Slow Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance.

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes.

EGLN1 Inhibition and Rerouting of α-Ketoglutarate Suffice for Remote Ischemic Protection.

Ischemic preconditioning is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent, more prolonged, ischemic insult. In remote ischemic preconditioning (RIPC), ischemia to one organ protects others organs at a distance. We created mouse models to ask if inhibition of the alpha-ketoglutarate (αKG)-dependent dioxygenase Egln1, which senses oxygen and regulates the hypoxia-inducible factor (HIF) transcription factor, could suffice to mediate local and remote ischemic preconditioning. Using somatic gene deletion and a pharmacological inhibitor, we found that inhibiting Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion (I/R) injury. Parabiosis experiments confirmed that RIPC in this latter model was mediated by a secreted factor. Egln1 loss causes accumulation of circulating αKG, which drives hepatic production and secretion of kynurenic acid (KYNA) that is necessary and sufficient to mediate cardiac ischemic protection in this setting.

Control of the mitotic cleavage plane by local epithelial topology.

For nearly 150 years, it has been recognized that cell shape strongly influences the orientation of the mitotic cleavage plane (e.g., Hofmeister, 1863). However, we still understand little about the complex interplay between cell shape and cleavage-plane orientation in epithelia, where polygonal cell geometries emerge from multiple factors, including cell packing, cell growth, and cell division itself. Here, using mechanical simulations, we show that the polygonal shapes of individual cells can systematically bias the long-axis orientations of their adjacent mitotic neighbors. Strikingly, analyses of both animal epithelia and plant epidermis confirm a robust and nearly identical correlation between local cell topology and cleavage-plane orientation in vivo. Using simple mathematics, we show that this effect derives from fundamental packing constraints. Our results suggest that local epithelial topology is a key determinant of cleavage-plane orientation, and that cleavage-plane bias may be a widespread property of polygonal cell sheets in plants and animals.

Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT.

The recognition that cytosolic mitochondrial DNA (mtDNA) activates cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) innate immune signaling has unlocked novel disease mechanisms. Here, an uncharacterized variant predicted to affect TOP1MT function, P193L, was discovered in a family with multiple early onset autoimmune diseases, including Systemic Lupus Erythematosus (SLE). Although there was no previous genetic association between TOP1MT and autoimmune disease, the role of TOP1MT as a regulator of mtDNA led us to investigate whether TOP1MT could mediate the release of mtDNA to the cytosol, where it could then activate the cGAS-STING innate immune pathway known to be activated in SLE and other autoimmune diseases. Through analysis of cells with reduced TOP1MT expression, we show that loss of TOP1MT results in release of mtDNA to the cytosol, which activates the cGAS-STING pathway. We also characterized the P193L variant for its ability to rescue several TOP1MT functions when expressed in TOP1MT knockout cells. We show that the P193L variant is not fully functional, as its re-expression at high levels was unable to rescue mitochondrial respiration deficits, and only showed partial rescue for other functions, including repletion of mtDNA replication following depletion, nucleoid size, steady state mtDNA transcripts levels and mitochondrial morphology. Additionally, expression of P193L at endogenous levels was unable to rescue mtDNA release-mediated cGAS-STING signaling. Overall, we report a link between TOP1MT and mtDNA release leading to cGAS-STING activation. Moreover, we show that the P193L variant has partial loss of function that may contribute to autoimmune disease susceptibility via cGAS-STING mediated activation of the innate immune system.

FLAIRR-Seq: A Method for Single-Molecule Resolution of Near Full-Length Antibody H Chain Repertoires.

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.

NITES, a nocturnal bladder score to aid diagnosis during the transition to older age care.

BACKGROUND: As adults transition to older age, bothersome nocturnal lower urinary tract symptoms (LUTS) become common. There is need for a reliable assessment metric to detect and measure specific symptoms. OBJECTIVE: To subject the nocturnal LUTS score for older individuals, Nocturia, Incontinence, Toileting and Enuresis Symptom Score (NITES), to psychometric analysis. MATERIAL AND METHODS: Factor analysis of the metric was conducted with completed questionnaires from 151 older individuals who were either admitted to a tertiary hospital or attending an outpatient continence clinic. Test re-test reliability involved 18 older community dwelling individuals attending a Geriatrician clinic completing the metric at two timepoints separated by at least 1 week. Intra-class correlation coefficients were determined for reliability of each factor and item. RESULTS: The NITES metric was completed by 98 hospitalized older individuals and 53 attending a continence clinic (mean age 83.2 years [SD 7.0]). Factor analysis demonstrated that one item had a floor effect and two items had poor endorsement. After test re-test reliability analysis, a further three items were removed: one due to poor correlation between timepoints and two demonstrating inadequate internal consistency. The final NITES metric is comprised of three factors: Sleep 4-items, Incontinence 4-items, and Personal Bother 2-items. A 4-item short form for symptom screening was extracted from the longer measure. CONCLUSION: The final NITES metric is a 10-item questionnaire with an embedded 4-item short symptom screen. It has utility utilized to detect nocturnal bladder symptoms in both community dwelling and hospitalized older adults.

Should hormone replacement therapy (any route of administration) be considered in all postmenopausal women with lower urinary tract symptoms? Report from the ICI-RS 2023.

AIMS: This International Consultation on Incontinence-Research Society report aims to summarize the evidence and uncertainties regarding the use of hormone replacement therapy by any route in the management of lower urinary tract symptoms (LUTS) including recurrent urinary tract infections (rUTI), with a review of special considerations for the elderly. Research question proposals to further this field have been highlighted. METHODS: An overview of the existing evidence, guidelines, and consensus regarding the use of topical or systemic estrogens in the management of LUTS. RESULTS: There are currently evidence and recommendations to offer topical estrogens to postmenopausal women with overactive bladder symptoms as well as postmenopausal women with rUTIs. Systemic estrogens however have been shown in a meta-analysis to have a negative effect on LUTS and, therefore are not currently recommended. CONCLUSIONS: Although available evidence and recommendations exist for the use of topical estrogens, few women are commenced on these in primary care. There remain large gaps still within our knowledge of the use of estrogens within the management of LUTS, particularly on when it should be commenced, the length of time treatment should be continued for, and barriers to prescribing.

The characteristics of the implicit body model of the trunk.

Knowing where the body is in space requires reference to a stored model of the size and shape of body parts, termed the body model. This study sought to investigate the characteristics of the implicit body model of the trunk by assessing the position sense of midline and lateral body landmarks. Sixty-nine healthy participants localised midline and lateral body landmarks on their thorax, waist and hips, with perceived positions of these landmarks compared to actual positions. This study demonstrates evidence of a significant distortion of the implicit body model of the trunk, presenting as a squatter trunk, wider at the waist and hips. A significant difference was found between perceived and actual location in the horizontal (x) and vertical (y) directions for the majority of trunk landmarks. Evidence of a rightward bias was noted in the perception of six of the nine body landmarks in the horizontal (x) direction, including all midline levels. In the vertical (y) direction, a substantial inferior bias was evident at the thorax and waist. The implicit body model of the trunk is shown to be distorted, with the lumbar spine (waist-to-hip region) held to be shorter and wider than reality.

Safety, timing, and outcomes of early post-operative cardiac catheterisation following congenital heart surgery.

OBJECTIVE: The safety of early post-operative cardiac catheterisation has been described following congenital heart surgery. Optimal timing of early post-operative cardiac catheterisation remains uncertain. The aim of this study was to describe the safety of early post-operative cardiac catheterisation and its impact on cardiac ICU and hospital length of stay, duration of mechanical ventilation, and extracorporeal support. METHODS: This single-centre retrospective cohort study compared clinical and outcome variables between "early" early post-operative cardiac catheterisation (less than 72 hours after surgery) and "late" early post-operative cardiac catheterisation (greater than 72 hours after surgery) groups using Chi-squared, Student's t, and log-rank test (or appropriate nonparametric test). RESULTS: In total, 132 patients were included, 22 (16.7%) "early" early post-operative cardiac catheterisation, and 110 (83.3%) "late" early post-operative cardiac catheterisation. Interventions were performed in 63 patients (51.5%), 7 (11.1%) early and 56 (88.9%) late. Complications of catheterisation occurred in seven (5.3%) patients, two early and five late. There were no major complications. Patients in the late group trended towards a longer stay in the cardiac ICU (19 days [7, 62] versus 11.5 days [7.2, 31.5], p = 0.6) and in the hospital (26 days [9.2, 68] versus 19 days [13.2, 41.8], p = 0.8) compared to the earlier group. CONCLUSION: "Early" early post-operative cardiac catheterisation was associated with an overall low rate of complications. Earlier catheterisations trended towards shorter cardiac ICU and hospital length of stays. Earlier catheterisations may lead to earlier recovery for patients not following an expected post-operative course.

Characterization of the immunoglobulin lambda chain locus from diverse populations reveals extensive genetic variation.

Immunoglobulins (IGs), crucial components of the adaptive immune system, are encoded by three genomic loci. However, the complexity of the IG loci severely limits the effective use of short read sequencing, limiting our knowledge of population diversity in these loci. We leveraged existing long read whole-genome sequencing (WGS) data, fosmid technology, and IG targeted single-molecule, real-time (SMRT) long-read sequencing (IG-Cap) to create haplotype-resolved assemblies of the IG Lambda (IGL) locus from 6 ethnically diverse individuals. In addition, we generated 10 diploid assemblies of IGL from a diverse cohort of individuals utilizing IG-Cap. From these 16 individuals, we identified significant allelic diversity, including 36 novel IGLV alleles. In addition, we observed highly elevated single nucleotide variation (SNV) in IGLV genes relative to IGL intergenic and genomic background SNV density. By comparing SNV calls between our high quality assemblies and existing short read datasets from the same individuals, we show a high propensity for false-positives in the short read datasets. Finally, for the first time, we nucleotide-resolved common 5-10 Kb duplications in the IGLC region that contain functional IGLJ and IGLC genes. Together these data represent a significant advancement in our understanding of genetic variation and population diversity in the IGL locus.

Bifunctional Small Molecules That Induce Nuclear Localization and Targeted Transcriptional Regulation.

The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better understand and potentially manipulate protein localization for therapeutic purposes, we engineered bifunctional compounds that bind to proteins in separate cellular compartments. We show these compounds induce nuclear import of cytosolic cargoes, using nuclear-localized BRD4 as a "carrier" for co-import and nuclear trapping of cytosolic proteins. We use this system to calculate kinetic constants for passive diffusion across the nuclear pore and demonstrate single-cell heterogeneity in response to these bifunctional molecules with cells requiring high carrier to cargo expression for complete import. We also observe incorporation of cargo into BRD4-containing condensates. Proteins shown to be substrates for nuclear transport include oncogenic mutant nucleophosmin (NPM1c) and mutant PI3K catalytic subunit alpha (PIK3CAE545K), suggesting potential applications to cancer treatment. In addition, we demonstrate that chemically induced localization of BRD4 to cytosolic-localized DNA-binding proteins, namely, IRF1 with a nuclear export signal, induces target gene expression. These results suggest that induced localization of proteins with bifunctional molecules enables the rewiring of cell circuitry, with significant implications for disease therapy.

DNA Methylation Signature for EZH2 Functionally Classifies Sequence Variants in Three PRC2 Complex Genes.

Weaver syndrome (WS), an overgrowth/intellectual disability syndrome (OGID), is caused by pathogenic variants in the histone methyltransferase EZH2, which encodes a core component of the Polycomb repressive complex-2 (PRC2). Using genome-wide DNA methylation (DNAm) data for 187 individuals with OGID and 969 control subjects, we show that pathogenic variants in EZH2 generate a highly specific and sensitive DNAm signature reflecting the phenotype of WS. This signature can be used to distinguish loss-of-function from gain-of-function missense variants and to detect somatic mosaicism. We also show that the signature can accurately classify sequence variants in EED and SUZ12, which encode two other core components of PRC2, and predict the presence of pathogenic variants in undiagnosed individuals with OGID. The discovery of a functionally relevant signature with utility for diagnostic classification of sequence variants in EZH2, EED, and SUZ12 supports the emerging paradigm shift for implementation of DNAm signatures into diagnostics and translational research.

Emicizumab for the treatment of acquired hemophilia A: Retrospective review of a single-institution experience.

INTRODUCTION: Acquired haemophilia A (AHA) is a rare and potentially life-threatening bleeding disorder arising from autoantibodies that inhibit coagulation factor VIII (FVIII). Treatment entails achieving haemostasis with bypassing agents or factor replacement, and eradication of the inhibitor with immunosuppressive therapy (IST). Due to the rarity of AHA, there are few prospective data to guide management. METHODS: We present a retrospective report of 11 AHA patients treated with emicizumab, a FVIII-mimetic bispecific antibody, administered at 3 mg/kg weekly for 4 weeks in conjunction with rituximab-based immunosuppressive therapy. The chromogenic FVIII inhibitor assay was used to assess for inhibitor eradication. RESULTS: The median follow-up was 13.9 months. The median number of days of additional haemostatic therapy or red blood cell transfusions after initiating emicizumab was 2 (range 0-15). The median was 0 days (range 0-8) for patients who did not require vascular embolization to achieve haemostasis. Eight patients achieved a complete remission (defined as recovery of FVIII activity to > 50% with a negative inhibitor test in the absence of haemostatic and IST); two patients achieved a partial remission (FVIII activity > 50% but with detectable inhibitor); one patient experienced refractory disease. One patient experienced rebleeding and two patients experienced inhibitor recurrence. No thrombotic, thrombotic microangiopathic or infectious complications occurred. CONCLUSION: Our observations suggest emicizumab can facilitate haemostasis for AHA patients and be combined with safer, lower-intensity immunosuppressive therapies to achieve remission.

Can we define the optimal postvoid residual volume at which intermittent catheterization should be recommended, and are there other measures that could guide an intermittent catheterization protocol: ICI-RS 2023.

AIMS: The postvoid residual (PVR) volume of urine in the bladder is widely used in clinical practice as a guide to initiate treatment, including clean-intermittent self-catheterization (CISC). It is often believed that an elevated PVR causes complications such as recurrent urinary tract infections (UTI) and renal failure. However, evidence for this is limited and identifying alternative measures to guide treatment decisions may optimize patient care. At the International Consultation on Incontinence Research Society (ICI-RS) meeting in 2023 a Think Tank addressed the question of whether we can define the optimal PVR at which CISC should be recommended, and whether there are other measures that could guide a CISC protocol. METHODS: The Think Tank conducted a literature review and expert consensus meeting focusing on current limitations in defining and measuring PVR, and highlighting other measures that may optimize selection for, and persistence with, CISC. RESULTS: There is no consensus on the threshold value of PVR that is considered "elevated" or "significant." There is a lack of standardization on terminology, and the normal range of PVR in different populations of different ages remains to be well-studied. The measurement of PVR is influenced by several factors, including intraindividual variation, timing and method of measurement. Furthermore, the evidence linking an elevated PVR with complications such as UTI and renal failure is mixed. Other measures, such as bladder voiding efficiency or urodynamic parameters, may be better at predicting such complications, and therefore may be more relevant at guiding a CISC protocol. CONCLUSIONS: There is a lack of high quality evidence to support PVR as a predictor for complications of UTI or renal failure. Threshold values for normal PVR in different populations are unknow, and so threshold values for "elevated" or "significant" PVR cannot be determined. Other factors, such as urodynamic findings, may be better at predicting complications and therefore guiding management decisions, and this remains to be studied. Areas for further research are proposed.

Pessaries for pelvic organ prolapse in women with severe dementia: an ethical challenge.

Pelvic organ prolapse is commonly treated with intravaginal devices to support the pelvic organs and maintain comfort. Pessaries generally require regular maintenance with removal, cleaning, and replacement. For women with severe dementia, this process can be extremely distressing. We present an illustrative case of a woman in whom the progression of her dementia led to a challenging ethical dilemma about continuing the use of a pessary and call for a conversation about these issues in the urogynaecology community.

A scoping review of risk factors for urinary incontinence in older men.

BACKGROUND: Most epidemiological studies have not systematically identified or categorized risk factors for urinary incontinence (UI) in older men, despite a higher prevalence than in younger men. Considering the burden of UI, an understanding of risk factors can inform cost-effective prevention/treatment programs. This scoping review aimed to identify and categorise risk factors for UI in older men, identify gaps in the evidence, and opportunities for future research. METHODS: The Joanna Briggs Institute (JBI) method for scoping reviews guided the conduct and reporting of this review alongside the Preferred Reporting Items for Systematic Reviews and Meta-analyses extension for scoping reviews checklist. JBI's Population, Concept, and Context approach framed the inclusion criteria (all evidence sources on UI risk factors that included older men [65 +]). We employed JBI's three-step search strategy, which included a limited initial search in Ovid MEDLINE, a detailed comprehensive database search, and a search of reference lists of included studies, Google Scholar and grey literature. There were no restrictions on language, study type, or publication date. Two independent reviewers screened, selected, and extracted eligible studies. Data were analyzed using descriptive statistics and qualitative content analysis. RESULTS: Forty-seven articles that met the inclusion criteria identified 98 risk factors across six categories. Behavioural risk factors, reported by only two studies, were the least investigated of all the categories, whereas medical factors/diseases were the most investigated. No genetic factors were documented. The top five risk factors were increasing age/advanced age (n = 12), Benign Prostatic Hyperplasia (n = 11), Diabetes Mellitus (n = 11), Detrusor overactivity (n = 10), limitation in physical function/ADL disability (n = 10), increased Body Mass Index (BMI)/overweight/obesity (n = 8), Dementia (n = 8), and Parkinson's disease (n = 7). CONCLUSION: There is a dearth of evidence to describe the role behavioural risk factors have in UI in older men. These factors may play a role in health promotion and disease prevention in this area. REGISTRATION: A protocol detailing the methods was developed and published, and is registered in the Open Science Framework [Feb 07 2023; https://osf.io/xsrge/ ].

Ventriculopleural shunt outcomes for pediatric hydrocephalus: a single-institution experience.

PURPOSE: The aim of this study is to analyze rates of ventriculopleural (VPL) shunt failure and complications among patients with pediatric hydrocephalus, and to analyze which factors may predict early (< 1 year) or late (> 1 year) VPL shunt failure in this sample. METHODS: A retrospective chart review was conducted of all consecutive VPL shunt placements from 2000 to 2019 at our institution. Data was collected on patient characteristics, shunt history, and shunt type. Primary endpoints include rates of VPL shunt survival and rates of symptomatic pleural effusion. The Kaplan-Meier method was used to calculate shunt survival, and Fisher's exact test and t-test were used to compare differences between categorical variables and means, respectively (p < 0.05). RESULTS: Thirty-one patients with pediatric hydrocephalus underwent VPL shunt placement (mean age 14.2 years). Of the 27 patients with long-term follow-up (mean 46 months), VPL shunt revision was required in 19, seven of which were due to pleural effusion. Overall shunt survival rates at 1, 3, 5, and 7 years were 76%, 62%, 55%, and 46%, respectively. Mean duration of shunt survival was 26.74 months. Overall pleural effusion rate was 26%. No patient-specific factors, including shunt valve type, were significantly associated with shunt survival, risk of early revision, or risk of pleural effusion. CONCLUSIONS: Our results are comparable to those reported in the literature and represent one of the largest case series on the topic. VPL shunts are a viable second-line option when ventriculoperitoneal (VP) shunt placement is not possible or desirable, though there are high rates of shunt revision and pleural effusion.

Severe obesity and global developmental delay in preschool children: Findings from a Canadian Paediatric Surveillance Program study.

Background: The co-presentation of severe obesity (SO) and global developmental delay (GDD) in Canadian preschool children has not been examined. However, SO and GDD may require syndromic diagnoses and unique management considerations. Objectives: To determine (1) minimum incidence; (2) age of onset and risk factors; and (3) health care utilization for co-presenting SO and GDD. Methods: Through the Canadian Paediatric Surveillance Program (CPSP), a monthly form was distributed to participants from February 2018 to January 2020 asking for reports of new cases of SO and GDD among children ≤5 years of age. We performed descriptive statistics for quantitative questions and qualitative content analysis for open-ended questions. Results: Forty-seven cases (64% male; 51% white; mean age: 3.5 ± 1.2 years) were included. Age of first weight concern was 2.5 ± 1.3 years and age of GDD diagnosis was 2.7 ± 1.4 years. Minimum incidence of SO and GDD was 3.3 cases per 100,000 for ≤5 years of age per year. Identified problems included school and/or behavioural problems (n = 17; 36%), snoring (n = 14; 30%), and asthma/recurrent wheeze (n = 10; 21%). Mothers of 32% of cases (n = 15) had obesity and 21% of cases (n = 10) received neonatal intensive care. Microarray was ordered for 57% (n = 27) of children. A variety of clinicians and services were accessed. As reported by CPSP participants, challenges faced by families and health service access were barriers to care. Conclusion: Children with SO and GDD have multiple comorbidities, and require early identification and referral to appropriate services. These cases may also benefit from additional testing to rule out known genetic obesity syndromes.