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BACKGROUND/AIM: Cigarette smoke has been shown to induce a phenotype in humans known as "acquired cystic fibrosis". This occurs because the cystic fibrosis transmembrane conductance regulator (CFTR) functions are impaired systemically due to the deleterious effects of smoke components. Elucidation of cigarette smoke effects on the tracheal epithelium is important. The aim of this study was to develop an ex vivo sheep tracheal model to investigate tracheal ion function. In this model, the epithelial sodium channel (ENaC) is inhibited after exposure to cigarette smoke extract (CSE) as a proof of principle. MATERIALS AND METHODS: Tracheas were isolated from healthy sheep and the tracheal epithelium was surgically excised. Tissues were mounted in Ussing chambers and the short circuit current (Isc) was measured after incubation with 5% CSE in PBS or PBS alone for 30 min. The function of ENaC was investigated by the addition of amiloride (10-5M) apically. Western blot analysis was performed to assess differences in ENaC quantity after CSE exposure. Some specimens were stained with H&E for detection of histological alterations. RESULTS: The amiloride effect on normal epithelium led to a significant decrease in Isc [ΔI=33±5.92 µA/cm2; p<0.001 versus control experiments (ΔI=1.44±0.71 µA/cm2)]. After incubation with CSE, ENaC Isc was significantly reduced (ΔI=14.80±1.96 µA/cm2; p<0.001). No differences in αENaC expression were observed between CSE-exposed and normal tracheal epithelium. Histological images post CSE incubation revealed decreases in the height of the epithelium, with basal cell hyperplasia and loss of ciliated cells. CONCLUSION: Reduced ENaC inhibition by amiloride after CSE incubation could be due to alterations in the tracheal epithelium.
Subject(s)
Epithelial Sodium Channels , Trachea , Animals , Epithelial Sodium Channels/metabolism , Sheep , Trachea/metabolism , Trachea/drug effects , Trachea/pathology , Pilot Projects , Smoke/adverse effects , Amiloride/pharmacology , Respiratory Mucosa/metabolism , Respiratory Mucosa/drug effects , Respiratory Mucosa/pathology , Epithelium/drug effects , Epithelium/metabolism , Epithelium/pathologyABSTRACT
In patients with lung cancer (LC), understanding factors that impact the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike antibody (SAb) titers over time is critical, but challenging, due to evolving treatments, infections, vaccinations, and health status. The objective was to develop a time-dependent regression model elucidating individual contributions of factors influencing SAb levels in LC patients using a prospective, longitudinal, multi-institutional cohort study initiated in January 2021. The study evaluated 296 LC patients-median age 69; 55% female; 50% stage IV. Blood samples were collected every three months to measure SAb levels using FDA-approved ELISA. Asymptomatic and unreported infections were documented through measurement of anti-nucleocapsid Ab levels (Meso Scale Discovery). Associations between clinical characteristics and titers were evaluated using a time-dependent linear regression model with a generalized estimating equation (GEE), considering time-independent variables (age, sex, ethnicity, smoking history, histology, and stage) and time-dependent variables (booster vaccinations, SARS-CoV-2 infections, cancer treatment, steroid use, and influenza vaccination). Significant time-dependent effects increasing titer levels were observed for prior SARS-CoV-2 infection (p < 0.001) and vaccination/boosters (p < 0.001). Steroid use (p = 0.043) and chemotherapy (p = 0.033) reduced titer levels. Influenza vaccination was associated with increased SAb levels (p < 0.001), independent of SARS-CoV-2 vaccine boosters. Prior smoking significantly decreased titers in females (p = 0.001). Age showed no association with titers. This GEE-based linear regression model unveiled the nuanced impact of multiple variables on patient anti-spike Ab levels over time. After controlling for the major influences of vaccine and SARS-CoV-2 infections, chemotherapy and steroid use were found to have negatively affected titers. Smoking in females significantly decreased titers. Surprisingly, influenza vaccinations were also significantly associated, likely indirectly, with improved SARS-CoV-2 titers.
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The immune system encodes information about the severity of a pathogenic threat in the quantity and type of memory cells it forms. This encoding emerges from lymphocyte decisions to maintain or lose self-renewal and memory potential during a challenge. By tracking CD8+ T cells at the single-cell and clonal lineage level using time-resolved transcriptomics, quantitative live imaging, and an acute infection model, we find that T cells will maintain or lose memory potential early after antigen recognition. However, following pathogen clearance, T cells may regain memory potential if initially lost. Mechanistically, this flexibility is implemented by a stochastic cis-epigenetic switch that tunably and reversibly silences the memory regulator, TCF1, in response to stimulation. Mathematical modeling shows how this flexibility allows memory T cell numbers to scale robustly with pathogen virulence and immune response magnitudes. We propose that flexibility and stochasticity in cellular decisions ensure optimal immune responses against diverse threats.
Subject(s)
CD8-Positive T-Lymphocytes , Memory T Cells , Epigenesis, Genetic , Clone Cells , Immunologic Memory , Cell DifferentiationABSTRACT
Virus specific PD-1+ TCF-1+ TOX+ stem-like CD8+ T cells are essential for maintaining T cell responses during chronic infection and are also critical for PD-1 directed immunotherapy. In this study we have used the mouse model of chronic LCMV infection to examine when these virus specific stem-like CD8+ T cells are generated during the course of chronic infection and what is the role of antigen in maintaining the stem-like program. We found that these stem-like CD8+ T cells are generated early (day 5) during chronic infection and that antigen is essential for maintaining their stem-like program. This early generation of stem-like CD8+ T cells suggested that the fate commitment to this cell population was agnostic to the eventual outcome of infection and the immune system prepares a priori for a potential chronic infection. Indeed, we found that an identical virus specific stem-cell like CD8+ T cell population was also generated during acute LCMV infection but these cells were lost once the virus was cleared. To determine the fate of these early PD-1+TCF-1+TOX+ stem-like CD8+ T cells that are generated during both acute and chronic LCMV infection we set up two reciprocal adoptive transfer experiments. In the first experiment we transferred day 5 stem-like CD8+ T cells from chronically infected into acutely infected mice and examined their differentiation after viral clearance. We found that these early stem-like CD8+ T cells downregulated canonical markers of the chronic stem-like CD8+ T cells and expressed markers (CD127 and CD62L) associated with central memory CD8+ T cells. In the second experiment, we transferred day 5 stem-like cells from acutely infected mice into chronically infected mice and found that these CD8+ T cells could function like resource cells after transfer into a chronic environment by generating effector CD8+ T cells in both lymphoid and non-lymphoid tissues while also maintaining the number of stem-like CD8+ T cells. These findings provide insight into the generation and maintenance of virus specific stem-like CD8+ T cells that play a critical role in chronic viral infection. In particular, our study highlights the early generation of stem-like CD8+ T cells and their ability to adapt to either an acute or chronic infection. These findings are of broad significance since these novel stem-like CD8+ T cells play an important role in not only viral infections but also in cancer and autoimmunity.
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BACKGROUND: DNA methylation, one of the most stable forms of epigenetic modification is associated with the development and progression of coronary artery disease (CAD). Our previously reported study on epigenome-wide microarray analysis showed significantly methylated CpG sites. Top 5 significant CpGs from HLA gene were selected and analysed by Pyrosequencing (PSQ) to determine their association with severity of CAD. METHODS: Blood samples of 50-age matched angiographically CAD positive male cases with 50 angiographically CAD negative male controls were subjected to lipid profile estimation and PSQ for methylation level analysis. Findings and subgroup analysis were evaluated by Mann-Whitney U; Kruskal-Wallis' rank test and two-way ANOVA by MedCalc (v19.6). RESULTS: Methylation levels in HLA-DQA1 for cg10217052 was 78.5 (37-85) and 76.5 (24-84); cg09411910 was 81 (72.0 to 93.0) and 81.5 (50.0 to 89.0) in cases and controls respectively. Levels in HLA-DQB1-cg03344051, were 28.88 + 9.41 for cases and 30.36 + 9.37 in controls. For HLA-DRB1-cg07889003, levels in cases and controls were 15.5 (5.00-39.00) and 10.5 (5.00-29.0); while in cg08269402 were 52 (16-65) and 42.5 (17-61) respectively. No association was observed between methylation levels and lipid profile. cg03344051, cg07889003 and cg08269402 were significantly differentiated in double or triple vessel disease (DVD or TVD) as compared to single vessel disease (SVD) suggesting an increase in the extent of methylation with the increase in CAD severity. CONCLUSION: The present study shows significant increase in the extent of methylation in 3 CpG sites in DVD/TVD cases as compared to SVD cases. Additionally, a novel site, cg07889003 identified in our discovery phase has shown association with the severity of CAD.
Subject(s)
Coronary Artery Disease , Vascular Diseases , Humans , Male , Coronary Artery Disease/genetics , DNA Methylation/genetics , Epigenesis, Genetic/genetics , LipidsABSTRACT
Peritoneal dialysis (PD) and prolonged exposure to PD fluids (PDF) induce peritoneal membrane (PM) fibrosis and hypervascularity, leading to functional PM degeneration. 2-deoxy-glucose (2-DG) has shown potential as PM antifibrotic by inhibiting hyper-glycolysis induced mesothelial-to-mesenchymal transition (MMT). We investigated whether administration of 2-DG with several PDF affects the permeability of mesothelial and endothelial barrier of the PM. The antifibrotic effect of 2-DG was confirmed by the gel contraction assay with embedded mesothelial (MeT-5A) or endothelial (EA.hy926) cells cultured in Dianeal® 2.5 % (CPDF), BicaVera® 2.3 % (BPDF), Balance® 2.3 % (LPDF) with/without 2-DG addition (0.2 mM), and qPCR for αSMA, CDH2 genes. Moreover, 2-DG effect was tested on the permeability of monolayers of mesothelial and endothelial cells by monitoring the transmembrane resistance (RTM), FITC-dextran (10, 70 kDa) diffusion and mRNA expression levels of CLDN-1 to -5, ZO1, SGLT1, and SGLT2 genes. Contractility of MeT-5A cells in CPDF/2-DG was decreased, accompanied by αSMA (0.17 ± 0.03) and CDH2 (2.92 ± 0.29) gene expression fold changes. Changes in αSMA, CDH2 were found in EA.hy926 cells, though αSMA also decreased under LPDF/2-DG incubation (0.42 ± 0.02). Overall, 2-DG mitigated the PDF-induced alterations in mesothelial and endothelial barrier function as shown by RTM, dextran transport and expression levels of the CLDN-1 to -5, ZO1, and SGLT2. Thus, supplementation of PDF with 2-DG not only reduces MMT but also improves functional permeability characteristics of the PM mesothelial and endothelial barrier.
Subject(s)
Peritoneal Dialysis , Peritoneal Fibrosis , Humans , Sodium-Glucose Transporter 2/metabolism , Deoxyglucose/pharmacology , Deoxyglucose/metabolism , Endothelial Cells , Peritoneal Dialysis/adverse effects , Peritoneum/pathology , Dialysis Solutions/metabolism , Dialysis Solutions/pharmacology , Peritoneal Fibrosis/metabolism , Glucose/metabolism , Epithelial Cells/metabolism , Cells, CulturedABSTRACT
INTRODUCTION: Peritoneal dialysis (PD) is a life maintaining treatment in patients with end-stage renal disease. Its chronic application leads to peritoneal mesothelial layer denudation and fibrotic transformation along with vascular activation of inflammatory pathways. The impact of different PD fluids (PDF) on mesothelial and endothelial cell function and repair mechanisms are not comprehensively described. MATERIALS AND METHODS: Mesothelial (MeT-5A) and endothelial cells (EA.hy926) were cultured in 1:1 ratio with cell medium and different PDF (icodextrin-based, amino acid-based, and glucose-based). Cell adhesion, cell migration, and cell proliferation in 2D and spheroid formation and collagen gel contraction assays in 3D cell cultures were performed. RESULTS: Cell proliferation and cell-mediated gel contraction were both significantly decreased in all conditions. 3D spheroid formation was significantly reduced with icodextrin and amino acid PDF, but unchanged with glucose PDF. Adhesion was significantly increased by amino acid PDF in mesothelial cells and decreased by icodextrin and amino acid PDF in endothelial cells. Migration capacity was significantly decreased in mesothelial cells by all three PDF, while endothelial cells remained unaffected. CONCLUSIONS: In 3D phenotypes the effects of PDF are more uniform in both mesothelial and endothelial cells, mitigating spheroid formation and gel contraction. On the contrary, effects on 2D phenotypes are more uniform in the icodextrin and amino acid PDF as opposed to glucose ones and affect mesothelial cells more variably. 2D and 3D comparative assessments of PDF effects on the main peritoneal membrane cell barriers, the mesothelial and endothelial, could provide useful translational information for PD studies.
Subject(s)
Endothelial Cells , Peritoneal Dialysis , Humans , Icodextrin/metabolism , Icodextrin/pharmacology , Dialysis Solutions/adverse effects , Dialysis Solutions/metabolism , Peritoneum/metabolism , Phenotype , Amino Acids/metabolism , Amino Acids/pharmacology , Glucose/pharmacology , Glucose/metabolism , Cells, Cultured , Epithelial CellsABSTRACT
This study reports that most patients with NSCLC had a significant increase in the nAb response to the currently circulating Omicron variants after bivalent booster vaccination and had Ab titers comparable to healthy participants. Interestingly, though the durability of the nAb response persisted in most of the healthy participants, patients with NSCLC had significantly reduced nAb titers after 4-6 months of vaccination. Our data highlight the importance of COVID-19 bivalent booster vaccination as the standard of care for patients with NSCLC given the evolution of new variants of concern.
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Arterial lines are routinely used for hemodynamic monitoring and blood sampling in the operating room and in cardiac surgery intensive care unit. The complications related to arterial line insertion are very low; the knowledge of the relevant artery anatomy, skills and the experience of the operator and selection of a right size cannula plays a vital role in reducing morbidity related to arterial line insertion. We describe extensive superficial and deep necrosis of lower limb following arterial cannula insertion in a preterm neonate undergoing arterial switch procedure and discuss measures to prevent such a complication.
Subject(s)
Arterial Switch Operation , Transposition of Great Vessels , Infant, Newborn , Humans , Arterial Switch Operation/adverse effects , Transposition of Great Vessels/surgery , Transposition of Great Vessels/complications , Arteries , Lower Extremity , CatheterizationABSTRACT
Patients diagnosed with lung cancer (LC) exhibit increased susceptibility to SARS-CoV-2 infection. Rodilla et al. monitor the levels of plasma anti-nucleocapsid antibodies within a cohort of fully vaccinated LC patients and reveal that the actual infection rate is nearly twice the documented rate, indicating a significant prevalence of unreported cases.
Subject(s)
COVID-19 , Lung Neoplasms , Humans , SARS-CoV-2 , Nucleocapsid , Immunologic Tests , COVID-19 TestingABSTRACT
BACKGROUND: Malignant pleural mesothelioma (MPM), a rare and aggressive pleural tumor, has significant histological and molecular heterogeneity. Primary Cilium (PC), an organelle of emerging importance in malignancies, has been scarcely investigated in MPM. A critical molecular complex for the PC function is the BBSome and here we aimed at assessing its expression patterns in ordinary 2D and spheroid 3D cell cultures. METHODS: A human benign mesothelial cell line (MeT-5A), MPM cell lines (M14K, epithelioid MPM; MSTO, biphasic MPM), and primary MPM cells (pMPM) were used. Primers specific for the human BBS1, 2, 4, 5, 7, 9, 18 transcripts were designed, and quantitative real-time PCR (qRT-PCR) was done with ß-actin as the gene of reference. The relative gene expression across 2D and 3D cultures was analyzed by the expression factor (mean of 1/ΔCt values). With the 2-∆∆Ct method the gene expression fold changes were assessed from qRT-PCR data. Molecular changes using the PC-modulating drugs ammonium sulfate (AS) and lithium chloride (LC) were also determined. RESULTS: PC was present in all cells used in the study at approximately 15% of the observed area. BBSome transcripts were differentially expressed in different dimensions of cell culture (2D vs. 3D) in all cell lines and pMPM. Treatment with AS and LC affected the expression of the ciliary BBS2 and BBS18 genes in the benign as well as in the MPM cells. CONCLUSIONS: These data indicate distinct BBSome molecular profiles in human benign and MPM cells cultured in 2D and 3D dimensions and support the notion that PC genes should be investigated as potential MPM therapeutic targets.
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Blast pathogen, Magnaporthe spp., that infects ancient millet crops such pearl millet, finger millet, foxtail millet, barnyard millet, and rice was isolated from different locations of blast hotspots in India using single spore isolation technique and 136 pure isolates were established. Numerous growth characteristics were captured via morphogenesis analysis. Among the 10 investigated virulent genes, we could amplify MPS1 (TTK Protein Kinase) and Mlc (Myosin Regulatory Light Chain edc4) in majority of tested isolates, regardless of the crop and region where they were collected, indicating that these may be crucial for their virulence. Additionally, among the four avirulence (Avr) genes studied, Avr-Pizt had the highest frequency of occurrence, followed by Avr-Pia. It is noteworthy to mention that Avr-Pik was present in the least number of isolates (9) and was completely absent from the blast isolates from finger millet, foxtail millet, and barnyard millet. A comparison at the molecular level between virulent and avirulent isolates indicated observably large variation both across (44%) and within (56%) them. The 136 Magnaporthe spp isolates were divided into four groups using molecular markers. Regardless of their geographic distribution, host plants, or tissues affected, the data indicate that the prevalence of numerous pathotypes and virulence factors at the field level, which may lead to a high degree of pathogenic variation. This research could be used for the strategic deployment of resistant genes to develop blast disease-resistant cultivars in rice, pearl millet, finger millet, foxtail millet, and barnyard millet.
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Objective: Snake bite-induced elevation of serum LDH and CRP-1 is considered as useful biomarkers of hemotoxic. The snake venom contains proteins and may result in various envenomation such as bleeding, inflammation, and pain, cytotoxic, cardiotoxic, or neurotoxic effects. This in silico study was aimed to screen the snake venom proteins and to find out the most interactive hemotoxic venom protein against LDH and CRP-1 proteins as biomarkers. Materials and Methods: To validate the hypothesis of the prospective interaction of snake venom proteins, molecular docking analysis was used in the current work by deploying a cutting-edge docking program. Snake venom peptides were screened from literature and both peptide as well as target protein were obtained from PDB. HDOCK online server was used for the molecular docking analysis of target proteins with hemotoxic snake venom peptides. Further, the toxicity properties of each docked complex of target proteins were subjected for ADME/T analysis. Results: The selected snake venom peptides were subjected to molecular docking study and the results generated from computational-based approach reveals that all the hematotoxin snake venom proteins are interactive with LDH and CRP-1 peptide. Further, this study indicates that snake venom metalloproteinase (SVMPS) peptide may be considered as the best interactive protein with both LDH and CRP-1 proteins; also, ADME/T screening revealed that all docked complex are safe and follow toxicity properties. Conclusion: This in silico study clearly shows that the greatest interaction of SVMPS peptide with LDH and CRP-1 may be due to strong binding in the active site of the target proteins LDH and CRP-1 with SVMPS. Results, further, confirmed LDH and CRP-1 as potential biomarkers against hemotoxic snake venoms. This study should be validated by in vitro and in vivo analysis as well as specific species snake venom should be assessed. For further studies, SVMPS can be consider as therapeutic point of view.
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INTRODUCTION: Primary cilium (PC) is a single non-motile antenna-like organelle composed of a microtubule core axon originating from the mother centriole of the centrosome. The PC is universal in all mammalian cells and protrudes to the extracellular environment receiving mechanochemical cues that it transmits in the cell. AIM: To investigate the role of PC in mesothelial malignancy in the context of two-dimensional (2D) and three-dimensional (3D) phenotypes. MATERIALS AND METHODS: The effect of pharmacological deciliation [using ammonium sulphate (AS) or chloral hydrate (CH)] and PC elongation [using lithium chloride (LC)] on cell viability, adhesion, and migration (2D cultures) as well as in mesothelial sphere formation, spheroid invasion and collagen gel contraction (3D cultures) was investigated in benign mesothelial MeT-5A cells and in malignant pleural mesothelioma (MPM) cell lines, M14K (epithelioid) and MSTO (biphasic), and primary malignant pleural mesothelioma cells (pMPM). RESULTS: Pharmacological deciliation or elongation of the PC significantly affected cell viability, adhesion, migration, spheroid formation, spheroid invasion and collagen gel contraction in MeT-5A, M14K, MSTO cell lines and in pMPM cells compared to controls (no drug treatment). CONCLUSIONS: Our findings indicate a pivotal role of the PC in functional phenotypes of benign mesothelial cells and MPM cells.
Subject(s)
Lung Neoplasms , Mesothelioma, Malignant , Mesothelioma , Pleural Neoplasms , Animals , Mesothelioma, Malignant/pathology , Mesothelioma/metabolism , Pleura/metabolism , Pleura/pathology , Cilia/metabolism , Pleural Neoplasms/metabolism , Cell Line, Tumor , Lung Neoplasms/pathology , MammalsABSTRACT
Poultry (Gallus domesticus) farming plays an important role as an income generating enterprise in a developing country like Nepal, contributing more than 4% to the national Gross Domestic Product (GDP). Newcastle Disease (ND) is a major poultry disease affecting both commercial and backyard poultry production worldwide. There were more than 90 reported ND outbreaks in Nepal in 2018 with over 74,986 birds being affected. ND is responsible for over 7% of total poultry mortality in the country. Recent outbreaks of ND in 2021 affected many farms throughout Nepal and caused massive loss in poultry production. ND is caused by a single-stranded ribonucleic acid (RNA) virus that presents very similar clinical symptoms as Influenza A (commonly known as bird flu) adding much complexity to clinical disease identification and intervention. We conducted a nationwide ND and Influenza A (IA) prevalence study, collecting samples from representative commercial and backyard poultry farms from across the major poultry production hubs of Nepal. We used both serological and molecular assessments to determine disease exposure history and identification of strains of ND Virus (NDV). Of the 40 commercial farms tested, both NDV (n = 28, 70%) and IAV (n = 11, 27.5%) antibodies were detected in majority of the samples. In the backyard farms (n = 36), sero-prevalence of NDV and IAV were 17.5% (n = 7) and 7.5% (n = 3) respectively. Genotype II NDV was present in most of the commercial farms, which was likely due to live vaccine usage. We detected never reported Genotype I NDV in two backyard farm samples. Our investigation into 2021 ND outbreak implicated Genotype VII.2 NDV strain as the causative pathogen. Additionally, we developed a Tablet formulation of the thermostable I2-NDV vaccine (Ranigoldunga™) and assessed its efficacy on various (mixed) breeds of chicken (Gallus domesticus). Ranigoldunga™ demonstrated an overall efficacy >85% with a stability of 30 days at room temperature (25°C). The intraocularly administered vaccine was highly effective in preventing ND, including Genotype VII.2 NDV strain.
Subject(s)
Influenza, Human , Newcastle Disease , Poultry Diseases , Animals , Humans , Newcastle Disease/prevention & control , Poultry , Nepal , Newcastle disease virus/genetics , Chickens , Vaccines, Attenuated , GenotypeABSTRACT
BACKGROUND: No human rabies post-exposure prophylaxis (PEP) failure has been documented in the United States using modern cell culture-based vaccines. In January 2021, an 84-year-old male died from rabies 6 months after being bitten by a rabid bat despite receiving timely rabies PEP. We investigated the cause of breakthrough infection. METHODS: We reviewed medical records, laboratory results, and autopsy findings and performed whole-genome sequencing (WGS) to compare patient and bat virus sequences. Storage, administration, and integrity of PEP biologics administered to the patient were assessed; samples from leftover rabies immunoglobulin were evaluated for potency. We conducted risk assessments for persons potentially exposed to the bat and for close patient contacts. RESULTS: Rabies virus antibodies present in serum and cerebrospinal fluid were nonneutralizing. Antemortem blood testing revealed that the patient had unrecognized monoclonal gammopathy of unknown significance. Autopsy findings showed rabies meningoencephalitis and metastatic prostatic adenocarcinoma. Rabies virus sequences from the patient and the offending bat were identical by WGS. No deviations were identified in potency, quality control, administration, or storage of administered PEP. Of 332 persons assessed for potential rabies exposure to the case patient, 3 (0.9%) warranted PEP. CONCLUSIONS: This is the first reported failure of rabies PEP in the Western Hemisphere using a cell culture-based vaccine. Host-mediated primary vaccine failure attributed to previously unrecognized impaired immunity is the most likely explanation for this breakthrough infection. Clinicians should consider measuring rabies neutralizing antibody titers after completion of PEP if there is any suspicion for immunocompromise.
Subject(s)
Rabies Vaccines , Rabies , Male , Humans , Aged, 80 and over , Rabies/prevention & control , Minnesota , Post-Exposure Prophylaxis/methods , Antibodies, ViralABSTRACT
Understanding disease burden and transmission dynamics in resource-limited, low-income countries like Nepal are often challenging due to inadequate surveillance systems. These issues are exacerbated by limited access to diagnostic and research facilities throughout the country. Nepal has one of the highest COVID-19 case rates (915 cases per 100,000 people) in South Asia, with densely-populated Kathmandu experiencing the highest number of cases. Swiftly identifying case clusters (hotspots) and introducing effective intervention programs is crucial to mounting an effective containment strategy. The rapid identification of circulating SARS-CoV-2 variants can also provide important information on viral evolution and epidemiology. Genomic-based environmental surveillance can help in the early detection of outbreaks before clinical cases are recognized and identify viral micro-diversity that can be used for designing real-time risk-based interventions. This research aimed to develop a genomic-based environmental surveillance system by detecting and characterizing SARS-CoV-2 in sewage samples of Kathmandu using portable next-generation DNA sequencing devices. Out of 22 sites in the Kathmandu Valley from June to August 2020, sewage samples from 16 (80%) sites had detectable SARS-CoV-2. A heatmap was created to visualize the presence of SARS-CoV-2 infection in the community based on viral load intensity and corresponding geospatial data. Further, 47 mutations were observed in the SARS-CoV-2 genome. Some detected mutations (n = 9, 22%) were novel at the time of data analysis and yet to be reported in the global database, with one indicating a frameshift deletion in the spike gene. SNP analysis revealed possibility of assessing circulating major/minor variant diversity on environmental samples based on key mutations. Our study demonstrated the feasibility of rapidly obtaining vital information on community transmission and disease dynamics of SARS-CoV-2 using genomic-based environmental surveillance.
Subject(s)
COVID-19 , Sewage , Humans , SARS-CoV-2/genetics , COVID-19/diagnosis , COVID-19/epidemiology , GenomicsABSTRACT
Improvements in tumor immunotherapies depend on better understanding of the anti-tumor T cell response. By studying human tumor-draining lymph nodes (TDLNs), we found that activated CD8+ T cells in TDLNs shared functional, transcriptional, and epigenetic traits with TCF1+ stem-like cells in the tumor. The phenotype and TCR overlap suggested that these TDLN cells were precursors to tumor-resident stem-like CD8+ T cells. Murine tumor models revealed that tumor-specific CD8+ T cells were activated in TDLNs but lacked an effector phenotype. These stem-like cells migrated into the tumor, where additional co-stimulation from antigen-presenting cells drove effector differentiation. This model of CD8+ T cell activation in response to cancer is different from that of canonical CD8+ T cell activation to acute viruses, and it proposes two stages of tumor-specific CD8+ T cell activation: initial activation in TDLNs and subsequent effector program acquisition within the tumor after additional co-stimulation.