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Background Blastomycosis, coccidioidomycosis, and histoplasmosis are environmentally acquired fungal diseases that clinically resemble bacterial and viral community-acquired pneumonia and require laboratory testing for diagnosis. Patients frequently present to primary care and experience diagnostic delays when a fungal etiology is not initially suspected. Current national-level public health surveillance for these diseases is limited and does not include laboratory data, so nationwide testing practices are unknown. Methods We identified laboratory tests for blastomycosis, coccidioidomycosis, and histoplasmosis ordered during 1 March 2019–29 February 2024 and performed within a major national commercial laboratory system. We analyzed test results, patient and healthcare provider features, reasons for testing, and temporal trends. Results Results included 5693 Blastomyces complement fixation tests (of those, 12% were positive), 71 858 immunodiffusion tests (0.1% positive), and 1186 serum enzyme immunoassay (EIA) tests (11% positive); 154 989 Coccidioides EIA immunoglobulin M results (5% positive) and 154 968 immunoglobulin G results (8% positive); and 46 346 Histoplasma complement fixation tests (30% positive), 49 062 immunodiffusion tests (1% positive), 35 506 serum EIA tests (4% positive), and 82 489 urine EIA tests (2% positive). Most histoplasmosis (58%–74%) and blastomycosis (42%–68%) tests were ordered from hospitals, whereas coccidioidomycosis tests were most frequently ordered by primary care providers (40%). A yearly average of 2727 positive tests were ordered by healthcare providers in states without public health surveillance for these diseases. Conclusions Blastomycosis, coccidioidomycosis, and histoplasmosis are likely underdetected in primary care settings or by public health surveillance. Increased testing by primary care providers and expanded surveillance are needed to reduce disease burden. [ABSTRACT FROM AUTHOR]

Ferritin, widely present in liver and spleen tissue, is considered as a serological biomarker for liver diseases and cancers. The detection of ferritin may be an important tool in health diagnosis. In this study, 14 non-immunized chicken spleens were utilized to construct a single-chain fragment (scFv) phage library. After 4 rounds of panning, 7 unique clones were obtained. The optimal clone was further screened and combined with NanoLuc luciferase (Nluc) as a dual functional immunoprobe to bioluminescent enzyme immunoassay (BLEIA), which was twice as sensitive as its parental scFv-based double-sandwich enzyme-linked immunoassay (ds-ELISA). The cross-reactivity analysis revealed that the proposed methods were highly selective and suitable for clinical detection. To further verify the performance of the immunoassays, serum samples were tested by the proposed methods and a commercial ELISA kit, and there was a good correlation between the results. These results suggested that scFv fused with Nluc might be a powerful dual functional tool for rapid, practically reliable, and highly sensitive ferritin detection.

To determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) residual levels in tilapia fish, chemiluminescent enzyme immunoassay (CLEIA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. At first, VH and VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against LMG, and then thoroughly by database-assisted sequence analysis. Finally, the productive VH and VL were assembled to an intact scFv sequence and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step CLEIA against LMG was developed with a half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of 1.3 and 0.04 ng/mL, respectively. The validation results of this novel competitive CLEIA was in line with those obtained by classical HPLC method for determination of total MG in spiked and field incurred samples.

The isolation of nanobodies (Nbs) from phage display libraries is an increasingly effective approach for the generation of new biorecognition elements, which can be used to develop immunoassays. In this study, highly specific Nbs against the Alternaria mycotoxin tenuazonic acid (TeA) were isolated from an immune nanobody phage display library using a stringent biopanning strategy. The obtained Nbs were characterized by classical enzyme-linked immunosorbent assay (ELISA), and the best one Nb-3F9 was fused with nanoluciferase to prepare an advanced bifunctional fusion named nanobody-nanoluciferase (Nb-Nluc). In order to improve the sensitivity and reduce the assay time, two different kinds of luminescent strategies including chemiluminescent enzyme immunoassay (CLEIA) and bioluminescent enzyme immunoassay (BLEIA) were established, respectively, on the basis of the single Nb and the fusion protein Nb-Nluc for TeA detection. The two-step CLEIA was developed on the basis of the same nanobody as ELISA, only with simple substrate replacement from 3,3',5,5'-tetramethylbenzidine (TMB) to luminol. In contrast with CLEIA, the novel BLEIA was conducted in one-step new strategy on the basis of Nb-Nluc and bioluminescent substrate coelenterazine-h (CTZ-h). Their half maximal inhibitory concentration (IC50) values were similar to 8.6 ng/mL for CLEIA and 9.3 ng/mL for BLEIA, which was a 6-fold improvement in sensitivity compared with that of ELISA (IC50 of 54.8 ng/mL). Both of the two assays provided satisfactory recoveries ranging from 80.1%-113.5% in real samples, which showed better selectivity for TeA analogues and other common mycotoxins. These results suggested that Nbs and Nb-Nluc could be used as useful reagents for immunodetection and that the developed CLEIA/BLEIA have great potential for TeA analysis.

Tetrabromobisphenol A (TBBPA) is a flame retardant and has become a widely concerning environmental pollutant. An ultrasensitive nanobody-based immunoassay was developed to monitor the exposure of TBBPA in sediment. First, the anti-TBBPA nanobody was fused with nanoluciferase, and then a one-step bioluminescent enzyme immunoassay (BLEIA) was developed with high sensitivity for TBBPA, with a maximum half inhibition concentration (IC50) at 187 pg/mL. Although approximately 10-fold higher sensitivity can be achieved by this developed BLEIA than by the classical two-step ELISA (IC50 at 1778 pg/mL), it is still a challenge to detect trace TBBPA in sediment samples reliably due to the relatively high matrix effect. To further improve the performance of this one-step BLEIA, a C4b-binding protein (C4BP) was inserted as a self-assembling linker between the nanobody and nanoluciferase. Therefore, a heptamer fusion containing seven binders and seven tracers was generated. This reagent improved the binding capacity and signal amplification. The one-step heptamer plus BLEIA based on this immune-reagent shows an additional 7-fold improvement of sensitivity, with the IC50 of 28.9 pg/mL and the limit of detection as low as 2.5 pg/mL. The proposed assay was further applied to determine the trace TBBPA in sediment, and the recovery was within 92-103%. Taking advantage of this heptamer fusion, one-step BLEIA can serve as a powerful tool for fast detection of trace TBBPA in the sediment samples.

BackgroundNo studies have evaluated the cost-effectiveness of single and two-step different diagnostic test strategies for Clostridioides difficile infection (CDI), including direct and indirect costs.ObjectiveTo evaluate the cost-effectiveness of commonly available diagnostic tests for CDI including nucleic acid amplification testing (NAAT) alone, glutamate dehydrogenase followed by enzyme immunoassay for toxin (GDH/EIA), GDH then NAAT (GDH/NAAT), and NAAT then EIA (NAAT/EIA).DesignDecision tree model from the US societal perspective with inputs derived from the literature. Willingness-to-pay threshold was set at $150,000 per quality-adjusted life year (QALY) gained. To assess the impact of uncertainty in model inputs on the findings, we performed one-way and probabilistic sensitivity analyses.ParticipantsWe conducted the analysis to represent a population aged 65 years old with diarrhea who received a CDI diagnostic test.Main measuresIncremental cost-effectiveness ratios (ICER) and incremental net monetary benefits (INMB).Key resultsNAAT alone was the most cost-effective approach overall; GDH/NAAT was the most cost-effective two-step option. NAAT alone led to the highest QALYs gained, at an incremental cost of $54,547 (vs. GDH/NAAT), $55,410 (vs. GDH/EIA), and $50,231 (vs. NAAT/EIA) per QALY gained. NAAT/EIA was not cost-effective compared to any other strategy. GDH/NAAT resulted in a higher QALY compared to GDH/EIA, at an incremental cost of $96,841 per QALY gained. Variability in the likelihood of comorbidities, CDI probability, and age at disease onset did not substantially change the results. One-way sensitivity analyses showed that results were most sensitive to likelihood of recurrence, followed by CDI mortality rate and probability of severe CDI. Probabilistic sensitivity analyses explored known uncertainties in the base case and confirmed the robustness of the results.ConclusionsNAAT alone and GDH/NAAT (among the two-step options) were the most cost-effective diagnostic test approaches for CDI.

A 2014 report evaluating accuracy of serologic testing for transfusion-transmissible viruses at African blood center laboratories found sensitivities of 92%, 87%, and 90% for detecting infections with human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV), respectively (1). Following substantial investments in national blood transfusion service (NBTS) laboratories, in 2017 investigators tested proficiency at 84 blood center laboratories (29 NBTS and 55 non-NBTS) in seven African countries. A blinded panel of 25 plasma samples was shipped to each participating laboratory for testing with their usual protocols based on rapid diagnostic tests (RDTs) (2) and third and fourth generation enzyme immunoassays (EIA-3 and EIA-4). Sensitivity and specificity were estimated using separate regression models that clustered assays by laboratory and adjusted for assay type and NBTS laboratory status. Mean specificities were ≥95% for all three viruses; however, mean sensitivities were 97% for HIV-positive, 76% for HBV-positive, and 80% for HCV-positive samples. Testing sensitivities for all viruses were high when EIA-3 assays were used (≥97%). Lower sensitivities for HBV-positive samples and HCV-positive samples were associated with assay types other than EIA-3, used primarily by non-NBTS laboratories. Proficiency for HIV testing has improved following international investments, but proficiency remains suboptimal for HBV and HCV testing. In sub-Saharan African blood centers, the quality of rapid tests used for HBV and HCV screening needs to be improved or their use discouraged in favor of EIA-3 tests.

Although dicamba has long been one of the most widely used selective herbicides, some U.S. states have banned the sale and use of dicamba because of farmers complaints of drift and damage to nonresistant crops. To prevent illegal use of dicamba and allow monitoring of nonresistant crops, a rapid and sensitive method for detection of dicamba is critical. In this paper, three novel dicamba haptens with an aldehyde group were synthesized, conjugated to the carrier protein via a reductive-amination procedure and an indirect competitive chemiluminescent enzyme immunoassay (CLEIA) for dicamba was developed. The assay showed an IC50 of 0.874 ng/mL which was over 15 times lower than that of the conventional enzyme immunoassay. The immunoassay was used to quantify dicamba concentrations in field samples of soil and soybean obtained from fields sprayed with dicamba. The developed CLEIA showed an excellent correlation with LC-MS analysis in spike-and-recovery studies, as well as in real samples. The recovery of dicamba ranged from 86 to 108% in plant samples and from 105 to 107% in soil samples. Thus, this assay is a rapid and simple analytical tool for detecting and quantifying dicamba levels in environmental samples and potentially a great tool for on-site crop and field monitoring.

Nanoluciferase (Nluc), the smallest luciferase known, was used as the fusion partner with a nanobody against aflatoxin B1 to develop a bioluminescent enzyme immunoassay (BLEIA) for detection of the aflatoxin B1 in cereal. Nanobody (clone G8) against aflatoxin B1 was fused with nanoluciferase and cloned into a pET22b expression vector, and then transformed into Escherichia coli. The nanobody fusion gene contained a hexahistidine tag for purification by immobilized metal affinity chromatography, yielding a biologically active fusion protein. The fusion protein G8-Nluc retained binding properties of the original nanobody. Concentration of the coelenterazine substrate and buffer composition were also optimized to provide high intensity and long half-life of the luminescent signal. The G8-Nluc was used as a detection antibody to establish a competitive bioluminescent ELISA for the detection of aflatoxin B1 in cereals successfully. Compared to classical ELISA, this novel assay showed more than 20-fold improvement in detection sensitivity, with an IC50 value of 0.41 ng/mL and linear range from 0.10 to 1.64 ng/mL. In addition, the entire BLEIA detection procedure can be completed in one step within 2 h, from sample preparation to data analysis. These results suggest that nanobody fragments fused with nanoluciferase might serve as useful and highly sensitive dual functional reagents for the development of rapid and highly sensitive immunoanalytical methods.

Chronic hepatitis B is a major cause of liver disease worldwide and is currently incurable. Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is a key form of the virus responsible for its persistence and is the transcriptional template for all viral transcripts. The field is focussed on methods to clear HBV cccDNA but this been limited by technical difficulties in its quantification due to: identical sequence to other forms of HBV DNA; low copy number per cell; and high resistance to denaturation by heat, leading to difficulty using polymerase chain reaction or hybridization methods for detection. A number of assays have been developed in order to overcome these hurdles either directly or detecting cccDNA levels indirectly via its transcriptional products. In this review, we summarize the approaches to cccDNA quantification that are currently used, and outline key open questions in the cccDNA biology field which remain to be answered due to the limitations of current methods.

3-Phenoxybenzoic acid (3-PBA) is a human urinary metabolite of many pyrethroid insecticides and can be used as a biomarker to monitor human exposure to these pesticides. A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for detecting 3-PBA on the basis of a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The anti-3-PBA Nb-AP fusion protein was expressed and purified. The 50% inhibitory concentration (IC50) and linear range of dc-FEIA were 0.082 and 0.015-0.447 ng/mL, respectively, with a detection limit of 0.011 ng/mL. The IC50 of dc-FEIA was improved by nearly ten times compared with those of one-step and three-step direct competitive enzyme-linked immunosorbent assay (dc-ELISA). Spiked urine samples were detected by both dc-FEIA and liquid chromatography-mass spectrometry (LC-MS), and the results showed good consistency between the two analysis methods, indicating the reliability of dc-FEIA based on the Nb-AP fusion protein for detecting 3-PBA in urine.

Cortisol, and other glucocorticoids, are routinely used as markers of physiological stress in wildlife. Typically, stress activates the hypothalamic-pituitary-adrenal (HPA) axis, with adrenocorticotropic hormone (ACTH) signaling the adrenal glands to release cortisol. Nevertheless, recent anecdotes in captive Coquerel's sifakas ( Propithecus coquereli ), strepsirrhine primates that are difficult to maintain under human care, may challenge the assumption that physiological stress universally increases circulating cortisol. Here, the authors ask if low circulating cortisol and minimal response to adrenal stimulation might be hallmarks of outwardly healthy sifakas in captivity. Comparative ACTH stimulation or control tests were performed in 10 Coquerel's sifakas and six ring-tailed lemurs ( Lemur catta ) at the Duke Lemur Center (DLC). At baseline, sifakas had average cortisol concentrations of just 0.67 µg/dl, whereas those of ring-tailed lemurs averaged 12.53 µg/dl. Stressful pre-experiment procedures, including kenneling and handling, activated the HPA axis in ring-tailed lemurs, masking further cortisol release from ACTH administration; however, neither these procedures nor exogenous ACTH raised cortisol concentrations in sifakas. Additionally, cortisol in dozens of serum samples from DLC sifakas banked over 17 yr was assayed. Across samples, cortisol concentrations averaged just 0.49 µg/dl and did not vary by animal sex, age, or housing condition. Comparable samples from two individual sifakas in sepsis at the end of life (4.28 and 21.88 µg/dl) indicate that the assay does capture meaningful variation in cortisol in captive sifakas, although robust biological validation is needed. Currently there is a lack of comparative data from wild Coquerel's sifakas that might determine if these unusual endocrine patterns are characteristic of the species or a function of captivity. If the latter, chronic stress in captivity could lead to a downregulated HPA axis, with persistent hypocortisolism perhaps contributing to the Coquerel's sifaka's susceptibility to infection under human management.

Background & objective: This study aims to assess the effect of cooling helmet during surgery on neuron specific enolase (NSE) and delirium levels following open heart surgery patients with cardiopulmonary bypass (CPB) using a heart-lung machine. Methodology: This double-blind randomized clinical trial took place from October to December 2021 in a tertiary care hospital. The research used a modified cooling helmet to decrease brain temperature. Inclusion criteria were adult patients who were scheduled for open heart surgery with CPB. The subjects were randomized into two groups: patients with the cooling helmet on as the treatment group (n = 12) and the patients with the non-cooling helmet on as the control group (n = 13). The differences between NSE and delirium levels in both groups were assessed at specific times. Results: NSE levels in the treatment group were lower than the control group (7.13 ± 7.63 vs. 12.49 ± 6.81; P < 0.05). Regarding the delirium, no statistically significant difference was found in both groups (P > 0.05). Conclusion: The hypothermia effect of the cooling helmet is associated with a decrease of neuron specific enolase levels, but it did not significantly correlate to prevent the delirium after open heart surgery patients with the cardiopulmonary bypass machine. [ABSTRACT FROM AUTHOR]

BackgroundAssays that detect HIV antigen (Ag) and antibody (Ab) can be used to screen for HIV infection.ObjectivesTo compare the performance of the BioPlex 2200 HIV Ag-Ab assay and two other Ag/Ab combination assays for detection of acute HIV infection.Study designSamples were obtained from 24 individuals (18 from the US, 6 from South Africa); these individuals were classified as having acute infection based on the following criteria: positive qualitative RNA assay; two negative rapid tests; negative discriminatory test. The samples were tested with the BioPlex assay, the ARCHITECT HIV Ag/Ab Combo test, the Bio-Rad GS HIV Combo Ag-Ab EIA test, and a viral load assay.ResultsTwelve (50.0%) of 24 samples had RNA detected only ( > 40 to 13,476 copies/mL). Ten (43.5%) samples had reactive results with all three Ag/Ab assays, one sample was reactive with the ARCHITECT and Bio-Rad assays, and one sample was reactive with the Bio-Rad and BioPlex assays. The 11 samples that were reactive with the BioPlex assay had viral loads from 83,010 to >750,000 copies/mL; 9/11 samples were classified as Ag positive/Ab negative by the BioPlex assay.ConclusionsDetection of acute HIV infection was similar for the BioPlex assay and two other Ag/Ab assays. All three tests were less sensitive than a qualitative RNA assay and only detected HIV Ag when the viral load was high. The BioPlex assay detected acute infection in about half of the cases, and identified most of those infections as Ag positive/Ab negative.

BackgroundCentral nervous system (CNS) histoplasmosis is a life-threatening condition and represents a diagnostic and therapeutic challenge. Isolation of Histoplasma capsulatum from cerebrospinal fluid (CSF) or brain tissue is diagnostic; however, culture is insensitive and slow growth may result in significant treatment delay. We performed a retrospective multicenter study to evaluate the sensitivity and specificity of a new anti-Histoplasma antibody enzyme immunoassay (EIA) for the detection of IgG and IgM antibody in the CSF for diagnosis of CNS histoplasmosis, the primary objective of the study. The secondary objective was to determine the effect of improvements in the Histoplasma galactomannan antigen detection EIA on the diagnosis of Histoplasma meningitis.MethodsResidual CSF specimens from patients with Histoplasma meningitis and controls were tested for Histoplasma antigen and anti-Histoplasma immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody using assays developed at MiraVista Diagnostics.ResultsA total of 50 cases and 157 controls were evaluated. Fifty percent of patients with CNS histoplasmosis were immunocompromised, 14% had other medical conditions, and 36% were healthy. Histoplasma antigen was detected in CSF in 78% of cases and the specificity was 97%. Anti-Histoplasma IgG or IgM antibody was detected in 82% of cases and the specificity was 93%. The sensitivity of detection of antibody by currently available serologic testing including immunodiffusion and complement fixation was 51% and the specificity was 96%. Testing for both CSF antigen and antibody by EIA was the most sensitive approach, detecting 98% of cases.ConclusionsTesting CSF for anti-Histoplasma IgG and IgM antibody complements antigen detection and improves the sensitivity for diagnosis of Histoplasma meningitis.

Brainstem encephalitis (BE) associated with Mycoplasma pneumoniae in adults is rare, and the diagnosis is challenging. We describe an uncommon case of BE in an immunocompetent patient. A 43-year-old, otherwise healthy woman visited our emergency department with high fever and a sore throat, and 3 days later she returned with an altered drowsy mental status. Magnetic resonance imaging displayed diffuse swelling in bilateral cerebral regions involving the bilateral pons. The sera tested positive for the immunoglobulin (Ig) M antibody against M. Pneumoniae as detected by an enzyme immunoassay (EIA), and on hospital day 10, the level of IgM index against M. pneumoniae further increased from 1.5 to 2.1. We changed the antibiotic regimen from vancomycin and ceftriaxone to clarithromycin based on detection of M. pneumoniae, and we added intravenous immunoglobulin. After one month, the patient fully recovered from the neurological deficits. A follow-up brain magnetic resonance imaging was performed, which showed completely resolved lesions. Particle agglutination assay (PA) and EIA are both largely used to diagnose M. pneumoniae. Compared to the PA test, the EIA test could be a reliable tool because it separately measures IgM and IgG antibodies. We diagnosed BE associated with M. pneumoniae through EIA with an increasing level of IgM in the acute and subacute paired sera. Early treatment with macrolide antibiotics resulted in a good outcome.

ObjectiveHundreds of scientific publications are produced annually that involve the measurement of cortisol in saliva. Intra- and inter-laboratory variation in salivary cortisol results has the potential to contribute to cross-study inconsistencies in findings, and the perception that salivary cortisol results are unreliable. This study rigorously estimates sources of measurement variability in the assay of salivary cortisol within and between established international academic-based laboratories that specialize in saliva analyses. One hundred young adults (Mean age: 23.10 years; 62 females) donated 2 mL of whole saliva by passive drool. Each sample was split into multiple- 100 µL aliquots and immediately frozen. One aliquot of each of the 100 participants' saliva was transported to academic laboratories (N = 9) in the United States, Canada, UK, and Germany and assayed for cortisol by the same commercially available immunoassay.Results1.76% of the variance in salivary cortisol levels was attributable to differences between duplicate assays of the same sample within laboratories, 7.93% of the variance was associated with differences between laboratories, and 90.31% to differences between samples. In established-qualified laboratories, measurement error of salivary cortisol is minimal, and inter-laboratory differences in measurement are unlikely to have a major influence on the determined values.

Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT. Instead, certain Provincial laboratories continue to screen samples using a chemical stain-based test (Sellers stain test, SST). This test has low diagnostic sensitivity, with negative SST-tested samples being forwarded to the nearest National laboratory resulting in significant delays for completion of testing and considerable additional costs. This study sought to develop a cost-effective and diagnostically-accurate immunoperoxidase antigen detection (RIAD) test for rabies that can be readily and quickly performed by the resource-constrained Provincial laboratories. This would reduce the burden on the National laboratories and allow more rapid diagnoses and implementation of post-exposure prophylaxis. The RIAD test was evaluated using brain smears fixed with acetone or formalin and its performance was validated by comparison with established rabies diagnostic tests used in Indonesia, including the SST and FAT. A proficiency testing panel was distributed between Provincial laboratories to assess the reproducibility of the test. The performance of the RIAD test was improved by using acetone fixation of brain smears rather than formalin fixation such that it was of equivalent accuracy to that of the World Organisation for Animal Health (OIE)-recommended FAT, with both tests returning median diagnostic sensitivity and specificity values of 0.989 and 0.993, respectively. The RIAD test and FAT had higher diagnostic sensitivity than the SST (median = 0.562). Proficiency testing using a panel of 6 coded samples distributed to 16 laboratories showed that the RIAD test had good reproducibility with an overall agreement of 97%. This study describes the successful development, characterisation and use of a novel RIAD test and its fitness for purpose as a screening test for use in provincial Indonesian veterinary laboratories.

BackgroundAbout half of patients with papillary thyroid cancer have tumours with activating BRAF(V600E) mutations. Vemurafenib, an oncogenic BRAF kinase inhibitor approved for BRAF-positive melanoma, showed clinical benefit in three patients with BRAF(V600E)-positive papillary thyroid cancer in a phase 1 trial. We aimed to establish the activity of vemurafenib in patients with BRAF(V600E)-positive papillary thyroid cancer.MethodsWe did an open-label, non-randomised, phase 2 trial at ten academic centres and hospitals worldwide in patients aged 18 years or older with histologically confirmed recurrent or metastatic papillary thyroid cancer refractory to radioactive iodine and positive for the BRAF(V600E) mutation. Participants either had never received a multikinase inhibitor targeting VEGFR (cohort 1) or had been treated previously with a VEGFR multikinase inhibitor (cohort 2). Patients received vemurafenib 960 mg orally twice daily. The primary endpoint was investigator-assessed best overall response in cohort 1 (confirmed on two assessments 4 weeks or longer apart). Analyses were planned to have a minimum median follow-up of 15 months (data cutoff April 18, 2014) and were done in safety, intention-to-treat, and per-protocol populations. This trial is closed and is registered at ClinicalTrials.gov, number NCT01286753.FindingsBetween June 23, 2011, and Jan 15, 2013, 51 patients were enrolled to the study, 26 in cohort 1 and 25 in cohort 2. Median duration of follow-up was 18·8 months (IQR 14·2-26·0) in cohort 1 and 12·0 months (6·7-20·3) in cohort 2. Partial responses were recorded in ten of 26 patients in cohort 1 (best overall response 38·5%, 95% CI 20·2-59·4). Grade 3 or 4 adverse events were recorded in 17 (65%) of 26 patients in cohort 1 and 17 (68%) of 25 patients in cohort 2; the most common grade 3 and 4 adverse events were squamous cell carcinoma of the skin (seven [27%] in cohort 1, five [20%] in cohort 2), lymphopenia (two [8%] in each cohort), and increased γ-glutamyltransferase (one [4%] in cohort 1, three [12%] in cohort 2). Two individuals in cohort 2 died due to adverse events, one from dyspnoea and one from multiorgan failure, but neither was treatment related. Serious adverse events were reported for 16 (62%) of 26 patients in cohort 1 and 17 (68%) of 25 patients in cohort 2.InterpretationVemurafenib showed antitumour activity in patients with progressive, BRAF(V600E)-positive papillary thyroid cancer refractory to radioactive iodine who had never been treated with a multikinase inhibitor. As such, this agent represents a potential new treatment option for these patients.FundingF Hoffmann-La Roche.

A critical function for skin is that when damaged it must simultaneously identify the nature of the injury, repair barrier function, and limit the intrusion of pathogenic organisms. These needs are carried out through the detection of damage-associated molecular patterns (DAMPs) and a response that includes secretion of cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). In this study, we analyzed how non-coding double-stranded RNA (dsRNAs) act as a DAMP in the skin and how the human cathelicidin AMP LL-37 might influence growth factor production in response to this DAMP. dsRNA alone significantly increased the expression of multiple growth factors in keratinocytes, endothelial cells, and fibroblasts. Furthermore, RNA sequencing transcriptome analysis found that multiple growth factors increase when cells are exposed to both LL-37 and dsRNA, a condition that mimics normal wounding. Quantitative PCR and/or ELISA validated that growth factors expressed by keratinocytes in these conditions included, but were not limited to, basic fibroblast growth factor (FGF2), heparin-binding EGF-like growth factor (HBEGF), vascular endothelial growth factor C (VEGFC), betacellulin (BTC), EGF, epiregulin (EREG), and other members of the transforming growth factor β superfamily. These results identify a novel role for DAMPs and AMPs in the stimulation of repair and highlight the complex interactions involved in the wound environment.

Human pluripotent stem cells (hPSCs) are sensitive to DNA damage and undergo rapid apoptosis compared to their differentiated progeny cells. Here, we explore the underlying mechanisms for the increased apoptotic sensitivity of hPSCs that helps to determine pluripotent stem cell fate. Apoptosis was induced by exposure to actinomycin D, etoposide, or tunicamycin, with each agent triggering a distinct apoptotic pathway. We show that hPSCs are more sensitive to all three types of apoptosis induction than are lineage-non-specific, retinoic-acid-differentiated hPSCs. Also, Bax activation and pro-apoptotic mitochondrial intermembrane space protein release, which are required to initiate the mitochondria-mediated apoptosis pathway, are more rapid in hPSCs than in retinoic-acid-differentiated hPSCs. Surprisingly, Bak and not Bax is essential for actinomycin-D-induced apoptosis in human embryonic stem cells. Finally, P53 is degraded rapidly in an ubiquitin-proteasome-dependent pathway in hPSCs at steady state but quickly accumulates and induces apoptosis when Mdm2 function is impaired. Rapid degradation of P53 ensures the survival of healthy hPSCs but avails these cells for immediate apoptosis upon cellular damage by P53 stabilization. Altogether, we provide an underlying, interconnected molecular mechanism that primes hPSCs for quick clearance by apoptosis to eliminate hPSCs with unrepaired genome alterations and preserves organismal genomic integrity during the early critical stages of human embryonic development.

BackgroundThe colony stimulating factor 1 receptor (CSF1R) ligands, CSF1 and interleukin-34, and the KIT ligand, stem cell factor, are expressed in glioblastoma (GB). Microglia, macrophages, blood vessels, and tumor cells also express CSF1R, and depletion of the microglia reduces tumor burden and invasive capacity. PLX3397 is an oral, small molecule that selectively inhibits CSF1R and KIT, penetrates the blood-brain barrier in model systems, and represents a novel approach for clinical development.MethodsWe conducted a phase II study in patients with recurrent GB. The primary endpoint was 6-month progression-free survival (PFS6). Secondary endpoints included overall survival response rate, safety, and plasma/tumor tissue pharmacokinetics. Exploratory endpoints included pharmacodynamic measures of drug effect in blood and tumor tissue.ResultsA total of 37 patients were enrolled, with 13 treated prior to a planned surgical resection (Cohort 1) and 24 treated without surgery (Cohort 2). PLX3397 was given at an oral dose of 1000 mg daily and was well tolerated. The primary efficacy endpoint of PFS6 was only 8.6%, with no objective responses. Pharmacokinetic endpoints revealed a median maximal concentration (Cmax) of 8090 ng/mL, with a time to attain Cmax of 2 hour in plasma. Tumor tissue obtained after 7 days of drug exposure revealed a median drug level of 5500 ng/g. Pharmacodynamic changes included an increase in colony stimulating factor 1 and reduced CD14(dim)/CD16+ monocytes in plasma compared with pretreatment baseline values.ConclusionPLX3397 was well tolerated and readily crossed the blood-tumor barrier but showed no efficacy. Additional studies are ongoing, testing combination strategies and potential biomarkers to identify patients with greater likelihood of response.

Background/Aims In serological tests for Helicobacter pylori (H. pylori), an enzyme-linked immunosorbent assay (GENEDIA®) and a solid-phase, two-step chemiluminescent enzyme immunoassay (IMMULITE®), which are easy to perform, inexpensive, and widely available, are commonly used. However, local validation of the test performance of IMMULITE® is required. This study aimed to examine the performance of IMMULITE® in comparison with that of GENEDIA® in a Korean health checkup population. Materials and Methods The sera of 300 subjects among those who underwent health checkup were analyzed using IMMULITE®, and results were compared with those of GENEDIA®. The two serological tests were compared for their ability to predict atrophic gastritis (AG) or intestinal metaplasia (IM) on endoscopy. Results We found significant correlation (Pearson correlation coefficient=0.903, P

Smoking is a major risk factor for developing pancreatic adenocarcinoma (PDAC); however, little is known about the mechanisms involved. Here we employed a genetic animal model of early stages of PDAC that overexpresses oncogenic Kras in the pancreas to investigate the mechanisms of smoking-induced promotion of the disease in vivo. We confirmed the regulation of the interactions between the tumor microenvironment cells using in vitro cellular systems. Aerial exposure to cigarette smoke stimulated development of pancreatic intraepithelial neaoplasia (PanIN) lesions associated with a tumor microenvironment-containing features of human PDAC including fibrosis, activated stellate cells, M2-macrophages and markers of epithelial-mesenchymal transition (EMT). The pro-cancer effects of smoking were prevented by Histone Deacetylase HDAC I/II inhibitor Saha. Smoking decreased histone acetylation associated with recruitment of and phenotypic changes in macrophages; which in turn, stimulated survival and induction of EMT of the pre-cancer and cancer cells. The interaction between the cancer cells and macrophages is mediated by IL-6 produced under the regulation of HDAC3 translocation to the nucleus in the cancer cells. Pharmacological and molecular inhibitions of HDAC3 decreased IL-6 levels in cancer cells. IL-6 stimulated the macrophage phenotype change through regulation of the IL-4 receptor level of the macrophage. This study demonstrates a novel pathway of interaction between cancer cells and tumor promoting macrophages involving HDAC3 and IL-6. It further demonstrates that targeting HDAC3 prevents progression of the disease and could provide a strategy for treating the disease considering that the HDAC inhibitor we used is FDA approved for a different disease.

Parkinson's disease (PD) is one of the most common movement disorders, and currently there is no effective treatment that can slow disease progression. Preserving and enhancing DA neuron survival is increasingly regarded as the most promising therapeutic strategy for treating PD. IRX4204 is a second generation retinoid X receptor (RXR) agonist that has no cross reactivity with retinoic acid receptors, farnesoid X receptor, liver X receptors or peroxisome proliferator-activated receptor PPARγ. We found that IRX4204 promotes the survival and maintenance of nigral dopaminergic (DA) neurons in a dose-dependent manner in primary mesencephalic cultures. Brain bioavailability studies demonstrate that IRX4204 can cross the blood brain barrier and reach the brain at nM concentration. Oral administration of IRX4204 can activate nuclear receptor Nurr1 downstream signaling in the substantia nigra (SN) andattenuate neurochemical and motor deficits in a rat model of PD. Our study suggests that IRX4204 represents a novel, potent and selective pharmacological means to activate cellular RXR-Nurr1 signaling and promote SN DA neuron survival in PD prevention and/or treatment.