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The purpose of this study was to identify the patient, injury, and treatment factors associated with infection of bicondylar plateau fractures and to evaluate whether center variation exists.Retrospective review.Eighteen academic trauma centers.A total of 1,287 patients with 1,297 OTA type 41-C bicondylar tibia plateau fractures who underwent open reduction and internal fixation were included. Exclusion criteria were follow-up less than 120 days, insufficient documentation, and definitive treatment only with external fixation.Open reduction and internal fixation.Superficial and deep infection.One hundred one patients (7.8%) developed an infection. In multivariate regression analysis, diabetes (DM) (OR [odds ratio] 3.24; P ≤ 0.001), alcohol abuse (EtOH) (OR 1.8; P = 0.040), dual plating (OR 1.8; P ≤ 0.001), and temporary external fixation (OR 2.07; P = 0.013) were associated with infection. In a risk-adjusted model, we found center variation in infection rates (P = 0.030).In a large series of patients undergoing open reduction and internal fixation of bicondylar plateau fractures, the infection rate was 7.8%. Infection was associated with DM, EtOH, combined dual plating, and temporary external fixation. Center expertise may also play a role because one center had a statistically lower rate and two trended toward higher rates after adjusting for confounders.Level IV-Therapeutic retrospective cohort study.

Background: Health literacy research and practice are constantly evolving. Recent inquiry has highlighted the burdensome literacy demands faced by people with serious mental illness (SMI). Systems, organizational operations, and structures can play a role in decreasing literacy demand, thereby reducing unnecessary challenges for people with SMI. Brief Description of Activity: An organizational health literacy assessment was conducted to explore literacy demands in one mental health shelter and revealed best practice action steps for improving the literacy environment. Implementation: The assessment included an exploration of the shelter environment using The Health Literacy Environment Activity Packet, First Impressions & Walking Interview, and a commonly used shelter document using the Simple Measure of Gobbledygook (SMOG), Suitability Assessment of Materials (SAM), and Centers for Disease Control and Prevention (CDC) Index. Results: The literacy demands of the shelter environment and a frequently used document exceeded the literacy skills of people with SMI. Environment assessment revealed environmental facilitators (e.g., welcoming atmosphere) and barriers (e.g., unclear signage). Document assessment also revealed facilitating factors and barriers. SMOG scores ranged from 11.25 to 11.80 (median: 11.38), meaning 11th to 12th grade-level reading skills are required to understand, use, and take action on the document's content. A SAM score of 50% (adequate) and a CDC Index score of 42.1 (revise and improve) reveal materials contain both facilitating factors (e.g., chunked sections) and barriers (e.g., jargon, mismatched graphics) to use. Lessons Learned: The mismatch between system demands and the literacy skills of people with SMI is more profound than that of the general United States population. Organizational health literacy assessments are achievable and useful for both immediate and long-term action aimed at understanding and improving the organizational health literacy of mental health spaces. Further work is needed to explore the role of behavioral health services in addressing the institutional and programmatic literacy demands that inhibit treatment and recovery. [ HLRP: Health Literacy Research and Practice . 2022;6(3):e167–e174. ] Plain Language Summary: An organizational health literacy assessment reveals how system demands can be changed to better serve intended users. Engaging in mental health, recovery, and treatment services requires complex literacy skills. Generally, the U.S. adult population does not have the skills to meet such demands, and this is especially true among public mental health service users.

BACKGROUND: US immigration policy changes may affect health care use among Latinx children. We hypothesized that January 2017 restrictive immigration executive actions would lead to decreased health care use among Latinx children. METHODS: We used controlled interrupted time series to estimate the effect of executive actions on outpatient cancellation or no-show rates from October 2016 to March 2017 (“immigration action period”) among Latinx children in 4 health care systems in North Carolina. We included control groups of (1) non-Latinx children and (2) Latinx children from the same period in the previous year (“control period”) to account for natural trends such as seasonality. RESULTS: In the immigration action period, 114 627 children contributed 314 092 appointments. In the control period, 107 657 children contributed 295 993 appointments. Relative to the control period, there was an immediate 5.7% (95% confidence interval [CI]: 0.40%–10.9%) decrease in cancellation rates among all Latinx children, but no sustained change in trend of cancellations and no change in no-show rates after executive immigration actions. Among uninsured Latinx children, there was an immediate 12.7% (95% CI: 2.3%–23.1%) decrease in cancellations; however, cancellations then increased by 2.4% (95% CI: 0.89%–3.9%) per week after immigration actions, an absolute increase of 15.5 cancellations per 100 appointments made. CONCLUSIONS: There was a sustained increase in cancellations among uninsured Latinx children after immigration actions, suggesting decreased health care use among uninsured Latinx children. Continued monitoring of effects of immigration policy on child health is needed, along with measures to ensure that all children receive necessary health care.

This cohort study evaluates the first 22 months of implementation of the Cherokee Nation Heath Services community-based program designed to eliminate hepatitis C virus (HCV) infection., Key Points Question Can a community-based tribal hepatitis C virus (HCV) elimination program be implemented successfully in a rural setting? Findings In this cohort study, first-time HCV screening coverage increased from 20.9% to 38.2% from 3 years before to 22 months into implementation of the Cherokee Nation Health Services HCV elimination program. Identification, treatment, and cure of newly identified current HCV infections increased. Meaning In this study, implementation of a community-based HCV elimination program was associated with an improved cascade of care; this information may serve other organizations planning to implement similar programs in large rural areas., Importance In 2019, hepatitis C virus (HCV) infection contributed to more deaths in the US than 60 other notifiable infectious diseases combined. The incidence of and mortality associated with HCV infection are highest among American Indian and Alaska Native individuals. Objective To evaluate the association of the Cherokee Nation (CN) HCV elimination program with each element of the cascade of care: HCV screening, linkage to care, treatment, and cure. Design, Setting, and Participants This cohort study used data from the CN Health Services (CNHS), which serves approximately 132 000 American Indian and Alaska Native individuals residing in the 14-county CN reservation in rural northeastern Oklahoma. Data from the first 22 months of implementation (November 1, 2015, to August 31, 2017) of an HCV elimination program were compared with those from the pre–elimination program period (October 1, 2012, to October 31, 2015). The analysis included American Indian and Alaska Native individuals aged 20 to 69 years who accessed care through the CNHS between October 1, 2012, and August 31, 2017. Cure data were recorded through April 15, 2018. Exposure The CN HCV elimination program. Main Outcomes and Measures The main outcomes were the proportions of the population screened for HCV, diagnosed with current HCV infection, linked to care, treated, and cured during the initial 22 months of the elimination program period and the pre–elimination program period. Data from electronic health records and an HCV treatment database were analyzed. The cumulative incidence of HCV infection in this population was estimated using bayesian analyses. Results Among the 74 039 eligible individuals accessing care during the elimination program period, the mean (SD) age was 36.0 (13.5) years and 55.9% were women. From the pre–elimination program period to the elimination program period, first-time HCV screening coverage increased from 20.9% to 38.2%, and identification of current HCV infection and treatment in newly screened individuals increased from a mean (SD) of 170 (40) per year to 244 (4) per year and a mean of 95 (133) per year to 215 (9) per year, respectively. During the implementation period, of the 793 individuals with current HCV infection accessing the CNHS, 664 were evaluated (83.7%), 394 (59.3%) initiated treatment, and 335 (85.0%) had documented cure. In less than 2 years, the 85% 3-year goal was reached for cure (85.0%), and the goal for linkage to care was nearly reached (83.7%), whereas screening (44.1%) and treatment initiation (59.3%) required more time and resources. Conclusions and Relevance This cohort study found that after 22 months of implementation, the CNHS community-based HCV elimination program was associated with an improved cascade of care. The facilitators and lessons learned in this program may be useful to other organizations planning similar programs.

Stakeholder engagement, specifically integration of patient and family perspectives about what matters, is increasingly recognized as a critical component of patient-centered healthcare delivery. This study describes a structured approach to and evaluation of stakeholder engagement in the development of novel sexual and reproductive health (SRH) educational resources for adolescent and young adult (AYA) women with cystic fibrosis (CF). Key stakeholders participated in a systematic series of steps to iteratively develop and adapt patient educational resources. Process measures (stakeholder recruitment, composition, co-learning, and transparency) and proximal outcomes of stakeholder involvement (impact on the development process and satisfaction) were measured via recorded stakeholder recommendations and a stakeholder survey. Seventeen stakeholders participated in seven group and two patient-only conference calls. The majority of stakeholders understood their roles, had their expectations met or exceeded, and were satisfied with the frequency and quality of engagement in the project. All stakeholders in attendance provided multiple concrete recommen-dations during the development process. Stakeholders explored the motivations of AYA women with CF related to SRH and agreed that the ideal resource should be online. After reviewing the design of existing resources, stakeholders decided by consensus to partner with a pre-existing young women’s health website and created 11 CF-specific SRH guides. This study illuminates a path for a formal process of stakeholder engagement and evaluation in educational resource development centered on the SRH care needs of AYA women with CF. Similar systematic, planned processes could be extended to other populations and aspects of healthcare.

Background COPD patients have increased risk of pneumonia when treated with fluticasone propionate (FP), whereas this is generally not the case with budesonide (BUD) treatment. We hypothesized that BUD and FP differentially affect the expression of immune defense genes. Methods Human bronchial epithelial 16HBE cells and air-liquid interface (ALI)-cultured primary bronchial epithelial cells (PBECs) were pre-treated with clinically equipotent concentrations of BUD or FP (0.16–16 nM BUD and 0.1–10 nM FP), and the expression of immune defense genes was studied at baseline and after exposure to rhinovirus (RV16). Results Using microfluidic cards, we observed that both BUD and FP significantly suppressed CXCL8, IFNB1 and S100A8 mRNA expression in unstimulated 16HBE cells. Interestingly, BUD, but not FP, significantly increased lactotransferrin (LTF) expression. The difference between the effect of BUD and FP on LTF expression was statistically significant and confirmed by qPCR and at the protein level by western blotting. RV16 infection of ALI-cultured PBECs significantly increased the expression of CCL20, IFNB1 and S100A8, but not of LTF or CAMP/LL-37. In these RV16-exposed cells, LTF expression was again significantly higher upon pre-treatment with BUD than with FP. The same was observed for S100A8, but not for CCL20, IFNB1 or CAMP/LL-37 expression. Conclusions Treatment of human bronchial epithelial cells with BUD results in significantly higher expression of specific immune defense genes than treatment with FP. The differential regulation of these immune defense genes may help to explain the clinical observation that BUD and FP treatment differ with respect to the risk of developing pneumonia in COPD.

Inhaled corticosteroids (ICSs) treatment combined with long-acting β2-adrenoceptor agonists (LABAs) reduces the risk of exacerbations in COPD, but the use of ICSs is associated with increased incidence of pneumonia. There are indications that this association is stronger for fluticasone propionate than for budesonide. We have examined systematic reviews assessing the risk of pneumonia associated with fluticasone propionate and budesonide COPD therapy. Compared with placebo or LABAs, we found that fluticasone propionate was associated with 43%–78% increased risk of pneumonia, while only slightly increased risk or no risk was found for budesonide. We have evaluated conceivable mechanisms which may explain this difference and suggest that the higher pneumonia risk with fluticasone propionate treatment is caused by greater and more protracted immunosuppressive effects locally in the airways/lungs. These effects are due to the much slower dissolution of fluticasone propionate particles in airway luminal fluid, resulting in a slower uptake into the airway tissue and a much longer presence of fluticasone propionate in airway epithelial lining fluid.

A wide range of concentrations are frequently observed when measuring drugs of abuse in urine toxicology samples; this is especially true for amphetamine and methamphetamine. Routine liquid chromatography-tandem mass spectrometry confirmatory methods commonly anchored at a 50 ng/mL lower limit of quantitation can span approximately a 100-fold concentration range before regions of non-linearity are reached deteriorating accurate quantitation and qualitative assessments. In our experience, approximately a quarter of amphetamine and methamphetamine positive samples are above a 5,000 ng/mL upper limit of quantitation and thus require reanalysis with dilution for accurate quantitative and acceptable qualitative results. We present here the development of an analytical method capable of accurately quantifying samples with concentrations spanning several orders of magnitude without the need for sample dilution and reanalysis. For each analyte the major isotopes were monitored for analysis through the lower concentration ranges (50-5,000 ng/mL), and the naturally occurring, low probability 13C2 isotopes were monitored for the analysis of the high concentration samples (5,000-100,000 ng/mL amphetamine and 5,000-200,000 ng/mL methamphetamine). The method simultaneously monitors transitions for the molecules containing only 12C and 13C2 isotopologues eliminating the need for re-extraction and reanalysis of high concentration samples.

Background: Glucocorticoids act on the glucocorticoid receptor (GR; NR3C1) to resolve inflammation and, as inhaled corticosteroids (ICS), are the cornerstone of treatment for asthma. However, reduced efficacy in severe disease or exacerbations indicates a need to improve ICS actions. Methods: Glucocorticoid-driven transcriptomes were compared using PrimeView microarrays between primary human bronchial epithelial (HBE) cells and the model cell lines, pulmonary type II A549 and bronchial epithelial BEAS-2B cells. Results: In BEAS-2B cells, budesonide induced (≥2-fold, P ≤ 0.05) or, in a more delayed fashion, repressed (≤0.5-fold, P ≤ 0.05) the expression of 63, 133, 240, and 257 or 15, 56, 236, and 344 mRNAs at 1, 2, 6, and 18 h, respectively. Within the early-induced mRNAs were multiple transcriptional activators and repressors, thereby providing mechanisms for the subsequent modulation of gene expression. Using the above criteria, 17 (BCL6, BIRC3, CEBPD, ERRFI1, FBXL16, FKBP5, GADD45B, IRS2, KLF9, PDK4, PER1, RGCC, RGS2, SEC14L2, SLC16A12, TFCP2L1, TSC22D3) induced and 8 (ARL4C, FLRT2, IER3, IL11, PLAUR, SEMA3A, SLC4A7, SOX9) repressed mRNAs were common between A549, BEAS-2B and HBE cells at 6 h. As absolute gene expression change showed greater commonality, lowering the cut-off (≥1.25 or ≤ 0.8-fold) within these groups produced 93 induced and 82 repressed genes in common. Since large changes in few mRNAs and/or small changes in many mRNAs may drive function, gene ontology (GO)/pathway analyses were performed using both stringency criteria. Budesonide-induced genes showed GO term enrichment for positive and negative regulation of transcription, signaling, proliferation, apoptosis, and movement, as well as FOXO and PI3K-Akt signaling pathways. Repressed genes were enriched for inflammatory signaling pathways (TNF, NF-κB) and GO terms for cytokine activity, chemotaxis and cell signaling. Reduced growth factor expression and effects on proliferation and apoptosis were highlighted. Conclusions: While glucocorticoids repress mRNAs associated with inflammation, prior induction of transcriptional activators and repressors may explain longer-term responses to these agents. Furthermore, positive and negative effects on signaling, proliferation, migration and apoptosis were revealed. Since many such gene expression changes occurred in human airways post-ICS inhalation, the effects observed in cell lines and primary HBE cells in vitro may be relevant to ICS in vivo.

The recent increase in illicit opioids sold on the black market, cut into heroin and masqueraded as prescription pills prompts a significant public health concern. Most designer opioids possess unknown potencies and unknown pharmacokinetics and their unregulated, variable dosages lead to rashes of overdoses. Additionally, many of the designer opioids, especially the fentanyl analogs are significantly more potent than heroin. High-profile cases involving overdoses of U-47700 and carfentanil have been reported in the media; however, the true prevalence of these and other designer opioids is unknown. Independent LC-MS-MS screen and confirmation methods have been developed and validated to identify and quantify fentanyl, and 18 designer opioids and their metabolites; methods were then exercised on urine specimens from contract pain management clients. Assuming patients in a pain management program may have a higher probability to seek out self-medication, samples from pain management patients were investigated for designer opioids. Similarly, pain management patients identified as using heroin may be more likely to experiment with or be accidentally exposed to designer opioids, specimens screening positive for the heroin metabolite 6-acetylmorphine were specifically chosen for designer opioid screening. Within this small group of pain management and heroin-positive samples, nine designer opioids were detected at a total prevalence of 25%. When screening random pain management samples not positive for heroin, a considerably lower percentage of samples (

Students entering North Carolina public schools must have documentation of a health assessment and immunizations by the 30th day of class, or they are excluded from attending school until they submit documentation. A community collaborative was essential in decreasing the number of students excluded from school for noncompliant documentation in the Winston-Salem Forsyth County school district.

Urine drug testing is common for workplace drug testing, prescription management, emergency medicine and the criminal justice system. Unsurprisingly, with the significant consequences based upon the results of urine drug testing, a donor in need of concealing the contents of their sample is highly motivated to cheat the process. Procedures and safeguards ensuring sample validity are well known, and include measuring sample temperature at the time of collection, and laboratory measurements of creatinine, specific gravity and pH. Synthetic urine samples are available and are designed to deceive all aspects of urine drug testing, including validity testing. These samples are sophisticated enough to contain biological levels of creatinine, and are at a physiological pH and specific gravity. The goal of our research was to develop new procedures designed to distinguish authentic samples from masquerading synthetic samples. We aimed to identify substances in commercial synthetic urines not expected to be present in a biological sample distinguishing fake specimens. Additionally, we aimed to identify and employ endogenous compounds in addition to creatinine for identifying biological samples. We successfully identified two compounds present in synthetic urines that are not present in biological samples and use them as markers of invalidity. Four new endogenous markers for validity were successfully evaluated. Validity assessment was further aided by monitoring metabolites of nicotine and caffeine. When the method was applied to patient samples, 2% of samples were identified as inconsistent with natural urine samples, even though they met the current acceptance criteria for creatinine, pH and specific gravity.

An estimated 3.5 million persons in the United States are living with hepatitis C virus (HCV) infection, resulting in approximately 20,000 deaths each year, primarily from cirrhosis or hepatocellular carcinoma (1,2). American Indian/Alaska Native (AI/AN) populations have the highest incidence of acute HCV infection among all U.S. racial/ethnic groups and are at greater risk for HCV-related mortality compared with the general population (3). In 2013, new antiviral drugs became available that make possible 8-12 week treatment regimens with fewer adverse events and are able to achieve sustained virologic response (SVR) in >90% of treated patients (4), equivalent to a cure of HCV infection. Also of note, HCV testing recommendations were expanded in 2012 by CDC and in 2013 by the U.S. Preventive Services Task Force to include one-time testing of persons born during 1945-1965 (the "baby boomer" cohort) in addition to anyone at increased risk for HCV infection (5,6). Given the availability of new HCV drugs, expanded testing recommendations, and high incidence of HCV infection in AI/AN populations, in October 2012, Cherokee Nation Health Services (CNHS) implemented a tribal HCV testing policy.* As part of the policy, CNHS added a reminder in the electronic health record (EHR) for clinical decision support and provided HCV education to primary care clinicians. From October 2012 to July 2015, among 92,012 persons with at least one CNHS clinic encounter, the cumulative number who received HCV screening for the first time increased from 3,337 (3.6%) to 16,772 (18.2%). The largest percentage of HCV screening was among persons born during 1945-1965. Of 715 persons who tested positive for HCV antibodies, 488 (68.3%) were tested for HCV RNA; among those 488 persons, 388 (79.5%) were RNA positive and were thus confirmed to have chronic HCV infection. Treatment was initiated for 223 (57.5%) of the 388 with chronic infection; 201 (90.1%) completed treatment, of whom 180 (89.6%) achieved SVR. CNHS has successfully increased HCV testing and treatment and is now collaborating with CDC and other external partners to develop an HCV elimination program for the Cherokee Nation that might serve as a model for similar settings.

The inflammatory processes associated with pulmonary disorders remains incompletely understood. CCAAT/enhancer-binding protein (C/EBP)β is implicated in inflammatory lung disorders as well as in β(2)-adrenoceptor signaling. We hypothesized that C/EBPβ in the lung epithelium contributes to lipopolysaccharide (LPS)-induced airway neutrophilia and expression of neutrophil chemoattractant chemokine (C-X-C) motif ligand (CXCL)1, as well as the suppressive effects of long-acting β(2)-agonists (LABAs) and glucocorticoids (GCs). To investigate this, mice with a lung epithelial-specific deletion of C/EBPβ (Cebpb(ΔLE)) and control littermates (Cebpb(fl/fl)) were pre-treated with a LABA, formoterol and/or a GC, budesonide, and challenged with LPS. Inflammatory cell recruitment in bronchoalveolar lavage (BAL) fluid and pulmonary expression of inflammatory mediators were investigated. In addition, the ability of formoterol to increase C/EBP transactivation was assessed in vitro. LPS-challenged Cebpb(ΔLE) mice exhibited fewer BAL neutrophils and lower pulmonary expression of CXCL1 versus Cebpb(fl/fl) mice. Suppression of LPS-induced neutrophilia by formoterol was impaired in Cebpb(ΔLE) mice and Cxcl1 expression was increased. However, suppression of the neutrophilia by budesonide with/without formoterol was preserved. Further studies indicated that C/EBP transactivation was increased by the cAMP elevating agent forskolin and formoterol in a β(2)-adrenoceptor dependent manner. Thus, C/EBPβ in the lung epithelium contributes to LPS-induced CXCL1 expression and airway neutrophilia as well as to the suppressive effects of formoterol. Reduced C/EBPβ activity, observed in smokers with chronic obstructive pulmonary disease, may impair the responsiveness to LABAs when used without GCs.

Rationale Patients with COPD have an increased risk for community-acquired pneumonia, which is further increased by inhaled corticosteroids. Objective To assess effects of the corticosteroids, budesonide and fluticasone propionate, on macrophage bacterial responses in COPD. Methods Monocyte-derived macrophages (MDMs) generated from blood monocytes from 10 non-smoker controls (NoS), 20 smokers without COPD (Sm), and 40 subjects with moderate to severe COPD (21 ex-smokers (COPD-ES) and 19 current smokers (COPD-S)) were pre-treated with budesonide or fluticasone (10 nM—1 μM) and challenged with live non-typeable Haemophilus influenzae (NTHI) or Streptococcus pneumoniae (SP). Cell surface bacterial recognition receptor expression (flow cytometry) and cytokine release (bead array) were analyzed. Results NTHI and SP reduced bacterial recognition receptor expression on MDMs from COPD and Sm, but not NoS (except TLR4). SR-AI and MARCO were reduced by both NTHI and SP, whereas other receptors by either NTHI or SP. Among COPD subjects, COPD-ES demonstrated a greater number of reductions as compared to COPD-S. NTHI reduced SR-AI, MARCO, CD11b, CD35 and CD206 in COPD-ES while only SR-AI and CD11b in COPD-S. SP reduced SRA-1, CD1d, TLR2 and TLR4 in both COPD-ES and COPD-S, and reduced MARCO and CD93 only in COPD-ES. All receptors reduced in COPD by NTHI and most by SP, were also reduced in Sm. Budesonide counteracted the receptor reductions induced by both NTHI (CD206 p = 0.03, MARCO p = 0.08) and SP (SR-AI p = 0.02) in COPD-ES. Fluticasone counteracted only SP-induced reductions in TLR2 (p = 0.008 COPD-ES and p = 0.04 COPD-S) and TLR4 (p = 0.02 COPD-ES). Cytokine release was equivalently reduced by both corticosteroids. Conclusions Reduction in macrophage bacterial recognition receptors during bacterial exposure could provide a mechanism for the increased pneumonia risk in COPD. Differential effects of budesonide and fluticasone propionate on macrophage bacterial recognition receptor expression may contribute to the higher pneumonia incidence reported with fluticasone propionate.

The reasons for enhanced deposition of extracellular matrix in the airways of asthmatic patients and the subsequent consequences on lung function are uncertain. Here, we investigated the synthesis of procollagen I and proteoglycans, the activity of various metalloproteinases (MMPs) and the production of their inhibitor TIMP-1 in biopsy-derived bronchial fibroblasts from eight patients with stable mild-to-moderate asthma, and how they are related to patients' lung function and airway hyperreactivity (AHR). Following 24-h fibroblast incubation in 0.4% serum, procollagen I carboxyterminal propeptide (PICP), TIMP-1 and MMP-1 in cell media were analysed by ELISA, MMP-2, MMP-3, MMP-9 by zymography and total proteoglycan production by [(35)S]-sulphate-incorporation/ion chromatography. Patients' FEV(1)% predicted and methacholine log PD(20) negatively correlated with PICP synthesized by patients' bronchial fibroblasts (r = -0.74 and r = -0.71, respectively). PICP and proteoglycan amounts positively correlated (0.8

We have defined some of the mechanisms by which the kinase inhibitor lapatinib kills HCT116 cells. Lapatinib inhibited radiation-induced activation of ERBB1/2, extracellular signal-regulated kinases 1/2, and AKT, and radiosensitized HCT116 cells. Prolonged incubation of HCT116 cells with lapatinib caused cell killing followed by outgrowth of lapatinib-adapted cells. Adapted cells were resistant to serum starvation-induced cell killing and were cross-resistant to multiple therapeutic drugs. Lapatinib was competent to inhibit basal and epidermal growth factor (EGF)-stimulated ERBB1 phosphorylation in adapted cells. Coexpression of dominant-negative ERBB1 and dominant-negative ERBB2 inhibited basal and EGF-stimulated ERBB1 and ERBB2 phosphorylation in parental and adapted cells. However, in neither parental nor adapted cells did expression of dominant-negative ERBB1 and dominant-negative ERBB2 recapitulate the cell death-promoting effects of lapatinib. Adapted cells had increased expression of MCL-1, decreased expression of BAX, and decreased activation of BAX and BAK. Overexpression of BCL-XL protected parental cells from lapatinib toxicity. Knockdown of MCL-1 expression enhanced lapatinib toxicity in adapted cells that was reverted by knockdown of BAK expression. Inhibition of caspase function modestly reduced lapatinib toxicity in parental cells, whereas knockdown of apoptosis-inducing factor expression suppressed lapatinib toxicity. Thus, in HCT116 cells, lapatinib adaptation can be mediated by altered expression of pro- and antiapoptotic proteins that maintain mitochondrial function.

Transforming growth factor beta (TGFbeta) plays a dual role in oncogenesis, acting as both a tumor suppressor and a tumor promoter. These disparate processes of suppression and promotion are mediated primarily by Smad and non-Smad signaling, respectively. A central issue in understanding the role of TGFbeta in the progression of epithelial cancers is the elucidation of the mechanisms underlying activation of non-Smad signaling cascades. Because the potent lipid mediator sphingosine-1-phosphate (S1P) has been shown to transactivate the TGFbeta receptor and activate Smad3, we examined its role in TGFbeta activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and promotion of migration and invasion of esophageal cancer cells. Both S1P and TGFbeta activate ERK1/2, but only TGFbeta activates Smad3. Both ligands promoted ERK1/2-dependent migration and invasion. Furthermore, TGFbeta rapidly increased S1P, which was required for TGFbeta-induced ERK1/2 activation, as well as migration and invasion, since downregulation of sphingosine kinases, the enzymes that produce S1P, inhibited these responses. Finally, our data demonstrate that TGFbeta activation of ERK1/2, as well as induction of migration and invasion, is mediated at least in part by ligation of the S1P receptor, S1PR2. Thus, these studies provide the first evidence that TGFbeta activation of sphingosine kinases and formation of S1P contribute to non-Smad signaling and could be important for progression of esophageal cancer.

This article reviews the experiences of programs designed to provide access to prevention of mother-to-child transmission services with the goal of improving services in resource-constrained settings. The article reports new data from the Elizabeth Glaser Pediatric AIDS Foundation's prevention of mother-to-child transmission program in sub-Saharan Africa, which has provided human immunodeficiency virus testing to more than 1,300,000 pregnant women and antiretroviral prophylaxis to 134,000 human immunodeficiency virus-infected pregnant women and more than 78,000 human immunodeficiency virus-exposed infants. Review of qualitative program data highlights the practical innovations that sites are implementing to improve the uptake of prevention of mother-to-child transmission services. Recommendations discussed include opt-out counseling and testing, rapid human immunodeficiency virus testing in antenatal care, counseling and testing in maternity, and provision of antiretroviral prophylaxis for mother and infant at the time of human immunodeficiency virus testing. Successful programmatic innovations need to be disseminated widely as more aggressive prevention strategies must be implemented to increase access to more than 10% of pregnant women worldwide.

Background: Improved asthma control by combinations of inhaled glucocorticosteroids (GCs) and long-acting β2-agonists (LABAs) includes a reduced frequency and severity of exacerbations. In view of the association of exacerbations with increased airway inflammation, the question has arisen as to whether LABAs are able to complement the known anti-inflammatory activity of GCs. To address this, we studied the effects of a LABA, formoterol (FORM), and a GC, budesonide (BUD), alone and in combination, on bronchial epithelial cell-mediated eosinophil superoxide production in vitro.Methods: We employed 2 experimental approaches. First, superoxide production by human eosinophils incubated with conditioned medium (CM) from human bronchial epithelial cells cultured for 24 h with vehicle, BUD, FORM or BUD + FORM was measured (Epi/Eos assay). Second, eosinophils were stimulated with vehicle-CM to which the drugs were added (Eos assay). Superoxide production was determined as the superoxide dismutase-inhibitable reduction of ferricytochrome C. Results: CM increased eosinophil superoxide generation (p < 0.01) and epithelial-derived granulocyte macrophage colony-stimulating factor was the mediator responsible. In both assays, FORM dose-dependently inhibited eosinophil superoxide similarly and in the same concentration range as BUD. The BUD + FORM combination was more effective than BUD alone, and it completely inhibited CM-induced superoxide production in the Epi/Eos assay, suggesting complementary effects of both drugs on bronchial epithelial cells and eosinophils. Conclusions: The cooperative, inhibitory effects of BUD and FORM on eosinophils and bronchial epithelial cells, in terms of their effects on eosinophil superoxide production, may represent a possible mechanism for the enhanced anti-inflammatory efficacy of BUD and FORM combination therapy of asthma.

Background We have recently reported that protein phosphate 2A (PP2A) inactivation resulted in increased phosphorylation of the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase 1 (JNK1) and glucocorticoid receptors (GR) at Ser226, thereby reducing GR nuclear translocation and causing corticosteroid insensitivity in severe asthmatics. Protein tyrosine phosphatases (PTPs) are also known to be critically involved in the regulation of MAPKs, such as JNK and therefore potentially associated with GR function. The aim of study was to elucidate the involvement of MAPK-PTPs (PTP-RR, PTP-N5 and PTP-N7), which can dephosphorylate MAPKs, in the regulation of corticosteroid sensitivity. Methods Corticosteroid sensitivity, GR nuclear translocation, phosphorylation levels of GR-Ser226, JNK1 and PP2A catalytic subunit (PP2AC)-Tyr307 and protein expression levels and activities of PTP-RR and PP2AC were evaluated in U937 cells and/or peripheral blood mononuclear cells (PBMCs). Knock-down effects of MAPK-PTPs using siRNA were also evaluated. Results Knock-down of PTP-RR, but not of PTP-N5 or PTP-N7 impaired corticosteroid sensitivity, induced GR-Ser226 phosphorylation and reduced GR nuclear translocation. Under IL-2/IL-4-induced corticosteroid insensitivity, PTP-RR expression, activity and associations with JNK1 and GR were reduced but PTP-RR activity was restored by formoterol. Also in PBMCs from severe asthmatic patients, PTP-RR and JNK1 expression were reduced and GR-Ser226 phosphorylation increased. Furthermore, PTP-RR was associated with PP2A. PTP-RR reduction enhanced PP2AC-Tyr307 phosphorylation leading to impairment of PP2A expression and activity. Conclusions We demonstrated that with corticosteroid insensitivity PTP-RR fails to reduce phosphorylation of JNK1 and GR-Ser226, resulting in down-regulation of GR nuclear translocation. Reduced PTP-RR may represent a novel cause of corticosteroid insensitivity in severe asthmatics. Electronic supplementary material The online version of this article (doi:10.1186/s12931-016-0349-0) contains supplementary material, which is available to authorized users.

The majority of children infected with human immunodeficiency virus live in resource-constrained settings and die without an established diagnosis. Definitive laboratory diagnosis in children younger than 12-18 months requires virologic testing; however, antibody testing is often the only option available. Antibody testing provides a definitive diagnosis in older children but is frequently not used. Children meeting clinical criteria should be treated regardless of availability of laboratory diagnoses.

ObjectiveTo describe the infant feeding practices and attitudes of women who used prevention of mother-to-child transmission of HIV (PMTCT) services in rural Zimbabwe.DesignA cross-sectional study including structured interviews and focus group discussions was conducted between June 2003 and February 2004.SettingThe study took place in Murambinda Mission Hospital (Buhera District, Manicaland Province), the first site offering PMTCT services in rural Zimbabwe.SubjectsThe interviews targeted HIV-infected and HIV-negative women who received prenatal HIV counselling and testing and minimal infant feeding counselling, and who delivered between 15 August 2001 and 15 February 2003. The focus groups were conducted among young and elderly men and women.ResultsOverall, 71 HIV-infected and 93 HIV-negative mothers were interviewed in clinics or at home. Most infants (97%) had ever been breast-fed. HIV-negative mothers introduced fluids/foods other than breast milk significantly sooner than HIV-infected mothers (median 4.0 vs. 6.0 months, P = 0.005). Infants born to HIV-negative mothers were weaned significantly later than HIV-exposed infants (median 19.0 vs. 6.0 months, P = 10−5). More than 90% of mothers reported that breast-feeding their infant was a personal decision, a third of whom also mentioned having taken into account health workers' messages.ConclusionThe HIV-infected mothers interviewed were gradually implementing infant feeding practices recommended in the context of HIV. Increased infant feeding support capacity in resource-limited rural populations is required, i.e. training of counselling staff, decentralised follow-up and weaning support.

The objective was to evaluate the influence of a prevention of mother-to-child transmission of HIV advocacy and mobilization campaign on awareness and knowledge levels within the community. We used a knowledge attitudes and practices survey to collect baseline data in November 2002 and again in July 2004 for evaluation purposes. A total of 351 women attending healthcare services were interviewed each time. The proportion of women aware of the service increased from 48.0% in 2002 to 82.8% in 2004 (OR = 4.9 95% CI 3.3-7.3 P = 0.001). Exclusive breastfeeding was identified as a mother-to-child transmission risk factor by 27.1% in 2002 and by 55.8% of respondents in 2004 (OR = 2.9 CI 2.1-4.2 P = 0.001). Although most interviewees agreed that the prevention of sexual transmission of HIV was the best strategy for prevention of mother-to-child transmission of HIV (88.3% in 2002 and 96.4% in 2004) few reported having ever used a male condom (24.8% in 2002 vs. 29.8% in 2004 P = 0.16). Prevention of mother-to-child transmission of HIV strategies at both individual and community level were still insufficiently understood and applied. Targeted educational messages and communication for social change need to be combined. Knowledge attitudes and practices surveys can be used to monitor programme progress. (authors)

Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-joining (NHEJ) of DNA double-strand breaks. Promyelocytic HL-60 cells express a variant form of Ku resulting in enhanced radiation sensitivity. This raises the question if low efficiency of NHEJ, instrumental for the cellular repair of oxidative damage, is a normal characteristic of myeloid differentiation. Here we confirmed the complete lack of DNA-PK in PMN protein extracts, and the expression of the truncated Ku86 variant form in HL-60. However, this degradation of DNA-PK was shown to be due to a DNA-PK-degrading protease in PMN and HL-60. In addition, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, suggesting the previously reported absence in PMN of DNA-PK to be an artefact. The levels of Ku86 and DNA-PKcs were much reduced in PMN, as compared with that of the lymphocytes, whereas HL-60 displayed a markedly elevated DNA-PK concentration. In conclusion, our findings provide evidence of reduced, not depleted expression of DNA-PK during the mature stages of myeloid differentiation.

NK cells are innate immune lymphocytes that express a vast repertoire of germ-line encoded receptors for target recognition. These receptors include inhibitory and activating proteins, among the latter of which is CD16, a low affinity binding Fc receptor. Here, we show that human NK cells expand in response to stimulation with various tumor cell lines. We further demonstrate that the tumor-derived expansion of NK cells is accompanied by rapid, cell-dependent, changes in CD16 expression levels. We show that in NK cells expanded in response to the EBV-transformed cell line 721.221, CD16 is shed and therefore approximately half of the expanded 721.221-derived NK-cell population does not express CD16. We also show, in contrast, that in response to 1106mel cells, CD16 expression is maintained on the cell surface of the expanded NK cells due to an antibody-dependent mechanism. Our results may provide a basis for the selective expansion of NK cells that may be used for tumor immunotherapy.

Paclitaxel (Taxol)-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death) plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim(-/-) MEFs (mouse embryonic fibroblasts), the bim(-/-) mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak (-/-) MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. However, paclitaxel sensitivity was not affected in bax(-/-) MEFs or in human breast cancer cells in which BAX was down-regulated, suggesting that paclitaxel-induced apoptosis is BAK-dependent, but BAX-independent. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A Cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that Cdk activation at mitotic arrest could induce subsequent MCL-1 degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance.

We previously found that reduced glutathione (GSH) or a mixture of GSH/glutathione disulfide (GSSG) potentiated platelet aggregation. We here report that GSSG, when added to platelets alone, also potentiates platelet aggregation. Most of the GSSG was converted to GSH by a flavoprotein-dependent platelet surface mechanism. This provided an appropriate redox potential for platelet activation. The addition of GSSG to platelets generated sulfhydryls in the β subunit of the αIIbβ3 fibrinogen receptor, suggesting a mechanism for facilitation of agonist-induced platelet activation.

Exacerbations of asthma, a chronic inflammatory respiratory disease, are associated with viral upper respiratory tract infections involving human rhinovirus. Although glucocorticoids (corticosteroids) effectively control airways inflammation in many asthmatics, human rhinovirus-associated exacerbations show reduced glucocorticoid responsiveness. Using human bronchial epithelial BEAS-2B cells, we show that human rhinovirus reduced glucocorticoid-inducible activation of glucocorticoid response element (GRE) reporter systems in a time- and concentration-dependent manner. The synthetic double-stranded viral RNA mimetic, polyinosinic:polycytidylic acid (poly(I:C)), also reduced activation of GRE reporter systems in BEAS-2B and pulmonary A549 cells. In addition, poly(I:C) decreased transcription from cAMP response element (CRE)-, TATA-, simian virus 40- and nuclear factor-kappa B (NF-κB)-dependent reporter systems. The effects of poly(I:C) on GRE-reporter activation were countered by the long-acting β2-adrenoceptor agonists, formoterol and salmeterol. Likewise, increased expression of the gene cyclin-dependent kinase inhibitor 1C (CDKN1C; p57(KIP2)) by dexamethasone was reduced by poly(I:C), but was substantially enhanced by the addition of formoterol. Poly(I:C) induced the expression of interleukin-8 (IL8; CXCL8) and this was significantly decreased by dexamethasone, formoterol or their combination. This confirms that not all transcriptional responses were attenuated by poly(I:C) and that decreased glucocorticoid-dependent transcription can be counteracted by the addition of long-acting β2-adrenoceptor agonists. These data show how human rhinovirus may attenuate glucocorticoid-induced transcription to reduce anti-inflammatory activity. However, addition of long-acting β2-adrenoceptor agonist to the glucocorticoid functionally restored this response and shows how glucocorticoid plus long-acting β2-adrenoceptor agonist combinations may prove beneficial during virus-induced exacerbations of asthma.

Bradykinin (BK) induces differentiation of lung fibroblasts into myofibroblasts, which play an important role in extracellular matrix remodeling in the airways of asthmatic patients. It is unclear whether this process is affected by antiasthma therapies. Here, we evaluated whether a glucocorticoid, budesonide (BUD), and a long-acting β2-agonist, formoterol (FM), either alone or in combination, modified BK-induced lung fibroblast differentiation, and affected the BK-activated intracellular signaling pathways.Human fetal lung fibroblasts were incubated with BUD (0.001-0.1 μM) and/or FM (0.0001-0.1 μM) before exposure to BK (0.1 or 1 μM). Fibroblast differentiation into α-smooth-muscle-actin-positive (α-SMA⁺) myofibroblasts, BK2 receptor (B2R) expression, extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation (p-ERK1/2), intracellular Ca²⁺ concentration ([Ca²⁺]i), and p65 nuclear factor kappa B translocation were evaluated.BUD (0.1 μM) and FM (0.1 μM), either alone or in combination, completely inhibited BK-induced α-SMA protein expression and decreased the numbers of α-SMA⁺ fibroblasts, with a clear reduction in α-SMA stress fibers organization. BUD also completely inhibited the increase of B2R, whereas FM with or without BUD had no effect. BK-induced increases of [Ca²⁺]i and p-ERK1/2 were significantly reduced to similar levels by BUD and FM, either alone or in combination, whereas p65 translocation was completely inhibited by all treatments.Both BUD and FM, either alone or in combination, effectively inhibited the BK-induced differentiation of fibroblasts into α-SMA⁺ myofibroblasts and the intracellular signaling pathways involved in fibroblast activation. These results suggest that BUD and FM combination therapy has potential to inhibit fibroblast-dependent matrix remodeling in the airways of asthmatic patients.

Long-acting β2-adrenoceptor agonists (LABAs) are reported to enhance anti-inflammatory effects of corticosteroids in vitro and in vivo, although the molecular mechanisms have not yet been elucidated. We investigated the role of serine/threonine protein phosphatase 2A (PP2A) on regulation of corticosteroid sensitivity via inhibition of glucocorticoid receptor (GR) phosphorylation as the target of formoterol, an LABA. Corticosteroid sensitivity was determined as IC50 to dexamethasone (Dex) on TNFα-induced IL-8 release in a U937 monocytic cell line (Dex-IC50). Phosphorylation levels of GR-Ser226 and c-Jun N-terminal kinase (JNK) were determined by western-blotting. Phosphatase activity of immunopurified PP2A was measured by fluorescence-based assay. Exposure to IL-2/IL-4 for 48 h decreased Dex sensitivity with a concomitant increase of GR phosphorylation at Ser226 with JNK1 activation. Formoterol restored Dex sensitivity by inhibiting phosphorylation of GR-Ser226 and JNK1. PP2A inhibition by okadaic acid, a phosphatase inhibitor, abrogated formoterol-mediated effects. In addition, formoterol enhanced PP2A activity in intact or IL-2/IL-4 treated U937 cells and human peripheral blood mononuclear cells. In addition, PP2A activation by formoterol was not antagonized by ICI-118551, and formoterol could activate PP2A directly in cell free system. Taken together, formoterol increases corticosteroid sensitivity via activation of PP2A in receptor independent manner, explaining its benefits as add-on therapy for the treatment of corticosteroid-insensitive diseases, such as severe asthma.

Liver X receptors (LXRs) alpha and beta are nuclear receptors, which form obligate heterodimers with the retinoid X receptor (RXR). The LXRs regulate both redundantly and non-redundantly the transcription of genes controlling cholesterol metabolism and transport as well as lipogenesis. Previously, we showed that mutations in putative N-terminal nuclear localization sequences (NLSs) within both LXRs inhibit nuclear import. Through in vitro studies, we show here that these NLSs bind importin alpha and are both necessary and sufficient for the nuclear import of LXRs. Imaging, transactivation, and electro-mobility shift experiments show that RXR rescues the nuclear import of the LXRalpha NLS mutant yet does not restore its transcriptional activity despite intact DNA binding. In contrast, RXR partially rescues the import of the LXRbeta NLS mutant, but has no effect on its transcriptional activity due to the loss of DNA binding. Experiments with NLS mutant RXR confirmed that RXR may dominate the nuclear import of the RXR/LXRalpha heterodimer, whereas LXRbeta dominates the nuclear import of the RXR/LXRbeta heterodimer. Intriguingly, our data indicate differences between LXRalpha and LXRbeta in their interaction with RXR and in the role their NLSs play in transactivating functions independent of nuclear import.

Proteoglycans contribute to extracellular matrix remodeling in asthmatic airways. We investigated the effects of budesonide, a glucocorticoid, and formoterol, a long-acting beta2-adrenergic agonist, on serum-induced proteoglycan production by human lung fibroblasts. In 10% serum, total proteoglycan production was increased 1.5-fold (P0.01) compared with basal production in 0.4% serum. Budesonide (10(-8) M) reduced this increase by 44% (P0.01) and, whereas formoterol (10(-10)-10(-8) M) had no inhibitory effects, the drug combination abolished the increase (P0.01) without affecting fibroblast proliferation. This synergistic effect required functional glucocorticoid and beta-adrenergic receptors. The production of the proteoglycans decorin, biglycan, perlecan, and versican was increased 2.5- to 5-fold (P0.01) in 10% serum. Combination treatment with budesonide (10(-8) M) and formoterol (10(-10) M) abolished this increase to a significantly greater extent than either drug alone. In 10% serum, only versican mRNA was increased 1.4-fold (P0.05), whereas decorin mRNA was reduced to 39% (P0.01) of basal expression. These serum effects were counteracted by the drug combination, but there were no significant differences between the combination and either drug alone. Thus, the budesonide and formoterol combination seems to synergistically control serum-induced proteoglycan production, primarily at the post-transcriptional level. In conclusion, the proteoglycan upregulation characteristic of asthmatic airways may be limited by combination therapy with budesonide and formoterol.

OBJECTIVE To report on activities and lessons learned during the first 18 months of a rural programme of prevention of mother-to-child transmission of HIV (PMTCT) in Zimbabwe. METHODS The PMTCT services were introduced in Murambinda Mission Hospital (120 beds), Buhera, in 2001. Programme strategies consisted in recruiting counselling staff, training health professionals, improving mother-child health (MCH) facilities and conducting information, education and communication activities within the community to address HIV/AIDS awareness and stigma. The following components were implemented within MCH services: voluntary counselling and testing of HIV using rapid testing, nevirapine short regimen proposed to all HIV-infected mothers identified and their newborns, support to exclusive breastfeeding for 6- and 18-month mother-child follow-up. Routine monitoring data collected from August 2001 to February 2003 were used to estimate programme uptake. RESULTS Of 2471 pregnant women using antenatal services, 2298 were pre-test counselled, the acceptance of HIV testing reached 92.9%. Of the women who decided to take an HIV test, 1588 (74.3%) returned to collect their result. Overall HIV prevalence was 20.4% (n = 437); 326 of the HIV-positive women were counselled and 104 (24%) received complete mother-child antiretroviral prophylaxis. CONCLUSIONS Acceptability of HIV testing after counselling has remained above 90% since the onset of the programme. Collection of test results and mother-child follow-up are among the most challenging activities of the programme. A district approach and community participation are critical to develop PMTCT programmes in rural settings, even with reasonably good MCH services.

The Nissen fundoplication is the most popular laparoscopic operation performed for the surgical treatment of gastroesophageal reflux disease (GERD). However, for patients in whom esophageal peristalsis is documented to be weak preoperatively, use of a partial wrap, or Toupet procedure, has often been used as an alternative to lessen the potential for postoperative dysphagia. Recent reports have criticized the Toupet procedure as having a higher long-term failure rate than the Nissen approach, especially for patients with severe forms of GERD. We reviewed our experience performing laparoscopic antireflux surgery over a 7-year period and compared the results of patients undergoing laparoscopic Nissen versus Toupet procedures. All procedures were performed at our institution by a single surgeon. Data recorded included preoperative demographic data, preoperative disease parameters, perioperative data, postoperative course, and symptom scores. Follow-up was based on a combination of medical records and phone interviews. There were 142 patients with complete records allowing review for this study. Of these, 118 underwent 122 Nissen fundoplications and 26 underwent 27 Toupet fundoplications. Selection of the procedure was based on preoperative manometric studies. There were seven reoperations. Seven of the patients (28%) who underwent Toupet procedures had severe GERD, a percentage comparable to the Nissen group (31.6%). Preoperative parameters were comparable for both groups, although the Toupet patients had lower average preoperative LES pressures (9.79 mmHg) than did the Nissen patients (16.1 mmHg, P0.05). The operative duration, operative blood loss, morbidity, length of hospitalization, need for reoperation, and efficacy in terms of relieving symptoms (average follow-up = 27.5 months) were comparable for both groups. Based on this experience, the Toupet procedure seems safe and effective in treating the symptoms of GERD, including patients with severe forms of the disease. We recommend its selective use in patients with preoperative esophageal hypomotility who are undergoing laparoscopic antireflux surgery.

Background: Since their introduction in the 1970s, inhaled corticosteroids (ICSs) have been used to control airway inflammation associated with asthma. Budesonide is one of the ICSs recommended as first-line therapy for mild to moderate persistent asthma. Objective: This article describes the esterification of budesonide and how it results in prolonged, location-specific retention of drug in the airways, allowing once-daily dosing. Results: Studies conducted over the past decade have shown that budesonide forms reversible fatty acid esters within the cells of airway tissue, resulting in the formation of an intracellular depot pool of inactive drug. As the intracellular concentration of free budesonide decreases, these budesonide esters are hydrolyzed back to their active state. This process increases budesonide's retention in the airways, prolongs its duration of action, and lowers the risk of systemic effects. Conclusions: By extending budesonide's local anti-inflammatory effect and increasing its airway selectivity, the esterification process appears to contribute to the drug's efficacy, particularly during once-daily administration. Reducing the number of required daily inhalations may increase patient compliance with asthma therapy, although this remains to be evaluated.

Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt’s lymphoma (Daudi) cDNA library inserted into the hightiter retroviral vector pFB.

The inhibition of blood platelet aggregation and secretion was studied using covalent thiol reagents, maleimides, or mercuribenzoates, or using inhibitors of protein disulfide isomerase (PDI), bacitracin or antibodies to PDI. As expected, both types of inhibitors were effective against stimulation by normal physiologic stimuli. On the other hand, when stimulation was initiated with the peptide LSARLAF, that specifically activates the integrin alphaIIbbeta3 (the fibrinogen receptor), the PDI inhibitors were without effect. LSARLAF-induced aggregation was, however, inhibited by the sulfhydryl reagents. To further investigate the role of sulfhydryl-containing proteins and alphaIIbbeta3, platelets were labeled with membrane-impermeant sulfhydryl reagents. Nine bands were found labeled on gel electrophoresis. Two of the labeled bands were identified as alphaIIb and beta3. The conclusions are that while PDI is required for platelet aggregation and secretion, an additional sulfhydryl-dependent step or protein is also required. This latter reaction occurs at the level of alphaIIbbeta3. In distinction to most literature reports, at least a subpopulation of alphaIIbbeta3 contains free sulfhydryl groups, consistent with the possibility that it is a substrate for PDI or part of the sulfhydryl-dependent response.

In this study, purified preparations of platelet protein disulfide isomerase (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to demonstrate that PDI catalyzes formation of vitronectin-thrombin-AT complexes. Complex formation requires reduced glutathione (GSH) and can be prevented by N-ethymaleimide, and the formed complex is dissociated by reducing agents such as mercaptoethanol. No vitronectin-thrombin complex formed in the absence of AT, indicating that the thrombin-AT complex is an obligate intermediate in the reaction. Under optimal conditions, the majority of the thrombin-AT is incorporated into the complex in 60 min. Thrombospondin-1, known to form disulfide-linked complexes with thrombin-AT [Milev, Y., and Essex, D. W. (1999) Arch. Biochem. Biophys. 361, 120-126], competes with vitronectin for thrombin-AT in the low-Ca(2+) environment that favors the active form of thrombospondin. The results presented here may also explain previous studies showing that vitronectin-thrombin-AT complexes form better in plasma (which contains PDI) than with purified proteins (where PDI was not used). We were able to purify a PDI from plasma that was immunologically identical to the platelet enzyme. We used the scrambled RNase assay to show that added purified PDI can function in a plasma environment. Complex formation in plasma was inhibited by inhibitors of PDI. PDI was released from the platelet surface in a soluble form at high pH (around the physiologic range), suggesting a source of the plasma PDI. In summary, these studies indicate that PDI functions to form disulfide-linked complexes of vitronectin with thrombin-AT.

Background Combination therapy with budesonide and formoterol reduces exacerbations of asthma, which are closely associated with human rhinovirus (RV) infections in both children and adults. These data suggest that budesonide and formoterol inhibit virus-induced inflammatory responses of airway epithelial cells. Methods To test this hypothesis, bronchial epithelial (BE) cells were obtained from airway brushings of 8 subjects with moderate-to-severe allergic asthma and 9 with neither asthma nor respiratory allergies. Cultured BE cells were incubated for 24 hours with budesonide (1.77 μM), formoterol (0.1 μM), both, or neither, and then inoculated with RV-16 (5×106 plaque forming units [PFU]/mL). After 24 hours, viral replication (RV RNA), cytokine secretion (CXCL8, CXCL10, TNFα, IFN-β, IL-28) and mRNA expression (CXCL8, CXCL10, TNF, IFNB1, IL28A&B) were analyzed. Results RV infection induced CXCL10 protein secretion and IFNB1 and IL28 mRNA expression. Drug treatments significantly inhibited secretion of CXCL10 in mock-infected, but not RV-infected, BE cells, and inhibited secretion of TNFα under both conditions. Neither budesonide nor formoterol, alone or in combination, significantly affected viral replication, nor did they inhibit RV-induced upregulation of IFNB1 and IL28 mRNA. Overall, RV replication was positively related to CXCL10 secretion and induction of IFNB1 and IL28 mRNA, but the positive relationship between RV RNA and CXCL10 secretion was stronger in normal subjects than in subjects with asthma. Conclusions Budesonide and formoterol can inhibit BE cell inflammatory responses in vitro without interfering with viral replication or production of interferons. These effects could potentially contribute to beneficial effects of budesonide/formoterol combination therapy in preventing RV-induced asthma exacerbations.

Glucocorticoids (GCs) are common components of many chemotherapeutic regimens for lymphoid malignancies. GC-induced apoptosis involves an intrinsic mitochondria-dependent pathway. BIM (BCL-2-interacting mediator of cell death), a BCL-2 homology 3-only pro-apoptotic protein, is upregulated by dexamethasone (Dex) treatment in acute lymphoblastic leukemia cells and has an essential role in Dex-induced apoptosis. It has been indicated that Dex-induced BIM is regulated mainly by transcription, however, the molecular mechanisms including responsible transcription factors are unclear. In this study, we found that Dex treatment induced transcription factor Runx2 and c-Jun in parallel with BIM induction. Dex-induced BIM and apoptosis were decreased in cells harboring dominant-negative c-Jun and were increased in cells with c-Jun overexpression. Cells harboring short hairpin RNA for Runx2 also decreased BIM induction and apoptosis. On the Bim promoter, c-Jun bound to and activated the AP-1-binding site at about -2.7 kb from the transcription start site. Treatment with RU486, a GC receptor antagonist, blocked Dex-induced Runx2, c-Jun and BIM induction, as well as apoptosis. Furthermore, pretreatment with SB203580, a p38-mitogen-activated protein kinase (MAPK) inhibitor, decreased Dex-induced Runx2, c-Jun and BIM, suggesting that p38-MAPK activation is upstream of the induction of these molecules. In conclusion, we identified the critical signaling pathway for GC-induced apoptosis, and targeting these molecules may be an alternative approach to overcome GC-resistance in leukemia treatment.

The radial variability of extracellular single muscle fibre action potential (SFAP) field was studied on the basis of an SFAP theoretical model originally constructed by Rosenfalck (1969). Amplitude and time parameters of the SFAP as a function of the radial distance r less than or equal to 10 mm from the fibre were described by simple mathematical expressions. The results obtained were compared with existing experimental data from different authors and discussed in detail.

SummaryTo investigate the mechanisms of enhanced airway deposition of subepithelial collagen in asthma and its sensitivity to drug therapy with combination of an inhaled glucocorticosteroid (GC) and a long-acting β2-agonist (LABA), a cell model system involving bronchial fibroblasts derived from biopsies from patients with stable mild-to-moderate asthma has been used. To mimic unstable conditions and severe asthma, fibroblasts were stimulated ex vivo with TGFβ1. Primary fibroblasts established from central bronchial biopsies from 8 asthmatic patients were incubated for 24 h with 0.4% serum or TGFβ1 (10 ng/ml) with/without the GC budesonide (BUD; 10 nM) and/or the LABA formoterol (FORM; 0.1 nM). Procollagen peptide I (PICP), metalloproteinase (MMP)-1 and tissue inhibitor of MMPs (TIMP-1) were determined in culture media using ELISA while the activity of MMP-2, -3, -9 by zymography. Metabolically labeled proteoglycans, biglycan and decorin, associated with collagen fibrillation/deposition, were separated using chromatography and SDS-PAGE. The levels of PICP and biglycan were increased 2-fold by TGFβ1 (p

In the airways of asthmatic patients, activated fibroblasts account for an excessive matrix production including proteoglycans (PGs). Transforming growth factor-beta (TGFbeta), metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play key roles in matrix turnover. It is unclear whether asthma therapy with combination of inhaled glucocorticoids and long-acting beta(2)-agonists affects metalloproteolytic equilibrium and by that counteracts airway fibrosis. The effects of the glucocorticoid, budesonide, and the long-acting beta(2)-agonist, formoterol, on the PG production and the activity of PGs' main regulators: MMP-3, MMP-9, MMP-2 and TIMP-1 were investigated in human lung fibroblasts (HFL-1) treated for 24h with TGFbeta1 (10 ng/ml) without/with budesonide (10(-9) to 10(-6)M) and/or formoterol (10(-11) to 10(-6)M). TGFbeta1 significantly increased production of PGs and TIMP-1, and the activity of MMP-3, MMP-9 and MMP-2. Concurrent budesonide/formoterol combination counteracted the enhanced: PG and TIMP-1 production, MMP-9 activity and MMP-9/TIMP-1 ratio, whereas MMP-2 and MMP-3 were not affected and so their ratios to TIMP-1 were significantly increased. Budesonide or formoterol alone achieved equal effects as budesonide/formoterol on MMP-9 and MMP-9/TIMP-1 ratio but had no effects on TIMP-1, MMP-2 or MMP-3. In the formoterol absence, higher budesonide concentrations were required to reduce the PG production, whereas formoterol alone had no effects. These results suggest that the budesonide/formoterol combination enhanced metalloproteolytic activity of human lung fibroblasts via a synergistic decrease of TIMP-1, and that this mechanism may be involved in the synergistic inhibition of the TGFbeta1-induced PG production. This implies that budesonide/formoterol combination therapy can counteract excessive matrix production and thus pathological airway fibrotic remodeling in asthma.