Search

Antibodies can undergo a variety of covalent and non-covalent degradation reactions that have adverse effects on efficacy, safety, manufacture and storage. We had identified an antibody to Angiopoietin 2 (Ang2 mAb) that neutralizes Ang2 binding to its receptor in vitro and inhibits tumor growth in vivo. Despite favorable pharmacological activity, the Ang2 mAb preparations were heterogeneous, aggregated rapidly and were poorly expressed. Here, we report the engineering of the antibody variable and constant domains to generate an antibody with reduced propensity to aggregate, enhanced homogeneity, 11°C elevated T(m), 26-fold improved level of expression and retained activity. The engineered molecule, MEDI-3617, is now compatible with the large scale material supply required for clinical trials and is currently being evaluated in Phase 1 in cancer patients. This is the first report to describe the stability engineering of a therapeutic antibody addressing non canonical cysteine residues and the design strategy reported here is generally applicable to other therapeutic antibodies and proteins.

Thirty-three infants aged ∼2 months had serial stool samples collected after receipt of Rotarix® vaccine dose 1, and were assessed for shedding of porcine circovirus type 1 DNA and Rotavirus group A RNA by molecular methods. We did not find strong evidence that porcine circovirus type 1 replication occurred. Porcine circovirus type 1 genome with the same sequence as that in Rotarix® was detected in a few infants as late as day ≥ 13; while this timing could suggest there may have been replication and not just transient passage through the gastrointestinal tract, the lack of increase in copy number in any infant supports transient passage and there are inherent limitations to the results. We found that 21% of infants did not shed Rotarix® RVA RNA beyond the day 3 sample, which may suggest lack of vaccine virus replication. Of the infants in whom Rotarix RVA RNA shedding continued, peak copy numbers were reached on days 3–5 for ∼40%, and after day 5 in ∼60%, and shedding can be prolonged (≥ 45 days).

Background Nonsevere diarrheal disease in Nepal represents a large burden of illness. Identification of the specific disease-causing pathogens will help target the appropriate control measures. Methods Infants aged 6 weeks to 12 months were recruited from 5 health facilities in eastern, central, and western Nepal between August 2012 and August 2013. The diarrhea arm included infants with mild or moderate diarrhea treatable in an outpatient setting; the nondiarrhea arm included healthy infants who presented for immunization visits or had a mild nondiarrheal illness. Stool samples were tested for 15 pathogens with a multiplex polymerase chain reaction (PCR) assay and real-time reverse-transcription (RT)-PCR assays for rotavirus and norovirus. Rotavirus- and norovirus-positive specimens were genotyped. We calculated attributable fractions (AFs) to estimate the pathogen-specific burden of diarrhea and adjusted for facility, age, stunting, wasting, and presence of other pathogens. Results We tested 307 diarrheal and 358 nondiarrheal specimens. Pathogens were detected more commonly in diarrheal specimens (164 of 307 [53.4%]) than in nondiarrheal specimens (113 of 358 [31.6%]) (P < .001). Rotavirus (AF, 23.9% [95% confidence interval (CI), 14.9%-32.8%]), Salmonella (AF, 12.4% [95% CI, 6.6%-17.8%]), and Campylobacter (AF, 5.6% [95% CI, 1.3%-9.8%]) contributed most to the burden of disease. In these diarrheal specimens, the most common genotypes for rotavirus were G12P[6] (27 of 82 [32.9%]) and G1P[8] (16 of 82 [19.5%]) and for norovirus were GII.4 Sydney (9 of 26 [34.6%]) and GII.7 (5 of 26 [19.2%]). Conclusions The results of this study indicate that the introduction of a rotavirus vaccine in Nepal will likely decrease outpatient diarrheal disease burden in infants younger than 1 year, but interventions to detect and target other pathogens, such as Salmonella and Campylobacter spp, should also be considered.

Affinity- and stability-engineered variants of CTLA4-Ig fusion molecules with enhanced pharmacokinetic profiles could yield improved therapies with the potential of higher efficacy and greater convenience to patients. In this study, to our knowledge, we have, for the first time, used in vitro evolution to simultaneously optimize CTLA4 affinity and stability. We selected for improved binding to both ligands, CD80 and CD86, and screened as dimeric Fc fusions directly in functional assays to identify variants with stronger suppression of in vitro T cell activation. The majority of CTLA4 molecules showing the largest potency gains in primary in vitro and ex vivo human cell assays, using PBMCs from type 1 diabetes patients, had significant improvements in CD80, but only modest gains in CD86 binding. We furthermore observed different potency rankings between our lead molecule MEDI5265, abatacept, and belatacept, depending on which type of APC was used, with MEDI5265 consistently being the most potent. We then created fusions of both stability- and potency-optimized CTLA4 moieties with human Fc variants conferring extended plasma t1/2. In a cynomolgus model of T cell–dependent Ab response, the CTLA4-Ig variant MEDI5265 could be formulated at >100 mg/ml for s.c. administration and showed superior efficacy and significantly prolonged serum t1/2. The combination of higher stability and potency with prolonged pharmacokinetics could be compatible with very infrequent, s.c. dosing while maintaining a similar level of immune suppression to more frequently and i.v. administered licensed therapies.

Background Group A rotaviruses (RVA) are a significant cause of pediatric gastroenteritis worldwide. The New Vaccine Surveillance Network (NVSN) has conducted active surveillance for RVA at pediatric hospitals and emergency departments at 3-7 geographically diverse sites in the United States since 2006. Methods Over 6 consecutive years, from 2008 to 2013, 1523 samples from NVSN sites that were tested positive by a Rotaclone enzyme immunoassay were submitted to the Centers for Disease Control and Prevention for genotyping. Results In the 2009, 2010, and 2011 seasons, genotype G3P[8] was the predominant genotype throughout the network, with a 46%-84% prevalence. In the 2012 season, G12P[8] replaced G3P[8] as the most common genotype, with a 70% prevalence, and this trend persisted in 2013 (68.0% prevalence). Vaccine (RotaTeq; Rotarix) strains were detected in 0.6%-3.4% of genotyped samples each season. Uncommon and unusual strains (eg, G8P[4], G3P[24], G2P[8], G3P[4], G3P[6], G24P[14], G4P[6], and G9P[4]) were detected sporadically over the study period. Year, study site, and race were found to be significant predictors of genotype. Conclusions Continued active surveillance is needed to monitor RVA genotypes in the United States and to detect potential changes since vaccine licensure.

To compare the effectiveness of different approaches to nutrition education in diabetes self-management education and support (DSME/S).We randomized 150 adults with type 2 diabetes to either certified diabetes educator (CDE)-delivered DSME/S with carbohydrate gram counting or the modified plate method versus general health education. The primary outcome was change in HbA1C over 6 months.At 6 months, HbA1C improved within the plate method [-0.83% (-1.29, -0.33), P0.001] and carbohydrate counting [-0.63% (-1.03, -0.18), P=0.04] groups but not the control group [P=0.34]. Change in HbA1C from baseline between the control and intervention groups was not significant at 6 months (carbohydrate counting, P=0.36; modified plate method, P=0.08). In a pre-specified subgroup analysis of patients with a baseline HbA1C 7-10%, change in HbA1C from baseline improved in the carbohydrate counting [-0.86% (-1.47, -0.26), P=0.006] and plate method groups [-0.76% (-1.33, -0.19), P=0.01] compared to controls.CDE-delivered DSME/S focused on carbohydrate counting or the modified plate method improved glycemic control in patients with an initial HbA1C between 7 and 10%.Both carbohydrate counting and the modified plate method improve glycemic control as part of DSME/S.

Systemic sclerosis (SSc) is a debilitating inflammatory and fibrotic disease that affects the skin and internal organs. Although the pathophysiology of SSc remains poorly characterized, mononuclear cells, mainly macrophages and T cells, have been implicated in inflammation and fibrosis. Inducible costimulator (ICOS), which is expressed on a subset of memory T helper (TH) and T follicular helper (TFH) cells, has been shown to be increased in SSc and associated with disease pathology. However, the identity of the relevant ICOS+ T cells and their contribution to inflammation and fibrosis in SSc are still unknown. We show that CD4+ ICOS-expressing T cells with a TFH-like phenotype infiltrate the skin of patients with SSc and are correlated with dermal fibrosis and clinical disease status. ICOS+ TFH-like cells were found to be increased in the skin of graft-versus-host disease (GVHD)-SSc mice and contributed to dermal fibrosis via an interleukin-21- and matrix metalloproteinase 12-dependent mechanism. Administration of an anti-ICOS antibody to GVHD-SSc mice prevented the expansion of ICOS+ TFH-like cells and inhibited inflammation and dermal fibrosis. Interleukin-21 neutralization in GVHD-SSc mice blocked disease pathogenesis by reducing skin fibrosis. These results identify ICOS+ TFH-like profibrotic cells as key drivers of fibrosis in a GVHD-SSc model and suggest that inhibition of these cells could offer therapeutic benefit for SSc.

The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.

Organizations have different levels of readiness to implement change in the patient care process. The Hypertension Telemedicine Nurse Implementation Project for Veterans (HTN‐IMPROVE) is an example of an innovation that seeks to enhance delivery of care for patients with hypertension. We describe the link between organizational readiness for change (ORC), assessed as the project began, and barriers and facilitators occurring during the process of implementing a primary care innovation. Each of 3 Veterans Affairs medical centers provided a half‐time nurse and implemented a nurse‐delivered, telephone‐based self‐management support program for patients with uncontrolled hypertension. As the program was starting, we assessed the ORC and factors associated with ORC. On the basis of consensus of medical center and research partners, we enumerated implementation process barriers and facilitators. The primary ORC barrier was unclear long‐term commitment of nursing to provide continued resources to the program. Three related barriers included the need to address: (1) competing organizational demands, (2) differing mechanisms to integrate new interventions into existing workload, and (3) methods for referring patients to disease and self‐management support programs. Prior to full implementation, however, stakeholders identified a high level of commitment to conduct nurse‐delivered interventions fully using their skills. There was also a significant commitment from the core implementation team and a desire to improve patient outcomes. These facilitators were observed during the implementation of HTN‐IMPROVE. As demonstrated by the link between barriers to and facilitators of implementation anticipated though the evaluation of ORC and what was actually observed during the process of implementation, this project demonstrates the practical utility of assessing ORC prior to embarking on the implementation of significant new clinical innovations.

Oxytocin is a neuropeptide released during birth and lactation, but also in response to social interaction and stressors. Exogenously administered oxytocin in animals augments acute drug effects, drug reward, tolerance, and withdrawal for a range of drugs and alcohol. The effects of oxytocin administration can be directly caused by oxytocin or by its bilateral interactions with numerous systems, including the dopamine system, hypothalamic–pituitary–adrenal axis, and immune system. The status of the endogenous oxytocin system might also enhance or reduce susceptibility to addiction through its interaction with these systems. Individual differences in the endogenous oxytocin system may therefore affect susceptibility to addiction. Consistent with this notion, preclinical research has demonstrated that boosting the activity of the endogenous oxytocin system might “inoculate” against future vulnerability to drug abuse. The first human trials of intranasal oxytocin in the treatment of addiction are now under way and the results are eagerly awaited.

Laboratory rats display pronounced defensive behaviors when confronted with a range of cat-derived stimuli, including collars worn by a cat, cloths rubbed on a cat, and cat fur. One possible explanation of this phenomenon (the “kairomone hypothesis”) is that rats derive a survival advantage by eavesdropping on signals used by cats to communicate with each other. Cats are known to rub their bodies on objects at strategic environmental locations to signal their identity and mating potential to other cats. The current study assessed the sensitivity of laboratory rats to these body rubbings. In Experiment 1, food deprived Sprague–Dawley rats were trained to consume food pellets in one arm of a Y maze. On test day a damp cloth was placed near the food pellets that had been rubbed on a location (wall) where a cat had recently engaged in body rubbing. A control cloth and a collar worn by the cat were also tested. The presence of both the body rubbing residue and the cat collar increased latency to eat and decreased amount of food eaten. The disruption of consummatory behavior in the test environment was still evident 24 h later in the absence of odor stimuli. Experiment 2 tested the reaction of naive Wistar rats to body rubbings using a paradigm in which rats were given the opportunity to hide. Relative to a control condition, rats exposed to a cotton pad wiped on a cat body rubbing location showed increased hiding behavior, decreased exploration and reduced stimulus approach and investigation. These defensive responses persisted for up to 4 days following a single stimulus exposure. These results suggest that rats eavesdrop readily on body rubbings cats use for identification purposes, providing further support for a kairomone hypothesis of predator odor avoidance.

The quality of sequencing results depends greatly upon the quality and purity of the template as well as the purity of the fluorescently labeled products generated by cycle sequencing. Numerous approaches have been used for purification of cycle sequencing products, including alcohol precipitation, affinity-based chromatography, size exclusion chromatography, commercially-available proprietary methods, and paramagnetic bead technology. In this paper, we describe an affordable paramagnetic technology method using BioMag Carboxyl beads. Compared to other well-established, proprietary methods for purification of cycle sequencing products, this method produced consistently good results, with a very low reagent cost and short procedure time.

In many prey species aggregation of individuals is a defensive strategy commonly employed in response to predators and predator-related cues. However, very little work has explored this adaptive response in laboratory rats. It is known that individual rats show characteristic defensive responses to predator odors, such as hiding, avoidance, inhibition of foraging, feeding and reproduction, and risk assessment directed toward the odor source. However, whether these species-typical responses in individuals are altered in the presence of other conspecifics is yet to be characterized. The present study therefore examined the defensive response of groups of two rats (dyads) or four rats (quads) to two unconditioned stressors: bright ambient light and cat odor (a 2 g ball of cat fur). The dyads and quads were formed from familiar cage mates and test sessions (20 min) occurred in a large open arena (1200 mm2) to which the rats had been extensively habituated under dark conditions. The results showed that when quads of rats were exposed to either cat odor or bright light in this arena, they showed characteristic increases in close social proximity, termed “huddling”. A tight grouping of 3 (triplet) or 4 (quad) rats was commonly seen in response to cat fur, while triplets were more commonly seen in response to bright light. Interestingly there was no evidence for increased social proximity in dyads exposed to either stressor, only in quads. However, cat odor caused other signs of fear (such as decreased locomotor activity and increased defecation) in both quads and dyads. It is concluded that huddling is a rodent defensive strategy in rats when anxiogenic stimuli are encountered by larger groups of rats.

In 2009, three children were hospitalized in Rochester, NY, with sequence-confirmed G8P[4] rotavirus gastroenteritis—the first U.S. detection of this uncommon strain more typically found in Africa. Continued monitoring of G8P[4] and other rotavirus genotypes not represented in current vaccines is essential to assess whether vaccination will result in an increase in prevalence of these strains.

OBJECTIVE To test the association of family history of diabetes with the adoption of diabetes risk–reducing behaviors and whether this association is strengthened by physician advice or commonly known factors associated with diabetes risk. RESEARCH DESIGN AND METHODS We used cross-sectional data from the 2005–2008 National Health and Nutrition Examination Survey (NHANES) to examine the effects of family history of diabetes on the adoption of selected risk-reducing behaviors in 8,598 adults (aged ≥20 years) without diabetes. We used multiple logistic regression to model three risk reduction behaviors (controlling or losing weight, increasing physical activity, and reducing the amount of dietary fat or calories) with family history of diabetes. RESULTS Overall, 36.2% of U.S. adults without diabetes had a family history of diabetes. Among them, ~39.8% reported receiving advice from a physician during the past year regarding any of the three selected behaviors compared with 29.2% of participants with no family history (P < 0.01). In univariate analysis, adults with a family history of diabetes were more likely to perform these risk-reducing behaviors compared with adults without a family history. Physician advice was strongly associated with each of the behavioral changes (P < 0.01), and this did not differ by family history of diabetes. CONCLUSIONS Familial risk for diabetes and physician advice both independently influence the adoption of diabetes risk–reducing behaviors. However, fewer than half of participants with familial risk reported receiving physician advice for adopting these behaviors.

Mephedrone (4-methylmethcathinone) is a novel recreational drug that has rapidly increased in popularity in recent years. Users report mephedrone as having the stimulant-like qualities of methamphetamine and cocaine, combined with the prosocial, entactogenic effects of 3,4-methylenedioxymethamphetamine (MDMA). Anecdotal and case study reports indicate that mephedrone may have the potential to engender compulsive patterns of use as well as toxicity in overdose. However, there have been almost no neuropharmacological investigations of the drug up to this point. Here we examined the effects of two different mephedrone doses [15 and 30 mg/kg, intraperitoneal (IP)] relative to the well-known stimulant methamphetamine (2 mg/kg IP) in adolescent rats. Rats were injected, assessed for locomotor activity for 60 minutes and then tested in a 10-minute social preference test (measuring time spent in close proximity to a real rat versus a dummy rat). Their brains were then processed using Fos immunohistochemistry to determine patterns of brain activation. Results showed that mephedrone caused profound locomotor hyperactivity at both dose levels while tending to reduce social preference. Patterns of Fos expression with mephedrone resembled a combination of those observed with methamphetamine and MDMA, with particularly strong Fos expression in the cortex, dorsal and ventral striatum, ventral tegmental area (typical of both MDMA and methamphetamine) and supraoptic nucleus (typical of MDMA). These results demonstrate for the first time the powerful stimulant effects of mephedrone in animal models and its capacity to activate mesolimbic regions. These results also provide some empirical basis to user reports that mephedrone subjectively resembles a MDMA/methamphetamine hybrid.

Human cluster of differentiation (CD) antigen 19 is a B cell-specific surface antigen and an attractive target for therapeutic monoclonal antibody (mAb) approaches to treat malignancies of B cell origin. MEDI-551 is an affinity-optimized and afucosylated CD19 mAb with enhanced antibody-dependent cellular cytotoxicity (ADCC). The results from in vitro ADCC assays with Natural Killer cells as effector cells, demonstrate that MEDI-551 is effective at lower mAb doses than rituximab with multiple cell lines as well as primary chronic lymphocytic leukaemia and acute lymphoblastic leukaemia samples. Targeting CD19 with MEDI-551 was also effective in several severe combined immunodeficiency lymphoma models. Furthermore, the combination of MEDI-551 with rituximab resulted in prolonged suppression of tumour growth, demonstrating that therapeutic mAbs with overlapping effector function can be combined for greater tumour growth inhibition. Together, the data demonstrate that MEDI-551 has potent antitumour activity in preclinical models of B cell malignancies. The results also suggest that the combination of the ADCC-enhanced CD19 mAb with an anti-CD20 mAb could be a novel approach for the treatment of B cell lymphomas.

The differentiation of therapeutic monoclonal antibodies in an increasingly competitive landscape requires optimization of clinical efficacy combined with increased patient convenience. We describe here the generation of MEDI5117, a human anti-interleukin (IL)-6 antibody generated by variable domain engineering, to achieve subpicomolar affinity for IL-6, combined with Fc (fragment crystallizable) engineering to enhance pharmacokinetic half-life. MEDI5117 was shown to be highly potent in disease-relevant cellular assays. The pharmacokinetics of MEDI5117 were evaluated and compared to those of its progenitor, CAT6001, in a single-dose study in cynomolgus monkeys. The antibodies were administered, either subcutaneously or intravenously, as a single dose of 5 mg/kg. The half-life of MEDI5117 was extended by approximately 3-fold, and clearance was reduced by approximately 4-fold when compared to CAT6001. MEDI5117 therefore represents a potential 'next-generation' antibody; future studies are planned to determine the potential for affinity-driven efficacy and/or less frequent administration.

This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit. The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases of tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at concentrations of ≤10 6 CFU/mL. Real-time PCR analysis using a multitarget 5′ nuclease assay for F. tularensis revealed that the PCR sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4 methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2 automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis .

In late February 2008, law enforcement officials in Las Vegas, Nevada, discovered in a hotel room, a copy of The Anarchist Cookbook, suspected castor beans and a "white powder" thought to be a preparation of ricin. Ricin is a deadly toxin from the seed of the castor bean plant (Ricinus communis). The United States regulates the possession, use, and transfer of ricin and it is the only substance considered a warfare agent in both the Chemical and the Biological Weapons Conventions. Six samples obtained from the hotel room were analyzed by laboratories at the Centers for Disease Control and Prevention using a panel of biological and mass spectrometric assays. The biological assays (real time-PCR, time resolved fluorescence and cytotoxicity) provided presumptive evidence of active ricin in each of the samples. This initial screen was followed by an in-depth analysis using a novel, state-of-the-art mass spectrometry-based ricin functional assay and high sensitivity tandem mass spectrometry for protein identification. Mass spectrometric analysis positively identified ricin and confirmed that in each of the samples it was enzymatically active. The tandem mass spectrometry analysis used here is the most selective method available to detect ricin toxin. In each sample, ricin was unequivocally identified along with other R. communis plant proteins, including the highly homologous protein RCA120. Although database searches using tandem mass spectra acquired from the samples indicated that additional controlled substances were not present in these samples, the mass spectrometric results did provide extensive detail about the sample contents. To the best of our knowledge following a review of the available literature, this report describes the most detailed analysis of a white powder for a public health or forensic investigation involving ricin.

In spite of accelerating human genome discoveries in a wide variety of diseases of public health significance, the promise of personalized health care and disease prevention based on genomics has lagged behind. In a time of limited resources, public health agencies must continue to focus on implementing programs that can improve health and prevent disease now. Nevertheless, public health has an important and assertive leadership role in addressing the promise and pitfalls of human genomics for population health. Such efforts are needed not only to implement what is known in genomics to improve health but also to reduce potential harm and create the infrastructure needed to derive health benefits in the future.

After discovery in the early 1980s, Rotavirus A serotype G9 was detected infrequently for almost a decade. Since the mid-1990s, however, serotype G9 has emerged to become a globally common strain linked to the introduction of a single, new genetic variant of G9 VP7 gene. Studies have demonstrated that genetically divergent G9 strains co-circulated at low frequency with the emerging variants. Examples include unique U.S. G9 strains Om46/Hu/USA/1998 and Om67/Hu/USA/1998, isolated in Omaha during the 1997–1998 rotavirus season, that are more closely related phylogenetically to reference strains from the 1980s than to most emerging G9 strains from the U.S. and globally. Here, we sequenced the VP7 full open reading frame for all available G9 strains (n = 12) identified in Omaha during 1996–2000 seasons to investigate their epidemiology and evolution. In addition, the full or partial length open reading frames of the remaining 10 genes for five divergent Om46-like strains and one modern G9 variant were sequenced to evaluate their potential origin. Our findings suggest that Om46-like G9 strains may have been introduced into humans recently, perhaps in 1997–1998 when it was first detected, and the presumed original host of this VP7 gene variant may have been an animal species based on the unexpected detection of porcine rotavirus related NSP2 gene in the genome. The relatively high fitness of Om46-like strains during the 1997–1998 rotavirus season, 1 year after the globally important G9 variant was documented to be already spreading in the study area and other sites of the United States, appears to parallel findings on seasonal replacement of various genetic and antigenic variants of other common human rotavirus antigen specificities.

Knowledge from early outbreaks is limited regarding the virus detection and illness duration of the 2009 pandemic influenza A (H1N1) infections. During the period from April to May 2009 in Texas, we collected serial nasopharyngeal (NP) and stool specimens from 35 participants, testing by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) and culture. The participants were aged 2 months to 71 years; 25 (71%) were under 18. The median duration of measured fever was 3.0 days and of virus detection in NP specimens was 4.2 days; however, few specimens were collected between days 5-9. The duration of virus detection (4.2 days) was similar to the duration of fever (3.5 days) (RR, 1.14; 95% CI, .66-1.95; P = .8), but was shorter than the duration of cough (11.0 days) (RR, .41; 95% CI, .24―.68; P < .001). We detected viral RNA in two participants' stools. All cultures were negative. This investigation suggests that the duration of virus detection was likely similar to the seasonal influenza virus.

Background Viruses are frequently implicated in acute gastroenteritis (AGE) outbreaks, yet the endemic burden of norovirus and rotavirus disease in adult populations is not well characterized. In 2016, we implemented a multisite AGE surveillance platform capturing cases and controls in 4 VA hospitals (Atlanta, Bronx, Houston, and Los Angeles), collectively serving >320,000 patients annually. Methods Inpatient AGE cases and age- and time-matched controls were identified through prospective screening of admissions via standardized case definitions. Outpatient cases were passively identified using stool samples submitted for routine clinical microbiological diagnostics. Samples were tested with the FilmArray Gastrointestinal Panel, followed by genotyping of virus positives. Incidence was estimated using population denominators of unique patients served annually by site. Results From December 1, 2016 to February 28, 2018, 875 cases (496 inpatients, 379 outpatients), and 374 controls were enrolled. Norovirus and rotavirus prevalence was highest among outpatient AGE cases (11.6% and 2.9%, respectively) followed by inpatient cases (3.4% and 1.6%, respectively); few controls were positive (norovirus, 1.3%; rotavirus, 0%). Norovirus-associated inpatient incidence was 15.2 per 100,000 population (range by site: 10.7–19.9/100,000) and rotavirus-associated inpatient incidence was 7.5 per 100,000 population (range by site: 0–12.8/100,000). The predominant norovirus genotype was GII.P16-GII.4 Sydney (50%), and rotavirus genotype was G12P[8] (83%). Norovirus was detected every calendar month and peaked in December–January, while rotavirus peaked in April. Nine deaths were documented among AGE inpatient cases, including one norovirus-associated death. Conclusion Implementation of a multisite AGE surveillance platform captured a wide spectrum of illness for norovirus and rotavirus in US Veterans including outpatient visits, inpatient hospitalizations, and one norovirus-associated death. Norovirus was the leading viral pathogen and was detected year-round. Ongoing surveillance using this platform will allow for further characterization of the pathogen distribution and associated AGE disease burden in adults. Disclosures V. Marconi, ViiV: Investigator, Research support and Salary. Bayer: Investigator, Research support. Gilead: Investigator, Research support.

JCAR017 is a CD19-directed 41BB chimeric antigen receptor (CAR) T cell product administered in a defined composition at a precise dose of CD8 and CD4 CAR T cells. JCAR017 manufacturing employs process controls that enable precise independent control of the infused dose of CD8 and CD4 cells. Preliminary safety data from JCAR017 administered in relapsed/refractory B cell non-Hodgkin lymphoma (NHL) demonstrated lower rates of cytokine release syndrome (CRS) and neurotoxicity (NT) compared to those reported for other CD19-directed CAR T cell therapies with heterogeneity in total infused dose and CD8 and CD4 composition. Multiple patient factors and blood biomarkers have been identified that correlate with JCAR017 CAR T cell in vivo expansion, antitumor activity, and toxicity (Heipel M et al. and Siddiqi T et al. ASH 2017). However, little is known regarding the contribution of CAR T cell product differentiation state to pharmacokinetics (PK), clinical outcome, or toxicity. Product characterization is executed on JCAR017 to define the CAR T cell memory phenotype composition and antigen-specific function. Product attributes were assessed for relationships with clinical response, safety, and PK through univariate, multivariate, and machine learning-based analyses. JCAR017 memory T cell composition demonstrated strong relationships with cytokine production profile observed following in vitro CD19 stimulation of the CAR T cell drug product. Specifically, products with elevated frequencies of CCR7+ central memory T cells exhibited increased production of IL-2 (Spearman ρ=0.55, P Citation Format: Ryan P. Larson, Rachel Lower, Todd DeVries, Yue Jiang, Ronald J. Hause, Rich Getto, Brian Christin, Nathan K. Yee, Michael A. Bowen, Clinton Weber, Daniel Li, Tina Albertson, Claire Sutherland, Christopher G. Ramsborg. Defined cell composition and precise control over JCAR017 dose enables identification of relationships between chimeric antigen receptor T cell product attributes, pharmacokinetics, and clinical endpoints in NHL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 960.

The pan B-cell surface antigen CD19 is an attractive target for therapeutic monoclonal antibody (mAb) approaches. We have generated a new afucosylated anti-human (hu)CD19 mAb, MEDI-551, with increased affinity to human FcγRIIIA and mouse FcγRIV and enhanced antibody-dependent cellular cytotoxicity (ADCC). During in vitro ADCC assays with B-cell lines, MEDI-551 is effective at much lower mAb concentrations than the fucosylated parental mAb anti-CD19-2. Furthermore, the afucosylated CD19 mAb MEDI-551 depleted B cells from normal donor peripheral blood mononuclear cell samples in an autologous ADCC assay, as well as blood and tissue B cells in human CD19/CD20 double transgenic (Tg) mice at lower concentrations than that of the positive control mAb rituximab. In huCD19/CD20 Tg mice, both macrophage-mediated phagocytosis and complement-dependent cytotoxicity contribute to depletion with rituximab; MEDI-551 did not require complement for maximal B-cell depletion. Furthermore, extended B-cell depletion from the blood and spleen was achieved with MEDI-551, which is probably explained by bone marrow B-cell depletion in huCD19/CD20 Tg mice relative to the control mAb rituximab. In summary, MEDI-551 has potent B-cell-depleting activity in vitro and in vivo and may be a promising new approach for the treatment of B-cell malignancies and autoimmune diseases.

Definitive identification of Brucella species remains a challenge due to the high degree of genetic homology shared within the genus. We report the development of a molecular technique which utilizes real-time PCR followed by high-resolution melt (HRM) curve analysis to reliably type members of this genus. Using a panel of seven primer sets, we tested 153 Brucella spp. isolates with >99% accuracy compared to traditional techniques. This assay provides a useful diagnostic tool that can rapidly type Brucella isolates and has the potential to detect novel species. This approach may also prove helpful for clinical, epidemiological and veterinary investigations.

IN BRIEF Improving health care transition in adolescents and young adults with diabetes is a challenge shared by both pediatric and adult practitioners. This article provides an overview of health care transitions, reviews selected approaches to transition, and provides recommendations for health care providers to facilitate and optimize transition in adolescents with diabetes.

The treatment of psoriasis has undergone a revolution with the advent of biologic therapies, including infliximab, etanercept, adalimumab, efalizumab, and alefacept. These medications are designed to target specific components of the immune system and are a major technological advancement over traditional immunosuppressive medications. These usually being well tolerated are being found useful in a growing number of immune-mediated diseases, psoriasis being just one example. The newest biologic, ustekinumab, is directed against the p40 subunit of the IL-12 and IL-23 cytokines. It has provided a new avenue of therapy for an array of T-cell-mediated diseases. Biologics are generally safe; however, there has been concern over the risk of lymphoma with use of these agents. All anti-TNF-alpha agents have been associated with a variety of serious and "routine" opportunistic infections.

This study asked whether latent class modeling methods and multiple ratings of the same cases might permit quantification of the accuracy of forensic assessments. Five evaluators examined 156 redacted court reports concerning criminal defendants who had undergone hospitalization for evaluation or restoration of their adjudicative competence. Evaluators rated each defendant's Dusky-defined competence to stand trial on a five-point scale as well as each defendant's understanding of, appreciation of, and reasoning about criminal proceedings. Having multiple ratings per defendant made it possible to estimate accuracy parameters using maximum likelihood and Bayesian approaches, despite the absence of any "gold standard" for the defendants' true competence status. Evaluators appeared to be very accurate, though this finding should be viewed with caution.

Machupo virus and Chapare virus are members of the Tacaribe serocomplex (virus family Arenaviridae) and etiological agents of hemorrhagic fever in humans in Bolivia. The nucleotide sequences of the complete Z genes, a large fragment of the RNA-dependent RNA polymerase genes, the complete glycoprotein precursor genes, and the complete nucleocapsid protein genes of 8 strains of Machupo virus were determined to increase our knowledge of the genetic diversity among the Bolivian arenaviruses. The results of analyses of the predicted amino acid sequences of the glycoproteins of the Machupo virus strains and Chapare virus strain 200001071 indicated that immune plasma from hemorrhagic fever cases caused by Machupo virus may prove beneficial in the treatment of Bolivian hemorrhagic fever but not hemorrhagic fever caused by Chapare virus.

The IBM POWER6™ microprocessor is a 790 million-transistor chip that runs at a clock frequency of greater than 4 GHz. The complexity and size of the POWER6 microprocessor, together with its high operating frequency, present a number of significant challenges. This paper describes the physical design and design methodology of the POWER6 processor. Emphasis is placed on aspects of the design methodology, technology, clock distribution, integration, chip analysis, power and performance, random logic macro (RLM), and design data management processes that enabled the design to be completed and the project goals to be met.

In this article, we contend that viewing organizations through an accountability lens (in terms of source, focus, salience, and intensity) helps illuminate issues of governance and ethical dilemmas common to most individuals at work. We also propose that disconnects between aspects of accountability may pressure individuals to behave unethically and seek to rationalize their behaviors. We suggest that accountability is not only an organizational requirement, but also a perceptual lens that can be used to observe and understand behavior in, and of, organizations. As such, we demonstrate herein how to make better sense of functional and dysfunctional behavior in organizations by applying the accountability lens. A key component of this accountability lens is the notion of an accountability environment: those aspects of an individual’s immediate work environment that directly affect the subjective interpretation and experience of felt accountability. The notion that individuals perceive and interpret their accountabilities subjectively is critical to understanding why multiple employees can behave differently (and sometimes unethically) under the same accountability conditions.

Toxigenic Vibrio cholerae, the etiological agent of cholera, is a natural inhabitant of the marine environment and causes severe diarrheal disease affecting thousands of people each year in developing countries. It is the subject of extensive testing of shrimp produced and exported from these countries. We report the development of a real time PCR (qPCR) assay to detect the gene encoding cholera toxin, ctxA, found in toxigenic V. cholerae strains. This assay was tested against DNA isolated from soil samples collected from diverse locations in the US, a panel of eukaryotic DNA from various sources, and prokaryotic DNA from closely related and unrelated bacterial sources. Only Vibrio strains known to contain ctxA generated a fluorescent signal with the 5' nuclease probe targeting the ctxA gene, thus confirming the specificity of the assay. In addition, the assay was quantitative in pure culture across a six-log dynamic range down to

During the 2008–2009 rotavirus season of the Centers for Disease Control and Prevention New Vaccine Surveillance Network, one case of paediatric acute gastroenteritis associated with a rotavirus G14P[24] strain was identified. This was the first detection of the genotype G14 and P[24] in humans, and the first detection of the G14P[24] combination. To gain an insight into the origins and the evolution of this strain, we determined the complete ORF sequences of all 11 genes. A majority of the genes identified were similar to the simian strain TUCH, except for the VP1 and VP7 genes that clustered only distantly with the bovine and equine strains, respectively. In addition, this strain carried AU-1-like NSP2 and NSP4 genes. Using codon-partitioning and protein-based phylogenetic approaches, we determined that the VP7 genotype of strain 2009727118 was actually G3; therefore, the proposed full genomic classification of the 2009727118 strain is G3-P[24]-I9-R2-C3-M3-A9-N3-T3-E3-H6. These findings indicate the possibility that the 2009727118 strain originated by interspecies transmission and multiple reassortment events involving human, bovine and equine rotaviruses, resulting in the introduction of some genes into the genome of simian rotaviruses. Additionally, we found evidence of mutational saturation in the third codon position of the VP7 ORF which presented an issue with homoplasy in phylogenetic analyses.

A globally-coordinated phase out of all type 2 containing oral polio vaccine (OPV) is planned for April 2016 during which bivalent 1+3 OPV (bOPV) will replace trivalent OPV (tOPV) in routine immunization schedules and campaigns. Diarrhea impairs the immune response to tOPV, but the effect of diarrhea on bOPV is unknown.Infants aged 6 weeks to 11 months, who had received3 doses of OPV and had mild-moderate diarrhea or no diarrhea, were recruited at five health facilities in Nepal. Neutralizing antibody titers to poliovirus types 1 and 3 were measured before and 28 days after bOPV administration. The effect of diarrhea and other factors on seroconversion or boosting in antibody titers to poliovirus was assessed by multivariable analysis.Infants with diarrhea, versus those without diarrhea, had reduced response for poliovirus types 1 (56% [87/156] vs 66% [109/164]) and 3 (34% [70/209] vs 52% [122/236]). After adjusting for other factors, infants with diarrhea had significantly reduced response for type 3 (odds ratio [OR]=0.44, 95% CI 0.29-0.68), as did infants with5 loose stools per day (OR=0.36, 95% CI 0.21-0.62).Diarrhea reduced the immune response to bOPV. Provision of additional doses of polio vaccine is necessary to maintain high population immunity in areas with high prevalence of diarrheal disease.This study is registered at clinicaltrials.gov as NCT01559636.

In this article, we share our experience in establishing a clinic-based practice for MR imaging-guided interventions. Clinic resources and operational logistics are described and our institutional cost analysis for supporting the clinic activity is provided. We highlight the overall value of the clinic model in transitioning the field of interventional MR imaging from the "proof-of-concept" to the "working model" era and engage in a detailed discussion of our experience with the positive impact of the clinic on streamlining the procedural workflow, increasing awareness of the technology, expanding referral bases, and boosting the satisfaction of both patients and referring services.

A genetic polymorphism affecting FUT2 secretor status in approximately one-quarter of humans of European descent affects the expression of histo-blood group antigens on the mucosal epithelia of human respiratory, genitourinary, and digestive tracts. These histo-blood group antigens serve as host receptor sites necessary for attachment and infection of some pathogens, including norovirus.We investigated whether an association exists between FUT2 secretor status and laboratory-confirmed rotavirus infections in US children.Multicenter case-control observational study involving active surveillance at 6 US pediatric medical institutions in the inpatient and emergency department clinical settings. We enrolled 1564 children younger than 5 years with acute gastroenteritis (diarrhea and/or vomiting) and 818 healthy controls frequency matched by age and month, from December 1, 2011, through March 31, 2013.Paired fecal-saliva specimens were tested for rotavirus and for secretor status. Comparisons were made between rotavirus test-positive cases and healthy controls stratified by ethnicity and vaccination status. Adjusted multivariable analyses assessed the preventive association of secretor status against severe rotavirus gastroenteritis.One (0.5%) of 189 rotavirus test-positive cases was a nonsecretor, compared with 188 (23%) of 818 healthy control participants (P .001). Healthy control participants of Hispanic ethnicity were significantly less likely to be nonsecretors (13%) compared with healthy children who were not of Hispanic ethnicity (25%) (P .001). After controlling for vaccination and other factors, children with the nonsecretor FUT2 polymorphism appeared statistically protected (98% [95% CI, 84%-100%]) against severe rotavirus gastroenteritis.Severe rotavirus gastroenteritis was virtually absent among US children who had a genetic polymorphism that inactivates FUT2 expression on the intestinal epithelium. We observed a strong epidemiologic association among children with rotavirus gastroenteritis compared with healthy control participants. The exact cellular mechanism behind this epidemiologic association remains unclear, but evidence suggests that it may be rotavirus genotype specific. The lower prevalence of nonsecretors among Hispanic children may translate to an enhanced burden of rotavirus gastroenteritis among this group. Our findings may have bearing on our full understanding of rotavirus infections and the effects of vaccination in diverse populations.

Techniques such as the real-time reverse transcription-polymerase chain reaction (qRT-PCR) can detect RNA in samples with a low viral load. However, these amplicons typically are either too short or at insufficient concentrations for use in subsequent sequencing reactions for genotyping and detection confirmation. The assay developed in this study detects rotavirus G genotypes and P genotypes with viral loads as low as 6.2 and 8.2 copies per reaction, respectively. The assay was validated using a panel of 91 stool samples, 32 reference rotavirus strains, and 6 non-target enteric virus samples.

Objective Using case-control methodology, we measured the vaccine effectiveness (VE) of the 2-dose monovalent rotavirus vaccine (RV1) and 3-dose pentavalent rotavirus vaccine (RV5) series given in infancy against rotavirus disease resulting in hospital emergency department or inpatient care. Study design Children were eligible for enrollment if they presented to any 1 of 3 hospitals in Atlanta, Georgia with diarrhea ≤10 days duration during January-June 2013 and were born after RV1 introduction. Stool samples were tested for rotavirus by enzyme immunoassay and immunization records were obtained from providers and the state electronic immunization information system. Case-subjects (children testing rotavirus antigen-positive) were compared with children testing rotavirus antigen-negative. Results Overall, 98 rotavirus-case subjects and 175 rotavirus-negative controls were enrolled. Genotype G12P[8] predominated (n = 87, 89%). The VE of 2 RV1 doses was 84% (95% CI 38, 96) among children aged 8-23 months and 82% (95% CI 41, 95) among children aged ≥24 months. For the same age groups, the VE of 3 RV5 doses was 80% (95% CI 27, 95) and 87% (95% CI 22, 98), respectively. Conclusions Under routine use, the RV1 and RV5 series were both effective against moderate-to-severe rotavirus disease during a G12P[8] season, and both vaccines demonstrated sustained protection beyond the first 2 years of life.

Protein secretion and vesicular trafficking in mammalian cells rely on several key lipids including sphingolipids, phospholipids, and neutral lipids crucial to protein processing and other intracellular events. Proteins interact with these lipids to alter the shape of lipid bilayer, thereby playing a pivotal role in cellular sorting. Although some efforts have elucidated the role of these components, extensive studies are needed to further decipher the protein-lipid interactions along with the effect of membrane curvature and rafts in sorting of proteins. The regulatory role of proteins in subcellular localization and metabolism of lipids also needs to be described. Recent studies on the role of lipid-protein interactions in modulating membrane shape, signal transduction, and vesicular trafficking are presented in this review.

This is a report of the complete genomic sequence of a rare rotavirus group A G8-P[14]-I2-R3-C2-M2-A3-N2-T6-E2-H3 strain detected in a stool sample from a 57-year-old subject.

We report the genomic characterization of a rare human G8P[14] rotavirus strain, identified in a stool sample from Guatemala (GTM) during routine rotavirus surveillance. This strain was designated as RV A/Human-wt/GTM/2009726790/2009/G8P[14], with a genomic constellation of G8-P[14]-I2-R2-C2-M2-A13-N2-T6-E2-H3. The VP4 gene occupied lineage VII within the P[14] genotype. Phylogenetic analysis of each genome segment revealed close relatedness to several zoonotic simian, guanaco and bovine strains. Our findings suggest that strain RVA/Human-wt/GTM/2009726790/2009/G8P[14] is an example of a direct zoonotic transmission event. The results of this study reinforce the potential role of interspecies transmission and reassortment in generating novel and rare rotavirus strains which infect humans.