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Four facets of leaving an abusive relationship are reviewed: (a) factors related to initially leaving an abusive partner; (b) the process of leaving an abusive relationship; (c) the psychological well-being of survivors after leaving; and (d) the predictors of this well-being. The conceptual and methodological limitations of studies in each of these areas are presented. Consistently found predictors of leaving include both material and psychological factors. Because battered women typically undergo several shifts in their thinking about the abuse before leaving permanently, research on leaving as a process is highlighted. A stress-process framework is used to explain the seemingly paradoxical finding that some women just out of the abusive relationship may have greater psychological difficulties than those who are still in it. For those experiencing the most stress, psychological health can worsen over time. Researchers and practitioners need to pay more attention to the plight of women who have left abusive partners. [ABSTRACT FROM AUTHOR]

Objective: To better understand the microbial profile of complicated parapneumonic effusions and empyema, and to evaluate whether antimicrobial selection would differ if guided by targeted metagenomic sequencing (tMGS) vs conventional cultures (CCs) alone., Patients and Methods: We analyzed the pleural fluid of a cohort of 47 patients undergoing thoracentesis from January 1, 2017 to August 31, 2019, to characterize their microbial profile. All samples underwent 16S ribosomal ribonucleic acid gene polymerase chain reaction, followed by tMGS., Results: Pleural space infection was deemed clinically present in 20 of the 47 (43%) participants. Of those, n=7 (35%) had positive pleural fluid cultures and n=14 (70%) had positive tMGS results. The organisms identified by tMGS were concordant with CCs; however, tMGS detected additional bacterial species over CCs alone. Streptococcus and Staphylococcus species were the most common organisms identified, with Streptococcus intermedius/constellatus identified in 5 patients . Polymicrobial infections were found in 6 of the 20 patients, with anaerobes being the most common organisms identified in these cases., Conclusion: Streptococci and staphylococci were the most common organisms identified in infected pleural fluid. Anaerobes were common in polymicrobial infections. When compared with CCs, tMGS had higher sensitivity than CCs. Targeted metagenomic sequencing identified additional organisms, not identified by CCs, with associated potential management implications., Competing Interests: Dr Patel reports receiving grants from ContraFect, TenNor Therapeutics Limited, and BioFire. Dr Patel is a consultant for PhAST, Torus Biosystems, Day Zero Diagnostics, Mammoth Biosciences, and HealthTrackRx; monies are paid to Mayo Clinic. Mayo Clinic and Dr Patel have a relationship with Pathogenomix. Dr Patel has research supported by Adaptive Phage Therapeutics. Mayo Clinic has a royalty-bearing know-how agreement and equity in Adaptive Phage Therapeutics. Dr Patel is also a consultant for Netflix, Abbott Laboratories, Oxford Nanopore Technologies, and CARB-X. In addition, Dr Patel has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication, with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued. Dr Patel receives honoraria from the NBME, Up-to-Date and the Infectious Diseases Board Review Course. Dr Samhouri reports providing consultation (unpaid) to AI Therapeutics that is unrelated to the submitted work. Dr Carmona reports providing consultation to Boehringer Ingelheim for sarcoidosis that is unrelated to the submitted work., (© 2023 The Authors.)

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease, a severe gastroenteritis of ruminants. This study developed a model cell culture system to rapidly screen MAP mutants with vaccine potential for apoptosis. Two wild-type strains, a transposon mutant, and two deletion mutant MAP strains (MOI of 10 with 1.2 × 10 6 CFU) were tested in murine RAW 264.7 macrophages to determine if they induce apoptosis and/or necrosis. Both deletion mutants were previously shown to be attenuated and immunogenic in primary bovine macrophages. All strains had similar growth rates, but cell morphology indicated that both deletion mutants were elongated with cell wall bulging. Cell death kinetics were followed by a real-time cellular assay to measure luminescence (apoptosis) and fluorescence (necrosis). A 6 h infection period was the appropriate time to assess apoptosis that was followed by secondary necrosis. Apoptosis was also quantified via DAPI-stained nuclear morphology and validated via flow cytometry. The combined analysis confirmed the hypothesis that candidate vaccine deletion mutants are pro-apoptotic in RAW 264.7 cells. In conclusion, the increased apoptosis seen in the deletion mutants correlates with the attenuated phenotype and immunogenicity observed in bovine macrophages, a property associated with good vaccine candidates.

Previous research has shown that the use of clickers in the classroom enhances student engagement. However, few studies have investigated how the type of clicker question may influence learning outcomes. To explore this, we compared the effects of lower-order cognitive skill (LOCS) and higher-order cognitive skill (HOCS) clicker questions on later exam performance in a biology course. During class time, students were presented with clicker questions directly related to unit content. Half of the content units were taught with LOCS, the other half with HOCS. To ensure that type of content did not influence results, the cognitive level of the clicker questions was counterbalanced across two semesters. The exams included a mix of LOCS and HOCS for each content unit. We also investigated the possible moderating effects of student perceptions on the relationship between type of clicker question and exam performance using student surveys. We found that using HOCS clicker questions significantly affects student learning. Practice with HOCS clicker questions improved performance on LOCS exam questions but not on HOCS exam questions. Students ranked lecture with clickers as a preferred and most helpful teaching methodology. Overall, these results suggest that practice with HOCS questions is engaging and gives students practice recalling content to "solve" a problem, thereby encoding low-level information and preparing them for higher-order thinking activities., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 Anderson et al.)

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary disease characterized by aberrant tissue remodeling, formation of scar tissue within the lungs and continuous loss of lung function. The areas of fibrosis seen in lungs of IPF patients share many features with normal aging lung including cellular senescence. The contribution of the immune system to the etiology of IPF remains poorly understood. Evidence obtained from animal models and human studies suggests that innate and adaptive immune processes can orchestrate existing fibrotic responses. Currently, there is only modest effective pharmacotherapy for IPF. Mesenchymal stem cells (MSCs)-based therapies have emerged as a potential option treatment of IPF. This study characterizes the functionality of autologous MSCs for use as an IPF therapy and presents an attempt to determine whether the disease occurring in the lungs is associated with an alterated immune system., Methods: Comprehensive characterization of autologous adipose-derived MSCs (aMSCs) from 5 IPF patient and 5 age- and gender-matched healthy controls (HC) was done using flow cytometry, PCR (ddPCR), multiplex Luminex xMAP technology, confocal microscopy self-renewal capacity and osteogenic differentiation. Additionally, multi-parameter quantitative flow cytometry of unmanipulated whole blood of 15 IPF patients and 87 (30 age- and gender-matched) HC was used to analyze 110 peripheral phenotypes to determine disease-associated changes in the immune system., Results: There are no differences between autologous aMSCs from IPF patients and HC in their stem cell properties, self-renewal capacity, osteogenic differentiation, secretome content, cell cycle inhibitor marker levels and mitochondrial health. IPF patients had altered peripheral blood immunophenotype including reduced B cells subsets, increased T cell subsets and increased granulocytes demonstrating disease-associated alterations in the immune system., Conclusions: Our results indicate that there are no differences in aMSC properties from IPF patients and HC, suggesting that autologous aMSCs may be an acceptable option for IPF therapy. The altered immune system of IPF patients may be a valuable biomarker for disease burden and monitoring therapeutic response., (© 2022. The Author(s).)

Gene expression depends on the binding of transcription factors with DNA regulatory sequences. The level of accessibility for these sequences varies between cells and cell types. Until recently, using the Tn5 assay for transposase-accessible chromatin for sequencing (ATAC-seq) technology allowed assessing the profiles of chromatin from an entire organ or, when coupled with the isolation of nuclei tagged in specific cell types (INTACT) method, from a cell-type. Recently, ATAC-seq experiments were conducted at the level of individual plant nuclei. Applying single nuclei ATAC-seq (sNucATAC-seq) technology to thousands of individual cells revealed more finely tuned profiles of chromatin accessibility. In this manuscript, we describe a method to isolate nuclei fom plant roots and green tissues, permeabilize the nuclear membrane using detergent to allow the penetration of the Tn5 transposase, and re-suspend them in a nuclei resuspension buffer compatible with the construction of sNucATAC-seq libraries using the 10× Genomic's Chromium technology. This protocol was successfully applied on Arabidopsis thaliana and Glycine max root nuclei., Competing Interests: Competing interestsThe authors have no competing interests., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

Objective: The purposes of this study were to identify and describe the attributes of excellence and innovation in professional pediatric physical therapy education and develop a conceptual framework identifying dimensions of excellence., Methods: A multimethod case study design based on a grounded theory framework was used. Data collection included review of artifacts and field interviews (individual and focus group). A constant-comparative method for within case and across case was used for data analysis to verify and revise coding schemes, identify categories and subcategories, revise emerging themes, and develop a conceptual framework., Results: Based on results of a predetermined grading rubric, 6 of 17 self-nominated academic sites were selected representing diverse institution types (public/private, Carnegie classification, size) and geographic locations. Pedagogical approaches and method of content delivery varied among programs; all used the essential core competencies. The core pediatric faculty member(s) were Board Certified Clinical Specialists. A conceptual framework was developed based on 4 key dimensions: Culture of Excellence, Exemplary Pediatric Faculty, Pedagogy, and Child and Family as Teacher (CFT), and 16 related elements., Conclusions: CFT is a unique and nonnegotiable dimension of excellence in pediatric physical therapy education, highlighting the partnership between the learner, child, family, and pediatric faculty member. CFT intersects with the other dimensions and integrates their elements (ie, faculty characteristics, contributions from the child and family, use of instructional strategies) to effectively prepare future pediatric physical therapists. Based on the pervasiveness of this dimension in teaching and learning across all programs, this may be the signature pedagogy of pediatric physical therapy education., Impact: Results of this study are important to professional physical therapist education administrators, pediatric academic faculty, and clinical educators because they represent an understanding of the attributes of excellence. The model can serve as a guide for best practice in pediatric physical therapy education., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Physical Therapy Association. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

B-cell activation is increasingly linked to numerous fibrotic lung diseases, and it is well known that aggregates of lymphocytes form in the lung of many of these patients. Activation of B-cells by pattern recognition receptors (PRRs) drives the release of inflammatory cytokines, chemokines, and metalloproteases important in the pathophysiology of pulmonary fibrosis. However, the specific mechanisms of B-cell activation in patients with idiopathic pulmonary fibrosis (IPF) are poorly understood. Herein, we have demonstrated that B-cell activation by microbial antigens contributes to the inflammatory and profibrotic milieu seen in patients with IPF. B-cell stimulation by CpG and β-glucan via PRRs resulted in activation of mTOR-dependent and independent pathways. Moreover, we showed that the B-cell-secreted inflammatory milieu is specific to the inducing antigen and causes differential fibroblast migration and activation. B-cell responses to infectious agents and subsequent B-cell-mediated fibroblast activation are modifiable by antifibrotics, but each seems to exert a specific and different effect. These results suggest that, upon PRR activation by microbial antigens, B-cells can contribute to the inflammatory and fibrotic changes seen in patients with IPF, and antifibrotics are able to at least partially reverse these responses.

Diaphragmatic dysfunction is a rare cause of dyspnea that can lead to hypercapnic respiratory failure. A variety of causes of diaphragmatic dysfunction have been identified, including myopathies and neuropathies, the latter of which can be further subdivided into infectious, idiopathic, demyelinating, malignant, and iatrogenic etiologies. Now, in the era of immune checkpoint inhibitors (ICIs), case reports of immune-mediated phrenic nerve palsies have been described. This diagnosis can be challenging to make, as dyspnea is a common presenting complaint and immune-mediated palsy of the phrenic nerve is a rarely described complication of ICI therapy. At Mayo Clinic, 3 patients with diaphragmatic dysfunction in the setting of ICI therapy were successfully treated without mortality. This case series describes the presentation, diagnoses, and management of these patients and their clinical outcomes.

Aims: This cross-sectional study investigated how typically developing children performed on a version of the Dynamic Gait Index, modified for children (Pediatric Modified DGI). Methods: Seventy-four typically developing children, ages 7-18 years, were evaluated using the Pediatric Modified DGI. Scores were analyzed in three age groups using descriptive statistics for age-related performance. The Kruskal-Wallis and Mann-Whitney U tests were used to evaluate group differences. Results: Children performed differently on the Pediatric Modified DGI between the three groups (Kruskal-Wallis H (2) = 18.819, p < .05). Mann-Whitney U post hoc testing identified significant differences between the 7 to 10 and the 15 to 18-year-old groups ( U  = 128.5, p = .000, r = -.54) and between the 11 to 14 and the 15 to 18-year-old groups ( U  = 139, p = .001, r = -.46). Pediatric modified DGI scores were notably different between the three age groups on three of the items: horizontal head turn, vertical head turn, and pivot turn. Conclusions: Children's age appears to impact performance on the pediatric modified DGI, which should be considered when using this tool to examine functional mobility. Future studies should explore functional mobility assessments that consider sensory system development.

Purpose: The purpose of this scoping review was to identify and map current evidence that underpins excellence in pediatric physical therapy education., Methods: An extensive review was conducted of literature published over a 27-year period regarding pediatric physical therapy/medical/allied health education., Results: Thirty articles were reviewed and analyzed. Articles were classified as levels of evidence 4 to 7. Three major overarching concepts emerged: faculty characteristics, curriculum, and pedagogy., Conclusions: Few published studies with methodological rigor exist on this topic. More research and higher quality studies are needed to identify exemplar programs, curriculum, and pedagogy. The key concepts identified in this review will help inform the definition of excellence, as it is perceived through the lens of pediatric physical therapy educators, clinicians, and students.

Purpose: The reliability and validity of a survey tool that aims to assess and stratify patient care services provided by ambulatory care pharmacists were evaluated., Methods: The Tool for Assessing Ambulatory Care Pharmacist Practice (TAAPP) was developed by updating the Pharmaceutical Care Clinical Pharmacist Questionnaire. The TAAPP is organized into 2 sections that include 5 domains derived from the Pharmacists' Patient Care Process (PPCP). The first section of the TAAPP gathers the demographic information of the respondents as well as practice site characteristics. The second section aims to assess the activities that ambulatory care pharmacists participate in when providing direct patient care, stratified by PPCP domains. After the TAAPP was created, face validity was established by the study investigators and content validity was confirmed by 5 experts in ambulatory care pharmacy. Lastly, a reliability study was conducted and included pharmacists providing ambulatory care services in outpatient clinics who had been working at their clinical practice site for at least 2 years. The survey was disseminated electronically through a national pharmacy organization listserver., Results: The results of this pilot study support both face and content validity of the TAAPP survey as well as internal consistency reliability of the TAAPP scores when used to evaluate pharmaceutical practices of ambulatory care pharmacists practicing in outpatient clinics throughout the United States., Conclusion: Internal consistency reliability testing demonstrated that the TAAPP scores were reliable with a Cronbach's α of >0.7 for each domain and the TAAPP overall., Competing Interests: DisclosuresThe authors have declared no potential conflicts of interest., (Copyright © 2018 by the American Society of Health-System Pharmacists, Inc. All rights reserved.)

Background: At the Section on Pediatrics Education Summit in July 2012, consensus was achieved on 5 essential core competencies (ECCs) that represent a knowledge base essential to all graduates of professional physical therapist education programs., Key Points: This article offers suggestions for how clinical instructors (CIs) might use the ECCs to identify student needs and guide student learning during a pediatric clinical education experience. Pediatric CIs potentially might choose to use the ECCs as a reference tool in clinical education to help (1) organize and develop general, clinic-specific clinical education objectives, (2) develop and plan individualized student learning experiences, (3) identify student needs, and (4) show progression of student learning from beginner to intermediate to entry level., Conclusions: The ECCs may offer CIs insights into the role of pediatric clinical education in professional physical therapist education.

In this follow-up study the authors examine the impact of two projects that provided volunteer practitioners with extensive training in trauma-focused cognitive-behavioral therapy (TF-CBT). The objective was to gain empirical data about clinicians' knowledge of the model and implementation 10-15 months after training. The 30 respondents achieved a mean knowledge score of 80% for eight hypothetical clinical vignettes, positive results for clients' attendance at initial appointments, parental involvement, and low dropout rates, but somewhat lower use of child and parent trauma work. Use of selected TF-CBT components was strong, with lowest rates for child and parent trauma narrative and modifying cognitions. Intentions to use the model were high. Implications and recommendations are discussed.

In this article the authors report on a collaborative effort to disseminate trauma-focused cognitive-behavioral therapy (TF-CBT) with community practitioners (N = 33). They discuss the evolution and planning of this local initiative and the research component to assess practitioners' self-efficacy at the basic and advanced levels of training. Self-efficacy scores increased significantly and were positively associated with practice experience. Independent implementation effort was low due to low referral numbers. Final ratings were high for competence, the value of consultation sessions, and future use of the model. The results offer insight into this approach to dissemination and implementation.

Background: Delayed, abnormal language is a common feature of autism and language therapy often a significant component of recommended treatment. However, as with other disorders with a language component, we know surprisingly little about the language trajectories and how varied these might be across different children. Thus, we know little about how and when language problems might resolve, whether there are periods of relative stability or lack of change and what periods might offer more favourable circumstances for intervention., Methods: Expressive and receptive language was measured on six occasions between age 2 and 19 on a cohort of 192 children initially referred for autism. Latent class growth models were fitted to characterize the patterns of heterogeneous development., Results: Latent class growth analysis identified seven classes. Between age 6 and 19, all classes tracked in parallel. Between ages 2 and 6, development was more heterogeneous with considerable variation in relative progress. In all groups, receptive and expressive language developed very largely in tandem., Conclusions: The results confirmed previous analysis of children with specific language impairment where progress beyond age 6 was remarkably uniform. Greater variation was evident before this age with some groups making clearly better or worse progress compared to others. While this developmental heterogeneity may simply be a reflection of variation in preexisting and unchanging biological disposition, it may also reflect, at least in part, greater sensitivity in the early years to environments that are more or less supportive of language development. These findings contribute to the case for the importance of early intervention., (© 2014 The Authors. Journal of Child Psychology and Psychiatry published by John Wiley & Sons Ltd on behalf of Association for Child and Adolescent Mental Health.)

Background: The range of outcomes for young adults with Autism Spectrum Disorders (ASD) and the early childhood factors associated with this diversity have implications for clinicians and scientists., Methods: This prospective study provided a unique opportunity to predict outcome 17 years later for a relatively large sample of children diagnosed with ASD at 2 years old. Diagnostic and psychometric instruments were administered between 2 and 19 with data from 2, 3, and 19 included in this study. Clinicians administered tests without knowledge of previous assessments whenever possible. Caregivers provided additional information through questionnaires., Results: Significant intellectual disabilities at 19 were predicted by age 2 about 85% of the time from VIQ and NVIQ scores together, though prediction of young adult outcome for youths with average or higher intelligence was more complex. By 19, 9% of participants had largely overcome core difficulties associated with ASD and no longer retained a diagnosis. These youths with Very Positive Outcomes were more likely to have participated in treatment and had a greater reduction in repetitive behaviors between age 2 and 3 compared to other Cognitively Able youths (VIQ ≥70) with ASD. Very Positive Outcome youths did not differ phenotypically from Cognitively Able ASD individuals at 2 but both groups differed from Cognitively Less Able individuals (VIQ <70)., Conclusion: Those most at risk for intellectual disabilities and ASD can be reliably identified at an early age to receive comprehensive treatment. Findings also suggest that some cognitively able children with ASD who participate in early intervention have very positive outcomes, although replication with randomized, larger samples is needed. In order to improve understanding of very positive outcomes in ASD, future research will need to identify how variations in child characteristics and environmental factors contribute to the nature and timing of growth across individuals and areas of development., (© 2013 The Authors. Journal of Child Psychology and Psychiatry © 2013 Association for Child and Adolescent Mental Health.)

Chronic lymphocytic leukemia (CLL) is a prevalent B-cell neoplasia that is often preceded by a more benign monoclonal CD5(+) B-cell lymphocytosis. We previously generated transgenic mice expressing catalytically inactive RAG1 (dominant-negative recombination activating gene 1 [dnRAG1] mice) that develop an early-onset indolent CD5(+) B-cell lymphocytosis attributed to a defect in secondary V(D)J rearrangements initiated to edit autoreactive B-cell receptor (BCR) specificity. Hypothesizing that CD5(+) B cells in these animals represent potential CLL precursors, we crossed dnRAG1 mice with CLL-prone Eμ-TCL1 mice to determine whether dnRAG1 expression in Eμ-TCL1 mice accelerates CLL onset. Consistent with this hypothesis, CD5(+) B-cell expansion and CLL progression occurred more rapidly in double-transgenic mice compared with Eμ-TCL1 mice. Nevertheless, CD5(+) B cells in the 2 mouse strains exhibited close similarities in phenotype, immunoglobulin gene usage, and mutation status, and expression of genes associated with immune tolerance and BCR signaling. Gene expression profiling further revealed a potential role for prolactin signaling in regulating BCR editing. These results suggest a model in which benign accumulation of CD5(+) B cells can be initiated through a failure to successfully edit autoreactive BCR specificity and may, in turn, progress to CLL upon introduction of additional genetic mutations.

Objective: With the physical therapy (PT) professions' advancement to the clinical doctorate degree and the promotion of autonomous practice, exemplary professional conduct is an expectation of the PT profession. PT education programs are being challenged to develop methods to teach and assess professional behavior., Participants: Forty-three PT students (11 male and 32 female, ages 20-28 years) completed the APTA Professionalism in Physical Therapy: Core Values Self-Assessment (PPTCVSA) after their first 3 week clinical experience and again after their final clinical experience., Methods: A mixed design ANOVA compared participants' total scores and individual Core Value scores on the Professionalism in Physical Therapy: Core Values Self-Assessment (PPTCVSA) after 3 and 33 weeks of clinical education. The effects of gender, age, and undergraduate area of study on growth in professionalism scores were also investigated., Results: Total PPTCVSA scores and individual Core Value scores on professionalism (accountability, altruism, compassion/caring, excellence, integrity, professional duty, and social responsibility) were higher after 33 weeks compared to scores after 3 weeks of clinical education. Female student's total professionalism scores were higher than male student's scores on both the first and second self-assessments. In addition, female students scored themselves higher than their male peers on accountability, excellence, integrity, and professional duty., Conclusions: Improved scores on the PPTCVSA indicate that physical therapy education is playing an important role in the development of professional behavior, knowledge, and application in practice.

The N-terminus of full-length RAG1, though dispensable for RAG1/2 cleavage activity, is required for efficient V(D)J recombination. This region supports RING E3 ubiquitin ligase activity in vitro, but whether full-length RAG1 functions as a single subunit or a multi-subunit E3 ligase in vivo is unclear. We show the multi-subunit cullin RING E3 ligase complex VprBP/DDB1/Cul4A/Roc1 associates with full-length RAG1 through VprBP. This complex is assembled into RAG protein-DNA complexes, and supports in-vitro ubiquitylation activity that is insensitive to RAG1 RING domain mutations. Conditional B lineage-specific VprBP disruption arrests B-cell development at the pro-B-to-pre-B cell transition, but this block is bypassed by expressing rearranged immunoglobulin transgenes. Mice with a conditional VprBP disruption show modest reduction of D-J(H) rearrangement, whereas V(H)-DJ(H) and V(κ)-J(κ) rearrangements are severely impaired. D-J(H) coding joints from VprBP-insufficent mice show longer junctional nucleotide insertions and a higher mutation frequency in D and J segments than normal. These data suggest full-length RAG1 recruits a cullin RING E3 ligase complex to ubiquitylate an unknown protein(s) to limit error-prone repair during V(D)J recombination.

During their development, B lymphocytes undergo V(D)J recombination events and selection processes that, if successfully completed, produce mature B cells expressing a non-self-reactive B-cell receptor (BCR). Primary V(D)J rearrangements yield self-reactive B cells at high frequency, triggering attempts to remove, silence, or reprogramme them through deletion, anergy induction, or secondary V(D)J recombination (receptor editing), respectively. In principle, expressing a catalytically inactive V(D)J recombinase during a developmental stage in which V(D)J rearrangement is initiated may impair this process. To test this idea, we generated transgenic mice expressing a RAG1 active site mutant (dnRAG1 mice); RAG1 transcript was elevated in splenic, but not bone marrow, B cells in dnRAG1 mice relative to wild-type mice. The dnRAG1 mice accumulate splenic B cells with a B1-like phenotype that exhibit defects in B-cell activation, and are clonally diverse, yet repertoire restricted with a bias toward Jκ1 gene segment usage. The dnRAG1 mice show evidence of impaired B-cell development at the immature-to-mature transition, immunoglobulin deficiency, and poorer immune responses to thymus-independent antigens. Interestingly, dnRAG1 mice expressing the anti-dsDNA 3H9H56R heavy chain fail to accumulate splenic B1-like cells, yet retain peritoneal B1 cells. Instead, these mice show an expanded marginal zone compartment, but no difference is detected in the frequency of heavy chain gene replacement. Taken together, these data suggest a model in which dnRAG1 expression impairs secondary V(D)J recombination. As a result, selection and/or differentiation processes are altered in a way that promotes expansion of B1-like B cells in the spleen., (© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.)

Numerous drugs such as clopidogrel have been developed to reduce coagulation or inhibit platelet function. The hepatic cytochrome P450 (CYP) pathway is involved in the conversion of clopidogrel to its active metabolite. A recent black-box warning was included in the clopidogrel package insert indicating a significant clinical link between specific CYP2C19 genetic variants and poor metabolism of clopidogrel. Of these variants, *2 and *3 are the most common and are associated with complete loss of enzyme activity. In patients who are carriers of a CYP2C19 *2 or *3 allele, the conversion of clopidogrel to its active metabolite may be reduced, which can lead to ischemic events and negative consequence for the patient. We examined the ability of the Verigene CLO assay (Nanosphere, Northbrook, IL) to identify CYP2C19 *2 and *3 polymorphisms in 1,286 unique whole blood samples. The Verigene CLO assay accurately identified homozygous and heterozygous *2 and *3 phenotypes with a specificity of 100% and a final call rate of 99.7%. The assay is fully automated and can produce a result in approximately 3.5 hours.

The current study prospectively examined trajectories of change in symptoms of irritability, hyperactivity, and social withdrawal, as well as predictors of such behaviors, for ages 9-18 years for youths with autism spectrum disorder and a comparison group with nonspectrum developmental delays. Children with more severe core features of autism had consistently higher irritability and hyperactivity scores over time than those with broader autism spectrum disorder and nonspectrum delays. Across all diagnoses, behaviors related to hyperactivity showed the greatest improvement. Social withdrawal worsened with age for a substantial proportion of youths with autism spectrum disorder but not for the nonspectrum comparison group. Compared with youths without autism spectrum disorder, children with the disorder showed greater heterogeneity in trajectories for maladaptive behaviors.

Seasonal epidemics of influenza and respiratory syncytial virus are responsible for significant morbidity and mortality worldwide. Infrequently, novel or reemergent strains of influenza A virus have caused rapid, severe global pandemics resulting in millions of fatalities. The ability to efficiently and accurately detect and differentiate respiratory viruses is paramount for effective treatment, infection control, and epidemiological surveillance. We evaluated the ability of two FDA-cleared nucleic acid-based tests, the semiautomated respiratory virus nucleic acid test (VRNAT) and the fully automated respiratory virus nucleic acid test SP (RVNAT(SP)) (Nanosphere Inc., Northbrook, IL) to detect influenza A virus, influenza B virus, and respiratory syncytial virus A and B (RSV A/B) from clinical nasopharyngeal swab specimens. Detection of viral RNA in both tests is based on nucleic acid amplification followed by hybridization to capture probes immobilized on a glass slide. A novel technology utilizing gold nanoparticle-conjugated probes is utilized to detect the presence of captured target DNA. This microarray-based approach to detection has proven to be more sensitive than the traditional culture/direct fluorescent-antibody assay (DFA) method for detecting RSV and influenza viruses in clinical specimens, including the novel 2009 H1N1 strain. Specifically, we report 98.0% sensitivity and 96.5% specificity for the VRNAT compared to culture/DFA. Further, the VRNAT detected virus in an additional 58% of specimens that were culture negative. These data were confirmed using bidirectional sequencing. Evaluation of the fully automated RVNAT(SP), which is built on the same detection technology as the VRNAT but contains an updated processor enabling complete automation, revealed the two tests to be functionally equivalent. Thus, the RVNAT(SP) is a fully automated sample-to-result test capable of reliable detection of select respiratory viruses directly from clinical specimens in 3.5 h.

Cigarette smoking adversely affects the immune system, and is a risk factor for developing osteoporosis. How smoking contributes to osteoporosis is unclear, but since lymphocytes help maintain bone homeostasis and lymphocyte depletion results in bone loss, one potential mechanism for how smoke exposure promotes osteoporosis is by reducing bone marrow lymphocytes. Since the risk for developing osteoporosis is reportedly greater in smokers with polymorphisms in LRP5, a gene involved in canonical Wnt signaling that regulates bone metabolism, smoking-induced effects on lymphocytes may be influenced by Lrp5 functionality. To test these possibilities, we examined how the duration and cessation of cigarette smoke exposure affects lymphocyte distribution and function in normal mice and mice predisposed to low or high bone mass due to disruption or mutation of Lrp5. We find that, independent of genotype, mice exposed to cigarette smoke for 3-12 weeks showed a significant reduction in bone marrow B220(+)CD43(-) B cells and splenic transitional T1 B cells, and exhibited a splenic CD4(+):CD8(+) T-cell ratio that was skewed toward CD8(+) T cells. Smoke exposure had little or no effect on other lymphocyte subsets or on lymphocyte function ex vivo. Interestingly, these differences were no longer apparent after 6 weeks without smoke exposure, except in mice with high bone mass where bone marrow B220(+)CD43(-) B cells failed to fully recover. These data provide the first evidence that smoke exposure reduces bone marrow B cells, providing a plausible mechanism for how smoking contributes to osteoporosis.

The proteoglycan decorin putatively inhibits cell adhesion and cell migration on various extracellular matrix substrates through interactions with beta(1) integrins. This study, therefore, examined the adhesive, migration, and proliferative characteristics of decorin knockout (Dcn(-/-)) murine embryonic fibroblasts compared to wild-type controls on collagen-coated, fibronectin-coated, and uncoated tissue culture plates. The Dcn(-/-) cells showed significantly greater proliferation than wild-type controls on all substrates. The Dcn(-/-) cells also showed significantly greater adhesion to both collagen and fibronectin; both cell types showed greater adhesion to collagen. The addition of exogenous decorin had a differential effect on adhesion to collagen between cell types, but not on fibronectin. For collagen, blocking either alpha(2) or beta(1) integrin subunits significantly reduced adhesion for Dcn(-/-) cells; whereas for fibronectin, blocking either the alpha(5) or beta(1) integrin subunits reduced adhesion for both cell types. Decorin and the alpha(5)beta(1) integrin may have lesser roles in adhesion to fibronectin than previously presumed. Finally, compared to wild-type cells, Dcn(-/-) cells showed greater migration on both uncoated and collagen substrates. This study demonstrates that decorin affects the biology of various integrins that participate in cell proliferation, adhesion, and migration on various substrates.

Adaptive social skills were assessed longitudinally at approximately ages 2, 3, 5, 9, and 13 years in a sample of 192 children with a clinical diagnosis of autism (n = 93), PDD-NOS (n = 51), or nonspectrum developmental disabilities (n = 46) at age 2. Growth curve analyses with SAS proc mixed were used to analyze social trajectories over time. Both individual characteristics and environmental resources emerged as key predictors of adaptive social behavior outcome. The gap between children with autism and the other two diagnostic groups widened with time as the social skills of the latter groups improved at a higher rate. However, within diagnostic groups, improvement ranged from minimal to very dramatic. Children with autism most at risk for problems with social adaptive abilities later in life can be identified with considerable accuracy at a very young age so they can be targeted for appropriate early intervention services.

Verbal skills were assessed at approximately ages 2, 3, 5, and 9 years for 206 children with a clinical diagnosis of autism (n = 98), pervasive developmental disorders-not otherwise specified (PDD-NOS; n = 58), or nonspectrum developmental disabilities (n = 50). Growth curve analyses were used to analyze verbal skills trajectories over time. Nonverbal IQ and joint attention emerged as strong positive predictors of verbal outcome. The gap between the autism and other 2 groups widened with time as the latter improved at a higher rate. However, there was considerable variability within diagnostic groups. Children with autism most at risk for more serious language impairments later in life can be identified with considerable accuracy at a very young age, while improvement can range from minimal to dramatic.

The bronchial arteries, which provide the systemic arterial supply to the lungs, are involved in a variety of disease processes in humans, including congenital disorders, infection, and pulmonary thromboembolism. In these conditions, the bronchial arteries hypertrophy and bronchial blood flow increases. Consequently, in many disorders, such as bronchiectasis, the bronchial arteries are a frequent source of haemoptysis, which may be massive and life-threatening. Evaluation of the bronchial circulation has typically required invasive imaging with angiography to determine the location of bleeding. Non-invasive assessment of bronchial arterial anatomy and morphology is currently being investigated with the use of helical computed tomography (CT). We evaluated eight patients with various lung diseases with helical CT (GE Medical Systems, LS16, Milwaukee, WI) to determine the imaging features of the bronchial circulation. Non-ionic contrast medium (iopromide) was injected intravenously (80-1000ml/30s) and scanning was triggered once contrast material was present in the pulmonary artery (average delay=15s) or ascending aorta (average delay=20s). Detector collimation (16-row unit) was 10mm. Imaging parameters included a section thickness of 0.6mm, kilovolt peak of 120, 150-440mA, pitch factor of 1.375, matrix of 512x512, and tube rotation time of 0.8s. The images were reconstructed and scanned isotropically (Advantage Workstation 4.1,GE Medical Systems). We conclude that helical computed tomography may provide a non-invasive means of evaluating the bronchial arteries and their role in pulmonary disease processes.

The purpose of this study is two-fold: (1) to examine skeletal muscle function in a rat model of midthoracic contusion spinal cord injury (SCI) and (2) to evaluate the therapeutic influence of a short bout (1 week) of treadmill locomotor training on soleus muscle function (peak force, fatigability, contractile properties, fiber types), size (fiber area), and motor deficit and recovery (BBB scores) after SCI. The rats were injured with a moderate T8 spinal cord contusion and were assigned to either receive treadmill locomotor training (TM), starting 1 week after SCI for 5 consecutive days (20 min/trial, 2 trials/day) or not to receive any exercise intervention (no TM). Locomotor training resulted in a significant improvement in overall locomotor function (32% improvement in BBB scores) when compared to no TM. Also, the injured animals that trained for 1 week had 38% greater peak soleus tetanic forces (p < 0.05), a 9% decrease in muscle fatigue (p < 0.05), 23% larger muscle fiber CSA (p < 0.05), and decreased immunoexpression of fast heavy chain fiber types than did rats receiving no TM. In addition, there was a good correlation (0.704) between the BBB scores of injured animals and peak soleus muscle force regardless of group assignment. No significant differences were seen in twitch or time to peak tension values across groups. Collectively, these results indicate that 1 week of treadmill locomotor training, initiated early after SCI, can significantly improve motor recovery following SCI. The magnitude of these changes is remarkable considering the relatively short training interval and clearly illustrates the potential that initiating treadmill locomotor training shortly after injury may have on countering some of the functional deficits resulting from SCI.

Magnetic resonance (MR) is frequently used to study structural and biochemical properties of skeletal muscle. Changes in proton transverse relaxation (T2) properties have been used to study muscle cellular damage, as well as muscle activation during exercise protocols. In this study, we implemented MR imaging to characterize the T2 relaxation properties of rat hindlimb muscles following spinal cord injury (SCI) and locomotor training. After moderate midthoracic contusion SCI, Sprague-Dawley rats were assigned to either treadmill training, cycle training or an untrained group. T2 weighted images were obtained and mean muscle T2 times were calculated in the tibialis anterior, soleus, and gastrocnemius (GAS) muscles at pre-injury as well as at 1, 2, 4, 8, and 12 weeks post-injury. Following SCI, hindlimb muscles in untrained animals showed a significant increase in muscle T2, with the most dramatic shift (+5.46 ms) observed in soleus muscle at 1 week post-SCI. Subsequently, all muscle groups showed a spontaneous recovery in muscle T2 with normalized T2 values in the GAS and tibilias anterior muscles at 4 weeks and the soleus at 12 weeks post-SCI. Both training paradigms, treadmill and cycling training, accelerated the recovery of soleus muscle T2. As a result, soleus muscle T2 recovered back to pre-injury values within 3 weeks of training in both training groups. Finally, in vitro histological assessments of rat skeletal muscles demonstrated that there was no apparent muscle injury in any of the muscles studied at 1 week post-SCI.

RAG-1 and RAG-2 initiate V(D)J recombination by cleaving DNA at recombination signal sequences through sequential nicking and transesterification reactions to yield blunt signal ends and coding ends terminating in a DNA hairpin structure. Ubiquitous DNA repair factors then mediate the rejoining of broken DNA. V(D)J recombination adheres to the 12/23 rule, which limits rearrangement to signal sequences bearing different lengths of DNA (12 or 23 base pairs) between the conserved heptamer and nonamer sequences to which the RAG proteins bind. Both RAG proteins have been subjected to extensive mutagenesis, revealing residues required for one or both cleavage steps or involved in the DNA end-joining process. Gain-of-function RAG mutants remain unidentified. Here, we report a novel RAG-1 mutation, E649A, that supports elevated cleavage activity in vitro by preferentially enhancing hairpin formation. DNA binding activity and the catalysis of other DNA strand transfer reactions, such as transposition, are not substantially affected by the RAG-1 mutation. However, 12/23-regulated synapsis does not strongly stimulate the cleavage activity of a RAG complex containing E649A RAG-1, unlike its wild-type counterpart. Interestingly, wild-type and E649A RAG-1 support similar levels of cleavage and recombination of plasmid substrates containing a 12/23 pair of signal sequences in cell culture; however, E649A RAG-1 supports about threefold more cleavage and recombination than wild-type RAG-1 on 12/12 plasmid substrates. These data suggest that the E649A RAG-1 mutation may interfere with the RAG proteins' ability to sense 12/23-regulated synapsis.

Two lymphoid cell-specific proteins, called RAG-1 and RAG-2, initiate the process of antigen receptor gene rearrangement, termed V(D)J recombination, by assembling a protein-DNA complex with two recombination signal sequences (RSSs), each of which adjoins a different receptor gene segment, and then introducing a DNA double strand break at the end of each RSS. The study of RAG-RSS complex assembly and activity has been facilitated by the development of methods to purify the RAG proteins and members of the HMG-box family of high mobility group proteins such as HMGB1 that promote RAG binding and cleavage activity in vitro. This chapter describes the purification of recombinant truncated and full-length RAG-1 and RAG-2 expressed transiently in mammalian cells, as well as the purification of bacterially expressed full-length HMGB1. In addition, it details several experimental procedures used in our laboratory to study RAG-RSS complex formation and function in vitro.

Several lines of reasoning suggest that the phosphorylated axonal form of the neurofilament subunit NF-H is likely to be released from damaged and diseased neurons in significant amounts. Detection of this protein in serum or CSF might therefore provide information about the presence and degree of neuronal loss. We therefore developed a sensitive NF-H ELISA capable of detecting picogram quantities of phosphorylated NF-H (pNF-H). This assay showed that soluble pNF-H immunoreactivity is readily detectable in the sera of adult rats following various types of experimental spinal cord injury (SCI) and traumatic brain injury (TBI), but is undetectable in the sera of normal animals. Here we describe details of the time course and extent of serum pNF-H expression following experimental SCI and TBI. Following SCI, serum pNF-H showed an initial peak of expression at 16h and a second, usually larger, peak at 3 days. Following TBI, lower levels of serum pNF-H were detected with a peak at 2 days post-injury. We also show that the higher levels of pNF-H released from injured spinal cord as compared to brain are in line with the approximately 20-fold higher levels of pNF-H present in spinal cord. These findings suggest that serum levels of pNF-H immunoreactivity may be used to conveniently monitor neuronal damage and degeneration in experimental and presumably clinical situations.

Mechanical spinal cord injury (SCI) initiates a cascade of pathochemical and pathophysiological events, collectively known as the secondary injury. There has been a long-standing interest in understanding the activation and involvement of proteases in this secondary injury process. Several proteases including the calpains, caspases and matrix metalloproteinases are activated by perturbations to the spinal cord and have been linked to cell death following SCI and in other models of CNS disease and insult. Cathepsin B (Cath B), a potent lysosomal protease, has also been implicated in the pathology of CNS diseases including brain tumors, Alzheimer's disease, amyotrophic lateral sclerosis and stroke. Previously, we reported significant increases in Cath B mRNA and protein expression following contusion-SCI. This characterization of Cath B continues with the experiments reported herein, which were designed to examine Cath B enzymatic activity and cellular localization following contusion-SCI in the rat. Cath B enzymatic activity was significantly increased in the injury epicenter at 5 and 7 days post-injury and was highly correlated with increases in the active forms of the Cath B protein reported earlier. Furthermore, the immunohistochemical analyses revealed that the post-injury increases in expression and enzymatic activity at the injury epicenter were due to the presence of a large and diverse population of inflammatory cells. However, in areas adjacent to the injury epicenter, it appears that parenchymal neurons may also contribute to these increases. Our findings coupled with the documented role of Cath B in other CNS pathologies make this potent protease an attractive candidate for involvement in the tissue destruction associated with the secondary injury cascade following SCI.

We provide the first data that cathepsin B (Cath B), a lysosomal cysteine protease, is up-regulated following contusion-spinal cord injury (SCI). Following T12 laminectomy and moderate contusion, Cath B mRNA and protein expression profiles were examined from 2 to 168 h post-injury in rats using real-time PCR and immunoblots, respectively. Contusion injury significantly increased [mRNA]Cath B in the injury site and adjacent segments over sham injury levels. While the largest [mRNA]Cath B induction (20-fold over naive) was seen in the injury site, the caudal segment routinely yielded [mRNA]Cath B levels greater than 10-fold over naive. Interestingly, sham injury animals also experienced mRNA induction at several time points at the injury site and in segments rostral and caudal to the injury site. Contusion injury also significantly elevated levels of Cath B proenzyme protein (37 kDa) over sham injury in the injury site (48, 72 and 168 h post-injury). Furthermore, significant protein increases of single and double chain Cath B (both active forms) occurred at the injury site at 72 and 168 h post-injury. Similar significant increases in Cath B protein levels were seen in areas adjacent to the injury site. The induction of Cath B mRNA and protein expression following contusion injury is previously undescribed and suggests that Cath B may potentially be involved in the secondary injury cascade, perhaps for as long as 1 week post-injury.

Aggrecan is a chondroitin sulfate (CS)/keratan sulfate (KS)-substituted proteoglycan (PG) abundant in cartilage which is also present within the mammalian embryonic, adult, and injured adult central nervous system (CNS). Although its role within the CNS is not clear, cell culture studies show that when substituted with CS, aggrecan inhibits neurite extension. To better understand the inhibitory effect of aggrecan on injured adult axons in vivo, we developed a model to independently test intact aggrecan and CS-depleted aggrecan core glycoprotein. Acute rat spinal cord hemisection cavities were filled with a growth-promoting matrix, Matrigel, and severed dorsal rootlets were placed into this matrix. This created an assay in which axons readily grew. The extent of ingrowth in this baseline assay was compared to the ingrowth in Matrigel loaded with intact aggrecan or the purified core glycoprotein of aggrecan. Our results show that both intact aggrecan and equivalent concentrations of the core glycoprotein component significantly inhibit axonal growth in this model system. These results confirm that aggrecan can inhibit the growth of adult axons in vivo and suggest that the inhibitory effects of aggrecan may be mediated, at least in part, by structures located on the core glycoprotein in the absence of the bulk of the CS chains.

Free radical-mediated mechanisms of cellular damage have been implicated in the early stages of spinal cord injury (SCI). Manganese superoxide dismutase (MnSOD) is a potent scavenger of superoxide radicals and likely serves an important cytoprotective role in preventing cellular damage after SCI. We have evaluated the expression of MnSOD to address its role during the early events of SCI using a well-established rat contusion model. Northern analysis showed a rapid induction of MnSOD mRNA between 2 and 6 h post injury. Observed time-dependent increases in MnSOD message was maximal 6 h post injury over that of MnSOD mRNA levels induced by laminectomy alone. Immunoblot and immunohistochemical analysis demonstrated increased expression of MnSOD protein 24 h after SCI with localization primarily within neurons. Interestingly, laminectomy alone also caused an induction of MnSOD gene and protein expression. To evaluate one potential mechanism of MnSOD induction, we microinjected the naive spinal cord with IL-1beta, which caused a similar fold induction of MnSOD mRNA levels by 6 h as observed with SCI, thus implicating it as a potential inducer of MnSOD during SCI. In summary, these results demonstrate that this potent cytoprotective antioxidant enzyme is rapidly and significantly induced as a consequence of SCI.

Increases in intracellular calcium and subsequent activation of calcium-activated proteases (e.g., calpains) may play a critical role in central nervous system injury. Several studies have implicated calpain activation following subarachnoid hemorrhage (SAH). This study evaluated the effect of a calpain inhibitor administration following SAH in the rat on behavioral deficits (postinjury days 1-5, employing a battery of well-characterized assessment tasks), and blood-brain barrier permeability changes (48 h post-SAH, quantifying the microvascular alterations according to the extravasation of protein-bound Evans Blue using a spectrophotofluorimetric technique). Rats were injected with 400 microl of autologous blood into the cisterna magna to induce SAH. Within 5 min after the surgical procedure, Calpain Inhibitor II or vehicle was continuously administered intravenously for 2 days. Results indicated that Calpain Inhibitor II treatment after SAH significantly improved (a) beam balance time (day 1, p < 0.05), but not beam balance score, (b) latency to traverse the beam on days 1-4 (day 1-3, p < 0.001; day 4, p < 0.01), and (c) loss in body weight on days 4-5 (p < 0.05). Evans Blue dye extravasation was significantly less in SAH Calpain Inhibitor II-treated rats compared to SAH vehicle-treated rats in seven out of the eight brain regions studied (p < 0.001, 0.01, and 0.05). These results suggest that pharmacological inhibition of a relatively selective, membrane-permeant calpain inhibitor can significantly reduce some pathophysiological SAH consequences, and indicate that the inhibition of calpain may be a beneficial therapeutic approach to reduce post-SAH global brain dysfunction.

Transplantation of fetal spinal cord (FSC) tissue has demonstrated significant potential in animal models for achieving partial anatomical and functional restoration following spinal cord injury (SCI). To determine whether this strategy can eventually be translated to humans with SCI, a pilot safety and feasibility study was initiated in patients with progressive posttraumatic syringomyelia (PPTS). A total of eight patients with PPTS have been enrolled to date, and this report presents findings for the first two patients through 18 months postoperative. The study design included detailed assessments of each subject at multiple pre- and postoperative time points. Outcome data were then compared with each subject's own baseline. The surgical protocol included detethering, cyst drainage, and implantation of 6-9-week postconception human FSC tissue. Immunosuppression with cyclosporine was initiated a few days prior to surgery and continued for 6 months postoperatively. Key outcome measures included: serial magnetic resonance imaging (MRI) exams, standardized measures of neurological impairment and functional disability, detailed pain assessment, and extensive neurophysiological testing. Through 18 months, the first two patients have been stable neurologically and the MRIs have shown evidence of solid tissue at the graft sites, without evidence of donor tissue overgrowth. Although it is still too soon to draw any firm conclusions, the findings from the initial two patients in this study suggest that intraspinal grafting of human FSC tissue is both feasible and safe.