The radial mode matching (RMM) method has been used to calculate accurately the microwave field distribution of the TE0 1 1 mode in a spherical EPR cavity containing a linear aqueous sample, in order to understand in detail the factors affecting sensitivity in EPR measurements at X band. Specific details of the experiment were included in the calculations, such as the cavity geometry, the presence of a quartz dewar, the size of the aqueous sample, and the sample’s dielectric properties. From the field distribution, several key physical parameters were calculated, including cavity Q, filling factor, mean microwave magnetic field at the sample, and cavity efficiency parameter Λ. The dependence of EPR signal intensity on sample diameter for a cylindrical aqueous sample was calculated and measured experimentally for non-saturated and half-saturated samples. The optimal aqueous sample diameter was determined for both cases. The impact of sample temperature, conductivity, and cavity Q on sensitivity of EPR is discussed.
EPR; Water; Sensitivity; Cavity; Field distribution
We characterized HCV treatment knowledge, experience and barriers in a cohort of community-based injection drug users (IDUs) in Baltimore, MD. In 2005, a questionnaire on HCV treatment knowledge, experience and barriers was administered to HCV-infected IDUs. Self-reported treatment was confirmed from medical records. Of 597 participants, 71% were male, 95% African-American, 31% HIV co-infected and 94% were infected with HCV genotype 1; 70% were aware that treatment was available, but only 22% understood that HCV could be cured. Of 418 who had heard of treatment, 86 (21%) reported an evaluation by a provider that included a discussion of treatment of whom 30 refused treatment, 20 deferred and 36 reported initiating treatment (6% overall). The most common reasons for refusal were related to treatment-related perceptions and a low perceived need of treatment. Compared to those who had discussed treatment with their provider, those who had not were more likely to be injecting drugs, less likely to have health insurance, and less knowledgeable about treatment. Low HCV treatment effectiveness was observed in this IDU population. Comprehensive integrated care strategies that incorporate education, case-management and peer support are needed to improve care and treatment of HCV-infected IDUs.
Hepatitis C virus; Injection drug use; Antiviral therapy; Health care access
To evaluate the relationship between regional body composition and liver disease (fibrosis or steatosis) in HIV/HCV co-infected individuals.
Whole body dual-energy X-ray absorptiometry (DXA) was performed in 173 HIV/HCV co-infected persons within 12 months of a liver biopsy. Significant fibrosis was defined as a METAVIR stage greater than 1. Steatosis was graded as: 0, none; 1, steatosis involving less than 5% of hepatocytes; 2, 5–29%; 3, 30–60%; 4 greater than 60%, and was defined as more than 0. Poisson regression with robust variance was used to estimate prevalence ratios of the outcome measures.
The population was 62% male and 84% black with a median body mass index of 25.2 kg/m2 (interquartile range 22.5, 29.3 kg/m2). No differences in regional body fat or fat distribution were observed in 42 patients with significant fibrosis compared to others with less fibrosis. However, the 77 individuals (45%) with steatosis had greater central fat than those without steatosis [prevalence ratio 1.04 per kg trunk fat; 95% confidence interval (CI) 1.04, 1.11], after adjusting for hepatic fibrosis (prevalence ratio 1.77; 95% CI 1.29, 2.42), uncontrolled HIV replication (viral load >400 copies/ml) (prevalence ratio 1.57; 95% CI 1.12, 2.22), age, sex, race and diabetes mellitus.
In HIV/HCV co-infected individuals, measures of regional body fat or fat distribution were not associated with hepatic fibrosis. In contrast, increased central adiposity by DXA, as well as concomitant fibrosis and uncontrolled HIV, were associated with hepatic steatosis. The extent to which weight loss and effective antiretroviral therapy can reduce the risk of steatosis deserves further investigation.
body composition; hepatic fibrosis; hepatitis C; HIV; steatosis
Liver disease continues to represent a critical mediator of morbidity and mortality in those with human immunodeficiency virus (HIV) infection. The frequent presence and overlap of concomitant injurious processes, including hepatitis C virus and hepatitis B virus infections, hepatoxicity associated with antiretroviral therapeutic agents, alcohol, and other toxins, in the setting of immunosuppression lead to rapid fibrotic progression and early development of end-stage liver disease. This conference summary describes the proceedings of a state-of-the-art gathering of international experts designed to highlight the status of current research in epidemiology, natural history, pathogenesis, and treatment of HIV and liver disease.
Chronic infection with hepatitis C virus (HCV) is a common cause of liver cirrhosis and cancer. We performed RNA-sequencing in primary human hepatocytes activated with synthetic dsRNA to mimic HCV infection. Upstream of IFNL3 (IL28B) on chromosome 19q13.13, we discovered a novel, transiently induced region that harbors dinucleotide variant ss469415590 (TT/ΔG), which is in high linkage disequilibrium with rs12979860, a genetic marker strongly associated with HCV clearance. ss469415590-ΔG is a frame-shift variant that creates a novel primate-specific gene, designated interferon lambda 4 (IFNL4), which encodes a protein of moderate similarity with IFNL3. Compared to rs12979860, ss469415590 is more strongly associated with HCV clearance in individuals of African ancestry, whereas it provides comparable information in Europeans and Asians. Transient over-expression of IFNL4 in a hepatoma cell line induced STAT1/STAT2 phosphorylation and expression of interferon-stimulated genes. Our findings provide new insights into the genetic regulation of HCV clearance and its clinical management.
Fractures and cirrhosis are major causes of morbidity and mortality among HIV–HCV-coinfected individuals. It is not known whether vitamin D deficiency is associated with these outcomes.
Between 2005 and 2007, 116 HIV–HCV- coinfected individuals underwent dual-energy X-ray absorptiometry within 1 year of a liver biopsy. 25-Hydroxy-vitamin D (25OHD) and parathyroid hormone were measured from archived samples. Low bone mineral density (BMD) was defined as BMD≥2 standard deviations lower than age-, sex- and race-matched controls (Z-score ≤−2.0) at the total hip, femoral neck or lumbar spine. Histological fibrosis staging was assessed according to the METAVIR system (0 [no fibrosis] to 4 [cirrhosis]).
The cohort was 87% African-American and 63% male. The median age (IQR) was 49.9 years (46.5–53.3). A total of 89% had a CD4+ T-cell count >200 cells/mm3 and 64% were receiving HAART. The median 25OHD was 19 ng/ ml (IQR 11.0–26.0). Hypovitaminosis D (25OHD≤15 ng/ml) was present in 41% and secondary hyperparathyroidism, defined by parathyroid hormone >65 pg/ml, was present in 24%. In total, 27% had low BMD (Z-score ≤−2) at the spine, femoral neck or total hip, and 39% had significant hepatic fibrosis (METAVIR≥2). In multivariate analysis, vitamin D deficiency was not associated with significant fibrosis or with BMD at any site.
Vitamin D deficiency was highly prevalent in this mostly African-American HIV–HCV-coinfected population, but was not related to BMD or liver disease severity. These data suggest that efforts to increase vitamin D levels in this population may not improve bone or liver outcomes.
Arsenic (+3 oxidation state) methyltransferase (AS3MT) is the key enzyme in the pathway for methylation of arsenicals. A common polymorphism in the AS3MT gene that replaces a threonyl residue in position 287 with a methionyl residue (AS3MT/M287T) occurs at a frequency of about 10% among populations worldwide. Here, we compared catalytic properties of recombinant human wild-type (wt) AS3MT and AS3MT/M287T in reaction mixtures containing S-adenosylmethionine, arsenite (iAsIII) or methylarsonous acid (MAsIII) as substrates and endogenous or synthetic reductants, including glutathione (GSH), a thioredoxin reductase (TR)/thioredoxin (Trx)/NADPH reducing system, or tris (2-carboxyethyl) phosphine hydrochloride (TCEP). With either TR/Trx/NADPH or TCEP, wtAS3MT or AS3MT/M287T catalyzed conversion of iAsIII to MAsIII, methylarsonic acid (MAsV), dimethylarsinous acid (DMAsIII), and dimethylarsinic acid (DMAsV); MAsIII was converted to DMAsIII and DMAsV. Although neither enzyme required GSH to support methylation of iAsIII or MAsIII, addition of 1 mM GSH decreased Km and increased Vmax estimates for either substrate in reaction mixtures containing TR/Trx/NADPH. Without GSH, Vmax and Km values were significantly lower for AS3MT/M287T than for wtAS3MT. In the presence of 1 mM GSH, significantly more DMAsIII was produced from iAsIII in reactions catalyzed by the M287T variant than in wtAS3MT-catalyzed reactions. Thus, 1 mM GSH modulates AS3MT activity, increasing both methylation rates and yield of DMAsIII. AS3MT genotype exemplified by differences in regulation of wtAS3MT and AS3MT/M287T-catalyzed reactions by GSH may contribute to differences in the phenotype for arsenic methylation and, ultimately, to differences in the disease susceptibility in individuals chronically exposed to inorganic arsenic.
Arsenic methylation; AS3MT polymorphism; thioredoxin reductase; glutathione
Myosin’s affinities for nucleotides and actin are reciprocal. Actin-binding substantially reduces the affinity of ATP for myosin, but the effect of actin on myosin’s ADP affinity is quite variable among myosin isoforms, serving as the principal mechanism for tuning the actomyosin system to specific physiological purposes. To understand the structural basis of this variable relationship between actin and ADP binding, we studied several constructs of the catalytic domain of Dictyostelium myosin II, varying their length (from the N-terminal origin) and cysteine content. The constructs varied considerably in their actin-activated ATPase activity and in the effect of actin on ADP affinity. Actin had no significant effect on ADP affinity for a single-cysteine catalytic domain construct, a double-cysteine construct partially restored the actin-dependence of ADP binding, and restoration of all native Cys restored it further, but full restoration of function (similar to that of skeletal muscle myosin II) was obtained only by adding all native Cys and an artificial lever arm extension. Pyrene-actin fluorescence confirmed these effects on ADP binding to actomyosin. We conclude that myosin’s Cys content and lever arm both allosterically modulate the reciprocal affinities of myosin for ADP and actin, a key determinant of the biological functions of myosin isoforms.
actin; cysteine; lever arm; ADP; fluorescence
Next-generation sequencing (NGS) studies in cancer are limited by the amount, quality and purity of tissue samples. In this situation, primary xenografts have proven useful preclinical models. However, the presence of mouse-derived stromal cells represents a technical challenge to their use in NGS studies. We examined this problem in an established primary xenograft model of small cell lung cancer (SCLC), a malignancy often diagnosed from small biopsy or needle aspirate samples. Using an in silico strategy that assign reads according to species-of-origin, we prospectively compared NGS data from primary xenograft models with matched cell lines and with published datasets. We show here that low-coverage whole-genome analysis demonstrated remarkable concordance between published genome data and internal controls, despite the presence of mouse genomic DNA. Exome capture sequencing revealed that this enrichment procedure was highly species-specific, with less than 4% of reads aligning to the mouse genome. Human-specific expression profiling with RNA-Seq replicated array-based gene expression experiments, whereas mouse-specific transcript profiles correlated with published datasets from human cancer stroma. We conclude that primary xenografts represent a useful platform for complex NGS analysis in cancer research for tumours with limited sample resources, or those with prominent stromal cell populations.
The reaction of Ni(COD)2, IPr, and nitrile affords dimeric [Ni(IPr)RCN]2 in high yields. X-ray analysis revealed these species display simultaneous η1- and η2-nitrile binding modes. These dimers are catalytically competent in the formation of pyridines from the cycloaddition of diynes and nitriles. Kinetic analysis showed the reaction to be first order in [Ni(IPr)RCN]2, zeroth order in added IPr, zeroth order in nitrile, and zeroth order in diyne. Extensive stoichiometric competition studies were performed, and selective incorporation of the exogenous, not dimer bound, nitrile was observed. Post cycloaddition, the dimeric state was found to be largely preserved. Nitrile and ligand exchange experiments were performed and found to be inoperative in the catalytic cycle. These observations suggest a mechanism whereby the catalyst is activated by partial dimer-opening followed by binding of exogenous nitrile and subsequent oxidative heterocoupling.
Nickel; Carbene; Cycloaddition; Nitrile; Catalysis; Mechanism; Bimetallic
Hepatitis C virus (HCV) infections occur worldwide and either spontaneously resolve or persist and markedly increase the person’s lifetime risk of cirrhosis and hepatocellular carcinoma. Although HCV persistence occurs more often in persons of African ancestry and in persons with a genetic variant near IL28B, the genetic basis is not well understood.
To evaluate the host genetic basis for spontaneous resolution of HCV infection.
Two-stage genome wide association study (GWAS).
13 international multicenter study sites.
919 individuals with serum HCV antibodies but no HCV RNA (spontaneous resolution) and 1482 individuals with serum HCV antibodies and RNA (persistence).
Frequencies of 792,721 SNPs.
Differences in allele frequencies between persons with spontaneous resolution and persistence were identified on chromosomes 19q13.13 and 6p21.32. On chromosome 19, allele frequency differences localized near IL28B and included rs12979860 (overall per-allele OR = 0.45, P = 2.17 × 10−30) and 10 additional SNPs spanning 55,000 bases. On chromosome 6, allele frequency differences localized near genes for class II human leukocyte antigens (HLA) and included rs4273729 (overall per-allele OR= 0.59, P = 1.71 × 10−16) near DQB1*03:01 and an additional 116 SNPs spanning 1,090,000 base pairs. The associations in chromosomes 19 and 6 were independent, additive, and explain an estimated 14.9% (95% CI: 8.5–22.6%) of the variation in HCV resolution in those of European-Ancestry, and 15.8% (95% CI:4.4–31.0%) in individuals of African-Ancestry. Replication of the chromosome 6 SNP, rs4272729 in an additional 746 individuals confirmed the findings (p=0.015).
Epigenetic effects were not studied.
IL28B and HLA class II are independently associated with spontaneous resolution of HCV infection and SNPs marking IL28B and DQB1*03:01 may explain ~15% of spontaneous resolution of HCV infection.
Genetic-modification strategies are currently being developed to reduce the transmission of vector-borne diseases, including African trypanosomiasis. For tsetse, the vector of African trypanosomiasis, a paratransgenic strategy is being considered: this approach involves modification of the commensal symbiotic bacteria Sodalis to express trypanosome-resistance-conferring products. Modified Sodalis can then be driven into the tsetse population by cytoplasmic incompatibility (CI) from Wolbachia bacteria. To evaluate the effectiveness of this paratransgenic strategy in controlling African trypanosomiasis, we developed a three-species mathematical model of trypanosomiasis transmission among tsetse, humans, and animal reservoir hosts. Using empirical estimates of CI parameters, we found that paratransgenic tsetse have the potential to eliminate trypanosomiasis, provided that any extra mortality caused by Wolbachia colonization is low, that the paratransgene is effective at protecting against trypanosome transmission, and that the target tsetse species comprises a large majority of the tsetse population in the release location.
African sleeping sickness is a fatal disease occurring in sub-Saharan Africa. The parasites that cause African sleeping sickness are transmitted between humans and livestock by the tsetse fly. Controlling the spread of the parasite by tsetse flies has been proposed as a promising strategy for reducing the incidence of sleeping sickness. One potential control method relies on releasing genetically modified tsetse that are resistant to carrying the sleeping sickness parasite. For this strategy to be successful, resistant tsetse must be able to invade the susceptible tsetse population. Here, we used a mathematical model to assess the feasibility of such a strategy and the implications for sleeping sickness prevalence in humans and livestock. We found that the strategy has the potential to eliminate sleeping sickness, provided that the genetic modification is effective at protecting against trypanosome transmission and provided that the target tsetse species comprises a large majority of the tsetse population in the release location.
Chronic hepatitis C virus (HCV) infection is complicated by hepatic fibrosis. Hypothesizing that early fibrogenic signals may originate in cells susceptible to HCV infection, hepatocyte gene expression was analyzed from persons with chronic HCV at different stages of liver fibrosis. Four HCV-infected subjects with pre-cirrhotic liver fibrosis (Ishak fibrosis 3–5) were matched for age, race, and gender to five HCV-infected subjects with no evidence of fibrosis (Ishak fibrosis 0). Hepatocytes from each subject were isolated from liver biopsies using laser capture microdissection. Transcriptome profiling was performed on hepatocyte RNA using hybridization arrays. We found that hepatocytes in pre-cirrhotic fibrosis were depleted for genes involved in small molecule and drug metabolism, especially butyrylcholinesterase (BCHE), a gene involved in the metabolism of drugs of abuse. Differential expression of BCHE was validated in the same tissues and cross-sectionally in an expanded cohort of 143 HCV-infected individuals. In a longitudinal study, serum BCHE activity were already decreased at study inception in 19 fibrosis progressors compared to 20 fibrosis non-progressors (p<0.05). Non-progressors also had decreased BCHE activity over time compared to initial values, but these evolved a median (range) 8.6 (7.8–11.4) years after the study period inception(p<0.05). Laser captured portal tracts were enriched for immune related genes when compared to hepatocytes but pre-cirrhotic livers lost this enrichment.
Overall, we found that chronic HCV is associated with hepatocyte BCHE loss years before hepatic synthetic function is impaired. These results indicate that BCHE may be involved in the pathogenesis of HCV-related fibrosis among injection drug users.
butyrylcholinesterase; Hepacivirus; chronic HCV infection; laser capture microdissection; portal tract
The study aimed to examine attitudes of individuals diagnosed with sarcoma and their family members towards genetics, genomic research and incidental information arising as a result of participating in genetic research.
A questionnaire was administered to 1200 individuals from the International Sarcoma Kindred Study (ISKS). Respondents were divided into three groups: individuals affected with sarcoma (probands), their spouses and family members.
Approximately half of all research participants felt positively towards new discoveries in human genetics. Overall, more were positive in their attitudes towards genetic testing for inherited conditions (60%) but family members were less so. Older participants reported more highly positive attitudes more often than younger participants. Males were less likely to feel positive about new genetic discoveries and more likely to believe they could modify genetic risk by altering lifestyle factors. Almost all ISKS participants believed participants would like to be given ancillary information arising as a result of participating in genetic research.
The only difference between the study groups was the decreased likelihood of family members being highly positive about genetic testing. This may be important if predictive testing for sarcoma becomes available. Generally ISKS research participants supported the notion of returning incidental genetic information to research participants.
Sarcomas are a key feature of Li-Fraumeni and related syndromes (LFS/LFL), associated with germline TP53 mutations. Current penetrance estimates for TP53 mutations are subject to significant ascertainment bias. The International Sarcoma Kindred Study is a clinic-based, prospective cohort of adult-onset sarcoma cases, without regard to family history. The entire cohort was screened for mutations in TP53 using high-resolution melting analysis and Sanger sequencing, and multiplex-ligation-dependent probe amplification and targeted massively parallel sequencing for copy number changes. Pathogenic TP53 mutations were detected in blood DNA of 20/559 sarcoma probands (3.6%); 17 were germline and 3 appeared to be somatically acquired. Of the germline carriers, one appeared to be mosaic, detectable in the tumor and blood, but not epithelial tissues. Germline mutation carriers were more likely to have multiple cancers (47% vs 15% for non-carriers, P = 3.0×10−3), and earlier cancer onset (33 vs 48 years, P = 1.19×10−3). The median survival of mutation carriers following first cancer diagnosis was not significantly different from non-carriers. Only 10/17 (59%) pedigrees met classical or Chompret criteria for LFS. In summary, germline TP53 mutations are not rare in adult patients with sarcoma, with implications for screening, surveillance, treatment and genetic counselling of carriers and family members.
The hepatitis C virus (HCV) direct antiviral treatment era is underway. Although there are some important differences, it is likely that the experience with HCV will be similar in many respects to what already occurred with HIV. This paper considers seven important lessons learned with HIV and the degree to which they should be anticipated with HCV.
Persons with HIV infection have been reported to develop age-related diseases at younger ages than those without HIV. Whether this finding is related to HIV infection or failure to control for other risk factors is unknown.
To investigate whether persons with HIV infection develop hepatitis C virus (HCV)–related liver disease at younger ages than similar persons without HIV.
Comparison of the severity of liver fibrosis by age among persons who have HCV with and without HIV followed concurrently in the same protocol.
Observational cohort from Baltimore, Maryland, participating in the ALIVE (AIDS Linked to the IntraVenous Experience) study.
1176 current and former injection drug users with antibodies to HCV.
Liver fibrosis assessed semiannually from 2006 to 2011 by elastography (FibroScan, Echosens, Paris, France) and using previously validated thresholds for clinically significant fibrosis and cirrhosis; concurrent assessment of medical history, alcohol and illicit drug use, HCV RNA levels, hepatitis B virus surface antigen level, body mass index, and (for those with HIV) CD4+ lymphocyte count and HIV RNA levels.
Among 1176 participants with antibodies to HCV, the median age was 49 years and 34% were coinfected with HIV and HCV. Participants contributed 5634 valid liver fibrosis measurements. The prevalence of clinically significant fibrosis without cirrhosis (12.9% vs. 9.5%) and of cirrhosis (19.5% vs. 11.0%) was greater in persons coinfected with HIV and HCV than in those with only HCV (P < 0.001). Increasing age and HIV infection were independently associated with liver fibrosis, as were daily alcohol use, chronic hepatitis B virus infection, body mass index greater than 25 kg/m2, and greater plasma HCV RNA levels. When these factors were kept constant, persons with HIV had liver fibrosis measurements equal to those of persons without HIV, who were, on average, 9.2 years older.
The process of liver fibrosis began before the study in most persons.
In this cohort, persons who have HCV with HIV have liver fibrosis stages similar to those without HIV who are nearly a decade older.
Several recent studies have supported relations between infant behavior (alertness and responsiveness) and nutrition (e.g. Dempsey 2008, Wachs et al 2005) in addition to investigating infant behavior within the context of changes in iron status over time (e.g. Black et al. 2004, Murray-Kolb & Beard 2009). Existing research is typically limited to investigation of the effects of a single vitamin or mineral and no studies have been found that examined the influence that early alertness and responsiveness have on growth in early infancy, despite the fact that relations between behavior and nutritional status may be bidirectional (Hulthén 2003). The current study used a sample of Ethiopian infants and investigated anthropometrics, hemoglobin, the frequency of alertness, and the frequency of responsiveness at 6 and 9 months of age. Six-month weight-for-age predicted 9-month frequency of alertness, while 6-month hemoglobin predicted 9-month frequency of responsiveness. Compared to responsive infants, non-responsive infants at 6 months remained more non-responsive at 9 months, though weight-for-age for both groups converged at 9 months. Results support relations between nutrition and behavior (alertness and responsiveness) and provide evidence of a potentially useful tool (the Laboratory Temperament Assessment Battery [Lab-TAB]) that was adapted to evaluate these relations in Ethiopia.
development; infant growth; hemoglobin; alertness; responsiveness; international nutrition
There are few published data characterizing patterns of liver stiffness measurements (LSMs) among HCV-infected persons and their potential impact on clinical decisions (for example, deferring treatment and hepatocellular carcinoma surveillance).
A total of 591 HCV-infected injection drug users (IDUs) in a community-based cohort had four LSMs. We used semi-parametric latent class growth modelling to identify patterns, which then became a gold standard against which we characterized validity of information from the initial measurements.
Median age was 49, 68% were male, 92% African-American and 33% HIV-coinfected. The median LSM at visit 1 was 6.7 kPa (IQR 5.3–8.8). Over a median 1.75 years, LSM measures were stable; median change between visits was 0 kPa (IQR -1.4–1.7). Only 3% had evidence of fibrosis progression. Other groups included stable patterns of 1) no fibrosis (59%), 2) moderate fibrosis (21%), 3) severe fibrosis (7%) and 4) cirrhosis (9%). Individuals with fibrosis progression were more likely to be HIV-infected than those with stable low fibrosis (P<0.001). The diagnostic accuracy of the first LSM for identification of need for cancer surveillance (cirrhosis ≥12.3 kPa) was high (positive predictive value = 97%). Although no single low LSM had high negative predictive value for significant fibrosis (metavir <2), individuals with two or more low results rarely had progression.
These data underscore the stability of liver fibrosis in a cohort of predominantly African-American HCV-infected persons over 1.75 years, support using LSMs to monitor untreated persons at risk for progression and assess need for hepatocellular carcinoma surveillance.
Limited data exist regarding the outcomes of adjuvant therapy in younger patients with stage II and III colon cancer. We examined disease-free survival (DFS), overall survival (OS), recurrence-free interval (RFI), and grade 3+ adverse events (AEs) in younger patients in the 33,574 patient Adjuvant Colon Cancer Endpoints Group data set.
Patients and Methods
Individual patient data from 24 randomized phase III clinical trials were obtained for survival outcomes, which included 10 clinical trials for AE outcomes. Two age-based cutoff points were used to define younger patients: age younger than 40 years and younger than 50 years. Adjuvant therapy benefit analyses were limited to the nine clinical trials in which the investigational chemotherapeutic arm demonstrated benefit.
One thousand seven hundred fifty-eight patients (5.2%) were younger than 40 years, 5,817 patients (17.3%) were younger than 50 years, and only 299 patients (0.9%) were younger than 30 years. No meaningful differences in sex or stage were noted in younger versus older patients. Younger and older patients did not differ in RFI (age, < 40 years: hazard ratio [HR], 1.0; P = .62 and age < 50 years: HR, 1.02; P = .35). Younger patients (both cutoff points), had longer OS and DFS than older patients. In trials demonstrating adjuvant therapy benefit, similar DFS benefit was observed by age. Younger patients experienced less leukopenia and stomatitis, but more frequent nausea/vomiting.
Among patients on clinical trials, younger and older patients with stage II and III colon cancer had similar RFI and adjuvant therapy benefit. Younger patients have longer OS and DFS, which is likely primarily because of fewer competing causes of death. Adjuvant therapy is beneficial for colon cancer in patients younger than 50 years who meet typical clinical trial eligibility criteria.
We have used time-resolved phosphorescence anisotropy (TPA) of actin to evaluate domains of dystrophin and utrophin, with implications for gene therapy in muscular dystrophy. Dystrophin and its homolog utrophin bind to cytoskeletal actin to form mechanical linkages that prevent muscular damage. Because these proteins are too large for most gene therapy vectors, much effort is currently devoted to smaller constructs. We previously used TPA to show that dystrophin and utrophin both have a paradoxical effect on actin rotational dynamics -- restricting amplitude while increasing rate, thus increasing resilience, with utrophin more effective than dystrophin. Here we have evaluated individual domains of these proteins. We found that a “mini-dystrophin,” lacking one of the two actin-binding domains, is less effective than dystrophin in regulating actin dynamics, correlating with its moderate effectiveness in rescuing the dystrophic phenotype in mice. In contrast, we found that a “micro-utrophin,” with more extensive internal deletions, is as effective as full-length dystrophin in the regulation of actin dynamics. Each of utrophin’s actin-binding domains promotes resilience in actin, while dystrophin constructs require the presence of both actin-binding domains and the CT domain for full function. This work supports the use of a utrophin template for gene or protein therapy designs. Resilience of the actin-protein complex, measured by TPA, correlates remarkably well with previous reports of functional rescue by dystrophin and utrophin constructs in mdx mice. We propose the use of TPA as an in vitro method to aid in the design and testing of emerging gene therapy constructs.
time-resolved phosphorescence anisotropy; TPA; muscular dystrophy; gene therapy
In cross-sectional analysis of 287 human immunodeficiency virus–infected persons with high hepatitis C virus prevalence, liver fibrosis defined by elastography was associated with higher CD4+ T-cell percentages relative to absolute CD4+ number; this discordance was most apparent in persons with marked lymphopenia.
Background. Cirrhosis of the liver can induce splenic sequestration of peripheral blood cells, recently suggested to reduce the number but not percentage of circulating CD4+ T cells in persons uninfected with human immunodeficiency virus (HIV). We investigated whether earlier stages of liver fibrosis prior to cirrhosis were associated with discordance between CD4 count (CD4N) and CD4 percentage (CD4%) in HIV-infected patients.
Methods. In cross-sectional analysis of 287 HIV-infected participants of the AIDS Linked to the Intravenous Experience cohort, we evaluated CD4N, CD4%, and transient elastography staging of liver fibrosis. High CD4+ lymphocyte discordance was defined as higher CD4% relative to CD4N based on accepted clinical cutoffs; multivariable logistic regression was used to determine covariates associated with discordance.
Results. Of 287 participants, 99 (34.4%) had high CD4+ discordance, which increased to 76% of 114 participants with marked lymphopenia (total lymphocyte count [TLC] ≤1200 cells/μL). In multivariable analysis, the odds of having high CD4+ discordance was increased in persons with significant liver fibrosis compared to those without fibrosis (odds ratio, 1.69; 95% confidence interval [CI], .95–2.96); the odds ratio of discordance increased to 2.66 (95% CI, 1.11–6.40) among the subset of participants with TLC ≤1200 cells/μL. The odds for discordance associated with cirrhosis were of similar magnitude as those observed with significant fibrosis.
Conclusions. In HIV-infected persons, liver fibrosis is associated with discordant peripheral CD4+ lymphocyte results, especially in the setting of marked lymphopenia. Clinicians should also consider CD4% when interpreting absolute CD4+ counts of HIV-infected persons with known or suspected liver disease, particularly if TLC is <1200 cells/μL.
We have used time-resolved phosphorescence anisotropy (TPA) to investigate the effects of essential light chain (ELC) isoforms (A1 and A2) on the interaction of skeletal muscle myosin with actin, in order to relate structural dynamics to previously reported functional effects. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes S1A1 and S1A2 by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin but substantially higher apparent actin affinity, resulting in higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin’s final anisotropy cooperatively and to a similar extent, indicating similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (S) state of actomyosin. In contrast, in the presence of saturating ATP, S1A1 increased actin anisotropy much more than S1A2 and with greater cooperativity, indicating that S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin. We conclude that during the active interaction of actin and ATP with myosin, S1A1 is more effective at stabilizing the S state (probably the force-generating state) of actomyosin, while S1A2 tends to stabilize the weak-binding (non-force-generating) W state. When a mixture of isoenzymes is present, S1A1 is dominant in its effects on actin dynamics. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, with significant implications for the contractile function of actomyosin.