Major Lymphocyte Subpopulations Research Articles

Understanding the relevance of immunological markers in severe multisystem inflammatory syndrome in children through machine learning

Objectives: Multisystem inflammatory syndrome in children (MIS-C) is a late manifestation of SARS-CoV-2 infection, which presents with symptoms ranging from milder mucocutaneous and gastrointestinal symptoms to severe cardiovascular and neurological manifestations. We studied the clinical, biochemical, hematological, and immunological characteristics of MIS-C patients to understand this disease entity and to identify markers of severe disease. Materials and Methods: Twenty-four MIS-C patients, four acute COVID-19 infections, and ten healthy controls (HC) from a tertiary care pediatric hospital in Mumbai were enrolled in the study. Clinical, biochemical, hematological, and immunological parameters comprising major lymphocyte, neutrophil, and monocyte subpopulations and key activator and inhibitory markers were studied. Statistical Analysis: All parameters were compared between the healthy, COVID and MIS-C groups at Day 0, 7 and 14 using non-parametric statistical tests. Machine learning tools were used for multivariate data analysis to identify the immunological parameters that could help predict severe disease. Results: NKp46pos NK cell (%), CD11 positive eosinophil (%), D-dimer, and Tim3pos Tc (%) were identified as the most important markers that could help predict severe disease, with NKp46pos NK cells as the top contributor. A disease severity metric utilizing these markers can be used to identify patients who are likely to have a severe course of disease. Conclusions: NK cells directly contribute to disease severity in MIS-C. As the JAK-STAT pathway is known to be important for NK cell development, maturation, and function, ruxolitinib, which is a JAK1/JAK2 inhibitor, might be beneficial in the management of this condition.

Preservation of functionality, immunophenotype, and recovery of HIV RNA from PBMCs cryopreserved for more than 20 years.

Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and in-vitro functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation. PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced. All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence. This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.

Features of the immune status and comorbidities in children with alopecia areata

Increased incidence of alopecia has been noted in children at the present time. Participation of autoimmune (immunopathological) mechanisms in pathogenesis of this disease necessitates additional study of immune status and characteristics of comorbid pathologies. The aim of our study was to specify the features of immune status and comorbidities in children with alopecia areata. The observation group consisted of children with various types of alopecia areata (n = 57), a comparison group included children without clinical manifestations of alopecia or a history of alopecia (n = 157). We performed a comparative evaluation of major lymphocyte subpopulations (CD3+, CD3+CD4+, CD3+CD8+, CD3+CD19+), interleukins (IL-4, IL-6), immunoglobulins (IgA, IgM, IgG), parameters of phagocytic activity (absolute phagocytosis, percentage of phagocytosis, phagocytic number and phagocytic index) and IgE to house dust and cat hair. Analysis of comorbidities was also performed. Statistical processing was carried out with Jamovi software. We have found that the focal clinical form of alopecia prevailed over the subtotal and total forms by 1.8 times (p = 0.033) in the observation group rather than in comparison group, with common variable immunodeficiency being more often (1.4-fold), chronic tonsillitis (3.9-fold), allergic rhinitis (3.9-fold) and autoimmune thyroiditis, which was absent in the comparison group. Among the children with alopecia, disturbances of the T-cell link were revealed, i.e., a higher median levels of the relative and absolute numbers of CD3+CD4+ cells (p = 0.001 to 0.003), larger proportions of elevated values for IgA (4.1-fold), IgM (7.3-fold), IgG (13.2-fold) with p-levels of 0.0001 to 0.0008; increased IL-4 (8.1-fold) and IL-6 (4.6-fold), with p = 0.002-0.004, along with medium and relatively strong correlations with alopecia. In children with alopecia, we have determined a 3.3-fold proportion of reduced values of absolute phagocytosis and 3.7-fold reduced percentage of phagocytosis (p = 0.0012 to 0.028), with an sufficient correlation (weak to moderate strength) with alopecia, as well as lower values of the median phagocytic index and phagocytic number in the observation group (p < 0.001) associated with a average-strength correlation. Hence, an imbalance of the immune system components was revealed in children with alopecia which manifested with signs of immune hyperfunction, characteristic, e.g., of autoimmune and allergic processes, accompanied by more frequent registration of autoimmune thyroiditis and allergic rhinitis. Moreover, distinct signs of immune deficiency, are found, characterized by a decrease in phagocytic activity and higher incidence of common variable immunodeficiency and chronic tonsillitis.

CD4-1 and CD4-2 single positive are two major CD4 lymphocyte subpopulations in ginbuna crucian carp Carassius auratus langsdorfii

CD4-1 and CD4-2 single positive are two major CD4 lymphocyte subpopulations in ginbuna crucian carp Carassius auratus langsdorfii

CD4-1 and CD4-2 single positive are two major CD4 lymphocyte subpopulations in ginbuna crucian carp Carassius auratus langsdorfii.

In this study, we established a murine cell line that expresses ginbuna crucian carp (ginbuna) CD4-2 and used it to develop an anti-CD4-2 monoclonal antibody (mAb). An established mAb, named D5, showed good reactivities to BALB/3T3 cells expressing CD4-2 and a lymphocyte population in the ginbuna leukocytes. Gene expression analysis showed that D5+ cells express CD4-2 and TCRβ genes but not CD4-1 and IgM genes, meanwhile May Grunwald-Giemsa staining of sorted D5+ cells had the typical morphology of lymphocytes. Two-color immunofluorescence analysis with anti-CD4-1 mAb (6D1) and anti-CD4-2 mAb (D5) by flow cytometry revealed that the percentages of CD4-1 single positive (SP) and CD4-2 SP lymphocytes were comparatively higher than CD4-1/CD4-2 double positive (CD4 DP) lymphocytes in all tissues examined in ginbuna. The highest percentages of CD4-2 SP cells (∼40%) were found in the thymus, while the head-kidney exhibited the highest percentages of CD4-1 SP (∼30%) and CD4 DP (∼5%) cells. These findings indicated that ginbuna CD4+ lymphocyte population consists of two major subpopulations (CD4-1 SP and CD4-2 SP) and a minor subset (CD4 DP).

Immunomodulatory Aspects of Therapeutic Plasma Exchange in Neurological Disorders—A Pilot Study

Therapeutic plasma exchange (TPE) is used for drug-resistant neuroimmunological disorders, but its mechanism of action remains poorly understood. We therefore prospectively explored changes in soluble, humoral, and cellular immune components associated with TPE. We included ten patients with neurological autoimmune disorders that underwent TPE and assessed a panel of clinically relevant pathogen-specific antibodies, total serum immunoglobulin (Ig) levels, interleukin-6 (IL-6, pg/mL), C-reactive protein (CRP, mg/dL), procalcitonin (PCT, µg/L) and major lymphocyte subpopulations (cells/µL). Blood was collected prior to TPE (pre-TPE, baseline), immediately after TPE (post-TPE), as well as five weeks (follow-up1) and 130 days (follow-up2) following TPE. Pathogen-specific antibody levels were reduced by -86% (p < 0.05) post-TPE and recovered to 55% (follow-up1) and 101% (follow-up2). Ig subclasses were reduced by -70-89% (p < 0.0001) post-TPE with subsequent complete (IgM/IgA) and incomplete (IgG) recovery throughout the follow-ups. Mean IL-6 and CRP concentrations increased by a factor of 3-4 at post-TPE (p > 0.05) while PCT remained unaffected. We found no alterations in B- and T-cell populations. No adverse events related to TPE occurred. TPE induced a profound but transient reduction in circulating antibodies, while the investigated soluble immune components were not washed out. Future studies should explore the effects of TPE on particular cytokines and assess inflammatory lymphocyte lineages to illuminate the mode of action of TPE beyond autoantibody removal.

Local cellular immunity in colorectal cancer depending on tumor site.

e15613 Background: Colorectal cancer (CRC) is the third most commonly diagnosed cancer. More than 60% of all cases of CRC are colon cancer (CC). The role of individual units of the immune system in the development of this tumor is ambiguous. The purpose of this study was to characterize local populations and subpopulations of immunocompetent cells in colorectal cancer with different tumor locations. Methods: The study included 50 CC (adenocarcinoma) patients aged 35-86 years, women n = 26 (52%). Stage I tumors were registered in 4 patients (8%), stage II 25 (50%), stage III 21 (42%). 20 patients (40%) had right-sided CC (group 1), 9 (18%) left-sided CC (group 2), and 21 (42%) sigmoid CC (group 3). All patients received surgical treatment. Cell suspensions were obtained from tumor (TT) and peritumoral tissues (PT) (1-3 cm from the tumor), and then processed with an antibody panel in accordance with the manufacturer instructions (Becton Dickinson, USA) to identify the main populations and subpopulations of leukocytes and lymphocytes. The relative number of major populations and subpopulations of lymphocytes was determined on the BD FACSCanto flow cytometer. Results: A decrease in lymphocytic infiltration was noted left-sided tumors and sigmoid tumors compared to right-sided CC, by 46% and 51%, respectively. The percentage of CD3+ cells was almost the same regardless of the colon tumor location, and the number of main populations of T lymphocytes differed: in group 3, the content of CD4+ cells was 21% higher, and CD8+ cells were 22% lower compared with group 1, while group 2 had no differences. Group 2 differed in the tumor infiltration with both NK and NKT lymphocytes, which were higher by 25% and 22%, respectively, than in group 1. An increase in NK cells was noted in the sigmoid colon (group 3), and the relative number of NKT lymphocytes decreased. A common feature of TT in groups 2 and 3 was an increase in the content of B lymphocytes by 98% and 133%, respectively. PT of group 2 showed a decrease by 72%, 33%, 66%, and 46% in the relative number of lymphocytes, CD4+, NKT and B lymphocytes compared with group 1, together with an increased content of NK and CD8+ lymphocytes. PT in patients of group 3 had a decrease in the number of total and NK lymphocytes by 46% and 26%, respectively, and a significant increase by 85% in the content of CD8+ cells, with no changes in CD3+, CD4+ cells, B and NKT lymphocytes. Conclusions: The local immune status of CRC patients demonstrated a number of differences: right-sided tumors were characterized by a higher T-lymphocytic infiltration with the same tendency in the perifocal tissues, while left-sided and sigmoid tumors showed higher B-lymphocytic infiltration, which can help in the disease prognosis and choice of treatment strategy.

Immune response in new coronavirus infection COVID-19 in children and adults

Introduction. It is known that COVID-19 occurs more often in adult patients, especially if they have concomitant somatic diseases, children are at less risk of developing it. The aim of this work is to evaluate and compare immune response parameters in new coronavirus infection COVID-19 in children and adults. Materials and methods. The results of the examination of 56 adults 19-55 years old and 57 children 14-18 years old were included in the work. The examination results of COVID-19 patients were compared with similar data in practically healthy individuals of the corresponding age groups. All patients underwent laboratory tests to determine the following parameters: presence of SARS-CoV-2 RNA, general blood test parameters, major lymphocyte subpopulations, level of total immunoglobulins (IgM, IgG, IgA), number of CIC, absorbance and bactericidal activity of leukocytes.Results. The proportion of positive findings for SARS-CoV-2 RNA in different age groups ranged from 13.6% to 25.8%. General and specific patterns of immune response in patients of different age groups were established. Common features were an increase in the number of circulating immune complexes and activated T-lymphocytes. Differences were noted in the level of serum immunoglobulins IgM and IgG, neutrophil uptake activity, the number of monocytes, as well as in the level of different subpopulations of lymphocytes. Discussion. In adults, changes in the adaptive immune response, including the cellular level, are predominantly expressed. In children, there are signs of inefficiency of innate mechanisms of immune responses. Conclusion. The dynamics of the number of examined and positive findings correlate with similar figures in Russia and have a two-wave pattern. Increased number of circulating immune complexes and activated T-lymphocytes is typical for all patients with new coronavirus infection COVID-19 regardless of age, which is a sign of acute infection-inflammatory process development and insufficiently effective elimination of antigen (pathogen).

Comparison of the composition of lymphocyte subpopulations in non-relapse and relapse patients with squamous cell carcinoma of the head and neck before, during radiochemotherapy and in the follow-up period: a multicenter prospective study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG)

BackgroundRadiochemotherapy (RCT) has been shown to induce changes in immune cell homeostasis which might affect antitumor immune responses. In the present study, we aimed to compare the composition and kinetics of major lymphocyte subsets in the periphery of patients with non-locoregional recurrent (n = 23) and locoregional recurrent (n = 9) squamous cell carcinoma of the head and neck (SCCHN) upon primary RCT.MethodsEDTA-blood of non-locoregional recurrent SCCHN patients was collected before (t0), after application of 20–30 Gy (t1), in the follow-up period 3 (t2) and 6 months (t3) after RCT. In patients with locoregional recurrence blood samples were taken at t0, t1, t2 and at the time of recurrence (t5). EDTA-blood of age-related, healthy volunteers (n = 22) served as a control (Ctrl). Major lymphocyte subpopulations were phenotyped by multiparameter flow cytometry.ResultsPatients with non-recurrent SCCHN had significantly lower proportions of CD19+ B cells compared to healthy individuals before start of any therapy (t0) that dropped further until 3 months after RCT (t2), but reached initial levels 6 months after RCT (t3). The proportion of CD3+ T and CD3+/CD4+ T helper cells continuously decreased between t0 and t3, whereas that of CD8+ cytotoxic T cells and CD3+/CD56+ NK-like T cells (NKT) gradually increased in the same period of time in non-recurrent patients. The percentage of CD4+/CD25+/FoxP3+ regulatory T cells (Tregs) decreased directly after RCT, but increased above initial levels in the follow-up period 3 (t2) and 6 (t3) months after RCT. Patients with locoregional recurrence showed similar trends with respect to B, T cells and Tregs between t0 and t5. CD4+ T helper cells remained stably low between t0 and t5 in patients with locoregional recurrence compared to Ctrl. NKT/NK cell subsets (CD56+/CD69+, CD3−/CD56+, CD3−/CD94+, CD3−/NKG2D+, CD3−/NKp30+, CD3−/NKp46+) increased continuously up to 6 months after RCT (t0-t3) in patients without locoregional recurrence, whereas in patients with locoregional recurrence, these subsets remained stably low until time of recurrence (t5).ConclusionMonitoring the kinetics of lymphocyte subpopulations especially activatory NK cells before and after RCT might provide a clue with respect to the development of an early locoregional recurrence in patients with SCCHN. However, studies with larger patient cohorts are needed.Trial registrationObservational Study on Biomarkers in Head and Neck Cancer (HNprädBio), NCT02059668. Registered on 11 February 2014, https://clinicaltrials.gov/ct2/show/NCT02059668.

Phenotypic profile of peripheral blood lymphocytes in patients with inflammatory bowel diseases

Mechanisms of recognition and effector responses of immune system upon initiation and maintenance of immune-mediated inflammation and tissue damage in inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis (UC), have been actively studied over recent time. Existing evidence suggests these diseases to be caused by abnormal immune response against intestinal flora microorganisms in genetically susceptible individuals. Despite available data on the features of immune disorders in ulcerative colitis and Crohn’s disease, there are still many questions about the involved minor lymphocyte subpopulations and contribution of various functionally active molecules, which play a key role in recognition and initiation of the immune response and can be considered biomarkers of a pathological process in inflammatory bowel diseases. The populations of T lymphocytes with γδT cell receptor, B1 cells and NK T lymphocytes are of greatest interest, as well as functionally active TLRs (Toll-like receptors), CD89, CD314, etc. Due to substantial progress in studying the nature of recognition and activation of the immune cells, the paper presents phenotypic and functional characteristics of major and minor subpopulations of peripheral blood lymphocytes observed in 25 patients treated at the Surgery Department of the State Institution “Minsk Regional Clinical Hospital” (Republic of Belarus) from 2018 to 2020. The detected changes in peripheral blood lymphocyte phenotype of inflammatory bowel diseases patients suggest distinct immunological profiles prevailing in the damage mechanisms in Crohn’s disease and ulcerative colitis. I.e., in Crohn’s disease patients, B1 lymphocytes with CD19+CD5+ and NK cells in combination with increased CD56bright population, as well as NK T cells with anti-inflammatory and regulatory activity are involved into genesis of the disease. In ulcerative colitis, T, B, NK lymphocytes with pro-inflammatory phenotype and T lymphocytes with γδ T cell receptor may play a pathogenetic role in maintenance of chronic inflammation. With respect to functional significance of activating receptors, the number of TLR4- и CD89-positive cells may be used for developing immunological criteria/ biomarkers of therapeutic efficacy of new drugs. Studying interactions between innate and adaptive immunity will open new perspectives in understanding immunological disorders associated with chronic gastrointestinal inflammation.

Features of immune status in patients with metastatic and glial brain tumors at the preparatory stage of radiotherapy

Background. Studies carried out in recent decades have shown that immune cells are essential participants in the cancer process as well as cancerrelated inflammation. Focus has been increased on understanding the way how immune cells affect a tumor at different stages of the disease: early neoplastic transformation, clinically detected tumors, metastatic spread, and at surgery and radiotherapy stages.&#x0D; Purpose – assessing the status of the immune system in patients with brain tumors before radiation therapy and radiosurgery and comparing the features of immunity in metastatic and glial brain tumors.&#x0D; Materials and methods. The study presents the immunogram findings of 61 patients. Out of those: 18 patients with primary glial tumors and 23 patients with secondary metastatic tumors to the brain. The outcomes of 20 conditionally healthy non-cancer patients are presented as a control group. The age of patients is 24–75. All patients were histologically diagnosed with the tumor. Surgery was performed 1.0–3.0 years before the examination. Assessment of the immune system in patients with brain tumors was performed taking into account the cellular, humoral and phagocytic component of innate immunity. When assessing cellular immunity, the relative and absolute count of major lymphocyte subpopulations, such as CD3+ – general T-lymphocytes, CD4+ – T-lymphocytes-helpers, CD8+ – cytotoxic lymphocytes, CD16+ – natural killer lymphocytes, CD19+-B-lymphocytes, were calculated. Determining the humoral parameters included an assessment of quantitative values of IgG, IgM and IgA. Quantitative assessment of the phagocytic component of innate immunity included phagocytic activity of neutrophils (i. e. NBT test (Nitroblue Tetrazolium test), inducing (Zymosanum) and spontaneous neutrophil myeloperoxidase activity).&#x0D; Results. When comparing the immune parameters of the number of T- and B-subpopulations of lymphocytes in patients with primary malignant brain tumors and secondary metastatic tumors, no statistically significant difference has been detected between these params. Glioblastomas show higher levels of СD4+- and CD8+-lymphocytes in comparison with other tumour groups as well as higher levels of IgG and IgA than in other tumors, while IgM concentration is almost at the same level in three groups of patients. There is a tendency for reducing IgG and IgM level in the blood of patients with metastatic tumors. Both groups of cancer patients under study show inhibition of myeloperoxidase activity of neutrophils in the setting of maintaining the function of NBT cell activity.&#x0D; Conclusions. According to the findings obtained via studying immunological indicators of brain tumors, both metastatic and primary malignant glial ones, there are partial changes in various immune system components such as cellular, humoral and phagocytic activity. However, no statistically significant difference was detected between immune status indicators, that substantiates the need for further study of this issue. At the stage of preparation for radiation therapy, no significant changes in the immune system of the patients with brain tumors, that would make such treatment impossible and be consiered as one of contraindications, are observed.

High levels of SARS-CoV-2-specific T cells with restricted functionality in severe courses of COVID-19.

BACKGROUNDPatients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) differ in the severity of disease. We hypothesized that characteristics of SARS-CoV-2–specific immunity correlate with disease severity.METHODSIn this study, SARS-CoV-2–specific T cells and antibodies were characterized in uninfected controls and patients with different coronavirus disease 2019 (COVID-19) disease severity. SARS-CoV-2–specific T cells were flow cytometrically quantified after stimulation with SARS-CoV-2 peptide pools and analyzed for expression of cytokines (IFN-γ, IL-2, and TNF-α) and markers for activation, proliferation, and functional anergy. SARS-CoV-2–specific IgG and IgA antibodies were quantified using ELISA. Moreover, global characteristics of lymphocyte subpopulations were compared between patient groups and uninfected controls.RESULTSDespite severe lymphopenia affecting all major lymphocyte subpopulations, patients with severe disease mounted significantly higher levels of SARS-CoV-2–specific T cells as compared with convalescent individuals. SARS-CoV-2–specific CD4+ T cells dominated over CD8+ T cells and closely correlated with the number of plasmablasts and SARS-CoV-2–specific IgA and IgG levels. Unlike in convalescent patients, SARS-CoV-2–specific T cells in patients with severe disease showed marked alterations in phenotypical and functional properties, which also extended to CD4+ and CD8+ T cells in general.CONCLUSIONGiven the strong induction of specific immunity to control viral replication in patients with severe disease, the functionally altered characteristics may result from the need for contraction of specific and general immunity to counteract excessive immunopathology in the lung.FUNDINGThe study was supported by institutional funds to MS and in part by grants of Saarland University, the State of Saarland, and the Rolf M. Schwiete Stiftung.

Major and minor lymphocytes subpopulations in peripheral blood and cerebrospinal fluid of children with meningitis

Introduction. The analysis of current publications indicates at our insufficient understanding of subpopulation composition of lymphocytes in peripheral blood and cerebrospinal fluid (CSF) during pediatric neuroinfectious diseases. It has been found that the main lymphocyte populations are divided into many small (minor) subpopulations.The purpose of this research was to assess percentage of major and minor blood and CSF lymphocyte subsets in children with aseptic viral meningitis (AM) or bacterial purulent meningitis (BM).Materials and methods. Phenotyping of blood and CSF lymphocytes of children aged from 4 months to 17 years diagnosed with AM (n = 86) and BM (n = 39) was carried out by using flow cytometry. As a comparison group, we analyzed peripheral blood and CSF samples collected from children with acute respiratory viral infections (ARVIs) associated with syndrome of meningism (n = 27). There was evaluated percentage of the major cell subpopulations (CD3+ T-lymphocytes, T-helpers — CD3+CD4+ Th, cytotoxic T-lymphocytes — CD3+CD8+ CTL, natural killer cells — CD3-CD16+CD56+ NK, B-cells — CD3-CD19+), as well as minor lymphocyte subsets (double positive (DP) (CD3+CD4+CD8+), double negative (DN) (CD3+CD4-CD8-) T-cells, NKT (CD3+CD16+CD56+), CD3-CD8+ NK, CD3+CD8dim and CD3+CD8 8bright).Results. It was found that the acute period of BM and AM vs. the comparison group (ARVI) was characterized by significant differences in the blood and CSF composition of major and minor lymphocyte subsets. In particular, blood T-cells, Th, CTL, NK, NKT, DN, CD3-CD8+ NK, CD3+CD8bright and CD3+CD8dim dominated in parallel with significantly lowered B-cell frequency in AM vs. BM. In the CSF of children with AM, T-cells and Th prevailed, whereas count of B-cells and CD3-CD8+ NK was lower compared to those in BM. In addition, further differences were revealed in CSF and blood cell subset composition depending on nosological entity, while maintaining differences in some major and minor lymphocyte subpopulations lacked in the comparison group. Calculating the CSF/blood ratio for the major and minor lymphocyte subsets uncovered the prevalence for the majority of cell subpopulations (the coefficients ranged from 1.2 to 16.4) in the CSF of the comparison group (ARVI), except B-cells, NK and CD3-CD8+ NK (coefficients ranged from 0.07 to 0.31). AM and BM were featured with various changes in the CSF/blood ratio found for most of the studied subpopulations in the acute period as well as the recovery phase highlighted with characteristic traits for each nosological form.Conclusion. The data obtained indicate about finding specific features in the activation of systemic and intrathecal immune response during viral and bacterial meningitis in children, which may be used as an additional differential diagnostic criterion.

Vaccination in children with chronic severe neutropenia – review of recommendations and a practical approach

While the management of childhood neutropenia associated with a modifiable factor should be appropriate for the primary cause, there are misconceptions regarding the management of severe congenital neutropenia, immune neutropenia and cases classified as “idiopathic”. Antibiotic prophylaxis or granulocyte-colony stimulating factor (G-CSF) are prescribed by specialists in pediatric hematology or immunology, whereas immunization may be conducted by primary care physicians should clear recommendations by provided. There is a belief that severe neutropenia, as an immunodeficiency, is associated with compromised effectiveness and increased rate of complications of immunization. The immunization might be delayed or omitted, increasing the risk of unnecessary infection. We discuss the available data and recommendations regarding vaccination of children with chronic severe neutropenia. While there are virtually no studies addressing the safety and effectiveness of vaccination in neutropenia, expert opinions provide information on immunization policy in “phagocytic cells defects” or explicitly neutropenia. There are no contraindications for inactivated vaccines in neutropenia. Live bacterial vaccines are contraindicated. While in general the vaccination with live viral vaccines is encouraged, occasionally neutropenia might be associated with defects of adaptive immunity, which would preclude the administration of such vaccines. Although this should be easily phenotypically identified, we propose assessing immunoglobulin levels and performing a low-cost flow cytometry test for major lymphocyte subpopulations to exclude significant defects in adaptive immunity before administration of live viral vaccines to such patients. This can improve the adherence of patients’ guardians and physicians to proposed vaccination policy and the professional and legal safety associated with the procedure.

MORPHO-FUNCTIONAL CHANGES OF THYMUS AND CONTENTS OF BLOOD LYMPHOCYTE SUBPOPULATIONS IN FEMALE WISTAR RATS WITH DIFFERENT RESISTANCE TO HYPOXIA IN SYSTEMIC INFLAMMATORY RESPONSE

Hypoxia and immune reactions are closely interrelated at molecular, cellular and organism levels, and the individuals differ in resistance to oxygen deficiency. Animals with high and low resistance to hypoxia have different adaptive capabilities and predisposition to the development of inflammatory diseases. Data on the individual characteristics of hypoxia resistance in female laboratory animals and humans, and its relationship to immune system reactions in both normal conditions and inflammatory diseases are not available in the literature. It is known, however, that acute infectious and inflammatory diseases develop at lesser rates and are less severe in women and female laboratory animals than in males, which can be explained by higher resistance of females to hypoxia. The aim of our study is to reveal the features of morpho-functional thymus changes, and subpopulation of peripheral blood lymphocytes in systemic inflammatory response induced by LPS administration to female Wistar rats with different resistance to hypoxia. Resistance of mature female Wistar rats to hypoxia was determined as a survival period in a ventilated lowpressure chamber simulating high altitude condition (11 500 m). The rats with a lifetime “at high altitude” of &gt; 180 s have been classified as highly resistant to hypoxia, and the animals surviving for &lt; 20 seconds were designated low-resistant. One month after determining the hypoxia resistance, the females were injected intraperitoneally with E. coli O26:B6 lipopolysaccharide (LPS) at a dose of 1.5 mg/kg during the dioestrus phase. The animals were withdrawn from the experiment by i/m Zoletyl injection (15 mg/kg) one day after LPS administration. The relative volume fractions of thymic cortex and medulla were evaluated; the areas of necrosis were determined in the liver, and the number of neutrophils in the interalveolar septa was counted in the lungs. The serum contents of corticosterone, testosterone, TGF-β were determined. A flow cytometry evaluation of the relative and absolute numbers was performed for major subpopulations of lymphocytes in peripheral blood. The number of apoptotically dying cells of the thymus was assessed. For statistical processing of the obtained data, the Statistica 8.0 software was applied, using criteria of multiple comparisons by Kruskal–Wallis and Dann. The differences were considered statistically significant at p &lt; 0.05.In both high- and low-resistant to hypoxia females, the development of a systemic inflammatory -response was accompanied by a moderately severe thymic involution, apoptosis of thymocytes, an increase in the absolute number of NK, and rise of testosterone and corticosterone contents. LPS injection into low-resistant rats, if compared to females highly resistant to hypoxia, led to more severe manifestations of systemic inflammation, i.e., a pronounced inflammatory reaction in the lungs and a more extensive liver necrotic area accompanied by increased absolute numbers of regulatory T lymphocytes and T helper cells, and more pronounced thymic accidental involution with apoptotic death of thymocytes. Systemic manifestations of inflammation were less pronounced in hypoxia-resistant female rats, which was apparently associated with activation of lymphocyte migration from the thymus and blood to the inflammation focus, and development of more effective immune response.Conclusion: immune reactions in the systemic inflammatory response induced by LPS in female Wistar rats depend on individual resistance to hypoxia. These data should be used to develop approaches to personalized therapy of infectious and inflammatory diseases in women.

Assessment of the Parameters of Adaptive Cell-Mediated Immunity in Naïve Common Marmosets (Callithrix jacchus)

Common marmosets are small New World primates that have been increasingly used in biomedical research. This report presents efficient protocols for assessment of the parameters of adaptive cell-mediated immunity in common marmosets, including the major subpopulations of lymphocytes and main markers of T- and B-cell maturation and activation using flow cytometry with a multicolor panel of fluorescently labelled antibodies. Blood samples from eight common marmosets were stained with fluorescently labeled monoclonal antibodies against their population markers (CD45, CD3, CD20, CD4, CD8) and lymphocyte maturation and activation markers (CD69, CD62L, CD45RO, CD107a and CD27) and analyzed by flow cytometry. Within the CD45+ population, 22.7±5.5% cells were CD3- CD20+ and 67.6±6.3% were CD3+CD20-. The CD3+ subpopulation included 55.7±5.5% CD3+CD4+CD8- and 34.3±3.7% CD3+CD4-CD8+ cells. Activation and maturation markers were expressed in the following lymphocyte proportions: CD62L on 54.0±10.7% of CD3+CD4+ cells and 74.4±12.1% of CD3+CD8+ cells; CD69 on 2.7±1.2% of CD3+CD4+ cells and 1.2±0.5% of CD3+CD8+ cells; CD45RO on 1.6±0.6% of CD3+CD4+ cells and 1.8±0.7% of CD3+CD8+ cells; CD107a on 0.7±0.5% of CD3+CD4+ cells and 0.5±0.3% of CD3+CD8+ cells; CD27 on 94.6±2.1% of CD3+ cells and 8.9±3.9% CD20+ cells. Female and male subjects differed in the percentage of CD3+CD4+CD45RO+ cells (1.9±0.5 in females vs 1.1±0.2 in males; p < 0.05). The percentage of CD20+CD27+ cells was found to highly correlate with animals' age (r = 0.923, p < 0.005). The basal parameters of adaptive cell-mediated immunity in naïve healthy marmosets without markers of systemic immune activation were obtained. These parameters and the described procedures are crucial in documenting the changes induced in common marmosets by prophylactic and therapeutic immune interventions.

INDIСATORS OF THE LYMPHOCYTE SUBSETS AS EFFICICIENCY PREDICTORS OF THERAPY WITH INHIBITORS OF TNFα IN CHILDREN WITH INFLAMMATORY BOWEL DISEASE

(IBD), who were for the first time treated with TNFα blocker (infliximab). Our aim was to determine prognostic informative value of the immunological parameters in order to assess the treatment efficiency. A comprehensive research included seventy children with IBD from 12 to 18 years old in the course of specific treatment (49 children with CD, 21 children with UC).The comparison group consisted of fifty healthy children of similar age who were subjected to a similar detailed examination. The patients were divided into two groups, depending on their therapeutic response following 1 year of biological therapy: the first group showed a persistent positive effect of the drug, and the second group exhibited only unstable effects of the treatment. We determined the contents of major and small subpopulations of peripheral blood lymphocytes before the first administration of infliximab. Immunophenotyping was performed by multicolor flow cytometry (FC 500), using the CD45, CD3, CD4, CD8, CD19, CD16, CD56, HLA-DR, CD5, CD161, CD127, CD25, and CD294 markers.We have revealed that the content of B lymphocytes was significantly reduced in children with unstable effects of therapy. By contrast, the B lymphocyte levels in children with persistent positive therapeutic effect did not differ from the comparison group. Analysis of the composition of the B lymphocyte profile showed an imbalance in the B1-to-B2 cell ratio, with decreased of B1 cell counts in IBD patients against the comparison group. In addition, the patients with unstable therapeutic effect showed a significant decrease in B2 cell numbers compared with a group with persistent effect and comparison group. The numbers of NK cells in IBD patients were found to be reduced against the comparison group. Assessment of T lymphocytes subsets revealed a number of features in the patients with minimal therapeutic effects, i.e., an increased level of activated T helper cells (CD4+CD25+CD127high) and Th17 lymphocytes (CD3+CD4+CD161+), as compared to children with stable effect of treatment and to the comparison group. Moreover, in children with minimal effects of therapy, the levels of Tregs within T-helper cell subsets were significantly higher than in the comparison group. By means of ROC analysis, we have identified most informative parameters for the groups with minimal versus persistent therapeutic effect, and showed a good quality for a discrimination model involving relative amount of Th17 cells, activated T helper cells and B lymphocytes. The number of Тh17 lymphocytes (% CD3+CD4+ lymphocytes) allowed to predict the effect of therapy with a TNFα blocker with high probability. The present study enables us to propose cellular immunity testing, as a promising tool for monitoring clinical state of IBD patients.

A Model of Chronic Exposure to Unpredictable Mild Socio-Environmental Stressors Replicates Some Spaceflight-Induced Immunological Changes.

During spaceflight, astronauts face radiations, mechanical, and socio-environmental stressors. To determine the impact of chronic socio-environmental stressors on immunity, we exposed adult male mice to chronic unpredictable mild psychosocial and environmental stressors (CUMS model) for 3 weeks. This duration was chosen to simulate a long flight at the human scale. Our data show that this combination of stressors induces an increase of serum IgA, a reduction of normalized splenic mass and tends to reduce the production of pro-inflammatory cytokines, as previously reported during or after space missions. However, CUMS did not modify major splenic lymphocyte sub-populations and the proliferative responses of splenocytes suggesting that these changes could be due to other factors such as gravity changes. Thus, CUMS, which is an easy to implement model, could contribute to deepen our understanding of some spaceflight-associated immune alterations and could be useful to test countermeasures.

Lymphocyte subpopulations, levels of interferon, and expression of their receptors in patients with chronic hepatitis B and C: Correlation with the species of viruses and the degree of liver fibrosis

To determine whether there is a correlation of the composition of circulating lymphocyte subpopulations, the serum concentrations of interferon (IFN)-α, IFN-γ, and IFN-λ3, and the lymphocyte expression of types I and II IFN receptors with the species of a disease pathogen and the degree of liver fibrosis (LF) in patients with chronic hepatitis B (CHB) and in those with chronic hepatitis C (CHC). The investigation enrolled 44 patients with CHC, 9 patients with CHB, and 13 clinically healthy donors. The degree of LF in the patients was determined using transient elastography. The composition of peripheral blood lymphocyte subpopulations was examined; the concentrations of IFN-α, IFN-γ, and IL-28B were estimated. Lymphocyte counts were higher in patients with CHC and in those with CHB than those in healthy donors; and the number of neutrophils was lower. There were no differences between the groups in the composition of lymphocyte subpopulations with the exception of the number of CD3+CD4+ cells, which in patients with CHC was larger than in those with CHB. In CHC and CHB patients, the counts of CD118+ lymphocytes were higher than those in healthy donors. Patients with CHB and those with CHC did not differ between themselves and from healthy donors in the expression of CD119 on the lymphocytes. In CHC patients, the relative CD119+ cell counts were higher between CD4+ lymphocytes than those in healthy donors. The serum levels of IFN-α and IFN-γ in CHC and CHB patients were similar, but higher in healthy donors. The concentration of IL-28B genotype in patients with CHC was twice as high as in those with CHB, but the differences were statistically insignificant. The number of lymphocytes increased with the progression of fibrosis; that of neutrophils decreased. There was an inverse relationship between platelet counts and LF severity. Multiple comparisons of the clusters of patients with different degrees of LF revealed no differences in the number of major lymphocyte subpopulations. However, the number of CD3+CD16+CD56+ natural killer-like T (NKT) cells correlated with fibrosis severity. Patients with different degrees of LF showed no differences in the proportion of CD118- and CD119 cells between lymphocytes and in the serum levels of IFN-α, IFN-γ, and IL-28B levels. Patients with grade IV LF displayed a higher proportion of CD4+CD119+ lymphocytes between CD45+ cells than did those with grade III LF. Several new clinical and laboratory trends were identified and the nature and extent of previously described hematological and immunological changes were clarified in CHC or CHB patients with various degrees of LF. Some indicators may be used as additional criteria for the prognosis of the above forms of hepatitis, and a number of newly described facts suggest that it is necessary to revise the protective/phlogogenic value of types I, II, and III IFNs in chronic viral hepatitis C and B.

Comparison of major lymphocyte subpopulations and recent thymic emigrants in patients with ataxia telangiectasia and age-matched healthy groups

BackgroundAtaxia telangiectasia (A-T) is a genetic disorder caused by the homozygous mutation of the A-T mutated gene. It is frequently associated with variable degrees of cellular and humoral immunodeficiency. However, the immune defects in A-T patients are not well characterized. To the best of our knowledge, no studies have focused on the major lymphocyte subpopulations and recent thymic emigrants of A-T patients in comparison with age-matched healthy controls. MethodsFollowing the European Society for Immunodeficiencies criteria, 17 patients diagnosed with A-The, and 12 age-matched healthy children were assigned to the study. Both patients and healthy controls were grouped as 1–5, 6–10, 11–15, and 15+ years. By using a flow cytometer, major lymphocyte subpopulations and CD4+CD45RA+CD31+ recent thymic emigrants were determined as percentage and absolute cell numbers and compared. ResultsNo significant differences in all lymphocyte subpopulations were observed between the age groups of A-T patients. Compared to the healthy controls, there was a decrease in T cells, effector memory T4 cells, B cells, naïve B cells, naïve T4 cells, switched B cells, and recent thymic emigrants and an increase in active T8 cells and non-switched B cells in the percentage and absolute number of some cell populations in the A-T group. ConclusionsThis study showed that effector functions in some cell lymphocyte populations were decreased in A-T patients.