Clinical Decision Support for Patients Presenting With Large Vessel Occlusion.

A single center had a collaborative, multidisciplinary review to determine how to best implement new acute ischemic stroke trials involving large vessel occlusions. A flow diagram process map was created for clinical decision support. Patients were divided into four groups based upon size of infarct and timing of presentation. The process map, available in the electronic health record (EHR) for clinicians to reference, guides the selection of patients for endovascular therapy with neuroimaging. In addition, the process map offers guidance for discussions with families and patients experiencing large vessel occlusions with both small and large core infarcts. This manuscript describes the process of creating the process map through a multidisciplinary review and discussion, with points of controversy and how these were addressed.

A novel optimized pre-embedding antibody-labelling correlative light electron microscopy technique.

In the intricate environment of a cell, many studies seek to discover the location of specific events or objects of interest. Advances in microscopy in recent years have allowed for high detail views of specific areas of cells of interest using correlative light electron microscopy (CLEM). While this powerful technique allows for the correlation of a specific area of fluorescence on a confocal microscope with that same area in an electron microscope, it is most often used to study tagged proteins of interest. This method adapts the correlative method for use with antibody labelling. We have shown that some cellular structures are more sensitive than others to this process and that this can be a useful technique for laboratories where tagged proteins or viruses, or dedicated CLEM instruments are not available.

Viral PIC-pocketing: RSV sequestration of translational preinitiation complexes into bi-phasic biomolecular condensates.

Orthopneumoviruses characteristically form membrane-less cytoplasmic inclusion bodies (IBs) wherein RNA replication and transcription occur. Here, we report a strategy whereby the orthopneumoviruses sequester various components of the translational preinitiation complex machinery into viral inclusion bodies to facilitate translation of their own mRNAs-PIC-pocketing. Electron microscopy of respiratory syncytial virus (RSV)-infected cells revealed bi-phasic organization of IBs, specifically, spherical "droplets" nested within the larger inclusion. Using correlative light and electron microscopy, combined with fluorescence in situ hybridization, we showed that the observed bi-phasic morphology represents functional compartmentalization of the inclusion body and that these domains are synonymous with the previously reported inclusion body-associated granules (IBAGs). Detailed analysis demonstrated that IBAGs concentrate nascent viral mRNA, the viral M2-1 protein as well as components of eukaryotic translation initiation factors (eIF), eIF4F and eIF3, and 40S complexes involved in translation initiation. Interestingly, although ribopuromycylation-based imaging indicates that the majority of viral mRNA translation occurs in the cytoplasm, there was some evidence for intra-IBAG translation, consistent with the likely presence of ribosomes in a subset of IBAGs imaged by electron microscopy. Mass spectrometry analysis of sub-cellular fractions from RSV-infected cells identified significant modification of the cellular translation machinery; however, interestingly, ribopuromycylation assays showed no changes to global levels of translation. The mechanistic basis for this pathway was subsequently determined to involve the viral M2-1 protein interacting with eIF4G, likely to facilitate its transport between the cytoplasm and the separate phases of the viral inclusion body. In summary, our data show that these viral organelles function to spatially regulate early steps in viral translation within a highly selective bi-phasic biomolecular condensate. IMPORTANCE: Respiratory syncytial viruses (RSVs) of cows and humans are a significant cause of morbidity and mortality in their respective populations. These RNA viruses replicate in the infected cells by compartmentalizing the cell's cytoplasm into distinct viral microdomains called inclusion bodies (IBs). In this paper, we show that these IBs are further compartmentalized into smaller structures that have significantly different density, as observed by electron microscopy. Within smaller intra-IB structures, we observed ribosomal components and evidence for active translation. These findings highlight that RSV may additionally compartmentalize translation to favor its own replication in the cell. These data contribute to our understanding of how RNA viruses hijack the cell to favor replication of their own genomes and may provide new targets for antiviral therapeutics in vivo.

Improving Seizure Frequency Documentation and Classification.

Background and Objectives: Accurate and reliable seizure data are essential for evaluating treatment strategies and tracking the quality of care in epilepsy clinics. This quality improvement project aimed to increase seizure documentation (i.e., documentation of seizure frequency from 80% to 100%, date of last seizure from 35% to 50%, and International League Against Epilepsy (ILAE) seizure classification from 35% to at least 50%) over 6 months. Methods: We surveyed 7 epileptologists to determine their perceived seizure frequency, ILAE classification, and date of last seizure documentation habits. Baseline data were collected weekly from September to December 2021. Subsequently, we implemented a newly created flowsheet in our Electronic Health Record (EHR) based on the Epilepsy Learning Healthcare System (ELHS) Case Report Forms to increase seizure documentation in a standardized way. Two epileptologists tested this flowsheet tool in their epilepsy clinics between February 2022 and July 2022. Data were collected weekly and compared with documentation from other epileptologists within the same group. Results: Epileptologists at our center believed they documented seizure frequency for 84%-87% of clinic visits, which aligned with baseline data collection, showing they recorded seizure frequency for 83% of clinic visits. Epileptologists believed they documented ILAE classification for 47%-52% of clinic visits, and baseline data showed this was documented in 33% of clinic visits. They also reported documenting the date of the last seizure for 52%-63% of clinic visits, but this occurred in only 35% of clinic visits. After implementing the new flowsheet, documentation increased to nearly 100% for all fields being completed by the providers who tested the flowsheet. Discussion: We demonstrated that by implementing an easy-to-use standardized EHR documentation tool, our documentation of critical metrics, as defined by the ELHS, improved dramatically. This shows that simple and practical interventions can substantially improve clinically meaningful documentation.

Use of a targeted, computer/web-based guided self-help psychoeducation toolkit for distressing hallucinations (MUSE) in people with an at-risk mental state for psychosis: protocol for a randomised controlled feasibility trial.

INTRODUCTION: Individuals who access at-risk mental state (ARMS) services often have unusual sensory experiences and levels of distress that lead them to seek help. The Managing Unusual Sensory Experiences (MUSE) treatment is a brief symptom targeted intervention that draws on psychological explanations to help account for unusual experiences. Practitioners use formulation and behavioural experiments to support individuals to make sense of their experiences and enhance coping strategies. The primary objective of this feasibility trial is to resolve key uncertainties before a definitive trial and inform parameters of a future fully powered trial. METHODS AND ANALYSIS: 88 participants aged 14-35 accepted into ARMS services, experiencing hallucinations/unusual sensory experiences which are considered by the patient to be a key target problem will be recruited from UK National Health Service (NHS) sites and randomised using 1:1 allocation (stratified by site, gender, and age) to either 6-8 sessions of MUSE or time-matched treatment as usual. Participants and therapists will be unblinded, research assessors are blinded. Blinded assessment will occur at baseline, 12 weeks and 20 weeks postrandomisation. Data will be reported in line with Consolidated Standards of Reporting Trials. Primary trial outcomes are feasibility outcomes, primary participant outcomes are functioning and hallucinations. Additional analysis will investigate potential psychological mechanisms and secondary mental well-being outcomes. Trial progression criteria follows signal of efficacy and uses an analytical framework with a traffic-light system to determine viability of a future trial. Subsequent analysis of the NHS England Mental Health Services Data Set 3 years postrandomisation will assess long-term transition to psychosis. ETHICS AND DISSEMINATION: This trial has received Research Ethics Committee approval (Newcastle North Tyneside 1 REC; 23/NE/0032). Participants provide written informed consent; young people provide assent with parental consent. Dissemination will be to ARMS Services, participants, public and patient forums, peer-reviewed publications and conferences. TRIAL REGISTRATION NUMBER: ISRCTN58558617.

Results of a Phase II Trial of Allogeneic Hematopoietic Stem Cell Transplantation Using 90Y-Ibritumomab Tiuxetan (Zevalin) in Combination With Fludarabine and Melphalan in Patients With High-Risk B-Cell Non-Hodgkin's Lymphoma.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (alloHCT) is potentially curative for relapsed/refractory (r/r) B-cell non-Hodgkin's lymphoma (B-cell NHL). However, relapse remains a major cause of treatment failure, especially in patients with either positron emission tomography (PET)-positive and/or chemoresistant disease prior to alloHCT. 90Y-ibritumomab tiuxetan (Zevalin) is a radiolabeled anti-CD20 antibody which is a safe and effective therapy in multiple histologic subtypes of B-cell NHL and has also been incorporated in both autologous HCT (autoHCT) and alloHCT conditioning regimens. OBJECTIVES: The purpose of this study was to evaluate the efficacy and confirm the safety of the radiolabeled anti-CD20 antibody ibritumomab tiuxetan (Zevalin) combined with the reduced intensity conditioning (RIC) regimen of fludarabine and melphalan (Flu/Mel) in patients with high-risk B-cell NHL. STUDY DESIGN: We conducted a phase II trial (NCT00577278) of Zevalin with Flu/Mel in patients with high-risk B-cell NHL. We enrolled 41 patients from October 2007 to April 2014, all of whom had either a fully matched sibling or 8/8 or 7/8 matched unrelated donor (MUD). Patients received 111In-Zevalin (5.0 mCi) on day -21 pre-HCT, followed by 90Y-Zevalin (0.4 mCi/kg) on day -14. Fludarabine (25 mg/m2 daily) was given from days -9 to -5 and melphalan (140 mg/m2) was administered on day -4. All patients received rituximab 250 mg/m2 on day +8 and an additional dose on either day +1 or -21 depending on the baseline rituximab level. Patients with a low rituximab level were given rituximab on days -21 and -15. All patients received tacrolimus/sirolimus (T/S) with or without methotrexate (MTX) for graft-versus-host disease (GVHD) prophylaxis starting on day -3, and stem cells were infused on day 0. RESULTS: The 2-year overall survival (OS) and progression-free survival (PFS) for all patients were 63% and 61%, respectively. The incidence of relapse at 2 years was 20%. Nonrelapse mortality (NRM) at day +100 and 1 year were 5% and 12%, respectively. The overall cumulative incidence of grade II-IV and III-IV acute GVHD (aGVHD) were 44% and 15%, respectively. Extensive chronic GVHD (cGVHD) occurred in 44% of patients. On univariate analysis, histology (diffuse large B cell lymphoma (DLBCL) vs. others) was negatively predictive for OS (P = .0013) and PFS (P = .0004), while histology (DLBCL vs. others, P = .0128) predicted for relapse. PET positivity pre-HCT did not correlate with any of the efficacy endpoints. CONCLUSION: Addition of Zevalin to Flu/Mel is safe and effective in high-risk NHL and met the prespecific endpoint. Results were suboptimal in patients with DLBCL.

2-Hydroxypropyl-ß-Cyclodextrin Treatment Induces Modest Immune Activation in Healthy Rhesus Macaques.

Experimental simian immunodeficiency virus (SIV) infection of Asian macaques is an excellent model for HIV disease progression and therapeutic development. Recent coformulations of nucleoside analogs and an integrase inhibitor have been used for parenteral antiretroviral (ARV) administration in SIV-infected macaques, successfully resulting in undetectable plasma SIV RNA. In a cohort of SIVmac239-infected macaques, we recently observed that administration of coformulated ARVs resulted in an unexpected increase in plasma levels of soluble CD14 (sCD14), associated with stimulation of myeloid cells. We hypothesized that the coformulation solubilizing agent Kleptose (2-hydroxypropyl-ß-cyclodextrin [HPßCD]) may induce inflammation with myeloid cell activation and the release of sCD14. Herein, we stimulated peripheral blood mononuclear cells (PBMCs) from healthy macaques with HPßCD from different commercial sources and evaluated inflammatory cytokine production in vitro. Treatment of PBMCs resulted in increased sCD14 release and myeloid cell interleukin-1ß (IL-1ß) production-with stimulation varying significantly by HPßCD source-and destabilized lymphocyte CCR5 surface expression. We further treated healthy macaques with Kleptose alone. In vivo, we observed modestly increased myeloid cell activation in response to Kleptose treatment without significant perturbation of the immunological transcriptome or epigenome. Our results demonstrate a need for vehicle-only controls and highlight immunological perturbations that can occur when using HPßCD in pharmaceutical coformulations. IMPORTANCE SIV infection of nonhuman primates is the principal model system for assessing HIV disease progression and therapeutic development. HPßCD has recently been incorporated as a solubilizing agent in coformulations of ARVs in SIV-infected nonhuman primates. Although HPßCD has historically been considered inert, recent findings suggest that HPßCD may contribute to inflammation. Herein, we investigate the contribution of HPßCD to healthy macaque inflammation in vitro and in vivo. We observe that HPßCD causes an induction of sCD14 and IL-1ß from myeloid cells in vitro and demonstrate that HPßCD stimulatory capacity varies by commercial source. In vivo, we observe modest myeloid cell activation in blood and bronchoalveolar lavage specimens absent systemic immune activation. From our findings, it is unclear whether HPßCD stimulation may improve or diminish immune reconstitution in ARV-treated lentiviral infections. Our results demonstrate a need for vehicle-only controls and highlight immunological perturbations that can occur when using HPßCD in pharmaceutical coformulations.

Maternal diet modulates the infant microbiome and intestinal Flt3L necessary for dendritic cell development and immunity to respiratory infection.

Poor maternal diet during pregnancy is a risk factor for severe lower respiratory infections (sLRIs) in the offspring, but the underlying mechanisms remain elusive. Here, we demonstrate that in mice a maternal low-fiber diet (LFD) led to enhanced LRI severity in infants because of delayed plasmacytoid dendritic cell (pDC) recruitment and perturbation of regulatory T cell expansion in the lungs. LFD altered the composition of the maternal milk microbiome and assembling infant gut microbiome. These microbial changes reduced the secretion of the DC growth factor Flt3L by neonatal intestinal epithelial cells and impaired downstream pDC hematopoiesis. Therapy with a propionate-producing bacteria isolated from the milk of high-fiber diet-fed mothers, or supplementation with propionate, conferred protection against sLRI by restoring gut Flt3L expression and pDC hematopoiesis. Our findings identify a microbiome-dependent Flt3L axis in the gut that promotes pDC hematopoiesis in early life and confers disease resistance against sLRIs.

Characteristics and clinical needs of young tertiary students attending a specialist mood disorders clinic.

OBJECTIVE: To investigate the clinical characteristics of tertiary students and non-students attending a specialist clinic for severe mood disorders. METHOD: Medical record audit of clients discharged from the Youth Mood Clinic (YMC). Data extracted included depressive symptomatology, suicidal ideation, self-harm, suicide attempt, tertiary education engagement, drop-out and deferral. RESULTS: Data from 131 clients (M age = 19.58 years, SD = 2.66) were analysed, including 46 tertiary students. Relative to non-students, at intake, tertiary students reported more severe depressive symptomatology (d = 0.43). They were more likely to experience suicidal ideation at intake (V = 0.23), and during treatment (V = 0.18). Tertiary students were also more likely to be living separately to their family of origin (V = 0.20) but were less likely to have experienced parental separation (V = 0.19). 21.73% of tertiary students dropped out or deferred study during care. CONCLUSION: In this cohort, those engaged in tertiary education experience more severe depression and more commonly experienced suicidal ideation. These young people require targeted support for their mental health while they undertake tertiary education.

Experimental bacterial dysbiosis with consequent immune alterations increase intrarectal SIV acquisition susceptibility.

Variations in the composition of the intestinal bacterial microbiome correlate with acquisition of some sexually transmitted pathogens. To experimentally assess the contribution of intestinal dysbiosis to rectal lentiviral acquisition, we induce dysbiosis in rhesus macaques (RMs) with the antibiotic vancomycin prior to repeated low-dose intrarectal challenge with simian immunodeficiency virus (SIV) SIVmac239X. Vancomycin administration reduces T helper 17 (TH17) and TH22 frequencies, increases expression of host bacterial sensors and antibacterial peptides, and increases numbers of transmitted-founder (T/F) variants detected upon SIV acquisition. We observe that SIV acquisition does not correlate with measures of dysbiosis but rather associates with perturbations in the host antimicrobial program. These findings establish a functional association between the intestinal microbiome and susceptibility to lentiviral acquisition across the rectal epithelial barrier.

Macrophage migration inhibitory factor promotes glucocorticoid resistance of neutrophilic inflammation in a murine model of severe asthma.

BACKGROUND: Severe neutrophilic asthma is resistant to treatment with glucocorticoids. The immunomodulatory protein macrophage migration inhibitory factor (MIF) promotes neutrophil recruitment to the lung and antagonises responses to glucocorticoids. We hypothesised that MIF promotes glucocorticoid resistance of neutrophilic inflammation in severe asthma. METHODS: We examined whether sputum MIF protein correlated with clinical and molecular characteristics of severe neutrophilic asthma in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. We also investigated whether MIF regulates neutrophilic inflammation and glucocorticoid responsiveness in a murine model of severe asthma in vivo. RESULTS: MIF protein levels positively correlated with the number of exacerbations in the previous year, sputum neutrophils and oral corticosteroid use across all U-BIOPRED subjects. Further analysis of MIF protein expression according to U-BIOPRED-defined transcriptomic-associated clusters (TACs) revealed increased MIF protein and a corresponding decrease in annexin-A1 protein in TAC2, which is most closely associated with airway neutrophilia and NLRP3 inflammasome activation. In a murine model of severe asthma, treatment with the MIF antagonist ISO-1 significantly inhibited neutrophilic inflammation and increased glucocorticoid responsiveness. Coimmunoprecipitation studies using lung tissue lysates demonstrated that MIF directly interacts with and cleaves annexin-A1, potentially reducing its biological activity. CONCLUSION: Our data suggest that MIF promotes glucocorticoid-resistance of neutrophilic inflammation by reducing the biological activity of annexin-A1, a potent glucocorticoid-regulated protein that inhibits neutrophil accumulation at sites of inflammation. This represents a previously unrecognised role for MIF in the regulation of inflammation and points to MIF as a potential therapeutic target for the management of severe neutrophilic asthma.

Favorable Activity and Safety Profile of Memory-Enriched CD19-Targeted Chimeric Antigen Receptor T-Cell Therapy in Adults with High-Risk Relapsed/Refractory ALL.

PURPOSE: A phase I/II study evaluating the safety and activity of memory-enriched CD19-directed chimeric antigen receptor (CD19-CAR) T cells in adults with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: In phase I, we tested sequentially two cell populations for CAR transduction: (i) central memory (Tcm) or (ii) naïve, stem, and central memory (Tn/mem) T cells. The study employed an activity constrained for toxicity design to determine the recommended phase II dose (RP2D), which was tested in phase II. RESULTS: The Tcm cohort was closed early due to lack of activity. The 200 ×106 Tn/mem-derived CD19-CAR T-cell dose was found to be safe and active, and was declared the RP2D. At RP2D, 58 participants underwent leukapheresis and 46 received CD19-CAR T cells. Median age for treated participants was 38 years (range, 22-72). Twenty-nine (63%) participants had relapsed post-allogeneic hematopoietic cell transplantation (alloHCT), 18 (39%) had Philadelphia-like (Ph-like) genotype, and 16 (35%) had extramedullary disease (EMD) at lymphodepletion (LD). Three (7%) participants had grade 3 cytokine release syndrome (CRS), and none had grade ≥ 4 CRS. Eight (17%) participants had grade ≥ 3 neurotoxicity, including one fatal cerebral edema. Forty (87%) patients achieved complete remission (CR)/CR with incomplete hematologic recovery, 2 (4%) progressed, and 4 (9%) were unevaluable for response. Among 42 response-evaluable participants, 16/17 with Ph-like ALL and 13/15 with EMD at LD responded. Twenty-one (53%) responders underwent alloHCT consolidation, which was associated with improved relapse-free survival (adjusted HR = 0.16; 95% confidence interval, 0.05-0.48; P = 0.001). CONCLUSIONS: Tn/mem-derived CD19-CAR T cells were safe and active, including in Ph-like ALL and EMD. See related commentary by El Marabti and Abdel-Wahab, p. 694.

First-In-Human Pilot PET Immunoimaging Study of 64Cu-Anti-Carcinoembryonic Antigen Monoclonal Antibody (hT84.66-M5A) in Patients with Carcinoembryonic Antigen-Producing Cancers.

Background: PET imaging using radiolabeled immunoconstructs shows promise in cancer detection and in assessing tumor response to therapies. The authors report the first-in-human pilot study evaluating M5A, a humanized anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb), radiolabeled with 64Cu in patients with CEA-expressing malignancies. The purpose of this pilot study was to identify the preferred patient population for further evaluation of this agent in an expanded trial. Methods: Patients with CEA-expressing primary or metastatic cancer received 64Cu-DOTA-hT84.66-M5A with imaging performed at 1 and 2 days postinfusion. 64Cu-DOTA-hT84.66-M5A PET scan findings were correlated with CT, MRI, and/or FDG PET scans and with histopathologic findings from planned surgery or biopsy performed postscan. Results: Twenty patients received 64Cu-DOTA-hT84.66-M5A. Twelve patients demonstrated positive images, which were confirmed in 10 patients as tumor by standard-of-care (SOC) imaging, biopsy, or surgical findings. Four of the 8 patients with negative imaging were confirmed as true negative, with the remaining 4 patients having disease demonstrated by SOC imaging or surgery. All 5 patients with locally advanced rectal cancer underwent planned biopsy or surgery after 64Cu-DOTA-hT84.66-M5A imaging (4 patients imaged 6-8 weeks after completing neoadjuvant chemotherapy and radiation therapy) and demonstrated a high concordance between biopsy findings and 64Cu-DOTA-hT84.66-M5A PET scan results. Three patients demonstrated positive uptake at the primary site later confirmed by biopsy and at surgery as residual disease. Two patients with negative scans each demonstrated complete pathologic response. In 5 patients with medullary thyroid cancer, 64Cu-DOTA-hT84.66-M5A identified disease not seen on initial CT scans in 3 patients, later confirmed to be disease by subsequent surgery or MRI. Conclusions: 64Cu-DOTA-hT84.66-M5A demonstrates promise in tumor detection, particularly in patients with locally advanced rectal cancer and medullary thyroid cancer. A successor trial in locally advanced rectal cancer has been initiated to further evaluate this agent's ability to define tumor extent before and assess disease response after neoadjuvant chemotherapy and radiotherapy. clinical trial.gov (NCT02293954).

The barriers and facilitators of HIV-exposed infant testing as perceived by HIV-positive mothers in Botswana: A qualitative study.

BACKGROUND: Despite high rates of HIV testing and enrolment of HIV-positive pregnant women on antiretroviral therapy in Botswana, coverage for HIV-exposed infant (HEI) testing remains suboptimal. Many factors can contribute to suboptimal HEI testing rates, but they have seldom been thoroughly investigated in Botswana. Therefore, the aim of this study was to explore the experiences and perspectives of HIV-positive mothers on the barriers and facilitators of HEI testing to inform interventions to promote HEI testing in Botswana. METHODS: We conducted focus group discussions (FGDs) with HIV-positive mothers who gave birth in 2016 at the three largest public hospitals in Botswana. FGDs were held in Maun, Francistown, and Gaborone from September 2019 to March 2020. The maximum variation sampling method was used to select the participants using information that was abstracted from birth registers and other medical records at the study sites. Mothers were asked to describe their HEI testing experiences, what made it easy or difficult for them to return the HEI for testing, and what needs to be done to improve HEI testing in Botswana. A thematic approach was used to analyse the data. RESULTS: Fifteen FGDs with 142 mothers (aged 21-52 years) were held. Participants identified several facilitators to HEI testing, including a mother with adequate knowledge of PMTCT, intensive tracking of HEI by healthcare workers (HCWs), positive attitudes of HCWs toward clients, and social support from significant others. Staff shortages at health care facilities, frequent stock-outs of HIV test kits, fear of stigma, fear of positive test results for the child, and transportation challenges were identified as key barriers to HEI testing. Increasing staffing at healthcare facilities, having adequate supplies of HIV test kits, enhanced HEI tracking, easing access to HEI testing services in rural areas, and providing quality PMTCT education were among the proposed interventions to promote HEI testing. CONCLUSION: Optimizing HEI testing in Botswana will require multi-level interventions at the policy, health system, community, interpersonal, and individual levels.

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates.

Antigen-specific CD8+ T cells play a key role in the host's antiviral response. T cells recognize viral epitopes via the T cell receptor (TCR), which contains the complementarity-determining region-3 (CDR3), comprising the variable, diversity and joining regions of the TCRß gene. During chronic simian immunodeficiency virus (SIV) infection of Asian macaque nonhuman primates, tissue-specific clonotypes are identifiable among SIV-specific CD8+ T cells. Here, we sought to determine level of antigen exposure responsible for the tissue-specific clonotypic structure. We examined whether the priming event and/or chronic antigen exposure is response for tissue-specific TCR repertoires. We evaluated the TCR repertoire of SIV-specific CD8+ T cells after acute antigen exposure following inoculation with a SIV DNA vaccine, longitudinally during the acute and chronic phases of SIV, and after administration of antiretrovirals (ARVs). Finally, we assessed the TCR repertoire of cytomegalovirus (CMV)-specific CD8+ T cells to establish if TCR tissue-specificity is shared among viruses that chronically replicate. TCR sequences unique to anatomical sites were identified after acute antigen exposure via vaccination and upon acute SIV infection. Tissue-specific clones also persisted into chronic infection and the clonotypic structure continued to evolve after ARV administration. Finally, tissue-specific clones were also observed in CMV-specific CD8+ T cells. Together, these data suggest that acute antigen priming is sufficient to induce tissue-specific clones and that this clonal hierarchy can persist when antigen loads are naturally or therapeutically reduced, providing mechanistic insight into tissue-residency.

Cross-species experiments reveal widespread cochlear neural damage in normal hearing.

Animal models suggest that cochlear afferent nerve endings may be more vulnerable than sensory hair cells to damage from acoustic overexposure and aging. Because neural degeneration without hair-cell loss cannot be detected in standard clinical audiometry, whether such damage occurs in humans is hotly debated. Here, we address this debate through co-ordinated experiments in at-risk humans and a wild-type chinchilla model. Cochlear neuropathy leads to large and sustained reductions of the wideband middle-ear muscle reflex in chinchillas. Analogously, human wideband reflex measures revealed distinct damage patterns in middle age, and in young individuals with histories of high acoustic exposure. Analysis of an independent large public dataset and additional measurements using clinical equipment corroborated the patterns revealed by our targeted cross-species experiments. Taken together, our results suggest that cochlear neural damage is widespread even in populations with clinically normal hearing.

MLKL Regulates Rapid Cell Death-independent HMGB1 Release in RSV Infected Airway Epithelial Cells.

Respiratory syncytial virus (RSV)-induced bronchiolitis is a significant contributor to infant morbidity and mortality. Previously, we identified that necroptosis, a pro-inflammatory form of cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3, and mixed lineage kinase domain like protein (MLKL), occurs in RSV-infected human airway epithelial cells (hAECs), mediating the release of the alarmin high mobility group box 1 (HMGB1). Here, we show that RSV infection of hAECs induces the biphasic release of HMGB1 at 6 ("early") and 24 ("late") hours post infection (hpi). The early phase of HMGB1 release at 6 hpi is cell death-independent, however, this release is nonetheless attenuated by inhibition of MLKL (primarily associated with necroptosis). The early release of HMGB1 promotes the late phase of HMGB1 release via the activation of RAGE (receptor for advanced glycation endproducts) and occurs with cell death. Treatment of hAECS with exogenous HMGB1 combined with a pan-caspase inhibitor induces hAEC necroptosis, and is attenuated by the RAGE antagonist, FPS-ZM1. Together, these findings demonstrate that RSV infection of hAECs leads to the early release of HMGB1, followed by a paracrine feed-forward amplification loop that further increases HMGB1 levels and promotes cell death. As the inhibition of MLKL or targeting of HMGB1/RAGE pathway attenuates the release of pro-inflammatory HMGB1 and decreases viral load, this suggests that the pharmacological targeting of these pathways may be of benefit for the treatment of severe RSV bronchiolitis.

Butyrate administration is not sufficient to improve immune reconstitution in antiretroviral-treated SIV-infected macaques.

Defective gastrointestinal barrier function and, in turn, microbial translocation have been identified as significant contributors to persistent inflammation in antiretroviral (ARV)-treated people living with HIV. Metabolic supplementation of short-chain fatty acids (SCFAs), generally produced by the commensal microbiome, may improve these outcomes. Butyrate is a SCFA that is essential for the development and maintenance of intestinal immunity and has a known role in supporting epithelial integrity. Herein we assessed whether supplementation with the dietary supplement sodium butyrate would improve immune reconstitution and reduce inflammation in ARV-treated, simian immunodeficiency virus (SIV)-infected rhesus macaques. We demonstrate that butyrate supplementation does not significantly improve immune reconstitution, with no differences observed in systemic CD4+ T-cell frequencies, T-cell functionality or immune activation, microbial translocation, or transcriptional regulation. Our findings demonstrate that oral administration of sodium butyrate is insufficient to reduce persistent inflammation and microbial translocation in ARV-treated, SIV-infected macaques, suggesting that this therapeutic may not reduce co-morbidities and co-mortalities in treated people living with HIV.

Preparation of Immunofluorescently Labeled Tissue Sections for Imaging at Low and High Magnifications in the Confocal Microscope.

The confocal laser scanning microscope allows us to examine tissue sections in greater detail than a widefield fluorescence microscope. However, this requires samples to be better preserved than standard cryostat sections, which are not usually aldehyde-fixed. Thick sections (approximately 70 µm) of formaldehyde-fixed tissue can be cut using a vibrating microtome and subsequently labeled with primary and secondary fluorescent antibodies and/or fluorescent stains. When imaged in the confocal microscope, these samples allow us to collect high-resolution images, detailing the intracellular location of multiple proteins and structures. In this chapter, we describe the technique used to prepare vibrating microtome sections, using porcine tissue infected with African swine fever virus as an example. This technique can easily be applied to any animal tissue with any suitable combination of antibodies, depending on the hypothesis.