Relationships between Isomeric Metabolism and Regioselective Toxicity of Hydroxychrysenes in Embryos of Japanese Medaka (Oryzias latipes).

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are ubiquitous contaminants that can be formed through oxidation of parent PAHs. Our previous studies found 2-hydroxychrysene (2-OHCHR) to be significantly more toxic to Japanese medaka embryos than 6-hydroxychrysene (6-OHCHR), an example of regioselective toxicity. We have also previously identified a sensitive developmental window to 2-OHCHR toxicity that closely coincided with liver development, leading us to hypothesize that differences in metabolism may play a role in the regioselective toxicity. To test this hypothesis, Japanese medaka embryos were treated with each isomer for 24 h during liver development (52-76 hpf). Although 6-OHCHR was absorbed 97.2 ± 0.18% faster than 2-OHCHR, it was eliminated 57.7 ± 0.36% faster as a glucuronide conjugate. Pretreatment with cytochrome P450 inhibitor, ketoconazole, reduced anemia by 96.8 ± 3.19% and mortality by 95.2 ± 4.76% in 2-OHCHR treatments. Formation of chrysene-1,2-diol (1,2-CAT) was also reduced by 64.4 ± 2.14% by ketoconazole pretreatment. While pretreatment with UDP-glucuronosyltransferase inhibitor, nilotinib, reduced glucuronidation of 2-OHCHR by 52.4 ± 2.55% and of 6-OHCHR by 63.7 ± 3.19%, it did not alter toxicity for either compound. These results indicate that CYP-mediated activation, potentially to 1,2-CAT, may explain the isomeric differences in developmental toxicity of 2-OHCHR.

Role of anti-HBs in functional cure of HBeAg+ chronic hepatitis B patients infected with HBV genotype A.

BACKGROUND & AIMS: HBsAg-specific antibody responses are difficult to detect during chronic hepatitis B infection (CHB) and are often overlooked. The aim of this study was to examine whether anti-HBs may be involved in functional cure (FC) by profiling anti-HBs responses in patients with CHB using a panel of specific assays. METHODS: Longitudinal serum samples were obtained from 25 patients with CHB who were infected with HBV genotype A and were undergoing nucleos(t)ide analogue (NA) treatment: 14 achieved FC while 11 remained infected (non-FC). Anti-HBs immune complexes (HBsAg-IC), FcγRIIIa dimer binding, epitope specificity and neutralisation efficacy were measured. RESULTS: HBsAg-IC peaks were detected prior to HBsAg loss in 10/14 FC patients. These HBsAg-IC peaks overlapped with either an alanine aminotransferase (ALT) flare (8/10 patients), or a rise in ALT (2/10 patients). HBsAg-IC peaks were detected in 7/11 non-FC patients, but were not associated with an ALT flare. FCγRIIIa binding was detected in 9/14 FC patients, independent from detection of overlapping HBsAg-IC/ALT peaks. FC patients had stable HBsAg epitope occupancy across the study, whereas non-FC patients had a reduction in HBsAg epitope occupancy within the first 12-24 weeks of NA treatment. Convalescent sera from FC patients recognised more HBsAg epitopes and neutralised HBV infection more potently than anti-HBs derived from vaccinees. Neutralisation potency appeared to increase post-HBsAg loss in 4/5 FC patients examined. CONCLUSIONS: Using these assays, we confirm that anti-HBs responses are present and fluctuate over time in this cohort of patients with HBeAg+ CHB, who were infected with HBV genotype A and treated with NAs. Key anti-HBs profiles associated with either FC or failure to achieve FC were also identified, suggesting a role for anti-HBs responses in FC. LAY SUMMARY: Using a panel of assays to characterise hepatitis B surface antibody (anti-HBs) responses in a group of patients with chronic hepatitis B, we identified anti-HBs profiles associated with either functional cure, or failure to achieve functional cure. Functional cure was associated with immune complex peaks which overlapped with alanine aminotransferase flares. Conversely, in those who did not achieve functional cure, immune complex peaks were present, but were not associated with alanine aminotransferase flares, and a decline in anti-HBs diversity was observed early during treatment.

Predicting HBsAg clearance in genotype A chronic hepatitis B using HBsAg epitope profiling: A biomarker for functional cure.

BACKGROUND AND AIM: Functional cure is the major goal of chronic hepatitis B (CHB) therapy though few biomarkers predict this outcome. HBsAg epitope occupancy can be influenced by therapeutic and immune pressure. The aim of this study was to map the HBsAg epitope profiles during long-term nucleos(t)ide analogue therapy in patients with genotype A CHB, in the context of HBsAg loss (SL)/seroconversion. METHODS: We evaluated 25 genotype A CHB patients in the GS-US-174-0103 trial of HBeAg-positive CHB patients treated with tenofovir or adefovir for 4 years, 14 who achieved SL whilst 11 had no change. We epitope mapped the major domains of HBsAg to identify those patients with HBsAg clearance profile (CP) (loss of binding at both loops 1 and 2 epitopes of the 'a' determinant) vs non-clearance profile (no change in epitope recognition, or loss of epitope binding at one loop only), correlating this to on-treatment HBsAg responses. Complexed anti-HBs was also measured. RESULTS: Analysis of the HBsAg epitope profiles of the 25 patients at baseline identified no predictive correlation with SL. In contrast, analysis at week 48 and end of study (week 192) or prior to SL identified significant predictive associations between development of HBsAg CPs and outcome of functional cure. The detection of a CP also correlated with the development of an alanine aminotransferase flare and detection of anti-HBs complexed with HBsAg. CONCLUSION: The detection of HBsAg CPs by epitope mapping represents a novel viral biomarker, reflecting an emerging anti-HBs selection pressure prior to functional cure.

Deep sequencing shows that HBV basal core promoter and precore variants reduce the likelihood of HBsAg loss following tenofovir disoproxil fumarate therapy in HBeAg-positive chronic hepatitis B.

OBJECTIVE: Hepatitis B e antigen (HBeAg) seroconversion and hepatitis B surface antigen (HBsAg) loss are important clinical outcomes for patients with chronic hepatitis B (CHB) treated with antiviral therapy. To date, there have been few studies that have evaluated viral sequence markers predicting serological response to nucleos(t)ide analogue (NA) treatment. DESIGN: We used next-generation sequencing (NGS) and quantitative HBV serology (HBeAg and HBsAg) to identify viral sequence markers associated with serological response to long-term tenofovir disoproxil fumarate therapy among HBeAg-positive patients. In the GS-US-174-0103 study, approximately half the patients seroconverted to anti-HBe by week 192 and 11% of patients exhibited HBsAg loss, the closest outcome to functional cure. The frequency of HBV variants that have previously been associated with HBV clinical outcomes was evaluated. HBV viral diversity in baseline sequences generated by NGS was calculated using Shannon entropy. RESULTS: NGS analysis of HBV sequences from 157 patients infected with genotypes A to D showed the frequency of variants in the basal core promoter (BCP) and precore (PC) regions varied by genotype and that these mutations were associated with the absence of HBsAg loss. This was the case even when mutations were present at frequencies below the threshold of detection by population sequencing. Increased viral diversity across the HBV genome as determined by NGS was also associated with reduced likelihood of HBsAg loss. CONCLUSION: Patients with detectable BCP and/or PC variants and higher viral diversity have a lower probability of HBsAg loss during long-term NA therapy. Strategies to achieve functional cure of HBV infection through combination therapy should consider using NGS to stratify patients according to BCP/PC sequence. Consideration should also be given to earlier initiation of therapy prior to the emergence of BCP/PC variants. TRIAL REGISTRATION NUMBER: NCT00116805; Post result.

Nuclear expression of Lyn, a Src family kinase member, is associated with poor prognosis in renal cancer patients.

BACKGROUND: 8000 cases of renal cancer are diagnosed each year in the UK, with a five-year survival rate of 50%. Treatment options are limited; a potential therapeutic target is the Src family kinases (SFKs). SFKs have roles in multiple oncogenic processes and promote metastases in solid tumours. The aim of this study was to investigate SFKs as potential therapeutic targets for clear cell renal cell carcinoma (ccRCC). METHODS: SFKs expression was assessed in a tissue microarray consisting of 192 ccRCC patients with full clinical follow-up. SFK inhibitors, dasatinib and saracatinib, were assessed in early ccRCC cell lines, 786-O and 769-P and a metastatic ccRCC cell line, ACHN (± Src) for effects on protein expression, apoptosis, proliferation and wound healing. RESULTS: High nuclear expression of Lyn and the downstream marker of activation, paxillin, were associated with decreased patient survival. Conversely, high cytoplasmic expression of other SFK members and downstream marker of activation, focal adhesion kinase (FAK) were associated with increased patient survival. Treatment of non-metastatic 786-O and 769-P cells with dasatinib, dose dependently reduced SFK activation, shown via SFK (Y(419)) and FAK (Y(861)) phosphorylation, with no effect in metastatic ACHN cells. Dasatinib also increased apoptosis, while decreasing proliferation and migration in 786-O and 769-P cell lines, both in the presence and absence of Src protein. CONCLUSIONS: Our data suggests that nuclear Lyn is a potential therapeutic target for ccRCC and dasatinib affects cellular functions associated with cancer progression via a Src kinase independent mechanism.

Lung transplant waitlist mortality: height as a predictor of poor outcomes.

The LAS was designed to minimize pretransplant mortality while maximizing post-transplant outcome. Recipients <12 are not allocated lungs based on LAS. Waitlist mortality has decreased for those >12, but not <12, suggesting this population may be disadvantaged. To identify predictors of waitlist mortality, a retrospective analysis of the UNOS database was performed since implementation of the LAS. There were 16,973 patients listed for lung transplant in the United States; 12,070 (71.1%) were transplanted, and 2498 (14.7%) patients died or were removed from the wait list. Significantly more pediatric patients died or were removed compared with adults (22.0% vs. 14.4%, p < 0.01). In multivariate analysis, in addition to higher LAS at time of listing (adj. HR1.058, 1.055-1.060), shorter height (1.008, 1.006-1.010), male gender (1.210, 1.110-1.319), and requiring ECMO (1.613, 1.202-2.163) were associated with pretransplant mortality. Post-transplant survival was not affected by height. The current age cutoff may impose limitations within the current lung allocation system in the United States. Height is an independent predictor of waitlist mortality and may be a valuable factor for the development of a comprehensive lung allocation system.

Association of left ventricular systolic function and vasopressor support with survival following pediatric out-of-hospital cardiac arrest.

OBJECTIVES: To characterize the association of hospital discharge survival with left ventricular systolic function evaluated by transthoracic echocardiography and vasoactive infusion support following return of spontaneous circulation after pediatric out-of-hospital cardiac arrest. DESIGN: Retrospective case series. SETTING: Single-center tertiary care pediatric cardiac arrest and critical care referral center. PATIENTS: Consecutive out-of-hospital cardiac arrest patients less than 18 years surviving to PICU admission who had a transthoracic echocardiography obtained by the clinical team within 24 hours of admission from January 2006 to May 2012. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Fifty-eight patients had a post-return of spontaneous circulation transthoracic echocardiography performed within 24 hours of admission. The median time from return of spontaneous circulation to echo was 6.5 hours (interquartile range, 4.7, 15.0 hr). Left ventricular systolic function was decreased in 24 of 58 patients (41%). The mortality rate was 67% (39 of 58). Thirty-six patients (62%) received vasoactive infusions at the time of transthoracic echocardiography, and increased vasopressor inotropic score was associated with increased mortality on univariate analysis (p < 0.001). After controlling for defibrillation, vasopressor inotropic score, and interaction between vasopressor inotropic score and left ventricular systolic function, decreased left ventricular systolic function was associated with increased mortality (odds ratio, 13.7; 95% CI, 1.54-122). CONCLUSIONS: In patients receiving transthoracic echocardiography within the first 24 hours following return of spontaneous circulation after pediatric out-of-hospital cardiac arrest, decreased left ventricular systolic function and vasopressor use were common. Decreased left ventricular systolic function was associated with increased mortality.

Local immune response predicts survival in patients with thick (t4) melanomas.

BACKGROUND: Tumor infiltrating lymphocytes (TIL) and histological regression in primary melanoma are generally considered indicators of the local immune response but their roles as prognostic factors have been variably reported. We examined the prognostic role of these variables in patients with high risk (T4) primary melanomas in a large series of patients with long-term follow-up. METHODS: From a prospectively maintained cohort of patients diagnosed between 1971 and 2004, 161 patients were retrospectively identified with primary thick melanomas (>4 mm), no clinical evidence of regional nodal disease (RND) at diagnosis and complete histopathologic data. Univariate and multivariate Cox regression models were performed to identify clinical and histopathologic predictors of disease-specific survival (DSS) and to identify subgroups with differential survival. RESULTS: Factors significantly associated with decreased DSS by univariate analysis included male gender, age ≥ 60 years, axial anatomic location, presence of ulceration, RND, absence of TIL, and presence of regression. In the final multivariate model, TIL and regression, as interacting variables, and RND status remained significantly associated with DSS. In the presence of TIL, concomitant regression was associated with significantly worse survival (p ≤ 0.0001). In the absence of TIL, there was no effect of regression on survival (p = 0.324). CONCLUSIONS: Primary TIL and regression status and RND status are independently associated with melanoma-specific survival in patients with T4 melanomas; presence of TIL in the primary melanoma with concomitant radial growth phase regression is associated with a poor prognosis and may reflect an ineffective local regional immune response.

IL28B genotype is not useful for predicting treatment outcome in Asian chronic hepatitis B patients treated with pegylated interferon-α.

BACKGROUND AND AIM: IL28B genotype predicts response to pegylated interferon (peg-IFN)-based therapy in chronic hepatitis C. However, the utility of IL28B genotyping in chronic hepatitis B (CHB) cohorts treated with peg-IFN is unclear. It was investigated whether IL28B genotype is associated with peg-IFN treatment outcomes in a predominantly Asian CHB cohort. METHODS: This was a retrospective analysis of CHB patients treated with 48 weeks of peg-IFN monotherapy. IL28B genotype (rs12979860) was determined (TaqMan allelic discrimination kit). Baseline hepatitis B virus (HBV)-DNA, alanine aminotransferase, and liver histology were available. The primary end-points were HBV e antigen (HBeAg) seroconversion with HBV-DNA < 2000 IU/mL 24 weeks post-therapy (HBeAg-positive patients) and HBV-DNA < 2000 IU/mL 24 weeks after peg-IFN (HBeAg-negative patients). The association between IL28B genotype and peg-IFN outcomes was analyzed. RESULTS: IL28B genotype was determined for 96 patients. Eighty-eight percent were Asian, 62% were HBeAg positive, and 13% were METAVIR stage F3-4. Median follow-up time was 39.3 months. The majority of patients carried the CC IL28B genotype (84%). IL28B genotype did not differ according to HBeAg status. The primary end-points were achieved in 27% of HBeAg-positive and 61% of HBeAg-negative patients. There was no association between IL28B genotype and the primary end-point in either group. Furthermore, there was no difference in HBeAg loss alone, HBV surface antigen, alanine aminotransferase normalization, or on-treatment HBV-DNA levels according to IL28B genotype. CONCLUSIONS: In the context of a small possible effect size and high frequency in Asian populations, IL28B genotyping is likely to have, at best, limited clinical utility for predicting peg-IFN treatment outcome for CHB patients in the Asia-Pacific region.

Immunogenicity of Wild Type and Mutant Hepatitis B Surface Antigen Virus-like Particles (VLPs) in Mice with Pre-Existing Immunity against the Wild Type Vector.

Virus-like particles (VLPs), composed of the small hepatitis B virus surface antigen (HBsAgS), are the antigenic components of the hepatitis B virus (HBV) vaccine and represent the backbones for a chimeric anti-malaria vaccine and various vaccine candidates. Biological vectors have to face pre-existing anti-vector immune responses due to previous immune exposure. Vector recognition after natural infections or vaccinations can result in unwarranted outcomes, with compromising effects on clinical outcomes. In order to evaluate the impact of a pre-existing anti-HBsAgS immune response, we developed mutant VLPs composed of subunits with reduced HBsAgS-specific antigenicity. The insertion of a Plasmodium falciparum circumsporozoite protein (CSP)-derived epitope as a read-out allowed the assessment of wild type (wt) and mutant VLPs in the context of a pre-existing immune response. Mutant and wt VLP platforms with a CSP-epitope insert are immunogenic and have the ability to generate anti-CSP antibody responses in both naïve BALB/c mice and mice with a pre-existing anti-HBsAgS immune response, but with superior anti-CSP responses in mice with a pre-existing immunity. The data indicate that previous HBsAgS exposure facilitates enhanced antibody responses against foreign epitopes delivered by the HBsAgS platform, and, in this context, the state of immune sensitization alters the outcome of subsequent vaccinations.

A xenograft mouse model coupled with in-depth plasma proteome analysis facilitates identification of novel serum biomarkers for human ovarian cancer.

Proteomics discovery of novel cancer serum biomarkers is hindered by the great complexity of serum, patient-to-patient variability, and triggering by the tumor of an acute-phase inflammatory reaction. This host response alters many serum protein levels in cancer patients, but these changes have low specificity as they can be triggered by diverse causes. We addressed these hurdles by utilizing a xenograft mouse model coupled with an in-depth 4-D protein profiling method to identify human proteins in the mouse serum. This strategy ensures that identified putative biomarkers are shed by the tumor, and detection of low-abundance proteins shed by the tumor is enhanced because the mouse blood volume is more than a thousand times smaller than that of a human. Using TOV-112D ovarian tumors, more than 200 human proteins were identified in the mouse serum, including novel candidate biomarkers and proteins previously reported to be elevated in either ovarian tumors or the blood of ovarian cancer patients. Subsequent quantitation of selected putative biomarkers in human sera using label-free multiple reaction monitoring (MRM) mass spectrometry (MS) showed that chloride intracellular channel 1, the mature form of cathepsin D, and peroxiredoxin 6 were elevated significantly in sera from ovarian carcinoma patients.

Incorporating hearing screening to an otolaryngology surgical mission in the rural Dominican Republic.

INTRODUCTION: Hearing loss disproportionately affects low- and middle-income countries. Children with undiagnosed hearing loss may have difficulty with learning, language development, and behavior. The aim of this study was to understand the extent of hearing loss and common otologic disorders among school-age children in the rural western region of the Dominican Republic and to chronical the early stages of a limited-resource, locally-sustained hearing screening program in tandem with a bi-annual surgical mission. METHODS: Hearing screenings were performed for 528 school-age children (1056 ears, age 5-17 years old) over 5 days in a village hospital in Peralta, DR. Testing initially included otoscopy and screening audiometry. Children who referred or could not be conditioned underwent distortion product otoacoustic emissions (OAEs), and tympanometry. Children who referred following both screening audiometry and OAEs were considered to have hearing loss. Those with normal tympanograms were considered potential hearing aid candidates. RESULTS: Abnormal ear examination/otoscopic results were present in 43 children (8.1%) and included: microtia/atresia, impacted cerumen, ear canal foreign body, serous otitis media, otitis externa, and tympanosclerosis. 55 of 528 school-age children referred following screening audiometry and 7 were unable to condition. Of these 62 children, 56 tolerated OAEs and 20 referred following OAEs (3.8%). Fourteen children had type B or C tympanogram and 6 school-age children who were determined to have chronic otitis media with effusion (COME) underwent myringotomy and pressure equalization tube placement. Ten of 528 children (1.9%) had normal tympanometry and otoscopy, and referred following screening audiometry and OAEs suggesting the patients may be potential hearing aid candidates. CONCLUSIONS: The prevalence of hearing loss in this cohort of children in the rural, western Dominican Republic was high at roughly 4% with roughly 2% of children being potential hearing aid candidates. Nearly 10% of children screened had an abnormal otologic examination; sometimes easily remedied by otolaryngologic intervention. With the support of local leadership, it is feasible to incorporate hearing services into otolaryngology outreach and build locally sustainable programs.

Defining the specificity and function of a human neutralizing antibody for Hepatitis B virus.

Hepatitis B Virus (HBV) is a hepadnavirus that is the principal pathogen underlying viral liver disease in human populations. In this study, we describe the isolation and characterization of a fully human monoclonal antibody for HBV. This HuMab was isolated by a combinatorial screen of the memory B-cell repertoire from an acute/recovered HBV-infected patient. Lead candidate selection was based upon strong binding and neutralizing activity for live HBV. We provide a detailed biochemical/biophysical, and subclass characterization of its specificity and affinity against all of the principal HBV genotypes combined with a functional analysis of its in vitro activity. We also demonstrate its potential as a prophylaxis/therapy in vivo using human liver chimeric mouse models for HBV infection. These data have important implications for our understanding of natural human immunity to HBV and suggest that this potentially represents a new antibody-based anti-viral candidate for prophylaxis and/or therapy for HBV infection.

Self-Organized Amphiphiles Are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments.

Analysis of membrane protein topography using fast photochemical oxidation of proteins (FPOP) has been reported in recent years but is still underrepresented in literature. Based on the hydroxyl radical reactivity of lipids and other amphiphiles, it is believed that the membrane environment acts as a hydroxyl radical scavenger decreasing effective hydroxyl radical doses and resulting in less observed oxidation of proteins. We found no significant change in bulk solvent radical scavenging activity upon the addition of disrupted cellular membranes up to 25600 cells/µL using an inline radical dosimeter. We confirmed the nonscavenging nature of the membrane in bulk solution with the FPOP results of a soluble model protein in the presence of cell membranes, which showed no significant difference in oxidation with or without membranes. The use of detergents revealed that, while soluble detergent below the critical micelle concentration is a potent hydroxyl radical scavenger, additional detergent has little to no hydroxyl radical scavenging effect once the critical micelle concentration is reached. Examination of both an extracellular peptide of the integral membrane protein bacteriorhodopsin as well as a novel hydroxyl radical dosimeter tethered to a Triton X-series amphiphile indicate that proximity to the membrane surface greatly decreases reaction with hydroxyl radicals, even though the oxidation target is equally solvent accessible. These results suggest that the observed reduced oxidation of solvent-accessible surfaces of integral membrane proteins is due to the high local concentration of radical scavengers in the membrane or membrane mimetics competing for the local concentration of hydroxyl radicals.

The familial Parkinsonism gene LRRK2 regulates neurite process morphology.

Mutations in LRRK2 underlie an autosomal-dominant, inherited form of Parkinson's disease (PD) that mimics the clinical features of the common "sporadic" form of PD. The LRRK2 protein includes putative GTPase, protein kinase, WD40 repeat, and leucine-rich repeat (LRR) domains of unknown function. Here we show that PD-associated LRRK2 mutations display disinhibited kinase activity and induce a progressive reduction in neurite length and branching both in primary neuronal cultures and in the intact rodent CNS. In contrast, LRRK2 deficiency leads to increased neurite length and branching. Neurons that express PD-associated LRRK2 mutations additionally harbor prominent phospho-tau-positive inclusions with lysosomal characteristics and ultimately undergo apoptosis.

The role of BRAF mutation and p53 inactivation during transformation of a subpopulation of primary human melanocytes.

Melanocytic nevi frequently harbor oncogenic BRAF mutations, but only a minority progress to melanoma. In human melanocytes, persistent BRAF(V600E) expression triggers oncogene-induced senescence, which implies that bypass of oncogene-induced senescence is necessary for malignant transformation of melanocytes. We show that a subpopulation of primary human melanocytes with persistent expression of BRAF(V600E) do not enter oncogene-induced senescence, but instead survive despite heightened MAPK activity. Disruption of the p53 pathway using short-hairpin RNA initiated rapid growth of these V600E(+) melanocytes in vitro. The resultant V600E(+)/p53(sh) melanocytes grew anchorage-independently in soft agar, formed pigmented lesions reminiscent of in situ melanoma in artificial skin reconstructs, and were weakly tumorigenic in vivo. Array comparative genomic hybridization analysis demonstrated that the transformed melanocytes acquired a substantial deletion in chromosome 13, which encodes the Rb1 tumor suppressor gene. Gene expression profiling study of nevi and melanomas showed that p53 target genes were differentially expressed in melanomas compared with nevi, suggesting a dysfunctional p53 pathway in melanoma in vivo. In summary, these data demonstrate that a subpopulation of melanocytes possesses the ability to survive BRAF(V600E)-induced senescence, and suggest that p53 inactivation may promote malignant transformation of these cells.

Increased immunogenicity of surviving tumor cells enables cooperation between liposomal doxorubicin and IL-18.

BACKGROUND: Liposomal doxorubicin (Doxil) is a cytotoxic chemotherapy drug with a favorable hematologic toxicity profile. Its active drug, doxorubicin, has interesting immunomodulatory properties. Here, the effects of Doxil on surviving tumor cell immunophenotype were investigated. METHODS: Using ID8 murine ovarian cancer cells, the immunomodulatory effects of Doxil were studied by measuring its impact on ovarian cancer cell expression of MHC class-I and Fas, and susceptibility to immune attack in vitro. To evaluate the ability of Doxil to cooperate with cancer immunotherapy, the interaction between Doxil and Interleukin 18 (IL-18), a pleiotropic immunostimulatory cytokine, was investigated in vivo in mice bearing ID8-Vegf tumors. RESULTS: While Doxil killed ID8 tumor cells in a dose-dependent manner, tumor cells escaping Doxil-induced apoptosis upregulated surface expression of MHC-I and Fas, and were sensitized to CTL killing and Fas-mediated death in vitro. We therefore tested the hypothesis that the combination of immunotherapy with Doxil provides positive interactions. Combination IL-18 and Doxil significantly suppressed tumor growth compared with either monotherapy in vivo and uniquely resulted in complete tumor regression and long term antitumor protection in a significant proportion of mice. CONCLUSION: These data demonstrate that Doxil favorably changes the immunophenotype of a large fraction of the tumor that escapes direct killing thus creating an opportunity to expand tumor killing by immunotherapy, which can be capitalized through addition of IL-18 in vivo.

Reduction in hepatitis C-related liver disease associated with GB virus C in human immunodeficiency virus coinfection.

BACKGROUND & AIMS: It has been reported that GB virus C infection (GBV-C) leads to improved morbidity and mortality in patients with human immunodeficiency virus (HIV) infection. However, GBV-C has no effect on the course of liver disease in hepatitis C virus (HCV) monoinfection. The aim of the study was to determine the influence of GBV-C infection on liver disease in patients with HCV/HIV coinfection. METHODS: Data on 158 HCV/HIV patients were collected from January 1996 to October 2005. Two plasma specimens, collected at least 18 months apart, were tested for GBV-C RNA by reverse transcription-polymerase chain reaction with primers to the NS5B gene and confirmed using E2 gene primers and sequencing. Antibodies to GBV-C E2 protein were also determined. Liver-related morbidity and mortality were assessed from patient records. RESULTS: Fifty-seven of 158 (36%) patients had GBV-C RNA and 94 (59%) had evidence of exposure to GBV-C based on combined polymerase chain reaction and antibody results. Thirty-four (21%) patients had features of cirrhosis, with 20 having compensated and 14 having decompensated cirrhosis. Active GBV-C RNA was significantly associated with a reduction in cirrhosis, both compensated and decompensated in multivariate analysis (hazard ratio, 0.27; 95% confidence interval, 0.08-0.88; P = .03), as well as in analysis for cirrhosis-free survival vs duration of HCV infection (P = .006). No significant effect on liver-related or overall survival was observed. CONCLUSIONS: In these HCV/HIV-coinfected patients, GBV-C RNA was associated with a significant reduction in the severity of HCV-related liver disease.

DNA microarrays detect effects of soil contamination on Arabidopsis thaliana gene expression.

Soil contamination, such as heavy metals and benzene compounds, is a widespread problem on military installations. It is important to be able to determine the effects of soil contamination before any adverse effects appear in organisms in surrounding areas. We examined gene expression in Arabidopsis thaliana grown in soil from three sites at the Radford Army Ammunition Plant in Radford, Virginia, USA, using DNA microarrays. We analyzed soil, germination, and growth rate to compare with the microarray data. Soil contamination affected both external phenotype and gene expression. Plants grown in soil with high levels of contaminants were chloritic and were smaller than control plants grown in potting soil. Plants grown in soil with the highest copper concentration had the lowest growth rates and had genes up-regulated across several functional groups. Plants grown in soils with elevated lead had many genes down-regulated that were related to photosystem II, metabolism, cellular transport, and protein synthesis. Genes consistently up-regulated across most microarrays were genes related to photosystem I, genes related to water deprivation and oxidative stress response, heat shock proteins, and toxin catabolism genes such as glutathiones. DNA microarrays, in concert with a model genetic organism such as A. thaliana, were an effective assessment tool to determine the presence of toxic substances in soil at a site used for the production of military explosives.

Neuroprognostication after pediatric cardiac arrest.

BACKGROUND: Management decisions and parental counseling after pediatric cardiac arrest depend on the ability of physicians to make accurate and timely predictions regarding neurological recovery. We evaluated neurologists and intensivists performing neuroprognostication after cardiac arrest to determine prediction agreement, accuracy, and confidence. METHODS: Pediatric neurologists (n = 10) and intensivists (n = 9) reviewed 18 cases of children successfully resuscitated from a cardiac arrest and managed in the pediatric intensive care unit. Cases were sequentially presented (after arrest day 1, days 2-4, and days 5-7), with updated examinations, neurophysiologic data, and neuroimaging data. At each time period, physicians predicted outcome by Pediatric Cerebral Performance Category and specified prediction confidence. RESULTS: Predicted discharge Pediatric Cerebral Performance Category versus actual hospital discharge Pediatric Cerebral Performance Category outcomes were compared. Exact (Predicted Pediatric Cerebral Performance Category - Actual Pediatric Cerebral Performance Category = 0) and close (Predicted Pediatric Cerebral Performance Category - Actual Pediatric Cerebral Performance Category = ±1) outcome prediction accuracies for all physicians improved over successive periods (P < 0.05). Prediction accuracy did not differ significantly between physician groups at any period or overall. Agreement improved over time among neurologists (day 1 Kappa [κ], 0.28; days 2-4 κ, 0.43; days 5-7 κ, 0.68) and among intensivists (day 1 κ, 0.30; days 2-4 κ, 0.44; days 5-7 κ, 0.57). Prediction confidence increased over time (P < 0.001) and did not differ between physician groups. CONCLUSIONS: Inter-rater agreement among neurologists and among intensivists improved over time and reached moderate levels. For all physicians, prediction accuracy and confidence improved over time. Further prospective research is needed to better characterize how physicians objectively and subjectively estimate neurological recovery after acute brain injury.