Radiation and anti-PD-L1 synergize by stimulating a stem-like T cell population in the tumor-draining lymph node.

Radiotherapy (RT) and anti-PD-L1 synergize to enhance local and distant (abscopal) tumor control. However, clinical results in humans have been variable. With the goal of improving clinical outcomes, we investigated the underlying synergistic mechanism focusing on a CD8+ PD-1+ Tcf-1+ stem-like T cell subset in the tumor-draining lymph node (TdLN). Using murine melanoma models, we found that RT + anti-PD-L1 induces a novel differentiation program in the TdLN stem-like population which leads to their expansion and differentiation into effector cells within the tumor. Our data indicate that optimal synergy between RT + anti-PD-L1 is dependent on the TdLN stem-like T cell population as either blockade of TdLN egress or specific stem-like T cell depletion reduced tumor control. Together, these data demonstrate a multistep stimulation of stem-like T cells following combination therapy which is initiated in the TdLN and completed in the tumor.

Microengineered In Vitro Assays for Screening and Sorting Manufactured Therapeutic T Cells.

Adoptively transferred T cells constitute a major class of current and emergent cellular immunotherapies for the treatment of disease, including but not limited to cancer. Although key advancements in molecular recognition, genetic engineering, and manufacturing have dramatically enhanced their translational potential, therapeutic potency remains limited by poor homing and infiltration of transferred cells within target host tissues. In vitro microengineered homing assays with precise control over micromechanical and biological cues can address these shortcomings by enabling interrogation, screening, sorting, and optimization of therapeutic T cells based on their homing capacity. In this article, the working principles, application, and integration of microengineered homing assays for the mechanistic study of biophysical and biomolecular cues relevant to homing of therapeutic T cells are reviewed. The potential for these platforms to enable scalable enrichment and screening of next-generation manufactured T cell therapies for cancer is also discussed.

High-Sensitivity Cardiac Troponin I Enhances Preeclampsia Prediction Beyond Maternal Factors and the sFlt-1/PlGF Ratio.

BACKGROUND: Preeclampsia shares numerous risk factors with cardiovascular diseases. Here, we aimed to assess the potential utility of high-sensitivity cardiac troponin I (hs-cTnI) values during pregnancy in predicting preeclampsia occurrence. METHODS: This study measured hs-cTnI levels in 3721 blood samples of 2245 pregnant women from 4 international, prospective cohorts. Three analytical approaches were used: (1) a cross-sectional analysis of all women using a single blood sample, (2) a longitudinal analysis of hs-cTnI trajectories in women with multiple samples, and (3) analyses of prediction models incorporating hs-cTnI, maternal factors, and the sFlt-1 (soluble fms-like tyrosine kinase 1)/PlGF (placental growth factor) ratio. RESULTS: Women with hs-cTnI levels in the upper quarter had higher odds ratios for preeclampsia occurrence compared with women with levels in the lower quarter. Associations were driven by preterm preeclampsia (odds ratio, 5.78 [95% CI, 2.73-12.26]) and remained significant when using hs-cTnI as a continuous variable adjusted for confounders. Between-trimester hs-cTnI trajectories were independent of subsequent preeclampsia occurrence. A prediction model incorporating a practical hs-cTnI level of detection cutoff (≥1.9 pg/mL) alongside maternal factors provided comparable performance with the sFlt-1/PlGF ratio. A comprehensive model including sFlt-1/PlGF, maternal factors, and hs-cTnI provided added value (cross-validated area under the receiver operator characteristic, 0.78 [95% CI, 0.73-0.82]) above the sFlt-1/PlGF ratio alone (cross-validated area under the receiver operator characteristic, 0.70 [95% CI, 0.65-0.76]; P=0.027). As assessed by likelihood ratio tests, the addition of hs-cTnI to each prediction model significantly improved the respective prediction model not incorporating hs-cTnI, particularly for preterm preeclampsia. Net reclassification improvement analyses indicated that incorporating hs-cTnI improved risk prediction predominantly by correctly reclassifying women with subsequent preeclampsia occurrence. CONCLUSIONS: These exploratory findings uncover a potential role for hs-cTnI as a complementary biomarker in the prediction of preeclampsia. After validation in prospective studies, hs-cTnI, alongside maternal factors, may either be considered as a substitute for angiogenic biomarkers in health care systems where they are sparce or unavailable, or as an enhancement to established prediction models using angiogenic markers.

Mammalian cysteine-rich secretory proteins interact with plasma membrane Ca2+ exporter PMCA4b.

BACKGROUND: Mammalian cysteine-rich secretory proteins (CRISPs) are predominantly expressed in the male reproductive tract. Knockout mice lacking two or more CRISPs show defects in sperm transport, sperm-egg interaction and Ca2+ homeostasis. CRISPs play redundant and specific roles via their binding partners. To understand this, a comprehensive analysis of CRISP interactome needs to be undertaken. OBJECTIVES: This study aimed to analyse CRISP4 binding partners on the plasma membrane of rat caudal spermatozoa. MATERIALS AND METHODS: Total proteins from rat caudal spermatozoa were subjected to immunoprecipitation using anti-CRISP4 antibody followed by liquid chromatography-mass spectrophotometry analysis. Plasma membrane localised proteins were shortlisted, and a key target was validated by co-immunoprecipitation and co-localisation. Co-transfection followed by co-immunoprecipitation was carried out for studying the interaction of full-length as well as deletion mutants of CRISPs with human plasma membrane calcium ATPase, isoform b (hPMCA4b). Calcium assays were performed using Fura-2-AM. The cholesterol binding ability of different CRISPs was evaluated in silico. RESULTS: The membrane-specific interactome of rat CRISP4 (rCRISP4) from caudal spermatozoa revealed PMCA4b as a novel binding partner, and their interaction was validated in rat spermatozoa. Human CRISP1 (hCRISP1) and hCRISP3 also interacted with PMCA4b via the N-terminal domain. Interestingly, hCRISP1 and rCRISP4 delayed PMCA4b-mediated calcium extrusion but hCRISP3 did not. In silico analysis demonstrated that hCRISP1 and rCRISP4 have higher binding affinity towards cholesterol than hCRISP3. The secretion profile of different CRISPs also showed that the ratio of secreted to cell-associated proteins was highest for hCRISP3. CONCLUSION: Our study identifies PMCA4b as a target of multiple mammalian CRISPs and unravels a new role of CRISPs in regulating calcium homeostasis. Differences in the interaction of different CRISPs with cholesterol may regulate their enrichment in the lipid rafts and redistribution in the membrane post-capacitation, thereby affecting their interaction with PMCA4b.

Genomic and computational analysis of four novel variants of MPL gene in Congenital Amegakaryocytic Thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare, genetic, autosomal recessive disorder characterized by severe thrombocytopenia, due to inefficient bone marrow megakaryopoiesis eventually leading to aplasia. Majority of the cases are due to homozygous or compound heterozygous mutations in MPL gene encoding for thrombopoietin (THPO) receptor protein. CAMT can be diagnosed at early phase of life, with major complication of transfusion dependency and hematopoietic transplantation as only curative treatment. We have investigated the sequence variations in MPL gene of 7 bone marrow failure (BMF) subjects, who presented with clinically diverse phenotypes, through next generation sequencing (NGS). Plasma THPO levels were estimated using ELISA. Insilico sequence and structure-based analyses were performed to understand the structural and functional implications of mutations, identified through NGS. We studied 7 CAMT subjects suspected of BMF, who presented with severe thrombocytopenia followed by pancytopenia, bleeding manifestation and physical anomalies. The plasma THPO levels were significantly elevated (p<0.05) in all the cases. Molecular analysis by NGS identified 9 genomic mutations in MPL gene. These included 7 non-synonymous substitution, 1 nonsense substitution and 1 in-del mutations, of which 4 are novel mutations. Insilico analysis predicted damaging effects on THPO-R and its reduced affinity for THPO for all the identified mutations. CAMT is a rare disorder with diverse clinical phenotypes and diagnosis is challenging. The elevated plasma THPO levels should be considered for the primary diagnosis and prognosis of the disease. However, molecular analysis of MPL gene is important for the diagnosis and management of the disease through genetic counselling. Though the cytokines, THPO-R agonist are used for the treatment of CAMT, HSCT is the only curative therapy.

Sustained release hydrogel for durable locoregional chemoimmunotherapy for BRAF-mutated melanoma.

The wide prevalence of BRAF mutations in diagnosed melanomas drove the clinical advancement of BRAF inhibitors in combination with immune checkpoint blockade for treatment of advanced disease. However, deficits in therapeutic potencies and safety profiles motivate the development of more effective strategies that improve the combination therapy's therapeutic index. Herein, we demonstrate the benefits of a locoregional chemoimmunotherapy delivery system, a novel thermosensitive hydrogel comprised of gelatin and Pluronic® F127 components already widely used in humans in both commercial and clinical products, for the co-delivery of a small molecule BRAF inhibitor with immune checkpoint blockade antibody for the treatment of BRAF-mutated melanoma. In vivo evaluation of administration route and immune checkpoint target effects revealed intratumoral administration of antagonistic programmed cell death protein 1 antibody (aPD-1) lead to potent antitumor therapy in combination with BRAF inhibitor vemurafenib. The thermosensitive F127-g-Gelatin hydrogel that was evaluated in multiple murine models of BRAF-mutated melanoma that facilitated prolonged local drug release within the tumor (>1 week) substantially improved local immunomodulation, tumor control, rates of tumor response, and animal survival. Thermosensitive F127-g-Gelatin hydrogels thus improve upon the clinical benefits of vemurafenib and aPD-1 in a locoregional chemoimmunotherapy approach for the treatment of BRAF-mutated melanoma.

Pro-inflammatory cytokines IL-1α, IL-6 and TNF-α in major depressive disorder: Sex-specific associations with psychological symptoms.

The pro-inflammatory cytokines IL-1α, IL-6 and TNF-α are associated with major depressive disorder, psychological distress, cardiovascular health and obesity. However, there is limited research that has examined multiple associations between these variables, particularly among individuals with major depressive disorder who are treatment free, in comparison with a control cohort, and including analyses of sex differences. In this study, data were analysed from 60 individuals with major depressive disorder and 60 controls, including plasma IL-1α, IL-6 and TNF-α, adiposity measures (body mass index, waist circumference), cardiovascular health indices (blood pressure, heart rate) and psychological symptoms (depressive severity, anxiety, hostility, stress). The cytokines were compared by group and sex and correlated with measures of adiposity, cardiovascular health indices and psychological health. Plasma IL-1α and IL-6 were higher in major depressive disorder group versus control, but with a sex interaction for IL-6, with this group difference only among females. TNF-α did not differ between groups. IL-1α and IL-6 correlated with depressive severity, anxiety, hostility and stress, whereas TNF-α correlated only with anxiety and hostility. Psychopathology was associated with IL-1α in males only and with IL-6 and TNF-α in females only. None of the cytokines correlated with body mass index, waist circumference, blood pressure or heart rate. The result of group by sex interaction for IL-6 and sex-specific associations between pro-inflammatory cytokines and psychometrics could be aetiologically important in depression interventions and treatments for females versus males, warranting further investigation.

Methodological reflections on the recruitment and engagement of people for cancer screening research in Wales.

Cancer is a major threat to public health and the second leading cause of death globally. Population-based cancer screening is an effective way to improve the early detection of a cancer and reduce mortality. Factors associated with participation in cancer screening have been increasingly explored in research. The challenges to undertaking such research are evident, but there is little discussion about how to address such challenges. This article discusses methodological issues associated with the recruitment and engagement of participants in research, drawing upon our experience of undertaking research exploring the support needs of people residing in Newport West, Wales, to participate in breast, bowel, and cervical screening programs. Four key areas were addressed: sampling issues, language barriers, IT issues, and time demand for participation. The paper highlights the importance of ongoing community engagement, the provision of appropriate study materials, and the adaption to different data collection modes to meet participants' needs to participate in research, thus enabling people who are usually excluded from research to have a voice and make a significant contribution to research.

FSHR activation through small molecule modulators: Mechanistic insights from MD simulations.

Follicle-stimulating hormone receptor (FSHR) is a glycoprotein hormone receptor that plays a vital role in reproduction, cancer progression and osteoporosis. Owing to its therapeutic importance, several small molecule modulators have been identified by researchers through high throughput studies that usually include virtual screening of chemical libraries followed by in vitro validation through radio-ligand binding assays, cAMP accumulation and luciferase-based luminescence assays. The binding site of these modulators and structural changes that accompany modulator binding remains elusive. Here, we address these aspects through molecular docking and MD simulations on well-studied FSHR modulators and comparing the domain motions between agonist/FSH bound and antagonist bound FSHR structures. It was observed that agonist and antagonist modulators bind to the same site, but interact with distinct residues in transmembrane domain(TMD). FSHR(TMD) residues Ile522, Ala595, Ile602 and Val604 were found to interact only with agonist. Notably, these residues are conserved in the close homolog luteinizing hormone/choriogonadotropin receptor (LHCGR) and participate in interaction with its agonist Org43553. We observed distinctly prominent domain motions and conformational changes in TM helices 3, 4 and 6 for agonist bound FSHR structure. These structural changes have also been reported for LHCGR, and few GPCR members suggesting an important and well conserved mechanism of GPHR activation that could be exploited for design of novel modulators.

Adhesion analysis via a tumor vasculature-like microfluidic device identifies CD8+ T cells with enhanced tumor homing to improve cell therapy.

CD8+ T cell recruitment to the tumor microenvironment is critical for the success of adoptive cell therapy (ACT). Unfortunately, only a small fraction of transferred cells home to solid tumors. Adhesive ligand-receptor interactions have been implicated in CD8+ T cell homing; however, there is a lack of understanding of how CD8+ T cells interact with tumor vasculature-expressed adhesive ligands under the influence of hemodynamic flow. Here, the capacity of CD8+ T cells to home to melanomas is modeled ex vivo using an engineered microfluidic device that recapitulates the hemodynamic microenvironment of the tumor vasculature. Adoptively transferred CD8+ T cells with enhanced adhesion in flow in vitro and tumor homing in vivo improve tumor control by ACT in combination with immune checkpoint blockade. These results show that engineered microfluidic devices can model the microenvironment of the tumor vasculature to identify subsets of T cells with enhanced tumor infiltrating capabilities, a key limitation in ACT.

Food addiction, hormones and blood biomarkers in humans: A systematic literature review.

BACKGROUND: Food addiction may play a role in rising obesity rates in connection with obesogenic environments and processed food availability, however the concept of food addiction remains controversial. While animal studies show evidence for addictive processes in relation to processed foods, most human studies are psychologically focussed and there is a need to better understand evidence for biological mechanisms of food addiction in humans. Several key hormones are implicated in models of food addiction, due to their key roles in feeding, energy metabolism, stress and addictive behaviours. This systematic literature review examines evidence for relationships between food addiction, hormones and other blood biomarkers. METHODS: A series of literature searches was performed in Scopus, PsychInfo, MedLine, ProQuest, CINAHL and Web of Science. A total of 3111 articles were found, of which 1045 were duplicates. Articles were included if they contained a psychometric measurement of food addiction, such as the Yale Food Addiction Scale, as well as addressed the association between FA and hormones or blood biomarkers in humans. Articles were assessed for eligibility by two independent reviewers. RESULTS: Sixteen studies were identified that examined relationships between food addiction and blood biomarkers, published between 2015 and 2021. Significant findings were reported for leptin, ghrelin, cortisol, insulin and glucose, oxytocin, cholesterol, plasma dopamine, thyroid stimulating hormone (TSH), haemoglobin A1c (HbA1c), triglyceride (TG), amylin, tumour necrosis factor alpha (TNF- α) and cholecystokinin (CCK). Methodological issues included small sample sizes and variation in obesity status, sex and mental health-related comorbidities. Due to methodological limitations, definite connections between FA, hormones and other blood biomarkers cannot yet be determined. CONCLUSION: This systematic review identified preliminary evidence linking FA symptoms to hormones and other blood biomarkers related to feeding, addiction, and stress. However, due to the small number of studies and methodological limitations, further research is needed to evaluate biopsychosocial models of FA and to resolve controversies.

Visualizing Inside Conduits-Intraoperative Screening of Grafts by Optical Coherence Tomography.

PURPOSE: Saphenous vein graft (SVG) failure is a complex phenomenon, with technical, biologic, and local factors contributing to early and medium- and long-term failure after coronary artery bypass graft. Both technical and conduit factors may have significant impact on early SVG failure. DESCRIPTION: We review the complex factors that play a pathogenic role in SVG failure, followed by review of the existing literature on potential utility of high-definition optical coherence tomography (OCT) in comprehensive intraoperative assessment of SVGs. EVALUATION: We describe a new technique for intraoperative acquisition of OCT images in the harvested SVGs and introduce a classification system for pathologic processes that can be detected in the harvested SVG conduits by OCT. CONCLUSIONS: The potential impact on early graft failure of the exclusion of segments of SVGs that are less than optimal (ie, containing fibroatheroma, retained thrombus, sclerotic valves, or procurement injury) will be examined in the randomized controlled OCTOCAB (Intraoperative Optical Coherence Tomography of the Saphenous Vein Conduit in Patients Undergoing Coronary Artery Bypass Surgery) trial.

Targeting vasoactive intestinal peptide-mediated signaling enhances response to immune checkpoint therapy in pancreatic ductal adenocarcinoma.

A paucity of effector T cells within tumors renders pancreatic ductal adenocarcinoma (PDAC) resistant to immune checkpoint therapies. While several under-development approaches target immune-suppressive cells in the tumor microenvironment, there is less focus on improving T cell function. Here we show that inhibiting vasoactive intestinal peptide receptor (VIP-R) signaling enhances anti-tumor immunity in murine PDAC models. In silico data mining and immunohistochemistry analysis of primary tumors indicate overexpression of the neuropeptide vasoactive intestinal peptide (VIP) in human PDAC tumors. Elevated VIP levels are also present in PDAC patient plasma and supernatants of cultured PDAC cells. Furthermore, T cells up-regulate VIP receptors after activation, identifying the VIP signaling pathway as a potential target to enhance T cell function. In mouse PDAC models, VIP-R antagonist peptides synergize with anti-PD-1 antibody treatment in improving T cell recruitment into the tumors, activation of tumor-antigen-specific T cells, and inhibition of T cell exhaustion. In contrast to the limited single-agent activity of anti-PD1 antibodies or VIP-R antagonist peptides, combining both therapies eliminate tumors in up to 40% of animals. Furthermore, tumor-free mice resist tumor re-challenge, indicating anti-cancer immunological memory generation. VIP-R signaling thus represents a tumor-protective immune-modulatory pathway that is targetable in PDAC.

Elicitation of stem-like CD8+ T cell responses via lymph node-targeted chemoimmunotherapy evokes systemic tumor control.

BACKGROUND: Tumor-draining lymph nodes (TdLNs) are critical in the regulation of local and systemic antitumor T cell immunity and are implicated in coordinating responses to immunomodulatory therapies. METHODS: Biomaterial nanoparticles that deliver chemotherapeutic drug paclitaxel to TdLNs were leveraged to explore its effects in combination and immune checkpoint blockade (ICB) antibody immunotherapy to determine the benefit of TdLN-directed chemoimmunotherapy on tumor control. RESULTS: Accumulation of immunotherapeutic drugs in combination within TdLNs synergistically enhanced systemic T cell responses that led to improved control of local and disseminated disease and enhanced survival in multiple murine breast tumor models. CONCLUSIONS: These findings suggest a previously underappreciated role of secondary lymphoid tissues in mediating effects of chemoimmunotherapy and demonstrate the potential for nanotechnology to unleashing drug synergies via lymph node targeted delivery to elicit improved response of breast and other cancers.

Opportunities for Nitric Oxide in Potentiating Cancer Immunotherapy.

Despite nearly 30 years of development and recent highlights of nitric oxide (NO) donors and NO delivery systems in anticancer therapy, the limited understanding of exogenous NO's effects on the immune system has prevented their advancement into clinical use. In particular, the effects of exogenously delivered NO differing from that of endogenous NO has obscured how the potential and functions of NO in anticancer therapy may be estimated and exploited despite the accumulating evidence of NO's cancer therapy-potentiating effects on the immune system. After introducing their fundamentals and characteristics, this review discusses the current mechanistic understanding of NO donors and delivery systems in modulating the immunogenicity of cancer cells as well as the differentiation and functions of innate and adaptive immune cells. Lastly, the potential for the complex modulatory effects of NO with the immune system to be leveraged for therapeutic applications is discussed in the context of recent advancements in the implementation of NO delivery systems for anticancer immunotherapy applications. SIGNIFICANCE STATEMENT: Despite a 30-year history and recent highlights of nitric oxide (NO) donors and delivery systems as anticancer therapeutics, their clinical translation has been limited. Increasing evidence of the complex interactions between NO and the immune system has revealed both the potential and hurdles in their clinical translation. This review summarizes the effects of exogenous NO on cancer and immune cells in vitro and elaborates these effects in the context of recent reports exploiting NO delivery systems in vivo in cancer therapy applications.

Management of Spontaneous Neck Hematoma in Patients on Anticoagulant Therapy: Our Experience from a Tertiary Care Center.

Spontaneous neck hematoma is a rare but life threatening condition which poses a challenge in clinical decision making. With the unsupervised outpatient use of oral anticoagulants, including newer generation ones and the thromboprophylaxis in  Covid-19 treatment protocol, the risk of developing spontaneous neck hematoma is high. In this context, our case series aimed at studying the clinicopathological profile, treatment options and outcome in patients presented with spontaneous neck hematoma in a tertiary care center. A retrospective chart review was done between the years 2010-2021, and three cases of spontaneous neck hematoma associated with anticoagulation therapy were identified. Based on our experience, we recommend a custom tailored approach to management of spontaneous neck hematoma.

Tumor Vascular Remodeling Affects Molecular Dissemination to Lymph Node and Systemic Leukocytes.

Angiogenic and lymphangiogenic remodeling has long been accepted as a hallmark of cancer development and progression; however, the impacts of this remodeling on immunological responses, which are paramount to the responses to immunotherapeutic treatments, are underexplored. As immunotherapies represent one of the most promising new classes of cancer therapy, in this study, we explore the effects of angiogenic and lymphangiogenic normalization on dissemination of molecules injected into the tumor microenvironment to immune cells in lymph nodes draining the tumor as well as in systemically distributed tissues. A system of fluorescent tracers, size-matched to biomolecules of interest, was implemented to track different mechanisms of tumor transport and access to immune cells. This revealed that the presence of a tumor, and either angiogenic or lymphangiogenic remodeling, altered local retention of model biomolecules, trended toward normalizing dissemination to systemic organs, and modified access to lymph node-resident immune cells in manners dependent on mechanism of transport. More specifically, active cell migration by skin-derived antigen presenting cells was enhanced by both the presence of a tumor and lymphangiogenic normalization, while both angiogenic and lymphangiogenic normalization restored patterns of immune cell access to passively draining species. As a whole, this work uncovers the potential ramifications of tumor-induced angiogenesis and lymphangiogenesis, along with impacts of interrogation into these pathways, on access of tumor-derived species to immune cells. Impact Statement Angiogenic and lymphangiogenic normalization strategies have been utilized clinically to interrogate tumor vasculature with some success. In the age of immunotherapy, the impacts of these therapeutic interventions on immune remodeling are unclear. This work utilizes mouse models of angiogenic and lymphangiogenic normalization, along with a system of fluorescently tagged tracers, to uncover the impacts of angiogenesis and lymphangiogenesis on access of tumor-derived species to immune cell subsets within various organs.