Restoration of innate and adaptive immune responses by HCV viral inhibition with an induction approach using natural interferon-beta in chronic hepatitis C.

Chronic hepatitis C (CHC) is a serious medical problem necessitating more effective treatment. This study investigated the hypothesis that an induction approach with nIFN-beta for 24 weeks followed by PEG-IFN-alpha+ribavirin (standard of care: SOC) for 48 weeks (novel combination treatment: NCT) would increase the initial virologic response rate and restore innate and adaptive immune responses in CHC. Seven CHC patients with a high viral load and genotype 1b were treated with NCT. Serum cytokine and chemokine levels were evaluated during NCT. NCT prevented viral escape and breakthrough resulting in persistent viral clearance of HCVRNA. IL-15 was increased at the end of induction therapy in both early virologic responders (EAVRs) and late virologic responders (LAVRs); CXCL-8, CXCL-10, and CCL-4 levels were significantly decreased (P < 0.05) in EAVR but not in LAVR during NCT, and IL-12 increased significantly (P < 0.05) and CXCL-8 decreased significantly (P < 0.05) after the end of NCT in EAVR but not in LAVR. NCT prevented viral breakthrough with viral clearance leading to improvement of innate and adaptive immunity resulting in a sustained virologic response (SVR). NCT (n = 8) achieved a higher SVR rate than SOC (n = 8) in difficult-to-treat CHC patients with genotype 1 and high viral loads.

Oxidative stress in diabetic nephropathy.

Diabetic nephropathy is a leading cause of end-stage renal failure worldwide. Its morphologic characteristics include glomerular hypertrophy, basement membrane thickening, mesangial expansion, tubular atrophy, interstitial fibrosis and arteriolar thickening. All of these are part and parcel of microvascular complications of diabetes. A large body of evidence indicates that oxidative stress is the common denominator link for the major pathways involved in the development and progression of diabetic micro- as well as macro-vascular complications of diabetes. There are a number of macromolecules that have been implicated for increased generation of reactive oxygen species (ROS), such as, NAD(P)H oxidase, advanced glycation end products (AGE), defects in polyol pathway, uncoupled nitric oxide synthase (NOS) and mitochondrial respiratory chain via oxidative phosphorylation. Excess amounts of ROS modulate activation of protein kinase C, mitogen-activated protein kinases, and various cytokines and transcription factors which eventually cause increased expression of extracellular matrix (ECM) genes with progression to fibrosis and end stage renal disease. Activation of renin-angiotensin system (RAS) further worsens the renal injury induced by ROS in diabetic nephropathy. Buffering the generation of ROS may sound a promising therapeutic to ameliorate renal damage from diabetic nephropathy, however, various studies have demonstrated minimal reno-protection by these agents. Interruption in the RAS has yielded much better results in terms of reno-protection and progression of diabetic nephropathy. In this review various aspects of oxidative stress coupled with the damage induced by RAS are discussed with the anticipation to yield an impetus for designing new generation of specific antioxidants that are potentially more effective to reduce reno-vascular complications of diabetes.

Relationship between total and regional bone mineral density and menopausal state, body composition and life style factors in overweight Japanese women.

OBJECTIVE: To investigate whether menopausal state, body composition and lifestyle factors influence total and regional bone mineral density in overweight Japanese women. DESIGN: Cross-sectional study of women who were recruited to the weight reduction program held at community-based health promotion center in Tokyo area. SUBJECTS: A total of 178 women with a mean age of 48 y old (20-69 y) with a clear menstrual history and BMI over 24. MEASUREMENTS: Total, regional and lumbar spine bone mineral density (BMD) and body composition were measured using DXA (Lunar). Menstrual history was taken by a questionnaire and walking steps per day and energy intake were measured. Physical fitness was assessed by cardio-respiratory fitness and leg extension power. Subjects were divided into pre-menopausal and post-menopausal groups. RESULTS: Pre-menopausal group had significantly higher total body BMD as well as regional BMD than post-menopausal group. However, no differences in BMI, percentage fat and fat mass (FM) were seen between the two groups. The multiple regression analysis stepwise method revealed that total and regional BMD correlated with menopausal state and total FM independently. Total and regional BMD did not correlate with total non-fat soft tissue mass (NFSM), energy intake, walking steps or physical fitness levels. Trunk and lower extremities BMD correlated with corresponding regional FM and NFSM, and upper extremities BMD correlated with only corresponding body part NFSM after adjusting menopausal state. CONCLUSION: Total and regional BMD had strong negative correlation with menopausal state rather than total FM in overweight Japanese women. Weight-bearing site BMD correlated with corresponding body part FM and NFSM and non-weight bearing site BMD only correlated with corresponding body part NFSM after adjusting for menopausal state.

Detection of hepatitis C virus in the bile and bile duct epithelial cells of hepatitis C virus-infected patients.

The pathobiology of hepatitis C virus (HCV) in the biliary system has not been clarified yet, although bile duct damage is a histological finding characteristic of chronic hepatitis C. In this study, we examined whether HCV infects bile ducts and is released into the bile. Twelve patients positive for serum HCV antibody were examined in this study, and eight were seropositive for HCV RNA by polymerase chain reaction (PCR). For those who underwent abdominal surgery, the bile was aspirated from the gall bladders by fine needle puncture. Five underwent wedge liver biopsy. Series of saline-diluted bile were assayed for HCV RNA by PCR to determine the HCV RNA titers. To examine HCV expression in the biliary system, the liver specimens were immunostained using monoclonal antibodies to the HCV proteins. HCV RNA was detected in the bile of 5 patients with high serum HCV RNA load (> or = 2.5 Meq/mL). Comparison of viral loads between serum and bile revealed that the HCV RNA level in the bile was as high as that in serum. Furthermore, immunohistological study showed that bile duct epithelial cells were infected with HCV. In contrast, HCV was not found in either the bile or bile duct of patients seronegative for HCV RNA or with low serum HCV load (< or = 1.1 Meq/mL). These findings suggest that the biliary system is involved in the pathobiology of HCV and that the bile is as highly infectious as the serum.

Ectopic activation of the transcription promoter for the testis-specific mouse Pgk-2 gene on elimination of a cis-acting upstream DNA region.

Transgenic mice carrying the coding sequence of beta-galactosidase, for which expression was driven by various upstream regions including the transcription promoter of the testis-specific mouse Pgk-2 gene, were generated. Expression of beta-galactosidase mRNA driven by the region between nucleotide positions -1404 and +61, with respect to the transcription initiation site numbered +1, was examined by reverse transcription-mediated polymerase chain reaction, blot hybridization and in situ hybridization, and compared with that of endogenous Pgk-2 mRNA. The results revealed that the 1.4kb DNA region is sufficient for determining the organ-specific, developmental stage-specific and spermatogenic stage-specific transcription of the mouse Pgk-2 gene. When the region between -684 and +61 was used to generate transgenic mice, beta-galactosidase mRNA was detectable not only in the testis, but also in other organs such as brain and lung. However, the timing and cell-type specificity of testicular expression of beta-galactosidase mRNA were retained in these mice. Because the region between -1404 and -685 repressed the Pgk-2 promoter in somatic cell-derived cell lines, it is suggested that the organ specificity of Pgk-2 transcription is achieved at least partly by negative regulation.

Human leukocyte antigen DRB1 1302 protects against bile duct damage and portal lymphocyte infiltration in patients with chronic hepatitis C.

BACKGROUND/AIMS: To confirm the immune reaction of hosts in chronic hepatitis C, we examined the association of human leukocyte antigen (HLA) DR with the histopathological outcome including bile duct damage and steatosis, which are characteristic of hepatitis C virus (HCV) infection. METHODS: One hundred and fifty-five patients with chronic HCV infection were examined. The pathological appearance of liver biopsy specimens was evaluated by both Knodell's histological activity index and examination of bile duct damage and steatosis. HLA DRB1 was determined by the polymerase chain reaction sequence-specific oligonucleotide probe method. RESULTS: HLA DRB1 1302 was found with significantly higher frequency in patients without than with bile duct damage (34.8% vs. 4.7%, p=0.0001, p corrected by Bonferroni's inequality method=0.002). It was also found more frequently in patients without marked portal lymphocyte infiltration (28.6% vs. 7.7%, p=0.0015, p corrected by Bonferroni's method=0.03). HLA DRB1 1101 was found more frequently in patients without than with piecemeal necrosis (p=0.004). In contrast, the frequency of HLA DRB1 1502 tended to be higher in patients with than without piecemeal necrosis and marked portal lymphocyte infiltration (p=0.015 and p=0.03, respectively). HLA DRB1 1201 and 0802 were seen more frequently in bile duct damage-negative (p=0.02) and piecemeal necrosis-negative patients (p=0.03), respectively. Interestingly, serum HCV levels of HLA DRB1 1302-positive patients were significantly higher than those of 1302-negative patients (mean: 7.7 Meq/ml vs. 3.1 Meq/ml, p=0.0007). CONCLUSION: These findings suggest that some histopathological changes in chronically HCV-infected livers could be caused by the host's immune reaction regulated by HLA DR.

Spermatocyte-specific gene excision by targeted expression of Cre recombinase.

Transgenic mice carrying the coding sequence of the Cre recombinase, whose expression was driven by the spermatocyte-specific Pgk-2 promoter, were generated. These mice were crossed with a reporter transgenic line, which produces beta-galactosidase depending on the occurrence of loxP-mediated DNA recombination. When DNA of the offspring was analyzed by PCR and Southern blotting, signals that appear after the recombination were detectable only in the testis. Histochemical analyses revealed that beta-galactosidase was present in spermatocytes and spermatogenic cells at later differentiation stages. However, the distribution of the protein was not uniform in all spermatocytes. Analyses of genomic DNA of the next generation indicated that recombination took place in about 70% of spermatogenic cells. From these results, we concluded that this transgenic line possessing Pgk-2-driven expression of the Cre recombinase should be useful for identifying spermatogenic genes that function at or after the spermatocyte stage.

Short-term cardiovascular responses to rapid whole-body tilting during exercise.

Our objective was to characterize the responses of heart rate (HR) and arterial blood pressure (BP) to changes in posture during concomitant dynamic leg exercise. Ten men performed dynamic leg exercise at 50, 100, and 150 W and were rapidly and repeatedly tilted between supine (0 degrees ) and upright (80 degrees ) positions at 2-min intervals. Continuous recordings of BP and HR were made, and changes in central blood volume were estimated from transthoracic impedance. Short-lasting increases in BP were observed immediately upon tilting from the upright to the supine position (down-tilt), averaging +18 mmHg (50 W) to +31 mmHg (150 W), and there were equally short-lasting decreases in BP, ranging from -26 to -38 mmHg upon tilting from supine to upright (up-tilt). These components occurred for all pressure parameters (systolic, mean, diastolic, and pulse pressures). We propose that these transients reflect mainly tilt-induced changes in total peripheral resistance resulting from decreases and increases of the efficiency of the venous muscle pump. After 3-4 s (down-tilt) and 7-11 s (up-tilt) there were large HR transients in a direction opposite to the pressure transients. These HR transients were larger during the down-tilt (-15 to -26 beats. min(-1)) than during the up-tilt (+13 to +17 beats. min(-1)), and increased in amplitude with work intensity during the down-tilt. The tilt-induced HR fluctuations could be modelled as a basically linear function of an arterial baroreflex input from a site half-way between the heart and the carotid sinus, and with varying contributions of fast vagal and slow sympathetic HR responses resulting in attenuated tachycardic responses to hypotensive stimuli during exercise.

Hoffmann-reflex is delayed during 6 degree head-down tilt with balanced traction.

BACKGROUND: Increased spinal height due to the lack of of axial compression on spinal structures in microgravity may stretch the spinal cord, cauda equina, nerve roots, and paraspinal tissues. HYPOTHESIS: Exposure to simulated microgravity causes dysfunction of nerve roots so that the synaptic portion of the Achilles tendon reflex is delayed. METHODS: Six healthy male subjects were randomly divided into two groups with three in each group. The subjects in the first group underwent horizontal bed rest (HBR) for three days. After a two week interval they underwent bed rest in a position of head-down tilt with balanced traction (HDT). So that each subject could serve as his own control, the second group was treated identically but in opposite order. Bilateral F waves and H-reflexes were measured daily (18:30-20:30) on all subjects placed in a prone position. RESULTS: By means of ANOVA, differences between HDT and HBR were observed only in M-latency and F-ratio, not in F-latency, central latency, and H-latency. Differences during the course of the bed rest were observed in M-latency and H-latency only. Tibial H latency was significantly lengthened in HDT group on day 2 and 3, although no significant difference between HDT and HBR was observed. CONCLUSION: The monosynaptic reflex assessed by H-reflex was delayed during 6 degree HDT with traction. The exact mechanism of this delay and whether the change was due to lengthening of the lower part of the vertebrae remain to be clarified.

Cardiovascular reserve to orthostatic stress in active-living elders.

A preliminary field survey of cardiovascular responses during orthostatic stress in active-living very old elders over 70 years old in Japan was conducted. The subjects were 36 active-living elders (age; mean=77.8 yrs, 70-88 yrs) in rural Japan (mountain area), and 9 healthy young persons (mean 22 yrs). During short-term (2min) orthostatic stress, active-living elders (>70 years old) had age-dependent orthostatic tachycardia and hypertension (by increased pulse pressure), and significantly decreased heart rate fluctuations with maintained blood pressure fluctuations. We conclude that cardiovascular changes with aging itself do not necessarily cause orthostatic intolerance.

Relatively shorter but intensive isotonic exercise in legs prevented the decrease in maximal aerobic power after 20 days of head down tilt bed rest in man.

As a countermeasures for the decrease in maximal oxygen uptake (VO2max) during prolong simulated weightlessness, it has been suggested that intensive isotonic legs exercise was useful. The reason is possibly that a dynamic and intensive resistance exercise training makes to maintain not only the volume of exercising muscles but also cardiac output. For the reason, it has been studied the effects of 3 different exercise training on VO2max during simulated weightlessness.

Degraded diet induced thermogenesis induced by 20 days of 6 degrees head down bed rest and effect of resistance training.

In the 21st century, our dream of living in a space will finally become reality. To understand the effects of microgravity on energy expenditure and possible countermeasure against them is important to maintain human health in space. Diet Induced Thermogenesis (DIT) is an increase in energy expenditure after ingestion of a meal, and accounts for 10% of total daily energy expenditure. Recently, the important role of DIT in regulating obesity is suggested. Basal Energy Expenditure (BEE), on the other hand, is the minimum energy expenditure required to keep on living, and accounts for 60-75% of total energy expenditure. Despite the importance of DIT, no information about DIT in microgravity and possible effects of exercise training is available. Therefore, we aimed to investigate, first, the effects of 20-days of 6 degrees head down tilting bed rest (HDBR) on postabsorptive energy expenditure (PEE), DIT, and BEE. Second, the effects of resistance training during HDBR on DIT and BEE.

Effects of physiological salt water injection after 20 days of head down tilt bed rest on maximal aerobic power in young males.

There is such a speculation that if the decrease in plasma volume (PV) makes central blood volume (CBV) and stroke volume (SV) reduce during prolonged BR, but if the changes in red cell volume (RCV) is little and a physiological salt water (PSW) volume matched to a body fluid loss is injected, then the increased PV would make CBV increase and the increase would contribute to prevent the decrease of SVmax as well as maximal cardiac output (COmax ). As a result, maximal oxygen uptake (VO2max ) would be come up to the before BR level. In the present study, to investigate the speculation, maximal exercise test immediately following the injection of PSW after prolonged head down tilt bed rest (HDTBR) was performed and compared to the before BR level in young males.

Effect of acute saline infusion on the cardiovascular deconditioning after 20-days head-down tilt bedrest.

Recent study showed that dehydration induced by prolonged exercise significantly impaired orthostatic tolerance with tachycardia and hypotension, and subsequent rehydration restored it. As physiological changes during exposure to actual or simulated microgravity can be viewed as dehydration process, we hypothesized that rehydration after prolonged bedrest may be a promising candidate to countermeasure not only fluid volume loss but also autonomic nervous deconditioning. Therefore, the purpose of present study was to determine the effect of acute rehydration by saline infusion on orthostatic cardiovascular deconditioning after bedrest.

Effect of a five minutes isokinetic one leg knee extension training on maximal oxygen uptake during 20 days of -6 degrees head-down tilting bed-rest in young male subjects.

Due to prolonged bed rest (BR), maximal oxygen uptake (VO2max) might be decreased by above -8%, even if a moderate supine bicycle exercise (SBE) was performed for 45 to 60 minutes every day during BR. However, the decrement of VO2max was lowered to below -5% by isometric training and using a reverse gradient garment. Further it was rather slightly increased by SBE training with gradually increasing load from 40% to 90% VO2max. The facts suggest the dependency of the decrease of VO2max during BR on exercising muscle mass (MM) and strength. In the present study, therefore, the effects of isokinetic knee extension (Ik-KnE) training on VO2max, exercising MM and muscle contraction strength during BR were investigated. Then, the relationships between changes in VO2max and the muscle mass and strength were considered.

Blood pressure and heart rate responses to sudden change of posture during 20 days of simulated microgravity (-6 degrees head-down tilt).

The cardiovascular function buffering the disturbance of blood pressure caused by postural changes may be deconditioned after exposure to microgravity (microG). However, total picture of the deconditioning including its longitudinal process is still unknown. The aim of this study was to determine time-dependent changes in the feedback regulation system of blood pressure as exposed to simulated microG (-6 degrees head-down tilt (HDT)) for 20 days.

Detection of hepatitis C virus RNA sequences in hepatocellular carcinoma and its precursors by microdissection polymerase chain reaction.

OBJECTIVE: Chronic hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinoma (HCC), but the mechanism of malignant transformation is unknown. To analyze the association of HCV with HCC, we developed a microdissection technique for the detection by polymerase chain reaction of positive (genomic)- and negative (replicative)-strand HCV RNA in histologically confirmed HCC and the surrounding cirrhotic and macroregenerative nodules. MATERIALS AND METHOD: Five HCCs and one macroregenerative nodule and the surrounding cirrhotic liver tissues of all cases were selected for this study. The method entails extraction of RNA from selected areas of formalin-fixed, hematoxylin-stained histologic sections, followed by strand-specific reverse-transcription double polymerase chain reaction and Southern blotting. RESULTS: Positive- and negative-strand HCV RNA sequences were detected in five of six tumors and the surrounding cirrhotic livers. CONCLUSIONS: These results verify the method of polymerase chain reaction detection of HCV RNA from histologically defined, selected lesions. In addition, the findings suggest that HCV RNA persists and replicates in hepatocytes during malignant transformation.

Effects of 20 days horizontal bed rest on mechanical efficiency during steady state exercise at mild-moderate work intensities in young subjects.

Delta efficiency(DE) at mild-moderate (31%-56% of maximum oxygen uptake) bicycle exercise in the upright sitting and in the supine position was measured in 10 young males and 5 females before and after 20 days bed rest (BR). Total muscle mass in the both legs(TMML) were measured by dual-energy x-ray absorptiometry. After BR, oxygen uptake decreased at all intensities during upright exercise and at 100 W during supine exercise (p<0.05) in the males but cardiac output (acetylene rebreathing) was almost unchanged. As defined as increase in work over the corresponding increase in energy liberation, DE significantly increased by 20.1% during upright exercise and 18.4% during supine exercise in males, but unchanged in females. TMML was decreased in both males and females. The differences in DE between males and females could not be explained in the present study. One possibility might be that the decreased DE was due to a simultaneous decrease in slow twitch muscle fiber content which might be responsible for the decreased TMML induced by BR in the males.

Baroreflex during exercise in different postures before and after 20-days bed rest.

Vagal-cardiac baroreflex functions in young healthy humans (n=6) were investigated in four different conditions; supine rest, seated rest, supine and seated exercise (50 watts) before and after 20-day horizontal bed rest. By selectively stimulating carotid baroreceptors using a neck pressure and suction technique, the primary finding was that the baroreflex sensitivity tuation at which we observed a tendency for an attenuation (0.05