Transferred antigen-specific T(H)17 but not T(H)1 cells induce crescentic glomerulonephritis in mice

To explore the role of antigen-specific CD4(+) T cells in glomerulonephritis, we administered ovalbumin 323-339 peptide conjugated to glomerular-binding polyclonal antibody and induced disease in RAG1(-/-) mice with CD4(+) T cells from OT2 × RAG1(-/-) mice. These OT2 × RAG1(-/-) mice have a transgenic T-cell receptor specific for this peptide. When CD4(+) T cells were primed in vivo, crescentic glomerulonephritis developed after 21 days in mice given peptide-conjugated glomerular-binding antibody but not unconjugated antibody control. We then investigated the relative roles of T(H)1 and T(H)17 cells, using Fab(2) fragments of glomerular-binding antibody to exclude a role for antibody in this model. T cells from OT2 × RAG1(-/-) mice were polarized in vitro, and T(H)1 or T(H)17 cell lines were injected into mice that were also given peptide-conjugated Fab(2) or unconjugated Fab(2) control, giving four experimental groups. After 21 days crescentic glomerulonephritis was seen in mice receiving T(H)17 cells and peptide-conjugated Fab(2) but in none of the other three groups. These results suggest that T(H)17 but not T(H)1 cells can induce crescentic glomerulonephritis.     

Memory precursor phenotype of CD8+ T cells reflects early antigenic experience rather than memory numbers in a model of localized acute influenza infection

The mechanistic basis of memory T‐cell development is poorly defined. Phenotypic markers that define precursors at effector stages have been characterized for acute systemic infections with high antigen load. We asked whether such markers can identify memory precursors from early effectors (d6) to late memory (>d500) for two immunodominant CD8⁺ responses during the course of a localized low‐load influenza infection in mice. CD8⁺ T cells stained with the D^bNP₃₆₆ and D^bPA₂₂₄ tetramers were characterized as IL‐7Rα^hi, IL‐7Rα^hiCD62L^hi or IL‐7Rα^hiKLRG1^lo. While the D^bNP₃₆₆‐ and D^bPA₂₂₄‐specific responses were comparable in size, decay kinetics and memory precursor frequency, their expansion characteristics differed. This correlated with a divergence in the IL‐7Rα^hi, IL‐7Rα^hiCD62L^hi and IL‐7Rα^hiKLRG1^lo phenotypes on effector, but not naïve, CD8⁺ populations. That effect was abrogated by priming with viruses engineered to present equivalent levels of NP₃₆₆ and PA₂₂₄ peptides, indicating that memory phenotypes reflect early antigenic experience rather than memory potential. Thus, the IL‐7Rα^hiKLRG1^lo phenotype had a poor predictive value in identifying memory precursors in the spleen and at the site of infection. Greater consistency in influenza‐specific IL‐7Rα^hiKLRG1^loCD8⁺ T‐cell numbers was found in draining lymph nodes, suggesting that this may be the preferential site for memory establishment and maintenance following localized virus infections.     

Participation of CD11c(+) leukocytes in methicillin-resistant Staphylococcus aureus clearance from the lung

Staphylococcus aureus causes especially severe pulmonary infection, associated with high morbidity and mortality. In addition to the effects of specific virulence factors, it appears that the intensity of the host proinflammatory response, particularly in the initial stages of infection, contributes substantially to pulmonary damage. We tested the hypothesis that the CD11c(+) leukocytes are important in the host response to pulmonary infection with methicillin-resistant S. aureus (MRSA) USA300. Clodronate-induced depletion of the alveolar macrophage population resulted in increased numbers of dendritic cells (DCs) and CD4(+) cells in bronchoalveolar lavage (BAL) fluid and was associated with significantly increased mortality by 18 h following S. aureus inoculation but had no effect on bacterial load or polymorphonuclear leukocyte (PMN) numbers in the lung. These clodronate-treated mice also had increased expression of interleukin-17A/F (IL-17A/F) and CXCL10 but not of gamma interferon (IFN-γ) or tumor necrosis factor (TNF). Depletion of the dendritic cell population in mice expressing a CD11c-enhanced green fluorescent protein (EGFP)-diphtheria toxin receptor (DTR) transgene was associated with an increased bacterial load in the lung but not increased mortality. Both DCs and airway epithelial cells produced CXCL9, -10, and -11 in response to S. aureus. Pretreatment of mice with an anti-CXCR3 antibody prior to inoculation with MRSA substantially reduced CD4(+) cells and decreased pulmonary inflammation at 18 h postinfection compared to pretreatment with an IgG control. The results of these experiments suggest that CD11c(+) cells, the induction of CXCR3 ligand expression, and subsequent CD4(+) cell recruitment have an important role in the pathogenesis of severe MRSA pulmonary infection.     

Change in self-protect ion and symptoms after dynamic psychotherapy: the influence of pretreatment motivation

The First Experimental Study of Transference-interpretations (FEST) is a dismantling, randomized clinical trial of the long-term effects of transference interpretation. This article looks at the influence of motivation on the ability to self-protect and symptoms. Patients were randomized to receive a moderate level of transference interpretations or no transference interpretation. Assessments were made at pretreatment, midtreatment, posttreatment, and at two follow-ups. The outcome measure, change in ability to self-protect, was cluster 4 of the Structural Analysis of Social Behavior (SASB) Long Form Intrex Introject Questionnaire, a measure close to the SASB-coded treatment process. Symptom change (SCL-90) was also analyzed. Pretreatment motivation was a significant moderator of change in the ability to self-protect and in symptom distress. Those with low motivation had a significant, positive, and long-term treatment effect of transference interpretation. Patients with low motivation showed a significant, negative effect of transference interpretation.     

Overexpression of SERPIN B3 promotes epithelial proliferation and lung fibrosis in mice

SERPIN B3/B4, members of the serpin superfamily, are fundamental for the control of proteolysis through a known inhibitory function of different proteases. Several studies have documented an important role of SERPIN B3 in the modulation of inflammation, programmed cell death and fibrosis. To confirm the role of SERPIN B3 in lung fibrosis and overall investigate its influence on epithelial dysfunction, a stratified controlled trial randomly assigning bleomycin (BLM) treatment was performed on both SERPIN B3 transgenic (TG) and wild-type (WT) mice. TG and WT animals were killed 48?h (group T48?h) and 20 days (group T20d) after BLM treatment. Lung fibrosis was assessed by histology and hydroxyproline measurement. Architectural remodeling, inflammation, epithelial apoptosis and proliferation were quantified. Moreover, the profibrogenetic cytokine transforming growth factor (TGF)-β, cathepsin K, L and S were also investigated. No significant differences were observed between TG and WT mice of group T48?h in any parameters. In group T20d, less inflammation and a significant increase in epithelial proliferation were detected in treated TG than WT mice despite a similar apoptotic index, thus resulting in a different apoptosis/proliferation imbalance with a significant gain of epithelial proliferation. Moreover, TG mice showed higher TGF-β expression and more extended fibrosis. General linear model analysis, applied on morphological data, showed that interaction between SERPIN B3 expression and treatment was mainly significant for fibrosis. This study provides in vivo evidence for a role of SERPIN B3 in inhibiting inflammation and favoring epithelial proliferation with increased TGF-β secretion and thus the likelihood of consequent fibrogenesis.     

Functional assessment in patients with osteoporotic wrist fractures treated with external fixation: a review of randomized trials

Introduction  Osteoporosis is a chronic and progressive condition that leads to decreased bone mass and skeletal fragility which may result in fractures, disability, pain, deformity and even death. Fractures of the wrist are the most common symptomatic fracture related to osteoporosis in which up to 80% of the persons with this fracture type have low bone mass. External fixation is minimally-invasive and is used in particular for the treatment of displaced, unstable fractures. Objective  The present systematic review will present functional outcome assessment in randomized controlled trials in the aged with distal radius fractures and treated with external fixation. Materials and methods  Multiple databases including Medline, EMBASE, CINAHL, AMED and OVID Healthstar were searched. MESH headings such as: “Radius fractures” or “wrist injuries” were used in combination with “randomized controlled trials”. Studies were included if the surgical treatment was external fixation, mean patient age was 50 years and over, and were in the English language. Results  The primary focus of wrist outcome assessments in patients treated with external fixation were based on traditional measures such as wrist range of movement and grip strength reflecting the need to shift the focus in future protocols towards measuring a patient’s functional ability, measuring the difficulty of task performance and compensatory mechanisms. Furthermore, it is essential to take into account more evidence of instrument development before clinicians can reliably choose the best measure to assess the aged treated with external fixation.     

Lessons from BWS twins: complex maternal and paternal hypomethylation and a common source of haematopoietic stem cells

The Beckwith–Wiedemann syndrome (BWS) is a growth disorder for which an increased frequency of monozygotic (MZ) twinning has been reported. With few exceptions, these twins are discordant for BWS and for females. Here, we describe the molecular and phenotypic analysis of 12 BWS twins and a triplet; seven twins are MZ, monochorionic and diamniotic, three twins are MZ, dichorionic and diamniotic and three twins are dizygotic. Twelve twins are female. In the majority of the twin pairs (11 of 13), the defect on chromosome 11p15 was hypomethylation of the paternal allele of DMR2. In 5 of 10 twins, there was additional hypomethylation of imprinted loci; in most cases, the loci affected were maternally methylated, but in two cases, hypomethylation of the paternally methylated DLK1 and H19 DMRs was detected, a novel finding in BWS. In buccal swabs of the MZ twins who share a placenta, the defect was present only in the affected twin; comparable hypomethylation in lymphocytes was detected in both the twins. The level of hypomethylation reached levels below 25%. The exchange of blood cells through vascular connections cannot fully explain the degree of hypomethylation found in the blood cell of the non-affected twin. We propose an additional mechanism through which sharing of aberrant methylation patterns in discordant twins, limited to blood cells, might occur. In a BWS-discordant MZ triplet, an intermediate level of demethylation was found in one of the non-affected sibs; this child showed mild signs of BWS. This finding supports the theory that a methylation error proceeds and possibly triggers the twinning process.     

The ClinSeq Project: Piloting large-scale genome sequencing for research in genomic medicine

ClinSeq is a pilot project to investigate the use of whole-genome sequencing as a tool for clinical research. By piloting the acquisition of large amounts of DNA sequence data from individual human subjects, we are fostering the development of hypothesis-generating approaches for performing research in genomic medicine, including the exploration of issues related to the genetic architecture of disease, implementation of genomic technology, informed consent, disclosure of genetic information, and archiving, analyzing, and displaying sequence data. In the initial phase of ClinSeq, we are enrolling roughly 1000 participants; the evaluation of each includes obtaining a detailed family and medical history, as well as a clinical evaluation. The participants are being consented broadly for research on many traits and for whole-genome sequencing. Initially, Sanger-based sequencing of 300–400 genes thought to be relevant to atherosclerosis is being performed, with the resulting data analyzed for rare, high-penetrance variants associated with specific clinical traits. The participants are also being consented to allow the contact of family members for additional studies of sequence variants to explore their potential association with specific phenotypes. Here, we present the general considerations in designing ClinSeq, preliminary results based on the generation of an initial 826 Mb of sequence data, the findings for several genes that serve as positive controls for the project, and our views about the potential implications of ClinSeq. The early experiences with ClinSeq illustrate how large-scale medical sequencing can be a practical, productive, and critical component of research in genomic medicine.Elucidating the sequence of the human genome (International Human Genome Sequencing Consortium 2001, 2004) and subsequent advances in DNA sequencing technologies (Mardis 2008) have the potential to dramatically improve the delivery of health care through the acquisition of genomic information about individual patients. However, much research will be needed to develop medical applications of genomics; for example, little is known about how to organize and implement large-scale medical sequencing (LSMS; i.e., systematic resequencing of human DNA) in a clinical context. Other approaches for applying high-throughput genomics to health care (e.g., assaying single-nucleotide polymorphisms and establishing gene-expression profiles) offer diagnostic promise; these are not further considered here, as our focus is on LSMS for studying the relationship of germline genomic variation to health and disease.We recently launched ClinSeq (http://genome.gov/20519355), a project that aims to apply LSMS within a clinical research environment to answer questions about the genetic basis of health, disease, and drug response. The application of genomic approaches (in particular LSMS) in a clinical research context is associated with a number of considerations that define key “dimensions” of any study: the number of subjects, the associated clinical data, and the breadth of genome covered (Fig. 1). Numerous detailed studies of single genes have been carried out; while often performed on many participants with significant amounts of phenotypic information, they are focused on a very small portion of the genome. The flurry of papers that describe recently generated whole-genome sequences (Levy et al. 2007; Bentley et al. 2008; Wang et al. 2008; Wheeler et al. 2008) has provided the first true individual genome sequences, including a modest amount of associated clinical data; however, the number of examples is small to date. Greater numbers are promised by the 1000 Genomes Project (http://www.1000genomes.org/), although no phenotypic information will be available for the individuals being studied. ClinSeq aims to model a more ideal study with respect to these three dimensions (Fig. 1), with the potential to further move toward the ultimate ideal as technology advances.Open in a separate windowFigure 1.A spatial conceptualization of research studies in genomic medicine. There are three key “dimensions” to consider when applying genomics to clinical research: genome breadth (the fraction of the genome that is interrogated), number of subjects or participants, and the associated clinical data about those individuals (including its depth, breadth, and rigor). While the ideal study would acquire whole-genome sequences from large numbers of extensively phenotyped subjects, this is currently impractical. Single-gene studies can involve a few or numerous subjects and extensive clinical data, but by definition involve the examination of only a single gene and thus occupy one wall of this space. The individual genomes that have recently been sequenced (Levy et al. 2007; Bentley et al. 2008; Wang et al. 2008; Wheeler et al. 2008) provide nearly complete genome breadth, but with limited clinical data; further, their limited subject numbers place them on another wall of this space. The 1000 Genomes Project (http://www.1000genomes.org/) is providing large subject numbers and extensive genome breadth, but no clinical data—positioning it on the floor of this space. ClinSeq aims to reside in the center of this space, having attributes of substantial subject size (n = 1000 initially), moderate genome breadth (∼400 genes initially, with plans for expanding this breadth), and substantial clinical data.The general aims of ClinSeq are to: (1) develop the infrastructure and approaches to acquire and analyze genome sequence from individual research participants; (2) pilot the use of LSMS to elucidate the genetic architecture underlying human traits; (3) provide an open, shared resource and environment for basic and clinical researchers to work collaboratively to perform research in genomic medicine; and (4) establish approaches for informed consent and the return of genetic information to subjects participating in LSMS studies. In pursuing these aims, our overriding goals include modeling whole-genome sequence acquisition in a manner that is practical for a clinical research setting, advancing our understanding of the genetic basis of important human diseases and traits, and establishing how to scale LSMS prior to the day when whole-genome sequencing becomes part of routine clinical practice. In this paper, we describe the ClinSeq study design, provide a snapshot of our very early data generation, and discuss the implications of this study for the nascent field of genomic medicine.