Neutrophil Cell Research Articles

NETosis in pulmonary pleomorphic carcinoma

AbstractPulmonary pleomorphic carcinoma (PC) is a rare non‐small‐cell lung carcinoma (NSCLC) with a poor prognosis, characterized by tumor necrosis (TN). NETosis is a form of neutrophil‐specific cell death, which is morphologically characterized by prominent neutrophil infiltration and cell detritus in the necrotic foci. Seventy‐six patients with pulmonary PC who underwent complete resection were enrolled. Tumor necrosis was evaluated using digitally scanned resected specimens. The regions of NETosis were quantified using citrullinated histone H3 (citH3)‐ and myeloperoxidase‐positive regions. We examined the association between the NETosis area and the prognostic outcomes and assessed the correlation between the NETosis area and systemic inflammation. Tumor necrosis was observed in 70 patients (92%). In all the cases, the TN region was accompanied by a citH3‐positive region. The patients with high NETosis area (n = 54) had significantly shorter overall survival than those with low NETosis area (n = 16) (p = 0.013). Furthermore, a high NETosis area was an independent poor prognostic factor in the multivariate analyses. Systemic inflammatory markers, including C‐reactive protein (CRP), CRP‐to‐albumin ratio, and neutrophil‐to‐lymphocyte ratio, were significantly higher in patients with high NETosis area than in those with low NETosis area. Furthermore, the levels of these inflammatory markers were significantly decreased postsurgery. This study shows that in surgically resected pulmonary PC, patients with high NETosis areas have higher systemic inflammation and worse prognosis.

Antibody isotyping and cytokine profiling in natural cases of Burkholderia mallei infection (glanders) in equines.

Immunological aspects of B. mallei infection were rarely studied in natural cases of equines. The present study was conducted to determine IgG, IgM, IgA and IgG (T) titre against recombinant Hcp1, TssA and TssB proteins and PilA-Hcp1-TssN-BipD and BpaB-BpaC- BMAA0553 chimeras and cytokine responses in glanders affected equine serum. The study was conducted on serum samples collected from 151 glanders positive equines which include horses (n=134), mules (n=16), and donkeys (n=01). The IgM, IgG, IgA and IgG (T) titre were determined against recombinant antigens by indirect ELISA and interleukin(IL)-1β, IL-17, IL-6, tumor necrosis factor alpha(TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interferon gamma (IFN-γ) responses were measured by commercial ELISA kits. The study showed that glanders affected equines elicited strong antibody response against Hcp1, moderate responses against TssA and TssB, and weak responses against two chimeras. Among the cytokines, IL-1β, MCP-1, IL-17 and IL-6 concentration were significantly higher in glanders affected equine serum. We found that IgG antibody titre was higher than IgM, IgG (T) and IgA isotypes and Hcp1 was most predominant antibody inducers in comparison to TssA, TssB, PilA-Hcp1-TssN-BipD and BpaB-BpaC-T2SS proteins. The elevated level of IL-1β, MCP-1, IL-17, IL-6, IFN-γ and TNF-α observed in this study support the important role of this cytokines for augmenting cellular defence by recruitment of macrophages, neutrophil and dendritic cells against B. mallei infection. Further studies should be conducted to determine memory cell responses in natural cases of equine glanders using recombinant B. mallei proteins for identifying well-characterized immuno-protective vaccine candidates.

Effect of acute exercise on gene expression in peripheral blood mononuclear cells of puberty children.

Studies have shown that acute exercise alters the expression of circulating neutrophil and peripheral blood mononuclear cell (PBMC) genes, affecting a wide range of processes from cellular growth to the promotion of inflammatory and anti-inflammatory responses. In this study, we identified differential gene expression associated with acute exercise and PBMC in puberty children through the GEO database. We utilized bioinformatics technology to analyze the impact of acute exercise on PBMC and identified key genes involving the puberty children. Combining two datasets of gene expression, we identified 197 shared differentially expressed genes (DEGs) in PBMC before and after exercise. We conducted pathway analysis of these DEGs and built a gene co-expression network. The identification of hub genes was achieved through the utilization of protein-protein interaction (PPI) networks. 268 shared DEGs were identified in the GSE11761 dataset in pre- and post-exercise groups, while 352 differential genes were identified between the pre- and post-exercise groups of females in the GSE14642 dataset. A total of 197 shared genes were identified between the two datasets. Seven core genes were identified by MCODE and CytoHubba, namely IL2RB, KLRD1, GZMB, TBX21, PRF1, HAVCR2, and GZMA. Bioinformatics techniques are effective in screening and analyzing PBMC-associated DEGs in children undergoing acute pre- and post-exercise puberty. Microarray analysis of PBMCs in children undergoing puberty can identify molecules that may predict acute exercise responses. This study provides insights for further exploration of the mechanisms and targets of acute exercise on the organism.

Abstract 4146302: NETosis Attenuating Nanoprobe Improves Survival after Cardiac Arrest

NETosis, the process of neutrophil cell death due to the formation and release of neutrophil extracellular traps (NETs) has been implicated in the pathogenesis of heart disease. NETs are composed of immunogenic DNA fragments and citrullinated histones. The role of NETosis in the context of cardiac arrest and resuscitation (CA/R) is however unknown. We previously reported the development of a Dextran-Thiazole Orange (DTO) nanoprobe, with nanomolar affinity for nucleic acids, that exerts acute anti-inflammatory effects and reduces ischemia-reperfusion injury in mice. We aimed here to determine whether the DTO nanoprobe would also bind to the nucleic acid in NETS, whether DTO could attenuate the intensity of NETosis, and whether this would improve outcomes and survival after cardiac arrest. C57Bl6 mice were arrested for 8 minutes before resuscitation. DTO or unmodified dextran (vehicle control) was injected at the time of resuscitation, and again 4 hours later (n=38 mice). Survival and neurological function were scored daily. Time-domain resolved (lifetime) and conventional fluorescence imaging was performed at 4 hours after resuscitation in 6 additional mice injected with DTO. NETosis was evaluated by protein blot in the hearts of cardiac arrest mice injected with DTO or control. DTO significantly (p<0.05) improved 10-day survival (Fig. A), mean survival (from 4 to 7 days, Fig. B), and neurological function (Fig. C) following CA/R. Lifetime imaging (Fig. D), but not conventional fluorescence imaging (Fig. E) revealed the presence of DTO in the injured heart, liver and kidneys. In the heart, DTO signal was significantly increased in CA/R compared to sham mice injected with DTO or CA/R mice injected with control dextran (Fig. F). Citrullinated H3 and HMGB-1 (High mobility group box 1, a chromatin protein) were both upregulated in CA/R, and were significantly attenuated by DTO injection (Fig. G-I). We show for the first time that NETosis plays a key role in cardiac arrest and resuscitation. DTO is capable of detecting NETosis as well as attenuating it, likely via a reduction in HMGB-1 activity. The modulation of NETosis may provide a new avenue to improve survival after cardiac arrest.

Multi-omics reveal immune microenvironment alterations in multiple myeloma and its precursor stages

Tumor immune microenvironmental alterations occur early in multiple myeloma (MM) development. In this study, we aim to systematically characterize the tumor immune microenvironment (TME) and the tumor-immune interactions from precursor stages, i.e., monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM), to newly diagnosed MM, comparing these to healthy donors. Using CIBERSORT, mass cytometry (CyTOF), and single-cell RNA sequencing (scRNA-Seq), we examined innate and adaptive immune changes across these stages. We found a decrease in granulocytes in the TME predicts MM outcomes. HLA-DR is reduced in CD16+ monocytes and plasmacytoid dendritic cells, while myeloid dendritic cells show decreased expression of stress and immune-response genes. NK cells and CD8+ T cells shift from a GZMK+ to a GZMB+ cytotoxic phenotype in the TME, with increased inhibitory markers TIM3 and TIGIT. In paired samples, the proportion and gene expression pattern in patient-specific GZMB+CD8+ T cells remain largely unchanged despite MM progression. Our findings provide a comprehensive immune landscape of MM and its precursors, offering insights into therapeutic strategies. Enhancing neutrophil and NK cell cytotoxicity, tumor antigen presentation, and CD8+ T cell versatility in precursor stages may prevent MM progression.

Neutrophil pyroptosis regulates corneal wound healing and post-injury neovascularisation.

The cornea is a unique structure that maintains its clarity by remaining avascular. Corneal injuries can lead to neovascularisation (CNV) and fibrosis and are the third most common cause of blindness worldwide. Corneal injuries induce an immune cell infiltration to initiate reparative processes. However, inflammation caused by sustained immune cell infiltration is known to be detrimental and can delay the healing process. This study was designed to understand the potential role of neutrophil and epithelial cell crosstalk in post-injury CNV. Western blotting and immunostaining assays demonstrated that neutrophils infiltrated corneas and underwent pyroptosis following acute alkali injury. In vivo studies showed that genetic ablation of Gasdermin D (GsdmD), a key effector of pyroptosis, enhanced corneal re-epithelialisation and suppressed post-injury CNV. In vitro co-culture experiments revealed that interleukin-1β (IL-1β) was released from pyroptotic neutrophils which suppressed migration of murine corneal epithelial cells. Real-time RT-PCR and immunostaining assays identified two factors, Wnt5a and soluble fms-like tyrosine kinase-1 (sflt-1), highly expressed in newly healed epithelial cells. sflt-1 is known to promote corneal avascularity. Bone marrow transplantation, antibody mediated neutrophil depletion, and pharmacological inhibition of pyroptosis promoted corneal wound healing and inhibited CNV in an in vivo murine corneal injury model. Taken together, our study reveals the importance of neutrophil/epithelium crosstalk and neutrophil pyroptosis in response to corneal injuries. Inhibition of neutrophil pyroptosis may serve as a potential treatment to promote corneal healing without CNV. Neutrophil pyroptosis delays re-epithelialization after corneal injury Compromised re-epithelialization promotes corneal neovascularization after injury Inhibition of post-injury pyroptosis could be an effective therapy to promote corneal wound healing.

Hypoxia-inducible factor-2α enhances neutrophil survival to promote cardiac injury following myocardial infarction.

Heart failure is a major cause of mortality following myocardial infarction. Neutrophils are among the first immune cells to accumulate in the infarcted region. Although beneficial functions of neutrophils in heart injury are now appreciated, neutrophils are also well known for their ability to exacerbate inflammation and promote tissue damage. Myocardial infarction induces hypoxia, where hypoxia-inducible factors (HIFs) are activated and play critical roles in cellular functions. In this context, the role of Hif2α in neutrophils during myocardial infarction is unknown. Here, we demonstrate that neutrophil Hif2α deletion markedly attenuates myocardial infarct size, improves cardiac function, reduces neutrophil survival and tissue accumulation, and correlates with increased macrophage engulfment rates. Mechanistic studies revealed that Hif2α promotes neutrophil survival through binding to hypoxia response element (HRE) in the promoter region of Birc2 to regulate expression of the prosurvival factor, cellular inhibitor of apoptosis protein-1 (cIAP1). Inhibition of cIAP1 in neutrophils using the pharmacological agent, Birinapant resulted in increased cell death, establishing a critical role of cIAP1 downstream of Hif2α in neutrophil survival. Taken together, our data demonstrate a protective effect of Hif2α deletion in neutrophils on cardiac injury outcomes through modulation of neutrophil cell survival.NEW & NOTEWORTHY Hif2α in neutrophils increases infarct size, cardiac dysfunction, and ventricular scar after myocardial infarction. Hif2α in neutrophils supports neutrophil survival via cIAP-1 signaling and delays macrophage engulfment.

Deciphering lipoprotein(a) atherogenicity with use of high-throughput proteomics

Abstract Background Accumulating observational and genetic evidence suggests that lipoprotein(a) [Lp(a)], is significantly more atherogenic than other apolipoprotein-B containing particles. Systemic inflammation is thought to mediate the atherogenic effects of Lp(a). Previous studies have predominantly focused on single inflammatory biomarkers, an approach that fails to capture the broader molecular response to elevated Lp(a) levels. Purpose The aim of this study was to derive a proteome-wide signature of plasma Lp(a) concentrations and subsequently identify the pathways through which Lp(a) influences the risk of coronary artery disease (CAD). Methods This study included 41,123 UK Biobank participants with baseline Lp(a) measurements who also underwent a proteomic assessment. Relative concentration of 2,920 proteins was quantified in EDTA plasma samples using a high-throughput proximity extension assay. Lp(a)-associated proteins were identified in linear regression models, adjusting for age, sex, body mass index, race, smoking status, estimated glomerular filtration rate, medication use, and apolipoprotein B. Proteins linked with Lp(a) concentration were then evaluated for their association with incident CAD risk using Cox proportional hazards models. Significant traits were further included in pathway enrichment analysis. Results Out of 2,920 analyzed proteins (Figure 1), 140 showed a positive association with Lp(a) concentration, while 336 exhibited a negative association after adjustment for multiple testing (False discovery rate < 0.05). The strongest positive associations were observed for apolipoprotein F, group 10 secretory phospholipase A, and C4b-binding protein beta chain. The strongest negative associations were observed for asporin, aminoacylase-1, and retinol-binding protein 5. Among 140 proteins positively associated with Lp(a), 49 were associated with an increased risk of CAD. Conversely, among the 336 proteins negatively associated with Lp(a), 10 were associated with a decreased risk of CAD. Pathway enrichment analysis (Figure 2) indicated that proteins linked with both Lp(a) levels and CAD risk predominantly upregulated pathways related to peptide-ligand receptor signalling, platelet degranulation, neutrophil degranulation, and cytosolic calcium response. Conversely, pathways related to HDL remodeling, PI3K/AKT, KIT, and nuclear receptor signalling were downregulated. Conclusions The greater atherogenic potential of Lp(a) may be attributable to the modulation of several molecular pathways, particularly those involving platelet and neutrophil responses, cell signalling, and lipoprotein metabolism.

Post-exchange neutrophil count, but not post-hematocrit, predicts endogenous erythropoiesis in patients with sickle cell disease undergoing chronic red cell exchange.

With chronic transfusion in sickle cell disease (SCD), equipoise exists regarding whether increasing the post-procedure hematocrit (Hct) suppresses endogenous erythropoiesis. Reticulocytosis predicts SCD morbidity and mortality, so this study's objective was to clarify the role of the post-procedure Hct in suppressing reticulocytosis and to identify other potential red cell exchange (RCE) parameters predictive of reticulocytosis. This retrospective analysis of 17 patients who underwent chronic RCE at a single institution between 2014 and 2022 examined both standard red cell exchanges (SRCE) and exchanges preceded by isovolemic hemodilution (IVH-RCE). Post-procedure parameters with biologic plausibility to influence the subsequent procedure's absolute reticulocyte count (sPre-ARC) were examined using regression modeling. Neither post-hematocrit, nor post-hemoglobin (Hb), nor ΔHb/day was associated with sPre-ARC or the change in HbS% per day (ΔHbS%/day). Concurrent Hb was predictive for SRCE but not IVH-RCE, where ARC trended lower than with SRCE. Male gender and post-procedure neutrophil and white cell counts were predictors of sPre-ARC, consistent with their associations with SCD morbidity and mortality. IVH-RCE had a stronger correlation than standard RCE between pre-Hct and neutrophil or white cell depletion. Although targeting a post-procedure Hct maintains a higher subsequent pre-procedure Hb and a lower sPre-HbS%, it does not lead to sustained suppression of reticulocytosis as measured by the sPre-ARC or the ΔHbS%/day. IVH-RCE or the addition of hydroxyurea could be considered in those patients with high reticulocyte, white blood cell, or neutrophil counts.

Effect of inflammatory factors on myocardial infarction

BackgroundCohort studies have increasingly shown associations between inflammatory markers and myocardial infarction (MI); however, the specific causal relationships between inflammatory markers and the development of MI remain unclear.Methods and resultsBy utilizing publicly accessible genome-wide association studies, we performed a two-sample Mendelian randomization (MR) analysis to explore the causal associations between inflammatory markers and myocardial infarction (MI). A random-effects inverse-variance weighted method was used to calculate effect estimates. The study included a total of 395,795 European participants for MI analysis and various sample sizes for inflammatory factors, ranging from 3,301 to 563,946 participants.Neutrophil count was found to increase the risk of MI (odds ratio [OR] = 1.08; 95% confidence interval [CI], 1.00–1.17; p = 0.04). C-reactive protein levels correlated positively with MI. No associations were observed with IL-1 beta, IL-6, IL-18, procalcitonin, TNF-α, total white cell count, or neutrophil percentage of white cells. Neutrophil count and C-reactive protein were inversely associated with lactate dehydrogenase: neutrophil cell count (OR 0.95; 95% CI, 0.93–0.98; p < 0.01) and C-reactive protein (OR 0.96; 95% CI, 0.92–1.00; p = 0.02). No associations of MI with myoglobin, troponin I, and creatine kinase-MB levels were found.ConclusionsThis two-sample MR analysis revealed a causal positive association of MI with neutrophil count, C-reactive protein level, and the myocardial injury marker lactate dehydrogenase. These results indicate that monitoring C-reactive protein and neutrophil counts may be useful in management of MI patients.

APEX1 in intestinal epithelium triggers neutrophil infiltration and intestinal barrier damage in ulcerative colitis

Ulcerative colitis (UC) can lead to the generation of large amounts of reactive oxygen species and DNA damage. DNA repair caused by base excision repair (BER) enzymes is an important mechanism for maintaining genomic integrity. However, the specific relationship between the function of BER enzymes and UC remains unclear. To address this, we conducted a study on non-cancerous colon tissue from patients with UC, focusing on the role of apurinic/apyrimidinic endonuclease 1 (APEX1) in BER to explore its significance in the progression of UC. Our research found that the expression of APEX1 in epithelium cells was significantly correlated to the severity of inflammatory bowel disease (IBD) and the infiltration and function of neutrophils in human UC and mouse models, particularly in relation to neutrophil extracellular traps (NETs) and the degranulation processes. APEX1 deficiency resulted in decreased production of the chemokines CXCL1 by the NF-κB pathway in epithelium cells, leading to reduced accumulation and activation of neutrophils associated with colitis in colon tissue, as well as decreased levels of IL-1β. Furthermore, APEX1 deficiency reduced symptoms of colitis by decreasing epithelial cell apoptosis and altering the gut microbiome. Studies related to the redox activity of APEX1 have shown that the combination of the redox inhibitor E3330 with 5-aminosalicylic acid (5-ASA) can effectively alleviate colitis, indicating that APEX1 has promising prospects for clinical treatment of IBD. APEX1 is required for interactions between neutrophil and intestinal epithelial cells. This study provided a mechanism demonstrating that APEX1 protein triggered the risk of UC by promoting neutrophil infiltration and compromising intestinal epithelial barrier function.

Effect of the injection of protoscolex antigens for hydatid cysts on some immunological parameters and the protection of injury

This study aimed to that capability of preparation of antigens from protoscolex found inside the hydatid cyst and likewise possibility of preparing antigen providing protection of the injury of this cysts. Cystic echinococcosis is the more important parasitic disease caused by a cestode tapeworm of species Echinococcus granulosus, spreading a lot in Iraq and the Eastern Mediterranean Countries in addition to its world distribution. The protoscolex found inside hydatid cysts results from the germinal layer of hydatid cyst isolated from people by the surgery section at the Fallujah teaching hospital to find out the extent of its influence on both of the cellular and humeral immunity. 20 white mice (Balb/c) were divided into two groups, the first group of white mice (Balb/ c) was injected subcutaneously with a concentration of 0.1ml of antigen and it measured antibodies IgG, IgA, IgM, IgE as well as injected with complement C3, C4 and the insulation of neutrophil cells and measuring percentage of phagocytosis, reduction of a dye NBT and viability of neutrophil cells and in the second group, the protoscolex was injected in the liver with a concentration 0.1ml to find out the extent of prevention provided by the antigen to prevent or to reduce injury. The results showed a significant increase for all types of antibodies and complements C3 , C4 and reduction of a dye NBT and phagocytosis especially at the time 60 minute and the results were not showing significant differences on viability of neutrophil cells. As for prevention of injury, the results have shown a significant difference at the level &lt; 0.05 and reached the percentage of prevention 56.4 % compared to the subordinate control. The results showed small size of the hydatid cyst in the experience parameters compared to the subordinate control.

Fabrication of a cell irradiation technique by alpha-particles using allyl diglycol carbonate (ADC) detector and micro-capillary tubes.

To study the impact of micro-alpha irradiation collimator with a specific design to irradiate specific normal or up-normal cells using capillary tubes and nuclear track detector type CR-39. The in vitro experimental study was conducted at radiobiology Lab, of the Physics department, Salahaddin University, Erbil, Iraq from February to April 2022. Several alpha irradiation collimators were calibrated using allyl diglycol carbonate to fabricate a suitable blood cell irradiation technique. Time of irradiation and alpha particle energy were calibrated. Healthy blood samples of Albino rats and cancer blood samples were used to assess the applicability of the fabricated cell irradiation technique. The Rats were divided into intervention and control groups. Data was analysed using SPSS software version 28. Of the 15 healthy, male Albino rats having a mean weight of 230±12g each, there were 12(80%) in the intervention group and 3(20%) in the control group. Microcapillary tubes with suitable diameters had high stability for deposition of a sufficient density of alpha particles on the surface of allyl diglycol carbonate and blood samples. The optimum time of irradiation that had a significant (p<0.05) effect was 20 sec corresponding to alpha energy 4.5MeV. Low irradiation time had significant impact of alpha particles on the average percentage of lymphocyte and neutrophil cells.

Unveiling the influence of circulating immune cells count on type 1 diabetes: Insight from bidirectional Mendelian randomization.

Observational studies have demonstrated an association between circulating immune cell and type 1 diabetes (T1D) risk. However, it is unknown whether this relationship is causal. Herein, we adopted a 2-sample bidirectional Mendelian randomization study to figure out whether circulating immune cell profiles causally impact T1D liability. Summary statistical data were obtained from genome-wide association study (GWAS) to investigate the causal relationship between white cell (WBC) count, 5 specific WBC count, and lymphocyte subtypes cell count and T1D risk. After false discovery rate (FDR) correction, the results indicated that lower lymphocyte cell count (odds ratio [OR] per 1 standard deviation [SD] decrease = 0.746, 95% confidence interval (CI): 0.673-0.828, PFDR = 0.036), and basophil cell count (OR per 1 SD decrease = 0.808, 95% CI: 0.700-0.932, PFDR = 0.010) were causally associated with T1D susceptibility. However, the absolute count of WBC, monocyte, neutrophil, eosinophil, and lymphocyte subtypes cell had no statistically significant effect on T1D risk. Taken together, this study indicates suggestive association between circulating immune cell count and T1D. Moreover, lower numbers of circulating lymphocyte and basophil cell were associated with the increased risk of T1D, which confirmed the immunity predisposition for T1D.

Identification of GPNMB in endometrial cancer based on pan-cancer analysis and in vitro validation

BackgroundGPNMB is a type I transmembrane protein, and emerging evidence supports the relationship between GPNMB and cancers.ObjectiveThrough a comprehensive pan-cancer analysis, we examined the expression levels, prognostic significance, and mutation profiles of GPNMB in different cancer types. Subsequently, utilizing in vitro experiments, we elucidated the impact of GPNMB in endometrial cancer (EC).MethodsTIMER2, GEPIA2, UALCAN and cBioPortal were used to analyze the expression pattern, prognostic values, and mutation status of GPNMB. HEC-1B and Ishikawa cells were used to conduct in vitro analyses of GPNMB overexpression. GeneMANIA and TIMER2 were used to evaluate the potential functions and correlations between GPNMB expression and tumor-infiltrating immune cells in EC.ResultsGPNMB was found to be highly expressed in multiple cancers, where it was associated with poor prognosis. Additionally, GPNMB was downregulated at both mRNA and protein levels in EC. Overexpression of GPNMB inhibited the proliferation, migration, and invasion of HEC-1B and Ishikawa cells. Functional analysis showed that GPNMB was enriched in pathways associated with regulation of plasma lipoprotein particle levels. The expression of GPNMB was positively connected with B cell, CD8+ T cell, CD4+ T cell, Macrophage, Neutrophil, and Dendritic cell levels.ConclusionThrough pan-cancer analysis, we identified the antitumor effect of GPNMB in EC and predicted the potential mechanisms between GPNMB expression and EC.

Serum Procalcitonin, Hematology Parameters, and Cell Morphology in Multiple Clinical Conditions and Sepsis.

The clinical value of procalcitonin (PCT) in infection diagnosis and antibiotic stewardship is still unclear. This study aimed to investigate the association between serum PCT and different clinical conditions as well as other infectious/inflammatory parameters in different septic patients in order to elucidate the value of PCT detection in infection management. Chemiluminescence immunoassay was used for serum PCT analysis. Hematology analysis was used for complete blood cell count. Digital automated cell morphology analysis was used for blood cell morphology examination. Blood, urine, and stool cultures were performed according to routine clinical laboratory standard operating procedures. C-reactive protein (CRP) was analyzed by immunoturbidimetry. Erythrocyte sedimentation rate test was performed using natural sedimentation methods. Outpatients, ICU patients, and patients under 2 years of age with respiratory infections had higher serum PCT levels. Septic patients had the highest-serum PCT levels and other infection indexes. PCT levels in the blood, urine, and stool culture-positive patients were significantly higher than in culture-negative patients. The neutrophil granulation and reactive lymphocytes were observed together with the PCT-level increments in different septic patients, and these alterations were lessened after treatment. There was no significant change in monocyte morphology between pre- and posttreatment septic patients. Serum PCT is associated with neutrophil cytotoxicity and lymphocyte morphology changes in sepsis; thus, the combination of neutrophil and lymphocyte digital cell morphology evaluations with PCT detection may be a useful examination for guiding the clinical management of sepsis.

Clinical variability of equine asthma phenotypes and analysis of diagnostic steps in phenotype differentiation

BackgroundEquine asthma is a common, non-infectious, chronic lung disease that affects up to 80% of the horse population. Strict phenotyping and identification of subclinically asthmatic horses can be challenging. The aim of this study was to describe equine asthma phenotypes (mild, moderate, and severe asthma) defined by BALF cytology and occurrence of clinical signs in a population of privately owned horses and to identify the variables and examination steps with best discriminative potential. The standardised examination protocol included clinical examinations, blood work, airway endoscopy with bronchoalveolar lavage fluid analysis, arterial blood gas analysis and radiography under clinical conditions performed by one veterinarian.ResultsOut of 26 horses, four were diagnosed with mild (subclinical), seven with moderate, and seven with severe asthma based on clinical examination and BALF cytology. Eight horses served as controls. Cough with history of coughing was the strongest variable in phenotype differentiation. Factor analysis revealed an increasing clinical variability with disease severity and an overlapping of clinical presentations between phenotypes. Elevated mast cell (4/4 horses) and neutrophil counts (3/4 horses) in bronchoalveolar lavage cytology differentiated mild asthmatic horses from healthy horses. Moderate and severe asthmatic horses were characterised by clinical signs and neutrophil counts.ConclusionsThe results indicate that medical history, clinical examination and bronchoalveolar lavage cytology are minimum indispensable steps to diagnose equine asthma and that phenotypes are clinically overlapping. A differentiation of three phenotypes without neutrophil and mast cell counts in bronchoalveolar lavage cytology is not sufficient for clinical diagnostics. A comparably exact diagnosis cannot be achieved by relying on alternative examinations used in this study. Screenings of inconspicuous horses with bronchoalveolar lavage can aid in diagnosing subclinically affected animals, however, group size was small, the procedure is invasive and clinical relevance of slightly elevated cells in bronchoalveolar lavage remains unclear. Clinical relevance could not be clarified in this study, since follow-up examinations or lung function testing were not performed.

Abstract A065: Spatial and single cell transcriptomics uncovers metabolic reprogramming and NETosis of neutrophils in human pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma is the most hypovascular solid tumor, imposing harsh constraints on tumor metabolism. Cancer cells adapt to reduced access to vascular-derived nutrients and O2 by cooperating with abundant stromal cells. Specifically, critical nutrients including alanine, proline, glycosaminoglycans, branched chain ketoacids, lysophosphatidic acid, and unsaturated fatty acids can be provided by fibroblasts in vitro and in vivo. However, the extent of metabolic support provided by other cell types in the PDAC TME is relatively unexplored. We hypothesized that cell types that are cooperating metabolically with cancer cells will be spatially positioned near cancer cells in these tumors. We performed Xenium spatial transcriptomics on 8 treatment-naive PDAC resections and generated a spatial atlas of nearly 700,000 cells across a range of expected cell types. We noted striking neutrophil aggregates, nucleating in the glands of classical-like malignant cells and elaborating extraglandularly. Cellular neighborhood analysis demonstrates neutrophil aggregation is strongly correlated with malignant cell proliferation and low endothelial content, suggesting neutrophils may be an important source of pro-tumoral, non-vascular nutrients in the PDAC TME. Reanalysis of published scRNAseq data revealed 3 neutrophil metabolic states in human PDAC tumors. Metabolic cluster 7 neutrophils were found only in PDAC patient samples but not healthy controls and were enriched for Type I interferon response as well as pentose phosphate pathway and iron uptake. Metabolic cluster 2 neutrophils were strikingly depleted from PDAC tumor samples compared to blood, suggesting decreased infiltration or increased cell death. Cluster 2 neutrophils were enriched for several catabolic pathways including glycogenolysis, autophagy, and proteasome, and selectively express the NETosis gene PADI4. Multiplexed immunofluorescence demonstrated that neutrophil aggregates in PDAC are citH3+/MPO+ large neutrophil extracellular traps (NETs). We reasoned pancreatic cancer cells could proliferate under tumor-like metabolic stress by repurposing amino acids liberated by autophagy, glucose liberated by glycogenolysis, or simply consuming bulk neutrophil dead cell debris via macropinocytosis. Using tumor interstitial fluid media (TIFM) and 0.5% O2 to mimic tumor-like metabolic stress, we cocultured pancreatic cancer cells with human primary neutrophils and HL60-derived neutrophil-like cells. However, under tumor-like metabolic conditions, coculture with human primary neutrophils failed to substantially increase cancer cell proliferation and NETotic neutrophils were cytotoxic. However, we noticed enhanced neutrophil viability in tumor-like metabolic conditions, suggesting metabolic regulation of neutrophil cell death. Together, these data shed light on the metabolic phenotypes and interactions with cancer cells of neutrophils in the PDAC tumor microenvironment. Citation Format: Carson D Poltorack, Sydney M Shaffer, M Celeste Simon. Spatial and single cell transcriptomics uncovers metabolic reprogramming and NETosis of neutrophils in human pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A065.

Identification of the shared gene MXD3 signatures and biological mechanism in patients with hip pain and prostate cancer.

Prostate cancer (PRAD) is recognized as having a significant effect on systemic illnesses. This study examined possible immune cells, metabolic pathways, and genes that may explain the interaction between PRAD and hip pain. We used information retrieved from the Cancer Genome Atlas and the Gene Expression Omnibus databases. To find common genes, we utilized differential expression analysis and weighted gene co-expression network analysis. The genes that were shared were subjected to pathway enrichment studies using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. Additionally, hub genes were analyzed using LASSO regression, and a receiver operating characteristic curve was generated based on the screening outcomes. The genes for the nodes were chosen in a protein-protein interaction network that was built. Single-sample gene-set enrichment analysis was performed to identify the differentially expressed genes. Immunohistochemistry staining confirmed hub gene expression, and single-sample gene-set enrichment analysis assessed immune cell infiltration. We concluded by comparing MAX dimerization protein 3 (MXD3) and MAX interactor 1 (MXI1) expression in tumor tissues using Uniform Manifold Approximation and Projection and violin plots in the Tumor lmmune Single-cell Hub database. After analyzing the intersection of the differentially expressed genes and weighted gene co-expression network analysis-significant module genes, we determined that MXD3 was the best shared diagnostic biomarker for PRAD and hip pain. One potential predictor of PRAD development was the MXI1 node gene, which was found in the protein-protein interaction network. The analyses revealed that MXD3 had a relatively positive correlation with neutrophil and T-helper cell infiltration levels, whereas MXI1 had a negative correlation with mast and Tgd cell levels. Tumors had lower levels of MXI1 expression and higher levels of MXD3 expression compared to normal tissues. Endothelial cells, induced pluripotent stem cells, and smooth muscle cells were all found to express MXI1. This is the first study to investigate the close genetic link between hip pain and PRAD using bioinformatics technologies. The 2 most significant genes involved in crosstalk between PRAD and hip pain were MXD3 and MXI1. The immunological responses triggered by T cells, mast cells, and neutrophils may be crucial in the relationship between PRAD and hip pain.

A systems serology approach to identifying key antibody correlates of protection from cerebral malaria in Malawian children

BackgroundPlasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins are expressed on the surface of infected erythrocytes, mediating parasite sequestration in the vasculature. PfEMP1 is a major target of protective antibodies, but the features of the antibody response are poorly defined.MethodsIn Malawian children with cerebral or uncomplicated malaria, we characterized the antibody response to 39 recombinant PfEMP1 Duffy binding like (DBL) domains or cysteine-rich interdomain regions (CIDRs) in detail, including measures of antibody classes, subclasses, and engagement with Fcγ receptors and complement. Using elastic net regularized logistic regression, we identified a combination of seven antibody targets and Fc features that best distinguished between children with cerebral and uncomplicated malaria. To confirm the role of the selected targets and Fc features, we measured antibody-dependent neutrophil and THP-1 cell phagocytosis of intercellular adhesion molecule-1 (ICAM-1) and endothelial protein C (EPCR) co-binding infected erythrocytes.ResultsThe selected features distinguished between children with cerebral and uncomplicated malaria with 87% accuracy (median, 80–96% interquartile range) and included antibody to well-characterized DBLβ3 domains and a less well-characterized CIDRγ12 domain. The abilities of antibodies to engage C1q and FcγRIIIb, rather than levels of IgG, correlated with protection. In line with a role of FcγRIIIb binding antibodies to DBLβ3 domains, antibody-dependent neutrophil phagocytosis of ICAM-1 and EPCR co-binding IE was higher in uncomplicated malaria (15% median, 8–38% interquartile range) compared to cerebral malaria (7%, 30–15%, p < 0.001).ConclusionsAntibodies associated with protection from cerebral malaria target a subset of PfEMP1 domains. The Fc features of protective antibody response include engagement of FcγRIIIb and C1q, and ability to induce antibody-dependent neutrophil phagocytosis of infected erythrocytes. Identifying the targets and Fc features of protective immunity could facilitate the development of PfEMP1-based therapeutics for cerebral malaria.