Ledipasvir-sofosbuvir and sofosbuvir plus ribavirin in patients with chronic hepatitis C and bleeding disorders.

INTRODUCTION: Chronic hepatitis C virus (HCV) infection is prevalent among patients with inherited bleeding disorders and is a leading cause of mortality in those with haemophilia. AIM: We evaluated the efficacy and safety of ledipasvir-sofosbuvir and sofosbuvir plus ribavirin in patients with chronic HCV genotype 1-4 infection and an inherited bleeding disorder. METHODS: Ledipasvir-sofosbuvir was administered for 12 weeks to patients with genotype 1 or 4 infection and for 12 or 24 weeks to treatment-experienced cirrhotic patients with genotype 1 infection. Patients with genotype 2 and 3 infection received sofosbuvir plus ribavirin for 12 and 24 weeks respectively. RESULTS: The majority of the 120 treated patients had a severe bleeding disorder (55%); overall, 65% of patients had haemophilia A and 26% of patients had haemophilia B; 22% were HIV coinfected. Sustained virologic response at 12 weeks posttreatment was 99% (98/99) in patients with genotype 1 or 4 infection; 100% (5/5) in treatment-experienced cirrhotic patients with genotype 1 infection; 100% (10/10) in patients with genotype 2 infection; and 83% (5/6) in patients with genotype 3 infection. There were no treatment discontinuations due to adverse events (AEs). The most frequent non-bleeding AEs were fatigue, headache, diarrhoea, nausea and insomnia. Bleeding AEs occurred in 22 patients, of which all but one were considered unrelated to treatment. CONCLUSION: Treatment with ledipasvir-sofosbuvir for patients with HCV genotype 1 or 4 infection or sofosbuvir plus ribavirin for patients with genotype 2 or 3 infection was highly effective and well tolerated among those with inherited bleeding disorders.

Once daily ledipasvir/sofosbuvir fixed-dose combination with ribavirin in patients with inherited bleeding disorders and hepatitis C genotype 1 infection.

AIM: People with inherited bleeding disorders have been disproportionally affected by HCV. We assessed the fixed-dose combination of the NS5A inhibitor ledipasvir (LDV) with the NS5B polymerase inhibitor sofosbuvir (SOF) with ribavirin (RBV) in patients with genotype 1 HCV and inherited bleeding disorders. METHODS: To be eligible, patients had to be over 18 years of age and have an inherited bleeding disorder. HCV treatment-naïve and -experienced patients could enrol. All patients received LDV 90 mg per SOF 400 mg once daily and weight-based RBV in a divided dose for 12 weeks. The primary efficacy endpoint was sustained virologic response (SVR), defined as HCV RNA below the limit of detection (15 IU mL-1 ) 12 weeks after the end of treatment (SVR12). RESULTS: Of the 14 patients enrolled, 8 (57%) had haemophilia A, 3 (21%) had haemophilia B and 2 (14%) had von Willebrand disease, and 1 (7%) had factor XIII deficiency. All 14 patients (100%, 95% CI: 77-100%) achieved SVR12. Treatment was well tolerated: all patients completed therapy, with mostly mild adverse events. No specific safety concerns associated with the patient's underlying bleeding disorders were noted. CONCLUSION: These results appear to suggest that people with HCV and inherited bleeding disorders can be safely and effectively treated with 12 weeks of LDV/SOF plus RBV.

Evolution of the HCV viral population from a patient with S282T detected at relapse after sofosbuvir monotherapy.

Clinical phase II/III studies of the nucleotide analogue HCV NS5B inhibitor sofosbuvir (SOF) have demonstrated high efficacy in HCV-infected patients in combination therapy. To date, resistance to SOF (S282T in NS5B) has rarely been detected in patients. In this study, we investigated the evolution of S282T viral variants detected in one HCV genotype 2b-infected patient who relapsed following 12 weeks of SOF monotherapy. Deep sequencing of the NS5B gene was performed on longitudinal plasma samples at baseline, days 2 and 3 on SOF, and longitudinal samples post-SOF treatment through week 48. Intrapatient HCV evolution was analysed by maximum-likelihood phylogenetic analysis. Deep sequencing analysis revealed a low level pre-existence of S282T at 0.05% of viral sequences (4/7755 reads) at baseline and 0.03% (6/23 415 reads) at day 2 on SOF. Viral relapse was detected at week 4 post-treatment where 99.8% of the viral population harboured S282T. Follow-up analysis determined that S282T levels diminished post-treatment reaching undetectable levels 24-48 weeks post-SOF. Phylogenetic analysis together with the persistence of unique post-treatment mutations in all post-SOF samples suggested that growth of wild type resulted from reversion of the S282T mutant to a wild type and not outgrowth of the baseline wild-type population. Our data suggest that a very low level of pre-existing S282T at baseline in this patient was enriched and transiently detected following SOF monotherapy. Despite relapse with drug resistance to SOF, this patient was successfully retreated with SOF plus ribavirin for 12 weeks and is now cured from HCV infection.

International guidelines for the diagnosis and management of hereditary haemorrhagic telangiectasia.

BACKGROUND: HHT is an autosomal dominant disease with an estimated prevalence of at least 1/5000 which can frequently be complicated by the presence of clinically significant arteriovenous malformations in the brain, lung, gastrointestinal tract and liver. HHT is under-diagnosed and families may be unaware of the available screening and treatment, leading to unnecessary stroke and life-threatening hemorrhage in children and adults. OBJECTIVE: The goal of this international HHT guidelines process was to develop evidence-informed consensus guidelines regarding the diagnosis of HHT and the prevention of HHT-related complications and treatment of symptomatic disease. METHODS: The overall guidelines process was developed using the AGREE framework, using a systematic search strategy and literature retrieval with incorporation of expert evidence in a structured consensus process where published literature was lacking. The Guidelines Working Group included experts (clinical and genetic) from eleven countries, in all aspects of HHT, guidelines methodologists, health care workers, health care administrators, HHT clinic staff, medical trainees, patient advocacy representatives and patients with HHT. The Working Group determined clinically relevant questions during the pre-conference process. The literature search was conducted using the OVID MEDLINE database, from 1966 to October 2006. The Working Group subsequently convened at the Guidelines Conference to partake in a structured consensus process using the evidence tables generated from the systematic searches. RESULTS: The outcome of the conference was the generation of 33 recommendations for the diagnosis and management of HHT, with at least 80% agreement amongst the expert panel for 30 of the 33 recommendations.

Contrast echocardiography for detection of pulmonary arteriovenous malformations.

BACKGROUND: Pulmonary arteriovenous malformations (PAVMs) lead to stroke, brain abscess, and hemorrhage in hereditary hemorrhagic telangiectasia (HHT). The current screening approach for PAVMs in HHT patients with chest radiograph (CXR) and oxygen shunt study has not been validated and is thought to be insensitive. We hypothesized that agitated saline contrast echocardiography (ECHO) would be a useful screening test for PAVMs. METHODS AND RESULTS: A total of 106 sequential HHT patients underwent screening for PAVMs with ECHO in a prospective study. If the test was positive, or if the CXR or shunt study suggested PAVMs, pulmonary angiography was performed. A positive ECHO was defined as appearance of bubbles in the left atrium after injection of agitated saline solution. A positive shunt study was defined as a partial pressure of oxygen in arterial blood <500 mm Hg while breathing 100% oxygen. The mean age was 41 years (range 15-80 years); 66% were female. Forty-four patients had positive ECHO. Forty-one of the 44 patients underwent angiography. Three patients declined further testing. Thirty-three of the 41 patients who underwent angiography were diagnosed with PAVMs. Of the 62 patients with a negative ECHO, 18 underwent angiography because of either a shunt study or CXR that was suggestive of PAVMs. Of these 18 patients, 2 had PAVMs. In the total population of 106 patients, 35 (33%) had PAVMs. ECHO was the only positive screening test in 11 of 35 (31%) patients. The diagnosis of PAVMs in these 11 patients would have otherwise been missed. CONCLUSIONS: ECHO is a useful screening tool for PAVMs in HHT.

Transcatheter embolotherapy of maternal pulmonary arteriovenous malformations during pregnancy.

STUDY OBJECTIVES: To determine if transcatheter embolotherapy is safe and effective for the treatment of pulmonary arteriovenous malformations during pregnancy. DESIGN: Prospective study. SETTING: Specialized hereditary hemorrhagic telangiectasia centers at Yale University School of Medicine and St. Michael's Hospital, University of Toronto. PATIENTS: Seven pregnant women (age range, 24 to 34 years; gestational age range, 16 to 36 weeks) undergoing transcatheter embolotherapy. INTERVENTIONS: Transcatheter embolotherapy in all patients. MEASUREMENTS AND RESULTS: Thirteen pulmonary arteriovenous malformations in seven patients were embolized with detachable silicone balloons and/or stainless steel coils without incident. The estimated fetal radiation dose ranged from < 50 to 220 mrad. No complications of pulmonary arteriovenous malformations occurred in any of the patients after transcatheter embolotherapy. The mothers went on to deliver healthy babies in all cases. CONCLUSIONS: Transcatheter embolotherapy of maternal pulmonary arteriovenous malformations performed by an experienced radiologist appears to be safe and effective after 16 weeks of gestational age.

Chimeras of the integrin beta subunit mid-region reveal regions required for heterodimer formation and for activation.

A central region of the beta2 integrin subunit, RN (residues D300 to C459), was replaced by the equivalent sequences from beta1 and beta7 to give the chimeras beta2RN1 and beta2RN7. Whilst the former construct failed to form heterodimer at the cell surface with alphaL, the later of these could be expressed together with the alphaL subunit to form a variant LFA-1. Based on recent modelling work, the RN region consists of two parts, one is the C-terminal end of the putative A-domain (RB, residues D300 to A359), and the other the mid-region (BN, residues Y360 to C459). Chimeras exchanging the two component regions were made. Of the four resultant chimeras, only the beta2RB1 chimera failed to support LFA-1 expression. Thus the beta1 specific residues of this region affect the interaction with the alphaL subunit. Whereas the alphaL/beta2RB7 LFA-1 variant is wildtype like with respect to ICAM-1 adhesion, the alphaLbeta2BN1 and alphaLbeta2BN7, as well as the alphaLbeta2RN7, variants are more adhesive than the wildtype. These results suggest that an authentic beta2 mid-region is, in part, required for maintaining the LFA-1 in a resting state.

Summary of Canadian guidelines for the initial management of community-acquired pneumonia: an evidence-based update by the Canadian Infectious Disease Society and the Canadian Thoracic Society.

Community-acquired pneumonia (CAP) is a serious illness with a significant impact on individual patients and society as a whole. Over the past several years, there have been significant advances in our knowledge and understanding of the etiology of the disease, and an appreciation of problems such as mixed infections and increasing antimicrobial resistance. The development of additional fluoroquinolone agents with enhanced activity against Streptococcus pneumoniae has been important as well. It was decided that the time had come to update and modify the previous CAP guidelines, which were published in 1993. The current guidelines represent a joint effort by the Canadian Infectious Disease Society and the Canadian Thoracic Society, and they address the etiology, diagnosis and initial management of CAP. The diagnostic section is based on the site of care, and the treatment section is organized according to whether one is dealing with outpatients, inpatients or nursing home patients.

Effect of integrin beta 2 subunit truncations on LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) assembly, surface expression, and function.

LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are members of the beta2 integrins involved in leukocyte function during immune and inflammatory responses. We aimed to determine a minimized beta2 subunit that forms functional LFA-1 and Mac-1. Using a series of truncated beta2 variants, we showed that the subregion Q23-D300 of the beta2 subunit is sufficient to combine with the alphaL and alphaM subunits intracellularly. However, only the beta2 variants terminating after Q444 promote cell surface expression of LFA-1 and Mac-1. Thus, the major cysteine-rich region and the three highly conserved cysteine residues at positions 445, 447, and 449 of the beta2 subunit are not required for LFA-1 and Mac-1 surface expression. The surface-expressed LFA-1 variants are constitutively active with respect to ICAM-1 adhesion and these variants express the activation reporter epitope of the mAb 24. In contrast, surface-expressed Mac-1, both the wild type and variants, require 0. 5 mM MnCl2 for adhesion to denatured BSA. These results suggest that the role of the beta2 subunit in LFA-1- and Mac-1-mediated adhesion may be different.

Diagnostic criteria for hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome).

Hereditary Hemorrhagic Telangiectasia (HHT) is easily recognized in individuals displaying the classical triad of epistaxis, telangiectasia, and a suitable family history, but the disease is more difficult to diagnosis in many patients. Serious consequences may result if visceral arteriovenous malformations, particularly in the pulmonary circulation, are unrecognized and left untreated. In spite of the identification of two of the disease-causing genes (endoglin and ALK-1), only a clinical diagnosis of HHT can be provided for the majority of individuals. On behalf of the Scientific Advisory Board of the HHT Foundation International, Inc., we present consensus clinical diagnostic criteria. The four criteria (epistaxes, telangiectasia, visceral lesions and an appropriate family history) are carefully delineated. The HHT diagnosis is definite if three criteria are present. A diagnosis of HHT cannot be established in patients with only two criteria, but should be recorded as possible or suspected to maintain a high index of clinical suspicion. If fewer than two criteria are present, HHT is unlikely, although children of affected individuals should be considered at risk in view of age-related penetration in this disorder. These criteria may be refined as molecular diagnostic tests become available in the next few years.

Diffuse pulmonary arteriovenous malformations: characteristics and prognosis.

OBJECTIVE: To study the clinical characteristics and prognosis of patients with diffuse pulmonary arteriovenous malformations (AVMs). DESIGN: Retrospective chart review of all patients (n = 16) with diffuse pulmonary AVMs seen at Yale New Haven Hospital, Johns Hopkins Hospital, and St. Michael's Hospital. Up-to-date follow-up information was obtained in all living patients. RESULTS: All patients were severely hypoxic. Neurologic complications (stroke or brain abscess) had occurred in 70% of patients by the time of diagnosis. During the follow-up period (mean, 6 years), three patients died and two others developed new neurologic complications. One of the deaths occurred perioperatively during lung transplantation. All patients underwent transcatheter embolotherapy of any large pulmonary AVMs. A selected group underwent pulmonary flow redistribution, a novel technique. Oxygenation did not improve significantly with embolotherapy of the larger AVMs, but there was a small significant improvement in those patients who underwent pulmonary flow redistribution. The majority (85%) of the living patients are currently working or studying full-time. CONCLUSIONS: Patients with diffuse pulmonary AVMs are at increased risk of neurologic complications. Transcatheter embolotherapy does not significantly improve the profound hypoxia, but it may reduce the risk of neurologic complications. Antibiotic prophylaxis is recommended for bacteremic procedures to prevent brain abscess. These patients can live for many years and lead productive lives. We do not recommend lung transplantation because survival with disease is difficult to predict and we have observed a perioperative transplant death.

Summary of Canadian guidelines for the initial management of community-acquired pneumonia: an evidence-based update by the Canadian Infectious Disease Society and the Canadian Thoracic Society.

Community-acquired pneumonia (CAP) is a serious illness with a significant impact on individual patients and society as a whole. Over the past several years, there have been significant advances in the knowledge and understanding of the etiology of the disease, and an appreciation of problems such as mixed infections and increasing antimicrobial resistance. The development of additional fluoroquinolone agents with enhanced activity against Streptococcus pneumoniae has been important as well.It was decided that the time had come to update and modify the previous CAP guidelines, which were published in 1993. The current guidelines represent a joint effort by the Canadian Infectious Diseases Society and the Canadian Thoracic Society, and they address the etiology, diagnosis and initial management of CAP. The diagnostic section is based on the site of care, and the treatment section is organized according to whether one is dealing with outpatients, inpatients or nursing home patients.

Spinal cord arteriovenous malformations in two patients with hereditary hemorrhagic telangiectasia.

We report two cases, in first cousins, of spinal arteriovenous malformations (AVMs) of the perimedullary fistula type and hereditary hemorrhagic telangiectasia (HHT). Spinal AVMs are a rare clinical presentation of HHT, but can be the first manifestation in a child with this disorder. The importance of considering a coexisting disorder of vascular dysplasia, such as HHT, when a child presents with a spinal AVM is discussed.

Pulmonary hypertension and cardiac function in adult cystic fibrosis: role of hypoxemia.

STUDY OBJECTIVES: To determine (1) the prevalence of pulmonary hypertension and cardiac dysfunction in adult cystic fibrosis (CF) patients with severe lung disease, (2) the relationship between these cardiovascular abnormalities and hypoxemia, and (3) the impact of subclinical pulmonary hypertension on survival. DESIGN: Single-blind, cross-sectional study. SETTING: Ambulatory clinic of the Adult CF program at a tertiary-level hospital. PATIENTS: Clinically stable patients with severe lung disease (FEV1 < 40% of predicted normal value) who were not receiving supplemental oxygen. A second cohort of patients in stable condition with less severe lung disease (FEV1 40 to 65% predicted) was also recruited to enable multivariate analysis for the determinants of pulmonary hypertension. MEASUREMENTS AND RESULTS: Eighteen patients with severe lung disease (FEV1 28 +/- 7% of predicted normal value) were initially studied. Each patient had overnight polysomnography, pulmonary function tests, and Doppler echocardiography. Arterial oxygen saturation (SaO2) was reduced during wakefulness (87.1 +/- 6.1%) and fell during sleep (84.0 +/- 6.6%) while transcutaneous PCO2 was normal during wakefulness (41.1 +/- 6.9 mm Hg) and increased during sleep (46.6 +/- 4.7 mm Hg). Left ventricular size, systolic function, and diastolic function were normal except in one patient who had had a previous silent myocardial infarction due to coronary artery disease. Qualitative assessment of right ventricular function was normal in all patients. Pulmonary artery systolic pressure (PASP) was increased (> 35 mm Hg) in seven patients without clinical evidence of cor pulmonale. Regression analysis was performed by combining these data with data from an additional 15 CF patients with moderately severe lung disease (FEV1 56.3 +/- 8.9% predicted normal) who were recruited to a modified study protocol that included overnight oximetry, pulmonary function tests, and Doppler echocardiography. None of these patients had evidence of hypoxemia and only three had mild elevation of PASP (36, 37, and 39 mm Hg). Linear regression analysis revealed that PASP was significantly correlated with FEV1 (r = -0.44; p = 0.013), and SaO2 during wakefulness (r =-0.60; p = 0.0003), during sleep (r = -0.56; p = 0.0008), and after 6 min of exercise (r = -0.75; p < 0.0001). Multivariate analysis revealed that awake SaO2 was a significantly better predictor of PASP than FEV1 (p = 0.0104). Clinical follow-up of the original cohort for up to 5 years revealed that mortality was significantly higher in those with pulmonary hypertension than those without pulmonary hypertension (p = 0.0129). CONCLUSIONS: In adult CF patients with severe stable lung disease, left and right ventricular function is well maintained in the absence of significant coronary artery disease; pulmonary hypertension develops in a significant proportion of patients and is strongly correlated with oxygen status, independent of lung function; and subclinical pulmonary hypertension is associated with an increased mortality.

A role for heterodimerization in nuclear localization of a homeodomain protein.

The A mating type genes of the mushroom Coprinus cinereus encode two families of dissimilar homeodomain proteins (HD1 and HD2). The proteins heterodimerize when mating cells fuse to generate a transcriptional regulator that promotes expression of genes required for early steps in sexual development. In previous work we showed that heterodimerization brings together different functional domains of the HD1 and HD2 proteins; a potential activation domain at the C terminus of the HD1 protein and an essential HD2 DNA-binding motif. Two predicted nuclear localization signals (NLS) are present in the HD1 protein but none are in the HD2 protein. We deleted each NLS separately from an HD1 protein and showed that one (NLS1) is essential for normal heterodimer function. Fusion of the NLS sequences to the C terminus of an HD2 protein compensated for their deletion from the HD1 protein partner and permitted the two modified proteins to form a functional transcriptional regulator. The nuclear targeting properties of the A protein NLS sequences were demonstrated by fusing the region that encodes them to the bacterial uidA (beta-glucuronidase) gene and showing that beta-glucuronidase expression localized to the nuclei of onion epidermal cells. These observations lead to the proposal that heterodimerization regulates entry of the active transcription factor complex to the nucleus.

The role of the cysteine-rich region of the beta2 integrin subunit in the leukocyte function-associated antigen-1 (LFA-1, alphaLbeta2, CD11a/CD18) heterodimer formation and ligand binding.

The cysteine-rich region (CRR) of the beta2 integrin subunit was replaced by that of beta1 to give the chimera beta2NV1. Beta2NV1 can combine with alphaL to form a variant leukocyte-function-associated antigen (LFA)-1 on COS cell surface, suggesting that the specificity of the beta2 interaction with alphaL does not lie in the CRR. Unlike those expressing wild-type LFA-1, COS cells expressing alphaL beta2NV1 are constitutively active in intercellular adhesion molecule (ICAM)-1 adhesion. These results suggest that activation of LFA-1 involves the release of an intramolecular constraint, which is maintained, in part, by the authentic beta2 CRR.