We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.
Intranasal administration has been widely used to investigate effects of the neuropeptides vasopressin and oxytocin on human behaviors and neurological disorders, but exactly what happens when these neuropeptides are administered intranasally is far from clear. In particular, it is not clear whether a physiological significant amount of peptide enters the brain to account for the observed effects. Here, we investigated whether intranasal administration of vasopressin and oxytocin to rats induces expression of the immediate-early gene product Fos in brain areas that are sensitive to centrally administered peptide, whether it alters neuronal activity in the way that centrally administered peptide does, and whether it affects behavior in ways expected from studies of centrally administered peptide. We found that, whereas intracerebroventricular (icv) injection of very low doses of vasopressin or oxytocin increased Fos expression in several distinct brain regions, intranasal administration of large doses of the peptides had no significant effect. In contrast to the effects of vasopressin applied topically to the main olfactory bulb, we saw no changes in the electrical activity of olfactory bulb mitral cells after intranasal vasopressin administration. In addition, vasopressin given intranasally had no significant effects on social recognition or short-term recognition memory. Finally, intranasal infusions of vasopressin had no significant effects on the parameters monitored on the elevated plus maze, a rodent model of anxiety. Our data in rats suggest that, after intranasal administration, significant amounts of vasopressin and oxytocin do not reach areas in the brain at levels sufficient to change immediate early gene expression, neural activity or behavior in the ways described for central administration of the peptides.
vasopressin; oxytocin; olfactory bulb intranasal; mitral cells; c-Fos; blood pressure; behavior
Older people with dementia have increased risk of nursing home (NH) use and higher Medicaid payments. Dementia’s impact on acute care use and Medicare payments is less well understood.
Identify trajectories of incident dementia and NH use, and (2) compare Medicare and Medicaid payments for persons having different trajectories.
Retrospective cohort of older patients who were screened for dementia in 2000–2004 and were tracked for five years. Trajectories were identified with latent class growth analysis.
3673 low-income persons age 65 or older without dementia at baseline.
Incident dementia diagnosis, comorbid conditions, dual eligibility, acute and long-term care use and payments based on Medicare and Medicaid claims, medical record systems, and administrative data.
Three trajectories were identified based on dementia incidence and short and long-term NH use: (1) high incidence of dementia with heavy NH use (5% of the cohort) averaging $56,111/year ($36,361 Medicare, $19,749 Medicaid); (2) high incidence of dementia with little or no NH use (16% of the cohort) averaging $16,206/year ($14,644 Medicare, $1,562 Medicaid); and (3) low incidence of dementia and little or no NH use (79% of the cohort) averaging $8,475/year ($7,558 Medicare, $917 Medicaid).
Dementia and its interaction with NH utilization are major drivers of publicly financed acute and long-term care payments. Medical providers in accountable care organizations and other health care reform efforts must effectively manage dementia care across the care continuum if they are to be financially viable.
dementia; nursing home; acute care; payment
Older Americans are facing an epidemic of chronic diseases and are thus exposed to anticholinergics (AC) that might negatively affect their risk of developing mild cognitive impairment (MCI) or dementia.
Investigate the association between impairment in cognitive function and previous AC exposure.
A retrospective cohort study.
Primary care clinics in Indianapolis, Indiana.
3690 older adults who have undergone cognitive assessment and had a one-year medication dispensing record.
Cognitive function was measured in two sequential steps; a two-step screening process followed by a formal diagnostic process for participants with positive screening results.
Three patterns of AC exposure were defined by the duration of AC exposure, the number of AC medications dispensed at the same time, and the severity of AC effects as determined by the Anticholinergic Cognitive Burden List.
In comparison to older adults with no anticholinergic exposure and after adjusting for age, race, gender, and underlying comorbidity, the odds ratio (OR) for having a diagnosis of MCI was 2.73 (95% confidence interval, CI; 1.27, 5.87) among older adults who were exposed to at least three possible anticholinergic for at least 90 days; and the OR for having dementia was 0.43 (95% CI; 0.10, 1.81).
Exposure to medications with severe anticholinergic cognitive burden may be a risk factor for developing MCI.
anticholinergics; cognitive impairment; dementia; mild cognitive impairment; elderly
Irritant hand dermatitis (IHD) is common in healthcare workers.
We studied endogenous irritant contact dermatitis threshold by patch testing, and exogenous factors such as season and hand washing for their association with IHD in healthcare workers.
Irritant patch testing with sodium lauryl sulfate (SLS), sodium hydroxide (NaOH) and benzalkonium chloride (BAK) at varying concentrations was measured in 113 healthcare workers. Examination for hand dermatitis occurred at one month intervals for a period of six months in the Midwestern US.
Positive patch testing to low concentration SLS was associated with IHD (p=0.0310) after adjusting for age, gender, ethnicity, season, history of childhood flexural dermatitis, mean indoor relative humidity, glove and hand sanitizer usage). Subjects with a positive patch test to SLS were 78% more likely to have occurrence of IHD (IRR=1.78, 95% CI: 0.92, 3.45). Hand washing frequency (≥ 10 times a day; IRR=1.55, 95% CI: 1.01, 2.39) and cold season (IRR=2.76, 95% CI: 1.35, 5.65) were associated with IHD. No association was found between history of childhood flexural dermatitis and IHD in this population.
Both genetic and environmental factors are important in the etiology of IHD and should be considered in designing strategies to protect, educate and treat susceptible individuals.
irritant contact dermatitis; patch testing; occupational; season; healthcare workers
In order to assess the safety of psychotropic medication use in children and adolescents, it is critical to be able to identify suicidal behaviors from medical claims data and distinguish them from other injuries. The purpose of this study was to develop an algorithm using administrative claims data to identify medically-treated suicidal behavior in a cohort of children and adolescents.
The cohort included 80,183 youth (6–18 years) enrolled in Tennessee’s Medicaid program from 1995–2006 who were prescribed antidepressants. Potential episodes of suicidal behavior were identified using external cause-of-injury codes (E-codes) and ICD-9-CM codes corresponding to the potential mechanisms of or injuries resulting from suicidal behavior. For each identified episode, medical records were reviewed to determine if the injury was self-inflicted and if intent to die was explicitly stated or could be inferred.
Medical records were reviewed for 2676 episodes of potential self-harm identified through claims data. Among 1162 episodes that were classified as suicidal behavior, 1117 (96%) had a claim for suicide & self-inflicted injury, poisoning by drugs, or both. The positive predictive value (PPV) of code groups to predict suicidal behavior ranged from 0–88% and improved when there was a concomitant hospitalization but with the limitation of excluding some episodes of confirmed suicidal behavior.
Nearly all episodes of confirmed suicidal behavior in this cohort of youth included an ICD-9-CM code for suicide or poisoning by drugs. An algorithm combining these ICD-9-CM codes and hospital stay greatly improved the PPV for identifying medically-treated suicidal behavior.
Suicidal behavior; Adolescence; Administrative claims
The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) respiratory infection in cystic fibrosis (CF) has increased dramatically over the last decade, and is now affecting approximately 25% of patients. Epidemiologic evidence suggests that persistent infection with MRSA results in an increased rate of decline in FEV1 and shortened survival. Currently, there are no conclusive studies demonstrating an effective and safe treatment protocol for persistent MRSA respiratory infection in CF.
The primary objective of this study is to evaluate the safety and efficacy of a 28-day course of vancomycin for inhalation in combination with oral antibiotics in eliminating MRSA from the respiratory tract of individuals with CF and persistent MRSA infection. This is a two-center, randomized, double-blind, comparator-controlled, parallel-group study with 1:1 assignment to either vancomycin for inhalation (250 mg twice a day) or taste-matched placebo for 28 days in individuals with cystic fibrosis. In addition, both groups will receive oral rifampin, a second oral antibiotic – trimethoprim/sulfamethoxazole (TMP/SMX) or doxycycline, protocol determined – mupirocin intranasal cream, and chlorhexidine body washes. Forty patients with persistent respiratory tract MRSA infection will be enrolled: 20 will be randomized to vancomycin for inhalation and 20 to a taste-matched placebo. The primary outcome will be the presence of MRSA in sputum respiratory tract cultures 1 month after the conclusion of treatment. Secondary outcomes include the efficacy of the intervention on: FEV1% predicted, patient reported outcomes, pulmonary exacerbations, and MRSA colony-forming units found in respiratory tract sample culture.
Results of this study will provide guidance to clinicians regarding the safety and effectiveness of a targeted eradication strategy for persistent MRSA infection in CF.
This trial is registered at ClinicalTrials.gov (NCT01594827, received 05/07/2012) and is funded by the Cystic Fibrosis Foundation (Grants: PMEP10K1 and PMEP11K1).
Cystic fibrosis; MRSA; Randomized controlled trial; Vancomycin
This cross-sectional study examined associations of occupational tasks with radiographic and symptomatic osteoarthritis (OA) in a community-based sample.
Participants from the Johnston County Osteoarthritis Project (n = 2729) self-reported the frequency of performing 10 specific occupational tasks at the longest job ever held (never/seldom/sometimes vs often/always) and lifetime exposure to jobs that required spending > 50% of their time doing 5 specific tasks or lifting 22, 44, or 110 pounds 10 times weekly. Multivariable logistic regression models examined associations of each occupational task separately with radiographic and symptomatic knee and hip OA, controlling for age, race, gender, body mass index, prior knee or hip injury, and smoking.
Radiographic hip and knee OA were not significantly associated with any occupational tasks, but several occupational tasks were associated with increased odds of both symptomatic knee and hip OA: lifting > 10 pounds, crawling, and doing heavy work while standing (OR 1.4–2.1). More occupational walking and standing and less sitting were also associated with symptomatic knee OA, and more bending/twisting/reaching was associated with symptomatic hip OA. Exposure to a greater number of physically demanding occupational tasks at the longest job was associated with greater odds of both symptomatic knee and hip OA.
Our results confirm an association of physically demanding occupational tasks with both symptomatic knee and hip OA, including several specific activities that increased the odds of OA in both joint groups. These tasks represent possibilities for identifying and targeting at-risk individuals with preventive interventions.
OSTEOARTHRITIS; OCCUPATIONS; HIP; KNEE
Dementia affects over 4 million people in the US and is frequently unrecognized and underdiagnosed in primary care. Routine dementia screening in primary care is not recommended by the US Preventive Services Task Force due to lack of empirical data on the benefits and harms of screening. This trial seeks to fill this gap and contribute information about the benefits, harms, and costs of routine screening for dementia in primary care.
Single-blinded, parallel, randomized controlled clinical trial with 1:1 allocation. A total of 4,000 individuals aged ≥65 years without a diagnosis of dementia, cognitive impairment, or serious mental illness receiving care at primary care practices within two cities in Indiana. Subjects will be randomized to either i) screening for dementia using the Memory Impairment Screen Telephone version or ii) no screening for dementia. Subjects who screen positive for dementia will be referred to the local Aging Brain Care program that delivers an evidence-based collaborative care model for dementia and depression. Research assistants will administer the 15-item Health Utility Index, Patient Health Questionnaire, Generalized Anxiety Disorder Scale, and Medical Outcomes Study at baseline, 1, 6, and 12 months. Information about advanced care planning will be collected at baseline and 12 months. All enrollees’ medical records will be reviewed to collect data on health care utilization and costs.
We have two primary hypotheses; first, in comparison to non-screened subjects, those who are screened and referred to a dementia collaborative care program will have a higher health-related quality of life as measured by the Health Utility Index at 12 months post-screening. Second, in comparison to non-screened subjects, those who are screened and referred to a dementia collaborative care program will not have higher depression or anxiety at one month post-screening as measured by the Patient Health Questionnaire and Generalized Anxiety Disorder Scale scales. Our secondary hypothesis is that screened subjects will have an Incremental Cost-Effectiveness Ratio below the maximum acceptable threshold of $60,000 per quality adjusted life year saved at 12 months.
Ongoing; registered on September 19, 2012. ClinicalTrials.gov Identifier: 2012 NCT01699503.
Alzheimer’s disease; Dementia screening; Dementia; Primary care
The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface.
The unregulated activity of inteins during expression and consequent side reactions during work-up limits their widespread use in biotechnology and chemical biology. Therefore, we exploited a mechanism-based approach to regulate intein autocatalysis for biotechnological application. The system, inspired by our previous structural studies, is based on reversible trapping of the intein’s catalytic cysteine residue through a disulfide bond. Using standard mutagenesis, the disulfide trap can be implemented to impart redox control over different inteins and for a variety of applications both in vitro and in Escherichia coli. Thereby, we first enhanced the output for bioconjugation in intein-mediated protein ligation, also referred to as expressed protein ligation, where precursor recovery and product yield were augmented fourfold to sixfold. Second, in bioseparation experiments, the redox trap boosted precursor recovery and product yield twofold. Finally, the disulfide-trap intein technology stimulated development of a novel bacterial redox sensor. This sensor reliably identified hyperoxic E. coli harboring mutations that disrupt the reductive pathways for thioredoxin and glutathione, against a background of wild-type cells.
redox regulation; expressed protein ligation; bioseparations; redox sensor
To examine cross-sectional baseline data from the Johnston County Osteoarthritis Project for the association between individual and community socioeconomic status (SES) measures with hip osteoarthritis (OA) outcomes.
We analyzed data on 3,087 individuals (68% Caucasian and 32% African American). Educational attainment and occupation were used as individual measures of SES. Census block group household poverty rate was used as a measure of community SES. Hip OA outcomes included radiographic OA (rOA) and symptomatic OA (sxOA) in one or both hip joints. Multivariable logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CI) for the association of each hip OA outcome with each SES variable separately, then with all SES measures simultaneously. Associations between hip OA outcomes and SES variables were evaluated for effect modification by race and gender.
Living in a community of high household poverty rate showed independent associations with hip rOA in one or both hips (OR=1.50; 95% CI=1.18–1.92) and bilateral (both hips) rOA (OR=1.87; 95% CI=1.32–2.66). Similar independent associations were found between low educational attainment among those with sxOA in one or both hips (OR=1.44; 95% CI=1.09, 1.91) or bilateral sxOA (OR=1.91; 95% CI=1.08–3.39), after adjusting for all SES measures simultaneously. No significant associations were observed between occupation and hip OA outcomes, nor did race or gender modify the associations.
Our data provide evidence that hip OA outcomes are associated with both education and community SES measures, associations which remained after adjustment for covariates and all SES measures.
The effectiveness of any new technology is typically measured in order to determine whether it successfully achieves equal or superior objectives over what is currently offered. Research in telemental health—in this article mainly referring to telepsychiatry and psychological services—has advanced rapidly since 2003, and a new effectiveness review is needed.
Materials and Methods:
The authors reviewed the published literature to synthesize information on what is and what is not effective related to telemental health. Terms for the search included, but were not limited to, telepsychiatry, effectiveness, mental health, e-health, videoconferencing, telemedicine, cost, access, and international.
Telemental health is effective for diagnosis and assessment across many populations (adult, child, geriatric, and ethnic) and for disorders in many settings (emergency, home health) and appears to be comparable to in-person care. In addition, this review has identified new models of care (i.e., collaborative care, asynchronous, mobile) with equally positive outcomes.
Telemental health is effective and increases access to care. Future directions suggest the need for more research on service models, specific disorders, the issues relevant to culture and language, and cost.
telepsychiatry; effectiveness; telemental health; videoconferencing; telemedicine
The relationship between late-life blood pressure (BP) and cognitive function in the elderly is poorly understood. Inconsistent results have been reported from existing studies. We report the results from a prospective cohort study on the association between BP and cognitive function in elderly African Americans.
Prospective cohort study conducted from 1997 to 2009.
Community-based study in Indianapolis.
3145 African Americans aged 65 years or older.
At each assessment, participants’ cognitive function was measured by the Community Screening Interview for Dementia score. Other measurements included BP, height, weight, education level, antihypertensive medication use, alcohol use, smoking and histories of chronic medical conditions.
5995 longitudinal assessments contributed by 2721 participants with complete independent variables were analyzed using a semiparametric mixed effects model. Systolic BP around 135 mmHg and diastolic BP around 80 mmHg were associated with optimal cognitive function after adjusting for other variables (P = 0.019). Weight loss with body mass index less than 30 kg/m2 was significantly related to poorer cognitive performance (P < 0.001). Older age at first assessment, lower education level, smoking, histories of depression, stroke and diabetes mellitus were related to worse cognitive function, while taking antihypertensive medication and drinking alcohol were associated with higher cognitive scores.
Both high and low BP levels were associated with poorer cognitive performance. A joint optimal region of systolic and diastolic BP for cognitive function has been identified, which may provide useful clinical information on optimal BP control in cognitive health and lead to improved quality of life for the elderly.
blood pressure; cognitive function; elderly
Pulmonary vein isolation (PVI) for atrial fibrillation (AF) is associated with a transient increased risk of thromboembolic and hemorrhagic events. We hypothesized that dabigatran can be safely used as an alternative to continuous warfarin for the peri-procedural anticoagulation in PVI.
Methods and Results
999 consecutive patients undergoing PVI were included; 376 patients were on dabigatran (150 mg) and 623 were on warfarin with therapeutic INR. Dabigatran was held 1 to 2 doses prior to PVI and restarted at the conclusion of the procedure or as soon as patients were transferred to the nursing floor. Propensity score matching was applied to generate a cohort of 344 patients in each group with balanced baseline data. Total hemorrhagic and thromboembolic complications were similar in both groups, before (3.2% vs 3.9%; p = 0.59), and after (3.2% vs 4.1%; p = 0.53) matching. Major hemorrhage occurred in 1.1% vs 1.6% (p = 0.48) before, and 1.2% vs 1.5% (p = 0.74) after matching in the dabigatran vs warfarin group respectively. A single thromboembolic event occurred in each of the dabigatran and warfarin groups. Despite higher doses of intra-procedural heparin, the mean ACT was significantly lower in patients who held dabigatran for 1 or 2 doses than those on warfarin.
Our study found no evidence to suggest a higher risk of thromboembolic or hemorrhagic complications with use of dabigatran for peri-procedural anticoagulation in patients undergoing PVI compared to uninterrupted warfarin therapy.
anticoagulants; fibrillation; ablation; catheter ablation; stroke
This multicenter, retrospective analysis assessed the efficacy and safety of ipilimumab in 33 patients with unresectable or metastatic mucosal melanoma. The study provides evidence that ipilimumab can result in durable antitumor effects in a subset of patients with mucosal melanoma, although the response rate was low.
The outcome of patients with mucosal melanoma treated with ipilimumab is not defined. To assess the efficacy and safety of ipilimumab in this melanoma subset, we performed a multicenter, retrospective analysis of 33 patients with unresectable or metastatic mucosal melanoma treated with ipilimumab. The clinical characteristics, treatments, toxicities, radiographic assessment of disease burden by central radiology review at each site, and mutational profiles of the patients' tumors were recorded. Available peripheral blood samples were used to assess humoral immunity against a panel of cancer-testis antigens and other antigens. By the immune-related response criteria of the 30 patients who underwent radiographic assessment after ipilimumab at approximately week 12, there were 1 immune-related complete response, 1 immune-related partial response, 6 immune-related stable disease, and 22 immune-related progressive disease. By the modified World Health Organization criteria, there were 1 immune-related complete response, 1 immune-related partial response, 5 immune-related stable disease, and 23 immune-related progressive disease. Immune-related adverse events (as graded by Common Terminology Criteria for Adverse Events version 4.0) consisted of six patients with rash (four grade 1, two grade 2), three patients with diarrhea (one grade 1, two grade 3), one patient with grade 1 thyroiditis, one patient with grade 3 hepatitis, and 1 patient with grade 2 hypophysitis. The median overall survival from the time of the first dose of ipilimumab was 6.4 months (range: 1.8–26.7 months). Several patients demonstrated serologic responses to cancer-testis antigens and other antigens. Durable responses to ipilimumab were observed, but the overall response rate was low. Additional investigation is necessary to clarify the role of ipilimumab in patients with mucosal melanoma.
Mucosal melanoma; Ipilimumab; CTLA-4; Immunotherapy; Cancer-testis antigens
Homebound seniors suffer from high levels of functional impairment and are high-cost users of acute medical services. This article describes a 7-year experience in building and sustaining a physician home visit program. The House Calls for Seniors program was established in 1999. The team includes a geriatrician, geriatrics nurse practitioner, and social worker. The program hosts trainees from multiple disciplines. The team provides care to 245 patients annually. In 2006, the healthcare system (62%), provider billing (36%), and philanthropy (2%) financed the annual program budget of $355,390. Over 7 years, the team has enrolled 468 older adults; the mean age was 80, 78% were women, and 64% were African American. One-third lived alone, and 39% were receiving Medicaid. Reflecting the disability of this cohort, 98% had impairment in at least one instrumental activity of daily living (mean 5.2), 71% had impairment in at least one activity of daily living (mean 2.6), 53% had a Mini-Mental State Examination score of 23 or less, 43% were receiving services from a home care agency, and 69% had at least one new geriatric syndrome diagnosed by the program. In the year after intake into the program, patients had an average of nine home visits; 21% were hospitalized, and 59% were seen in the emergency department. Consistent with the program goals, primary care, specialty care, and emergency department visits declined in the year after enrollment, whereas access and quality-of-care targets improved. An academic physician house calls program in partnership with a healthcare system can improve access to care for homebound frail older adults, improve quality of care and patient satisfaction, and provide a positive learning experience for trainees.
home visits; house calls; physicians
We examined whether occupational and household tasks contributed to differences in pain between African Americans and whites with radiographic knee osteoarthritis (OA).
Participants from the Johnston County Osteoarthritis Project self-reported the frequency (often/always vs never/seldom/sometimes) of performing 9 occupational tasks involving lower extremity joint loading at their longest job (N = 868) and current job (N = 273), as well as 8 household tasks ever performed (N = 811) and currently being performed (N = 767). The associations of the numbers of occupational or household tasks with the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain subscale were first examined in simple linear regression models. If significantly associated with greater pain, each of these was included in adjusted linear regression models to examine whether the association of race with pain remained statistically significant.
African Americans reported significantly greater WOMAC pain scores than whites. Exposures to more occupational tasks at the longest job and the current job were associated with greater WOMAC pain scores (p < 0.01). The association of race with greater pain scores remained statistically significant when controlling for occupational tasks at the longest job, but was reduced by 26% and no longer significant when controlling for the number of current occupational tasks. Exposures to an increasing number of household tasks were associated with lower pain scores and were not further analyzed.
Current performance of physically demanding occupational tasks contributed to racial differences in pain severity among individuals with knee OA. Better workplace policies to accommodate OA-related limitations may help to reduce racial differences in pain.
OSTEOARTHRITIS; OCCUPATIONS; KNEE
In this work, we developed a sensitive method to quantify cotinine (COT), norcotinine (NCOT), trans-3′-hydroxycotinine (OHCOT) and cotinine-N-oxide (COTNO) in rat plasma and brain tissue, using solid phase extraction (SPE), hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS). The linear range was 1–100 ng/ml for each analyte in rat plasma and brain homogenate (3–300 ng/g brain tissue). The method was validated with precision within 15% relative standard deviation (RSD) and accuracy within 15% relative error (RE). Stable isotope-labeled internal standards (IS) were used for all the analytes to achieve good reproducibility, minimizing the influence of recovery and matrix effects. This method can be used in future studies to simultaneously determine the concentrations of COT and three major metabolites in rat plasma and brain tissue.
cotinine; norcotinine; trans-3′-hydroxycotinine; cotinine-N-oxide; plasma; brain; hydrophilic interaction chromatography; tandem mass spectrometry
Deficiencies of lysosomal β-D-galactosidase can result in GM1 gangliosidosis, a severe neurodegenerative disease characterized by massive neuronal storage of GM1 ganglioside in the brain. Currently there are no available therapies that can even slow the progression of this disease. Enzyme enhancement therapy utilizes small molecules that can often cross the blood brain barrier, but are also often competitive inhibitors of their target enzyme. It is a promising new approach for treating diseases, often caused by missense mutations, associated with dramatically reduced levels of functionally folded enzyme. Despite a number of positive reports based on assays performed with patient cells, skepticism persists that an inhibitor-based treatment can increase mutant enzyme activity in vivo. To date no appropriate animal model, i.e., one that recapitulates a responsive human genotype and clinical phenotype, has been reported that could be used to validate enzyme enhancement therapy. In this report, we identify a novel enzyme enhancement-agent, N-nonyl-deoxygalactonojirimycin, that enhances the mutant β-galactosidase activity in the lysosomes of a number of patient cell lines containing a variety of missense mutations. We then demonstrate that treatment of cells from a previously described, naturally occurring feline model (that biochemically, clinically and molecularly closely mimics GM1 gangliosidosis in humans) with this molecule, results in a robust enhancement of their mutant lysosomal β-galactosidase activity. These data indicate that the feline model could be used to validate this therapeutic approach and determine the relationship between the disease stage at which this therapy is initiated and the maximum clinical benefits obtainable.
PMID: 22784478 CAMSID: cams2306
Lysosomal storage disease; β-D-galactosidase; Morquio disease type B; Endoplasmic reticulum quality control; Large animal model; Small molecule therapy
A major consequence of ICU-acquired weakness (ICUAW) is diaphragm weakness, which prolongs the duration of mechanical ventilation. Hyperglycemia (HG) is a risk factor for ICUAW. However, the mechanisms underlying HG-induced respiratory muscle weakness are not known. Excessive reactive oxygen species (ROS) injure multiple tissues during HG, but only one study suggests that excessive ROS generation may be linked to HG-induced diaphragm weakness. We hypothesized that HG-induced diaphragm dysfunction is mediated by excessive superoxide generation and that administration of a specific superoxide scavenger, polyethylene glycol superoxide dismutase (PEG-SOD), would ameliorate these effects.
HG was induced in rats using streptozotocin (60 mg/kg intravenously) and the following groups assessed at two weeks: controls, HG, HG + PEG-SOD (2,000U/kg/d intraperitoneally for seven days), and HG + denatured (dn)PEG-SOD (2000U/kg/d intraperitoneally for seven days). PEG-SOD and dnPEG-SOD were administered on day 8, we measured diaphragm specific force generation in muscle strips, force-pCa relationships in single permeabilized fibers, contractile protein content and indices of oxidative stress.
HG reduced diaphragm specific force generation, altered single fiber force-pCa relationships, depleted troponin T, and increased oxidative stress. PEG-SOD prevented HG-induced reductions in diaphragm specific force generation (for example 80 Hz force was 26.4 ± 0.9, 15.4 ± 0.9, 24.0 ± 1.5 and 14.9 ± 0.9 N/cm2 for control, HG, HG + PEG-SOD, and HG + dnPEG-SOD groups, respectively, P <0.001). PEG-SOD also restored HG-induced reductions in diaphragm single fiber force generation (for example, Fmax was 182.9 ± 1.8, 85.7 ± 2.0, 148.6 ± 2.4 and 90.9 ± 1.5 kPa in control, HG, HG + PEG-SOD, and HG + dnPEG-SOD groups, respectively, P <0.001). HG-induced troponin T depletion, protein nitrotyrosine formation, and carbonyl modifications were largely prevented by PEG-SOD.
HG-induced reductions in diaphragm force generation occur largely at the level of the contractile proteins, are associated with depletion of troponin T and increased indices of oxidative stress, findings not previously reported. Importantly, administration of PEG-SOD largely ablated these derangements, indicating that superoxide generation plays a major role in hyperglycemia-induced diaphragm dysfunction. This new mechanistic information could explain how HG alters diaphragm function during critical illness.
The association of trauma and PTSD with alcohol and cocaine use is explored to determine if there is additive risk associated with dual dependence. Data were collected from out-of-treatment women enrolled in an HIV-prevention study. Women who experienced a DSM-IV qualifying event (N=791) were stratified into four substance use groups based on lifetime alcohol and cocaine use. Women with lifetime co-morbid alcohol and cocaine dependence experienced significantly more traumatic events, had a higher prevalence of violent events and lifetime diagnosis of PTSD and PTSD-related impairment. There is added risk for associated trauma and subsequent PTSD among women who have dual substance dependence.
The Disease Activity Score based on 28 joints (DAS28) has been increasingly used in clinical practice and research studies of rheumatoid arthritis (RA). Studies have reported discordance between DAS28 based on erythrocyte sedimentation rate (ESR) versus C-reactive protein (CRP) in RA patients. However such comparison is lacking in African-Americans with RA.
This analysis included participants from the Consortium for the Longitudinal Evaluation of African Americans with Early Rheumatoid Arthritis (CLEAR) Registry which enrolls self-declared African-Americans with RA. Using tender and swollen joint counts separate ESR-based and CRP-based DAS28 scores (DAS28-ESR3 and DAS28-CRP3) were calculated, as were DAS28-ESR4 and DAS28-CRP4, which included the patient’s assessment of disease activity. The scores were compared using paired t-test, simple agreement and kappa, correlation coefficient and Bland-Altman plots.
Of the 233 included participants, 85% were women, mean age at enrollment was 52.6 years, and median disease duration at enrollment was 21 months. Mean DAS28-ESR3 was significantly higher than DAS28-CRP3 (4.8 vs. 3.9; p<0.001). Similarly, mean DAS28-ESR4 was significantly higher than DAS28-CRP4 (4.7 vs. 3.9; p<0.001). ESR-based DAS28 remained higher than CRP-based DAS28 even when stratified by age, sex, and disease duration. Overall agreement was not high between DAS28-ESR3 and DAS28-CRP3 (50%) or between DAS28-ESR4 and DAS28-CRP4 (59%). DAS28-CRP3 underestimated disease activity in 47% of the participants relative to DAS28-ESR3 and DAS28-CRP4 in 40% of the participants relative to DAS28-ESR4.
There was significant discordance between the ESR-based and CRP-based DAS28 which could impact clinical treatment decisions in African-Americans with RA.
DAS28; Rheumatoid Arthritis; African-Americans
Uveal melanoma exhibits a high incidence of metastases and no systemic therapy clearly improves outcomes. The anti-CTLA-4 antibody ipilimumab is a standard of care for metastatic melanoma; however, the clinical activity of CTLA-4 inhibition in patients with metastatic uveal melanoma is poorly defined.
To assess ipilimumab in this setting, we performed a multicenter, retrospective analysis of four hospitals in the United States and Europe. Clinical characteristics, toxicities and radiographic disease burden as determined by central, blinded radiology review were determined.
Thirty-nine patients were identified (34 treated with 3 mg/kg and 5 treated with 10 mg/kg). Using the immune-related response criteria and modified WHO criteria, response rate (RR) and combined response plus stable disease (SD) rate were assessed after 12 weeks, 23 weeks and total (median follow-up 50.4 weeks (12.6 months)). At week 12, the RR and response plus SD rate were 2.6% and 46.0%, at week 23: 2.6% and 28.2%. There was one complete response and one late partial response (at 100 weeks after initial SD) for irRR of 5.1%. Immune-related adverse events (irAE) were observed in 28 (71.8%) patients, with seven (17.9%) grade 3-4 events. irAEs were more frequent in patients receiving 10 mg/kg versus 3 mg/kg. The median overall survival from first dose of ipilimumab was 9.6 months (confidence interval 6.3-13.4 months, range: 1.6-41.6 months). Performance status, LDH and absolute lymphocyte count ≥1000 cells/μL at week 7 were significantly associated with survival.
In uveal melanoma, durable responses to ipilimumab and manageable toxicity were observed.
uveal melanoma; ipilimumab; CTLA-4; immunotherapy; absolute lymphocyte count