immunohistochemically investigated in 117 formalin-fixed paraffin-embedded liver bioptic samples obtained from an equal number of patients affected by chronic hepatitis (HCV = 76; HBV = 17; HBV + HDV = 14; cryptogenetic = 10); in addition, 10 autoptic specimens of normal liver were studied as control. The Lf immunoreactivity was evaluated by an intensity-distribution (ID) score. The Lf immunoexpression was observed in 88 out of 117 (75%) cases of chronic hepatitis; interestingly, all liver specimens from HBV hepatitis showed a constant Lf reactivity with the highest ID-score, whereas the evidence of Lf was encountered in 54/76 (71.1%) HCV as well as in 11/14 (78.6%) HDV chronic hepatitis, thus documenting a variable degree of Lf immunostaining in relation to different viruses. Moreover, in 6/10 (60%) cases of cryptogenetic hepatitis Lf immunoexpression was documented, whereas all normal liver controls were unreactive. In HCV specimens, the Lf nuclear immunoreactivity appeared to increase with the progression of the disease, with a greater expression in genotype 1. In contrast, no relationship among Lf ID-scores and different stages or grades of HBV, HDV or cryptogenetic hepatitis was encountered. This fact may suggest a role for Lf as an unspecific defensive agent in chronic inflammatory liver diseases, similarly to that elsewhere reported in other inflammatory tissue injuries.

Adult↗

PubMed2001

Oxidative stress causes nuclear factor-kappaB activation in acute hypovolemic hemorrhagic shock.

Nuclear Factor kappaB (NFkappaB) is an ubiquitous rapid response transcription factor involved in inflammatory reactions and exerts its action by expressing cytokines, chemokines, and cell adhesion molecules. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock. Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg. Hemorrhagic shocked rats died in 26.3 +/- 2.1 min following the discontinuance of bleeding, experienced a marked hypotension (mean arterial blood pressure = 20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (200 +/- 15 pg/ml, 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE; 1nM to 10 microM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-alpha mRNA in the liver (15-20 min after the end of bleeding) and enhanced plasma levels of 2,5-dihydroxybenzoic acid (2,5-DHBA; 6 +/- 2.2 microm), 2,3-dihydroxybenzoic acid (2,3-DHBA; 13 +/- 2.1 microm), both studied to evaluate OH(*) production. Electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory IkappaBalpha protein in the cytoplasm became decreased at 5 min after the end of bleeding. IRFI-042, a vitamin E analogue (20 mg/kg intraperitoneally 2 min after the end of bleeding), inhibited the loss of IkappaBalpha protein from the cytoplasm and blunted the increase in NF-kappaB binding activity. Furthermore IRFI-042 increased survival time (117.8 +/- 6.51 min; p <.01) and survival rate (vehicle = 0% and IRFI-042 = 80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (21 +/- 4.3 pg/ml), and restored to control values the hypo-reactivity to PE. Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-kappaB, likely through an increased production of reactive oxygen species. This experiment indicates that NF-kappaB-triggered inflammatory cascade becomes early activated during acute hemorrhage even in the absence of resuscitation procedures.

Animals↗

PubMed2001

Nuclear factor-kappaB as a target of cyclosporin in acute hypovolemic hemorrhagic shock.

BACKGROUND: Cyclosporin is an immunosuppressive drug that blocks Nuclear Factor kappaB (NF-kappaB) activation. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock and the effects of cyclosporin in this model of experimental shock. METHODS: Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg. Two minutes after bleeding cessation, animals received intravenously cyclosporin (1 mg kg(-1)) or its vehicle. Survival rate and survival time were evaluated for 120 min after bleeding was discontinued. Plasma TNF-alpha levels were investigated at different time points after bleeding cessation. Moreover we investigated levels of TNF-alpha mRNA in the liver, vascular reactivity, liver NF-kappaB binding activity and levels of the inhibitory protein IkappaBalpha in the cytoplasm. RESULTS: Hemorrhagic shocked rats died in 27+/-6 min following the cessation of bleeding, experienced a marked hypotension (mean arterial blood pressure=20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (208+/-22 pg ml(-1), 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE: 1 nM-10 microM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-alpha mRNA in the liver (15-20 min after the end of bleeding). Electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory protein IkappaBalpha in the cytoplasm decreased at 5 min after the end of bleeding. Cyclosporin inhibited the loss of IkappaBalpha protein from the cytoplasm and prevented NF-kappaB binding activity in the nucleus. Furthermore, cyclosporin increased survival time (118+/-7 min; P<0.01) and survival rate (vehicle=0% and cyclosporin=80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (28+/-7 pg ml(-1)), and restored to control values the hypo-reactivity to PE. CONCLUSIONS: Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-kappaB which triggers an inflammatory cascade leading to a fatal outcome. Cyclosporin blocks NF-kappaB activation and protects against hypovolemic hemorrhagic shock.

Animals↗

PubMed2001

Analysis of haemochromatosis gene mutations in a population from the Mediterranean Basin.

BACKGROUND/AIMS: The C282Y mutation in the haemochromatosis gene (HFE) located on chromosome 6 has been identified as the main genetic basis of hereditary haemochromatosis (HH). Two more mutations of that gene, H63D and S65C, appear to be associated with milder forms of HH. A high allele frequency for C282Y and H63D mutations was reported in populations from North Europe, while incomplete information is available for individuals from the Mediterranean Basin where C282Y homozygotes comprise a smaller percentage of HH cases. In this study we investigated the allele frequency of HFE mutations and the association between HFE mutations and cases of HH in a population from the South of Italy (Sicily and Calabria). In addition, we evaluated a possible association between HFE mutations and either chronic liver disease or type II diabetes. PATIENTS AND METHODS: Three hundred and twenty-seven individuals (654 chromosomes) were tested for C282Y, H63D and S65C mutations of the HFE gene by restriction fragment length polymorphism. Four had HH, 23 had hepatocellular carcinoma, 100 had chronic liver disease, 100 had type II diabetes, and 100 were healthy controls. RESULTS: Both C282Y and S65C mutations were each detected in one of the 654 chromosomes analysed (allele frequency=0.15%), while H63D change was found in 122 chromosomes (allele frequency=18.6%) and was equally distributed in all the categories examined. One healthy individual had compound heterozygosity for C282Y and H63D mutations. The frequency of C282Y in this Southern Italian sample was the lowest yet reported for a population of European origin. None of the four HH patients was either homozygous or heterozygous for C282Y. CONCLUSIONS: In Mediterranean populations from Southern Italy the C282Y mutation occurs sporadically and HFE polymorphisms seem to have little diagnostic relevance.

Chromosomes, Human, Pair 6↗

PubMed2001

The phytoestrogen alpha-zearalenol reverses endothelial dysfunction induced by oophorectomy in rats.

It has been shown recently that alpha-zearalenol, a resorcyclic acid lactone, prevents bone loss in a rat model of postmenopausal bone loss. We have therefore investigated the effects of this phytoestrogen on endothelial dysfunction induced by estrogen deficiency in rats. Female mature Sprague-Dawley rats underwent a bilateral oophorectomy (OVX rats). Sham-operated animals (sham OVX rats) were used as controls. Three weeks after surgery, animals were randomized to the following treatments: alpha-zearalenol (1 mg/kg/day, i.m., for 4 weeks), 17beta-estradiol (20 microg/kg/day, i.m., for 4 weeks), or their vehicle (100 microl, i.m., of cottonseed oil). Two other groups of rats were treated with alpha-zearalenol or 17beta-estradiol plus the pure estrogen receptor antagonist ICI 182780 (2.5 mg/kg/day, i.m., for 4 weeks). Mean arterial blood pressure (MAP), heart rate (HR), total plasma cholesterol, plasma estradiol, and plasma alpha-zearalenol were studied. We also investigated endothelial-dependent (acetylcholine, 10 nM to 10 microM) and endothelial-independent (sodium nitroprusside, 15 nM to 30 nM) relaxation of aortic rings, as well as N(G)-methyl-L-arginine (L-NMA: 10 to 100 microM)-induced vasoconstriction and calcium-dependent nitric oxide synthase (cNOS) activity in homogenates of lungs taken from both sham OVX rats and OVX rats. Untreated OVX rats had, compared with sham OVX animals, unchanged body weight, MAP, HR, and plasma cholesterol. In contrast oophorectomy reduced plasma estradiol levels (OVX, 2 +/- 0.5 pg/ml; sham OVX, 35 +/- 6 pg/ml), impaired endothelial-dependent relaxation and blunted L-NMA-induced contraction (L-NMA 100 microM: sham OVX, 2.7 +/- 0.3 g/mg tissue; OVX, 1.3 +/- 0.1 g/mg tissue). Moreover OVX rats showed a reduced calcium-dependent NO synthase (cNOS) activity. Treatment with alpha-zearalenol or with 17beta-estradiol reverted the endothelial dysfunction and increased cNOS activity in lung homogenates. These effects were abolished by the pure estrogen receptor antagonist ICI 182780. Our data suggest that alpha-zearalenol improves endothelial-dependent relaxation in OVX rats through an estrogen receptor-mediated effect.

Acetylcholine↗

PubMed2001

Reduction of carbon tetrachloride-induced rat liver injury by IRFI 042, a novel dual vitamin E-like antioxidant.

Carbon tetrachloride (CCl4 )-induced hepatotoxicity is likely the result of a CCl4 -induced free radical production which causes membrane lipid peroxidation and activation of transcription factors regulating both the TNF-alpha gene and the early-immediate genes involved in tissue regeneration. IRFI 042 is a novel vitamin E-like compound having a masked sulphydryl group in the aliphatic side chain. We studied the effect of IRFI 042 on CCl4 -induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of CCl4 (1 ml/kg in vegetal oil). Serum alanine aminotransferase (ALT) activity, liver malondialdehyde (MAL), hydroxyl radical formation (OH*), calculated indirectly by a trapping agent, hepatic reduced glutathione (GSH) concentration, plasma TNF-alpha, liver histology and hepatic mRNA levels for TNF-alpha were evaluated 48 h after CCl4 administration. Hepatic vitamin E (VE) levels were evaluated, in a separate group of animals, 2 h after CCl4 injection. A control group with vitamin E (100 mg/kg) was also treated in order to evaluate the differences versus the analogue treated groups. Intraperitoneal injection of carbon tetrachloride produced a marked increase in serum ALT activity (CCl4 = 404.61 +/- 10.33 U/L; Controls= 28.54 +/- 4.25 U/L), liver MAL (CCl4 = 0.67 +/- 0.16 nmol/mg protein; Controls= 0.13 +/- 0.06 nmol/mg protein), OH(7) levels assayed as 2,3-DHBA (CCl4 = 8.73 +/- 1.46 microM; Controls= 0.45 +/- 0.15 microM) and 2,5-DHBA (CCl4 = 24.61 +/- 3.32 microM; Controls= 2.75 +/- 0.93 microM), induced a severe depletion of GSH (CCl4 = 3.26 +/- 1.85 micromol/g protein; Controls= 17.82 +/- 3.13 micromol/g protein) and a marked decrease in VE levels (CCl4 = 5.67 +/- 1.22 nmol/g tissue; Controls= 13.47 +/- 3.21 nmol/g tissue), caused liver necrosis, increased plasma TNF-alpha levels (CCl4 = 57.36 +/- 13.24 IU/ml; Controls= 7.26 +/- 2.31 IU/ml) and enhanced hepatic mRNA for TNF-alpha (CCl4 = 19.22 +/- 4.38 a.u.; Controls= 0.76 +/- 0.36 a.u.). IRFI 042 (100 mg/kg, 30 min after CCl4 injection) blunted liver MAL (0.32 +/- 0.17 nmol/mg protein), decreased the serum levels of ALT (128.71 +/- 13.23 U/L), and restored the hepatic concentrations of VE (9.52 +/- 3.21 nmol/g tissue), inhibited OH* production (2,3-DHBA= 3.54 +/- 1.31 microM; 2,5-DHBA= 7.37 +/- 2.46 microM), restored the endogenous antioxidant GSH (12.77 +/- 3.73 mmol/g protein) and improved histology. Furthermore IRFI 042 treatment suppressed plasma TNF-alpha concentrations (31.47 +/- 18.25 IU/ml) and hepatic TNF-alpha mRNA levels (11.65 +/- 3.21 a.u.). The acute treatment with vitamin E failed to exert any protective effect against CCl4 -induced hepatotoxicity. These investigations suggest that IRFI 042 treatment may be of benefit during free radical-mediated liver injury.

Alanine Transaminase↗

PubMed2001

Cardiovascular effects of raloxifene hydrochloride.

Raloxifene hydrochloride binds to the estrogen receptor and shows tissue-selective effects; thus, it belongs to a class of drugs recently described as selective estrogen receptor modulators (SERMs). Tissue selectivity of raloxifene may be achieved through several mechanisms: the ligand structure, interaction of the ligand with different receptor subtypes in various tissues, and intracellular events after ligand binding. Raloxifene has estrogen-agonist effects on bone and lipids and estrogen antagonist effects on the breast and uterus. In addition to its well established effects on osteoporosis, recent preclinical and clinical findings suggest that raloxifene also possesses beneficial effects on the cardiovascular system. These findings indicated that raloxifene may have cardioprotective properties without an increased risk of cancer or other side effects. Raloxifene has been shown to reduce total and low-density lipoprotein cholesterol concentrations in plasma, an effect similar to that produced by estrogens. Unlike estrogens, however, raloxifene does not increase high-density lipoprotein cholesterol and triglyceride levels in plasma. Endothelium is thought to play an important role in the genesis of atherosclerosis. Several lines of evidence suggest that an intervention with endothelial function might influence the progression of coronary disease and the incidence of cardiovascular events. Raloxifene increases the nitric oxide/endothelin-1 ratio, and improves endothelium-dependent vasomotion in post-menopausal women to the same extent as estrogens. Furthermore, in two randomized trials on post-menopausal women raloxifene reduced homocysteine levels, another independent risk factor for the development of cardiovascular disease. Although estrogens remain the drugs of choice in the hormonal therapy of most postmenopausal women, raloxifene may represent and alternative in women who are at risk of coronary artery disease.

Animals↗

PubMed2001

Randomized, double-blind, placebo-controlled study on effects of raloxifene and hormone replacement therapy on plasma no concentrations, endothelin-1 levels, and endothelium-dependent vasodilation in postmenopausal women.

The endothelium is thought to play an important role in the genesis of atherosclerosis, and several lines of evidence suggest that the effect of an intervention on endothelial function might predict its involvement in coronary disease progression and in the rate of cardiovascular events. Estrogen has direct effects on the blood vessel wall, indicating that vascular endothelium may play a key role in the cardiovascular protective effects of hormone replacement therapy (HRT). Raloxifene relaxes coronary arteries in vitro by an estrogen receptor-dependent and NO-dependent mechanism, thus suggesting that this selective estrogen receptor modulator could also have beneficial effects on endothelial function. This study compared the effects of HRT and raloxifene on NO products, endothelin-1 plasma levels, and endothelium-dependent vasodilatation in postmenopausal women. Healthy postmenopausal women (n=90) were enrolled in a double-blind, randomized, placebo-controlled, 6-month trial. Women were randomly assigned to receive continuous HRT (1 mg 17beta-estradiol combined with 0.5 mg norethisterone acetate), raloxifene (60 mg/d), or placebo for 6 months. Flow-mediated endothelium-dependent vasodilation of the brachial artery, plasma NO concentrations, and endothelin levels were measured at baseline and after 6 months of therapy. The mean baseline level of NO breakdown products was 26.5+/-10.7 micromol/L and increased to 36.3+/-11.4 micromol/L after 6 months of treatment with raloxifene. The mean baseline plasma endothelin level was 17.3+/-8.9 pg/mL and decreased to 11.5+/-2.1 pg/mL after 6 months of treatment with the selective estrogen receptor modulator. The mean baseline ratio of NO (breakdown products) to endothelin was also significantly increased at the end of treatment with raloxifene. Postmenopausal women treated with HRT had similar changes in plasma nitrites/nitrates and endothelin levels as well as in the ratio of NO to endothelin. In contrast, these markers of endothelial function did not change in the placebo-treated women. Flow-mediated endothelium-dependent vasodilation of the brachial artery was 8.3+/-2.1% at baseline and increased to 12.3+/-2.1% after 6 months of treatment with raloxifene. HRT also caused a significant and similar increase in flow-mediated endothelium-dependent vasodilation. No change in flow-mediated vasodilation was observed in the participants treated with placebo. We conclude that raloxifene therapy and HRT influence endothelial function and improve flow-mediated endothelium-dependent vasodilation to a comparable extent in healthy postmenopausal women at least after a 6-month treatment period. However, further investigation is warranted to enhance our understanding of the mechanisms of the effect of raloxifene on vascular function and to determine whether its effect on endothelial function may contribute to the reduction in cardiovascular-related morbidity and mortality.

Aged↗

PubMed2001

Inhibition of lipid peroxidation restores impaired vascular endothelial growth factor expression and stimulates wound healing and angiogenesis in the genetically diabetic mouse.

Impaired wound healing is a well-documented phenomenon in experimental and clinical diabetes. Experimental evidence suggests that a defect in vascular endothelial growth factor (VEGF) regulation might be associated with wound-healing disorders. We studied the involvement of lipid peroxidation in the pathogenesis of altered VEGF expression in diabetes-related healing deficit by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db+/ db+ mice and their normal (db+/+m) littermates. Animals were then randomized to the following treatment: raxofelast (15 mg.kg(-1).day(-1) i.p.), an inhibitor of lipid peroxidation, or its vehicle (DMSO/NaCl 0.9%, 1:1 vol: vol). The animals were killed on different days (3, 6, and 12 days after skin injury), and the wounded skin tissues were used for histological evaluation, for analysis of conjugated dienes (CDs), as an index of lipid peroxidation and wound breaking strength. Furthermore, we studied the time course of VEGF mRNA expression throughout the skin-repair process (3, 6, and 12 days after skin injury), by means of reverse transcriptase-polymerase chain reaction, as well as the mature protein in the wounds. Diabetic mice showed impaired wound healing with delayed angiogenesis, low breaking strength, and increased wound CD content when compared with their normal littermates. In healthy control mice, a strong induction of VEGF mRNA was found between day 3 and day 6 after injury, while no significant VEGF mRNA expression was observed at day 12 after injury. In contrast, VEGF mRNA levels, after an initial increase (day 3), were significantly lower in diabetic mice than in normal littermates, and light induction of VEGF mRNA expression was also present at day 12 after injury. Similarly, the wound content of the angiogenic factor was markedly changed in diabetic mice. Administration of raxofelast did not modify the process of wound repair in normal mice, but significantly improved the impaired wound healing in diabetic mice through the stimulation of angiogenesis, re-epithelization, and synthesis and maturation of extracellular matrix. Moreover, raxofelast treatment significantly reduced wound CD levels and increased the breaking strength of the wound. Lastly, the inhibition of lipid peroxidation restored the defect in VEGF expression during the process of skin repair in diabetic mice and normalized the VEGF wound content. The current study provides evidence that lipid peroxidation inhibition restores wound healing to nearly normal levels in experimental diabetes-impaired wounds and normalizes the defect in VEGF regulation associated with diabetes-induced skin-repair disorders.

Animals↗

PubMed2000

The reduction of myocardial damage and leukocyte polymorphonuclear accumulation following coronary artery occlusion by the tyrosine kinase inhibitor tyrphostin AG 556.

We investigated the effects of tyrophostin AG 556, a tyrosine kinase inhibitor, on the phenomenon of leukocyte accumulation during ischaemia and reperfusion of the myocardium. Male anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5 h reperfusion (MI/R). Sham myocardial ischaemia-reperfusion rats (Sham MI/R) were used as controls. Myocardial necrosis, myocardial myeloperoxidase activity (MPO), serum creatinine phosphokinase activity (CPK) serum Tumor Necrosis Factor (TNF-alpha) and Interleukin 6 (IL-6), cardiac intercellular adhesion molecule-1 (ICAM-1) and TNF-alpha expression and myocardial contractility (left ventricle dP/dt(max)) were evaluated. Myocardial ischaemia plus reperfusion in untreated rats produced marked myocardial necrosis, increased serum CPK activity (196.5 +/- 19 U/100 ml, at the end of reperfusion) and myeloperoxidase activity (MPO, a marker of leukocyte accumulation) both in the area-at-risk (4.5 +/- 0.5 U/g/tissue) and in necrotic area (8.2 +/- 1.2 U/g/tissue), reduced myocardial contractility (1,706 +/- 52 mmHg/s, at the end of reperfusion) and induced a marked increase in the serum levels of TNF-alpha (1,950 +/- 97 pg/ml, at 1 h of reperfusion) and IL-6 (998 +/- 16 U/ml, at the end of reperfusion). Finally, myocardial ischaemia-reperfusion injury also increased cardiac mRNA for TNF-alpha and ICAM-1 in the myocardium-at risk. Tyrphostin AG 556 (0.5, 1 and 2 mg/kg subcutaneously 5 min after the onset of reperfusion) lowered myocardial necrosis and myeloperoxidase activity in the area-at-risk (1.5 +/- 0.2 U/g/tissue, following the highest dose) and in necrotic area (2.9 +/- 0.3 U/g/tissue following the highest dose), decreased serum CPK activity (96 +/- 9 U/100 ml, at the end of reperfusion), lowered serum TNF-alpha and IL-6, increased myocardial contractility (2,096 +/- 88 mmHg s, at the end of reperfusion) and reduced cardiac mRNA levels for TNF-alpha and ICAM-1. The present data suggest that tyrosine kinase inhibitors protect against myocardial ischaemia-reperfusion injury by reducing leukocyte accumulation to the ischaemic myocardium.

Animals↗

PubMed2000

IRFI 042, a novel dual vitamin E-like antioxidant, inhibits activation of nuclear factor-kappaB and reduces the inflammatory response in myocardial ischemia-reperfusion injury.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a ubiquitous rapid response transcription factor involved in inflammatory reactions which exerts its effect by expressing cytokines, chemokines, and cell adhesion molecules. Oxidative stress causes NF-kappaB activation. IRFI 042 is a novel dual vitamin E-like antioxidant and we, therefore, investigated its ability to protect the heart from oxidative stress and to halt the inflammatory response in a model of myocardial ischaemia-reperfusion injury. METHODS: Anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5-h reperfusion (MI/R). Sham myocardial ischaemia rats (sham-operated rats) were used as controls. Myocardial necrosis, cardiac output, cardiac and plasma vitamin E levels, myocardial malondialdehyde (MAL), cardiac SOD activity and elastase content (an index of leukocyte infiltration), hydroxyl radical (OH&z.ccirf;) formation, cardiac amount of mRNA codifying for ICAM-1 (evaluated by the means of reverse transcriptase polymerase chain reaction) and ICAM-1 immunostaining in the at-risk myocardium were investigated. NF-kappaB activation and the inhibitory protein of NF-kappaB, I-kappaBalpha, were also studied in at-risk myocardium by using electrophoretic mobility shift assay (EMSA) and Western blot analysis, respectively. RESULTS: The ischaemia-reperfusion model produced wide heart necrosis (area at risk-necrotic area=52+/-5%; necrotic area-left ventricle=28+/-3%), increased cardiac MAL, an index of lipid peroxidation (area at risk=62.5+/-3.9 nmol/g tissue; necrotic area=80.3+/-5.1 nmol/g tissue), induced tissue neutrophil infiltration, and caused a marked decrease in endogenous antioxidants. Furthermore, myocardial ischaemia plus reperfusion caused in the area at risk peak message for ICAM-1 at 3 h of reperfusion and increased cardiac ICAM-1 immunostaining at 5 h of reperfusion. NF-kappaB activation was also evident at 0.5 h of reperfusion and reached its maximum at 2 h of reperfusion. I-kappaBalpha was markedly decreased at 45 min of occlusion; peak reduction was observed at 1 h of reperfusion and thereafter it was gradually restored. Intraperitoneal administration of IRFI 042 (5, 10, 20 mg/kg, 5 min after reperfusion) reduced myocardial necrosis expressed as a percentage either of the area at risk (18+/-4%) or the total left ventricle (11+/-2%), and improved cardiac output. This treatment also limited membrane lipid peroxidation in the area at risk (MAL=14.3+/-2.5 nmol/g tissue) and in the necrotic area (MAL=26.5+/-3.7 nmol/g tissue), restored the endogenous antioxidants vitamin E and superoxide dismutase, and inhibited detrimental hydroxyl radical formation. Finally, IRFI 042 blocked the activation of NF-kappaB, reduced cardiac ICAM-1 expression, and blunted tissue elastase content, an index of leukocytes accumulation at the site of injury. CONCLUSIONS: Our data suggest that IRFI 042 is cardioprotective during myocardial infarction by limiting reperfusion-induced oxidative stress and by halting the inflammatory response.

Analysis of Variance↗

PubMed2000

Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats.

BACKGROUND: We investigated the effect of genistein, a phytoestrogen derived from a soy diet with a flavonoid chemical structure, on endothelial dysfunction induced by estrogen deficiency in rats. METHODS: Female mature Sprague-Dawley rats were subjected to a bilateral ovariectomy (OVX rats). Sham-operated animals (Sham OVX rats) were used as controls. Three weeks after surgery animals were randomized to the following treatments: genistein (0.2 mg/kg/day, s.c. for 4 weeks), 17 beta-estradiol (20 micrograms/kg/day, s.c. for 4 weeks) or their respective vehicles. Mean arterial blood pressure (MAP), heart rate (HR), total plasma cholesterol, plasma estradiol, plasma genistein levels and uterine weights were studied. Furthermore, we investigated acetylcholine (ACh 10 nM-10 microM) and sodium nitroprusside: (SN 15-30 nM) induced relaxation of aortic rings as well as NG-L-arginine (L-NMA: 10-100 microM) induced vasoconstriction in phenylephrine precontracted aortic segments and calcium-dependent nitric oxide synthase (cNOS) activity in homogenates of lungs taken from both sham OVX and OVX rats. RESULTS: Untreated OVX rats had, compared with sham OVX animals, unchanged body weight, MAP, HR and plasma cholesterol. In contrast ovariectomy impaired endothelial responses, blunted L-NMA induced contraction (L-NMA 100 microM: Sham OVX = 2.1 +/- 0.2 g/mg tissue; OVX = 1.7 +/- 0.4 g/mg tissue) and reduced cNOS activity. Treatment with 17 beta-estradiol increased the hormone plasma levels, reverted the endothelial dysfunction and increased cNOS activity in lung homogenates. Genistein supplementation enhanced the circulating levels of the phytoestrogen and affected NOS activity and endothelial dysfunction to the same extent. CONCLUSIONS: Our data suggest that genistein and 17 beta-estradiol show overlapping effects on experimental endothelial dysfunction.

Acetylcholine↗

PubMed2000

Quantification of intrahepatic hepatitis B virus (HBV) DNA in patients with chronic HBV infection.

No data are available about the amount of hepatitis B virus (HBV) genomes in liver of patients with chronic HBV infection. The aim of this study was to quantify the intrahepatic HBV DNA in hepatitis B surface antigen (HBsAg)-positive patients with either active or suppressed viral replication and in HBsAg-negative subjects with occult HBV infection. We optimized the Roche "Amplicor HBV Monitor" kit for quantifying liver HBV DNA and analyzed hepatic DNA extracts and serum samples from 19 HBs-Ag-positive and 43 HBsAg-negative individuals. Eight of the HBsAg carriers had active HBV replication, and for 3 of them we analyzed samples obtained before and at the end of 1 year of lamivudine treatment. Five hepatitis Delta virus (HDV) coinfected patients and 6 healthy HBsAg carriers had inhibited HBV activity. Among the HBsAg-negative subjects 21 had occult HBV infection and 22 had no evidence of HBV infection. The median of HBV genomes per microgram of liver DNA milliliter of serum was 34,500 to 2,620,000 in patients with active viral replication, 20,000 to 3,900, 000 before and 10,000 to 2,800 at the end of therapy in lamivudine-treated individuals, 9,800 to 600 in HDV-infected individuals, and 7,450 to 17,400 in healthy HBsAg carriers. These data indicate that cases with suppressed HBV activity, despite the very low levels of viremia, maintain a relatively high amount of intrahepatic viral genomes. This virus reservoir is likely involved in HBV reactivation, which is usually observed after stopping lamivudine treatment. Finally, the analysis of cases with occult HBV infection showed that the assay we used was able to specifically detect and quantify as few as 100 copies of viral genomes per microgram of liver DNA.

Adult↗

PubMed2000

Tacrolimus limits polymorphonuclear leucocyte accumulation and protects against myocardial ischaemia- reperfusion injury.

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the pathogenesis of either human and experimental myocardial ischaemia. Tacrolimus, formerly known as FK506, has been previously shown to display cardioprotective effects on experimental ischaemia/reperfusion-induced myocardial damage. This study investigated whether cardioprotection induced by tacrolimus in myocardial ischaemia-reperfusion (MI/R) injury might be due to inhibition of the nuclear factor kappa B (NF- kappaB) that in turn causes reduced cardiac ICAM-1 expression and blunted polymorphonuclear leukocyte accumulation. Anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5 h reperfusion (MI/R). Sham myocardial ischaemia-reperfusion rats (Sham MI/R) were used as controls. Myocardial necrosis, myocardial myeloperoxidase activity, serum creatine kinase (CK) activity, cardiac mRNA for ICAM-1 reverse-transcriptase polymerase chain reaction, the inhibitory protein of NF- kappaB I kappaB alpha (Western blot analysis) in the myocardium-at-risk, and left ventricle d P/d t(max)were evaluated. Myocardial ischaemia plus reperfusion in untreated rats produced marked myocardial necrosis, increased serum CK activity and myeloperoxidase activity (MPO, a marker of leukocyte accumulation) both in the area at risk and in the necrotic area, and reduced the left ventricle dP/d t(max). Furthermore, inhibitory protein I kappaB alpha levels decreased, and cardiac mRNA for ICAM-1 increased, after 0.5 and 5 h of reperfusion, respectively. Administration of tacrolimus (25, 50 and 100microg/kg as an i.v. infusion 5 min after reperfusion) lowered myocardial necrosis and myeloperoxidase activity in the area at risk and in necrotic area, decreased serum CK activity, increased left ventricle dP/d t(max), reduced the loss the of inhibitory protein I kappaB alpha and blunted the message for ICAM-1. The present data suggest that tacrolimus blocks the early activation of the transcription factor NF- kappaB, suppresses ICAM-1 gene activation, reduces leukocyte accumulation and protects against myocardial ischaemia-reperfusion injury.

Animals↗

PubMed2000

Occult hepatitis B virus infection.

Many studies have shown that hepatitis B virus infection may also occur in hepatitis B surface antigen-negative patients. This occult infection has been identified both in patients with cryptogenic liver disease and in patients with hepatitis C virus-related chronic hepatitis, and much evidence suggests that it may be a risk factor of hepatocellular carcinoma development. However several aspects of this occult infection remain unclear such as its prevalence and the factor(s) involved in the lack of circulating hepatitis B surface antigen. Moreover, it is uncertain whether the occult hepatitis B virus infection may contribute to chronic liver damage, considering that it is usually associated with a suppressed viral replication. Evidence and hypotheses concerning this fascinating field of bio-medical research are reviewed.

Carcinoma, Hepatocellular↗

PubMed2000

Protective effects of cyclosporin-A in splanchnic artery occlusion shock.

Cyclosporin A (CsA) is an immunosuppressant drug that inhibits nitric oxide (NO) synthase induction in vascular smooth muscle cells. Splanchnic artery occlusion (SAO) shock is a lethal type of shock characterized by a marked vascular dysfunction in which the L-arginine/nitric oxide pathway plays an important role. We investigated whether CsA exerts protective effects in SAO shock by interfering with the L-arginine/nitric oxide pathway. Male anaesthetized rats (n=156) were subjected to clamping of the splanchnic arteries for 45 min. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham operated animals were used as controls. SAO shocked rats had a decreased survival (86+/-6 min, while sham shocked rats survived more than 240 min), marked hypotension, increased serum levels of TNF-alpha, enhanced plasma nitrite/nitrate concentrations (75+/-7.1 microM; sham shocked rats=1.6+/-0.5 microM) and enhanced inducible NO synthase (iNOS) protein induction and activity in the aorta. Moreover aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM - 10 microM). CsA (0.25, 0.5 and 1 mg kg(-1), 5 min after reperfusion) increased survival rate (SAO+CsA=236+/-9 min following the highest dose), reverted the marked hypotension, reduced plasma nitrite/nitrate concentration (11+/-5.2 microM following the highest dose), restored to control values the hyporeactivity to PE, and blunted iNOS protein induction and activity in aortic rings. The present data indicate that in an experimental rat model CsA may have antishock properties related to inhibition of L-arginine/nitric oxide pathway.

Animals↗