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We utilized a disease progression model to predict the number of viraemic infections, cirrhotic cases, and liver-related deaths in the state of Rhode Island (RI) under four treatment scenarios: (1) current HCV treatment paradigm (about 215 patients treated annually, Medicaid reimbursement criteria fibrosis stage ⩾F3); (2) immediate scale-up of treatment (to 430 annually) and less restrictive Medicaid reimbursement criteria (fibrosis stage ⩾F2); (3) immediate treatment scale-up and no fibrosis stage-specific Medicaid reimbursement criteria (⩾F0); (4) an 'elimination' scenario (i.e. a continued treatment scale-up needed to achieve >90% reduction in viraemic cases by 2030). Under current treatment models, the number of cirrhotic cases and liver-related deaths will plateau and peak by 2030, respectively. Treatment scale-up with ⩾F2 and ⩾F0 fibrosis stage treatment criteria could reduce the number of cirrhotic cases by 21·7% and 10·0%, and the number of liver-related deaths by 19·3% and 7·4%, respectively by 2030. To achieve a >90% reduction in viraemic cases by 2030, over 2000 persons will need to be treated annually by 2020. This strategy could reduce cirrhosis cases and liver-related deaths by 78·9% and 72·4%, respectively by 2030. Increased HCV treatment uptake is needed to substantially reduce the burden of HCV by 2030 in Rhode Island.

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Transforming Growth Factor-ß (TGF-ß) and Epidermal Growth Factor (EGF) signaling pathways are both independently implicated as key regulators in tumor formation and progression. Here, we report that the tumor-associated overexpression of epidermal growth factor receptor (EGFR) desensitizes TGF-ß signaling and its cytostatic regulation through specific and persistent Stat3 activation and Smad7 induction in vivo. In human tumor cell lines, reduction of TGF-ß-mediated Smad2 phosphorylation, nuclear translocation and Smad3 target gene activation were observed when EGFR was overexpressed, but not in cells that expressed EGFR at normal levels. We identified Stat3, which is activated specifically and persistently by overexpressed EGFR, as a key signaling molecule responsible for the reduced TGF-ß sensitivity. Stable knockdown of Stat3 using small hairpin RNA(shRNA) in Head and Neck (HN5) and Epidermoid (A431) tumor cell lines resulted in reduced growth compared with control shRNA-transfected cells when grown as subcutaneous tumor xenografts. Furthermore, xenografts with Stat3 knockdown displayed increased Smad3 transcriptional activity, increased Smad2 phosphorylation and decreased Smad7 expression compared with control xenografts in vivo. Consistently, Smad7 mRNA and protein expression was also significantly reduced when EGFR activity was blocked by a specific tyrosine kinase inhibitor, AG1478, or in Stat3 knockdown tumors. Similarly, Smad7 knockdown also resulted in enhanced Smad3 transcriptional activity in vivo. Importantly, there was no uptake of subcutaneous HN5 xenografts with Smad7 knockdown. Taken together, we demonstrate here that targeting Stat3 or Smad7 for knockdown results in resensitization of TGF-ß's cytostatic regulation in vivo. Overall, these results establish EGFR/Stat3/Smad7/TGF-ß signaling axis driving tumor growth, which can be targeted therapeutically.

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AIMS: Duchenne muscular dystrophy (DMD) is usually associated with absent or nearly absent dystrophin expression at the sarcolemmal membrane. Quantification of very low levels of dystrophin signal in immunofluorescent studies of muscle biopsy sections presents a technical challenge. This is particularly true in the setting of proof-of-principle drug trials, in which the detection and quantification of what may be significant changes in levels of expression is important, even if absolute dystrophin levels remain low. METHODS: We have developed a method of image analysis that allows reliable and semi-automated immunofluorescent quantification of low-level dystrophin expression in sections co-stained for spectrin. Using a custom Metamorph script to create a contiguous region spectrin mask, we quantify dystrophin signal intensity only at pixels within the spectrin mask that presumably represent the sarcolemmal membrane. Using this method, we analysed muscle biopsy tissue from a series of patients with DMD, Becker muscular dystrophy, intermediate muscular dystrophy and normal control tissue. RESULTS: Analysis of serial sections on multiple days confirms reproducibility, and normalized dystrophin:spectrin intensity ratios (expressed as a percentage of normal control tissue) correlate well with the dystrophin expression levels as determined by Western blot analysis. CONCLUSION: This method offers a robust and reliable method of biomarker detection for trials of DMD therapies.

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Acute hepatitis C virus (HCV) infection is being acquired undetected among HIV-infected individuals. A practical way to regularly screen HIV-infected patients for acute HCV irrespective of perceived risk or symptoms is needed. We piloted implementation of an acute HCV screening strategy using routine HIV clinical care schedules and the least costly blood tests, in a Rhode Island HIV care center. Study participants had ongoing HCV risk, completed questionnaires encompassing risk behaviors and perception of risk, and were screened with quarterly alanine aminotransferase (ALT). ALT rise triggered HCV RNA testing, with pooled rather than individual specimen HCV RNA testing for underinsured participants. Participants were primarily older, college-educated men who have sex with men (MSM) with history of sexually transmitted infection other than HIV. One of 58 participants developed acute HCV in 50 person-years of observation for an annual incidence of 2.0% per year (95% confidence interval [CI] 0.05-11.1%). The majority (54%) of MSM did not perceive that traumatic sexual and drug practices they were engaging in put them at risk for HCV. Unprotected sex often occurred under the influence of drugs or alcohol. Self-reported HCV risk and participation in several risk behaviors declined during the study. It was possible to collect frequent ALTs in a busy HIV clinic with 71% of total projected ALTs obtained and 88% of participants having at least one ALT during the 9-month follow-up period. All instances of ALT rise led to reflexive HCV RNA testing. Tracking quarterly ALT for elevation to systematically prompt HCV RNA testing before seroconversion is a promising approach to screen for acute HCV in a real-world HIV clinical setting.

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REASONS FOR PERFORMING STUDY: Exercise depletes muscle glycogen stores, which could subsequently impair performance. Muscle glycogen replenishment is determined by substrate availability. OBJECTIVES: To study the effects of feeding meals of varying glycaemic responses on blood concentrations of substrates used for glycogenesis in horses with exercise-induced glycogen depletion. METHODS: In a 3-way crossover study, 7 horses received each of 3 isocaloric diets for 72 h after undergoing glycogen-depleting exercise: 1) a high soluble-carbohydrate diet, which induced a high-glycaemic (HGI) response; 2 and 3) a low soluble-carbohydrate or a mixed soluble-carbohydrate diet (control group), which both induced a similar low-to-moderate glycaemic (LGI) response. Muscle biopsies and venous samples were collected before and up to 72 h after exercise. RESULTS: Feeding HGI diet resulted in a higher (P<0.001) rate of muscle glycogenesis over 72 h compared to LGI diets. Plasma glycerol, triglyceride, lactate, serum NEFA and total protein concentrations, and haematocrit were significantly (P<0.001) higher after compared to before exercise. Whereas no significant overall dietary effect was observed on these metabolites over 72 h, there was a tendency for glycerol, NEFA and triglyceride concentrations to be lower for LGI compared to HGI diets over 6 h after exercise (P<0.05; 1, 6 and 4-6 h after exercise, respectively). CONCLUSIONS: These data suggest that horses fed LGI meals after exercise had limited lipid utilisation without any significant shift of substrate utilisation toward gluconeogenesis, which could have contributed to the slower rate of muscle glycogenesis compared to horses fed HGI diets.

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Muscle glycogen synthesis depends on glucose availability. This study was undertaken to determine the glycemic and insulinemic response of horses to equal amounts of hydrolyzable carbohydrates (starch and sugar) in the form of one of three grain meals or intragastric administration of a glucose solution. In a randomized crossover design, seven horses were fed each of three grain meals (cracked corn, steamed oat groats, or rolled barley) or were infused intragastrically with glucose solution at 2 g of hydrolyzable carbohydrate (starch plus sugar) per kilogram of BW. The quantity of hydrolyzable carbohydrate ingested was not different among all treatments (P = 0.70). Plasma glucose concentration peaked in all four treatments by 1.5 to 2 h after feeding. Plasma glucose concentration remained higher than baseline in oat groats or barley-fed horses throughout 8 h, whereas plasma glucose returned to baseline by 5 to 6 h in corn-fed horses or after glucose administration. Meal consumption was slower in oat groats-fed horses than in corn-fed ones, which may confound the glycemic and insulinemic responses observed after grain feeding. Plasma glucose area under the curve (AUC) was 63% both in corn and oat groats and 57% in barley-fed horses compared with that of horses administered glucose (P = 0.13). Serum immunoreactive insulin concentration peaked between 2 and 3 h after feeding or glucose administration, and barley-fed horses had lower serum immunoreactive insulin concentration by 3 to 4 h than corn-fed horses or after glucose administration (P < 0.05). We conclude, in horses, ingestion of oat groats, corn, and barley result in similar plasma glucose AUC and, compared with the glycemic index of 100 as the glucose reference, corn, oat groats, and barley had a glycemic index of approximately 60.

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High intensity exercise is associated with production of energy by both aerobic and anaerobic metabolism. Conditioning by repeated exercise increases the maximal rate of aerobic metabolism, aerobic capacity, of horses, but whether the maximal amount of energy provided by anaerobic metabolism, anaerobic capacity, can be increased by conditioning of horses is unknown. We, therefore, examined the effects of 10 weeks of regular (4-5 days/week) high intensity (92+/-3 % VO2max) exercise on accumulated oxygen deficit of 8 Standardbred horses that had been confined to box stalls for 12 weeks. Exercise conditioning resulted in increases of 17% in VO2max (P<0.001), 11% in the speed at which VO2max was achieved (P = 0.019) and 9% in the speed at 115% of VO2max (P = 0.003). During a high speed exercise test at 115% VO2max, sprint duration was 25% longer (P = 0.047), oxygen demand was 36% greater (P<0.001), oxygen consumption was 38% greater (P<0.001) and accumulated oxygen deficit was 27% higher (P = 0.040) than values before conditioning. VLa4 was 33% higher (P<0.05) after conditioning. There was no effect of conditioning on blood lactate concentration at the speed producing VO2max or at the end of the high speed exercise test. The rate of increase in muscle lactate concentration was greater (P = 0.006) in horses before conditioning. Muscle glycogen concentrations before exercise were 17% higher (P<0.05) after conditioning. Exercise resulted in nearly identical (P = 0.938) reductions in muscle glycogen concentrations before and after conditioning. There was no detectable effect of conditioning on muscle buffering capacity. These results are consistent with a conditioning-induced increase in both aerobic and anaerobic capacity of horses demonstrating that anaerobic capacity of horses can be increased by an appropriate conditioning programme that includes regular, high intensity exercise. Furthermore, increases in anaerobic capacity are not reflected in blood lactate concentrations measured during intense, exhaustive exercise or during recovery from such exercise.

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Injection drug users (IDUs) are a population at high risk for many diseases, including AIDS, and are clearly in need of medical and substance abuse treatment. Access to sterile syringes is critical for lowering the risk of transmission of HIV and other blood-borne pathogens among IDUs. Previously tried strategies include needle exchange programs and changing laws to allow the legal purchase and possession of syringes. An alternative strategy is to have physicians prescribe syringes to IDUs. To the best of our knowledge, this has previously been tried by only a few physicians in rare situations and never on a programmatic basis. This report describes the genesis of physician's syringe prescription in Rhode Island and some of the lessons learned to date. Because of the illicit nature of drug use, a tremendous amount of mistrust and fear on the part of IDUs often leads to poor interaction with the medical establishment. Prescription of syringes by a physician can serve as a tool for reaching out to a high-risk and often out-of-treatment population of drug users. It is a way for the health care community to tap into drug-using networks and bring those populations into a medical care system.

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Eucaryotic cells contain at least two general classes of oxygen-regulated nuclear genes: aerobic genes and hypoxic genes. Hypoxic genes are induced upon exposure to anoxia while aerobic genes are down-regulated. Recently, it has been reported that induction of some hypoxic nuclear genes in mammals and yeast requires mitochondrial respiration and that cytochrome-c oxidase functions as an oxygen sensor during this process. In this study, we have examined the role of the mitochondrion and cytochrome-c oxidase in the expression of yeast aerobic nuclear COX genes. We have found that the down-regulation of these genes in anoxic cells is reflected in reduced levels of their subunit polypeptides and that cytochrome-c oxidase subunits I, II, III, Vb, VI, VII, and VIIa are present in promitochondria from anoxic cells. By using nuclear cox mutants and mitochondrial rho(0) and mit(-) mutants, we have found that neither respiration nor cytochrome-c oxidase is required for the down-regulation of these genes in cells exposed to anoxia but that a mitochondrial genome is required for their full expression under both normoxic and anoxic conditions. This requirement for a mitochondrial genome is unrelated to the presence or absence of a functional holocytochrome-c oxidase. We have also found that the down-regulation of these genes in cells exposed to anoxia and the down-regulation that results from the absence of a mitochondrial genome are independent of one another. These findings indicate that the mitochondrial genome, acting independently of respiration and oxidative phosphorylation, affects the expression of the aerobic nuclear COX genes and suggest the existence of a signaling pathway from the mitochondrial genome to the nucleus.

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OBJECTIVE: To determine lactate breakpoint of horses and test for effects of training and dietary supplementation with corn oil on that breakpoint. ANIMALS: 7 healthy Arabian horses. PROCEDURES: Horses received a control diet (n = 4) or a diet supplemented with 10% corn oil (4). A training program, which comprised two 5-week conditioning periods with 1 week of rest, was initiated. Submaximal incremental exercise tests (IET) were conducted before the first and after both conditioning periods. Blood samples for determination of blood lactate and plasma glucose concentrations were collected 1 minute before IET and during the 15 seconds immediately preceding each speed change. Data collected were fit to one- and two-slope broken-line models and an exponential model. RESULTS: Good fits were obtained by application of the broken-line models (adjusted R2 > 0.92) to blood lactate concentration versus speed curves. Lactate breakpoints increased 41% after training. After training, slope 2 and peak blood lactate concentrations were greater in the corn oil group, compared with controls. Mean blood lactate concentration at the breakpoint was not affected by training or diet. Plasma glucose concentration versus speed curves also fit the broken-line models, and glucose breakpoints preceded lactate breakpoints by approximately 1 m/s in the second and third IET. CONCLUSIONS AND CLINICAL RELEVANCE: Lactate breakpoints can be determined for horses, using blood lactate concentration versus speed curves generated during submaximal IET and may be useful for assessing fitness and monitoring training programs in equine athletes.

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This study aimed to develop an alternative graduated compression bandage for the treatment of venous leg ulcers. Alternative bandage components were identified and assessed for optimal performance as a graduated multi-layer compression bandage. Subsequently the physical characteristics and clinical efficacy of the optimal bandage combination was prospectively examined. Ten healthy limbs were used to develop the optimal combination and 20 limbs with venous ulceration to compare the physical properties of the two bandage types. Subsequently 42 consecutive ulcerated limbs were prospectively treated to examine the efficacy of the new bandage combination. The new combination produced graduated median (range) sub-bandage pressures (mmHg) as follows: ankle 59 (42-100), calf 36 (27-67) and knee 35 (16-67). Over a seven-day period this combination maintained a comparable level of compression with the Charing Cross system, and achieved an overall healing rate at one year of 88%. The described combination should be brought to the attention of healthcare professionals treating venous ulcers as a possible alternative to other forms of multi-layer graduated compression bandages pending prospective, randomised clinical trials.

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Increases in blood [H+] and lactic acid [La-] attend fatigue. We applied Stewart's physiological model of acid-base status and simple regressions to assess the importance of independent variables and [La-] on [H+] during repeated sprints. Eight well-conditioned Arabians performed 9 sprints. Plasma from jugular vein samples was analysed for pH, PCO2, Na+, K+ and Cl-. Plasma [La-] was calculated from blood [La-], plasma [H+] from pH, SID from Na+, K+, Cl- and La-, Atot from pH, PCO2 and SID. Peaks for SID, PCO2 and [H+] were reached at sprint 1, -2 and -3, respectively. At sprint 3, the 5.7 nmol/l peak in [H+] was partitioned into 2.3, 2.7 and 0.7 nmol/l for Atot, PCO2 and SID, respectively. From sprint 3 to sprint 9, increases in Atot and decreases in SID tended to increase [H+] but were counteracted by a steady decrease in PCO2 that determined the progressive decrease in [H+]. Therefore PCO2 was the dominant determinant of [H+] during 9 repeated sprints, and the expected major effect of [La-] was moderated in the SID by opposing increases in [Na+] and [K+]. In the work-adapted phase (sprints 3-9), decreasing [H+] was correlated positively with PCO2 (r = 0.997, P < 0.001) but negatively with La- (r = -0.986, P < 0.001). Respiration was therefore completely compensating for the effects of metabolism on [H+]. During the transition from rest to sprint 3 (peak plasma [H+]), increasing [H+] was highly correlated (r = 0.99, P = 0.011) with [La-] but no other variable. The empirical and physiological analyses were consistent with one another during the work-adapted phase, but emphasis was placed on [La-] by the regression analysis, in contrast to PCO2 by the Stewart analysis, during the rest-work transition.

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Rectal temperature (Tre) is often used to adjust measurements of blood gases, but these adjusted measurements may not approximate temperatures during intense exercise at main sites of gas exchange: muscle and lung. To evaluate differences in blood gases between sites, temperatures (T) were measured with thermocouples in the rectum (re), in mixed venous blood (v), in gluteal muscle (mu), and on the skin (sk) in seven Arabian horses as they underwent an incremental exercise test on a treadmill. Blood samples were drawn from the carotid artery and pulmonary artery (mixed venous) 30 s before each increase in speed and during recovery. Blood gases and pH were measured at 37 degreesC, and all variables were adjusted to Tre, Tv, and Tmu. Adjusted variables during exercise and recovery were significantly different from each other at the three sites. Linear and polynomial equations described the time course of venous temperature and from Tre and Tsk during exercise and from Tsk during recovery. Interpretation of changes in muscle metabolism and gas exchanges based on blood-gas measurements is improved if they are adjusted appropriately to Tmu or Tv, which may be predicted from Tsk in addition to Tre during strenuous exercise and from Tsk during recovery.

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This trial was undertaken to examine the safety and efficacy of four-layer compared with short stretch compression bandages for the treatment of venous leg ulcers within the confines of a prospective, randomised, ethically approved trial. Fifty-three patients were recruited from a dedicated venous ulcer assessment clinic and their individual ulcerated limbs were randomised to receive either a four-layer bandage (FLB)(n = 32) or a short stretch bandage (SSB)(n = 32). The endpoint was a completely healed ulcer. However, if after 12 weeks of compression therapy no healing had been achieved, that limb was withdrawn from the study and deemed to have failed to heal with the prescribed bandage. Leg volume was measured using the multiple disc model at the first bandaging visit, 4 weeks later, and on ulcer healing. Complications arising during the study were recorded. Data from all limbs were analysed on an intention to treat basis; thus the three limbs not completing the protocol were included in the analysis. Of the 53 patients, 50 completed the protocol. At 1 year the healing rate was FLB 55% and SSB 57% (chi 2 = 0.0, df = 1, P = 1.0). Limbs in the FLB arm of the study sustained one minor complication, whereas SSB limbs sustained four significant complications. Leg volumes reduced significantly after 4 weeks of compression, but subsequent volume changes were insignificant. Ulcer healing rates were not influenced by the presence of deep venous reflux, post-thrombotic deep vein changes nor by ulcer duration. Although larger ulcers took longer to heal, the overall healing rates for large (> 10 cm2) and small (10 cm2 or less) ulcers were comparable. Four-layer and short stretch bandages were equally efficacious in healing venous ulcers independent of pattern of venous reflux, ulcer area or duration. FLB limbs sustained fewer complications than SSB.

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Seven Arabian horses performed a standard incremental exercise test on a high-speed treadmill at 6% slope then were randomly assigned to two diets, a control diet of ground hay and concentrates and a similar diet with 10% added fat (by weight). Horses were sprint-trained 4 d/wk, and two additional exercise tests were performed at 5-wk intervals. Heart rates and rectal temperatures were monitored and venous blood samples were collected at rest and at each speed increment. Whole blood was analyzed for glucose, lactate, and hemoglobin concentrations, and plasma was analyzed for pH, pCO2, albumin, total protein, and sodium, potassium, and chloride concentrations. Bicarbonate concentration ([HCO3-]) and strong ion difference ([SID]) were calculated, and total weak acid ([Atot]) was estimated from total protein. During exercise, there were increases in plasma sodium and potassium concentrations (P < .001), whole blood lactate and glucose (P < .001), and hemoglobin concentrations (P < .01). There were decreases in plasma pH, [HCO3-], and chloride concentrations (P < .001). The decrease in plasma pH was associated with changes in [SID] and [Atot] that combined to offset a decrease in pCO2. After sprint training, heart rates at rest and during submaximal exercise were decreased (P < .01), whereas heart rates at the end of exercise were increased (P < .05). Sprint training also increased workrate and estimated oxygen consumption at a heart rate of 200 beats/min (P < .001). Training increased the duration of exercise and the speed attained at the end of exercise (P < .05). Training increased the blood hemoglobin response to exercise and decreased the pCO2 response (P < .01). There were diet x training interactions for pH, pCO2, and [SID] (P < .05). Horses consuming the high-fat diet had higher blood glucose during both standard exercise tests and higher lactate concentrations at fatigue (P < .05) during the last test. Fat adaptation involving sprint training of horses may influence glucolysis at the level of pyruvate during an incremental exercise test.

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Retinoic acid (RA) in the form of isotretinoin (Accutane) and tretinoin (Retin-A) is a clinically important compound in the treatment of dermatologic disorders. However, it is also a potent teratogen associated with a number of serious congenital malformations. Generally, these malformations involve the craniofacial structures derived from the first and second branchial arches. To determine how altered gene expression may contribute to the observed RA-induced defects, pregnant LM/Bc mice were administered (5 mg/kg) all-trans RA on gestational day (GD) 8:12. First and second branchial arches were removed from control and teratogen-treated embryos on GD 10:00 10:12, or 12:00, processed by in situ transcription/aRNA techniques, and analyzed for alterations in gene expression. In these studies, a panel of 40 candidate genes that are known to be important in mammalian craniofacial development were examined. This analysis revealed significant differences in the expression level of the nicotinic acetylcholine receptor subunit alpha (NAChR), transforming growth factor beta 2 (TGF beta 2), type 1 cellular retinoid binding protein (CRBP-1), retinoic acid receptor gamma (RAR gamma), and cAMP response element binding protein (CREB). The alterations observed in the expression of these genes following RA exposure may prohibit normal morphogenetic processes within the second branchial arch and lead to the observed malformations.

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