Supplementing culture medium with the weak acid, 5,5-dimethyl-2,4-oxazolidinedione (DMO) limits the development of aneuploid mouse embryos through a Trp53-dependent mechanism.

PURPOSE: This study was designed to determine if DMO limits in vitro development of aneuploid-enriched mouse embryos by activating a Trp53-dependent mechanism. METHODS: Mouse cleavage-stage embryos were treated with reversine to induce aneuploidy or vehicle to generate controls, and then cultured in media supplemented with DMO to reduce the pH of the culture media. Embryo morphology was assessed by phase microscopy. Cell number, mitotic figures, and apoptotic bodies were revealed by staining fixed embryos with DAPI. mRNA levels of Trp53, Oct-4, and Cdx2 were monitored by quantitative polymerase chain reactions (qPCRs). The effect of Trp53 on the expression of Oct-4 and Cdx2 was assessed by depleting Trp53 using Trp53 siRNA. RESULTS: Aneuploid-enriched late-stage blastocysts were morphologically indistinguishable from control blastocysts but had fewer cells and reduced mRNA levels of Oct-4 and Cdx2. Adding 1 mM DMO to the culture media during the 8-cell to blastocyst transition reduced the formation of aneuploid-enriched late-stage blastocysts but not control blastocysts and further suppressed the levels of Oct-4 and Cdx2 mRNA. Trp53 RNA levels in aneuploid-enriched embryos that were exposed to DMO were > twofold higher than controls, and Trp53 siRNA levels reduced the levels of Trp53 and increased levels of Oct-4 and Cdx2 mRNA by > twofold. CONCLUSION: These studies suggest that the development of morphologically normal aneuploid-enriched mouse blastocysts can be inhibited by adding low amounts of DMO to the culture media, which results in elevated levels of Trp53 mRNA that suppresses Oct-4 and Cdx2 expression.

Systemic Lupus Erythematosus: Diagnosis and Treatment.

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects the cardiovascular, gastrointestinal, hematologic, integumentary, musculoskeletal, neuropsychiatric, pulmonary, renal, and reproductive systems. It is a chronic disease and may cause recurrent flare-ups without adequate treatment. The newest clinical criteria proposed by the European League Against Rheumatism/American College of Rheumatology in 2019 include an obligatory entry criterion of a positive antinuclear antibody titer of 1: 80 or greater. Management of SLE is directed at complete remission or low disease activity, minimizing the use of glucocorticoids, preventing flare-ups, and improving quality of life. Hydroxychloroquine is recommended for all patients with SLE to prevent flare-ups, organ damage, and thrombosis and increase long-term survival. Pregnant patients with SLE have an increased risk of spontaneous abortions, stillbirths, preeclampsia, and fetal growth restriction. Preconception counseling regarding risks, planning the timing of pregnancy, and a multidisciplinary approach play a major role in the management of SLE in patients contemplating pregnancy. All patients with SLE should receive ongoing education, counseling, and support. Those with mild SLE can be monitored by a primary care physician in conjunction with rheumatology. Patients with increased disease activity, complications, or adverse effects from treatment should be managed by a rheumatologist.

Social Media Reflection Assignment: a Simple Classroom Intervention To Help Students Examine Scientific Claims in Social Media.

Social media has the power to spread information faster than any other news source. The science community has experienced this firsthand during recent years, unfortunately to its detriment. When scientific and medical claims are made without responsible examination of scientific evidence, misinformation is allowed to spread. While all users are likely faced with misleading claims on social media, this is especially troublesome for young adults. As the most prevalent users, many in this age group have never known a time without social media. Educators have an opportunity to use social media as a real-world application to teach students how to critically analyze scientific and medical information. The Social Media Reflection Assignment (SMRA) was created to help students develop such scientific literacy skills. This intervention requires students to find social media posts that make scientific claims, citing published scientific data. Students locate the corresponding research article and describe the results in their own words. Finally, a comparison is drawn between scientific findings in the research article and the interpretation described in the social media post. Students are taught to judge whether social media claims are supported by the scientific evidence. This activity is adaptable and applicable in a variety of classroom settings, from upper-level majors courses to science courses for nonmajors to disciplines outside the sciences. Importantly, the SMRA helps students question claims in social media while training them to find and elucidate answers from reliable resources.

Neural-Cell-Intrinsic NF-κB Signaling Enhances Reovirus Virulence.

Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germ line ablation of NF-κB subunit p50. It is not known whether the proapoptotic function of NF-κB is mediated by neural-cell-intrinsic (neural-intrinsic) processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neural cells using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural cell NF-κB p65 (Nsp65-/-) survived. While viral loads in brains of WT and Nsp65-/- mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brains of WT mice displayed increased immunoreactivity for cleaved caspase-3, a marker of apoptosis, relative to Nsp65-/- mice. These data suggest that neural-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected brain cortices of WT and Nsp65-/- mice suggests that NF-κB activation in neuronal cells upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations. IMPORTANCE Viral encephalitis contributes to illness and death in children and adults worldwide and has limited treatment options. Identifying common host factors upregulated by neurotropic viruses can enhance an understanding of virus-induced neuropathogenesis and aid in development of therapeutics. Although many neurotropic viruses activate NF-κB during infection, mechanisms by which NF-κB regulates viral neuropathogenesis and contributes to viral encephalitis are not well understood. We established mice in which NF-κB expression is ablated in neural tissue to study the function of NF-κB in reovirus neurovirulence and identify genes activated by NF-κB in response to reovirus infection in the central nervous system. Encephalitis following reovirus infection was dampened in mice lacking neural cell NF-κB. Reovirus induced a chemokine profile in the brain that was dependent on NF-κB signaling and was similar to chemokine profiles elicited by other neurotropic viruses. These data suggest common underlying mechanisms of encephalitis caused by neurotropic viruses and potentially shared therapeutic targets.

Health disparities in preterm births.

Introduction: Black African American (B/AA) women have a 2-fold to 3-fold elevated risk compared with non-Hispanic White (W) women for preterm birth. Further, preterm birth is the leading cause of mortality among B/AA infants, and among survivors, preterm infant adverse health outcomes occur disproportionately in B/AA infants. Racial inequities in maternal and infant health continue to pose a public health crisis despite the discovery >100 years ago. The purpose of this study was to expand on reported preterm infant outcome disparities. A life-course approach, accumulation of lifelong stress, including discrimination, may explain social factors causing preterm birth rate and outcome inequities in B/AA mothers. Methods: Anthropometric measures and clinical treatment information for 197 consented participants were milled from electronic health records across 4 years. The Neonatal Infant Stressor Scale was used to tally acute and chronic painful/stressful procedures. Neurobehavioral differences were investigated using the Neonatal Intensive Care Unit (NICU) Network Neurobehavioral Scale. Results: B/AA mothers gave birth to preterm infants earlier than W mothers. NICU hospitalization stays were extended more than 2 weeks for the significantly smaller B/AA preterm infants in comparison to the age-matched W preterm infants. A higher number of chronic lifesaving procedures with demonstrated altered stress response patterns were recorded for B/AA preterm infants. Discussion: This cross-sectional analysis of preterm birth rates and preterm infant developmental and neurodevelopmental outcomes are presented in the context of NICU stress and pain, with attendant implications for infant mortality and future health disparities. Preterm birth rate and outcome inequities further support the need to develop interventions and policies that will reduce the impact of discrimination and improve social determinants of health for Black, Indigenous, and other People of Color.

Evidence-based guideline recommendations on multiparametric magnetic resonance imaging in the diagnosis of clinically significant prostate cancer: A Cancer Care Ontario updated clinical practice guideline.

INTRODUCTION: This clinical practice guideline is based on a systematic review to assess the use of multiparametric magnetic resonance imaging (mpMRI) in the diagnosis of clinically significant prostate cancer (csPCa) for biopsy-naive men and men with a prior negative transrectal ultrasound-guided systematic biopsy (TRUS-SB) at elevated risk. METHODS: The methods of the clinical practice guideline included searches to September of 2020 of MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials. Internal and external reviews were conducted. RESULTS: The recommendations are:Recommendation 1: For biopsy-naive patients at elevated risk of csPCa, mpMRI is recommended prior to biopsy in patients who are candidates for curative management with suspected clinically localized prostate cancer.- If the mpMRI is positive, mpMRI-targeted biopsy (TB) and TRUS-SB should be performed together to maximize detection of csPCa.- If the mpMRI is negative, consider forgoing any biopsy after discussion of the risks and benefits with the patient as part of shared decision-making and ongoing followup.Recommendation 2: In patients who had a prior negative TRUS-SB and demonstrate a high risk of having csPCa in whom curative management is being considered:- mpMRI should be performed.- If the mpMRI is positive, targeted biopsy should be performed. Concomitant TRUS-SB can be considered depending on the patient's risk profile and time since prior TRUS-SB biopsy.- If the mpMRI is negative, consider forgoing a TRUS-SB only after discussion of the risks and benefits with the patient as part of shared decision-making and ongoing followup.Recommendation 3: mpMRI should be performed and interpreted in compliance with the current Prostate Imaging Reporting & Data System (PI-RADS) guidelines.

Enteric viruses evoke broad host immune responses resembling those elicited by the bacterial microbiome.

The contributions of the viral component of the microbiome-the virome-to the development of innate and adaptive immunity are largely unknown. Here, we systematically defined the host response in mice to a panel of eukaryotic enteric viruses representing six different families. Infections with most of these viruses were asymptomatic in the mice, the magnitude and duration of which was dependent on the microbiota. Flow cytometric and transcriptional profiling of mice mono-associated with these viruses unveiled general adaptations by the host, such as lymphocyte differentiation and IL-22 signatures in the intestine, as well as numerous viral-strain-specific responses that persisted. Comparison with a dataset derived from analogous bacterial mono-association in mice identified bacterial species that evoke an immune response comparable with the viruses we examined. These results expand an understanding of the immune space occupied by the enteric virome and underscore the importance of viral exposure events.

The Addition of Lipid-Based Contrast Medium does not Inactivate the Detergent Sclerosant Sodium Tetradecyl Sulfate in-vitro.

PURPOSE: To investigate if the detergent sclerosant sodium tetradecyl sulfate (STS) is deactivated by the lipid-based contrast agent ethiodised oil. METHOD: 3% STS was mixed with ethiodised oil and room air in a 2:1:4 ratio in two luer lock syringes and a three way connector and agitated to make foam (the Tessari technique) to replicate the clinical use of the products. The assay of STS in the mixture was assessed using the British Pharmacopoeia method. Briefly this is a manual titration method where the solution of STS is mixed with an indicator solution and titrated with hyamine solution of known concentration; the concentration of the STS can then be calculated with the titration results. To further mimic the clinical environment with the presence of blood, the effect of adding increasing amounts of albumin to the STS-ethiodised oil mixture was assessed. RESULTS: The assay of STS in the solution after mixing with ethiodised oil was 3% indicating that the ethiodised oil did not deactivate the STS. The addition of albumin to the STS-contrast mixture resulted in near linear neutralisation of the STS with increasing concentrations in the same quantities as with STS alone. CONCLUSIONS: The mixture of the lipid-based contrast agent ethiodised oil with the detergent sclerosant STS did not affect the availability of the sclerosant. The continued use of STS-ethiodised oil in the management of vascular malformations can be supported.

Murine Norovirus Infection Induces TH1 Inflammatory Responses to Dietary Antigens.

Intestinal reovirus infection can trigger T helper 1 (TH1) immunity to dietary antigen, raising the question of whether other viruses can have a similar impact. Here we show that the acute CW3 strain of murine norovirus, but not the persistent CR6 strain, induces TH1 immunity to dietary antigen. This property of CW3 is dependent on its major capsid protein, a virulence determinant. Transcriptional profiling of mesenteric lymph nodes following infection reveals an immunopathological signature that does not segregate with protective immunity but with loss of oral tolerance, in which interferon regulatory factor 1 is critical. These data show that viral capacity to trigger specific inflammatory pathways at sites where T cell responses to dietary antigens take place interferes with the development of tolerance to an oral antigen. Collectively, these data provide a foundation for the development of therapeutic strategies to prevent TH1-mediated complex immune disorders triggered by viral infections.

An updated genetic map of Peromyscus with chromosomal assignment of linkage groups.

Species across the rodent genus Peromyscus have become prominent models for studying diverse mechanistic and evolutionary processes, including chromosome evolution, infectious disease transmission and human health, ecological adaptation, coat color variation, and parental care. Supporting such diverse research programs has been the development of genetic and genomic resources for species within this genus, including genome data, interspecific chromosome homologies, and a recently developed genetic map. Based on interspecific hybrids between the deer mouse (Peromyscus maniculatus bairdii) and the old-field, or beach, mouse (Peromyscus polionotus) and backcross progeny to Peromyscus maniculatus, a linkage map was developed based on 190 genes and 141 microsatellite loci. However, resolution of several linkage groups with respect to chromosome assignment was lacking and four chromosomes (8, 16, 20, and 21) were not clearly delineated with linkage data alone. The recent development of a high-density map for Peromyscus proved ineffective in resolving chromosome linkage for these four chromosomes. Herein we present an updated linkage map for Peromyscus maniculatus, including linkage group-chromosome assignments, using fluorescence in situ hybridization mapping of BACs and whole chromosome paints. We resolve the previously conflicting chromosome assignment of linkage groups to Chromosomes 8, 16, 20, and 21, and confirm the assignment of linkage groups to Chromosomes 18 and 22. This updated linkage map with validated chromosome assignment provides a solid foundation for chromosome nomenclature for this species.

Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection.

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease.IMPORTANCE Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Infection with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this virus in the development of celiac disease, an autoimmune intestinal disorder triggered by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal infection. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease.

Age-dependent susceptibility to reovirus encephalitis in mice is influenced by maturation of the type-I interferon response.

BackgroundInfants and young children are particularly susceptible to viral encephalitis; however, the mechanisms are unknown. We determined the age-dependent contribution of innate and adaptive immune functions to reovirus-induced encephalitis in mice.MethodsNewborn wild-type mice, 2-20 days of age, were inoculated with reovirus or diluent and monitored for mortality, weight gain, and viral load. Four- and fifteen-day-old IFNAR-/- and RAG2-/- mice were inoculated with reovirus and similarly monitored.ResultsWeight gain was impaired in mice inoculated with reovirus at 8 days of age or less. Clinical signs of encephalitis were detected in mice inoculated at 10 days of age or less. Mortality decreased when mice were inoculated after 6 days of age. Survival was ≤15% in wild type (WT), RAG2-/-, and IFNAR-/- mice inoculated at 4 days of age. All WT mice, 92% of RAG2-/- mice, and only 48% of IFNAR-/- mice survived following inoculation at 15 days of age.ConclusionsSusceptibility of mice to reovirus-induced disease decreases between 6 and 8 days of age. Enhanced reovirus virulence in IFNAR-/- mice relative to WT and RAG2-/- mice inoculated at 15 days of age suggests that maturation of the type-I interferon response contributes to age-related mortality following reovirus infection.

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease.

Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

Self-management education interventions for patients with cancer: a systematic review.

PURPOSE: This systematic review was intended to identify the effectiveness and inclusion of essential components of self-management education interventions to support patients with cancer in developing the skills needed for effective self-management of their disease and the acute or immediate, long-term, and late harmful effects of treatments. METHODS: Self-management education interventions were included if they were randomized controlled trials (RCTs) containing at least one of the eight core elements outlined by the research team. A systematic search was conducted in Ovid MEDLINE (2005 through April 2015), Embase (2005 to 2015, week 15), the Cochrane Database of Systematic Reviews (Issue 4, April 2015), CINAHL (2005 to 2015) and PsychINFO (2005 to 2015). Keywords searched include 'self-management patient education' or 'patient education'. RESULTS: Forty-two RCTs examining self-management education interventions for patients with cancer were identified. Heterogeneity of interventions precluded meta-analysis, but narrative qualitative synthesis suggested that self-management education interventions improve symptoms of fatigue, pain, depression, anxiety, emotional distress and quality of life. Results for specific combinations of core elements were inconclusive. Very few studies used the same combinations of core elements, and among those that did, results were conflicting. Thus, conclusions as to the components or elements of self-management education interventions associated with the strength of the effects could not be assessed by this review. CONCLUSION: Defining the core components of cancer self-management education and the fundamental elements for inclusion in supporting effective self-management will be critical to ensure consistent and effective provision of self-management support in the cancer system.

Expression of Ifnlr1 on Intestinal Epithelial Cells Is Critical to the Antiviral Effects of Interferon Lambda against Norovirus and Reovirus.

Lambda interferon (IFN-λ) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN-λ acts to exert its antiviral effects is unclear. Here, we sought to identify IFN-λ-responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-λ, Ifnlr1 We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN-λ antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN-λ in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Thus, the expression of Ifnlr1 by IECs is necessary for the response to both endogenous and exogenous IFN-λ. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN-λ by extending these findings in Rag1-deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1-null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. Thus, IECs are the critical cell type responding to IFN-λ to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN-λ-mediated control of enteric viral infection, and these findings provide insight into the mechanism of IFN-λ-mediated antiviral activity.IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-λ) control enteric viral infections in the gut and have been shown to cure mouse norovirus, a small-animal model for HNoVs. Using a genetic approach with conditional knockout mice, we identified IECs as the dominant IFN-λ-responsive cells in control of enteric virus infection in vivo Upon murine norovirus or reovirus infection, Ifnlr1 depletion in IECs largely recapitulated the phenotype seen in Ifnlr1-/- mice of higher intestinal tissue viral titers and increased viral shedding in the stool. Moreover, IFN-λ-mediated sterilizing immunity against murine norovirus requires the capacity of IECs to respond to IFN-λ. These findings clarify the mechanism of action of this cytokine and emphasize the therapeutic potential of IFN-λ for treating mucosal viral infections.

Pygopagus Conjoined Twins: A Neurophysiologic Intraoperative Monitoring Schema.

Conjoined twins occur in up to 1 in 50,000 live births with approximately 18% joined in a pygopagus configuration at the buttocks. Twins with this configuration display symptoms and carry surgical risks during separation related to the extent of their connection which can include anorectal, genitourinary, vertebral, and neural structures. Neurophysiologic intraoperative monitoring for these cases has been discussed in the literature with variable utility. The authors present a case of pygopagus twins with fused spinal cords and imperforate anus where the use of neurophysiologic intraoperative monitoring significantly impacted surgical decision-making in division of these critical structures.

Regulatory T Cell Responses in Participants with Type 1 Diabetes after a Single Dose of Interleukin-2: A Non-Randomised, Open Label, Adaptive Dose-Finding Trial.

BACKGROUND: Interleukin-2 (IL-2) has an essential role in the expansion and function of CD4+ regulatory T cells (Tregs). Tregs reduce tissue damage by limiting the immune response following infection and regulate autoreactive CD4+ effector T cells (Teffs) to prevent autoimmune diseases, such as type 1 diabetes (T1D). Genetic susceptibility to T1D causes alterations in the IL-2 pathway, a finding that supports Tregs as a cellular therapeutic target. Aldesleukin (Proleukin; recombinant human IL-2), which is administered at high doses to activate the immune system in cancer immunotherapy, is now being repositioned to treat inflammatory and autoimmune disorders at lower doses by targeting Tregs. METHODS AND FINDINGS: To define the aldesleukin dose response for Tregs and to find doses that increase Tregs physiologically for treatment of T1D, a statistical and systematic approach was taken by analysing the pharmacokinetics and pharmacodynamics of single doses of subcutaneous aldesleukin in the Adaptive Study of IL-2 Dose on Regulatory T Cells in Type 1 Diabetes (DILT1D), a single centre, non-randomised, open label, adaptive dose-finding trial with 40 adult participants with recently diagnosed T1D. The primary endpoint was the maximum percentage increase in Tregs (defined as CD3+CD4+CD25highCD127low) from the baseline frequency in each participant measured over the 7 d following treatment. There was an initial learning phase with five pairs of participants, each pair receiving one of five pre-assigned single doses from 0.04 × 106 to 1.5 × 106 IU/m2, in order to model the dose-response curve. Results from each participant were then incorporated into interim statistical modelling to target the two doses most likely to induce 10% and 20% increases in Treg frequencies. Primary analysis of the evaluable population (n = 39) found that the optimal doses of aldesleukin to induce 10% and 20% increases in Tregs were 0.101 × 106 IU/m2 (standard error [SE] = 0.078, 95% CI = -0.052, 0.254) and 0.497 × 106 IU/m2 (SE = 0.092, 95% CI = 0.316, 0.678), respectively. On analysis of secondary outcomes, using a highly sensitive IL-2 assay, the observed plasma concentrations of the drug at 90 min exceeded the hypothetical Treg-specific therapeutic window determined in vitro (0.015-0.24 IU/ml), even at the lowest doses (0.040 × 106 and 0.045 × 106 IU/m2) administered. A rapid decrease in Treg frequency in the circulation was observed at 90 min and at day 1, which was dose dependent (mean decrease 11.6%, SE = 2.3%, range 10.0%-48.2%, n = 37), rebounding at day 2 and increasing to frequencies above baseline over 7 d. Teffs, natural killer cells, and eosinophils also responded, with their frequencies rapidly and dose-dependently decreased in the blood, then returning to, or exceeding, pretreatment levels. Furthermore, there was a dose-dependent down modulation of one of the two signalling subunits of the IL-2 receptor, the ß chain (CD122) (mean decrease = 58.0%, SE = 2.8%, range 9.8%-85.5%, n = 33), on Tregs and a reduction in their sensitivity to aldesleukin at 90 min and day 1 and 2 post-treatment. Due to blood volume requirements as well as ethical and practical considerations, the study was limited to adults and to analysis of peripheral blood only. CONCLUSIONS: The DILT1D trial results, most notably the early altered trafficking and desensitisation of Tregs induced by a single ultra-low dose of aldesleukin that resolves within 2-3 d, inform the design of the next trial to determine a repeat dosing regimen aimed at establishing a steady-state Treg frequency increase of 20%-50%, with the eventual goal of preventing T1D. TRIAL REGISTRATION: ISRCTN Registry ISRCTN27852285; ClinicalTrials.gov NCT01827735.

Development of a reliable automated screening system to identify small molecules and biologics that promote human ß-cell regeneration.

Numerous compounds stimulate rodent ß-cell proliferation; however, translating these findings to human ß-cells remains a challenge. To examine human ß-cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human ß-cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently labeled ß-cells with high specificity using both nuclear and cytoplasmic markers. ß-Cells from each donor were assessed for their function and ability to enter the cell cycle by cotransduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens, including neurotransmitters, hormones, growth factors, and molecules, involved in adenosine and Tgf-1ß signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with the control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki-67+ ß-cells, whereas treatment with other compounds had limited to no effect on human ß-cell proliferation. This new scalable approach reduces the time and effort required for sensitive and specific evaluation of human ß-cell proliferation, thus allowing for increased testing of candidate human ß-cell mitogens.

Hospital incident command system: tool for a TJC accreditation survey.

The unannounced Joint Commission (TJC) accreditation survey can prove just as unpredictable and challenging as any other incident. In this article, the authors describe a plan developed by a hospital emergency response team that has proven successful in dealing with TJC and other surveys.