Adult-onset cystic fibrosis liver disease: Diagnosis and characterization of an underappreciated entity.

Cystic fibrosis (CF) liver disease (CFLD), a leading cause of death in CF, is mostly described in pediatric populations. Adult-onset CFLD lacks sufficient characterization and diagnostic tools. A cohort of CF patients without CFLD during childhood were followed for up to 38 years with serologic testing, imaging, and noninvasive fibrosis markers. Historical CFLD diagnostic criteria were compared with newly proposed CFLD criteria. Thirty-six CF patients were followed for a median of 24.5 years (interquartile range 15.6-32.9). By the last follow-up, 11 (31%) had died. With conventional criteria, 8 (22%) patients had CFLD; and by the new criteria, 17 (47%) had CFLD at a median age of 36.6 years (interquartile range 26.5-43.2). By the new criteria, those with CFLD had higher median alanine aminotransferase (42 versus 27, P = 0.005), aspartate aminotransferase (AST; 26 versus 21, P = 0.01), direct bilirubin (0.13 versus 0.1, P = 0.01), prothrombin time (14.4 versus 12.4, P = 0.002), and AST-to-platelet ratio index (0.31 versus 0.23, P = 0.003) over the last 2 years of follow-up. Subjects with a FibroScan >6.8 kPa had higher alanine aminotransferase (42 versus 28U/L, P = 0.02), AST (35 versus 25U/L, P = 0.02), AST-to-platelet ratio index (0.77 versus 0.25, P = 0.0004), and Fibrosis-4 index (2.14 versus 0.74, P = 0.0003) and lower platelet counts (205 versus 293, P = 0.02). One CFLD patient had nodular regenerative hyperplasia. Longitudinally, mean platelet counts significantly declined in the CFLD group (from 310 to 230 U/L, P = 0.0005). Deceased CFLD patients had lower platelet counts than those alive with CFLD (143 versus 258 U/L, P = 0.004) or those deceased with no CFLD (143 versus 327U/L, P = 0.006). CONCLUSION: Adult-onset CFLD may be more prevalent than previously described, which suggests a later wave of CFLD that impacts morbidity; routine liver tests, radiologic imaging, noninvasive fibrosis markers, and FibroScan can be used algorithmically to identify adult CFLD; and further evaluation in other CF cohorts should be performed for validation. (Hepatology 2017;66:591-601).

Chronic Administration of Hydroxyurea (HU) Benefits Caucasian Patients with Sickle-Beta Thalassemia.

In sickle cell disease (SCD), hydroxyurea (HU) treatment decreases the number of vaso-occlusive crisis (VOC) and acute chest syndrome (ACS) by increasing fetal hemoglobin (HbF). Data are lacking regarding the frequency of HU dose modification or whether sub-therapeutic doses (<15 mg/kg/day) are beneficial. We reviewed the medical records of 140 patients from 2010 to 2014. The laboratory parameters and SCD complications were compared between the first and last visits based on HU use. Fifty patients (36%) never took HU or suspended HU ("no HU" group). Among patients taking <15 mg/kg/day HU on their first visit, half remained at the same dose, and the other half increased to ≥15 mg/kg/day. Among patients taking ≥15 mg/kg/day, 17% decreased to <15 mg/kg/day, and 83% stayed at ≥15 mg/kg/day. The "no HU" group had fewer episodes of VOC and ACS. Both HU treatment groups had a reduction in both complications (p < 0.0001). This improvement was observed in all SCD phenotypes. The white blood cell (WBC) counts were found to be lower, and HbF increased in both HU groups (p = 0.004, 0.001). The maximal HbF response to HU in HbS/ß⁺-thalassemia was 20%, similar to those observed for HbSS (19%) and HbS/ß°-thalassemia (22%). HbS/ß⁺-thalassemia could have a similar disease severity as HbSS or HbS/ß°-thalassemia. Patients with HbS/ß°-thalassemia or HbS/ß⁺-thalassemia phenotypes responded to HU.

Gastrointestinal Manifestations of STAT3-Deficient Hyper-IgE Syndrome.

OBJECTIVE: STAT 3 deficiency (autosomal dominant hyper immunoglobulin E syndrome (AD-HIES)) is a primary immunodeficiency disorder with multi-organ involvement caused by dominant negative signal transducer and activator of transcription gene 3 (STAT3) mutations. We sought to describe the gastrointestinal (GI) manifestations of this disease. METHODS: Seventy subjects aged five to 60 years with a molecular diagnosis of AD-HIES were evaluated at the National Institutes of Health (NIH). Data collection involved a GI symptom questionnaire and retrospective chart review. RESULTS: In our cohort of 70 subjects, we found that 60% had GI symptoms (42/70). The most common manifestations were gastroesophageal reflux disease (GERD) observed in 41%, dysphagia in 31%, and abdominal pain in 24%. The most serious complications were food impaction in 13% and colonic perforation in 6%. Diffuse esophageal wall thickening in 74%, solid stool in the right colon in 50% (12/24), and hiatal hernia in 26% were the most prevalent radiologic findings. Esophagogastroduodenoscopy (EGD) demonstrated esophageal tortuosity in 35% (8/23), esophageal ulceration in 17% (4/23), esophageal strictures requiring dilation in 9% (2/23), and gastric ulceration in 17% (4/23). Esophageal eosinophilic infiltration was an unexpected histologic finding seen in 65% (11/17). CONCLUSION: The majority of AD-HIES subjects develop GI manifestations as part of their disease. Most notable are the symptoms and radiologic findings of GI dysmotility, as well as significant eosinophilic infiltration, concerning for a secondary eosinophilic esophagitis. These findings suggest that the STAT3 pathway may be implicated in a new mechanism for the pathogenesis of several GI disorders.

Gastrointestinal Features of Chronic Granulomatous Disease Found During Endoscopy.

BACKGROUND & AIMS: Chronic granulomatous disease (CGD) is an inherited disorder of the reduced nicotinamide adenine dinucleotide phosphate oxidase complex within phagocytic cells that predisposes people to bacterial and fungal infections. Approximately 40% of patients with CGD have gastrointestinal involvement. We aimed to characterize the endoscopic features of gastrointestinal CGD and define the role of endoscopy in patients. METHODS: We created a database of all patients with CGD seen at the National Institutes of Health from 1990 through 2010. We identified patients who had an endoscopy, and collected information from those with CGD-associated inflammatory bowel disease. We analyzed clinical data (demographic information and symptoms), endoscopic data (indication, preparation quality, degree of inflammation, mucosal findings, and complications), and pathologic data. RESULTS: A total of 211 endoscopies (96 esophagogastroduodenoscopies, 82 colonoscopies, and 33 flexible sigmoidoscopies) were performed at the National Institutes of Health on 78 patients with CGD. Esophageal, gastric, and duodenal inflammation were detected in 21%, 74%, and 37% of patients, respectively. Esophageal dysmotility and structural abnormalities were noted in 26%. Of the patients who had colonic CGD-inflammatory bowel disease, 74% had skip lesions and 93% had anorectal disease. Enteric fistulae were found in 18% of patients; 73% of these were perianal. Colonic strictures were observed in 24% of patients; 80% were in the anorectal area. CONCLUSIONS: Based on an analysis of clinical and endoscopic data from 78 patients, CGD-inflammatory bowel disease is a distinct entity, primarily involving the anus and rectum, with skip lesions in the remaining bowel. Bowel strictures and fistulae are present in a significant number of patients. Upper gastrointestinal tract inflammatory disease is common, although typically not as severe as colonic disease. Upper and lower endoscopies are important in characterizing the gastrointestinal features of CGD.

Clinical Features and Histology of Apolipoprotein L1-Associated Nephropathy in the FSGS Clinical Trial.

Genetic variants in apolipoprotein L1 (APOL1) confer risk for kidney disease. We sought to better define the phenotype of APOL1-associated nephropathy. The FSGS Clinical Trial involved 138 children and young adults who were randomized to cyclosporin or mycophenolate mofetil plus pulse oral dexamethasone with a primary outcome of proteinuria remission. DNA was available from 94 subjects who were genotyped for APOL1 renal risk variants, with two risk alleles comprising the risk genotype. Two APOL1 risk alleles were present in 27 subjects, of whom four subjects did not self-identify as African American, and 23 of 32 (72%) self-identified African Americans. Individuals with the APOL1 risk genotype tended to present at an older age and had significantly lower baseline eGFR, more segmental glomerulosclerosis and total glomerulosclerosis, and more tubular atrophy/interstitial fibrosis. There were differences in renal histology, particularly more collapsing variants in those with the risk genotype (P=0.02), although this association was confounded by age. APOL1 risk genotype did not affect response to either treatment regimen. Individuals with the risk genotype were more likely to progress to ESRD (P<0.01). In conclusion, APOL1 risk genotypes are common in African-American subjects with primary FSGS and may also be present in individuals who do not self-identify as African American. APOL1 risk status is associated with lower kidney function, more glomerulosclerosis and interstitial fibrosis, and greater propensity to progress to ESRD. The APOL1 risk genotype did not influence proteinuria responses to cyclosporin or mycophenolate mofetil/dexamethasone.

Durability of antibody response against hepatitis B virus in healthcare workers vaccinated as adults.

BACKGROUND: Follow-up studies of recipients of hepatitis B vaccine from endemic areas have reported loss of antibody to hepatitis B surface antigen (anti-HBs) in a high proportion of persons vaccinated at birth. In contrast, the long-term durability of antibody in persons vaccinated as adults in nonendemic areas is not well defined. We aimed to assess the durability of anti-HBs among healthcare workers (HCWs) vaccinated as adults and response to a booster among those without protective levels of antibody. METHODS: Adult HCWs aged 18-60 at the time of initial vaccination were recruited. All were tested for hepatitis B surface antigen (HBsAg), antibody to hepatitis B core antigen (anti-HBc), and anti-HBs level. HCWs with anti-HBs <12 mIU/mL were offered a booster and levels were measured 1, 7, and 21 days afterward. RESULTS: Anti-HBs levels were <12 mIU/mL in 9 of 50 (18%), 13 of 50 (26%), and 14 of 59 (24%) HCWs 10-15, 16-20, and >20 years postvaccination, respectively, (P = ns). Four HCWs were anti-HBc positive; none had HBsAg. By logistic regression, older age at vaccination was the only predictor of inadequate anti-HBs level (P = .0005). Thirty-four of 36 subjects with inadequate anti-HBs levels received a booster and 32 (94%) developed levels >12 mIU/mL within 3 weeks. CONCLUSIONS: Anti-HBs levels decrease after 10-31 years and fall below a level considered protective in approximately 25% of cases. The rapid and robust response to a booster vaccine suggests a long-lasting amnestic response. Hepatitis B vaccination provides long-term protection against hepatitis B and booster vaccination does not appear to be necessary in HCWs. Clinical Trials Registration. NCT01182311.

Characteristics of congenital hepatic fibrosis in a large cohort of patients with autosomal recessive polycystic kidney disease.

BACKGROUND & AIMS: Autosomal recessive polycystic kidney disease (ARPKD), the most common ciliopathy of childhood, is characterized by congenital hepatic fibrosis and progressive cystic degeneration of kidneys. We aimed to describe congenital hepatic fibrosis in patients with ARPKD, confirmed by detection of mutations in PKHD1. METHODS: Patients with ARPKD and congenital hepatic fibrosis were evaluated at the National Institutes of Health from 2003 to 2009. We analyzed clinical, molecular, and imaging data from 73 patients (age, 1-56 years; average, 12.7 ± 13.1 years) with kidney and liver involvement (based on clinical, imaging, or biopsy analyses) and mutations in PKHD1. RESULTS: Initial symptoms were liver related in 26% of patients, and others presented with kidney disease. One patient underwent liver and kidney transplantation, and 10 others received kidney transplants. Four presented with cholangitis and one with variceal bleeding. Sixty-nine percent of patients had enlarged left lobes on magnetic resonance imaging, 92% had increased liver echogenicity on ultrasonography, and 65% had splenomegaly. Splenomegaly started early in life; 60% of children younger than 5 years had enlarged spleens. Spleen volume had an inverse correlation with platelet count and prothrombin time but not with serum albumin level. Platelet count was the best predictor of spleen volume (area under the curve of 0.88905), and spleen length corrected for patient's height correlated inversely with platelet count (R(2) = 0.42, P < .0001). Spleen volume did not correlate with renal function or type of PKHD1 mutation. Twenty-two of 31 patients who underwent endoscopy were found to have varices. Five had variceal bleeding, and 2 had portosystemic shunts. Forty-percent had Caroli syndrome, and 30% had an isolated dilated common bile duct. CONCLUSIONS: Platelet count is the best predictor of the severity of portal hypertension, which has early onset but is underdiagnosed in patients with ARPKD. Seventy percent of patients with ARPKD have biliary abnormalities. Kidney and liver disease are independent, and variability in severity is not explainable by type of PKHD1 mutation; ClinicalTrials.gov number, NCT00068224.

AASLD clinical practice guidelines: a critical review of scientific evidence and evolving recommendations.

UNLABELLED: The American Association for the Study of Liver Diseases (AASLD) practice guidelines provide recommendations in diagnosing and managing patients with liver disease from available scientific evidence in combination with expert consensus opinions. The aim was to systematically review the evolution of recommendations from AASLD guidelines and identify gaps limiting the evidence-based foundations of these guidelines. Initial and current AASLD guidelines published from January 1998 to August 2012 were reviewed. The AGREE II instrument was used to evaluate rigor and transparency of guideline development. The number of recommendations, distribution of grades (strength or certainty), classes (benefit versus risk), and types of recommendations were evaluated. Whenever possible, multiple versions were evaluated for evolving scientific evidence. A total of 991 recommendations from 28 guidelines on 17 topics were evaluated. From initial to current guidelines, the total number of recommendations increased by 36% (512 to 699). The largest increases were from chronic hepatitis B virus (HBV) (+71), liver transplantation (+53), and autoimmune hepatitis (AIH) (+27). Most current recommendations are grade II (44%) and less than 20% are grade I. The AGREE II evaluation showed global improvement in guideline quality. Both HBV and chronic hepatitis C guidelines had greatest increases in grade I recommendations (+383% and +67%, respectively). The greatest increases in treatment recommendations were from HBV (grade I, +1,150%), liver transplantation (grade II, +112%), and AIH (grade III, +105%). CONCLUSION: Despite significant increases in the numbers of recommendations within AASLD practice guidelines over time, only a minority are supported by grade I evidence, highlighting the need for developing well-designed investigations to provide evidence for areas of uncertainty and improving the quality of future guidelines in hepatobiliary diseases.

Association of IL28B genotype with fibrosis progression and clinical outcomes in patients with chronic hepatitis C: a longitudinal analysis.

UNLABELLED: Interleukin (IL)28B polymorphisms are associated with spontaneous clearance of hepatitis C virus (HCV) infection and response to therapy. Whether IL28B genotype affects fibrosis progression or clinical outcome is unclear. Our aim was to study the relationship between IL28B genotype and both histological and clinical outcomes in patients with chronic hepatitis C (CHC). Hepatic fibrosis was scored using the Ishak (0-6) scale; progression was defined as a 2-point increase in Ishak score between biopsies. Multiple logistic and Cox regressions were used to identify variables associated with fibrosis progression. In all, 1,483 patients were included in a baseline cross-sectional analysis, from which 276 were eligible for a paired biopsy analysis (median time between biopsies 4 years), and 400 for a clinical outcome analysis. At baseline biopsy, patients with IL28B CC genotype had significantly higher portal inflammation (2.4 versus 2.2) and alanine aminotransferase (ALT) levels (133 versus 105 U/L; P < 0.05 for all). In the paired biopsy analysis, there was no difference in the frequency of fibrosis progression between patients with IL28B CC and non-CC genotypes (17% versus 23%). In logistic regression, only higher baseline alkaline phosphatase, lower platelets, and greater hepatic steatosis were associated with fibrosis progression. Patients with IL28B CC were twice as likely to develop adverse clinical outcomes compared to non-CC (32% versus 16%; P = 0.007). CONCLUSION: IL28B CC genotype was associated with greater hepatic necroinflammation, higher ALT, and worse clinical outcomes in CHC patients. This suggests that IL28B CC is associated with a state of enhanced immunity that, on the one hand, can promote viral clearance, but alternately can increase necroinflammation and hepatic decompensation without enhancing fibrosis progression.

Infusion of hemolyzed red blood cells within peripheral blood stem cell grafts in patients with and without sickle cell disease.

Peripheral blood stem cell (PBSC) infusions are associated with complications such as elevated blood pressure and decreased creatinine clearance. Patients with sickle cell disease experience similar manifestations, and some have postulated release of plasma-free hemoglobin with subsequent nitric oxide consumption as causative. We sought to evaluate whether the infusion of PBSC grafts containing lysed red blood cells (RBCs) leads to the toxicity observed in transplant subjects. We report a prospective cohort study of 60 subjects divided into 4 groups based on whether their infusions contained dimethyl sulfoxide (DMSO) and lysed RBCs, no DMSO and fresh RBCs, DMSO and no RBCs, or saline. Our primary end point, change in maximum blood pressure compared with baseline, was not significantly different among groups. Tricuspid regurgitant velocity and creatinine levels also did not differ significantly among groups. Our data do not support free hemoglobin as a significant contributor to toxicity associated with PBSC infusions. This study was registered at clinicaltrials.gov (NCT00631787).

Nonmyeloablative HLA-matched sibling allogeneic hematopoietic stem cell transplantation for severe sickle cell phenotype.

IMPORTANCE: Myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) is curative for children with severe sickle cell disease, but toxicity may be prohibitive for adults. Nonmyeloablative transplantation has been attempted with degrees of preparative regimen intensity, but graft rejection and graft-vs-host disease remain significant. OBJECTIVE: To determine the efficacy, safety, and outcome on end-organ function with this low-intensity regimen for sickle cell phenotype with or without thalassemia. DESIGN, SETTING, AND PARTICIPANTS: From July 16, 2004, to October 25, 2013, 30 patients aged 16-65 years with severe disease enrolled in this nonmyeloablative transplant study, consisting of alemtuzumab (1 mg/kg in divided doses), total-body irradiation (300 cGy), sirolimus, and infusion of unmanipulated filgrastim mobilized peripheral blood stem cells (5.5-31.7 × 10(6) cells/kg) from human leukocyte antigen-matched siblings. MAIN OUTCOMES AND MEASURES: The primary end point was treatment success at 1 year after the transplant, defined as a full donor-type hemoglobin for patients with sickle cell disease and transfusion independence for patients with thalassemia. The secondary end points were the level of donor leukocyte chimerism; incidence of acute and chronic graft-vs-host disease; and sickle cell-thalassemia disease-free survival, immunologic recovery, and changes in organ function, assessed by annual brain imaging, pulmonary function, echocardiographic image, and laboratory testing. RESULTS: Twenty-nine patients survived a median 3.4 years (range, 1-8.6), with no nonrelapse mortality. One patient died from intracranial bleeding after relapse. As of October 25, 2013, 26 patients (87%) had long-term stable donor engraftment without acute or chronic graft-vs-host disease. The mean donor T-cell level was 48% (95% CI, 34%-62%); the myeloid chimerism levels, 86% (95% CI, 70%-100%). Fifteen engrafted patients discontinued immunosuppression medication with continued stable donor chimerism and no graft-vs-host disease. The normalized hemoglobin and resolution of hemolysis among engrafted patients were accompanied by stabilization in brain imaging, a reduction of echocardiographic estimates of pulmonary pressure, and allowed for phlebotomy to reduce hepatic iron. The mean annual hospitalization rate was 3.23 (95% CI, 1.83-4.63) the year before, 0.63 (95% CI, 0.26-1.01) the first year after, 0.19 (95% CI, 0-0.45) the second year after, and 0.11 (95% CI, 0.04-0.19) the third year after transplant. For patients taking long-term narcotics, the mean use per week was 639 mg (95% CI, 220-1058) of intravenous morphine-equivalent dose the week of their transplants and 140 mg (95% CI, 56-225) 6 months after transplant. There were 38 serious adverse events: pain and related management, infections, abdominal events, and sirolimus related toxic effects. CONCLUSIONS AND RELEVANCE: Among 30 patients with sickle cell phenotype with or without thalassemia who underwent nonmyeloablative allogeneic HSCT, the rate of stable mixed-donor chimerism was high and allowed for complete replacement with circulating donor red blood cells among engrafted participants. Further accrual and follow-up are required to assess longer-term clinical outcomes, adverse events, and transplant tolerance. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00061568.

Discordance among transient elastography, aspartate aminotransferase to platelet ratio index, and histologic assessments of liver fibrosis in patients with chronic hepatitis C.

BACKGROUND & AIMS: Liver biopsy is the standard for assessing hepatic fibrosis. Ultrasound transient elastography (TE) and the aspartate aminotransferase to platelet ratio index (APRI) are validated, noninvasive tests for identifying patients with cirrhosis. We evaluated discordance among TE, APRI, and histology diagnoses of cirrhosis. METHODS: We analyzed findings from 109 patients with chronic hepatitis C who underwent TE within 6 months of liver biopsy at the US National Institutes of Health from 2006 to 2011. Fibrosis was scored using the Ishak scale (0-6). APRI scores were calculated using data collected on the day of the biopsy. Area under receiver operator characteristic curves for TE and APRI were calculated to distinguish patients with cirrhosis (Ishak scores, 5-6) from those without cirrhosis (Ishak scores, 0-4). The best cut-off value and corresponding positive predictive value (PPV) and negative predictive value (NPV) were selected. RESULTS: Based on biopsy analysis, 18% of the patients had no fibrosis, 52% had mild fibrosis, 17% had bridging fibrosis, and 13% had cirrhosis. A TE cut-off value of 13.1 kPa identified patients with cirrhosis with the highest level of accuracy (100% sensitivity, 89% specificity, 58% PPV, 100% NPV), as did an APRI cut-off value of 1.0 (79% sensitivity, 78% specificity, 34% PPV, 96% NPV). Results from TE and APRI were discordant for 28% of cases. TE identified all cases of cirrhosis and an additional 10 patients who were not found to have cirrhosis based on histology analysis; 7 of these patients had clinical or radiologic evidence of cirrhosis, indicating that the biopsy sample was not staged correctly. CONCLUSIONS: TE increases the accuracies of biopsy and APRI analyses in identifying patients with cirrhosis. TE also might be used to screen patients for cirrhosis and identify those who should be followed up for development of hepatocellular carcinoma and varices.

Liver stiffness increases acutely during sickle cell vaso-occlusive crisis.

Acute vaso-occlusive crisis (VOC) in sickle cell disease (SCD) is an important cause of end-organ damage. It is estimated that 10-39% of VOC occurs with hepatic involvement. Current assessments of hepatic involvement during VOC are unsatisfactory. We investigated transient elastography (TE) as a marker of hepatic involvement, its relationship with histology, and biochemical markers during VOC. SCD patients were evaluated with biochemical markers and TE at steady-state and during VOC. Change in TE and biochemical markers were correlated with length of hospital stay. When available, liver biopsy and tricuspid regurgitation velocity (TRV) at steady-state were correlated with TE. Twenty-three patients were evaluated (mean age = 34.3 years, standard deviation = 7.96). In 15 patients with liver biopsies, TE correlated with fibrosis (P = 0.01) and TRV (P = 0.0063), but not hepatic iron. Hemolysis biomarkers changed during VOC (P < 0.022), but not alanine aminotransferase (ALT). Paired comparison of TE at steady-state and during VOC showed an increased from 6.2 to 12.3 kPa (P = 0.0029). Increasing TE during VOC associated with increasing ALT and alkaline phosphatase (P = 0.0088 and 0.0099, respectively). At steady-state, increasing inflammation on biopsy (P = 0.0037) and TRV (P = 0.0075) correlated with increasing TE during VOC. Increased hospital stay was associated with higher ALT (P = 0.041), lower albumin (P = 0.046), hemoglobin/hematocrit (P < 0.0021) but not TE. TE may identify patients with hepatic involvement during VOC independent of biochemical measures. Increase in TE may reflect both hepatic passive congestion and hepatic involvement during VOC. TE may serve as a physiological biomarker for hepatic features of VOC.

Olfactomedin 4 suppresses prostate cancer cell growth and metastasis via negative interaction with cathepsin D and SDF-1.

The human olfactomedin 4 gene (OLFM4) encodes an olfactomedin-related glycoprotein. OLFM4 is normally expressed in a limited number of tissues, including the prostate, but its biological functions in prostate are largely unknown. In this study, we found that OLFM4 messenger RNA was reduced or undetectable in prostate cancer tissues and prostate cancer cell lines. To study the effects of OLFM4 on prostate cancer progression, we transfected PC-3 prostate cancer cells with OLFM4 to establish OLFM4-expressing PC-3 cell clones. The OLFM4-expressing PC-3 cell clones were found to have decreased proliferation and invasiveness compared with vector-transfected control PC-3 cells in vitro. In addition, nude mice injected with OLFM4-expressing PC-3 cells demonstrated reduced tumor growth and bone invasion and metastasis compared with mice injected with vector-transfected control cells. Mechanistic studies revealed that OLFM4 may exhibit its anticancer effects through regulating cell autophagy by targeting cathepsin D, as OLFM4 reduced cathepsin D protein levels and enzymatic activity and attenuated cathepsin D-induced cancer cell proliferation. In addition, overexpression of OLFM4 abrogated stromal cell derived factor-1 (SDF-1)-induced PC-3 cell invasiveness in a Matrigel invasion assay, partially through blocking SDF-1-mediated AKT phosphorylation. Coimmunoprecipitation and immunofluorescence staining studies in OLFM4-expressing PC-3 cells demonstrated a direct interaction between OLFM4 and cathepsin D or SDF-1. Taken together, these results suggest that OLFM4 negatively interacts with cathepsin D and SDF-1 and inhibits prostate cancer growth and bone metastasis.

Revisiting the adequacy of infection criteria recommended in VICH GL 7 for anthelmintic effectiveness studies: Retrospective simulations.

Studies conducted to support registration or approval of veterinary anthelmintics generally follow study design recommendations provided by the VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products), "Efficacy of Anthelmintics: General Requirements" (VICH GL7). For dose confirmation studies, VICH GL7 provides recommendations for determining that the control animals had an adequate infection "to permit the appropriate standards of efficacy to be met with acceptable statistical and biological certitude/confidence." In the simulation studies described in this report, we investigated the performance of one method, the statistical criterion given in Section 4.5 of VICH GL7, for evaluating the adequacy of infection in anthelmintic studies, in combination with the conventional criterion of a minimum of six adequately infected animals. We conducted numerical simulations, based on parasite data from previously conducted dose confirmation studies in dogs and cattle, to investigate how the statistical criterion impacts adequacy of infection determinations when used with the conventional criterion at various sample sizes. Simulation studies in common nematode species in both dogs and cattle indicated that under certain circumstances the statistical criterion can guard against overinterpreting the evaluation of adequacy of infection as sample size is increased. However, the statistical criterion may be overly restrictive for samples with adequate infection but containing multiple zero parasite counts and adding it to the conventional criterion does not provide any additional benefit when the sample contains no zero counts. It is important for investigators designing efficacy studies to understand the potential impact this criterion may have when establishing adequacy of infection criteria in study protocols.

Prospective Study of Withdrawal of Antiviral Therapy in Patients with Chronic Hepatitis B after Prolonged Virological Response.

Nucleoside analogue (NA) therapy for chronic hepatitis B (CHB) is associated with improved clinical outcomes, but usually requires long-term use. Whether treatment can be safely withdrawn and the factors associated with post-withdrawal outcome are not well defined. To assess long-term outcomes after stopping antiviral therapy, patients with hepatitis B e antigen (HBeAg)-negative CHB who had received antiviral therapy for 4 or more years with hepatitis B virus (HBV) DNA (≤100 IU/mL) were prospectively withdrawn from antiviral therapy and monitored monthly for the initial 6 months and every 3 months thereafter. Those with clinical relapse were retreated according to severity of relapse. Fifteen patients were withdrawn from lamivudine (4), adefovir (5), or a combination of the two (6) after a mean treatment duration of 8.4 years. The mean age was 45 years, 13 were male, and 8 were initially HBeAg-positive before treatment. After a mean follow-up of 6.6 years, outcomes differed by pretreatment HBeAg status. All patients who were HBeAg+ before treatment experienced virological relapse (8 of 8); 6 of 8 experienced clinical relapse; 4 of 8 had ALT flares; 5 of 8 required re-initiation of treatment, one of whom cleared hepatitis B surface antigen (HBsAg); and 3 of 8 remained off treatment, one of whom cleared HBsAg. In contrast, 4 of 7 patients who were HBeAg-negative before treatment experienced virological relapse, 3 of 7 experienced clinical relapse, and 1 of 7 had an alanine aminotransferase (ALT) flare. None restarted treatment, and 4 of 7 cleared HBsAg. Low pre-withdrawal HBsAg level was predictive of HBsAg loss. Conclusion: NA therapy can be safely withdrawn with long-term remission and high rates of HBsAg loss in most HBeAg-negative patients without cirrhosis. Patients who were initially HBeAg+ should not be withdrawn from treatment, because clinical relapse was frequent and often severe.

The O-glycosylated linker from the Trichoderma reesei Family 7 cellulase is a flexible, disordered protein.

Fungi and bacteria secrete glycoprotein cocktails to deconstruct cellulose. Cellulose-degrading enzymes (cellulases) are often modular, with catalytic domains for cellulose hydrolysis and carbohydrate-binding modules connected by linkers rich in serine and threonine with O-glycosylation. Few studies have probed the role that the linker and O-glycans play in catalysis. Since different expression and growth conditions produce different glycosylation patterns that affect enzyme activity, the structure-function relationships that glycosylation imparts to linkers are relevant for understanding cellulase mechanisms. Here, the linker of the Trichoderma reesei Family 7 cellobiohydrolase (Cel7A) is examined by simulation. Our results suggest that the Cel7A linker is an intrinsically disordered protein with and without glycosylation. Contrary to the predominant view, the O-glycosylation does not change the stiffness of the linker, as measured by the relative fluctuations in the end-to-end distance; rather, it provides a 16 Å extension, thus expanding the operating range of Cel7A. We explain observations from previous biochemical experiments in the light of results obtained here, and compare the Cel7A linker with linkers from other cellulases with sequence-based tools to predict disorder. This preliminary screen indicates that linkers from Family 7 enzymes from other genera and other cellulases within T. reesei may not be as disordered, warranting further study.

Cardiopulmonary complications leading to premature deaths in adult patients with sickle cell disease.

Sickle cell disease (SCD) is associated with early mortality. We sought to determine the incidence, cause, and risk factors for death in an adult population of patients with SCD. All patients aged >/=18 years seen at the Adult Sickle Cell Center at Duke University Medical Center between January 2000 and April 2005 were enrolled. Forty-three patients (21 males and 22 females) died during the study period. The median age of survival was 39 years for females (95% CI: 34-56), 40 years for males (95% CI: 34-48), and 40 years overall (95% CI: 35-48). Cardiac causes of death accounted for 25.6% (11/43 patients); pulmonary, 14.0% (six patients); other SCD related, 32.6% (14 patients); unknown, 14.0% (six patients); and others, 14.0% (six patients). Pulseless electrical activity arrest, pulmonary emboli, multiorgan failure, and stroke were the most frequent causes of death. Among the deceased patients, the most common premorbid conditions were cardiopulmonary: acute chest syndrome/pneumonia (58.1%), Pulmonary hypertension (pHTN; 41.9%), systemic HTN (25.6%), congestive heart failure (25.6%), myocardial infarction (20.9%), and arrhythmias (14.0%). Tricuspid regurgitant jet velocity was significantly higher (3.1 m/sec vs. 2.6 m/sec, P < 0.001) and hemoglobin significantly lower (8.3 g/dL vs. 9.2 g/dL, P < 0.05) in deceased patients when compared with patients who lived, respectively. With improved preventive and therapeutic advances, including hydroxyurea therapy, acute complications such as infection are no longer the leading cause of death; instead, causes of death and premorbid conditions are shifting to chronic cardiopulmonary complications. Further, arrhythmia leading to premature death is under-recognized in SCD and warrants further investigation.

Molecular dynamics simulation of ss-DNA translocation between copper nanoelectrodes incorporating electrode charge dynamics.

Molecular dynamics simulations have been performed to study the translocation of single-stranded (ss)-DNA through the nanoscale gap between the nanoscale electrodes of a proposed genomic sequencing device. Using a fixed gap width between the electrodes and a small sample segment of ss-DNA as initial starting points in this project, the effect of applied electric fields on translocation velocity was studied. To describe the electrostatic interactions of the water, ions, and ss-DNA with the nanoscale electrodes, we applied the electrode charge dynamics (ECD) method. Through the density profile and comparison of translocation velocities to extrapolated experimental data, we found the ECD potential to be a better descriptor of the metal/nonmetal electrostatic interactions compared to the commonly used universal force field (UFF). Translocation velocities obtained using the ECD potential were consistent with simulated bulk data.

Electrophoresis of ssDNA through nanoelectrode gaps from molecular dynamics: impact of gap width and chain length.

Molecular dynamics simulations were performed to study the translocation of single-stranded (ss) DNA through the nanoscale gap between the nanoscale electrodes of a proposed genomic sequencing device. An applied electric field forces the ssDNA to move in the direction of the nanoscale gap in platinum electrodes. A series of simulations utilizing eight different nanoscale gap distances as well as seven different nucleotide chain lengths were performed to determine the impact of these variables on the overall design of the sequencing device and the translocation behavior of ssDNA. The results clearly indicate a threshold value of the gap width below which the ssDNA will readily enter and traverse the nanoscale gap. Translocation velocities obtained for various chain lengths were consistent with simulated bulk data; however, successful translocation was inconsistent, possibly related to the sample's affinity for the metal electrodes. An attempt at overcoming this barrier was made through the implementation of shaped electrodes as well as pre-threading of the ssDNA sample.