Blood Leukocytes Research Articles

The impact of PD-L1 polymorphisms on the efficacy of immune checkpoint inhibitors depends on the tumor proportion score: a retrospective study

PurposeThis study aims to clarify the relationship between rs2282055, a single-nucleotide polymorphism (SNP) in programmed death-ligand 1 (PD-L1), and TPS. Polcaro et al. (2024) showed that rs822336, a SNP in PD-L1, predicts the effect of immune checkpoint inhibitors (ICIs). However, the study did not show a relationship between rs822336 and the tumor proportion score (TPS), which is currently used as a primary marker. Therefore, we examined this relationship.MethodPatients treated with immune checkpoint inhibitor monotherapy for non-small cell lung cancer at Kyoto University Hospital until January 2023, with TPS data and biological specimens available for SNP measurement, were eligible for this study. Genomic DNA was extracted from peripheral blood leukocytes. We used rs2282055, which is in linkage disequilibrium with rs822336, instead of rs822336, because of its distribution in the Asian patient population. We retrospectively extracted data on age, sex, smoking history, driver mutations, TPS, progression-free survival (PFS), and best response to ICI from medical records.ResultThe rs2282055 T/T genotype was associated with significantly better PFS in the TPS-negative population than in the other genotypes. In contrast, no differences were observed in TPS-positive patients.ConclusionThe rs2282055 genotype may help in selecting cases from the TPS-negative patient population that may benefit from ICI therapy.

Abstract TP361: Type 2 Diabetes Enhances Leukocytes And Brain Endothelial Cells Adhesion Interactions Following Ischemic Stroke

Introduction: Stroke is a leading cause of death and disability worldwide, with diabetes being a significant risk factor that exacerbates stroke outcomes. While previous studies have identified various cell populations characteristic of stroke and other neurological diseases through transcriptomic analysis of brain and blood cells, the interaction between leukocytes and brain endothelial cells during stroke remains inadequately understood. This study aimed to investigate these intercellular interactions in the context of diabetes and stroke. Methods: We utilized a permanent ischemic stroke model in leptin receptor-deficient db/db mice (a model for type 2 diabetes) and their non-diabetic counterparts, db/+ mice. Three days post-stroke, brain cells and circulating leukocytes were isolated and subjected to single-cell RNA sequencing (scRNAseq). The CellChat tool was employed to analyze the intercellular communication between these cells. Additionally, in vivo two-photon microscopy was used to observe real-time interactions between leukocytes and brain endothelial cells in cortical veins. Results: scRNAseq analysis revealed that diabetic mice had a higher proportion of myeloid cells in the blood compared to controls. Post-stroke, both diabetic and non-diabetic mice showed an increased presence of microglia and macrophages in the brain. CellChat analysis indicated that both diabetes and stroke significantly elevated the number and strength of cell-cell interactions, with the highest levels observed in the diabetic stroke group. Notably, interactions involving the integrin family and ICAM/VCAM, known for their roles in cell adhesion between blood leukocytes and brain endothelial cells, were particularly prominent in the diabetic mice. In vivo imaging confirmed significant leukocyte accumulation in the cortical veins immediately after stroke in both genotypes, with delayed clearance observed in the diabetic group. Conclusion: The findings suggest that diabetes exacerbates the inflammatory response in stroke by enhancing leukocyte-endothelial interactions, leading to prolonged leukocyte retention in the cerebral vasculature, which likely contributes to increased inflammation and worsened stroke outcomes in diabetic conditions.

Scale to predict risk for refractory septic shock based on a hybrid approach using machine learning and regression modeling.

To develop a scale to predict refractory septic shock (SS) based on clinical variables recorded during initial evaluations of patients. Multicenter retrospective study of data for patients with suspected infection registered in the Marketplace for Medical Information in Intensive Care (MIMIC-IV). These data were used for the development and internal validation of the refractory SS scale (RSSS). For external validation, we used retrospective data for 2 cohorts: 1) patients diagnosed with SS in an emergency department (ED cohort) whose data were registered in a Korean SS registry, and 2) patients diagnosed with SS in 6 hospital intensive care units (ICU cohort). A machine-learning automatic clinical scoring system (AutoScore) was used in the development phase. The performance of the RSSS in the validation cohorts was assessed with the area under the receiver operating characteristic curve (AUROC) for each. The primary outcome was the development of refractory SS within 24 hours of ICU admission. Refractory SS was defined by the need for a norepinephrine-equivalent dose greater than 0.5 µg/kg/min. We collected data for 29 618 patients from the MIMIC-IV registry, 3113 patients for the ED cohort, and 1015 for the ICU cohort. The RSSS had 6 predictors: serum lactate level, systolic blood pressure, heart rate, temperature, arterial pH, and leukocyte count. The scale's AUROCs were as follows: 0.873 (95% CI, 0.846-0.900) in the internal validation, 0.705 (95% CI, 0.678-0.733) in the ED cohort on arrival, 0.781 (95% CI, 0.757-0.805) in the ED cohort at the moment of diagnosing hypoperfusion or hypotension, and 0.822 (95% CI, 0.787-0.857) in the ICU cohort. Calibration was acceptable in all the cohorts. The RSSS had adequate diagnostic accuracy in multiple cohorts of patients diagnosed in the ED and ICU.

Abnormal Expression of Peripheral Blood Leukocyte Surface Markers in Retinopathy of Prematurity Patients

Abnormal Expression of Peripheral Blood Leukocyte Surface Markers in Retinopathy of Prematurity Patients

Complement-activated fragment Ba functions as an antibacterial protein and mediates immune responses in lower vertebrates.

The complement system plays an important role in antibacterial infection and immune regulation. Ba, an important complement component, is produced and released by the cleavage of complement factor B (CFB) during complement activation. However, the immune functions of Ba are unclear. In this study, we reported that recombinant Ba exerted direct bactericidal and immune regulatory effects. Recombinant Paralichthys olivaceus Ba (rPoBa) bound bacteria via interaction with the bacterial wall component lipopolysaccharide (LPS), resulting in bacterial membrane permeabilization and bacterial death. Furthermore, rPoBa exhibited bactericidal activity against Gram-negative bacteria in a manner that depended on concentration, time, temperature, pH, and metal ions. Structure prediction analysis showed that PoBa contained three distinct CCP domains. CCP1 was mainly responsible for binding to LPS, and both CCP1 and CCP3 might be required for bacterial membranous permeabilization. The bactericidal effects of Ba were observed only in lower vertebrates, with no such effects observed in mammals. In addition, rPoBa could protect P. olivaceus against Vibrio harveyi infection both in vitro and in vivo by significantly improving the immune activity of peripheral blood leukocytes and reducing tissue bacterial loads. Consistently, when PoCFB expression in P. olivaceus was knocked down, the PoBa production and complement activity were decreased, and bacterial replication was significantly enhanced. In conclusion, this study revealed that the complement-activated recombinant Ba fragment improved the immune defense against bacterial infection and provided a potential strategy to control disease in lower vertebrates.

Abstract TP341: Longitudinal Analysis of Peripheral Blood Dynamics During the First Week after Ischemic Stroke: Biomarkers and Correlation to Functional Outcome

Introduction: Peripheral blood-based biomarker development for neurological diseases such as ischemic stroke has traditionally focused on measuring alterations in leukocyte number and composition. Deeper immunophenotyping of cells using fluorescent probes that detect alterations among organelles and structures can provide a better understanding of the biological function of leukocytes after ischemic stroke. Methods: We evaluated the utility of BODIPY 493/503 and LysoTracker as fluorogenic probes to assess cellular changes in lipid content and lysosomal burden. Flow cytometric assessments were performed on peripheral mononuclear blood cells from healthy volunteers (HV, N=31), and from ischemic stroke patients (N=43) at 3d and 7d to capture the temporal profile of these biomarkers. Results: We found decreased frequency of CD3+ T cells 3d after stroke, which was also seen at 7d after stroke. We found CD4:CD8 ratios were significantly and persistently increased in stroke patients compared to HV controls. Within the CD4 subset, we found a persistent increase in the frequency of effector CD4 T cells after stroke, this finding is consistent with previous literature. Interestingly, we noted a delayed, but profound increase in lipid content of all cell types at 7d after stroke compared controls. This difference was more pronounced among females. Lysosomal content was differentially altered by cell type, with increased levels in CD4 naïve T cells, but decreased levels in CD4 central memory, CD8 naïve, CD8 effector, CD8 central and effector memory T cells after stroke. While we did not see an association with increase in severity at admission, high lipid content at the acute phase was significantly associated with increased disability at 3 months after hospitalization. Conclusion: Taken together, our findings provide greater context to the previously documented compositional changes in the blood leukocyte compartment after stroke. Ischemic stroke causes a pervasive increase in cellular lipidosis. Lysosomal content has also been significantly positively correlated to functional disability among stroke patients. Using cell-based functional assays with flow cytometric immunophenotyping may provide meaningful insights into biological health and function of circulating leukocytes after brain injury. Understanding the effects of these compositional changes seen in the acute phase in respect to functional outcomes should be a priority for researchers moving forward.

Leucocyte profile of blood in cows with purulent-necrotic lesions in the hoof area

The work is devoted to the study of the leukocyte profile of blood in animals with orthopedic diseases. Experimental studies were conducted at the Khmelevskoye LLC, Melekessky District, Ulyanovsk Region, during orthopedic medical examination. To achieve the goal set in the work, we formed 2 groups of animals: Group 1 - clinically healthy cows (n = 5). Group 2 - orthopedic diseases (n = 5). Blood samples were taken from all animals before morning feeding, the concentration of leukocytes was studied in the SIMPSON veterinary clinic (Samara) using a Mindray BC-30Vet hematological veterinary analyzer. The leukocyte formula of cattle blood was determined using Philipson staining, and based on the obtained data on the “white blood” parameters, the leukocyte indices were calculated: Garkavi Index, Breddeck Index, Krebs Index, Leukocyte Intoxication Index according to Ya.Ya. Kalf-Kalif, Leukocyte Intoxication Index according to V.K. Ostrovsky, Blood Leukocyte Shift Index, Lymphocyte-Granulocyte Index, Allergization Index according to V.S. Tikhonchuk, Neutrophil and Monocyte Ratio Index according to Zh.G. Mustafina.

Amphibian cellular immune response to chytridiomycosis at metamorphic climax

The fungal disease chytridiomycosis (caused by Batrachochytrium dendrobatidis [Bd]) is a primary contributor to amphibian declines. The frog metamorphic stages, characterised by extensive physiological reorganisation and energy expenditure, have heightened susceptibility to Bd. However, little is known about how these metamorphic stages respond immunologically to Bd infection. In this study, we examined Bd infection and the cellular immune response of Mixophyes fleayi at Gosner stages 40, 42 and 45, using blood smears and skin and liver histology. Although proportional differences were observed, the impact of Bd exposure appeared negligible prior to Gosner stage 45 (onset of morbidity), with no significant differences observed in absolute leukocyte counts for blood or liver samples between control and Bd-exposed groups at Gosner stages 40 and 42. Animals exhibiting clinical signs at Gosner stage 45 demonstrated significant elevation in liver leukocyte counts, blood neutrophil and monocyte counts and neutrophil-to-lymphocyte ratios. These findings are reminiscent of the amplified inflammatory response characteristic of immunopathology in clinically infected amphibians. Interestingly, a subset of exposed animals that had apparently cleared infections at Gosner stage 45 had similar blood leukocyte counts but reduced liver leukocyte counts compared to naïve controls. This could be a consequence of prior cellular consumption during pathogen removal or effective immune regulation via anti-inflammatory protective feedback mechanisms. We recommend targeted gene expression analyses (e.g. immunomodulatory cytokines) to establish the mechanisms responsible for the varied immune expression and infection outcomes across metamorphosis.

Peripheral blood leukocyte signatures as biomarkers in relapsed ovarian cancer patients receiving combined anti-CD73/anti-PD-L1 immunotherapy in arm A of the NSGO-OV-UMB1/ENGOT-OV30 trial.

Immune checkpoint inhibitors have demonstrated limited efficacy in overcoming immunosuppression in patients with epithelial ovarian cancer (EOC). Although certain patients experience long-term treatment benefit, reliable biomarkers for responder pre-selection and the distinction of dominant immunosuppressive mechanisms have yet to be identified. Here, we used a 40-marker suspension mass cytometry panel to comprehensively phenotype peripheral blood leukocytes sampled over time from patients with relapsed EOC who underwent combination oleclumab (anti-CD73) and durvalumab (anti-PD-L1) immunotherapy in the NSGO-OV-UMB1/ENGOT-OV30 trial. We found that survival duration was impacted by baseline abundances of total peripheral blood mononuclear cells. Longitudinal analyses revealed a significant increase in CD14+CD16- myeloid cells during treatment, with significant expansion of monocytic myeloid-derived suppressor cells occurring in patients with shorter progression-free survival, who additionally showed a continuous decrease in central memory T-cell abundances. All patients demonstrated significant PD-L1 upregulation over time on most T-cell subsets. Higher CD73 and IDO1 expression on certain leukocytes at baseline significantly positively correlated with longer progression-free survival. Overall, our study proposes potential biomarkers for EOC immunotherapy personalization and response monitoring; however, further validation in larger studies is needed.

P-231. Association Between the Use of the Pneumonia Panel and Clinical Outcomes in Patients with Ventilator-Associated Pneumonia in a Peruvian Hospital

Abstract Background Ventilator-associated pneumonia (VAP) is a public health problem due to its high mortality in low-income countries. Therefore, the pneumonia panel emerges as a microbiological alternative due to its rapid response, allowing an early and targeted antibiotic treatment, which could reduce morbidity and mortality. The objective of the study was to describe the association between the use of the pneumonia panel versus culture alone and its clinical impact on patients with VAP in the intensive care unit (ICU). Clinical characteristics and outcomes in patients with VAP in the ICU of a hospital in Peru, 2021-2022 *Means ± standard deviations were used. Differences were evaluated using Student's t test. †Frequencies were compared using the Chi-square test. ‡Frequencies were compared using Fisher's exact test. ¥Medians (25th and 75th percentiles) were used. Differences were evaluated using the equality of medians test. Methods This retrospective cohort included patients diagnosed with VAP in the ICU of a hospital in Lima, Peru from May 2021 to February 2022. All patients diagnosed by panel + culture (n=89) were included, and 89 controls diagnosed only by culture were randomly assigned. Adjusted regressions were used to evaluate the association with clinical outcomes. Isolated microorganisms and antimicrobial resistance genes in patients with VAP in the ICU of a hospital in Peru, 2021-2022 All microorganisms that can be detected by the FilmArray Plus pneumonia panel have been placed, and those that have been detected by culture have been added to the culture only group. *The FilmArray Plus Pneumonia Panel does not detect microorganisms such as Stenotrophomonas maltophilia, Achromobacter spp and Citrobacter koseri. Results The mean age was 47 years, 72.5% were men, 19.7% had diabetes and 16.3% had high blood pressure. The most identified microorganisms were: Acinetobacter calcoaceticus-baumannii complex (37.6%), Klebsiella pneumoniae group (34.2%), Pseudomonas aeruginosa (29.7%), Staphylococcus aureus (17.9%), Enterobacter cloacae complex (12.9%) and Escherichia coli (11.2%). The most frequent resistance genes were: CTX-M (30.3%) and NDM (15.7%). When comparing the "panel + culture" and "culture only" groups, leukocyte counts (15.1*10-3/uL versus vs. 12.3*10-3/uL) and C-reactive protein level (195 mg/L versus 132 mg/L) were higher in the panel-use group. Then, adjusted analyses found no statistically significant differences in mortality (RR: 0.56, 95% CI: 0.25 to 1.27), days on mechanical ventilation (MD: 4.74, 95% CI: -0.76 to 10.23), or days in the ICU (MD: 4.94, 95% CI: -1.09 to 10.97). However, the "panel + culture" group had more days of antibiotic therapy (MD: 2.00, 95% CI: 0.45 to 3.55), as well as a greater number of isolated microorganisms. Association between group and clinical outcomes in patients with VAP in the ICU of a hospital in Peru, 2021-2022 RR: Relative risk MD: Mean Difference The adjusted models were adjusted for diastolic blood pressure, fever, FiO2, leukocytes, and C-reactive protein (variables that had a p<0.20 association with group). *The days were counted after the NAV test was requested. Conclusion We found no differences in mortality, number of days in the ICU, and days on mechanical ventilation. However, there was evidence of a greater number of days with antibiotic therapy in the panel-use group. Antibiotic treatment in patients during the VAP episode in the ICU of a hospital in Peru, 2021-2022 * Chi-square test was used, except for imipenem + cilastatin in which the Fisher's exact test was used. Disclosures All Authors: No reported disclosures

P-1576. An Exploration of the Utility of Urinalysis Criteria in Predicting Urine Culture Positivity

Abstract Background Urinalysis (UA) testing with reflex to culture is commonly ordered and is considered a tool for both diagnostic and antibiotic stewardship. Criteria for culture delineate normal from abnormal values leading to use for diagnosis of urinary tract infections (UTI). However, urinalysis criteria which predict urine culture growth that correlate with UTI are not well elucidated. The objective of this study is to evaluate the utility of several different indicators in urinalysis as reflex criteria to urine culture. The area under the curves are: 0.618 for WBCs, 0.607 for nitrites, 0.570 for LE by semi-quantitative testing, and 0.479 for LE by numeric testing. Methods Retrospective review of 14,212 unique de-identified samples of paired urinalysis and urine culture collected in the Froedtert Health System in Milwaukee, Wisconsin from 9/22/22 to 8/23/23. All urine specimens were included regardless of collection method. Cultures were deemed to be positive if growing at least 100,000 colony-forming units (CFU) of a likely urinary pathogen and were evaluated for white blood cells (WBC), leukocyte esterase (LE), and nitrite positivity in the corresponding urinalysis. Results For each criterion, sensitivity and negative predictive values (NPV) were calculated at varying thresholds. A threshold of 5 WBCs provided the greatest NPV at 88.60%, and the greatest sensitivity at 89.76%. Next, receiver operating characteristic (ROC) curves were generated and the area under the curves were: 0.618 for WBCs, 0.607 for nitrites, 0.570 for LE by semi-quantitative testing, and 0.479 for LE by numeric testing. Conclusion Of the three urinalysis criteria, WBC had the best sensitivity and NPV but the ROC curves suggest that none of the tests predict significant urine culture growth well, emphasizing the importance of clinical context in both determining when to test and how to interpret. As diagnosis of a UTI does not simply rest on certain CFU growth or uropathogen on urine culture, but requires incorporation of history and physical findings, the next stage will be to correlate urine testing results with symptoms. Disclosures All Authors: No reported disclosures

P-2363. Efficacy Of SARS–CoV-2 Specific Antiviral Therapy for Enteroviral Myalgic Encephalomyelitis/ChronicFatigue Syndrome

Abstract Background Etiology remains elusive for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), and no treatment exists. Antivirals had no efficacy in randomized clinical trials (RCT) for Epstein-Barr Virus and HHV-6. Enteroviruses (EV) have been implicated, but no antivirals are available. Many patients who received SARS–CoV-2-specific antiviral drugs for acute Covid-19 (COV19) infection experienced significant improvement of prior ME/CFS symptoms. This study summarizes their responses to antivirals for SARS-Cov-2. Methods Neutralizing Antibody (NA) for Coxsackievirus B (CVB)1-6 and Echovirus 6, 7, 9, 11, 30 were done by ARUP lab. Enterovirus Protein (EVP) of Peripheral Blood Leukocytes (PBL) was determined by Western Blot. ME/CFS patients fulfilled Canadian consensus criteria, and had either elevated NA for enteroviruses and/or positive EVP in PBL. ME/CFS patients hospitalized for acute COV19 infection and patients without COV19, were given 5-10 days of IV Remdesivir (Rem) +/- immune modulators. Controls: 20 ME/CFS patients seen concurrently without Remdesivir treatment. Other ME/CFS patients (non-COV19) were given Nirmaltrelvir/Ritonavir (PAX) daily for 10 days +/- one repeat. The energy index (EI) was monitored by the patients before, during and after treatment. Significant improvement was defined as > 30%. Results 15/20 (75%) ME/CFS patients – 10/12 hospitalized, 5/8 non-COV19 patients – responded to IV Rem 2-6 weeks after infusions; remission lasted 6-8 weeks to 6-9 months before relapse. Of Controls: 2/20 had mild improvement (< 0.001, X 2 ). 104/200 (52%) of PAX-treated ME/CFS patients improved, often within 2-3 days; all relapsed within days to weeks after treatment. 66%, 33% and 44% of CVB4+, CVB3+, non-CVB3,4+ patients responded to treatment, respectively. EVP decreased and increased with clinical response and relapse. Conclusion Rem and PAX demonstrated clinical efficacy in ME/CFS patients with chronic enterovirus infections. Placebo-controlled RCT will be needed to clarify the role of antivirals in ME/CFS. Disclosures All Authors: No reported disclosures

Sleeve Gastrectomy and Gastric Bypass Impact in Patient's Metabolic, Gut Microbiome, and Immuno-inflammatory Profiles-A Comparative Study.

Bariatric surgery is the most long-term effective treatment option for severe obesity. The role of gut microbiome (GM) in either the development of obesity or in response to obesity management strategies has been a matter of debate. This study aims to compare the impact of two of the most popular procedures, sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (GB), on metabolic syndrome parameters and gut bacterial microbiome and in systemic immuno-inflammatory response. A prospective observational study enrolled 24 patients with severe obesity, 14 underwent SG and 10GB. Evaluations before (0M) and 6months (6M) after surgical procedures included clinical and biochemical parameters, expression of 17 immuno-inflammatory genes in peripheral blood leukocytes, and assessment of gut microbiome profile using 16s rRNA next-generation sequencing approach. Statistical significance was set to a p value < 0.05 with an FDR < 0.1. A significant and similar decrease in weight-associated parameters and for most metabolic markers was achieved with both surgeries. Considering the gut microbiome in the whole study population, there was an increase in alpha diversity at family-level taxa. Beta diversity between SG and GB at 6M showed near significant differences (p = 0.042) at genus levels. Analysis of the relative abundance of individual taxonomic groups highlighted differences between pre- and post-surgical treatment and between both approaches, namely, a higher representation of family Enterobacteriaceae and genera Veillonella and Enterobacteriaceae_unclassified after GB. Increased expression of immune-inflammatory genes was observed mainly for SG patients. We conclude that SG and GB have similar clinical and metabolic outcomes but different impacts in the gut bacterial microbiome. Results also suggest reactivation of immune response after bariatric surgery.

Leukocyte profile of laying hens’ blood under subchronic intake of sodium bromide with feed

The optimal physiological development and productivity of poultry depends on the intake of essential macroand microelements at levels that ensure the safety and quality of the resulting products (meat and eggs). It is important to note that both a lack and an excess of elements can lead to organ and system dysfunction. For instance, even small doses of bromine can cause hepatocyte dysfunction and disruptions to the endocrine and genitourinary systems. Additionally, the ability of these elements to accumulate in eggs and meat makes the products potentially dangerous for human consumption. Our work aimed to investigate the blood leukocyte profile of laying hens under subchronic intake of sodium bromide with feed and calculate integral leukocyte indices that allow assessing the state of the immune system and the degree of endogenous intoxication of the body. We also aimed to determine the strategy for further therapeutic or preventive measures. It was found that the intake of sodium bromide (by bromine) in the body of laying hens for 28 days in doses of 10.0 mg/kg, 50.0 mg/kg, and 250.0 mg/kg of feed leads to a significant increase in the Krebs index, leukocyte intoxication index, leukocyte shift index. This indicates changes in the functional activity of the immune system with the activation of the humoral immune system and decompensation of endogenous intoxication caused by the toxic effects of bromine. The significant decrease in the leukocyte index, neutrophil-monocyte ratio index, lymphocyte-monocyte ratio index, allergy index, and immunoreactivity index indicates a disruption in the interaction between the affective and effector links of immunity. This could potentially lead to cytokine deficiency, a reduction in the level of phagocytic defense, and an increased risk of immediate hypersensitivity.

Epigenome-wide association study of objectively measured physical activity in peripheral blood leukocytes

BackgroundFew studies have explored the association between DNA methylation and physical activity. The aim of this study was to evaluate the association of objectively measured hours of sedentary behavior (SB) and moderate physical activity (MPA) with DNA methylation. We further aimed to explore the association between SB or MPA related CpG sites and cardiometabolic traits, gene expression, and genetic variation.ResultsFor discovery, we performed cross sectional analyses in pregnant women from the Epigenetics in pregnancy (EPIPREG) sample with both DNA methylation (Illumina MethylationEPIC BeadChip) and objectively measured physical activity data (SenseWear™ Pro 3 armband) (European = 244, South Asian = 109). For EWAS of SB and MPA, two main models were designed: model (1) a linear mixed model adjusted for age, smoking, blood cell composition, including ancestry as random intercept, and model (2) which was additionally adjusted for the total number of steps per day. In model 1, we did not identify any CpG sites associated with neither SB nor MPA. In model 2, SB was positively associated (false discovery rate, FDR < 0.05) with two CpG sites within the VSX1 gene. Both CpG sites were positively associated with BMI and were associated with several genetic variants in cis. MPA was associated with 122 significant CpG sites at FDR < 0.05 (model 2). We further analyzed the ten most statistically significant MPA related CpG sites and found that they presented opposite associations with sedentary behavior and BMI. We were not able to replicate the SB and MPA-related CpG sites in the Avon Longitudinal Study of Parents and Children (ALSPAC). ALSPAC had available objectively measured physical activity data from Actigraph (without steps/day available) and leucocyte DNA methylation data collected during adolescence (n = 408, European).ConclusionThis study suggests associations of objectively measured SB and MPA with maternal DNA methylation in peripheral blood leukocytes, that needs to be confirmed in larger samples of similar study design.

P1309 Is there any association between genetic polymorphisms and response to Anti-TNFs in patients with inflammatory bowel disease ? Experience of a Tunisian IBD center

Abstract Background Although highly effective, primary failure and loss of response to anti tumor necrosis factors (anti-TNF) alpha in inflammatory bowel disease (IBD) are common and associated with poor clinical outcomes. Multiple factors, including genetics, have been involved in the response to anti TNF alpha therapy. Thus, the purpose of our study was to evaluate the impact of the polymorphism of the SNPs rs1799724 (TNF alpha), rs767455 (TNFRSF1A), and rs1061622 (TNFRSF1B) in the response to anti-TNF alpha treatment in Tunisian IBD patients. Methods We conducted a prospective study including patients followed for IBD and treated with anti-TNF alpha. We classified these patients into two groups: responders and non-responders to anti-TNF alpha. Genomic DNA was purified from peripheral blood leucocytes using the salting-out method. Subjects were genotyped for polymorphisms TNF (rs 1799724), TNFRSF1A (rs767455) and TNFRSF1B (rs1061622) using a variant of allele-specific PCR, Mutagenically Separated PCR (MS-PCR). Genotypic and allelic frequencies were compared between the two groups of patients. Results We included 61 IBD patients with a mean age of 38,9 years [18-64 years]. Forty six patients (75.4%) had CD and 15 (24.6%) had UC. In the CD group, 35 (76.1%) patients were treated with infliximab and 11(23.9%) with Adalimumab. Nineteen were responders to treatment (41,3%) and 27 (58.7%) experienced treatment failure: primary failure in 4 and loss of response in 23. Genetic analysis did not show any association between the response to anti TNF alpha treatment and SNPs rs1799724 (TNF alpha) (P= 0,12 ; OR = 1,9) and rs1061622 (TNFRSF1B) (P= 0,9; OR= 1,06). Although, the CT genotype of rs767455 (TNFRSF1A) was associated with non-response (OR= 3.5 with a 95% CI [0.98-12.4], p=0.049). In the UC group, 14 patients were treated with Infliximab and only one patient treated with Adalimumab. Seven patients responded to anti-TNF therapy (46.7%) and 8 were non-responders (53.3%) with 5 cases of primary failure and 3 cases of loss of response. We didn’t find any association between the SNPs rs1799724 (TNF alpha), rs767455 (TNFRSF1A), and rs1061622 (TNFRSF1B) and response to anti TNF alpha in the UC patients. Conclusion Among the three SNPs studied, only the rs767455 (TNFRSF1A) was shown to be associated with non-response to anti-TNF in chrohn’s disease patients. However, our results need to be validated in a larger sample of patients.

Identification and characterization of CD83 and CD276 as markers of dendritic cells in olive flounder (Paralichthys olivaceus).

Dendritic cells (DCs) play a pivotal role in activating naïve T-cells and bridging innate and adaptive immunity. This study aimed to identify and characterize CD83 and CD276 as potential markers for DCs in olive flounder (Paralichthys olivaceus). Specific antibodies against these markers were developed and used to analyze their distribution in peripheral blood leukocytes (PBLs) and intestinal tissues. Flow cytometry and immunohistochemistry revealed that CD83 and CD276 are expressed on DCs, with peak expression observed one week after oral administration of an inactivated viral hemorrhagic septicemia virus (VHSV) vaccine. Gene expression analysis further demonstrated significant activation of immune-related genes, including CD3, IgM, and TLRs, indicating that oral vaccine administration effectively activates the intestinal mucosal immune system. These findings provide valuable insights into fish immune responses and establish CD83 and CD276 as promising DC markers, contributing to the development of mucosal vaccine strategies in aquaculture.

FIRST DATA ON THE MOLECULAR GENETIC DIVERSITY OF HHV6B AND ITS IMPACT ON THE CLINICAL AND LABORATORY COURSE OF MONONUCLEOSIS-LIKE SYNDROME IN CHILDREN

Human herpes virus 6B (HHV6B) is a ubiquitous causative agent of mononucleosis-like syndrome (MLS) and other diseases. The diversity of HHV6B infection may be due to virus genetic polymorphism. Molecular genetic diversity of circulating HHV6B, as well as its impact on the clinical and laboratory features of HHV6B infection have not been studied. The aim of the work was to study the intraspecific diversity of HHV6B circulating among children in Nizhny Novgorod (Russia) region, and to investigate its effect on MLS clinical and laboratory features. Blood leukocytes and saliva DNA samples from healthy children and children with HHV6B infection were analyzed for HHV6B DNA detection. Original intraspecific HHV6B classification based on detecting signature nucleotide substitutions in the sequence of the U90 gene fragment, was used for virus typing. A relationship between HHV6B genovariant and clinical/laboratory MLS characteristics was studied using multivariate statistical methods. Circulation of four HHV6B genovariants was shown among children in Nizhny Novgorod region: HHV6B/GV2e, HHV6B/GV2b (dominant genovariants), HHV6B/GV1a and HHV6B/GV2a (minor genovariants). Children with MLS infected with HHV6B/GV2e genovariant were more likely to have laboratory signs of cytolysis and increased peripheral blood lymphocyte count. Children with MLS infected with the HHV6B/GV2b genovariant were more likely to have higher mature neutrophil level. An increase of HHV6B viral load in leukocytes was accompanied by aggravated intoxication symptoms. Co-infection with the Epstein-Barr virus (EBV) led to more severe symptoms of acute tonsillitis, hepatosplenomegaly, and lymphadenopathy. Patients with EBV were more likely to have higher ALAT and lymphocyte level along with decreased mature neutrophil count. In children with MLS, EBV coinfection caused smaller HHV6B viral load in blood leukocytes (in children with HHV6B DNA 10 or more copies/105 cells) and lower HHV6B DNA detection frequency in saliva (in children with HHV6B DNA less than 10 copies/105 leukocytes). The results show the influence of genetic HHV6B polymorphism upon the clinical and laboratory features of MLS in children, and also demonstrate that HHV6B/GV2e and EBV cause similar disease symptoms.

Analysis of IL10 gene promoter haplotypes and changes in mRNA expression and soluble levels in patients with basal cell carcinoma.

Interleukin-10 (IL-10) is an immunomodulatory molecule that may play an immunosuppressive role in nonmelanoma skin cancer (NMSC), specifically basal cell carcinoma (BCC). We analyzed the role of IL10 promoter variants in genetic determinants of BCC susceptibility and their association with IL10 mRNA and IL-10 serum levels. Three promoter variants (-1082 A > G, -819T > C, and -592 A > C) were examined in 250 BCC patients and 250 reference group (RG) individuals. IL10 relative gene expression was evaluated in total leucocytes in peripheral blood, and IL-10 levels were quantified in serum. The allelic and genotypic frequencies did not show significant differences between the groups. However, haplotype analysis revealed that the ATA haplotype was associated with a decreased risk of BCC (OR: 0.73, 95% CI 0.56-0.95, p = 0.02), whereas the ATC and ACA haplotypes were associated with an increased risk for the neoplasia (OR: 4.15, 95% CI 1.56-11.04, p < 0.01, and OR: 3.51, 95% CI 1.33-9.29, p < 0.01, respectively). BCC patients showed a 0.09-fold reduction in IL10 mRNA expression compared to the RG. Significant differences were in IL-10 median serum levels between the RG and BCC patients (0.4 vs 0.64pg/mL, p = 0.02). Significant median serum differences were also observed between low- and high-grade histopathological BCC (0.34 vs 1.35pg/mL, p = 0.02). The ATA, ATC, and ACA haplotypes, and serum IL-10 levels, could be markers of susceptibility to BCC in Western Mexican individuals. BCC patients had higher serum IL-10 levels than the RG, with levels increasing with the severity of lesions, confirming the role of IL-10 in immunosuppression in neoplasia.

DYNAMICS OF ALTERATIONS IN WHITE BLOOD CELL AND SEPSIS-ASSOCIATED HUMORAL FACTORS DURING SULFUR MUSTARD INTOXICATION

Introduction. Sulfur mustard (SM) is an important chemical warfare agent. Lack of antidote and pathogenetic means in the arsenal of modern medicine for the treatment of SM poisoning requires further studies on the pathogenesis and development of protective medical devices and treatment schemes. The aim is to assess the features of immune response and intestinal bacterial translocation during SM intoxication. Materials and methods. The experiments were performed with Chinchilla male rabbits. SM was injected to laboratory animals intramuscularly at a dose of 63mg/kg, amounting to 1.0 LD84. The test materials presented by blood samples collected by cardiac puncture and from the lateral ear vein. Cardiac blood was used to produce serum and subsequently to determine the level of the sepsis-associated humoral factors (lipopolysaccharide, sCD14, procalcitonin, tumor necrosis factor a, interleukins 6 and 10) by using enzyme-linked immunosorbent assay. Results. In the peripheral blood of rabbits there were found changes characterized by decreased total leukocyte count, a development of lymphocytopenia and monocytopenia, as well as two-phase granulocyte count dynamics. The blood leukocyte formula showed the most significant changes related to three cell subsets: segmentonuclear neutrophils, lymphocytes and plasmocytes. The analysis of blood serum for assessing level of the sepsis-associated humoral factors showed that the earliest pathobiochemical change was related to higher sCD14. On day 2 post-exposure, a simultaneous increase in the levels of tumor necrosis factor a, interleukins 6 and 10 was reported. The cytokine level was decreasing to baseline range starting from day 3 onwards, whereas the clinical manifestation of SM intoxication was increasing and reaching the maximum magnitude. The analysis of blood serum for lipopolysaccharide level showed its increase on day 3 and 4 post-exposure. Conclusion. The study data in the context of the proposed interpretation, allow to highlight the following phasing of pathological changes during an extremely severe SM intoxication: day 1 - myeloid phagocytic system activation and hyperphagocytosis; day 2 - systemic inflammatory and compensatory anti-inflammatory response syndromes; days 3-4 - «immune paralysis», intensified bacterial translocation, depletion of the endotoxin-binding system and «endotoxin aggression».