Peripheral Blood Leukocyte Composition Research Articles

Blood integrin and cytokine producing T cells are associated with stage and genetic risk score in age-related macular degeneration.

Age-related macular degeneration (AMD) remains a leading cause of vision loss in the geriatric population. There are age-related changes in peripheral blood leukocyte composition, but their significance for AMD remains unclear. We aimed to determine changes in immune cell populations in blood of AMD patients. A standardized 31 parameter flow cytometry analysis was conducted on peripheral blood mononuclear cells from 59 patients with early and advanced AMD and 39 controls without AMD older than 65 years. Fundus photography and optical coherence tomography were used to classify disease stages and a custom genotype array was used to compute an AMD genetic risk score based on 52 AMD disease risk variants (GRS-52). A generalized linear regression model corrected for age, sex, and smoking status revealed that AMD patients showed decreased frequencies of CD4-positive T helper cell population expressing Integrin Alpha E (CD103) (Padj = 0.019). We further noted that early AMD was characterized by increased interleukin-4 (IL-4)-producing CD4+ T helper cells (Padj = 0.013; <0.001), as well as IL-4-producing cytotoxic CD8+ T cells (Padj = 0.016; <0.001). Reclassification of samples based on the GRS-52 revealed that IL-17-producing T cells decreased incrementally across GRS-52 categories. In AMD, alterations in peripheral blood leukocyte populations are associated with genetic risk score and disease stage and include specifically IL-4 and IL-17A cytokine-producing and CD103 integrin expressing T cell populations.

The study of the pharmacological activity of a cream with a white willow bark extract on the model of contact non-allergic dermatitis

Despite the achievements of medicine, dermatological skin diseases are an urgent problem today as the incidence rate increases annually. A promising direction of modern pharmacotherapy of dermatoses is the creation of innovative etiotropic topical agents on a plant basis. Aim. To study the pharmacological activity of a combined cream with a white willow (Salix alba L.) bark extract and zinc perithionate under the conditional name “Pyrisalix” against the background of the experimental non-allergic dermatitis induced by turpentine oil. Materials and methods. As the study object, a cream made with the active substances of a thick extract of white willow bark and zinc perithionate on a cream base was used; it corresponded to the pharmacotechnological indicators by colloidal, thermal stability and the description of oil/water (20 % of the oil phase). The model of alternative chronic inflammation, non-allergic contact dermatitis, was induced in white non-linear rats by daily application of turpentine oil to the depilated area. The effectiveness of the “Pyrisalix” cream was evaluated based on the total leukocyte count, the peripheral blood leukocyte composition, erythrocyte sedimentation rate (ESR), including the leukocyte-ESR ratio index (LI-ESR), the skinfold thickness, and the skin cover condition. In our experiment, the phagocytic activity of granulocytic neutrophils in a leukocyte suspension obtained from heparinized blood was determined to assess the activity of nonspecific immune factors. Results. The emulsion base of the cream has been determined, according to which the product developed has thermal and colloidal stability. In the course of studying the pharmacological activity of the “Pyrisalix” cream on themodel of non-allergic skin inflammation based on the dynamics of local manifestations of contact dermatitis, hematological, and immunological parameters, it has been found that the “Pyrisalix” cream has a pronounced preventive and therapeutic effect due to its anti-inflammatory and immunological properties. In the experimental conditions, the anti-alterative action of the cream on the emulsion oil/water base studied was equal to or even significantly exceeded the activity of the reference drugs – “Fladex” ointment and “Psorikap” cream by the intensity of inhibition of inflammatory processes in the skin, reduction in total leukocyte count, and normalization of the functional activity of granulocytic neutrophils. Conclusions. The combined cream “Pyrisalix” on the emulsion oil/water base demonstrates a significant antiinflammatory activity on the model of non-allergic contact dermatitis. The combined cream “Pyrisalix” is a promising agent for further study in the treatment of dermatoses.

SEXUAL-DIMORPHISM IN HUMAN IMMUNE SYSTEM AGING

Abstract Differences in immune function and responses contribute to health- and life-span disparities between sexes. However, the role of sex in immune system aging is not well understood. Here, we characterize peripheral blood mononuclear cells from 172 healthy adults 22–93 years of age using ATAC-seq, RNA-seq and flow cytometry. These data reveal a shared epigenomic signature of aging including declining naïve T cells and increasing monocyte and cytotoxic cell functions. These changes are greater in magnitude in men and accompanied by a male-specific decline in B-cell-specific loci. Interestingly, genomic differences between sexes increase after age 65, with men having higher innate and pro-inflammatory activity and lower adaptive activity. In a separate cohort (n=39) we studied how older adults respond to two available pneumococcal vaccines: T-dependent Prevnar and T-independent Pneumovax. How older adults respond to these vaccines at the molecular and cellular level, and whether there are pre-vaccination predictors for vaccine responsiveness, is poorly understood. To address this, we characterized serum antibody responses and peripheral blood leukocyte composition using flow cytometry and transcriptional profiles using RNA-seq, in healthy older adults before and after vaccination. The vaccines induced comparable serological responses and increases in the number of ICOS+ T follicular helper cells. A shared plasmablast transcriptional signature (IGHG2, IGHA1, IGHA2) was induced by both vaccines ten days after vaccination, which is distinct from influenza vaccine responses. Importantly, the pre-vaccination (baseline) ratio of Th1/Th17 cells predicted responsiveness to Prevnar but not to Pneumovax. Interestingly, women had higher levels of Th1/Th17; and responded stronger to Prevnar compared to men. This study uncovered how older adults respond to different pneumococcal vaccines and demonstrated the significance of considering biological sex and the immune cell composition for precision vaccinology.

Abstract 27: Early dynamics in peripheral blood immune cell subsets and ctDNA are predictive of outcome to immunotherapy

Abstract Background: Treatment-induced anti-tumor immune response may be reflected in peripheral blood leukocyte composition and shifts. The relative abundance of immune cell subsets captured by neutrophil-to-lymphocyte ratio (NLR) is a promising biomarker of response to immune checkpoint inhibition (ICI). However early dynamics and integration with circulating tumor DNA (ctDNA) kinetics during ICI have not been comprehensively studied. Methods: We evaluated the predictive role of early dynamic changes of peripheral immune cell subsets in 239 ICI treated metastatic non-small cell lung cancers (mNSCLC) and combined those with ctDNA dynamics in 18 early stage NSCLCs (esNSCLC) treated with neoadjuvant ICI. Blood cell counts were analyzed at baseline, 4 weeks on treatment, and at first radiographic follow up (mNSCLC) or preoperatively (esNSCLC). mNSCLC patients without progression at 6 months were considered to have durable clinical benefit (DCB). We employed eXtreme Gradient Boosting, a decision-tree based machine learning algorithm, to integrate baseline values and early changes in immune cell subsets. To reduce modeling overfitting, we trained an ensemble of models, incorporating 10-fold cross-validation that included feature selection (training n=171) and tested the model in an unseen independent set (n=68). For esNSCLCs, major pathologic response (MPR; ≤10% residual tumor; RT) was determined. We performed Targeted Error Correction next generation sequencing on 60 serial plasma and matched leukocyte DNA samples to assess ctDNA clonal dynamics. Changes in immune cell subsets were correlated with RT, MPR, progression-free (PFS) and overall survival (OS). Results: Our machine learning integrative model predicted DCB with an area under the ROC curve (AUC) of 0.96 for training, 0.72 for cross-validation testing and 0.74 for unseen testing datasets. Feature importance analysis revealed that NLR at 4 weeks, first radiographic follow up, and relative change in NLR from baseline were the strongest predictors of outcome together with relative eosinophil and lymphocyte count. Our model's performance was superior to TMB (AUC=0.52) or PD-L1 (AUC=0.54). For esNSCLCs, change in NLR at 4 weeks after ICI initiation was predictive of tumor regression (p=0.02), such that those with MPR showed significant decrease in NLR. This was also predictive of PFS (log rank p=0.004) and OS (log rank p=0.02). Furthermore, higher eosinophil count at 4 weeks was correlated with MPR (p=0.006) and decreased RT (p=0.006). Immune cell subset kinetics were concordant with ctDNA clearance in all but two patients and importantly NLR dynamics at 4 weeks were predictive of MPR even when ctDNA was undetectable. Conclusions: Early changes in peripheral immune cell subsets together with ctDNA are reflective of anti-tumor immune response during ICI and may more accurately predict ICI response than currently used biomarkers. Citation Format: Michael Hwang, Jenna Canzoniero, Samuel Rosner, Guangfan Zhang, Mara Lanis, Lamia Rhymee, Alexandria Curry, Gavin Pereira, Kristen Marrone, Joshua Reuss, Jarushka Naidoo, Christine Hann, Vincent Lam, Benjamin Levy, David Ettinger, Patrick Forde, Julie Brahmer, Victor Velculescu, Tanguy Seiwert, Valsamo Anagnostou. Early dynamics in peripheral blood immune cell subsets and ctDNA are predictive of outcome to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 27.

Composition of peripheral blood leukocytes and immuno-competent bodies of the cross carp “Petrovsky” in different periods of the fishing season

Crossing is an effective method of increasing agricultural production. In addition to the productive qualities of the resulting crosses, it is important to maintain and enhance their immune resistance. Breeding for immunity to pathogens of various diseases provides a decrease in economic losses and an increase in breeding efficiency. Cross “Petrovsky” (breeding achievement No. 9153820) is a reciprocal hybrid of Anish Mirror and Chuvash scaled productive carp species. It has been established that this cross is superior to the parental forms in terms of growth intensity and survival of juveniles. The work is devoted to a comparative analysis of the leukocyte composition of peripheral blood and immunocompetent organs of the carp cross “Petrovsky” in different periods of the fish-breeding season. The studies were carried out in late May and early October 2019 in the Kirya breeding farm, Chuvashia (2nd fish breeding zone). In fish, peripheral blood was sampled from the tail vein and tissues of the spleen and head kidney. Blood smears and organ-imprinted smears were taken for the study. The preparations were examined under a light microscope and the leukocyte formula was determined. The study of leukograms of immunocompetent organs and tissues of fish helps to judge the functional state and immune resistance of fish. To determine the index of the abundance of leukocytes in a peripheral blood smear, 100 visual fields were examined in different areas. This indicator allows you to record the intensity of leukopoiesis and indirectly assess the level of leukocytes in a unit of blood volume. Studies have established differences in the proportion of certain types of leukocytes and the intensity of leukopoiesis in different periods of the fish-breeding season. Fish tissues and organs differed in the proportion of lymphocytes, monocytes, and neutrophils. The obtained data can be used in carrying out selection and breeding work aimed at increasing productivity and enhancing the resistance of fish to infectious diseases.

Влияние инсульт-ассоциированной пневмонии на исход острого каротидного ишемического инсульта

Aim: to study the stroke-associated pneumonia (SAP) effect on the outcome of ischemic stroke (IS) in the internal carotid artery system. Patients and Methods: 87 patients with IS underwent the follow-up study, of which 75 had no inflammatory bronchopulmonary complications (group 1), and 12 had pneumonia manifestation (group 2). The study was performed on days 1, 5, and 9 after IS, and 6 months and 12 months after discharge from the hospital. Neurological deficit severity (NIHSS, Barthel index) and inflammatory markers (peripheral blood leukocyte composition, C-reactive protein (CRP), ESR) were studied. Results: it was found that the presence of SAP was associated with increased mortality during the acute IS period (4 (33.1%) patients died in group 1 and 10 (13.3%) — in group 2, p&lt;0.05), greater severity of neurological deficits (63.3±5.3 and 71.5±4.0 points on the NIHSS scale, respectively, p&lt;0.05) and incapacitation (Barthel index — 63.3±5.3 and 71.5±4.0 points, respectively, p&lt;0.05) at the end of the inpatient treatment period. In group 2, signs of an inflammatory response were detected on day 5, and the values of the white blood cell shift index, ESR and CRP significantly (p&lt;0.05) differed from the initial values. During examination at 6 months and 12 months, there were no significant differences in these indicators between the groups. An association was established between the probability of SAP occurrence and the presence of swallowing disorders (r=0.672; p&lt;0.05), the age of patients (r=0.572; p&lt;0.05) and the presence of diabetes mellitus (r=0.522; p&lt;0.05). The studied laboratory inflammatory markers allow us to timely assume the occurrence of pulmonary pathology. Conclusion: timely diagnosis and prevention of SAP can reduce the risk of fatal outcome, facilitate rehabilitation measures, and improve early IS outcomes. KEYWORDS: ischemic stroke, cardioembolic stroke, atherothrombotic stroke, complications, acute period, inflammatory markers, strokeassociated pneumonia, long-term outcomes. FOR CITATION: Chipova D.T., Santikova L.V., Zhemukhov A.Ch. Impact of stroke-associated pneumonia on the outcome of acute ischemic stroke in internal carotid artery system. Russian Medical Inquiry. 2020;4(9):539–543. DOI: 10.32364/2587-6821-2020-4-9-539-543.

Methylomic profiles reveal sex-specific differences in leukocyte composition associated with post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is a debilitating mental disorder precipitated by trauma exposure. However, only some persons exposed to trauma develop PTSD. There are sex differences in risk; twice as many women as men develop a lifetime diagnosis of PTSD. Methylomic profiles derived from peripheral blood are well-suited for investigating PTSD because DNA methylation (DNAm) encodes individual response to trauma and may play a key role in the immune dysregulation characteristic of PTSD pathophysiology. In the current study, we leveraged recent methodological advances to investigate sex-specific differences in DNAm-based leukocyte composition that are associated with lifetime PTSD. We estimated leukocyte composition on a combined methylation array dataset (483 participants, ∼450 k CpG sites) consisting of two civilian cohorts, the Detroit Neighborhood Health Study and Grady Trauma Project. Sex-stratified Mann-Whitney U test and two-way ANCOVA revealed that lifetime PTSD was associated with significantly higher monocyte proportions in males, but not in females (Holm-adjusted p-val < 0.05). No difference in monocyte proportions was observed between current and remitted PTSD cases in males, suggesting that this sex-specific difference may reflect a long-standing trait of lifetime history of PTSD, rather than current state of PTSD. Associations with lifetime PTSD or PTSD status were not observed in any other leukocyte subtype and our finding in monocytes was confirmed using cell estimates based on a different deconvolution algorithm, suggesting that our sex-specific findings are robust across cell estimation approaches. Overall, our main finding of elevated monocyte proportions in males, but not in females with lifetime history of PTSD provides evidence for a sex-specific difference in peripheral blood leukocyte composition that is detectable in methylomic profiles and that may reflect long-standing changes associated with PTSD diagnosis.

Ascites bacterial burden and immune cell profile are associated with poor clinical outcomes in the absence of overt infection.

Bacterial infections, most commonly spontaneous bacterial peritonitis in patients with ascites, occur in one third of admitted patients with cirrhosis, and account for a 4-fold increase in mortality. Bacteria are isolated from less than 40% of ascites infections by culture, necessitating empirical antibiotic treatment, but culture-independent studies suggest bacteria are commonly present, even in the absence of overt infection. Widespread detection of low levels of bacteria in ascites, in the absence of peritonitis, suggests immune impairment may contribute to higher susceptibility to infection in cirrhotic patients. However, little is known about the role of ascites leukocyte composition and function in this context. We determined ascites bacterial composition by quantitative PCR and 16S rRNA gene sequencing in 25 patients with culture-negative, non-neutrocytic ascites, and compared microbiological data with ascites and peripheral blood leukocyte composition and phenotype. Bacterial DNA was detected in ascitic fluid from 23 of 25 patients, with significant positive correlations between bacterial DNA levels and poor 6-month clinical outcomes (death, readmission). Ascites leukocyte composition was variable, but dominated by macrophages or T lymphocytes, with lower numbers of B lymphocytes and natural killer cells. Consistent with the hypothesis that impaired innate immunity contributes to susceptibility to infection, high bacterial DNA burden was associated with reduced major histocompatibility complex class II expression on ascites (but not peripheral blood) monocytes/macrophages. These data indicate an association between the presence of ascites bacterial DNA and early death and readmission in patients with decompensated cirrhosis. They further suggest that impairment of innate immunity contributes to increased bacterial translocation, risk of peritonitis, or both.

Influences of age and sex on leukocytes of healthy horses and their ex vivo cytokine release

Leukocytes and their functional capacities are used extensively as biomarkers in immunological research. Commonly employed indicators concerning leukocytes are as follows: number, composition in blood, response to discrete stimuli, cytokine release, and morphometric characteristics. In order to employ leukocytes as biomarkers for disease and therapeutic monitoring, physiological variations and influencing factors on the parameters measured have to be considered. The aim of this report was to describe the ranges of selected leukocyte parameters in a sample of healthy horses and to analyse whether age, sex, breed, and sampling time point (time of day) influence peripheral blood leukocyte composition, cell morphology and release of cytokines ex vivo. Flow cytometric comparative characterisation of cell size and complexity in 24 healthy horses revealed significant variance. Similarly, basal release of selected cytokines by blood mononuclear cells also showed high variability [TNFα (65–16,624pg/ml), IFNγ (4–80U/ml), IL-4 (0–5069pg/ml), IL-10 (49–1862pg/ml), and IL-17 (4–1244U/ml)]. Each animal's age influenced leukocyte composition, cell morphology and cytokine release (TNFα, IL-4, IL-10) ex vivo. Geldings showed smaller monocytes and higher spontaneous production of IL-10 when compared to the mares included. The stimulation to spontaneous release ratios of TNFα, IL-4 and IL-17 differed in Warmblood and Thoroughbred types. Sampling time influenced leukocyte composition and cell morphology. In summary, many animal factors – age being the dominant one – should be considered for studies involving the analysis of equine leukocytes. In addition, high inter-individual variances argue for individual baseline measurements.

Combined inflammatory and metabolic defects reflected by reduced serum protein levels in patients with Buruli ulcer disease.

Buruli ulcer is a skin disease caused by Mycobacterium ulcerans that is spreading in tropical countries, with major public health and economic implications in West Africa. Multi-analyte profiling of serum proteins in patients and endemic controls revealed that Buruli ulcer disease down-regulates the circulating levels of a large array of inflammatory mediators, without impacting on the leukocyte composition of peripheral blood. Notably, several proteins contributing to acute phase reaction, lipid metabolism, coagulation and tissue remodelling were also impacted. Their down-regulation was selective and persisted after the elimination of bacteria with antibiotic therapy. It involved proteins with various functions and origins, suggesting that M. ulcerans infection causes global and chronic defects in the host's protein metabolism. Accordingly, patients had reduced levels of total serum proteins and blood urea, in the absence of signs of malnutrition, or functional failure of liver or kidney. Interestingly, slow healers had deeper metabolic and coagulation defects at the start of antibiotic therapy. In addition to providing novel insight into Buruli ulcer pathogenesis, our study therefore identifies a unique proteomic signature for this disease.

Factors underlying variable DNA methylation in a human community cohort

Epigenetics is emerging as an attractive mechanism to explain the persistent genomic embedding of early-life experiences. Tightly linked to chromatin, which packages DNA into chromosomes, epigenetic marks primarily serve to regulate the activity of genes. DNA methylation is the most accessible and characterized component of the many chromatin marks that constitute the epigenome, making it an ideal target for epigenetic studies in human populations. Here, using peripheral blood mononuclear cells collected from a community-based cohort stratified for early-life socioeconomic status, we measured DNA methylation in the promoter regions of more than 14,000 human genes. Using this approach, we broadly assessed and characterized epigenetic variation, identified some of the factors that sculpt the epigenome, and determined its functional relation to gene expression. We found that the leukocyte composition of peripheral blood covaried with patterns of DNA methylation at many sites, as did demographic factors, such as sex, age, and ethnicity. Furthermore, psychosocial factors, such as perceived stress, and cortisol output were associated with DNA methylation, as was early-life socioeconomic status. Interestingly, we determined that DNA methylation was strongly correlated to the ex vivo inflammatory response of peripheral blood mononuclear cells to stimulation with microbial products that engage Toll-like receptors. In contrast, our work found limited effects of DNA methylation marks on the expression of associated genes across individuals, suggesting a more complex relationship than anticipated.

BCR-ABL1 transcript levels increase in peripheral blood but not in granulocytes after physical exercise in patients with chronic myeloid leukemia

In chronic myeloid leukemia (CML) treatment response is determined by measurements of BCR-AB1L transcripts in peripheral blood by quantitative real-time PCR (qRT-PCR) and a 2–5 fold increase is considered a warning sign. The BCR-ABL1 gene is mainly expressed in myeloid cells whereas quantification of BCR-ABL1 is performed on the nucleated cell fraction of peripheral blood. Hence, leukocyte composition of the nucleated cell fraction may affect the result of BCR-ABL1 quantification. The aim of this study was to investigate if changes in leukocyte composition of peripheral blood had any effect on BCR-ABL1 transcript levels in CML patients. Six CML patients in complete cytogenetic remission (CCgR) performed a maximal physical exercise test. Blood samples were collected before exercise, at maximal exhaustion and after exercise. A biphasic increase in leukocyte count was observed and the relative proportion of granulocytes in peripheral blood changed significantly after exercise compared with baseline (p < 0.001). The BCR-ABL1 transcript level increased significantly following exercise, in nucleated cell fraction of peripheral blood (p < 0.05) but not in isolated granulocytes. In the nucleated cell fraction, the mean BCR-ABL1 transcript level was 3.3-fold (range 0.7–6.8) higher 180 min after exercise compared with baseline (p < 0.01). In conclusion, physical exercise induced significant increases in BCR-ABL1 transcript levels concomitant with changes in leukocyte content of peripheral blood. We therefore suggest that variations in leukocyte composition of peripheral blood, causing pre-analytic variations that affect BCR-ABL1 quantification, have to be accounted for. Consequently, small variations in BCR-ABL1 transcript levels should be interpreted cautiously in CML patients in CCgR.

Genetic Line Effect on Peripheral Blood Leukocyte Cell Surface Marker Expression in Chickens

To determine the role of genetics in baseline lymphocyte parameters, several distinct lines of chickens were examined for differences in peripheral blood leukocyte (PBL) populations. Four highly inbred chicken lines (MHC congenic Fayoumi lines M15.2 and M5.1, and MHC congenic Leghorn lines G-B1 and G-B2), two advanced intercrosses [F5 (Broiler × G-B2) and F5 (Broiler × M15.2)], and an outbred population of broilers were used. Leukocytes isolated from healthy adult birds were labeled with monoclonal antibodies: chCD3, chCD4, chCD8, chBu-1, and hCD14. Flow cytometry was used to determine the total percentage of positively labeled cells for each surface marker in a sample, as well as the mean fluorescent intensity, or surface marker density, of a labeled subset. Significant line differences for percentage positive CD3 T cells and the ratio of B cells:T cells (represented by the Bu-1:CD3 ratio) were found. The effect of line was also significant for CD3 and CD8 T cell receptor density. Effects of sex and MHC on PBL cell surface marker expression were not significant in the lines examined. This study demonstrates the effect of genetic line on resting leukocyte composition of peripheral blood in the chicken lines examined. Observed PBL differences add to our growing knowledge of the varied roles that immune system status (defined by specific cell populations) and genetic background have in determining susceptibility and disease progression in chickens.

A reduced peripheral blood CD4 + lymphocyte proportion is a consistent ageing phenotype

Peripheral blood leukocyte composition was measured in young, middle-aged and old C57BL/6J (B6) and BALB/cByJ (BALB) mice by flow cytometry to test the hypothesis that ageing is associated with declines in the proportions of peripheral blood T lymphocytes. In both B6 and BALB mice, increasing age is associated with a significant and continuous decline in the proportions of CD4 + lymphocytes, a moderate decline in the proportion of CD8 + lymphocytes, a significant increase in the proportion of Gr1 + granulocytes and an almost unchanged proportion of B lymphocytes. As expected, the proportion of CD44 low naive T lymphocytes decreased with age. Expression of Fas (CD95 +) on CD4 + and CD8 + lymphocytes showed no consistent change with age. We also measured peripheral blood CD4 + and CD8 + lymphocyte proportions in young and old A/J, CBA/CaJ, DBA/2J, DW/J and (DW×C3H) F1 mice. The CD4 + lymphocyte proportion decreased from young to old age in these strains by 56, 65, 72, 78 and 68%, respectively. The CD8 + lymphocyte proportion decreased moderately with age in all the inbred strains tested but not in the (DW×C3H) F1 hybrid. Thus, a reduced proportion of peripheral blood CD4 + lymphocytes is a consistent ageing phenotype in a wide range of Mus musculus strains.