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Patients are increasingly viewed as key stakeholders who can contribute in meaningful ways to clinical research and are emphasized in research funded by the Patient-Centered Outcomes Research Institute (PCORI). We are not aware of other peer-reviewed publications that report methods and outcomes of patient engagement to refine study design for a PCORI-sponsored clinical effectiveness trial. The aim of this report was to describe the process and outcomes of involving patients in the design of a clinical trial to promote adherence to supplemental oxygen therapy among patients with chronic obstructive pulmonary disease. In-person focus groups and individual discussions via telephone and email were used to elicit feedback to refine the intervention and clarify outcomes of highest importance to patients. A total of 25 patients and five caregivers provided feedback. Their feedback has informed decisions regarding the length of intervention sessions (20 min and in some cases longer was acceptable), the importance of including caregivers, and discussion topics (e.g., social discomfort about using oxygen in public, identifying personally relevant reasons to use oxygen, pulmonary rehabilitation). Multiple outcomes were rated as highly important to patients (physical function, fatigue, sleep, anxiety, depression, and ability to participate in social roles and activities), and the outcome that was ranked as most important varied by individual. Therefore, multiple patient-reported outcomes will be used as endpoints for the clinical trial. Patient involvement led to refinements of the intervention and clinical trial endpoints to better address the expressed needs and concerns of patients and caregivers.

The homopentameric rho1 GABA(C) receptor is a ligand-gated ion channel with a binding pocket for gamma-aminobutyric acid (GABA) at the interfaces of N-terminal extracellular domains. We combined evolutionary analysis, structural modeling, and experimental testing to study determinants of GABA(C) receptor assembly and channel gating. We estimated the posterior probability of selection pressure at amino acid residue sites measured as omega-values and built a comparative structural model, which identified several polar residues under strong selection pressure at the subunit interfaces that may form intersubunit hydrogen bonds or salt bridges. At three selected sites (R111, T151, and E55), mutations disrupting intersubunit interactions had strong effects on receptor folding, assembly, and function. We next examined the role of a predicted intersubunit salt bridge for residue pair R158-D204. The mutant R158D, where the positively charged residue is replaced by a negatively charged aspartate, yielded a partially degraded receptor and lacked membrane surface expression. The membrane surface expression was rescued by the double mutant R158D-D204R, where positive and negative charges are switched, although the mutant receptor was inactive. The single mutants R158A, D204R, and D204A exhibited diminished activities and altered kinetic profiles with fast recovery kinetics, suggesting that R158-D204 salt bridge perhaps stabilizes the open state of the GABA(C) receptor. Our results emphasize the functional importance of highly conserved polar residues at the protein-protein interfaces in GABA(C) rho1 receptors and demonstrate how the integration of computational and experimental approaches can aid discovery of functionally important interactions.

Objectives: To establish optimal culture conditions for fetal endothelial cells, and determine whether these can be used for preferential expansion of fetal cells from maternal blood. Methods: Human adult microvascular and umbilical vein endothelial cells were cultured in the presence of colony-stimulating factor-1 (CSF-1), placental growth factor (PlGF), and transforming growth factor-β1 (TGF-β1). The effect of each cytokine was assessed. We expanded peripheral blood mononuclear cells (PBMCs) from 18 pregnant women using the conditions most favorable to fetal cells; in specimens from women carrying male fetuses (n = 9), cell origin was determined by PCR (SRY locus). Results: The optimal concentrations of CSF-1, PlGF and TGF-β1 were 10, 100, and 5 ng/ml, respectively. PBMCs from maternal blood expanded in the presence or absence of the cytokines; PCR analysis showed no Y sequences in cultured maternal samples. Conclusion: Optimal concentrations of CSF-1, PlGF and TGF-β1 for preferential expansion of fetal endothelial cells were determined in model cultures. However, when these conditions were applied to maternal blood samples, no fetal cells could be detected based on PCR for SRY in women carrying male fetuses.

Objectives We hypothesized that fetal cells in maternal blood that do not respond to hematopoietic culture conditions represent endothelial cells. We investigated whether endothelial progenitor cells of fetal origin may be selected from maternal blood on the basis of their expression of CD133 or CD105 and expanded in culture. Methods Peripheral blood mononuclear cells from 16 pregnant women (gestational age: 11 to 24 weeks) were labeled with magnetic beads coupled to anti-CD133 or anti-CD105. Selection of labeled cells was performed using MACS. Resulting CD133+, CD105+, and CD133−/CD105− cell fractions were placed in culture in conditions favoring endothelial cells for 7 days (positive fractions) to 30 days (depleted fractions). Cells from women carrying male fetuses were analyzed by conventional PCR (SRY primers) for detection of male cells. Results Expansion of cells isolated from all subjects occurred in each of the cell fractions. No PCR products consistent with the presence of male cells were detected in women carrying male fetuses. Conclusion CD133+ and CD105+ cells isolated from maternal blood can be expanded in vitro under endothelial conditions. These cells appear to be of maternal, rather than fetal, origin. Copyright © 2004 John Wiley & Sons, Ltd.

The isolation and analysis of fetal cells from maternal blood would allow non-invasive prenatal genetic screening and diagnosis. Over the past decade, progress has been made towards this goal using various enrichment strategies and analysis by fluorescence in-situ hybridization with chromosome-specific probes and PCR. One method that is currently being explored involves culturing fetal cells. Developing conditions which allow the number of fetal-derived cells to expand in culture and the number of maternally derived cells to be suppressed in culture may lead to a new selection process for obtaining fetal cells. Culturing of fetal cells from maternal blood could make possible conventional metaphase analysis of fetal cells for diagnosis of chromosomal abnormalities.

The mechanism of attachment of circulating immune complexes (CIC) to glomerular basement membranes (GBM) has not yet been elucidated. Since it has been proposed that histone may be the ligand between GBM and DNA/anti-DNA CIC, we explored by ELISA and Western blots the nature of the interaction of IgG with histone on solid phase. Cognate binding of IgG anti-histone antibody was characteristically dependent on in its F(ab')(2) fragment and was inhibited by free histone. On the other hand, heat-aggregated IgG, a model for CIC, and IgG from most patients with idiopathic SLE had a characteristic noncognate binding behavior to histone: it was dependent on Fcgamma rather than on the F(ab')(2) fragment and was not effectively inhibited by free histones. Also, binding to histone of in vitro generated DNA/anti-DNA immune complexes was not dependent on DNA as a histone ligand, but on Fcgamma. Finally, there was good agreement between the binding of this IgG to histone and to C1q. We concluded that: (1) altered IgG and/or CIC bind to solid-phase-attached histone primarily through their Fcgamma and (2) CIC may mimic IgG antihistone antibodies in solid-phase immunoassays.

Approximately 20% of patients hospitalized for COPD exacerbations in the United States will be readmitted within 30 days. The Centers for Medicare and Medicaid Services has recently proposed to revise the Hospital Readmissions Reduction Program to financially penalize hospitals with high all-cause 30-day rehospitalization rates after a hospitalization for COPD exacerbation on or after October 1, 2014.To report the results of a systematic review of randomized clinical trials evaluating interventions to reduce the rehospitalizations after COPD exacerbations.Multiple electronic databases were systematically searched to identify relevant studies published between January 1966 and June 2013. Titles, abstracts, and, subsequently, full-text articles were assessed for eligibility. Each study was appraised using predefined criteria.Among 913 titles and abstracts screened, 5 studies (1,393 participants) met eligibility criteria. All studies had a primary outcome of rehospitalization at 6 or 12 months. No study examined 30-day rehospitalization as the primary outcome. Each study tested a different set of interventions. Two studies (one conducted in Canada and one conducted in Spain and Belgium) showed a decrease in all-cause rehospitalization over 12 months in the intervention group versus comparator group (mean number of hospitalizations per patient, 1.0 vs. 1.8; P = 0.01; percent hospitalized, 45 vs. 67%; P = 0.028; respectively). The only study conducted in the United States found a greater than twofold higher risk of mortality in the intervention group (17 vs. 7%, P = 0.003) but no significant difference in rehospitalizations. It was unclear which set of interventions was effective or harmful.The evidence base is inadequate to recommend specific interventions to reduce rehospitalizations in this population and does not justify penalizing hospitals for high 30-day rehospitalization rates after COPD exacerbations.

About 20% of patients hospitalized for chronic obstructive pulmonary disease (COPD) exacerbations are readmitted within 30 days. High 30-day risk-standardized readmission rates after COPD exacerbations will likely place hospitals at risk for financial penalties from the Centers for Medicare and Medicaid Services starting in fiscal year 2015. Factors contributing to hospital readmissions include healthcare quality, access to care, coordination of care between hospital and ambulatory settings, and factors linked to socioeconomic resources (e.g., social support, stable housing, transportation, and food). These concerns are exacerbated at minority-serving institutions, which provide a disproportionate share of care to patients with low socioeconomic resources. Solutions tailored to the needs of minority-serving institutions are urgently needed. We recommend research that will provide the evidence base for strategies to reduce readmissions at minority-serving institutions. Promising innovative approaches include using a nontraditional healthcare workforce, such as community health workers and peer-coaches, and telemedicine. These strategies have been successfully used in other conditions and need to be studied in patients with COPD.

GABAA receptors are ligand-gated ion channels that mediate inhibitory synaptic signaling in the CNS. Fluorescent probes with the ability to target these receptors can provide insights into receptor location, distribution and dynamics in live cells, while revealing abnormalities in their distribution and dynamics that could occur in a variety of diseases. We have developed fluorescent probes of GABAA receptors that are composed of a CdSe/ZnS core-shell nanocrystal (quantum dot; qdot) conjugated to pegylated derivatives of the GABA receptor agonists GABA and muscimol (GABA-qdots and muscimol-qdots, respectively). Quantitative fluorescence imaging was used to analyze the binding activity of these conjugates to α1β2γ2 GABAA and ρ1 GABAA receptors expressed in Xenopus oocytes. The selectivity of these conjugates for α1β2γ2 GABAA and ρ1 GABAA receptors was determined by their ability to compete with the antagonists bicuculline and methyl-(1,2,3,6-tetrahydropyridin-4-yl)phosphinic acid (TPMPA). Both GABA- and muscimol-qdots exhibited robust binding to both α1β2γ2 and ρ1 GABAA receptors. At α1β2γ2 receptors, pretreatment with bicuculline reduced conjugate binding by ≥8-fold on average, an extent far exceeding the reduction produced by TPMPA (~30%). Conversely, at ρ1 receptors, pretreatment with TPMPA inhibited binding by ~10-fold, an extent greatly exceeding the change produced by bicuculline (~50% or less). These results indicate specific binding of muscimol-qdots and GABA-qdots to α1β2γ2 GABAA and ρ1 GABAA receptors in a manner that preserves the respective pharmacological sensitivities of these receptors to TPMPA and bicuculline, and encourage the use of qdot-conjugated neurotransmitter analogs as labeling agents at GABAA receptors.

The GABA(C) receptor, a postsynaptic membrane receptor expressed prominently in the retina, is a ligand-gated ion channel that consists of a combination of ρ subunits. We report characterization of a novel guinea pig polyclonal antibody, termed GABA(C) Ab N-14, directed against a 14-mer peptide (N-14) of the extracellular domain of the human ρ1 subunit. The antibody exhibits high sensitivity for N-14 by ELISA. In Western blots, GABA(C) Ab N-14 shows reactivity with the ρ1 subunit of preparations obtained from ρ1 GABA(C)-expressing neuroblastoma cells, Xenopus oocytes, and mammalian retina and brain. Flow cytometry reveals a rightward shift in mean fluorescence intensity of GABA(C)-expressing neuroblastoma cells probed with GABA(C) Ab N-14. Immunostaining of neuroblastoma cells and oocytes with GABA(C) Ab N-14 yields fluorescence only with GABA(C)-expressing cells. Antibody binding has no effect on GABA-elicited membrane current responses. Immunostaining of human retinal sections shows preferential staining within the inner plexiform layer. GABA(C) Ab N-14 appears well suited for investigative studies of GABA(C) ρ1 subunit in retina and other neural tissues.

To generate monoclonal antibodies to the human ρ1 GABA(C) receptor, a ligand-gated chloride ion channel that is activated by the neurotransmitter γ-aminobutyric acid (GABA), we recovered the immunoglobulin variable heavy chain (V(H)) and light chain (V(L)) regions of a guinea pig immunized with a 14-mer peptide segment of the N-terminal extracellular domain of the ρ1 subunit. Oligonucleotide primers were designed and used to amplify the V(H) and V(L) regions of guinea pig RNA by the reverse transcriptase polymerase chain reaction. The amplified and cloned V(H) and V(L) regions were transferred together into a phagemid vector, yielding a library of 5×10(6) members, which displayed chimeric fragments of antigen binding (Fabs) with guinea pig variable and human constant regions fused to protein III of M13 bacteriophage. Through affinity selection of this phage-display library with the biotinylated 14-mer peptide segment of GABA(C), we isolated four different antibody fragments that bound specifically to the immunogenic peptide. Phage particles displaying two of these antibodies, but not negative controls, bound selectively to the surface of neuroblastoma cells expressing the ρ1 GABA(C) receptor. Such antibody fragments will be useful in future studies involving targeting of specific neural tissues that express the GABA(C) receptor.

OBJECTIVE: To determine whether primitive endothelial precursor cells are present in the peripheral blood of pregnant compared with nonpregnant subjects and whether these precursor cells are of fetal or maternal origin. METHODS: Peripheral blood mononuclear cells from 13 pregnant women in the second trimester and from ten nonpregnant women and men were cultured for 8-10 weeks under conditions that promoted endothelial cell development. Early outgrowth (1 week culture) and late outgrowth (4-6 weeks) colonies were observed, their endothelial nature was investigated, and fluorescence in situ hybridization was performed to determine the origin of the colonies from pregnant women's specimens. RESULTS: Peripheral blood mononuclear cell cultures from all pregnant women and all nonpregnant controls yielded early-outgrowth endothelial cells. Late-outgrowth endothelial cells were observed in 61.5% (eight of 13) of pregnant subjects, but in none of the ten nonpregnant controls (chi(2) test; P

The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

OBJECTIVE—To determine whether the reaction of rheumatoid factor (RF) with solid phase histone is due to the simultaneous presence of circulating immune complexes (CICs) or aggregated IgG. METHODS—Serum samples from 56 patients with seropositive rheumatoid arthritis (RA) and 50 random blood bank donors were used. Binding of immunoglobulins to histone was determined by enzyme linked immunosorbent assay (ELISA) and by western blots. Aggregated IgG was obtained by heating at 61oC for 30 minutes. RESULTS—Among the RA sera tested by ELISA, 54% were positive for histone binding by IgM, IgG, or IgA and 20% by IgM only. Heating of normal sera caused a significant enhancement in the binding of IgG to histone (p