Inhibition of Acid Sphingomyelinase Allows for Selective Targeting of CD4+ Conventional versus Foxp3+ Regulatory T Cells.

CD4+ Foxp3+ regulatory T cells (Tregs) depend on CD28 signaling for their survival and function, a receptor that has been previously shown to activate the acid sphingomyelinase (Asm)/ceramide system. In this article, we show that the basal and CD28-induced Asm activity is higher in Tregs than in conventional CD4+ T cells (Tconvs) of wild-type (wt) mice. In Asm-deficient (Smpd1-/-; Asm-/-) mice, as compared with wt mice, the frequency of Tregs among CD4+ T cells, turnover of the effector molecule CTLA-4, and their suppressive activity in vitro were increased. The biological significance of these findings was confirmed in our Treg-sensitive mouse model of measles virus (MV) CNS infection, in which we observed more infected neurons and less MV-specific CD8+ T cells in brains of Asm-/- mice compared with wt mice. In addition to genetic deficiency, treatment of wt mice with the Asm inhibitor amitriptyline recapitulated the phenotype of Asm-deficient mice because it also increased the frequency of Tregs among CD4+ T cells. Reduced absolute cell numbers of Tconvs after inhibitor treatment in vivo and extensive in vitro experiments revealed that Tregs are more resistant toward Asm inhibitor-induced cell death than Tconvs. Mechanistically, IL-2 was capable of providing crucial survival signals to the Tregs upon inhibitor treatment in vitro, shifting the Treg/Tconv ratio to the Treg side. Thus, our data indicate that Asm-inhibiting drugs should be further evaluated for the therapy of inflammatory and autoimmune disorders.

In vitro polyclonal activation of conventional T cells with a CD28 superagonist protects mice from acute graft versus host disease.

Upon transplantation of T cells from a CD28 superagonist (CD28-SA) treated donor into an irradiated allogeneic host, the CD28-SA-induced activation and expansion of Treg cells inhibits acute graft versus host disease (aGvHD), while not abrogating the desired graft versus tumor effect. Human peripheral blood CD4(+) T cells, however, harbor only very few Treg cells. Therefore, we studied whether polyclonal in vitro prestimulation of conventional, that is Treg -cell-depleted, CD4(+) T cells of C57BL/6 mice with CD28-SA-coated paramagnetic beads is sufficient to protect recipient BALB/c mice from aGvHD. CD28-SA prestimulation of conventional CD4(+) T cells efficiently protected BALB/c recipient mice from aGvHD and CD28-SA-stimulated CD4(+) and CD8(+) T cells were capable of mediating long-term protection from the BCL1 lymphoma. The recently completed successful phase I testing of the human CD28-SA TGN1412/TAB08 should greatly facilitate further development of this straightforward method into a novel immunotherapy for patients.

Equal Performance, Different Grade: Women's Performance in Discussion Perceived Worse Than Men's.

We investigate how men and women are evaluated in group discussions. In five studies (N = 761) using a variant of a Hidden Profile Task, we find that, when experimentally and/or statistically controlling for actual gender differences in behavior, the female performance in a group discussion is devalued in comparison to male performance. This was observed for fellow group members (Study 1) and outside observers (Studies 2-5), in both primarily student (Studies 1, 4, and 5) and mixed samples (Studies 2 and 3), for different measures of performance (perceived helpfulness of the contribution, for work-related competence), across different discussion formats (preformulated chat messages, open chat), and when controlling for the number of female group members (Study 5). In contrast to our hypothesis, we did not find a moderating effect of selection procedure in that women were devalued to a similar degree in both situations with a women's quota and without.

Efficacy and Safety of Roluperidone for the Treatment of Negative Symptoms of Schizophrenia.

BACKGROUND: This is a placebo-controlled multi-national trial of roluperidone, a compound with antagonist properties for 5-HT2A, sigma2, and α1A-adrenergic receptors, targeting negative symptoms in patients with schizophrenia. This trial follows a previous trial that demonstrated roluperidone superiority over placebo in a similar patient population. METHODS: Roluperidone 32 mg/day, roluperidone 64 mg/day, or placebo was administered for 12 weeks to 513 patients with schizophrenia with moderate to severe negative symptoms. The primary endpoint was the PANSS-derived Negative Symptom Factor Score (NSFS) and the key secondary endpoint was Personal and Social Performance scale (PSP) total score. RESULTS: NSFS scores were lower (improved) for roluperidone 64 mg compared to placebo and marginally missing statistical significance for the intent-to-treat (ITT) analysis data set (P ≤ .064), but reached nominal significance (P ≤ .044) for the modified-ITT (m-ITT) data set. Changes in PSP total score were statistically significantly better on roluperidone 64 mg compared to placebo for both ITT and m-ITT (P ≤ .021 and P ≤ .017, respectively). CONCLUSIONS: Results of this trial confirm the potential of roluperidone as a treatment of negative symptoms and improving everyday functioning in patients with schizophrenia. Study registration: Eudra-CT: 2017-003333-29; NCT03397134.

Fibroblast growth factor-2 deficiency causes defects in adult hippocampal neurogenesis, which are not rescued by exogenous fibroblast growth factor-2.

Neurogenesis within the adult brain is restricted to selected areas, one of which is the dentate gyrus (DG). Several growth factors have been reported to affect neurogenesis in the adult DG. However, a role of fibroblast growth factor-2 (FGF-2) in adult hippocampal neurogenesis has not been firmly established. We have analyzed neurogenesis in the DG using in vivo and in vitro approaches. FGF-2(-/-) mice revealed no alterations in the number of proliferating cells but a significant decrease in the numbers of newly generated neurons. Moreover, FGF-2 added to hippocampal slice cultures from FGF-2(-/-) mice was unable to rescue the phenotype. Although an increase in death of neurogenic cells in the FGF-2-deficient DG could not be specifically demonstrated, there was a massive increase in global cell death in FGF-2(-/-) hippocampal slice cultures compared with slices from wild-type mice. Cell death could not be prevented by addition of FGF-2. Neutralization of endogenous FGF-2 in hippocampal slices did not interfere with neurogenesis in a short-term paradigm. Together, our data suggest that FGF-2 is essentially required for maturation of new neurons in adult hippocampal neurogenesis but is likely to operate synergistically in combination with other mechanisms/growth factors.

Overcoming barriers to promotion for women and underrepresented in medicine faculty in academic emergency medicine.

Equity in the promotion of women and underrepresented minorities (URiM) is essential for the advancement of academic emergency medicine and the specialty as a whole. Forward-thinking healthcare organizations can best position themselves to optimally care for an increasingly diverse patient population and mentor trainees by championing increased diversity in senior faculty ranks, leadership, and governance roles. This article explores several potential solutions to addressing inequities that hinder the advancement of women and URiM faculty. It is intended to complement the recently approved American College of Emergency Physicians (ACEP) policy statement aimed at overcoming barriers to promotion of women and URiM faculty in academic emergency medicine. This policy statement was jointly released and supported by the Society for Academic Emergency Medicine (SAEM), American Academy of Emergency Medicine (AAEM), and the Association of Academic Chairs of Emergency Medicine (AACEM).

Zoonosis: Update on Existing and Emerging Vector-Borne Illnesses in the USA.

PURPOSE OF REVIEW: This review describes mosquito- and tick-borne diseases found in the Western Hemisphere. It focuses on emerging diseases and recent geographic shifts in the presence of disease vectors. RECENT FINDINGS: Mosquito and tick vectors have become more widespread as environmental conditions have become more favorable. Zika recently has emerged as a concern for fetal anomalies. West Nile Virus has become widespread. Lyme disease and other tick-borne diseases are more prevalent in areas previously inhospitable to these ticks. SUMMARY: Healthcare providers must consider the possibility of mosquito- and tick-borne diseases in broader geographic areas and council patients traveling to endemic areas on precautions against these diseases. Treatment for suspected cases of serious tick-borne illnesses should not be delayed pending culture results.

Pediatric hemoptysis with pulmonary hemorrhage and respiratory failure.

Hemoptysis is a rare complaint in the pediatric population. It is most commonly of infectious etiology and is rarely life threatening. However, there are rare life-threatening causes of pediatric hemoptysis, which should be included in the differential diagnoses of children presenting to the emergency department (ED) with this complaint. This study aims to present a case of pediatric hemoptysis, briefly discuss the differential diagnosis, and present a review of the causes, manifestations, and treatment for hemoptysis secondary to diffuse alveolar hemorrhage (DAH).

Effect of hip abduction and external rotation on femoral vein exposure for possible cannulation.

Femoral vein access is often required during resuscitation efforts and when other routes of intravenous access are difficult. This study evaluated by ultrasound the effect of abduction/external rotation of the hip on venous accessibility. This was a prospective repeated measurement study. The common femoral veins of 25 volunteers were scanned transversely inferior to the inguinal ligament with the leg straight and in external rotation/abduction. The diameter of the vein and percent accessible (not posterior to the femoral artery) were determined. Data were analyzed using repeated measures analysis of variance. The mean percentage of the femoral vein accessible with the leg in external rotation/abduction was greater than with the leg straight (82.6 +/- 20.3 vs. 70.4 +/- 26.3, respectively); p < 0.03. External rotation/abduction of the hip may improve the success rate of femoral vein cannulation by increasing the percentage of the femoral vein accessible.

Molecular cloning and functional expression of the murine noradrenaline transporter.

The cDNA of the murine noradrenaline transporter (mNAT) was cloned from the RNA of the placenta of a C57BL/6 mouse. The cloned mNAT differs from a previously published sequence in two amino acids within the C-terminal region. A cDNA obtained from an inbred mouse strain showed a further amino acid exchange (Ile(505)Val) within the fifth intracellular loop. The pharmacological properties of both, the wild-type mNAT and the variant (mNAT-I(505)V), were studied in human embryonic kidney HEK293 cells transfected with the corresponding cDNA. The kinetic constants for transport (K (m), V (max)) of [(3)H]noradrenaline ([(3)H]-NA) and binding (K (D), B (max)) of the selective NAT inhibitor [(3)H]nisoxetine were not different between the two isoforms; the mean kinetic constants amounted to about 4 microM and 120pmol/mg protein for K (m) and V (max) and 6nM and 18pmol/mg protein for K (D) and B (max), respectively. [(3)H]-NA transport by both isoforms showed the typical properties of an NAT because it was dependent on sodium and chloride and inhibited with almost identical K (i) values by various NAT substrates and inhibitors. The only significant pharmacological difference identified between the two mNAT isoforms was an about threefold higher affinity for cocaine of the very rare mNAT-I(505)V variant.

Pilot study to evaluate the accuracy of ultrasonography in confirming endotracheal tube placement.

STUDY OBJECTIVE: Visualization of the vocal cords and end-tidal capnography are the usual standards in confirming endotracheal tube placement. Vocal cord visualization is, however, not always possible, and capnography is not 100% reliable and requires ventilation of the lungs to confirm placement. The goal of this study is to determine the accuracy of ultrasonography for detecting endotracheal tube placement into the trachea and esophagus in real time. METHODS: This was a prospective, randomized, controlled study. Eligible patients were adults undergoing elective surgery requiring intubation. Exclusion criteria were a history of difficult intubation, abnormal airway anatomy, aspiration risk factors, and esophageal disease. Thirty-three patients were enrolled. After induction of anesthesia and neuromuscular blockade, the anesthesiologist placed the endotracheal tube in the trachea and esophagus in random order with direct laryngoscopy. During the intubations, a high-frequency, linear transducer was placed transversely on the neck at the suprasternal notch. Two emergency physicians, blinded to the order and performance of the intubations, independently recorded the location of the endotracheal tube according to the real-time ultrasonographic image. A 2-by-2 table was used to calculate sensitivity and specificity of the emergency physicians' ability to detect placement of the endotracheal tube. RESULTS: For each physician, the sensitivity for identifying the first intubation as tracheal was 100% (95% confidence interval [CI] 77% to 100%) with a specificity of 100% (95% CI 82% to 100%). One endotracheal tube was unintentionally placed twice in the esophagus, but both tube placements were identified as esophageal by the emergency physicians. CONCLUSION: In this pilot study, 2 emergency physicians experienced in ultrasonography accurately detected placement of endotracheal tubes during intubation with ultrasonography in select patients in the controlled environment of the operating room. Future studies should examine the use of ultrasonography to visualize endotracheal tube placement in real time by emergency physicians with less ultrasonographic training; use of the technique in the emergency department on a wider range of patients, including patients with difficult airways; and assessment of the utility of ultrasonography in confirmation of endotracheal tube position in already intubated patients.

Homologous versus heterologous interactions in the bicomponent staphylococcal gamma-haemolysin pore.

Staphylococcal gamma-haemolysin HlgA-HlgB forms a beta-barrel transmembrane pore in cells and in model membranes. The pore is formed by the oligomerization of two different proteins and a still debated number of monomers. To clarify the topology of the pore, we have mutated single residues - placed near the right and left interfaces of each monomer into cysteine. The mutants were labelled with fluorescent probes, forming a donor-acceptor pair for FRET (fluorescence resonance energy transfer). Heterologous couples (labelled on complementary left and right interfaces) displayed a marked FRET, suggesting extensive HlgA-HlgB or HlgB-HlgA contacts. Heterologous control couples (with both components labelled on the same side) showed absent or low FRET. We found the same result for the homologous couple formed by HlgA [i.e. HlgA-HlgA in the presence of wt (wild-type) HlgB]. The homologous HlgB couple (HlgB-HlgB labelled on left and right interfaces and in the presence of wt HlgA) displayed a transient, declining FRET, which may indicate fast formation of an intermediate that is consumed during pore formation. We conclude that bicomponent pores are assembled by alternating heterologous monomers.

Protection of Mice from Acute Graft-versus-Host Disease Requires CD28 Co-stimulation on Donor CD4+ Foxp3+ Regulatory T Cells.

Acute graft-versus-host disease (aGvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell plus T cell transplantation (allo-HSCT). In this study, we investigated the requirement for CD28 co-stimulation of donor CD4+ conventional (CD4+CD25-Foxp3-, Tconv) and regulatory (CD4+CD25+Foxp3+, Treg) T cells in aGvHD using tamoxifen-inducible CD28 knockout (iCD28KO) or wild-type (wt) littermates as donors of CD4+ Tconv and Treg. In the highly inflammatory C57BL/6 into BALB/c allo-HSCT transplantation model, CD28 depletion on donor CD4+ Tconv reduced clinical signs of aGvHD, but did not significantly prolong survival of the recipient mice. Selective depletion of CD28 on donor Treg did not abrogate protection of recipient mice from aGvHD until about day 20 after allo-HSCT. Later, however, the pool of CD28-depleted Treg drastically declined as compared to wt Treg. Consequently, only wt, but not CD28-deficient, Treg were able to continuously suppress aGvHD and induce long-term survival of the recipient mice. To our knowledge, this is the first study that specifically evaluates the impact of CD28 expression on donor Treg in aGvHD. Moreover, the delayed kinetics of aGvHD lethality after transplantation of iCD28KO Treg provides a novel animal model for similar disease courses found in patients after allo-HSCT.

Efficacy and Safety of MIN-101: A 12-Week Randomized, Double-Blind, Placebo-Controlled Trial of a New Drug in Development for the Treatment of Negative Symptoms in Schizophrenia.

OBJECTIVE: The authors assessed the efficacy, safety, and tolerability of MIN-101, a compound with affinities for sigma-2 and 5-HT2A receptors and no direct dopamine affinities, in comparison with placebo in treating negative symptoms in stabilized patients with schizophrenia. METHOD: The trial enrolled 244 patients who had been symptomatically stable for at least 3 months and had scores of at least 20 on the negative subscale of the Positive and Negative Syndrome Scale (PANSS). After at least 5 days' withdrawal from all antipsychotic medication, patients were randomly assigned to receive placebo or 32 mg/day or 64 mg/day of MIN-101 for 12 weeks. The primary outcome measure was the PANSS negative factor score (pentagonal structure model). Secondary outcome measures were PANSS total score and scores on the Clinical Global Impressions Scale (CGI), the Brief Negative Symptom Scale, the Brief Assessment of Cognition in Schizophrenia, and the Calgary Depression Scale for Schizophrenia. RESULTS: A statistically significant difference in PANSS negative factor score was observed, with lower scores for the MIN-101 32 mg/day and 64 mg/day groups compared with the placebo group (effect sizes, d=0.45 and d=0.57, respectively). Supporting these findings were similar effects on several of the secondary outcome measures, such as the PANSS negative symptom, total, and activation factor scores, the CGI severity item, and the Brief Negative Symptom Scale. There were no statistically significant differences in PANSS positive scale score between the MIN-101 and placebo groups. No clinically significant changes were observed in vital signs, routine laboratory values, weight, metabolic indices, and Abnormal Involuntary Movement Scale score. CONCLUSIONS: MIN-101 demonstrated statistically significant efficacy in reducing negative symptoms and good tolerability in stable schizophrenia patients.

Direct Binding of the pH-Regulated Protein 1 (Pra1) from Candida albicans Inhibits Cytokine Secretion by Mouse CD4+ T Cells.

Opportunistic infections with the saprophytic yeast Candida albicans are a major cause of morbidity in immunocompromised patients. While the interaction of cells and molecules of innate immunity with C. albicans has been studied to great depth, comparatively little is known about the modulation of adaptive immunity by C. albicans. In particular, direct interaction of proteins secreted by C. albicans with CD4+ T cells has not been studied in detail. In a first screening approach, we identified the pH-regulated antigen 1 (Pra1) as a molecule capable of directly binding to mouse CD4+ T cells in vitro. Binding of Pra1 to the T cell surface was enhanced by extracellular Zn2+ ions which Pra1 is known to scavenge from the host in order to supply the fungus with Zn2+. In vitro stimulation assays using highly purified mouse CD4+ T cells showed that Pra1 increased proliferation of CD4+ T cells in the presence of plate-bound anti-CD3 monoclonal antibody. In contrast, secretion of effector cytokines such as IFNγ and TNF by CD4+ T cells upon anti-CD3/ anti-CD28 mAb as well as cognate antigen stimulation was reduced in the presence of Pra1. By secreting Pra1 C. albicans, thus, directly modulates and partially controls CD4+ T cell responses as shown in our in vitro assays.

Assessing sleep architecture and continuity measures through the analysis of heart rate and wrist movement recordings in healthy subjects: comparison with results based on polysomnography.

OBJECTIVE: The objective of the study was to evaluate the reliability of a new methodology for assessing sleep architecture descriptors based on heart rate and body movement recordings. METHODS: Twelve healthy male and female subjects between 18 and 40 years of age, without sleep disorders and not taking any drug or medication that could affect sleep, were recorded continuously during five consecutive nights. Together with the standard polysomnography, heart rate was recorded with a Holter and wrist movements by actimetry. Of the 60 recorded nights, 48 artifact-free nights were analyzed by two independent and well-trained visual scorers according to the rules of the American Academy of Sleep Medicine. Sleep stages were assigned to every 30-s epoch. In parallel, the same nights were analyzed by the new methodology using only heart rate and actimetry data, allowing a 1-s epoch sleep stage classification. Sleep architecture was measured for 48 nights, independently for the two manual scorings and the automatic analysis. RESULTS: Over 42 nights, the intra-class correlation coefficient, used to assess the consistency or reproducibility of quantitative measurements made by different observers, was classified as excellent when all 12 descriptors were combined. Analyses of the individual descriptors showed excellent interclass correlation for eight and good for four of the 12. CONCLUSION: The automatic analysis of heart rate and body movement during sleep allows for the evaluation of sleep architecture and continuity that is equivalent to those obtained by manual scoring of polysomnography. The technique used here is simple and robust to allow for home sleep monitoring.

Hsp90 inhibition ameliorates CD4+ T cell-mediated acute Graft versus Host disease in mice.

INTRODUCTION: For many patients with leukemia only allogeneic bone marrow transplantion provides a chance of cure. Co-transplanted mature donor T cells mediate the desired Graft versus Tumor (GvT) effect required to destroy residual leukemic cells. The donor T cells very often, however, also attack healthy tissue of the patient inducing acute Graft versus Host Disease (aGvHD)-a potentially life-threatening complication. METHODS: Therefore, we used the well established C57BL/6 into BALB/c mouse aGvHD model to evaluate whether pharmacological inhibition of heat shock protein 90 (Hsp90) would protect the mice from aGvHD. RESULTS: Treatment of the BALB/c recipient mice from day 0 to +2 after allogeneic CD4+ T cell transplantation with the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) partially protected the mice from aGvHD. DMAG treatment was, however, insufficient to prolong overall survival of leukemia-bearing mice after transplantation of allogeneic CD4+ and CD8+ T cells. Ex vivo analyses and in vitro experiments revealed that DMAG primarily inhibits conventional CD4+ T cells with a relative resistance of CD4+ regulatory and CD8+ T cells toward Hsp90 inhibition. CONCLUSIONS: Our data, thus, suggest that Hsp90 inhibition might constitute a novel approach to reduce aGvHD in patients without abrogating the desired GvT effect.

The T cell-selective IL-2 mutant AIC284 mediates protection in a rat model of Multiple Sclerosis.

Targeting regulatory T cells (Treg cells) with interleukin-2 (IL-2) constitutes a novel therapeutic approach for autoimmunity. As anti-cancer therapy with IL-2 has revealed substantial toxicities a mutated human IL-2 molecule, termed AIC284 (formerly BAY 50-4798), has been developed to reduce these side effects. To assess whether AIC284 is efficacious in autoimmunity, we studied its therapeutic potential in an animal model for Multiple Sclerosis. Treatment of Lewis rats with AIC284 increased Treg cell numbers and protected the rats from Experimental Autoimmune Encephalomyelitis (EAE). AIC284 might, thus, also efficiently prevent progression of autoimmune diseases in humans.

Sonographic assessment of the epiglottis.

OBJECTIVES: The ideal diagnostic test for the diagnosis of epiglottitis would be simple, rapid, noninvasive, and highly accurate, performed at the bedside, and would not use ionizing radiation. The purpose of this study was to assess the utility of ultrasound to image the epiglottis and to determine the range of normal epiglottis diameter for men and women. METHODS: This was a prospective study of a convenience sample of 100 subjects between the ages of 18 and 50 years who had no known acute or chronic laryngeal diseases or surgeries. The anterior neck of each subject was scanned in both the long and short axis with a 5-10 MHz linear transducer. Sonographically, the epiglottis appeared as a curvilinear, hypoechoic structure with an echogenic pre-epiglottic space. The sonographic appearance of the epiglottis and the pre-epiglottic space were recorded and anteroposterior measurements of the epiglottis just distal to the hyoid bone were made. Comparisons between men and women were performed with use of a Student's t-test. Pearson's correlation analysis was performed to evaluate the relationship between subject height and epiglottic size. RESULTS: The epiglottis was visualized in all 100 subjects including 62 women and 38 men. The average patient age was 35.2 +/- 8.1 years. The epiglottic thickness was 2.39 +/- 0.15 mm. This was greater in men (2.49 +/- 0.13 mm) than in women (2.34 +/- 0.13 mm) (p < 0.001). There was moderate correlation between height and epiglottic thickness (R = 0.48). However, when this was analyzed separately for men and women, there was no significant correlation between epiglottic thickness and height. CONCLUSIONS: Bedside ultrasonography is easy to perform and can accurately evaluate the epiglottis. Further analysis should include patients with known epiglottic disease to assess the utility of this technique to detect pathologic enlargement.