A quality improvement initiative to reduce the time to initial maternal visit in the neonatal intensive care unit.

OBJECTIVE: We aimed to reduce the time interval between an infant's admission to the Neonatal Intensive Care Unit (NICU) and first maternal interaction. METHODS: We identified three key drivers: 1. Collaboration with Labor and Delivery, 2. Education of staff and parents, and 3. Improved documentation of maternal presence. We measured the time interval from NICU admission to the initial maternal presence. We followed length of stay as a balancing measure to assay whether use of remote televisitation impeded efficient parental teaching and delayed discharge. RESULTS: We reduced the time interval from an average of 19.7 h in February 2020 to 12.3 h in June 2021. We expanded an already existing televisitation program as a surrogate to in-person interaction during COVID-19 pandemic. Televisitation did not affect in-person parental presence or LOS. CONCLUSION: Our multidisciplinary efforts resulted in a significantly accelerated time to initial maternal presence and did not prolong LOS.

Remimazolam: A Retrospective Study of Initial Safety and Recovery Data in Diverse Procedural Sedation.

PURPOSE: The new ultra-short-acting benzodiazepine, remimazolam, offers a pharmacokinetic and pharmacodynamic advantage over commonly used procedural sedation medication. This retrospective study explored the real-world utilization of remimazolam during procedural sedation to support the development of a nurse sedation protocol. The primary outcome was to identify associations between recovery time, adverse reactions, and dose-response in expanded patient populations. METHODS: This study reviewed charts of 292 adult patients from 3 hospitals within one institution who received remimazolam during procedural sedation between June 1, 2021 and December 31, 2021. Data were analyzed using logistic and linear regression. FINDINGS: The median time to alert in patients receiving remimazolam alone was 12 minutes (interquartile range 10, 17) and increased when additional sedation medications were utilized. Receiving additional sedative medication significantly increased the odds of hypoxia (OR 2.77, 95% CI 1.30-5.91, P = 0.008) after adjusting for body mass index (BMI), American Society of Anesthesiologists physical status (ASA-PS), and total remimazolam dose. There was a 25% increase in odds of experiencing hypoxia for every 5 kg/m2 increase in BMI (95% CI 1.01-1.54, P = 0.037). IMPLICATIONS: Remimazolam presents as a promising option for nurse procedural sedation, offering minimal impact on hemodynamics and respirations, quick recovery, and no residual sedative effects.

Convergent alterations in the tumor microenvironment of MYC-driven human and murine prostate cancer.

The tissue microenvironment in prostate cancer is profoundly altered. While such alterations have been implicated in driving prostate cancer initiation and progression to aggressive disease, how prostate cancer cells and their precursors mediate those changes is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we performed extensive single-cell RNA-sequencing (scRNA-seq) and rigorous molecular pathology of the comparative biology between human prostate cancer and key time points in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues, with validation in a large external data set, revealed that cancer cell-intrinsic activation of MYC signaling was the top up-regulated pathway in human cancers, representing a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Likewise, numerous non-malignant cell states in the tumor microenvironment (TME), including non-cancerous epithelial, immune, and fibroblast cell compartments, were conserved across individuals, raising the possibility that these cell types may be a sequelae of the convergent MYC activation in the cancer cells. To test this hypothesis, we employed a GEMM of prostate epithelial cell-specific MYC activation in two mouse strains. Cell communication network and pathway analyses suggested that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogrammed the TME during carcinogenesis, leading to the emergence of cascading cell state alterations in neighboring epithelial, immune, and fibroblast cell types that paralleled key findings in human prostate cancer. Importantly, among these changes, the progression from a precursor-enriched to invasive-cancer-enriched state was accompanied by a cell-intrinsic switch from pro-immunogenic to immunosuppressive transcriptional programs with coinciding enrichment of immunosuppressive myeloid and Treg cells in the immune microenvironment. These findings implicate activation of MYC signaling in reshaping convergent aspects of the TME of prostate cancer as a common denominator across the otherwise well-documented molecular heterogeneity of human prostate cancer.

Corrigendum: Convergent antibody responses are associated with broad neutralization of hepatitis C virus.

[This corrects the article DOI: 10.3389/fimmu.2023.1135841.].

Convergent antibody responses are associated with broad neutralization of hepatitis C virus.

Introduction: Early development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known. Methods: To identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects. Results: We found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing. Discussion: Together, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.

Single-cell atlas of epithelial and stromal cell heterogeneity by lobe and strain in the mouse prostate.

BACKGROUND: Evaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered and exposure-based mouse models in different mouse strains and prostate lobes within each mouse strain, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood. METHODS: In this study, we used single-cell RNA-sequencing (scRNA-seq) methods to assess the single-cell transcriptomes of 6-month-old mouse prostates from two commonly used mouse strains, friend virus B/NIH jackson (FVB/NJ) (N = 2) and C57BL/6J (N = 3). For each mouse, the lobes of the prostate were dissected (anterior, dorsal, lateral, and ventral), and individual scRNA-seq libraries were generated. In situ and pathological analyses were used to explore the spatial and anatomical distributions of novel cell types and molecular markers defining these cell types. RESULTS: Data dimensionality reduction and clustering analysis of scRNA-seq data revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Gene set enrichment analysis comparing luminal cells by strain showed enrichment of proto-Oncogene targets in FVB/NJ mice. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2 and Atp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressing Chga, Chgb, and Syp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate. CONCLUSIONS: Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes.

Androgen receptor activity in prostate cancer dictates efficacy of bipolar androgen therapy through MYC.

Testosterone is the canonical growth factor of prostate cancer but can paradoxically suppress its growth when present at supraphysiological levels. We have previously demonstrated that the cyclical administration of supraphysiological androgen (SPA), termed bipolar androgen therapy (BAT), can result in tumor regression and clinical benefit for patients with castration-resistant prostate cancer. However, predictors and mechanisms of response and resistance have been ill defined. Here, we show that growth inhibition of prostate cancer models by SPA required high androgen receptor (AR) activity and were driven in part by downregulation of MYC. Using matched sequential patient biopsies, we show that high pretreatment AR activity predicted downregulation of MYC, improved clinical response, and prolonged progression-free and overall survival for patients on BAT. BAT induced strong downregulation of AR in all patients, which is shown to be a primary mechanism of acquired resistance to SPA. Acquired resistance was overcome by alternating SPA with the AR inhibitor enzalutamide, which induced adaptive upregulation of AR and resensitized prostate cancer to SPA. This work identifies high AR activity as a predictive biomarker of response to BAT and supports a treatment paradigm for prostate cancer involving alternating between AR inhibition and activation.

Increasing usage of mother's own milk in neonates at risk of neonatal abstinence syndrome: MOM-NAS quality improvement initiative.

OBJECTIVE: To increase the usage rate of mothers' own milk (MOM) among neonates with prenatal opioid exposure from a baseline average of 47% to an average of 75% over two years. STUDY DESIGN: Between October 2018 and December 2020, we implemented various Plan-Do-Study-Act cycles that involved engaging providers in postpartum counseling for mothers with opioid dependence, using electronic medical records to track the rate of counseling, providing NAS educational materials to parents, and establishing a rooming-in unit. Our outcome measure was the provision of MOM to eligible neonates, while our process measure was the rate of postpartum counseling. RESULTS: During this initiative, we witnessed a special cause variation with an increase in the usage rate of MOM from a baseline of 47% to a 27-month average of 85% by December 2020. CONCLUSION: A series of quality improvement efforts resulted in increased usage of MOM among infants at risk of NAS.

Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers.

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a 'barcode' to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein-Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.

Pervasive promoter hypermethylation of silenced TERT alleles in human cancers.

BACKGROUND: In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of TERT gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating TERT gene expression in cancer cells is as yet not fully understood. METHODS: Here, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines, including primary, immortalized and cancer cell types, as well as in control and reference samples. RESULTS: In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally found that hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells. CONCLUSIONS: Our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to attenuation of TERT activation in cancer cells. This type of fine tuning of TERT expression may account for the modest activation of TERT expression in most cancers.

Using the Nintendo Wii Fit and body weight support to improve aerobic capacity, balance, gait ability, and fear of falling: two case reports.

BACKGROUND & PURPOSE: Lower limb amputation in older adults has a significant impact on balance, gait, and cardiovascular fitness, resulting in diminished community participation. The purpose of this case study was to describe the effects of a balance training program utilizing the Nintendo Wii™ Fit (Nintendo of America, Inc, Redmond, Washington) balance board and body-weight supported gait training on aerobic capacity, balance, gait, and fear of falling in two persons with transfemoral amputation. CASE DESCRIPTIONS: Participant A, a 62 year-old male 32 months post traumatic transfemoral amputation, reported fear of falling and restrictions in community activity. Participant B, a 58 year-old male 9 years post transfemoral amputation, reported limited energy and balance deficits during advanced gait activities. INTERVENTION: 6-weeks, 2 supervised sessions per week included 20 minutes of Nintendo™ Wii Fit Balance gaming and 20 minutes of gait training using Body Weight Support. OUTCOMES: Measures included oxygen uptake efficiency slope (OUES), economy of movement, dynamic balance (Biodex platform system), Activities-Specific Balance Confidence (ABC) Scale, and spatial-temporal parameters of gait (GAITRite). Both participants demonstrated improvement in dynamic balance, balance confidence, economy of movement, and spatial-temporal parameters of gait. Participant A reduced the need for an assistive device during community ambulation. Participant B improved his aerobic capacity, indicated by an increase in OUES. DISCUSSION: This case study illustrated that the use of Nintendo Wii™ Fit training and Body Weight Support were effective interventions to achieve functional goals for improving balance confidence, reducing use of assistive devices, and increasing energy efficiency when ambulating with a transfemoral prosthesis.

Advanced cardiac life support instruction: do we know tomorrow what we know today?

This study compared two instructional and evaluation methods for teaching advanced cardiac life support (ACLS) to health care professionals who were taking the ACLS course for the first time. Outcomes of the instruction were measured on completion of the course and at 3 months and 6 months postinstruction to identify differences in participants' knowledge retention, skills competency, and self-efficacy in performing ACLS. In addition, satisfaction with the teaching method was evaluated. The two methods of teaching and evaluating competencies for ACLS were (1) traditional classroom instruction plus practice and evaluation with monitors (low-fidelity simulation); and (2) classroom instruction plus practice with high-fidelity patient simulators. Participants in the study were 148 health care professionals or health care students who were novices in ACLS preparation. Participants were recruited from a large Midwest school of nursing and school of medicine, a Midwest physicians' assistant program, and a not-for-profit hospital. The findings showed no significant differences in ACLS knowledge, skills, self-efficacy, or learner satisfaction immediately after instruction or at 3 to 9 months posttraining. Retention of ACLS knowledge and skills competency over time was low in both groups; recommendations and interventions are discussed based on the study results.

CpG oligodeoxynucleotides augment the murine immune response to the Yersinia pestis F1-V vaccine in bubonic and pneumonic models of plague.

The current U.S. Department of Defense candidate plague vaccine is a fusion between two Yersinia pestis proteins: the F1 capsular protein, and the low calcium response (Lcr) V-protein. We hypothesized that an immunomodulator, such as CpG oligodeoxynucleotide (ODN)s, could augment the immune response to the plague F1-V vaccine in a mouse model for plague. CpG ODNs significantly augmented the antibody response and efficacy of a single dose of the plague vaccine in murine bubonic and pneumonic models of plague. In the latter study, we also found an overall significant augmentation the immune response to the individual subunits of the plague vaccine by CpG ODN 2006. In a long-term, prime-boost study, CpG ODN induced a significant early augmentation of the IgG response to the vaccine. The presence of CpG ODN induced a significant increase in the IgG2a subclass response to the vaccine up to 5 months after the boost. Our studies showed that CpG ODNs significantly augmented the IgG antibody response to the plague vaccine, which increased the probability of survival in murine models of plague (P<0.0001).

Simple inhibitors of histone deacetylase activity that combine features of short-chain fatty acid and hydroxamic acid inhibitors.

Butyric acid and trichostatin A (TSA) are anti-cancer compounds that cause the upregulation of genes involved in differentiation and cell cycle regulation by inhibiting histone deacetylase (HDAC) activity. In this study we have synthesized and evaluated compounds that combine the bioavailability of short-chain fatty acids, like butyric acid, with the bidentate binding ability of TSA. A series of analogs were made to examine the effects of chain length, simple aromatic cap groups, and substituted hydroxamates on the compounds' ability to inhibit rat-liver HDAC using a fluorometric assay. In keeping with previous structure-activity relationships, the most effective inhibitors consisted of longer chains and hydroxamic acid groups. It was found that 5-phenylvaleric hydroxamic acid and 4-benzoylbutyric hydroxamic acid were the most potent inhibitors with IC50's of 5 microM and 133 microM respectively.

TIM-4 expressed on APCs induces T cell expansion and survival.

TIM (T cell, Ig, mucin) proteins can regulate T cell immune responses. Tim-4 mRNA is not expressed in T cells, but exclusively in APCs. Tim-4 is a ligand for Tim-1 and Tim-4.Ig fusion protein was shown to either inhibit or expand T cells. However, the molecular basis for such opposite effects was not defined. By generating mAbs, we show that expression of Tim-4 protein is restricted to CD11c(+) and CD11b(+) cells and is up-regulated upon activation. We show that Tim-4 specifically phosphorylates Tim-1 and induces T cell expansion by enhancing cell division and reducing apoptosis. Tim-4 also induces the phosphorylation of signaling molecules LAT, Akt, and ERK1/2 in T cells. Tim-4, expressed on APCs, is a costimulatory molecule that promotes T cell expansion and survival by cross-linking Tim-1 on T cells.

Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy.

Human immunodeficiency virus (HIV) persists in peripheral blood mononuclear cells despite sustained, undetectable plasma viremia resulting from long-term antiretroviral therapy. However, the source of persistent HIV in such infected individuals remains unclear. Given recent data suggesting high levels of viral replication and profound depletion of CD4(+) T cells in gut-associated lymphoid tissue (GALT) of animals infected with simian immunodeficiency virus and HIV-infected humans, we sought to determine the level of CD4(+) T cell depletion as well as the degree and extent of HIV persistence in the GALT of infected individuals who had been receiving effective antiviral therapy for prolonged periods of time. We demonstrate incomplete recoveries of CD4(+) T cells in the GALT of aviremic, HIV-infected individuals who had received up to 9.9 years of effective antiretroviral therapy. In addition, we demonstrate higher frequencies of HIV infection in GALT, compared with PBMCs, in these aviremic individuals and provide evidence for cross-infection between these 2 cellular compartments. Together, these data provide a possible mechanism for the maintenance of viral reservoirs revolving around the GALT of HIV-infected individuals despite long-term viral suppression and suggest that the GALT may play a major role in the persistence of HIV in such individuals.

Detection of the host immune response to Burkholderia mallei heat-shock proteins GroEL and DnaK in a glanders patient and infected mice.

We examined, by enzyme-linked immunosorbent assay and Western blot analysis, the host immune response to 2 heat-shock proteins (hsps) in a patient and mice previously infected with Burkholderia mallei. The patient was the first reported human glanders case in 50 years in the United States. The expression of the groEL and dnaK operons appeared to be dependent upon a sigma(32) RNA polymerase as suggested by conserved heat-shock promoter sequences, and the groESL operon may be negatively regulated by a controlling invert repeat of chaperone expression (CIRCE) site. In the antisera, the GroEL protein was found to be more immunoreactive than the DnaK protein in both a human patient and mice previously infected with B. mallei. Examination of the supernatant of a growing culture of B. mallei showed that more GroEL protein than DnaK protein was released from the cell. This may occur similarly within an infected host causing an elevated host immune response to the B. mallei hsps.

Impact of HIV on cell survival and antiviral activity of plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are important mediators of innate immunity that act mainly through secretion of interferon (IFN)-alpha. Previous studies have found that these cells can suppress HIV in vitro; additionally, pDCs have been shown to be severely reduced in the peripheral blood of HIV-infected individuals. In the present study, we sought to determine the ability of pDCs to directly suppress viral replication ex vivo and to delineate the potential mechanisms whereby pDCs are depleted in HIV-infected individuals. We demonstrate that activated pDCs strongly suppress HIV replication in autologous CD4(+) T cells via a mechanism involving IFN-alpha as well as other antiviral factors. Of note, unstimulated pDCs from infected individuals who maintain low levels of plasma viremia without antiretroviral therapy were able to suppress HIV ex vivo via a mechanism requiring cell-to-cell contact. Our data also demonstrate that death of pDCs by both apoptosis and necrosis is induced by fusion of HIV with pDCs. Taken together, our data suggest that pDCs play an important role in the control of HIV replication and that high levels of viral replication in vivo are associated with pDC cell death via apoptosis and necrosis. Elucidation of the mechanism by which pDCs suppress HIV replication in vivo may have clinically relevant implications for future therapeutic strategies.

TIM family of genes in immunity and tolerance.

T cells on activation differentiate into different subsets (Th1 or Th2) with distinct effector functions. These T cell subsets are primarily differentiated on the basis of the cytokines that they produce, however, we have identified a novel gene family called TIM (T cell, immunoglobulin, mucin domain-containing molecules), whose members are differentially expressed on Th1 and Th2 cells. Three of the family members (Tim-1, Tim-3, and Tim-4) are conserved between mouse and man. Genomic association of the TIM family and polymorphisms in both Tim-1 and Tim-3 in different immune-mediated diseases suggest that the family may have an important role in regulating immunity, both in terms of normal immune responses and in diseases like autoimmunity and asthma.