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Survival rates in non-small cell lung cancer (NSCLC) are low. Detection of circulating tumor DNA in liquid biopsy (plasma) is increasingly used to identify targeted therapies for clinically actionable mutations, including EGFR mutations in NSCLC. The cobas® EGFR Mutation Test v2 (cobas EGFR test) is FDA-approved for EGFR mutation detection in tissue or liquid biopsy from NSCLC. Standard K2EDTA tubes require plasma separation from blood within 4 to 8 hours; however, Roche Cell-Free DNA (cfDNA) Collection Tubes (Roche cfDNA tube) enable whole blood stability for up to 7 days prior to plasma separation. This analysis assessed performance of Roche cfDNA tubes with the cobas EGFR test for the detection of EGFR mutations in plasma from healthy donors or patients with NSCLC. Overall, test performance was equally robust with either blood collection tube, eg, regarding limit of detection, linearity, and reproducibility, making Roche cfDNA tubes suitable for routine clinical laboratory use in this setting. Importantly, the Roche cfDNA tubes provided more flexibility for specimen handling versus K2EDTA tubes, eg, in terms of tube mixing, plasma separation, and sample stability, and do not require processing of blood within 8 hours thereby increasing the reach of plasma biopsies in NSCLC., Competing Interests: TM, HH, NL, SJ, SS, and PO are employees of Roche Molecular Systems, Inc, and SS holds stock in F. Hoffmann-La Roche Ltd. AK, M Kohlmann, and XL-S are employees of and hold stock in AstraZeneca. JL was previously an employee of Roche Diagnostics and is currently an employee of Genentech, Inc., and holds stock in F. Hoffmann-La Roche Ltd. BS has received support for this manuscript from Roche Diagnostics. M Kiral was an employee of AstraZeneca and is currently an employee of Sanofi. All other authors declare no competing interests., (Copyright: © 2024 May et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Oncogenic fusion drivers are common in hematological cancers and are thus relevant targets of future CRISPR-Cas9-based treatment strategies. However, breakpoint-location variation in patients pose a challenge to traditional breakpoint-targeting CRISPR-Cas9-mediated disruption strategies. Here we present a new dual intron-targeting CRISPR-Cas9 treatment strategy, for targeting t(8;21) found in 5-10% of de novo acute myeloid leukemia (AML), which efficiently disrupts fusion genes without prior identification of breakpoint location. We show in vitro growth rate and proliferation reduction by 69 and 94% in AML t(8;21) Kasumi-1 cells, following dual intron-targeted disruption of RUNX1-RUNX1T1 compared to a non t(8;21) AML control. Furthermore, mice injected with RUNX1-RUNX1T1-disrupted Kasumi-1 cells had in vivo tumor growth reduction by 69 and 91% compared to controls. Demonstrating the feasibility of RUNX1-RUNX1T1 disruption, these findings were substantiated in isolated primary cells from a patient diagnosed with AML t(8;21). In conclusion, we demonstrate proof-of-principle of a dual intron-targeting CRISPR-Cas9 treatment strategy in AML t(8;21) without need for precise knowledge of the breakpoint location., (© 2023. The Author(s).)

Introduction: Bisexual male youth are more likely to engage in certain behaviors that contribute to HIV/STI transmission (e.g., substance use) than are heterosexual and gay male youth. However, sexuality education rarely addresses the unique needs of sexual minority youth, especially bisexual, pansexual, and queer (bi+) youth, and little is known about their sexuality education experiences and preferences. As such, the goal of this study was to examine bi+ male youth's experiences learning about sex and their preferences for sexuality education., Methods: In 2019, 56 bi+ male youth ages 14-17 were surveyed and interviewed about their sexuality education experiences and preferences. Participants identified as bisexual (64%), pansexual (27%), and queer (9%), were racially/ethnically diverse (39% white, 32% Latinx, 20% Black, 9% other races), and included cisgender (79%) and transgender (21%) male youth., Results: Participants described varied experiences with school-based sexuality education (e.g., none, abstinence only, covered sexual health in some way), but it rarely addressed their unique needs. They typically learned about sex by searching for information online and from sexually explicit media. Participants identified several topics they wanted to learn more about (e.g., sex with same-gender partners, anal sex, consent), but they typically believed they were prepared to have sex. Finally, some participants described benefits of tailoring sexuality education to their unique needs, while others described benefits of more inclusive programs., Conclusions and Policy Implications: Findings suggest that bi+ male youth do not receive adequate sexuality education to make informed decisions about safer sex, highlighting the critical need for reform., Competing Interests: Conflicts of interest: The authors have no conflicts of interest to declare that are relevant to the content of this article.

Organized mosquito control programs (MCP) in the United States have been protecting public health since the early 1900s. These programs utilize integrated mosquito management for surveillance and control measures to enhance quality of life and protect the public from mosquito-borne diseases. Because much of the equipment and insecticides are developed for agriculture, MCP are left to innovate and adapt what is available to accomplish their core missions. Unmanned aerial systems (UAS) are one such innovation that are quickly being adopted by MCP. The advantages of UAS are no longer conjectural. In addition to locating mosquito larval habitats, UAS affords MCP real-time imagery, improved accuracy of aerial insecticide applications, mosquito larval detection and sampling. UAS are also leveraged for applying larvicides to water in habitats that range in size from multi-acre wetlands to small containers in urban settings. Employing UAS can reduce staff exposure to hazards and the impact associated with the use of heavy equipment in sensitive habitats. UAS are utilized by MCP nationally and their use will continue to increase as technology advances and regulations change. Current impediments include a dearth of major UAS manufacturers of equipment that is tailor-made for mosquito control, pesticides that are optimized for application via UAS and regulations that limit the access of UAS to national airspace. This manuscript highlights the strengths and weaknesses of UAS within MCP, provides an update on systems and methods used, and charts the future direction of UAS technology within MCP tasked with public health protection., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cervical cancer are generally caused by a persistent infection with the oncogenic virus, HPV. Patients with HPV integration are more prone to develop cervical cancer than patients without integration. In this proof-of-concept study, we aimed to develop a sensitive method based on targeted amplicon based NGS for early and precise detection of high-risk HPV-genotypes that are highly associated with the development of cervical cancer. Furthermore, we aimed to investigate if amplicon based NGS allowed for HPV genotyping in cervical lesions and whether it could detect HPV integration. The cohort included a group of CIN3 + biopsies (n = 64), CIN2 samples that progressed (n = 5), CIN2 samples that regressed (n = 3), healthy controls (n = 10), and plasma samples (n = 10) from cervical cancer patients. Sequencing was performed using a custom targeted NGS panel designed to detect all 25 high-risk and probably high-risk and two low-risk HPV genotypes. The method was validated by the SPF 10 PCR-DEIA-LiPA 25 assay. In the cohort, the following HPV genotypes were identified: HPV-16, 18, 31, 33, 35, 45, 51, 52, 56, 58, and 59. When comparing the results from the SPF 10 PCR-DEIA-LiPA 25 analyses with the NGS analyses, there was close to a perfect agreement (K = 0.92) among the genotyped HPV types, while in the two cases with complete disagreement, a third assay was applied, and here the results of the NGS analyses were confirmed. Whereas multiple HPV types were detected by the SPF 10 PCR-DEIA-LiPA 25 assay, the NGS analysis clearly suggest that there is one predomentant HPV type. The NGS assay was capable of detecting HPV-16 in a previous false-negative sample classified by the INNO-LiPA assay, emphasizing the importance of including multiple regions of the HPV genome when genotyping. For the 10 plasma samples, our NGS analyses showed full agreement with the digital droplet PCR (ddPCR) analyses of HPV positive as well as negative plasma samples. Lastly, the custom panel was capable of detecting the integration of HPV-16 in the SiHa cell line. The HPV panel provides a highly cost-effective method for HPV detection and genotyping, as exemplified by a list price of around 75 € per sample. In conclusion, the current study demonstrates that targeted NGS is capable of detecting and genotyping HPV in both FFPE biopsies and plasma samples. This method provides for early diagnosis and prognosis of cervical cancer disease progression, thereby optimizing the potential of recovery and survival for these patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Antithrombin (AT) reduction has been shown to improve thrombin generation (TG) in haemophilia with or without inhibitors. As treatment with bypassing agents (BPAs) may be required in patients with breakthrough bleeding while receiving AT-lowering therapy, we assessed TG in platelet-poor plasma samples from haemophilia patients in the presence of BPA (recombinant activated factor VII [rFVIIa; 1.25 or 2.5 μg mL -1 ] or activated prothrombin complex concentrate [aPCC; 0.5 or 1 U mL -1 ]) and AT reduction (anti-AT antibody). Mean ± SEM baseline peak thrombin height was 19.9 ± 4.3 nM in plasma from haemophilia patients (n = 12) and 230.5 ± 9.8 nM in healthy males (n = 24). Reduced AT improved mean peak thrombin height in haemophilia patient plasma to 75.4 ± 17.4 nM. Spiking of 90% AT-reduced haemophilia patient plasma with 2.5 μg mL -1 rFVIIa or 1 U mL -1 aPCC increased the mean peak thrombin height to 82.5 ± 12 nM and 134.8 ± 18.7 nM, respectively. Peak thrombin levels did not exceed the range for healthy volunteers when plasma samples from haemophilia patients with in vitro AT reduction were treated with BPAs, suggesting the potential use of BPAs in conjunction with AT reduction. Further clinical investigations are needed to confirm the safety of this approach., Competing Interests: Declaration of competing interest GK received research support from Alnylam, BPL, Bayer, Baxter, Opko Biologics and Pfizer. GK served on advisory boards and received honoraria for lectures from Alnylam, Bayer, BioMarin, CSL, Opko Biologics, Pfizer Shire, Spark and Uniquore. TL has nothing to declare. AS, KQ, HVN and KM are/were Alnylam employees and have nothing to declare. AS is currently an employee of CAMP4 Therapeutics. KM is currently an employee of Syros Pharmaceuticals Inc. BS was an employee of Alnylam Pharmaceuticals and currently serves as a consultant; currently an employee of Codiak Biosciences., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: Tyrosine kinase inhibitors (TKIs) are first line treatment choices for patients with epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC). However, responses vary among patients, therefore good biomarkers predicting better responses are required. EGFR mutations are detected in the blood from patients as circulating tumour DNA (ctDNA). Studies have shown that clearing ctDNA during first line TKI treatment predicts outcomes for first and second generation TKI treatments. We aimed to investigate the effects on outcome measures of ctDNA clearing in subsequent treatment lines to treatment with the third generation TKI osimertinib., Methods: In total, 225 patients were included in a prospective, multicentre study, where consecutive blood samples were monitored for EGFR mutations during systemic treatment lines, using the Cobas® EGFR mutation test v2. This study focused on EGFR mutations in ctDNA of 82 systemically pre-treated patients receiving osimertinib., Results: Clearing all EGFR mutations from the blood after osimertinib treatment, significantly predicted progression-free survival, objective response rates and disease control rates. Primary sensitising EGFR mutations were found in ctDNA in 70 % of patients, and were accompanied by the T790 M mutation in nearly two thirds of cases. The T790 M mutation was cleared in all cases, while the accompanying sensitising mutations did not necessarily clear. However, T790 M clearing without simultaneously clearing of the primary sensitising mutation did not predict clinical responses. Neither the detection of T790 M before osimertinib treatment, nor the presence of EGFR mutations at the time of osimertinib initiation predicted clinical outcomes., Conclusion: The clearing of EGFR mutations in ctDNA after osimertinib treatment initiation in patients with advanced NSCLC is useful as a positive predictor of clinical outcome., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: Epidermal growth factor receptor (EGFR) mutations confer sensitivity to tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). However, a subset of patients has limited or no response. We investigated the initial dynamics of EGFR mutations detected in circulating tumour DNA (ctDNA) during treatment as a predictive marker of outcome., Methods: A total of 225 patients with advanced EGFR mutated NSCLC were included for consecutive blood sampling in this prospective multicentre study. Out of these, 146 patients received first line TKI and had a baseline blood sample available for EGFR mutation testing with the Cobas® EGFR mutation test V2. For examinations on clearing and clinical outcome, 98 patients who had detectable ctDNA at baseline and at least one follow-up blood sample were included., Results: For patients with EGFR mutations present in plasma at baseline, clearing of mutations from the blood during first line TKI served as a positive predictor for objective response rate (p = 0.0008), progression-free survival (PFS) (p < 0.0001) and overall survival (OS) (p < 0.0001). This was seen both for patients who cleared the ctDNA within the first 7 weeks of treatment and patients who cleared the ctDNA at a slower pace. Baseline mutation presence was a negative predictor for PFS (p = 0.0069) and OS (p = 0.0340)., Conclusion: The current study is the first to confirm, in a sizeable Caucasian cohort, that clearing of EGFR mutations predict outcome to first line TKI in patients with EGFR mutated NSCLC., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Both the interest and actual extent of use of point-of-care ultrasound, PoCUS, among general practitioners or family physicians are increasing and training is also increasingly implemented in residency programs. However, the amount of research within the field is still rather limited compared to what is seen within other specialties in which it has become more established, such as in the specialty of emergency medicine. An assumption is made that what is relevant for emergency medicine physicians and their populations is also relevant to the general practitioner, as both groups are generalists working in unselected populations. This systematic review aims to examine the extent of use and to identify clinical studies on the use of PoCUS by either general practitioners or emergency physicians on indications that are relevant for the former, both in their daily practice and in out-of-hours services., Methods: Systematic searches were done in PubMed/MEDLINE using terms related to general practice, emergency medicine, and ultrasound., Results: On the extent of use, we identified 19 articles, as well as 26 meta-analyses and 168 primary studies on the clinical use of PoCUS. We found variable, but generally low, use among general practitioners, while it seems to be thoroughly established in emergency medicine in North America, and increasingly also in the rest of the world. In terms of clinical studies, most were on diagnostic accuracy, and most organ systems were studied; the heart, lungs/thorax, vessels, abdominal and pelvic organs, obstetric ultrasound, the eye, soft tissue, and the musculoskeletal system. The studies found in general either high sensitivity or high specificity for the particular test studied, and in some cases high total accuracy and superiority to other established diagnostic imaging modalities. PoCUS also showed faster time to diagnosis and change in management in some studies., Conclusion: Our review shows that generalists can, given a certain level of pre-test probability, safely use PoCUS in a wide range of clinical settings to aid diagnosis and better the care of their patients.

Objectives: MiR-30b, miR-30c, miR-221 and miR-222 are known to induce gefitinib resistance in lung cancer cell lines with activation of mutations in the epidermal growth factor receptor (EGFR). However, the role of these four microRNAs in tyrosine kinase inhibitor (TKI)-resistance in non-small cell lung cancer (NSCLC) patients is unknown. Thus, the aim of this study was to investigate the predictive value of miR-30b, miR-30c, miR-221 and miR-222 in plasma from EGFR-mutated lung cancer patients receiving erlotinib., Materials and Methods: The cohort consisted of 29 EGFR-mutated lung cancer patients receiving erlotinib. Plasma levels of miR-30b, miR-30c, miR-221 and miR-222 were analyzed by qPCR from blood samples collected before treatment start. Plasma concentration of each microRNA was correlated to clinical outcome., Results: Plasma concentrations of miR-30b and miR-30c could be determined in all 29 patients. Low plasma concentrations of miR-30b and miR-30c showed significant correlation with superior progression-free survival (PFS) (miR-30b: HR = 0.303 [0.123-0.747], p < 0.05; miR-30c: HR = 0.264 [0.103-0.674], p < 0.05). Low plasma concentrations of miR-30c were also significantly correlated with superior overall survival (OS) (HR = 0.30 [0.094-0.954], p < 0.041)., Conclusion: High plasma concentrations of miR-30b and miR-30c predicted shorter PFS and OS. This implies that miR-30b and miR-30c could have clinical potential as biomarkers in EGFR-mutated lung cancer patients., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

In this article, we review Davanloo's metapsychology of the unconscious and how it can contribute to the current psychodynamic understanding and treatment of psychosis. In this framework, current attachment and emotions become connected with unconscious conflict-laden feelings about early attachment trauma at the core of the unconscious conflict. These conflict-laden feelings mobilize unconscious anxiety and defenses, which are alongside or, in and of themselves, constitute the entire picture of psychosis. Those patients with low emotional capacities are provided specific therapeutic techniques to bolster anxiety tolerance while those more defended patients are offered means to begin to accept and experience the feelings they have about present and past adverse experiences including those caused by psychosis itself. Case and case series research have shown this model to be clinically effective and cost effective as an adjunct to care. Case vignettes will describe the assessment of capacities and treatment frame for patients with a history of psychosis. Davanloo's metapsychology of the unconscious offers an important contribution to the current psychodynamic understanding of psychosis by considering the role of attachment, emotions and unconscious conflict and addressing these through specific psychodynamic interventions.

Background: Cell free DNA (cfDNA) has shown promising utility as prognostic biomarker for patients with colorectal cancer (CRC), with an ongoing need to optimize and validate the laboratory methodology. Here, we report our optimization and validation of a direct fluorescent assay and display the potential utility in patients with colorectal cancer., Methods: Plasma cfDNA was analyzed by a direct fluorescent assay (DFA) and compared to quantification by droplet digital PCR (ddPCR). For clinical validation, baseline blood samples were available for a total of 273 patients from six different Nordic trials, covering patients with locally advanced rectal cancer (n = 176, cohorts A + B), liver limited metastatic CRC (n = 75C + D) and wide spread metastatic CRC (n = 22 E + F)., Results: Validating the DFA analysis with ddPCR revealed a strong correlation with an R2 of 0.81. For the clinical cohorts, the levels of cfDNA were: 0.8 ng/uL (95%CI 0.75-0.83) (A + B), 0.93 ng/uL (95%CI 0.86-1.02) (C + D) and 1.2 ng/uL (95%CI 0.85-1.47) (E + F), respectively (p < 0.01). All cohorts of colorectal cancer had higher levels of cell free DNA than healthy individuals (n = 94) (p < 0.01)., Conclusion: Analysis of cell free DNA by a direct fluorescent assay could be an attractive laboratory option for a rapid inexpensive quantification of cell free DNA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non-small cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of a real-time PCR EGFR mutation test (cobas EGFR Mutation Test v2) to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per milliliter. The circulating tumor DNA was extracted by a cell-free circulating DNA sample preparation kit (cobas cfDNA Sample Preparation Kit), followed by duplicate analysis with the real-time PCR EGFR mutation test (Roche Molecular Systems, Pleasanton, CA). Lowest sensitivities were obtained for the c.2156G>C p.(Gly719Ala) and c.2573T>G p.(Leu858Arg) variants for the lowest target copies. For all other variants, sensitivities varied between 96.3% and 100.0%. All specificities were 98.8% to 100.0%. Coefficients of variation indicated good intralaboratory and interlaboratory repeatability and reproducibility but increased for decreasing concentrations. Prediction models revealed a significant correlation for all variants between the predefined copy number and the observed semiquantitative index values, which reflect the samples' plasma mutation load. This study demonstrates an overall robust performance of the real-time PCR EGFR mutation test kit in plasma. Prediction models may be applied to estimate the plasma mutation load for diagnostic or research purposes., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Plasma DNA-histone complexes and total free-plasma DNA have the potential to quantify the ischaemia-reperfusion damages occurring after cardiac arrest. Furthermore, DNA-histone complexes may have the potential of being a target for future treatment. The aim was to examine if plasma DNA-histone complexes and the levels of total free-plasma DNA were elevated in post-cardiac arrest patients compared with healthy individuals, and to examine if these biomarkers were capable of predicting mortality., Methods: We included 42 comatose out-of-hospital cardiac arrest patients and collected blood samples after 22, 46 and 70 h. Samples for DNA-histone complexes were quantified by Cell Death Detection ELISA plus . The total free-plasma DNA analyses were quantified with qPCR by analysing the Beta-2 microglobulin gene. The control group comprised 40 healthy individuals., Results: We found no difference in the level of DNA-histone complexes between the 22-h sample and healthy individuals (P = 0.10). In the 46-h sample, there was an increased level of DNA-histone complexes in non-survivors compared with survivors 30 days after the cardiac arrest (P < 0.01) and the area under the ROC curve was 0.78 (95% confidence interval: 0.59;0.96). The level of total free-plasma DNA was increased in the 22-h sample compared with healthy individuals (P < 0.001) but no significant difference was found between non-survivors and survivors 30 days after the cardiac arrest (all P ≥ 0.06)., Conclusion: An increased level of DNA-histone complexes was associated with increased mortality and that the level of total free-plasma DNA was elevated post-cardiac arrest., (© 2017 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Although many epidermal growth factor receptor (EGFR)-mutated lung cancer patients initially benefit from the EGFR-inhibitor erlotinib, all acquire resistance. So far, several mechanisms implicated in resistance have been identified, but the existence of multiple resistance mechanisms in parallel have only been sparsely investigated. In this study, we investigated parallel resistance mechanisms acquired by HCC827, an EGFR-mutated adenocarcinoma cell line dependent on EGFR activity and sensitive to erlotinib. The cell line was treated with erlotinib by stepwise escalation of the drug-concentration and erlotinib-resistant (HCC827ER) cells created. HCC827ER cells depicted a mixed epithelial and mesenchymal phenotype. To clarify potential parallel resistance mechanisms, 14 resistant subclones were established by limited dilution. Interestingly, all HCC827ER subclones harbored either a MET-amplification (6/14) or underwent EMT (8/14), mechanisms both found in previous studies, but not in co-occurrence. Both subclone-types were resistant to erlotinib, but only MET-subclones responded to the MET-inhibitors crizotinib and capmatinib. EMT-subclones on the other hand had markedly increased FGFR1 expression and responded to the FGFR-inhibitor AZD4547, whereas MET-subclones did not. Monitoring gene expression through the development of HCC827ER revealed upregulation of FGFR1 expression as an early response to erlotinib. In addition, FGFR1 expression increased upon short-term erlotinib treatment (48 h) identifying a physiological role immediately after erlotinib exposure. The high FGFR1 expression seen in EMT-subclones was stable even after five passages without erlotinib. Here we show, that parallel resistance mechanisms appear during erlotinib-resistance development in EGFR-mutated NSCLC cells and highlight a role for FGFR1 expression changes as an early response to erlotinib as well as a bypass-signaling mechanism.

Background: Use of exosomes as biomarkers in non-small cell lung cancer (NSCLC) is an intriguing approach in the liquid-biopsy era. Exosomes are nano-sized vesicles with membrane-bound proteins that reflect their originating cell. Prognostic biomarkers are needed to improve patient selection for optimal treatment. We here evaluate exosomes by protein phenotyping as a prognostic biomarker in NSCLC., Methods: Exosomes from plasma of 276 NSCLC patients were phenotyped using the Extracellular Vesicle Array; 49 antibodies captured the proteins on the exosomes, and a cocktail of biotin-conjugated antibodies binding the general exosome markers CD9, CD81 and CD63 was used to visualise the captured exosomes. For each individual membrane-bound protein, results were analysed based on presence, in a concentration-dependent manner, and correlated to overall survival (OS)., Results: The 49 proteins attached to the exosomal membrane were evaluated. NY-ESO-1, EGFR, PLAP, EpCam and Alix had a significant concentration-dependent impact on inferior OS. Due to multiple testing, NY-ESO-1 was the only marker that maintained a significant impact on inferior survival (hazard rate (HR) 1.78 95% (1.78-2.44); p = 0.0001) after Bonferroni correction. Results were adjusted for clinico-pathological characteristics, stage, histology, age, sex and performance status., Conclusion: We illustrate the promising aspects associated with the use of exosomal membrane-bound proteins as a biomarker and demonstrate that they are a strong prognostic biomarker in NSCLC., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Background. One of the major challenges affecting sarcoma treatment outcome, particularly that of metastatic disease, is resistance to chemotherapy. Cancer-initiating cells are considered a major contributor to this resistance. Methods. An immortalised nontransformed human stromal (mesenchymal) stem cell line hMSC-TERT4 and a transformed cell line hMSC-TERT20-CE8, known to form sarcoma-like tumours when implanted in immune-deficient mice, were used as models. Receptor tyrosine kinase (RTK) activation was analysed by RTK arrays and cellular viability after tyrosine kinases inhibitor (TKI) treatment with or without doxorubicin was assessed by MTS assay. Results. Initial results showed that the hMSC-TERT4 was more doxorubicin-sensitive while hMSC-TERT20-CE8 was less doxorubicin-sensitive evidenced by monitoring cell viability in the presence of doxorubicin at different doses. The epidermal growth factor receptor (EGFR) was activated in both cell lines. However hMSC-TERT20-CE8 exhibited significantly higher expression of the EGFR ligands. EGFR inhibitors such as erlotinib and afatinib alone or in combination with doxorubicin failed to further decrease cell viability of hMSC-TERT20-CE8. However, inhibition with the TKI dasatinib in combination with doxorubicin decreased cell viability of the hMSC-TERT20-CE8 cell line. Conclusion. Our results demonstrate that dasatinib, but not EGFR-directed treatment, can decrease cell viability of stromal cancer stem cells less sensitive to doxorubicin.

Hemophilia A and B are inherited bleeding disorders characterized by deficiencies in procoagulant factor VIII (FVIII) or factor IX (FIX), respectively. There remains a substantial unmet medical need in hemophilia, especially in patients with inhibitory antibodies against replacement factor therapy, for novel and improved therapeutic agents that can be used prophylactically to provide effective hemostasis. Guided by reports suggesting that co-inheritance of prothrombotic mutations may ameliorate the clinical phenotype in hemophilia, we developed an RNA interference (RNAi) therapeutic (ALN-AT3) targeting antithrombin (AT) as a means to promote hemostasis in hemophilia. When administered subcutaneously, ALN-AT3 showed potent, dose-dependent, and durable reduction of AT levels in wild-type mice, mice with hemophilia A, and nonhuman primates (NHPs). In NHPs, a 50% reduction in AT levels was achieved with weekly dosing at approximately 0.125 mg/kg, and a near-complete reduction in AT levels was achieved with weekly dosing at 1.5 mg/kg. Treatment with ALN-AT3 promoted hemostasis in mouse models of hemophilia and led to improved thrombin generation in an NHP model of hemophilia A with anti-factor VIII inhibitors. This investigational compound is currently in phase 1 clinical testing in subjects with hemophilia A or B.

Bernard-Soulier syndrome (BSS) is a rare severe autosomal recessive bleeding disorder. To date heterozygous carriers of BSS mutations have not been shown to have bleeding symptoms. We assessed bleeding using a semi-quantitative questionnaire, platelet parameters, PFA-100 closure times, ristocetin response, GP Ib/IX expression and VWF antigen in 14 BSS patients, 30 heterozygote carriers for related mutations and 29 controls. Eight mutations in GP1BA, GP1BB or GP9 were identified including four previously unknown pathogenic mutations. Subjects with BSS reported markedly more mucocutaneous bleeding than controls. Increased bleeding was also observed in heterozygotes. Compared to controls, patients with BSS had lower optical platelet counts (P < 0.001), CD61-platelet counts (P < 0.001) and higher mean platelet volume (17.7 vs. 7.8 fL, P < 0.001) and ristocetin response and closure times were unmeasurable. Heterozygotes had higher MPV (9.7 fL, P < 0.001) and lower platelet counts (P < 0.001) than controls but response to ristocetin and closure times were normal. The VWF was elevated in both BSS and in heterozygotes (P = 0.005). We conclude that heterozygotes for BSS mutations have lower platelet counts than controls and show a bleeding phenotype albeit much milder than in BSS. Both patients with BSS and heterozygote carriers of pathogenic mutations have raised VWF., (© 2014 Wiley Periodicals, Inc.)

Background: Effective treatment of acute bleeding episodes in patients with hemophilia B relies on factor IX recovery, with higher levels being more desirable, whereas prevention of bleeds with a prophylactic regimen depends on the half-life of the product. Lower recovery values have been reported following administration of recombinant FIX (rFIX) than following administration of plasma-derived FIX (pdFIX)., Objectives: To compare the pharmacokinetic and pharmacodynamic properties of rFIX and pdFIX in patients with hemophilia B., Methods: A prospective crossover study of nine patients with moderate to severe hemophilia B was performed. Following a washout period, 50 U kg(-1) FIX was administered, and blood samples were taken as per protocol up to 48 h postinfusion. Paired data were analyzed with the Wilcoxon signed rank test., Results: Mean peak recovery at 10 min postinfusion was 62.14 IU dL(-1) with pdFIX and 52.7 IU dL(-1) with rFIX (P = 0.08). Mean half-life was 16.6 h with pdFIX and 17.5 h with rFIX (P = 0.55). Maximum peak thrombin generation (PTG) was 35.9 nm with pdFIX and 28.9 nm with rFIX (P = 0.21). Administration of rFIX resulted in early PTG, whereas administration of pdFIX resulted in slightly later and sustained PTG. At 48 h, PTG was similar with pdFIX (19.0 nm) and rFIX (19.4 nm) (P = 0.91)., Conclusions: Patients experienced better recovery with pdFIX than with rFIX. pdFIX and rFIX had similar half-lives. Maximum PTG was higher for pdFIX; however, this difference did not reach statistical significance. The clinical impact of the slightly increased, delayed and sustained PTG with pdFIX requires further investigation., (© 2014 International Society on Thrombosis and Haemostasis.)

Background: HIV-1 shedding from the female genital tract is associated with increased sexual and perinatal transmission and has been broadly evaluated in cross-sectional studies. However, few longitudinal studies have evaluated how the immune microenvironment effects shedding., Methods: Thirty-nine HIV-1-infected women had blood, cervicovaginal lavage, and biopsies of the uterine cervix taken quarterly for up to 5 years. Cytokines/chemokines were quantified by Luminex assay in cervicovaginal lavage, and cellular phenotypes were characterized using immunohistochemistry in cervical biopsies. Comparisons of cytokine/chemokine concentrations and the percent of tissue staining positive for T cells were compared using generalized estimating equations between non-shedding and shedding visits across all women and within a subgroup of women who intermittently shed HIV-1., Results: Genital HIV-1 shedding was more common when plasma HIV-1 was detected. Cytokines associated with cell growth (interleukin-7), Th1 cells/inflammation (interleukin-12p70), and fractalkine were significantly increased at shedding visits compared with non-shedding visits within intermittent shedders and across all subjects. Within intermittent shedders and across all subjects, FOXP3 T cells were significantly decreased at shedding visits. However, there were significant increases in CD8 cells and proportions of CD8FOXP3 T cells associated with HIV-1 shedding., Conclusions: Within intermittent HIV-1 shedders, decreases in FOXP3 T cells at the shedding visit suggests that local HIV-1 replication leads to CD4 T-cell depletion, with increases in the proportion of CD8FOXP3 cells. HIV-1-infected cell loss may promote a cytokine milieu that maintains cellular homeostasis and increases immune suppressor cells in response to HIV-1 replication in the cervical tissues.

Background: Infants born to HIV-1 infected mothers may have increased risk for tuberculosis (TB), but the prevalence of TB infection in this population is undefined. In contrast to tuberculin skin tests that are confounded by recent bacille Calmette-Guérin (BCG) vaccination, TB interferon gamma release assays (IGRAs) do not cross-react with BCG and enable detection of TB infection in infancy., Methods: In a nested observational cohort of HIV-1 infected Kenyan mothers and their infants, we conducted T-SPOT.TB assays on cryopreserved peripheral blood mononuclear cells from 6-month-old infants without prior active TB. Maternal and infant correlates of infant TB infection were assessed., Results: One hundred and eight-two infants were tested with T-SPOT.TB. Of 128 infants with determinate T-SPOT.TB results, the prevalence of a positive T-SPOT.TB was 10.9% [95% confidence interval (CI): 6.1-17.7%]. All infants were BCG-vaccinated and 7.0% were HIV-1 infected. Positive infant T-SPOT.TB was associated with maternal active TB (odds ratio: 15.5, 95% CI: 1.3-184; P = 0.04) and prolonged infant fever (>1 month) (odds ratio: 18.8, 95% CI: 1.6-223; P = 0.03)., Conclusions: We observed a high prevalence of TB infection in 6-month-old HIV-1 exposed infants. Improved TB detection and prevention are warranted in HIV-1 exposed infants at high risk for active TB disease.

Women with Bernard-Soulier syndrome (BSS) are considered to be at high risk of serious bleeding during childbirth. Due to the frequently occurring platelet transfusion refractoriness, alternative prophylactic therapy is required. Using rotational thromboelastometry, we evaluated the whole blood coagulation profile of a pregnant woman with BSS before and after spiking ex vivo with different concentrations of recombinant activated factor VII (rFVIIa) and fibrinogen. As experiments suggested improved clotting with clinically applicable concentrations of both agents in a complementary manner, the findings were confirmed on blood from a non-pregnant woman and three men suffering from BSS. During delivery, bleeding refractory to platelets occurred and immediately following delivery she received both rFVIIa and fibrinogen intravenously. Immediate cessation of bleeding occurred, and no postpartum hemorrhage was seen. Another woman with BSS later also received the same rFVIIa and fibrinogen treatment prophylactically after delivery without any postpartum bleeding. Eventually, the first woman during her second delivery received the same treatment again prophylactically without any bleeding. No side effects were observed during these three deliveries. Our observations suggest that rFVIIa combined with fibrinogen may provide a beneficial clinical hemostatic effect partly by separate but complementary mechanisms.

Introduction: Rapid tests for rifampicin resistance may be useful for identifying isolates at high risk of drug resistance, including multidrug-resistant TB (MDR-TB). However, choice of diagnostic test and prevalence of rifampicin resistance may both impact a diagnostic strategy for identifying drug resistant-TB. We performed a systematic review to evaluate the performance of WHO-endorsed rapid tests for rifampicin resistance detection., Methods: We searched MEDLINE, Embase and the Cochrane Library through January 1, 2012. For each rapid test, we determined pooled sensitivity and specificity estimates using a hierarchical random effects model. Predictive values of the tests were determined at different prevalence rates of rifampicin resistance and MDR-TB., Results: We identified 60 publications involving six different tests (INNO-LiPA Rif. TB assay, Genotype MTBDR assay, Genotype MTBDRplus assay, Colorimetric Redox Indicator (CRI) assay, Nitrate Reductase Assay (NRA) and MODS tests): for all tests, negative predictive values were high when rifampicin resistance prevalence was ≤ 30%. However, positive predictive values were considerably reduced for the INNO-LiPA Rif. TB assay, the MTBDRplus assay and MODS when rifampicin resistance prevalence was < 5%., Limitations: In many studies, it was unclear whether patient selection or index test performance could have introduced bias. In addition, we were unable to evaluate critical concentration thresholds for the colorimetric tests., Discussion: Rapid tests for rifampicin resistance alone cannot accurately predict rifampicin resistance or MDR-TB in areas with a low prevalence of rifampicin resistance. However, in areas with a high prevalence of rifampicin resistance and MDR-TB, these tests may be a valuable component of an MDR-TB management strategy.

Objectives: The Florida Public Health Practice-Based Research Network conducted the study of Florida county health departments (CHDs) to assess relationships between self-assessed performance on essential services (ESs) and sources of funding., Methods: Primary data were collected using an online survey based on Public Health Accreditation Board standards for ES. Bivariate and multivariate analyses were conducted to assess the relationship of sources and amounts of revenue obtained from the Florida Department of Health financial system to responses to the survey of CHD capacity for ESs., Results: Self-assessed CHD performance for each ES varied extensively among the CHDs and across the 10 ESs, ranging from a high of 98% CHDs completely or almost completely meeting the standards for ES 2 (Investigating Problems and Hazards) to a low of 32% completely or almost completely meeting standards for ES 10 (Research/Evidence). Medicaid revenue and fees were positively correlated with some ESs. Per capita revenue support varied extensively among the CHDs., Conclusions: Revenue for ES is decreasing and is heavily reliant on noncategorical (discretionary) revenue. This study has important implications for continued reliance on ES as an organizing construct for public health.

Background: The tyrosine kinase receptor HER4 is a member of the epidermal growth factor receptor (EGFR) family. It plays diverse roles in cancer development and cancer progression and can both exert oncogenic and tumour-suppressive activities. Alternatively spliced isoforms of HER4 are critical to the different signalling possibilities of HER4., Methods: We use a splice-switching oligonucleotide (SSO) to direct the alternative splicing of HER4 from the CYT1 to the CYT2 isoform in HER4-expressing breast cancer cells., Results: Treatment with a target-specific SSO was accompanied by a decreased growth of the cells (P<0.0001). In addition, the SSO treatment induced a decreased activity of Akt. We confirmed the SSO-dependent switching of the HER4 isoform CYT1 to CYT2 expression in a xenografted mouse tumour model driven by subcutaneously injected MCF7 cells. We hence demonstrated the feasibility of SSO-directed splice-switching activity in vivo. Furthermore, the SSO treatment efficiently decreased the growth of the xenografted tumour (P=0.0014)., Conclusion: An SSO directing the splicing of HER4 towards the CYT2 isoform has an inhibitory effect of cancer cell growth in vitro and in vivo. These results may pave the way for the development of new anticancer drugs in HER4-deregulated cancers in humans.

The evaluation of the coagulation profile has used so far either clotting-based or chromogenic assays with different endpoints. Clotting-based techniques are the most used worldwide, and they certainly are useful for diagnosis of clotting factor deficiencies. However, the information provided is relatively limited, and therefore the individual profile of coagulation is poorly assessed. This is reflected by the weak correlation between the results of these assays and the clinical phenotype. Among the assays that benefited from technological advances, thrombin generation and thromboelastography are probably the most actively investigated, but they require specific instruments and are not fully automated. Their standardisation level is rapidly progressing, and they are progressively entering the clinical scene, with the attempt to provide additional information on the coagulation process and a meaningful clinical correlation. These inherited bleeding disorders frequently require replacement therapy using clotting factor concentrates that increase the plasma level of the missing clotting factor. The classical adjustment of the therapy is mainly based on the measurement of the plasma clotting activity of the protein administered. If one considers that a certain level of thrombin generated would predict clinical efficacy, monitoring of thrombin formation might offer new possibilities to individually predict the bleeding phenotype, select the most adapted therapeutic product and tailor the dose. The same holds true for thromboelastography/thromboelastometry which evaluate fibrin formation as well as clot resistance to fibrinolytic challenge, one step further down in the coagulation process. In this regard, these 2 assays could be seen as complementary in terms of information provided on the coagulation profile at the individual level., (© 2012 Blackwell Publishing Ltd.)

Objective: To audit the quality of obstetric management preceding emergency caesarean sections for prolonged labour., Design: A quality assurance analysis of a retrospective criterion-based audit supplemented by in-depth interviews with hospital staff., Setting: Two Tanzanian rural mission hospitals., Population: Audit of 144 cases of women undergoing caesarean sections for prolonged labour; in addition, eight staff members were interviewed., Methods: Criteria of realistic best practice were established, and the case files were audited and compared with these. Hospital staff were interviewed about what they felt might be the causes for the audit findings., Main Outcome Measures: Prevalence of suboptimal management and themes emerging from an analysis of the transcripts., Results: Suboptimal management was identified in most cases. Non-invasive interventions to potentially avoid operative delivery were inadequately used. When deciding on caesarean section, in 26% of the cases labour was not prolonged, and in 16% the membranes were still intact. Of the women with genuine prolonged labour, caesarean sections were performed with a fully dilated cervix in 36% of the cases. Vacuum extraction was not considered. Amongst the hospital staff interviewed, the awareness of evidence-based guidelines was poor. Word of mouth, personal experience, and fear, especially of HIV transmission, influenced management decisions., Conclusion: The lack of use and awareness of evidence-based guidelines led to misinterpretation of clinical signs, fear of simple interventions, and an excessive rate of emergency caesarean sections., (© 2012 The Authors BJOG An International Journal of Obstetrics and Gynaecology © 2012 RCOG.)

Background: Trauma-induced coagulopathy has a multifactorial aetiology. Coagulopathy is related to blood loss including consumption of clotting factors and platelets and haemodilution. Additionally hyperfibrinolysis, hypothermia, acidosis and metabolic changes affect the coagulation system., Methods: This is a review of pathophysiology and new treatment strategies for trauma-induced coagulopathy., Results: Paradigms are actively changing and there is still a shortage of data. The aim of any haemostatic therapy is to control bleeding and minimize blood loss and transfusion requirements. Transfusion of allogeneic blood products as well as trauma-induced coagulopathy cause increased morbidity and mortality. Current opinion is based on present studies and results from small case series, combined with findings from experimental studies in animals, in vitro studies and expert opinions, as opposed to large, randomized, placebo-controlled studies. A summary of new and emerging strategies, including medical infusion and blood products, to beneficially manipulate the coagulation system in the critically injured patient is suggested., Conclusion: Future treatment of trauma-induced coagulopathy may be based on systemic antifibrinolytics, local haemostatics and individualized point-of-care-guided rational use of coagulation factor concentrates such as fibrinogen, prothrombin complex concentrate, recombinant factor VIIa and factor XIII. The authors speculate that timely and rational use of coagulation factor concentrates will be more efficacious and safer than ratio-driven use of transfusion packages of allogeneic blood products. Copyright © 2011 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd., (Copyright © 2011 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

Background: We have previously developed (11)C-erlotinib as a new positron emission tomography (PET) tracer and shown that it accumulates in epidermal growth factor receptor (EGFR)-positive lung cancer xenografts in mice. Here, we present a study in patients with non-small cell lung cancer (NSCLC) investigating the feasibility of (11)C-erlotinib PET as a potential method for the identification of lung tumours accumulating erlotinib., Methods: Thirteen patients with NSCLC destined for erlotinib treatment were examined by contrast-enhanced computed tomography (CT), (11)C-erlotinib PET/low-dose CT and (18)F-fluoro-2-deoxy-D-glucose ((18)F-FDG) PET/low-dose CT before start of the erlotinib treatment. After 12 weeks treatment, they were examined by (18)F-FDG PET/contrast-enhanced CT for the assessment of clinical response., Results: Of the 13 patients included, 4 accumulated (11)C-erlotinib in one or more of their lung tumours or lymph-node metastases. Moreover, (11)C-erlotinib PET/CT identified lesions that were not visible on (18)F-FDG PET/CT. Of the four patients with accumulation of (11)C-erlotinib, one died before follow-up, whereas the other three showed a positive response to erlotinib treatment. Three of the nine patients with no accumulation died before follow-up, four showed progressive disease while two had stable disease after 12 weeks of treatment., Conclusion: Our data show a potential for (11)C-erlotinib PET/CT for visualizing NSCLC lung tumours, including lymph nodes not identified by (18)F-FDG PET/CT. Large clinical studies are now needed to explore to which extent pre-treatment (11)C-erlotinib PET/CT can predict erlotinib treatment response.

Laboratory evaluation of bleeding disorders has been performed with the standard clotting assays such as the PT and PTT for several decades. Our improved understanding of the process of blood coagulation has now revealed the important role played by the cellular elements such as platelets, monocytes and red blood cells. The need for a test that can assess clotting in a more 'global' manner, beyond the initiation of clot formation, has led to greater interest in assays such as thrombin generation and thromboelastography. Even though there are several publications using thromboelastography it remains a research tool as the methodology is not standardized. In an attempt to show reproducibility and consistency using thromboelastography, a group of investigators from different countries joined hands to form the TEG-ROTEM Working Group. Two studies were performed using PRP and FVIII deficient plasma and an intrinsic pathway activator. This article summarizes the results of the first international effort at standardization of thromboelastography. Both of the instruments using this technology (TEG(®) and ROTEM(®)) were used. Nine laboratories from countries around the globe participated in this effort. The results showed a significant inter-laboratory variance with CV's greater than 10%. Although these results were not satisfactory, this has been the first effort to standardize this methodology and significant work remains to be done to improve reliability and reproducibility. These studies were performed on PRP and the results may be more reliable when preformed on whole blood samples. We believe that it is important to continue this work so that we may investigate the usefulness and potential applications of thromboelastography in the evaluation of bleeding and thrombosis., (© 2011 Blackwell Publishing Ltd.)

Population-based studies suggest that repetitive cycling of weight loss and regain may be associated with future weight gain. Therefore, to better define the relationship between weight cycling, energy homeostasis, and future weight gain, we examined associations between frequent intentional weight loss and hormonal profiles in postmenopausal women. This cross-sectional study evaluated the relationship between a history of frequent weight loss and biomarkers, including serum glucose, insulin, leptin, and ghrelin, as well as sex steroid hormones. We hypothesized that frequent intentional weight loss would be associated with changes in normal appetite and body weight regulatory hormones, favoring increased appetite and weight gain. One hundred fifty-nine healthy, weight stable, sedentary, overweight, postmenopausal women who had been recruited for an exercise intervention participated in this study. History of intentional weight loss (frequency and magnitude) was assessed by questionnaire. Hormonal assays were performed by radioimmunoassay (insulin, leptin, ghrelin, estrogens, androgens, and dehydroepiandrosterone), chemiluminescence immunoassay (insulin-like growth factor-1), and immunometric assay (sex hormone binding globulin). Analysis of variance and regression analyses were used to investigate the relationship between weight loss history and metabolic hormones. A higher degree of weight cycling, characterized by the frequency of intentionally losing more than 10 lb, was associated with an appetite-stimulating hormonal profile, including higher concentrations of ghrelin (P trend = .04), lower glucose (P trend = .047), and to some extent, lower insulin (P trend = .08). Frequent weight loss was also associated with lower androgen concentrations, including androstenedione (P trend = .02), testosterone (P trend = .04), and free testosterone (P trend = .01). No independent associations between the concentrations of leptin or estrogens and weight cycling were observed. This study suggests that frequent intentional weight loss may affect hormones involved in energy regulation., (2010 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) mediates host resistance to severe malaria and other infectious diseases. NO production and mononuclear cell expression of the NO producing enzyme-inducible nitric oxide synthase (NOS2) have been associated with protection from severe falciparum malaria. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) and haplotypes in the NOS2 promoter, to identify associations of these haplotypes with malaria severity and to test the effects of these polymorphisms on promoter activity. We identified 34 SNPs in the proximal 7.3 kb region of the NOS2 promoter and inferred NOS2 promoter haplotypes based on genotyping 24 of these SNPs in a population of Tanzanian children with and without cerebral malaria. We identified 71 haplotypes; 24 of these haplotypes comprised 82% of the alleles. We determined whether NOS2 promoter haplotypes were associated with malaria severity in two groups of subjects from Dar es Salaam (N = 185 and N = 250) and in an inception cohort of children from Muheza-Tanga, Tanzania (N = 883). We did not find consistent associations of NOS2 promoter haplotypes with malaria severity or malarial anemia, although interpretation of these results was potentially limited by the sample size of each group. Furthermore, cytokine-induced NOS2 promoter activity determined using luciferase reporter constructs containing the proximal 7.3 kb region of the NOS2 promoter and the G-954C or C-1173T SNPs did not differ from NOS2 promoter constructs that lacked these polymorphisms. Taken together, these studies suggest that the relationship between NOS2 promoter polymorphisms and malaria severity is more complex than previously described.

Intermittent preventive treatment in pregnancy (IPTp) is used to prevent Plasmodium falciparum malaria. However, parasites resistant to the IPTp drug sulfadoxine-pyrimethamine (SP) have emerged worldwide, and infections with mixed resistant and susceptible parasites are exacerbated by pyrimethamine in mice. In a prospective delivery cohort in Muheza, Tanzania, we examined the effects of SP IPTp on parasite resistance alleles, parasite diversity, level of parasitemia, and inflammation in the placenta. IPTp use was associated with an increased fraction of parasites carrying the resistance allele at DHPS codon 581, an increase in the level of parasitemia, and more intense placental inflammation. The lowest mean level of parasite diversity and highest mean level of parasitemia occurred in women after recent IPTp use. These findings support a model of parasite release and facilitation, whereby the most highly resistant parasites out-compete less fit parasite populations and overgrow under drug pressure. Use of partially effective anti-malarial agents for IPTp may exacerbate malaria infections in the setting of widespread drug resistance.

Background: Epidemiologic studies suggest a reduced risk of breast cancer among women who regularly use aspirin; a plausible mechanism is through aspirin effect on mammographic breast density, a breast cancer risk factor, possibly mediated through aspirin interference with estrogen synthesis., Methods: In a 2-arm randomized placebo-controlled clinical trial, we evaluated the effects of 6-month administration of 325 mg/day aspirin on total mammographic breast dense area and percent of the mammographic breast image occupied by dense areas (% density) in 143 postmenopausal women. Eligible women, recruited from 2005 to 2007, were healthy, not taking hormone therapy, with elevated mammographic breast density (American College of Radiology Breast Imaging Reporting and Data System density category 2, 3, or 4) within 6 months before enrollment., Results: Women were a mean (SD) 59.5 (5.5) years. Geometric mean baseline percent density was 17.6% (95% confidence interval, 14.8-20.9) in women randomized to aspirin and 19.2% (95% confidence interval, 16.3-22.7) in women randomized to placebo. Percent density decreased in women randomized to aspirin by an absolute 0.8% versus an absolute decrease of 1.2% in controls (P = 0.84). Total breast area and dense area decreased to a similar degree in women assigned to aspirin and in those assigned to placebo, with no statistically significant differences between trial arms., Conclusions: A single daily administration of adult-dose aspirin for 6 months had no effect on mammographic density in postmenopausal women. If aspirin affects breast cancer risk in postmenopausal women, it may do so through alternative pathways than mammographic breast density.

Introduction: Inflammatory status may be an important prognostic factor for breast cancer. Correlates of markers of inflammation in breast cancer survivors have not been thoroughly evaluated., Methods: Using data from, the Health, Eating, Activity, and Lifestyle (HEAL) Study (a population-based, multiethnic prospective cohort study of female breast cancer patients) we evaluated the associations between circulating markers of inflammation (C-reactive protein [CRP] and serum amyloid A [SAA], measured approximately 31 months after diagnosis) and several demographic, lifestyle, and clinical characteristics in 741 disease-free breast cancer survivors. Analysis of variance and regression methods were used for statistical analyses of log-transformed values of CRP and SAA., Results: After adjusting for age, BMI, ethnicity, and study site, higher concentrations of CRP were associated with increasing concentration of SAA (P-trend < 0.0001), increasing age (P-trend < 0.0001), increasing BMI (P-trend < 0.0001), increasing waist circumference (P-trend < 0.0001), positive history of heart failure (P = 0.0007), decreasing physical activity (P-trend = 0.005), Hispanic ethnicity (P = 0.05 vs. non-Hispanic white), and current smoking (P = 0.03 vs. never smoking). Vitamin E supplementation (P = 0.0005), tamoxifen use (P = 0.008), and radiation treatment (compared to no chemotherapy or radiation; P = 0.04) were associated with reduced CRP. Associations of CRP with clinical characteristics were not significant in the adjusted models. In a multivariate analysis, CRP showed significant associations with waist circumference, BMI, age, history of heart failure, tamoxifen use, and vitamin E supplementation (R (2) = 0.35). Similar, yet fewer, associations were observed for SAA (R (2) = 0.19)., Conclusions: This study highlights important correlates of inflammatory status in breast cancer patients. Our results are consistent with those from similar studies of healthy women.

Preservation of normal joint function in patients with haemophilia is a goal of modern therapy. Regular injections of anti-haemophilic factor concentrate reduce the risk of joint bleeding, the optimal regimen for which remains under investigation. The goals of the experiment described here are: (i) to assess the capacity of a murine model of severe haemophilic arthropathy to predict the likelihood of success of a test product to prevent joint bleeding and the complications that follow and (ii) to compare the effectiveness of recombinant human activated factor VII (rFVIIa) to recombinant human factor VIII (rFVIII) to prevent acute joint bleeding in the mouse model of haemarthrosis. Mice lacking expression of FVIII received a single intravenous injection of human rFVIII (280 U kg(-1)), rFVIIa (10 mg kg(-1)) or vehicle prior to blunt trauma injury to the knee joint. Mice receiving rFVIII and rFVIIa developed less injury-induced joint bleeding, swelling and loss of range of motion compared to mice pretreated with vehicle. Despite the reduction in clinical symptoms, synovial hyperplasia was evident in all groups after 7 days although less pronounced in mice receiving rFVIII and rFVIIa. The data under these experimental conditions demonstrate: (i) that this model can be used to evaluate novel therapies designed to prevent joint bleeding (prophylaxis) and (ii) both rFVIII and rFVIIa reduced acute haemarthrosis but did not completely prevent synovitis, the sequelae of blood induced joint injury.

Complement activation resulting in significant increases of C4a split product may be a marker of postexertional malaise in individuals with chronic fatigue syndrome (CFS). This study focused on identification of the transcriptional control that may contribute to the increased C4a in CFS subjects after exercise. We used quantitative reverse-transcription polymerase chain reaction to evaluate differential expression of genes in the classical and lectin pathways in peripheral blood mononuclear cells (PBMCs). Calibrated expression values were normalized to the internal reference gene peptidylpropyl isomerase B (PPIB), the external reference gene ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL), or the geometric mean (GM) of the genes ribosomal protein, large, P0 (RPLP0) and phosphoglycerate kinase 1 (PGK1). All nine genes tested, except mannose-binding lectin 2 (MBL2), were expressed in PBMCs. At 1 hour postexercise, C4, mannan-binding lectin serine protease 2 (MASP2) and ficolin 1 (FCN1) transcripts were detected at higher levels (> or = 2-fold) in at least 50% (4 of 8) of CFS subjects and were detected in 88% (7 of 8) CFS subjects when subjects with overexpression of either C4 or MASP2 were combined. Only an increase in the MASP2 transcript was statistically significant (PPIB, P = 0.001; GM, P = 0.047; rbcL, P = 0.045). This result may be due to the significant but transient downregulation of MASP2 in control subjects (PPIB, P = 0.023; rbcL, P = 0.027). By 6 hours postexercise, MASP2 expression was similar in both groups. In conclusion, lectin pathway responded to exercise differentially in CFS than in control subjects. MASP2 down-regulation may act as an antiinflammatory acute-phase response in healthy subjects, whereas its elevated level may account for increased C4a and inflammation-mediated postexertional malaise in CFS subjects.

Experimental studies and case reports suggest a multifunctional role of leptin in immune function. However, clinical studies of leptin in healthy individuals with a comprehensive assessment of immunity are lacking. This study investigated associations between serum leptin concentrations and multiple biomarkers of cellular immunity and inflammation among 114 healthy postmenopausal, overweight, or obese women. Leptin was measured by RIA. C-reactive protein (CRP) and serum amyloid A (SAA) were measured by nephelometry. Flow cytometry was used to measure natural killer (NK) cell cytotoxicity and to enumerate and phenotype lymphocyte subsets. T-lymphocyte proliferation was assessed in response to phytohemagluttinin, as well as to anti-CD3 antibodies by the flow cytometric cell division tracking method. Multiple linear regression analysis with adjustment for confounding factors and log transformation, where appropriate, was used. Serum leptin concentrations were positively associated with serum CRP, SAA, and interleukin 6 (IL6) (P<0.0001, P=0.01, and P=0.04 respectively), more strongly among women with a body mass index (BMI) <30 kg/m(2). The associations were attenuated after adjustment for measured body composition, yet remained significant for CRP and SAA. No statistically significant associations were observed between leptin and NK cytotoxicity, lymphocyte subpopulations, or T-lymphocyte proliferation. This study fills an important gap in knowledge about the relationship between leptin concentrations and immune function in healthy individuals. Findings support an association between serum leptin and the inflammatory proteins CRP and SAA, which appears to be mediated only partly by adipose tissue. Our study does not support a link between leptin and other immune parameters among overweight or obese, but otherwise healthy postmenopausal women, perhaps because such effects are only present at low or deficient leptin concentrations.

Background: Little is known about vitamin D status in breast cancer survivors. This issue is important because vitamin D influences pathways related to carcinogenesis., Objective: The objective of this report was to describe and understand vitamin D status in a breast cancer survivor cohort., Design: Data are from the Health, Eating, Activity, and Lifestyle study. With the use of a cross-sectional design, we examined serum concentrations of 25-hydroxyvitamin D [25(OH)D] in 790 breast cancer survivors from western Washington state, New Mexico, and Los Angeles County. Cancer treatment data were obtained from Surveillance, Epidemiology, and End Results registries and medical records. Fasting blood, anthropometry, and lifestyle habits were collected after diagnosis and treatment. We examined distributions of 25(OH)D by race-ethnicity, season, geography, and clinical characteristics. Multivariate regression tested associations between 25(OH)D and stage of disease., Results: Five hundred ninety-seven (75.6%) of the women had low serum 25(OH)D, suggesting vitamin D insufficiency or frank deficiency. The overall mean (+/-SD) was 24.8 +/- 10.4 ng/mL, but it was lower for African Americans (18.1 +/- 8.7 ng/mL) and Hispanics (22.1 +/- 9.2 ng/mL). Women with localized (n = 424) or regional (n = 182) breast cancer had lower serum 25(OH)D than did women with in situ disease (n = 184) (P = 0.05 and P = 0.03, respectively). Multivariate regression models controlled for age, body mass index (in kg/m(2)), race-ethnicity, geography, season, physical activity, diet, and cancer treatments showed that stage of disease independently predicted serum 25(OH)D (P = 0.02)., Conclusions: In these breast cancer survivors, the prevalence of vitamin D insufficiency was high. Clinicians might consider monitoring vitamin D status in breast cancer patients, together with appropriate treatments, if necessary.

Cross-sectional studies suggest that moderate physical activity is associated with enhanced resting immune function; however, few randomized controlled trials have investigated this link. We investigated the effect of 12-mo aerobic exercise, relative to stretching control, on in vitro immune function in a randomized, controlled trial of 115 postmenopausal, overweight, or obese sedentary women, aged 50-75 yr. The exercise goal was > or =45 min/day, 5 days/wk. Control women participated in 1 day/wk stretching classes. Immune markers (natural killer cell cytotoxicity, T-lymphocyte proliferation, immune cell counts and phenotypes, and serum immunoglobulins) were assessed at baseline, 3 mo, and 12 mo under strict blood-draw criteria. General estimation equations evaluated intervention effects at 3 and 12 mo, controlling for baseline. Of the 115 women who began the trial, blood samples were available from 109 at 3 mo (95%) and 108 at 12 mo (94%). From baseline to 12 mo, the exercise group participated in 87% of the prescribed physical activity minutes per week and increased maximal O(2) uptake by 13.8%; controls experienced no change in fitness. The main outcomes, natural killer cell cytotoxicity and T-lymphocyte proliferation, did not differ between groups at 3 and 12 mo. Secondary outcome and subgroup (e.g., stratification by baseline categories of body mass index, immune status, C-reactive protein, and age) analyses did not show any clear patterns of association. This 12-mo randomized, controlled trial showed no effect of aerobic exercise on in vitro immune function, despite excellent retention, high adherence, and demonstrable efficacy of the exercise intervention.

Purpose: The effect of exercise on androgens in middle-aged to older men is poorly understood, and it could have implications for several aspects of health. This analysis was conducted to examine the effects of long-term aerobic exercise on serum sex hormones in middle-aged to older men., Methods: One hundred two sedentary men, ages 40-75 yr, were randomly assigned to a 12-month exercise intervention or a control group (no change in activity). The combined facility- and home-based exercise program consisted of moderate/vigorous-intensity aerobic activity for 60 min.d(-1), 6 d.wk(-1). Serum concentrations of testosterone, free testosterone, dihydrotestosterone (DHT), 3alpha-androstanediol glucuronide (3alpha-Diol-G), estradiol, free estradiol, and sex hormone-binding globulin (SHBG) were measured at baseline, 3, and 12 months., Results: Exercisers trained a mean of 370 min.wk(-1) (102% of goal), with only two dropouts. Cardiopulmonary fitness (.VO(2max)) increased 10.8% in exercisers and decreased by 1.8% in controls (P < 0.001). DHT increased 14.5% in exercisers versus 1.7% in controls at 3 months (P = 0.04); at 12 months, it remained 8.6% above baseline in exercisers versus a 3.1% decrease in controls (P = 0.03). SHBG increased 14.3% in exercisers versus 5.7% in controls at 3 months (P = 0.04); at 12 months, it remained 8.9% above baseline in exercisers versus 4.0% in controls (P = 0.13). There were significant trends toward increasing DHT and SHBG, with greater increases in .VO(2max) at 3 and 12 months in exercisers. No statistically significant differences were observed for testosterone, free testosterone, 3alpha-Diol-G, estradiol, or free estradiol in exercisers versus controls., Conclusions: A year-long, moderate-intensity aerobic exercise program increased DHT and SHBG, but it had no effect on other androgens in middle-aged to older men.

Background: Population-based data on the rate and outcome of complications related to blood donation are sparse., Study Design and Methods: Data from a survey conducted in 2003 in Aarhus County, Denmark, were used to assess the overall rate of donor complications. Additional nationwide data on moderate and severe donor complications were obtained from the Danish Register of Complications Related to Blood Donation, with records of all moderate and severe donor complications in Denmark occurring during the period 1997-2003., Results: In the regional survey, we identified 340 complications of any type among 41 274 donations, corresponding to a rate of 824/100,000 donations [95% confidence interval (CI): 741-916]. All complications were either needle injuries or vasovagal reactions. In the nationwide register, a total of 752 moderate and severe complications were recorded among 2,575,264 donations, corresponding to a rate of 29/100,000 donations (95% CI: 27-31). The rates of complications leading to long-term morbidity or disablement (> 5% loss of working capacity) were 5/100,000 donations (95% CI: 4.2-5.9) and 2.3/100,000 donations (95% CI: 1.8-2.9), respectively., Conclusion: The risk of complications related to blood donation is low. However, attention towards donor complications is warranted, given the non-negligible rate of complications resulting in long-term morbidity and disablement.