Fraction Of Neutrophils Research Articles

Phenotypical differences of neutrophils patrolling tumour-draining lymph nodes in head and neck cancer.

The complexity and heterogeneity of neutrophils are recognized, especially their roles in modulating inflammation and cancer immune responses. The detailed functions of neutrophils in human tumour-draining lymph nodes (TDLNs), specifically in the context of head and neck cancer, remain inadequately characterized. This study aims to delineate the phenotypic diversity of neutrophils in TDLNs, non-tumour-draining lymph nodes (nTDLNs) from patients with oral squamous cell carcinoma (OSCC), and to evaluate their correlation with clinical outcomes. A flow cytometry-based investigation. Neutrophils manifest a tissue-specific heterogeneity with significant phenotypic differences between compartments. A substantial fraction of neutrophils displayed an activated CD16highCD62Ldim profile in TDLNs, more prominent in patients with advanced T stages, implicating their involvement in the disease's progression. Notably, the presence of this activated neutrophil phenotype in TDLNs was strongly associated with poorer patient prognosis. The study confirms the heterogeneity of neutrophils in human TDLNs, aligning with findings from animal models but extending them to show clinical relevance in human disease. The correlation of neutrophil phenotypes with cancer progression and prognosis emphasizes the importance of these cells in the tumour-microenvironment. The data suggests a future possibility to develop targeted therapies that modulate the neutrophilic response in OSCC.

Exploring diagnostic biomarkers of type 2 cardio-renal syndrome based on secreted proteins and bioinformatics analysis

Chronic heart failure (CHF) can induce chronic kidney disease (CKD), called type 2 cardio-renal syndrome (CRS2). The mechanism is not completely clear, and there is a lack of early warning biomarkers of CKD in the context of CHF. Two CKD, one CHF-PBMC and four CHF-cardiac tissue expression profile datasets were obtained from GEO database. Differential expression analysis and WGCNA were used to detect CKD key genes and CHF-related secreted proteins. Protein–protein interaction (PPI), functional enrichment, and cMAP analysis reveal potential mechanisms and drugs of CHF-related CKD. Five machine learning algorithms were used to screen candidate biomarkers, construct a diagnostic nomogram for CKD and validate it in two external cohorts. Clinical serum samples were collected in our hospital to evaluate the correlation and diagnostic value of biomarkers and CKD. 225 CKD key genes and 316 CHF-related secreted proteins were identified. Four key subgroups, including 204 genes, were identified as CRS2-related pathogenic genes by PPI analysis. Enrichment analysis revealed that the identified subgroups exhibited significant enrichment in cytokine action, immune responses, and inflammatory processes. The cMAP analysis highlighted metiradone as a drug with greater potential for therapeutic intervention for CRS2. Utilizing five machine learning algorithms, three hub genes (CD48, COL3A1, LOXL1) were pinpointed as potential biomarkers for CKD, and a nomogram model was constructed. Receiver operating characteristic analysis demonstrated that the nomogram’s area under the curve (AUC) exceeded 0.80 in both the CKD combined dataset and two external cohorts. In addition, the three biomarkers were significantly correlated with the glomerular filtration rate, and the AUC of the model predicting disease progression was 0.944. Furthermore, analysis of immune cell infiltration indicated a correlation between the three biomarkers and the infiltration fraction of macrophages, neutrophils, and other immune cells in CKD. Our clinical cohort validated the expression patterns of three biomarkers in serum, and the diagnostic model achieved an AUC of 0.876. CHF has the potential to facilitate the progression of CKD via the release of cardiac and PBMC secreted proteins. Furthermore, CD48, COL3A1, and LOXL1 have been identified as potential biomarkers for the detection of CKD.

IFN-γ primes bone marrow neutrophils to acquire regulatory functions in severe viral respiratory infections.

Neutrophil subsets endowed with regulatory/suppressive properties are widely regarded as deleterious immune cells that can jeopardize antitumoral response and/or antimicrobial resistance. Here, we describe a sizeable fraction of neutrophils characterized by the expression of programmed death-ligand 1 (PD-L1) in biological fluids of humans and mice with severe viral respiratory infections (VRI). Biological and transcriptomic approaches indicated that VRI-driven PD-L1+ neutrophils are endowed with potent regulatory functions and reduced classical antimicrobial properties, as compared to their PD-L1- counterpart. VRI-induced regulatory PD-L1+ neutrophils were generated remotely in the bone marrow in an IFN-γ-dependent manner and were quickly mobilized into the inflamed lungs where they fulfilled their maturation. Neutrophil depletion and PD-L1 blockade during experimental VRI resulted in higher mortality, increased local inflammation, and reduced expression of resolving factors. These findings suggest that PD-L1+ neutrophils are important players in disease tolerance by mitigating local inflammation during severe VRI and that they may constitute relevant targets for future immune interventions.

A-307 Clinical utility of serum calprotectin in febrile infants presenting to the Emergency Department

Abstract Background Antimicrobial stewardship involves a delicate balance between the risk of undertreating individuals and the potential societal burden of overprescribing antimicrobials. However, early and accurate diagnosis of bacterial infections in critically ill patients is challenging, as the clinical manifestation is non-specific. Furthermore, the results from microbial cultures may be delayed, resulting in delayed antibiotic treatment, which is associated with increased mortality. Neutrophil activation is a major response to bacterial infection and calprotectin is the most abundant protein in the cytosolic fraction of neutrophils, shown as an early marker for neutrophil activation. The objective of this study was to evaluate the clinical performance of serum calprotectin in identifying bacterial infection in febrile infants presenting to an Emergency Department (ED) relative to other common biomarkers of infection. Methods 141 infants aged 28-90 days with suspected infectious disease presenting to the ED at The Hospital for Sick Children in Toronto, were included in this non-interventional investigation. All biomarkers except calprotectin were used in the routine clinical adjudication of the final diagnosis. Residual serum specimens collected as part of the standard care pathway were stored at -80°C prior to analysis of serum calprotectin using GCAL® assay (Gentian AS, Norway). Chart review was completed for key variables including, age (days), sex, chief complaint and temperature at ED presentation, and available laboratory test results (i.e., C-reactive protein and procalcitonin (Architect, Abbott Diagnostics), WBC count and neutrophil count (XN3000, Sysmex Europe GmbH), urinary leukocytes and nitrates, blood and/or urine bacterial culture). Final diagnosis, admission status, and antibiotic prescription at ED discharge were also extracted. Results Final discharge diagnosis in patient population included bacterial infection (n=23), viral infection (n=22), fever of unknown source (n=54) and other/unknown diagnosis (n=42). Antibiotics were prescribed in 36 cases, although bacterial infection could only be confirmed in 23 cases when final diagnosis was made. Statistically significant differences were observed in serum calprotectin (p<0.001), procalcitonin (p<0.001), and CRP (p<0.001) in patients with bacterial infections compared to patients with other discharge diagnoses. ROC curve analysis was performed for the identification of bacterial infections in the entire clinical cohort (n=141) and in only patients with laboratory confirmed bacterial or viral infection (n=41). Calprotectin and procalcitonin showed higher specificity than CRP and WBC count for differentiation between bacterial and viral infection in both cohorts. Performance of calprotectin in detection of bacterial infections was comparable to the performance of CRP, slightly better than performance of procalcitonin and superior to WBC count. Conclusions In this cohort, one third of the children that were prescribed antibiotics did not have confirmed bacterial infection. Hence, there is a need for adequate diagnostic tools to help discriminate between various kinds of infections. This study confirms serum calprotectin as a valuable biomarker for differentiation between types of infection and estimation of disease severity in febrile infants. Access to fast and accurate analysis of circulating calprotectin, combined with good clinical performance has the potential to make this biomarker a valuable complement to the current diagnostics of patients with bacterial infections and sepsis.

Protocols for circulating neutrophil depletion in neonatal C57Bl/6 mice.

Murine neonatal neutrophil depletion strategies have problems with achieving deep circulating neutrophil clearance and accurate residual neutrophil fraction detection. Boivin et al. demonstrated an isotype switch method of achieving profound neutrophil clearance using a combination of anti-Ly6G and anti-rat κ Ig light chain antibodies in adult C57Bl/6 mice, proven by extra- and intracellular Ly6G detection by flow cytometry. We adapted this technique to neonatal mice testing four neutrophil depletion strategies. Four protocols were tested: P3 Ly6G and P1-3 Ly6G (anti-Ly6G on postnatal days (P) 3 and 1-3 respectively), and P3 Dual and P1-3 Dual (anti-Ly6G and anti-rat κ Ig light chain on P3 and P1-3 respectively). Intracellular and extracellular Ly6G presence was detected using flow cytometry. Isotype control antibodies were used as controls. P1-3 Dual achieved significantly better neutrophil depletion than the P1-3 Ly6G or P3 Ly6G protocols (97% vs 74% and 97% vs 50%, respectively). The P3 Dual protocol alone was enough to achieve significantly better neutrophil clearance (93%) than any of the Ly6G alone protocols. The Ly6G alone protocols led to near-total elimination of extracellular Ly6G, but there was a significant presence of intracellular Ly6G in the CD45+ cell population, which evaded detection by extracellular Ly6G antibody-based detection methods. Anti-Ly6G antibody alone is ineffective in producing effective circulating neutrophil clearance in neonatal mice. Dual antibody-based neutrophil depletion strategies achieve>90% clearance in P4 mice. A single day of dual antibody treatment achieves 93% neutrophil depletion in neonatal mice and is an alternative to daily injections. Dual antibody-based neutrophil depletion effectively induces circulating neutrophil clearance in neonatal mice.

Features of the neutrophil granulocyte phenotype in children with infectious mononucleosis

The course of infectious diseases caused by viruses, and their common outcome is determined by the activity of the inflammatory reaction which occurs both at the local and systemic levels. However, the features of neutrophil functions during inflammatory reaction are virtually unknown in the patients with infectious mononucleosis (IM), caused by Epstein–Barr virus (EBV). Hence, the aim of our study was to evaluate some characteristics of phenotypic spectrum of blood neutrophils in children with IM. Patients and methods. We examined 84 children aged 3 to 11 years with EBV infection with moderate or severe clinical course of the disease. All patients exhibited a positive test for EBV DNA in blood lymphocytes and appropriate serological markers of acute EBV infection. The control group consisted of 40 conditionally healthy children at the similar age range. The study of neutrophil phenotype was carried out by flow cytometry using direct immunofluorescence of whole peripheral blood samples. A study of the neutrophil phenotype with a combination of two functional antigens (CD64 and CD32) has revealed that in children with IM, regardless of age, the main fraction of blood neutrophils are double-negative cells, whereas in healthy children it consists of CD64-CD32+ neutrophils. The main fraction of neutrophils in the paired combination of CD64 and CD11b antigens in sick children aged 3-6 and 7-11 years was similar to the healthy controls (CD64-CD11b+), but with a change in the content of minor cell fractions. The number of CD64-CD15+ neutrophils (main fraction of cells in healthy children) proved to be significantly reduced in the IM patients of both age groups. However, we have revealed a marked increase in the level of double-negative cells for the CD64 and CD15 antigens. At the same time, the content of double-negative neutrophils for these markers was also increased in IM children of both age groups. The cells with CD11b-CD15+ and CD11b+CD15+ phenotypes comprised the main fractions in IM, as studied by a paired combination of CD11b and CD15 antigens; in healthy children – only CD11b+CD15+ neutrophils are detected. The phenotypic changes of neutrophils during IM suggest a decreased migratory ability of cells with high activity of proinflammatory functions. The established ontogenetic features of the neutrophil phenotype are significantly changed in the children with IM, probably, due to specific immunopathogenesis of the viral infection. The detected changes in phenotypic composition of neutrophils associated with IM may be caused both by the features of protective reaction of innate immune cells and pathogenic effects of the virus itself upon blood neutrophils.

Epigenetic age oscillates during the day.

Since their introduction, epigenetic clocks have been extensively used in aging, human disease, and rejuvenation studies. In this article, we report an intriguing pattern: epigenetic age predictions display a 24-h periodicity. We tested a circadian blood sample collection using 17 epigenetic clocks addressing different aspects of aging. Thirteen clocks exhibited significant oscillations with the youngest and oldest age estimates around midnight and noon, respectively. In addition, daily oscillations were consistent with the changes of epigenetic age across different times of day observed in an independant populational dataset. While these oscillations can in part be attributed to variations in white blood cell type composition, cell count correction methods might not fully resolve the issue. Furthermore, some epigenetic clocks exhibited 24-h periodicity even in the purified fraction of neutrophils pointing at plausible contributions of intracellular epigenomic oscillations. Evidence for circadian variation in epigenetic clocks emphasizes the importance of the time-of-day for obtaining accurate estimates of epigenetic age.

Abstract LB329: Interleukin6 as a Potential Prognostic Marker and Therapeutic Target in Oral Squamous Cell Carcinoma

Abstract This study investigates the role of interleukin (IL)-6 in oral squamous cell carcinoma (OSCC), evaluating its potential as a prognostic marker and therapeutic target. This study involved 95 untreated OSCC patients, with pre-surgical serum IL-6 levels measured by Electro chemiluminescence immunoassay (ECLIA). Patients were categorized into high (78 patients) and low (17 patients) IL-6 groups based on a cutoff value of 8 pg/mL.A strong correlation was observed between higher IL-6 levels and lower overall survival (P<0.0001) and disease-free survival (P=0.0283). Serum IL-6 levels also showed a strong positive correlation with certain blood test markers such as neutrophil and lymphocyte fractions and C-reactive protein (CRP), and a negative correlation with albumin levels. These findings suggest a link between systemic inflammation, malnutrition, and the Inflammation-based prognostic score (IBPS).Further analysis through microarray assessed the gene expression in primary tumor tissues, revealing 513 genes with more than a two-fold change in the high IL-6 group compared to controls. Gene Ontology analysis indicated increased metabolic activity in the high IL-6 group and suppressed inflammation and immune-related molecules in the low IL-6 group.Immunohistochemical staining for IL-6 in tumor tissues showed its presence mainly in the stroma, particularly in cancer-associated fibroblasts (CAFs) identified by vimentin and α-SMA positivity. Both OSCC cell lines and CAFs established from OSCC tissues demonstrated high IL-6 secretion, especially in cell lines derived from lymph node metastases. Furthermore, cell lines from primary, lymph node, and lung metastases exhibited high IL-6 secretion, with HRAS mutations also identified. Knockdown of HRAS using siRNA reduced IL-6 secretion.Finally, in vivo experiments using an anti-IL-6 receptor antibody showed that blocking IL-6 signaling reduced the proliferation of OSCC cell lines.The study concludes that IL-6 is a useful prognostic and therapeutic target in OSCC. The wide applicability of IL-6 testing and the clinical use of anti-human IL-6R antibody drugs, combined with aggressive nutritional intervention, suggest a potential paradigm shift in OSCC treatment. Citation Format: Hiroyuki Goda, Tomoko Adachi, Koh-ichi Nakashiro, Nobuyuki Kuribayashi, Daisuke Uchida. Interleukin6 as a Potential Prognostic Marker and Therapeutic Target in Oral Squamous Cell Carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB329.

Lower respiratory tract single-cell RNA sequencing and neutrophil extracellular trap profiling of COVID-19-associated pulmonary aspergillosis: a single centre, retrospective, observational study

COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus affecting patients who are critically ill with COVID-19. The pathophysiology and the role of neutrophil extracellular traps (NETs) in this infection are largely unknown. We aimed to characterise the immune profile, with a focus on neutrophils and NET concentrations, of critically ill patients with COVID-19, with or without CAPA. We conducted a single-centre, retrospective, observational study in two patient cohorts, both recruited at University Hospitals Leuven, Belgium. We included adults aged 18 years or older who were admitted to the intensive care unit because of COVID-19 between March 31, 2020, and May 18, 2021, and who were included in the previous Contagious trial (NCT04327570). We investigated the immune cellular landscape of CAPA versus COVID-19 only by performing single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid. Bronchoalveolar lavage immune cell fractions were compared between patients with CAPA and patients with COVID-19 only. Additionally, wedetermined lower respiratory tract NET concentrations using biochemical assays in patients aged 18 years and older who were admitted to the intensive care unit because of severe COVID-19 between March 15, 2020, and Dec31, 2021, for whom bronchoalveolar lavage was available in the hospital biobank. Bronchoalveolar lavage NET concentrations were compared between patients with CAPA and patients with COVID-19 only and integrated with existing data on immune mediators in bronchoalveolar lavage and 90-day mortality. We performed scRNA-seq of bronchoalveolar lavage on 43 samples from 39 patients, of whom 36 patients (30male and six female; 14 with CAPA) were included in downstream analyses. We performed bronchoalveolar lavage NET analyses in 59 patients (46 male and 13 female), of whom 26 had CAPA. By scRNA-seq, patients with CAPA had significantly lower neutrophil fractions than patients with COVID-19 only (16% vs 33%; p=0·0020). The remaining neutrophils in patients with CAPA preferentially followed a hybrid maturation trajectory characterised by expression of genes linked to antigen presentation, with enhanced transcription of antifungal effector pathways. Patients with CAPA also showed depletion of mucosal-associated invariant T cells, reduced T helper 1 and T helper 17 differentiation, and transcriptional defects in specific aspects of antifungal immunity in macrophages andmonocytes. We observed increased formation of NETs in patients with CAPA compared with patients with COVID-19 only (DNA complexed with citrullinated histone H3 median 15 898 ng/mL [IQR 4588-86 419] vs 7062 ng/mL [775-14 088]; p=0·042), thereby explaining decreased neutrophil fractions by scRNA-seq. Low bronchoalveolar lavage NET concentrations were associated with increased 90-day mortality in patients with CAPA. Qualitative and quantitative disturbances in monocyte, macrophage, B-cell, and T-cell populations could predispose patients with severe COVID-19 to develop CAPA. Hybrid neutrophils form a specialised response to CAPA, and an adequate neutrophil response to CAPA is a major determinant for survival in these patients. Therefore, measuring bronchoalveolar lavage NETs could have diagnostic and prognostic value in patients with CAPA. Clinicians should be wary of aspergillosis when using immunomodulatory therapy that might inhibit NETosis to treat patients with severe COVID-19. Research Foundation Flanders, KU Leuven, UZ Leuven, VIB, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, la Caixa Foundation, the Flemish Government, and Horizon 2020.

Clinical and laboratory characteristics of newborns from mothers with gestational diabetes mellitus and COVID-19 during pregnancy

Data on the course of pregnancy complicated by SARS-CoV-2 infection, and the condition ofthe fetus and newborn are of great interest to neonatologists and pediatricians.Purpose. The study aimed at evaluating the clinical and laboratory features of the early neonatal period and individual health indicators in infancy in full-term newborns from mothers with gestational diabetes mellitus and COVID-19 during pregnancy.Materials and Methods. Two groups of full-term newborns whose mothers’ pregnancy was complicated by gestational diabetes mellitus were compared in matched pairs (each group included 80 patients). In the first group of newborns, COVID-19 was diagnosed in mothers during pregnancy, in the second group, COVID-19 was not observed in mothers. At the age of 9 months, an analysis of outpatient medical records was carried out and a questionnaire survey of mothers was conducted. The methods of collecting and processing information using the Microsoft Excel 2016 software package for Windows 7, IBM SPSS Statistics v22 were used. Results. COVID-19 infection during pregnancy is associated with a higher frequency of respiratory and neurological disorders in the newborn, and with problems of enteral feeding. In the peripheral blood, the indices fell within the limits of reference values, but there was a decrease in leukocytes, platelets and an increase in the fraction of segmented neutrophils. At the age of 9 months, the children of the main group had a larger body weight and head circumference, a higher incidence of chronic morbidity was noted, and the children were registered with a neurologist, cardiologist and endocrinologist.Conclusions. Clinical and laboratory features of early neonatal adaptation of children from mothers with gestational diabetes mellitus and COVID-19 during pregnancy were revealed. A more severe course of diabetic fetopathy has not been proven in children from women who suffered COVID-19 during pregnancy. The higher incidence of chronic morbidity in infancy requires further study.

Enhancement of Human Neutrophil Bactericidal Activity through ROS Regulation

Antimicrobial agents are vital for treating infectious diseases, but their widespread use has led to a concerning rise in drug-resistant bacteria. The development and overuse of new antimicrobial agents have paradoxically given rise to even more drug-resistant strains. If current trends persist, drug-resistant infections may become the leading global cause of mortality by 2050. However, current measures such as restricting antimicrobial use and promoting appropriate usage have not provided a definitive solution. Therefore, exploring alternative treatment approaches is essential. In this study, our research was based on a novel strategy that shifts the focus from targeting specific bacterial species to leveraging the defense mechanisms of human neutrophils. Neutrophils play a critical role in innate immunity by engulfing bacteria and generating reactive oxygen species (ROS) in phagosomes. The primary ROS producer in neutrophils is the NADPH oxidase (NOX) complex centered on gp91 phox (NOX2), a membrane protein of the NOX family. However, excessive ROS are harmful, necessitating negative regulation. Neutrophils contain a negative regulator of reactive oxygen species (NRROS), which regulates ROS production by competing with p22 phox for binding to NOX2 when inflammation go into ending. NRROS knockout mice have shown increased ROS production and bactericidal activity during bacterial infection, resulting in significantly prolonged survival compared to wild-type mice. If this mechanism could be applied to human cells to enhance bactericidal activity, it would eliminate the need to target specific bacterial species and could revolutionize the treatment of infectious diseases. We used human induced pluripotent stem cells (iPSC) as a model to validate the NRROS knockout effect in human cells, overcoming challenges in isolating peripheral blood neutrophils due to their limited lifespan, poor in vitro culture tolerance, and genetic editing complexities. We successfully generated NRROS-heterozygous knockout (hetero-KO) and NRROS-homozygous knockout (homo-KO) iPSC clones using genome editing techniques. Additionally, using the PiggyBac system, we established doxycycline-inducible NRROS expression clones (homo-KO+Dox) based on homo-KO clones. These iPSC were differentiated into neutrophils and evaluated for function. The isolated neutrophil fraction exhibited characteristic segmented nuclei in all clones. We measured ROS production and examined changes induced by NRROS knockout after stimulation. Immediate superoxide production showed no difference between wild-type and NRROS-KO clones. However, after 2 hours of stimulation, the NRROS-KO clones exhibited significantly elevated hydrogen peroxide production, indicating increased total ROS production, possibly due to delayed NOX complex degradation. Furthermore, we assessed the bactericidal capacity of iPSC-derived neutrophils against multidrug-resistant bacteria, revealing significantly enhanced bactericidal activity in NRROS-KO clones compared to wild-type clones. In conclusion, NRROS knockout in human neutrophils enhances bactericidal capacity through heightened ROS production, offering promising prospects for innovative infectious disease treatments. If successfully harnessed, this innovative treatment modality could revolutionize infectious disease management, combating drug-resistant infections, and improving global health outcomes.

Assessment of neutrophil NETs‑forming activity in patients with ulcerative colitis

The aim of the study. To determine the non-tossing capacity of neutrophils in patients with ulcerative colitis (UC).Materials and methods. Ten patients with UC (study group) were included in the study; the control group consisted of 22 healthy volunteers of comparable age to the study group. All patients with UC received treatment according to clinical guidelines. We determined the non-tumorforming ability of the isolated neutrophil fraction in vitro in both groups using an original technique developed by members of the authors' team (Invention Patent No. 2768152 C 1; dated 23.03.2022. Application No. 2021129097 dated 06.10.2021).Results. In patients with UC, after 15 minutes of in vitro stimulation with probiotic, there was a statistically significant increase in early netosis cells (p < 0.010), cloud-like traps (p < 0.001) and filamentous NVLs (p < 0.001) compared with the control group. Bacterial uptake rate by neutrophil traps in UC patients was statistically significantly lower than in controls (p < 0.010).Conclusions. All identified features of neutrophils non-trapping capacity in UC patients may indicate changes in granulocyte reactivity, probably, under the influence of cytokines and/or due to therapeutic intervention. The neutrophil traps formed had fewer protective effects and probably more damaging effects. Studying the influence of the role of netosis on the course and prognosis of UC is a promising research area.

A clinical prediction model to differentiate tuberculous spondylodiscitis from pyogenic spontaneous spondylodiscitis.

Microbiological diagnosis of tuberculous spondylodiscitis (TS) and pyogenic spontaneous spondylodiscitis (PS) is sometime difficult. This study aimed to identify the predictive factors for differentiating TS from PS using clinical characteristics, radiologic findings, and biomarkers, and to develop scoring system by using predictive factors to stratify the probability of TS. A retrospective single-center study. Demographics, clinical characteristics, laboratory findings and radiographic findings of patients, confirmed causative pathogens of PS or TS, were assessed for independent factors that associated with TS. The coefficients and odds ratio (OR) of the final model were estimated and used to construct the scoring scheme to identify patients with TS. There were 73 patients (51.8%) with TS and 68 patients (48.2%) with PS. TS was more frequently associated with younger age, history of tuberculous infection, longer duration of symptoms, no fever, thoracic spine involvement, ≥3 vertebrae involvement, presence of paraspinal abscess in magnetic-resonance-image (MRI), well-defined thin wall abscess, anterior subligamentous abscess, and lower biomarker levels included white blood cell (WBC) counts, erythrocyte-sedimentation-rate (ESR), neutrophil fraction, and C-reactive protein (all p < 0.05). Multivariate logistic regression analysis revealed significant predictors of TS included WBC ≤9,700/mm3 (odds ratio [OR] 13.11, 95% confidence interval [CI] 4.23-40.61), neutrophil fraction ≤78% (OR 4.93, 95% CI 1.59-15.30), ESR ≤92 mm/hr (OR 4.07, 95% CI 1.24-13.36) and presence of paraspinal abscess in MRI (OR 10.25, 95% CI 3.17-33.13), with an area under the curve of 0.921. The scoring system stratified the probability of TS into three categories: low, moderate, and high with a TS prevalence of 8.1%, 29.6%, and 82.2%, respectively. This prediction model incorporating WBC, neutrophil fraction counts, ESR and presence of paraspinal abscess accurately predicted the causative pathogens. The scoring scheme with combination of these biomarkers and radiologic features can be useful to differentiate TS from PS.

BSBM-06 UNRAVELING THE IMMUNE MICROENVIRONMENT OF BRAIN METASTASIS FROM LUNG ADENOCARCINOMA

Abstract Brain metastasis (BM) is a common complication of advanced lung adenocarcinoma (LUAD) and is associated with significantly poor prognosis and reduced quality of life. The immune system plays a critical role in the development and progression of BM, as well as in response to therapy. Herein, we used Next-Generation Sequencing (NGS) and array technologies to elucidate the immune landscape of LUAD-BM. NGS data from BM and primary tumors (T): RNA-Seq (n=24; 13 BM and 11 T), microarray (n=140; 63 BM and 77 T) were used for quantifying the immune cell fractions by CIBERSORT. Additionally, single-cell (scRNA-seq) data (n=57,774 cells; 23 BM samples) were used for data validation. Results showed that resting memory T CD4 comprised the largest cellular fraction (17.4%) of the total immune cells in BM. Furthermore, the fraction of resting dendritic cells (DC) was lower in BM vs. T, while the neutrophil fraction was higher (p&amp;lt;0.05). ScRNA-seq confirmed the presence of DC and neutrophils in BM, identifying multiple subsets of DC (cDC1, cDC2/MoDC, and DC3) that are highly conserved across solid human cancers with cDC2/MoDC as the most frequent subpopulation among dendritic cells. Therefore, our results show a different immune profile between BM and primary LUAD. The immune environment of LUAD-BM is highly suppressed, accompanied by enriched T cell subpopulations predicted as immature T cells. The identification of cDC2/MoDC subpopulations in LUAD-BM replicates the findings observed in BM from melanoma patients. It has been shown that cDC2 can interact with CD4+ T cells to promote anti-tumor immune responses. Our findings elucidate the profiles of LUAD-BM immune microenvironment, comprehensively identifying specific immune subpopulations with important roles in the establishment and progression of BM from LUAD.

Neutrophil activation status and low-density neutrophil fractions in patients with coronary artery disease

Neutrophil activation status and low-density neutrophil fractions in patients with coronary artery disease

Clinical genomic implications of transcriptional subtypes in pancreatic cancer.

4145 Background: Transcriptional profiling of pancreatic cancers (PC) has defined classical and basal subtypes; basal tumors have worse outcomes. Mesenchymal (MES) and neural-like progenitor (NRP) subtypes are increasingly recognized and enriched post-therapy. Initial data suggests worse outcomes to FOLFIRINOX (FFX) compared with gemcitabine nab-Paclitaxel (GnP) in basal tumors. Several clinical trials are ongoing to investigate this. Here, we examined the clinical implications of transcriptional subtypes in a large, real-world dataset. Methods: Retrospective IRB exempt, deidentified data was examined from NextGen DNA and RNA sequencing performed on PCs at Caris Life Sciences (Phoenix, AZ). Classical and basal cell states were identified using RNA-seq and the PurIST algorithm in a genomic cohort or GATA6 and KRT5 expression levels in a clinical cohort. Tumor microenvironment immune cell composition on RNA seq was performed using QuantiSeq. Survival was obtained from insurance claims data and calculated from first treatment date to last known contact. Kaplan-Meier estimates were calculated for patient cohorts. P values were adjusted using Benjamini-Hochberg correction. Results: A total of 7,250 PCs were profiled in the genomic cohort. 3,063 tumors (42.2%) were strongly classical (SC), 2,015 tumors (27.8%) were strongly basal (SB) and the remaining had mixed phenotypes. MES and NRP marker genes were significantly co-expressed with each other, with basal genes, and anti-correlated with classical genes. When compared to SC, SB had significantly higher mutation rates in KRAS (93% vs. 88%), TP53 (83% vs. 72%) and ARID1A (12% vs. 8%), whereas SMAD4 (23% vs. 17%) mutations were more common in SC (all q &lt; 0.05). There were no differences in mutation rates in homologous recombination or mismatch repair genes. SB had a significantly higher fraction of M1 macrophages (fold change [FC]: 1.14) and neutrophils (FC 1.16), whereas SC tumors had higher M2 macrophages (FC 1.18), NK (FC 1.2), and dendritic cells. Overall proportions of CD4/8 T cells were low and not different. Interestingly, SB had higher levels of PD-L1 by IHC (4.8% vs. 35%) and higher expression of immune exhaustion genes including CTLA4 (FC 1.19), TIM3 (FC 1.22) and PD-1 (FC 1.43) (all q &lt; 0.05). The clinical cohort had 1,623 patients. Basal tumors had an inferior survival (median survival: 8.2 months (mo) vs 13.3 mo (Hazard Ratio (HR) 0.67, p &lt; 0.00001)) and showed a significant improvement in outcomes when treated with upfront FFX vs GnP (n = 80 vs 90, Median: 15.8 vs 7.4 mos., HR 0.68, p = 0.021). This difference between FFX vs GnP was less pronounced in classical tumors (n = 70 vs 89, Median: 17.3 vs 15.4 mos, HR 0.70, p = 0.049). Conclusions: Our work represents the largest known real world molecular comparison of transcriptional subtypes of PC. Differential outcomes for patients with basal tumors treated with FFX versus GnP warrants further investigation in prospective studies.

Patients with schizophrenia and bipolar disorder display a similar global gene expression signature in whole blood that reflects elevated proportion of immature neutrophil cells with association to lipid changes

Schizophrenia (SCZ) and bipolar disorder (BD) share clinical characteristics, genetic susceptibility, and immune alterations. We aimed to identify differential transcriptional patterns in peripheral blood cells of patients with SCZ or BD versus healthy controls (HC). We analyzed microarray-based global gene expression data in whole blood from a cohort of SCZ (N = 329), BD (N = 203) and HC (N = 189). In total, 65 genes were significantly differentially expressed in SCZ and 125 in BD, as compared to HC, with similar ratio of up- and downregulated genes in both disorders. Among the top differentially expressed genes, we found an innate immunity signature that was shared between SCZ and BD, consisting of a cluster of upregulated genes (e.g., OLFM4, ELANE, BPI and MPO) that indicate an increased fraction of immature neutrophils. Several of these genes displayed sex differences in the expression pattern, and post-hoc analysis demonstrated a positive correlation with triglyceride and a negative correlation with HDL cholesterol. We found that many of the downregulated genes in SCZ and BD were associated with smoking. These findings of neutrophil granulocyte-associated transcriptome signatures in both SCZ and BD point at altered innate immunity pathways with association to lipid changes and potential for clinical translation.

Isochlorogenic acid A alleviates dextran sulfate sodium-induced ulcerative colitis in mice through STAT3/NF-кB pathway

Isochlorogenic acid A (ICGA-A) is a dicaffeoylquinic acid widely found in various medicinal plants or vegetables, such as Lonicerae japonicae Flos and chicory, and multiple properties of ICGA-A have been reported. However, the therapeutic effect of ICGA-A on colitis is not clear, and thus were investigated in our present study, as well as the underlying mechanisms. Here we found that ICGA-A alleviated clinical symptoms of dextran sodium sulfate (DSS) induced colitis model mice, including disease activity index (DAI) and histological damage. In addition, DSS-induced inflammation was significantly attenuated in mice given ICGA-A supplementation. ICGA-A reduced the fraction of neutrophils in peripheral blood and the infiltration of neutrophils and macrophages in colon tissue, and reduced pro-inflammatory cytokine production and tight junctions in mouse models. Furthermore, ICGA-A down-regulated expression of STAT3 and up-regulated the protein level of IκBα. Our in vitro studies confirmed that ICGA-A inhibited the mRNA expression of pro-inflammatory cytokines. ICGA-A blocked the phosphorylation of STAT3, p65, and IκBα, suppressed the expression STAT3 and p65. In addition, the present study also demonstrated that ICGA-A had no obvious toxicity on normal cells and organs. Taken together, we conclude that ICGA-A mitigates experimental ulcerative colitis (UC) at least in part by inhibiting the STAT3/NF-кB signaling pathways. Hence, ICGA-A may be a promising and effective drug for treating UC.

Transcriptome Analysis Reveals a Two-Gene Signature Links to Motor Progression and Alterations of Immune Cells in Parkinson's Disease.

The motor impairment in Parkinson's disease (PD) can be managed but effective treatments for stopping or slowing the disease process are lacking. The advent of transcriptomics studies in PD shed light on the development of promising measures to predict disease progression and discover novel therapeutic strategies. To reveal the potential role of transcripts in the motor impairment progression of patients with PD via transcriptome analysis. We separately analyzed the differentially expressed genes (DEGs) between PD cases and healthy controls in two cohorts using whole blood bulk transcriptome data. Based on the intersection of DEGs, we established a prognostic signature by regularized regression and Cox proportional hazards analysis. We further performed immune cell analysis and single-cell RNA sequencing analysis to study the biological features of this signature. We identified a two-gene-based prognostic signature that links to PD motor progression and the two-gene signature-derived risk score was associated with several types of immune cells in blood. Notably, the fraction of neutrophils increased 5% and CD4+ T cells decreased 7% in patients with high-risk scores compared to that in patients with low-risk scores, suggesting these two types of immune cells might play key roles in the prognosis of PD. We also observed the downregulated genes in PD patients with high-risk scores that enriched in PD-associated pathways from iPSC-derived dopaminergic neurons single-cell RNA sequencing analysis. We identified a two-gene signature linked to the motor progression in PD, which provides new insights into the motor prognosis of PD.

Identification of two molecular subtypes and a novel prognostic model of lung adenocarcinoma based on a cuproptosis-associated gene signature.

Lung adenocarcinoma is the most common subtype of lung cancer clinically, with high mortality and poor prognosis. Cuproptosis present a newly discovered mode of cell death characterized by aggregation of fatty acylated proteins, depletion of iron-sulfur clusterin, triggering of HSP70, and induction of intracellular toxic oxidative stress. However, the impact of cuproptosis on lung adenocarcinoma development, prognosis, and treatment has not been elucidated. By systematically analyzing the genetic alterations of 10 cuproptosis-related genes in lung adenocarcinoma, we found that CDKN2A, DLAT, LIAS, PDHA1, FDX1, GLS, and MTF1 were differentially expressed between lung cancer tissues and adjacent tissues. Based on the expression levels of 10 cuproptosis-related genes, we classified lung adenocarcinoma patients into two molecular subtypes using the Consensus clustering method, of which subtype 2 had a worse prognosis. Differential expression genes associated with prognosis between the two subtypes were obtained by differential analysis and survival analysis, and cox lasso regression was applied to construct a cuproptosis-related prognostic model. Its survival predicting ability was validated in three extrinsic validation cohorts. The results of multivariate cox analysis indicated that cuproptosis risk score was an independent prognostic predictor, and the mixed model formed by cupproptosis prognostic model combined with stage had more robust prognostic prediction accuracy. We found the differences in cell cycle, mitosis, and p53 signaling pathways between high- and low-risk groups according to GO and KEGG enrichment analysis. The results of immune microenvironment analysis showed that the enrichment score of activated dendritic cells, mast cells, and type 2 interferon response were down-regulated in the high-risk group, while the fraction of neutrophils and M0 macrophages were upregulated in the high-risk group. Compared with the high-risk group, subjects in the low-risk group had higher Immunophenoscore and may be more sensitive to immunotherapy. We identified seven chemotherapy agents may improve the curative effect in LUAD samples with higher risk score. Overall, we discovered that cuproptosis is closely related to the occurrence, prognosis, and treatment of lung adenocarcinoma. The cuproptosis prognostic model is a potential prognostic predictor and may provide new strategies for precision therapy in lung adenocarcinoma.