Next-generation muscle-directed gene therapy by in silico vector design.

There is an urgent need to develop the next-generation vectors for gene therapy of muscle disorders, given the relatively modest advances in clinical trials. These vectors should express substantially higher levels of the therapeutic transgene, enabling the use of lower and safer vector doses. In the current study, we identify potent muscle-specific transcriptional cis-regulatory modules (CRMs), containing clusters of transcription factor binding sites, using a genome-wide data-mining strategy. These novel muscle-specific CRMs result in a substantial increase in muscle-specific gene transcription (up to 400-fold) when delivered using adeno-associated viral vectors in mice. Significantly higher and sustained human micro-dystrophin and follistatin expression levels are attained than when conventional promoters are used. This results in robust phenotypic correction in dystrophic mice, without triggering apoptosis or evoking an immune response. This multidisciplinary approach has potentially broad implications for augmenting the efficacy and safety of muscle-directed gene therapy.

Diversity of hepatitis C virus infection among HIV-infected people who inject drugs in India.

The availability of generic direct acting antivirals (DAAs) for hepatitis C virus (HCV) treatment has prompted many low-and-middle-income countries to launch HCV elimination programs. Because the efficacy of some of these generic DAAs varies by HCV viral subtype, information on subtype distribution can contribute important information to these elimination programs. We conducted a cross-sectional serosurvey to characterize HCV subtype diversity among HIV positive people who inject drugs (PWID) across 14 cities in India. Of 801 HIV positive PWID sampled, 639 tested HCV antibody positive (78.9%). Among 105 samples sequenced, genotype 3 (58.1%) was the most commonly observed followed by genotype 1 (36.2%) and genotype 6 (5.7%). Of the genotype 3 infections, 65% were subtype 3a and 35% were subtype 3b. Of the genotype 1 infections, 94% were subtype 1a and 6% were subtype 1b. All genotype 6 samples were subtype 6n. There was some variability in genotype diversity depending on geographic region and PWID epidemic stage with greater diversity observed in older PWID epidemics. One sequence, HY018, did not cluster with any known reference sequences in phylogenetic analysis. Nearly 80% of HIV infected PWID across India are co-infected with HCV, and subtype prevalence and genetic diversity varied by region and PWID epidemic stage. HCV elimination programs in India will need to consider HCV subtype.

Human papillomavirus detection and comorbidity: critical issues in selection of patients with oropharyngeal cancer for treatment De-escalation trials.

BACKGROUND: The presence of human papillomavirus (HPV)-infection in oropharyngeal squamous cell carcinoma (OPSCC) is a major determinant in prognostic risk modeling. However, most risk models are based on clinical trials which only include a selected patient population. The clinical significance of HPV and other prognostic factors in patients with OPSCC remains to be evaluated in a large, unselected cohort, which also includes patients with stage I/II disease and patients with severe comorbidity. PATIENTS AND METHODS: All patients diagnosed with OPSCC in 2000-2006 in two Dutch university hospitals were included. The presence of an oncogenic HPV infection was determined by p16-immunostaining, followed by a high-risk HPV general primer 5+/6+ DNA PCR on the p16-positive cases. Cox regression analysis was carried out to compare survival rates between HPV-positive and HPV-negative patients and a prognostic model was generated by recursive partitioning. RESULTS: In total, 163 of 841 (19.4%) tumors were HPV-positive. Patients with HPV-positive OPSCC had a more favorable overall survival [73.5% versus 40.9% after 5 years; P < 0.001; hazard ratio = 0.34, 95% confidence interval (CI) 0.25-0.48] compared with patients with HPV-negative OPSCC. Patients with p16-positive but HPV DNA-negative tumors showed a significantly less favorable survival than patients with p16-positive and HPV DNA-positive tumors (P < 0.001). A prognostic model was developed in which patients were classified into three risk groups according to HPV status, nodal stage and comorbidity. [Harrell's concordance index of 0.68 (95% CI 0.65-0.71)]. CONCLUSIONS: Tumor HPV status is a strong and independent prognostic factor for survival among patients with OPSCC. A prognostic risk model was proposed, based on our large, unselected cohort of patients with HPV status, comorbidity and nodal stage being the important prognostic factors. In addition, this study emphasizes the importance of performing an HPV DNA-specific test besides p16-immunostaining.

The Derriford twelve commandments of emergency medicine: a model for good practice in a changing world, or a survival guide for new medical staff.

Time is a precious commodity and with more junior doctors coming through our departments for shorter periods of time it has been useful to lay down some ground rules to facilitate their induction. These are presented in the form of the twelve commandments of emergency medicine.

An evaluation of the GlideScope, a new video laryngoscope for difficult airways: a manikin study.

BACKGROUND AND OBJECTIVES: The GlideScope is a new video laryngoscope. The aim of our study was to assess its use compared to a Macintosh blade in airway scenarios on the Airman airway simulator. The scenarios were: 'normal' or resting state of the manikin, pharyngeal obstruction, cervical rigidity and tongue oedema. METHODS: Thirty anaesthetists from the Christchurch Anaesthetic Department attempted to pass a bougie or stylet through the vocal cords of the manikin with a size 3 Macintosh blade, then the GlideScope. View at laryngoscopy, ease of 'intubation' and whether they thought the GlideScope would be useful in clinical practice were recorded. RESULTS: Forty three percent (P = 0.02) found an improved view with the GlideScope in the pharyngeal obstruction scenario. In the other scenarios there was no significant difference in view. Reportage of ease of intubation showed no statistical difference in any scenarios. However, 93% of anaesthetists considered having the GlideScope would be useful if faced clinically with one or more of the studied scenarios. CONCLUSIONS: The GlideScope improved the view in one of three difficult airway situations when used by anaesthetists with no formal training in its use. No single airway device offers a solution to all scenarios, however, we consider that the GlideScope is a useful addition to the range of difficult airway devices available.

Chronic eosinophilic dermatitis associated with persistent feline herpes virus infection in cheetahs (Acinonyx jubatus).

A chronic ulcerative and eosinophilic dermatitis occurred in 20 captive cheetahs (Acinonyx jubatus) with persistent feline herpes virus 1 (FHV1) infection. Affected animals had erythematous, ulcerated plaques primarily on the face and forelegs in sites of contact with lachrymal and salivary secretions. The dermatitis was characterized by dense infiltrates of eosinophils and plasma cells and pseudoepitheliomatous hyperplasia. Rare keratinocytes within the lesions had nuclei with marginated chromatin and small eosinophilic inclusions composed of herpes virus nucleocapsids. Virus isolated from lesions was confirmed to be FHV1. Lesions persisted and progressed unless removed by cryoexcision. The occurrence of this unusual reaction to FHV1 in approximately 5% of captive North American cheetahs suggests a species propensity for a Th2-dominant response to herpes virus infection. This atypical immune reaction may indicate a heritable trait or modulation of the immune response by other factors such as chronic stress.

Acute effects of peritoneal dialysis on hemodynamics.

OBJECTIVES: To investigate the acute hemodynamic effects of peritoneal dialysis (PD) using the noninvasive Portapres technique [TNO Biomedical Instrumentation (TNO BMI); Amsterdam, The Netherlands]. DESIGN AND METHODS: Blood pressure was measured in 21 consecutive patients on continuous ambulatory PD during a standard peritoneal permeability analysis (SPA). Blood pressure, stroke volume, cardiac output, and total peripheral resistance were recorded and calculated using continuous finger pressure recordings with Portapres and Modelflow software (TNO BMI). The SPA consists of four phases: (1) drainage of night dwell dialysate, (2) instillation of a rinsing solution (1.36% glucose), (3) drainage of rinsing solution, and (4) instillation of the test solution (3.86% glucose to which dextran 70 has been added). RESULTS: Both systolic blood pressure (SBP) (7 +/- 9 mmHg, p < 0.005) and diastolic blood pressure (DBP) (5 +/- 6 mmHg, p < 0.01) increased during phase 2. Systolic BP and DBP increased further during phase 4 (SBP 8 +/- 14 mmHg, p < 0.05; DBP 6 +/- 8 mmHg, p < 0.005). These BP increases were caused by a rise in total peripheral resistance of 10% +/- 18% (p< 0.05) during phase 1, and 15% +/- 21% (p < 0.005) during phase 2. CONCLUSIONS: Instillation and dwell of a dialysis solution during PD causes a rise in blood pressure. This is caused by an increase in total peripheral resistance. Factors influencing total peripheral resistance could be a direct mechanical effect of dialysate on mesenteric resistance vessels or a temperature-related effect.

Pyrokinin neuropeptides in a crustacean. Isolation and identification in the white shrimp Penaeus vannamei.

Identification of substances able to elicit physiological or behavioural processes that are related to reproduction would greatly contribute to the domestication of commercially important crustaceans that do not reproduce easily in captivity. Crustaceans are thought to release urine signals used for chemical communication involved in courtship behaviour. In contrast to insects, very little is known about the endocrinological processes underlying this phenomenon. Therefore, an extract of 3500 central nervous systems of female white shrimp Penaeus vannamei was screened for myotropic activity in order to purify pyrokinin-like peptides that belong to the pyrokinin/PBAN neuropeptide family. Members of this family regulate reproductive processes in insects, including pheromone biosynthesis. Purification of these pyrokinins was achieved by a combination of reversed-phase and normal-phase chromatography. Subsequent characterization by mass spectrometry, Edman degradation and peptide synthesis resulted in the elucidation of two novel peptides. Pev-PK 1 has the primary sequence DFAFSPRL-NH(2) and a second peptide (Pev-PK 2) is characterized as the nonapeptide ADFAFNPRL-NH(2). Pev-PK 1 contains the typical FXPRL-NH(2) (X = G, S, T or V) C-terminal sequence that characterizes members of the versatile pyrokinin/PBAN family. Pev-PK 2 displays an Asn residue at the variable X position of the core pyrokinin sequence. These crustacean pyrokinins are the first to be found in a noninsect. The synthetic peptides display myotropic activity on the Leucophaea maderae as well as on the Astacus leptodactylus hindgut.

Silent ischaemia and hypertension.

For many years now, silent ischaemia has been recognized as a distinct clinical entity, and its relevance in different patient groups has been established. However, a number of basic questions have not been answered. In explaining the pathophysiology of silent ischaemia, factors affecting both the demand and the supply side are now being recognized. With the exception of certain well-defined groups, it is not clear why some patients are mostly symptomatic, while other patients are predominantly asymptomatic. There appear to be many factors influencing the ischaemic pain threshold. Studies investigating the prevalence of silent ischaemia show a remarkably high prevalence of silent ischaemia in different patient groups. Patients with hypertension but without coronary artery disease form a specific and vulnerable high-risk population that is particularly prone to silent ischaemia. Since changes at the macrovascular level are not responsible, various factors negatively influencing either cardiac supply or demand have been investigated. A reduced coronary reserve is central in explaining the increased prevalence of silent ischaemia in hypertensives. Left ventricular hypertrophy renders meaningful detection of ST segment changes difficult, but a possible solution dealing with this problem is offered by applying more stringent criteria in terms of minimal ST depression in the definition of ischaemia. The treatment of silent ischaemia is largely the same as for angina pectoris, but whether therapy should be directed at elimination of all ischaemic episodes or only of symptomatic episodes depends on further prospective work addressing this question.

Cytotoxicity and biotransformation of the anticancer drug perillyl alcohol in PC12 cells and in the rat.

The cytotoxic monoterpene perillyl alcohol (POH) has anticancer properties. We investigated its cytotoxicity in PC12 cells in relation to its biotransformation. POH is oxidized by alcohol dehydrogenase and aldehyde dehydrogenase to perillaldehyde (PCO) and perillic acid (PCOOH), respectively. Apoptosis was determined by cell cycle (subG(0)G(1)) analysis and AnnexinV staining followed by flow cytometry. PCO caused apoptosis at 200 microM, POH caused apoptosis from 500 microM on, while PCOOH had no effect. The caspase inhibitor zVAD prevented apoptosis. Inhibition of POH oxidation by 4-methylpyrazol did not prevent the apoptotic effect of POH indicating that POH itself is also apoptotic. To find out to what extent POH is metabolized to PCO, the metabolism of POH, PCO, and PCOOH was determined after intravenous injection in the rat and in isolated hepatocytes. Although PCO can form a glutathione conjugate(s), no indication of the formation of GSH conjugates was found either in vivo or in hepatocytes. About 70% of the dose was recovered as glucuronides in bile and urine. PCOOH generated only the acyl glucuronide, while POH and PCO formed both acyl and ether glucuronides. These results indicate that PCO is a major intermediary metabolite of POH in the rat in vivo and suggest that PCO may contribute to the anticancer effect of POH.

The kinin peptide family in invertebrates.

Kinins comprise a family of peptides that were first found in the central nervous system of insects and recently also in mollusks and crustaceans. After the isolation of the first members of the kinin family, the leukokinins from Leucophaea maderae, leukokinin-related peptides were found in the cricket Acheta domesticus and the locust Locusta migratoria, all through their ability to induce Leucophaea maderae hindgut contraction. Subsequently, kinins were found in the mosquitoes Culex salinarius and Aedes aegypti and in the earworm Helicoverpa zea. The first noninsect member of this family was isolated from a mollusk, the pond snail Lymnaea stagnalis. Most recently our group has isolated the first kinins from crustaceans. Six kinins were isolated from the white shrimp Penaeus vannamei. To date, 35 members of this family have been isolated. The first relatively small family of insect kinins has grown into an expanding and rather large family with members in insects, crustaceans, and mollusks. In this paper we discuss the kinin family in terms of method of isolation, structure, in vitro and in vivo activity, distribution, receptors, and signal transduction. We will compare the crustacean and insect members of the kinin family, using the data available on crustacea.

Heparin-induced thrombocytopenia and thrombosis: a prospective analysis of the incidence in patients with heart and cerebrovascular diseases.

Heparin-induced thrombocytopenia and/or thrombosis (HITT) are serious complications of heparin treatment. The incidence, as previously reported, varies widely and, in consequence, is not precisely known. Moreover, most reports only concern clinically defined heparin-induced thrombocytopenia. Therefore we carried out a prospective study of the incidence of serologically confirmed HITT. All patients admitted to the Departments of Cardiology and Neurology of our institution with an indication for treatment with therapeutic-dose intravenous unfractionated heparin were enrolled in the study. The patients were examined daily for the occurrence of thromboembolic complications. Regular platelet counts and tests for the presence of heparin-dependent antibodies were carried out using two different tests: a quantitative platelet factor 4/ heparin (PF4/hep) Elisa, and a functional test, the heparin-induced platelet activation assay (HIPAA). HITT was defined as a rapidly occurring (within 5 d) decrease of the platelet count from normal values of > 120 x 10(9)/l to < 60 x 10(9)/l or to < 100 x 10(9)/l if there was a rapid fall of >50% of starting value or >30% with concomitant acute thrombosis. The observed incidence of HITT was 1/358 patients (0.3%, 95% confidence limits 0.01-1.5%). However, Elisa PF4/hep specific IgG antibodies were demonstrated in nine (2.5%) and IgM antibodies in seven (2.0%) of 358 patients. 30/358 patients (8.4%) had platelet activating antibodies in the HIPAA. We conclude that the incidence of serologically confirmed HITT in this study is very low (0.3%) in patients with cardiac and neurologic diseases treated with intravenous unfractionated heparin. The frequency of heparin-dependent antibodies without concomitant occurrence of thrombocytopenia is much higher.

Heparin-induced thrombocytopenia with multiple cerebral infarctions simulating thrombotic thrombocytopenic purpura. A case report.

The authors describe a patient with stroke, treated with heparin for unstable angina, whose clinical features mimicked those of thrombotic thrombocytopenic purpura (TTP). His condition eventually proved to be caused by heparin-induced thrombocytopenia (HIT), complicated by thrombosis (HITT). The absence of microangiopathic hemolytic anemia should question the diagnosis in a presumed TTP patient. Early diagnosis of HITT is possible since recently two highly sensitive and specific tests have become available. Heparin treatment has to be stopped immediately if HITT is diagnosed. First-choice antithrombotic treatment in HITT patients is danaparoid.

Heparin-induced thrombocytopenia and thrombosis: a potential fatal complication in a routine treatment.

Heparin-induced thrombocytopenia (HIT) is a severe complication of heparin therapy. Life-threatening thromboembolism (HITT) may occur in a large number of patients with HIT. In this article diagnostic problems and the clinical course of 4 typical patients are described. Diagnosis was based on the occurrence of thrombocytopenia during heparin therapy and was confirmed in vitro by an ELISA to heparin-platelet factor 4 antibodies, heparin-induced platelet activation assay (HIPAA) or the platelet aggregation assay (PAA). Thrombotic complications developed in 2 patients, one of whom suffered a fatal embolism after accidentally rechallenging with low-dose heparin which was used to maintain the patency of an intravascular catheter. After discontinuation of heparin the thrombocyte count rapidly increased to normal values during treatment with the heparinoid danaparoid (Orgaran) without complications.