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Background: Prescription affordability is a key component of healthcare accessibility and a determinant of health outcomes. Prior studies indicate that up to 1 in 4 Americans report difficulty affording prescriptions.Objective(s): This study aims to identify factors associated with cost-based prescription refusal.Methods: We identified 17,869 study participants from the 2017 National Health Interview Survey who had been prescribed at least one medication in the past 12 months. The outcome was defined as inability to afford at least one prescription medication. Covariates included demographic data, medical history, and social attitudes. Logistic regression models were constructed to identify predictors of cost-based prescription refusal.Results: Among 8223 study participants, 8.1% reported the inability to afford at least one prescription medication in the past 12 months. Twenty-seven covariates were correlated with prescription unaffordability, and 8 were selected by the LASSO: Income (Odds ratio (OR) 0.55), Concerned About Bills (OR 2.0), Emergency Department Visits past 12 months (OR 1.33), Dissatisfaction with Medical Care (OR 1.3), Seeking Insurance Through the Health Insurance Marketplace (OR 1.26), Feeling Sad Most of the Time (OR 1.24), History of Asthma (OR 1.26) and History of Diabetes (OR 1.24).Conclusions: Prescription unaffordability remains a significant public health problem and is more common among low-income individuals and patients with, chronic medical conditions. [ABSTRACT FROM AUTHOR]

Background The epithelial-mesenchymal transition (EMT) is thought to contribute to the overall invasiveness of malignant cells. Expression of cluster of differentiation (CD) 44 and CD90 mark the mesenchymal state in multiple epithelial malignancies. Their role in lung cancer remains unclear, however. This study evaluated the prognostic significance of CD44 and CD90 coexpression in patients with resectable primary non-small cell lung cancer (NSCLC). Methods This was a nonconcurrent cohort study of patients with resectable NSCLC, stratified by the degree of expression of CD44/CD90 double-positive cells in their primary tumor. Flow cytometry was used for immunophenotyping of freshly isolated disaggregated tumor. We analyzed the relationship between expression of CD44/CD90 and relapse-free survival. Results We evaluated 37 patients (18 men; median age, 70 years) with NSCLC. For this group, the geometric mean proportion of cells coexpressing CD44/CD90 was 0.52%. Expression of CD44/CD90 was significantly elevated (24.4%, geometric mean) in 6 patients. The median relapse-free survival for patients with high CD44/CD90 coexpression was 7.7 months (95% confidence interval, 4.2 to 11.7) compared with 40 months (95% confidence interval, 18.2 to 77.8) for the group with low CD44/CD90 coexpression ( p = 0.00006 by Mantel log-rank test). The assessment of risk based upon CD44/CD90 expression status was not correlated with pathologic staging ( p = 0.073 by χ 2 ). Conclusions High expression of CD44 and CD90 was associated with significantly reduced relapse-free survival in NSCLC patients. These results suggest that CD44 and CD90 may be important markers of tumor progression in NSCLC. [ABSTRACT FROM AUTHOR]

Red blood cells (RBCs) can be labeled with N-hydroxysuccinimidobiotin (sulfo-NHS-biotin), which binds to cell surface proteins under aqueous conditions. Biotinylated RBCs can be safely infused and detected in peripheral blood samples using flow cytometry, using a fluorochrome-conjugated streptavidin (SA) detection reagent. Biotinylated RBCs have been used to track survival of transfused RBCs, and have applications in optimizing RBC storage and in understanding donor genetic, environmental and disease factors affecting RBC products. We have developed a closed-system, current good manufacturing practices (cGMP)–compliant procedure for biotinylation of RBCs and a quantitative flow cytometric assay to estimate the dose of cell-bound biotin delivered to the patient. Resulting products were characterized for variability, sterility, endotoxin, hemolysis, total dose of cell-bound biotin and stability. The density of biotin-labeling increased as a log-linear function of sulfo-NHS-biotin–labeling concentration, with greater variability at lower concentrations. The upper estimates of biotin doses in the average product (mean RBC content = 5.55 × 1011) were 9.8 and 73.0 µg for products labeled at 3 and 15 µg sulfo-NHS-biotin/mL of total reaction mixture (27 and 135 nmol/mL packed RBCs), respectively. All products were negative for bacterial and fungal growth at 14 days and were below the limit of endotoxin detection. Biotinylated RBCs were stable in vitro for up to 50 days after labeling. We have validated a closed-system procedure for biotinylating RBCs for investigational use. A standard operating procedure is presented in sufficient detail for implementation in a cGMP-compliant cell-processing facility. [ABSTRACT FROM AUTHOR]

Objective Vascular restenosis remains a major obstacle to long-term success after vascular intervention. Circulating progenitor cells have been implicated in restenosis, and yet it has remained unclear if these cells, particularly nonendothelial progenitors, have an active role in this pathologic process. We hypothesized that circulating CD34 + /c-kit + progenitors would increase after vascular injury, mirrored by changes in the injury signal, stromal cell-derived factor 1α (sdf1α). We further postulated that an antibody-based depletion would mitigate progenitor surge and, in turn, reduce restenosis in a murine model. Methods C57BL6 mice underwent wire injury of the femoral artery and were compared with mice with sham surgery and vessel ligation by flow cytometry as well as by sdf1α enzyme-linked immunosorbent assay of peripheral blood. Next, injured C57BL6 mice treated with a depleting antibody toward the progenitor marker sca-1 or with an isotype control were compared in terms of sdf1α as well as enumeration of progenitors. At 28 days, restenosis was quantified between sca-1- and isotype-treated animals. Results Wire injury generated an increase in sdf1α as well as a surge of CD34 + /c-kit + progenitors relative to nonsurgical controls ( P = .005). Treatment with sca-1 antibody ablated the peripheral surge compared with isotype-treated, injured animals ( P = .02), and sca progenitor depletion reduced the 28-day intima to media ratio in a statistically significant fashion compared with either nontreated ( P = .04) or isotype-treated ( P = .036) animals. Conclusions Our study has demonstrated that sca-1 antibody reduces both progenitor surge and vascular restenosis after endoluminal vascular injury in a murine model. This suggests that circulating progenitors play an active role in restenotic disease. [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp), a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family of transporter molecules, is responsible for maintaining low intracellular concentrations of a variety of extracellular compounds and xenobiotics, and for transport of various intracellular molecules. Many drugs are P-gp substrates and intracellular concentrations of these agents may be critical for drug action. Experience in oncology indicates that repeated exposure to P-gp substrate cytotoxic drugs leads to the selection of drug-resistant tumor cells that overexpress P-gp. Since immunosuppressive agents such as cyclosporine, tacrolimus, sirolimus and corticosteroids are substrates for P-gp and since T-cells also express P-gp, it is conceivable that an analogous mechanism exits for therapy-resistant graft rejection. As will be discussed in this article, P-gp may interfere with the response to immunosuppressive therapy through several distinct mechanisms, and as such may represent an attractive therapeutic target. [Copyright &y& Elsevier]

Background: Intimal hyperplasia (restenosis) is an exaggerated healing response leading to failure of half of vascular interventions. Increasing evidence suggests that circulating progenitor cells contribute to intimal pathology, and clinical studies have demonstrated a correlation between progenitor cells and the incidence of restenosis after cardiovascular interventions. The aims of this study were to characterize the temporal response of CD34+ progenitors following vascular injury in an ovine model and to evaluate an affinity pheresis approach to attenuate this response. Methods: An ovine model underwent either operative vascular injury or a nonvascular surgery (n = 3 per group). Blood was examined perioperatively over 2 weeks by flow cytometry. Next, an affinity pheresis approach to mediate systemic depletion of CD34 progenitors was designed. Custom agarose pheresis matrix with antibody affinity toward CD34 or an isotype control was evaluated in vitro. Next, following vascular injury, sheep underwent perioperative whole blood volume pheresis toward either the progenitor cell marker CD34 (n = 3) or an isotype control (n = 4) for 14 days. Animals were monitored by physical exam as well as complete blood counts. Cells recovered by pheresis were eluted and examined by flow cytometry. Results: Flow cytometry revealed a focal surge of circulating CD34 cells after vascular injury but not among surgical controls (P = .05). Toward the goal of an approach to attenuate the surge of CD34 progenitors, an evaluation of high-flow affinity matrix revealed efficacy in removal of progenitors from ovine blood in vitro. Next, a separate group of animals undergoing affinity pheresis after vascular injury was evaluated to mediate systemic depletion of CD34+ cells. Again, a surge of CD34+ cells was observed among isotype pheresis animals following vascular intervention but was attenuated over 20-fold by a CD34 pheresis approach (P = .029). Furthermore, an average of 77 million CD34-positive cells were eluted from the CD34 pheresis matrix. Despite multiple sessions of pheresis, complete blood counts remained essentially unchanged over 2 weeks. Conclusions: Despite evidence suggesting a role for CD34+ circulating progenitor cells in restenotic pathology, the temporal pattern of CD34 progenitors after vascular injury has not been previously defined. We have demonstrated a surge among circulating CD34+ cells that appears confined to procedures involving vascular injury and that this event seems to occur early after vascular injury. We further conclude that CD34 affinity pheresis attenuates the surge. This approach for direct depletion of progenitors may have important implications for the study of progenitors in vascular restenosis. [Copyright &y& Elsevier]

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. A Tn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein-labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and inoculum 1), ground beef outbreak O157:H7 strain 86-24hl 1 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 10³ or 107 CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 7, 14, 21, and 28. lnoculum 1 survived at significantly higher populations (P < 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates, lnoculum 2 applied at ca. 107 CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue. [ABSTRACT FROM AUTHOR]

In the mid-1990s, HTS labs were built with high-end automation for screening 10,000s of compounds. Compound Management labs continued to manually pick samples and invest in standalone equipment. The bottleneck for screening shifted from testing samples to the distribution of compounds. To solve this problem, Merck & Co., Inc. developed an automated compound distribution center. The facility uses automation from The Automation Partnership and a Merck-developed compound ordering system to provide solid and solution samples to Merck scientists worldwide. [Copyright &y& Elsevier]

Graft-versus-host disease (GvHD) is the major cause of morbidity and mortality following bone marrow transplantation (BMT). The goal of this study of 69 cyclosporin-treated, allogeneic BMT patients was to identify early clinical, laboratory, or histopathologic indicators of the development of progressive, fatal GvHD. Peak values within 100 d of allgeneic BMT for total bilirubin, stool volume in a day, clinical stage of cutaneous GvHD (based on extent of rash), and overall clinical stage of GvHD (based on a combination of graft-versus-host reactions in the skin, liver, and gastrointestinal tract) were most useful (p < 0.05, by logistic regression) in identifying those patients with clinically progressive and fatal average of 40 d or less after BMT. Each unit increase in peak clinical stage of rash (e.g., stage 2 versus stage 3) was associated with an odds ratio incremental risk of 5.8 for clinical progression of GvHD, and each tenfold increase in peak total bilirubin (e.g., 2 mg/dl versus 20 mg/dl) or stool output in a day (e.g., 100 cm3/d versus 1000 cm3/d) was associated with an incremental risk of 8.4 and 10.6, respectively, for a fatal outcome from GvHD. Number of exocytosed lymphocytes and dyskeratotic epidermal keratinocytes (DEK) per linear millimeter of epidermis, the presence of follicular involvement, and the degree of dermal perivascular lymphocytic infiltration in 121 skin biopsy specimens were not associated with the development of progressive or fatal GvHD. Pretransplant total body irradiation was associated (p = 0.03, by Mann-Whitney U testing) with an increased number of DEK in skin biopsy specimens taken less than 20 d after BMT. This study demonstrates that monitoring of total bilirubin, stool output, extent of rash, and overall clinical stage of GvHD is most useful during the first 40 d after BMT in formulating the prognosis of early acute GvHD in allogeneic BMT patients receiving cyclosporin. [ABSTRACT FROM AUTHOR]

Enteropathogenic Escherichia coli (EPEC) strains continue to cause severe and sometimes fatal infantile diarrhea, particularly in Africa. Increased efforts at diagnosis, defining the clinical spectrum of disease, understanding pathogenic mechanisms, and delineating immune responses are desperately needed to develop new strategies to combat EPEC. [Copyright &y& Elsevier]

Since the standardization of CD34 measurement by flow cytometry, predictors of leukapheresis CD34 yield have played a pivotal role in planning donor leukaphereses. We describe here a single institution's experience with a multivariate predictor that was used for 2,929 products without alteration for 20 years. The ordinary least squares regression model variables included log peripheral CD34 count, collection duration (3- versus 4-hours), collection number, donor sex, and transplant type. During the study period we changed flow cytometers twice and leukapheresis instruments once. During the Cobe Spectra era the predictor explained 90% of the variability in CD34 collection yield for autologous transplants (r2 = 0.90), and 70% for allogeneic transplants with an overall sensitivity to predict a CD34 yield of ≥ 1 × 106/kg of 97.7%, and specificity of 81.4%. Implemented prospectively with real-time result reporting, the model allowed us to predict CD34 yield with both 3- and 4-hour collection scenarios. Given this guidance, 3-hour collections were selected by the clinical team 25% of the time, saving patient leukapheresis time and resources. When faced with a prediction of < 1 × 106 CD34/kg, the clinical team chose to defer collection 72% of the time. In instances where leukapheresis was performed despite a poor predicted outcome, 85% of patients collected on the Cobe Spectra, and 92% of patients collected on the Optia, failed to collect at least 1 × 106 CD34/kg. A revised model is tested retrospectively on Optia data, and suggestions for further improvements are discussed. [Display omitted] • Performance of an empirical multivariate algorithm to predict leukapheresis CD34 yield was tracked in a single institution over 20 years, providing a definitive assessment of performance. • The algorithm was stable with high sensitivity (97.7%) and specificity (81.4%) over a 13-year period but required revision when Cobe Spectra leukapheresis instruments were replaced with the automated Terumo Spectra Optia system. • The relative merits of two different approaches to CD34 prediction (physical versus empirical) are discussed in Supplemental Materials , with practical examples. [ABSTRACT FROM AUTHOR]

Background aims. Adipose tissue represents a practical source of autologous mesenchymsl stromal cells (MSCs) and vascular-endothelial progenitor cells, available for regenerative therapy without in vitro expansion. One of the problems confronting the therapeutic application of such cells is how to immobilize them at the wound site. We evaluated in vitro the growth and differentiation of human adipose stromal vascular fraction (SVF) cells after delivery through the use of a fibrin spray system. Methods. SVF cells were harvested from four human adult patients undergoing elective abdominoplasty, through the use of the LipiVsge system. After collagenase digestion, mesenchymal and endothelial progenitor cells (pericytes, supra-adventitial stromal cells, endothelial progenitors) were quantified by flow cytometry before culture. SVF cells were applied to culture vessels by means of the Tisseel flbrin spray system. SVF cell growth and differention were documented by immunofluorescence staining and photomicrography. Results. SVF cells remained viable after application and were expanded up to 3 weeks, when they reached confluence and adipogenic differentiation. Under angiogenic conditions, SYF cells formed endothelial (vWF+, CD31+ and CD34+) tubules surrounded by CD14fl+ and a-smooth muscle sctin+ perivascular/stromal cells. Conclusions. Human adipose tissue is a rich source of autologous stem cells, which are readily available for regenerative applications such as wound healing, without in vitro expansion. Our results indicate that mesenchymal and endothelial progenitor cells, prepared in a closed system from unpassaged lipoaspirate samples, retain their growth and differentiation capacity when applied and immobilized on a substrate using a clinically approved fibrin sealant spray system. [ABSTRACT FROM AUTHOR]

A role for dendritic cells (DC) as critical regulators of immune reactivity has become increasingly recognized. There is evidence in rodent models that donor-derived DC, particularly in the immature state, can prolong organ allograft survival and even induce donor-specific tolerance. To allow the potential tolerogenic properties of these cells to be evaluated more fully with a view to clinical testing, it is necessary to identify DC subsets in nonhuman primates. We have identified the putative rhesus monkey equivalents of circulating human DC subset precursors as lineage−, HLA-DR+, CD123lo,CD11chi(pDC1) and lineage−, HLA-DR+, CD123hi,CD11clo(pDC2). Testing of these DC populations both in vitro and in vivo, as well as in transplant models in combination with conventional or experimental immunosuppressive reagents, will aid the development of novel strategies for the promotion of allo-antigen specific tolerance in transplantation. [Copyright &y& Elsevier]

Summary Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, biofilm formation, cellular adhesion, and horizontal gene transfer. However, many Gram-positive species, including Clostridium difficile , also produce type IV pili. Here, we identify the major subunit of the type IV pili of C. difficile , PilA1, and describe multiple 3D structures of PilA1, demonstrating the diversity found in three strains of C. difficile . We also model the incorporation of both PilA1 and a minor pilin, PilJ, into the pilus fiber. Although PilA1 contains no cysteine residues, and therefore cannot form the disulfide bonds found in all Gram-negative type IV pilins, it adopts unique strategies to achieve a typical pilin fold. The structures of PilA1 and PilJ exhibit similarities with the type IVb pilins from Gram-negative bacteria that suggest that the type IV pili of C. difficile are involved in microcolony formation. [ABSTRACT FROM AUTHOR]

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, cellular adhesion, and colonization. Recently, there has been an increased appreciation of the ability of Gram-positive species, including Clostridium difficile, to produce Type IV pili. Here we report the first three-dimensional structure of a Gram-positive Type IV pilin, PilJ, demonstrate its incorporation into Type IV pili, and offer insights into how the Type IV pili of C. difficile may assemble and function. PilJ has several unique structural features, including a dual-pilin fold and the incorporation of a structural zinc ion. We show that PilJ is incorporated into Type IV pili in C. difficile and present a model in which the incorporation of PilJ into pili exposes the C-terminal domain of PilJ to create a novel interaction surface. [ABSTRACT FROM AUTHOR]

Bundle-forming pili (BFP) are essential for the full virulence of enteropathogenic Escherichia coli (EPEC) because they are required for localized adherence to epithelial cells and auto-aggregation. We report the high resolution structure of bundlin, the monomer of BFP, solved by NMR. The structure reveals a new variation in the topology of type IVb pilins with significant differences in the composition and relative orientation of elements of secondary structure. In addition, the structural parameters of native BFP filaments were determined by electron microscopy after negative staining. The solution structure of bundlin was assembled according to these helical parameters to provide a plausible atomic resolution model for the BFP filament. We show that EPEC and Vibriocholerae type IVb pili display distinct differences in their monomer subunits consistent with data showing that bundlin and TcpA cannot complement each other, but assemble into filaments with similar helical organization. [ABSTRACT FROM AUTHOR]

Type IV pilus biogenesis, protein secretion, DNA transfer, and filamentous phage morphogenesis systems are thought to possess similar architectures and mechanisms. These multiprotein complexes include members of the Pule superfamily of putative NTPases that have extensive sequence similarity and probably similar functions as the energizers of macromolecular transport. We purified the Pule homologue BfpD of the enteropathogenic Escherichia coli bundle-forming pilus (BFP) biogenesis machine and characterized its ATPase activity, providing new insights into its mode of action. Numerous techniques revealed that BfpD forms hexamers in the presence of nucleotide. Hexameric BfpD displayed weak ATPase activity. We previously demonstrated that the N termini of membrane proteins BfpC and BfpE recruit BfpD to the cytoplasmic membrane. Here, we identified two BfpD-binding sites, BfpE39-76 and BfpE77-l14, in the N terminus of BfpE using a yeast two-hybrid system. Isothermal titration calorimetry and protease sensitivity assays showed that hexameric BfpD-ATPγS binds to BfoE77-114, whereas hexameric BfpD-ADP binds to BfpE39-76. Interestingly, the N terminus of BfpC and BfoE77-114 together increased the ATPase activity of hexameric BfpD over 1200-fold to a Vmax of 75.3 µmol of Pi min-1 mg-1, which exceeds by over 1200-fold the activity of other Pule family members. This augmented activity occurred only in the presence of Zn2+. We conclude that allosteric interactions between BfpD and BfpC and BfpE dramatically stimulate its ATPase activity. The differential nucleotide-dependent binding of hexameric BfpD to BfpE39-76 and BfpE77-114 suggests a model for the mechanism by which BfpD transduces mechanical energy to the biogenesis machine. [ABSTRACT FROM AUTHOR]

Patients with primary immune-deficiency (PID) may develop pulmonary complications and at that stage most are ineligible for either lung transplant (LTX) or bone marrow transplant (BMT) alone due to futility. We are conducting a first in human prospective trial, ClinicalTrials.gov: NCT01852370 where PID patients in lung failure -past the opportunity for curative BMT alone- may receive a 2 of 6 HLA antigen-matched cadaveric BOLT and minimum 8 weeks alter cryopreserved BMT from the same deceased UNOS donor. We hypothesized: 1) hematopoietic stem cells prepared from organ donor vertebral bodies (VB) would yield greater CD34+ cell dose compared to iliac crest aspirates-in part due to limited harvest time to avoid delay in the activity of organ recovery teams; 2) cryopreserved CD3/CD19 depleted bone marrow could generate durable hematopoiesis and donor derived immunity. Here we present the first 5 consecutive cases vertebral bone marrow (VBM) processing experience at Children's Hospital of Pittsburgh and subsequent CD3+/CD19+ cell depletion on the CliniMACS® device. With a threshold viability of 70% prior to cryopreservation the CD3/CD19-depleted product must contain 1 × 10E+6/kg CD34+ cells and 1 × 10E+5/kg CD3+ T cells to achieve durable engraftment and reduce the risk of GVHD respectively.® For details of pre/post processing, see Table. Since early 2017 we introduced chest/abdominal cavity wash with 1:1 saline-betadine mix and scrubbing T11-L4VB logs with chlora-prep that has eliminated bacterial contamination for the last 3 products. The first 2 were infused without adverse events following IRB/FDA approved antibiotic prophylaxis. Initially, processing of the VBs was arduous, using a scalpel and wire brush to clean the logs, then dissecting into small pieces with a mallet and osteotome. Since early 2017 a Stryker RemB saw has been used. A physical task that had taken 2 technologists 3 hours to complete has become almost effortless and one tech is freed up. The target pre-cryo CD34+ dose and viabilities have met target at all times, in fact CD34+ recovery has increased >70%, see Table. The entire process (dissection, crushing, filtering, CD3/CD19 depletion and cryopreservation) can be completed in 12 hours with 3 technologists. In summary, we have developed a complex sequence of procedures that even exceeded our target of 2E+06 CD34/kg pre-cryo dose (mean: 4.49E+06) leading to prompt and multilineage engraftment in 3 of the first 4 analyzable patients. [ABSTRACT FROM AUTHOR]