Lymphocyte Activation Markers Research Articles

In neonates with vitamin D deficiency, low lymphocyte activation markers are risk factors for infection

ABSTRACTBackground: Vitamin D has regulatory effects on different cells of the immune system and low levels are associated with several immune-mediated diseases.Aim: To investigate the association between neonatal 25-hydroxy vitamin D (25-OHD) level and the expression of lymphocyte activation markers (HLA-DR, CD69, CD25, CD45RA) on T-lymphocyte subpopulations and its impact in neonatal infection.Methods: 25-OHD level was measured in the cord blood of 56 neonates and their mothers using an enzyme immune-assay method. Based on the 25-OHD level, infants were categorised into four groups: severe deficiency (n = 7), moderate deficiency (n = 21), mild deficiency (n = 15) and normal 25-OHD level (n = 13). Mothers were classified into deficient (n = 18), insufficient (n = 21) and normal levels (n = 17). T-lymphocyte subpopulations and lymphocyte activation markers were investigated using flow cytometry.Results: There was a positive correlation between maternal and cord blood 25-OHD levels (r = 0.503, p = 0.001). The group with severe 25-OHD deficiency had the significantly lowest level of total lymphocytes, CD3+ T lymphocytes, CD4+ T-helper and CD8+ T-cytotoxic lymphocytes and CD4+CD45RA+ naïve T-cells compared with the other groups. The frequencies of CD8+CD25+, CD4+CD25+ and CD4+HLA-DR+ activated T-lymphocytes were significantly lower in the severe, moderate and mild deficiency groups than in the normal group. Seven of 43 (16.27%) infants with 25-OHD deficiency were admitted with sepsis to the neonatal intensive care unit and there were no cases of sepsis in the normal 25-OHD group.Conclusion: Vitamin D deficiency is associated with a reduction of lymphocyte subsets and altered T-lymphocyte activation which are considered to be risk factors for neonatal infection.

ВИВЧЕННЯ ДИНАМІКИ РІВНІВ CD4+CD25+, CD4+HLA-DR4+ ТА CD4+CD95+ ЛІМФОЦИТІВ СПЕРМИ ІНФЕРТИЛЬНИХ ЧОЛОВІКІВ З ХРОНІЧНИМ ЗАПАЛЕННЯМ УРОГЕНІТАЛЬНОГО ТРАКТУ ПІД ВПЛИВОМ НУКЛЕКСУ

Вступ. У практиці сімейних лікарів почастішали випадки звернень чоловіків із порушенням запліднювальної функції. Ретельний збір анамнезу дозволяє запідозрити, що зниження фертильності у чоловіків супроводжується латентним та персистуючим перебігом урогенітальних інфекцій, що дуже ускладнює їх своєчасну діаг­ностику.Мета – дослідження маркерів активації лімфоцитів сперми чоловіків з малосимптомними формами хронічної інфекції урогенітального тракту в залежності від фертильності та впливу нуклексу на їх рівні.Матеріал і методи. Було досліджено сперму 60 чоловіків, хворих на малосимптомні форми хронічного запалення урогенітального тракту (ХЗУТ); І групу склали 32 пацієнти, в яких діагностували ускладнення – інфертильність, у ІІ групу ввійшли 28 фертильних пацієнтів.Висновки. 1. Вивчення маркерів активації лімфоцитів сперми у чоловіків, хворих на малосимптомні форми хронічної інфекції урогенітального тракту, вказує на різнопланове збільшення їх кількості залежно від фертильності.2. Діагностично важливим є визначення співвідношення CD25+/CD95+ для прогнозування розвитку інфертильності. Збільшення цього індексу вказує на порушення процесів апоптозу патологічних форм генеративних клітин, що призводить до їх накопичення у спермі.3. Імунокорегуюча терапія з використанням нуклексу приводить до нормалізації співвідношення CD25+/CD95+, до зменшення патологічних форм сперматозоїдів й, зрештою, до збільшення запліднювального потенціалу.

Visceral leishmaniasis: A novel nuclear envelope protein ‘nucleoporins-93 (NUP-93)’ from Leishmania donovani prompts macrophage signaling for T-cell activation towards host protective immune response

The shift of macrophage and T-cell repertoires towards proinflammatory cytokine signalling ensures the generation of host-protective machinery that is otherwise compromised in cases of the intracellular Leishmania parasite. Different groups have attempted to restore host protective immunity. These vaccine candidates showed good responses and protective effects in murine models, but they generally failed during human trials. In the present study, we evaluated the effect of 97 kDa recombinant nucleoporin-93 of Leishmania donovani (rLd-NUP93) on mononuclear cells in healthy and treated visceral leishmaniasis (VL) patients and on THP-1 cell lines. rLd-NUP93 stimulation increased the expression of the early lymphocyte activation marker CD69 on CD4+ and CD8+ T cells. The expression of the host protective pro-inflammatory cytokines IFN-γ, IL-12 and TNF-α was increased, with a corresponding down-regulation of IL-10 and TGF-β upon rLd-NUP93 stimulation. This immune polarization resulted in the up-regulation of NF-κB p50 with scant expression of SMAD-4. Augmenting lymphocyte proliferation upon priming with rLd-NUP93 ensured its potential for activation and generation of strong T-cell mediated immune responses. This stimulation extended the leishmanicidal activity of macrophages by releasing high amounts of reactive oxygen species (ROS). Further, the leishmanicidal activity of macrophages was intensified by the elevated production of nitric oxide (NO). The fact that this antigen was earlier reported in circulating immune complexes of VL patients highlights its antigenic importance. In addition, in silico analysis suggested the presence of MHC class I and II-restricted epitopes that proficiently trigger CD8+ and CD4+ T-cells, respectively. This study reported that rLd-NUP93 was an effective immunoprophylactic agent that can be explored in future vaccine design.

PO-257 The immunoreactivity of patients with cervical cancer on the background of extracorporeal immunopharmacotherapy (EIPHT)

IntroductionThe aim of the study was to study the state of cellular and humoral factors of immunity of cervical cancer patients on the background of using extracorporeal immunopharmacotherapy.Material and methods136 patients with cervical cancer T2-3N0-1M0 stages. With cervical cancer IIA, the stage of the disease was observed in 18.7% of cases, IIB - in 20.9%, IIIA - in 16.0% and IIIB stage - in 44.4% of cases. The largest part of the patients was female cervical cancer at stage IIIB. Depending on the methods of therapy in the complex treatment: group 1–36 patients with cervical cancer before treatment; 46 patients with cervical cancer who received EIPHT with thymalin; group 3–54 patients with cervical cancer who did not receive EIPHT. Also 34 persons of the control group with normative values. The study of the immune system was carried out at the admission of patients to the hospital before the immunotherapy course and immediately a few days before discharge from the hospital. All patients with cervical cancer received standard polychemotherapy according to the protocol of the department, a complex treatment including a two-stage combined radiation therapy. Immunological studies included the study of cellular and humoral parameters of the immune system of patients with cervical cancer. The EIPHT method was designed primarily to reduce toxic effects after chemotherapy and radiotherapy, as well as improve the overall condition after chemotherapy and radiation therapy.Results and discussionsThe analysis showed that patients with cervical cancer was found a T-cell immunodeficiency, which was associated with CD4+ - deficiency against the background of an increase in CD8 +and increased activation of lymphocytes with the predominance of apoptosis of immunocompetent cells. After carrying out EIPHT with thymalin, activation of the T-cell link of immunity is observed. The revealed imbalance of the humoral link of immunity before and after polychemotherapy and radiotherapy was associated with an imbalance of the main serum immunoglobulins and an elevated CIC level of small and large values. Against the background of the use of EIPHT with thymalin, normalisation of humoral factors of immunity and activation markers of lymphocytes is observed.ConclusionThus, the method of EIPHT in our case turned out to be promising in the treatment of cervical cancer due to the possibility of removing the effects of cancer and chemoradiation intoxication, as well as activating its own system of antitumor protection of the organism.

Оптимізація тактики ведення вагітності та ранніх передчасних пологів у жінок після застосування допоміжних репродуктивних технологій

The objective: reduction of the frequency of perinatal complications in women with early preterm labor after the ART, based on the improvement and implementation of the algorithm for diagnostic and therapeutic and prophylactic measures. Materials and methods.We analyzed the course of 130 single-pregnancy pregnancy women with restored fertility after the use of therapeutic assisted reproductive technologies programs that ended with early premature births. The study of the subpopulation composition of lymphocytes and the content of activation markers of peripheral blood lymphocytes was performed using the method of quadratic color laser flow cytometry and a set of monoclonal antibodies. The concentration of cytokines IFN- γ, IL-4, IL-17A, IL-17F, IL-21 and IL-22 in serum of pregnant women was determined by solid-phase immunoassay analysis. The material for studying and analyzing the morpho-functional state of the fetoplacental complex was the results of pathomorphological study of the litter in all cases of early premature birth. By our own research, we established the frequency, structure and leading causes of early premature birth after assisted reproductive technologies, optimized the tactics of conducting induced pregnancies of high obstetric risk, formulated practical recommendations for implementation in practical medicine of Ukraine. Results. For introduction into practical medicine of Ukraine with the purpose of decreasing the frequency of perinatal complications and effective provision of obstetric and gynecological care, we recommend that the following provisions be included in the clinical protocol for the management of pregnancy of high obstetric risk in women after assisted reproductive technologies: 1. To predict the risk of early premature birth in women after ancillary reproductive technologies, the diagnostic algorithm should additionally include the definition of the main indicators of the serum cytokine profile: proinflammatory serum cytokines IFN- γ, IL-2, IL-12, IL-18; anti-inflammatory regulatory cytokine IL-10; quantitative indicators of NK cells with the phenotype CD3-CD16+CD56+, CD94+ lymphocytes and peripheral blood CD71 transferrin. 2. Use of medication correction – Micronized progesterone (200 mg/day). 3. At the gestational age of 28–33 weeks, with the premature rupture of the membranes optimal and effective is the expectant management of pregnancy for 5 days with subsequent delivery of women through the natural birth canal, which makes it possible to achieve the maximum possible degree of maturity of the fetus with a minimum risk of ascending infection in newborns. Conclusions. The effectiveness of our improved algorithm is a significant reduction in women after assisted reproductive technologies, early preterm labor (2.8 times), placental dysfunction (2.3 times), premature rupture of the membranes (in 2, 1 time), fetal distress (3,6 times), abdominal degeneration (2.9 times) and implementation of intraaminal infections (2.6 times; p<0.05). The total morbidity of newborns in the early neonatal period decreased 2.4 times, and perinatal losses were absent. Key words: assisted reproductive technologies, RPP, PRPO, markers of activation of lymphocytes, cytokines.

Human Herpes Virus 8 in HIV-1 infected individuals receiving cancer chemotherapy and stem cell transplantation.

BackgroundHuman Herpes Virus 8 (HHV8) can cause Kaposi’s Sarcoma (KS) in immunosuppressed individuals. However, little is known about the association between chemotherapy or hematopoietic stem cell transplantation (HSCT), circulating HHV8 DNA levels, and clinical KS in HIV-1-infected individuals with various malignancies. Therefore, we examined the associations between various malignancies, systemic cancer chemotherapy, T cell phenotypes, and circulating HHV8 DNA in 29 HIV-1-infected participants with concomitant KS or other cancer diagnoses.MethodsWe quantified HHV8 plasma viral loads and cell-associated HHV8 DNA and determined the relationship between circulating HHV8 DNA and lymphocyte counts, and markers of early and late lymphocyte activation, proliferation and exhaustion.ResultsThere were no significant differences in plasma HHV8 DNA levels between baseline and post-chemotherapy time points or with the presence or absence of clinical KS. However, in two participants circulating HHV8 DNA increased following treatment for KS or HSCT for lymphoma,. We observed an approximately 2-log10 reduction in plasma HHV8 DNA in an individual with KS and multicentric Castleman disease following rituximab monotherapy. Although individuals with clinical KS had lower mean CD4+ T cell counts and percentages as expected, there were no significant associations with these factors and plasma HHV8 levels. We identified increased proportions of CD8+ and CD4+ T cells expressing CD69 (P = 0.01 & P = 0.04 respectively), and increased CD57 expression on CD4+ T cells (P = 0.003) in participants with detectable HHV8.ConclusionThese results suggest there is a complex relationship between circulating HHV8 DNA and tissue-based disease in HIV-1 and HHV8 co-infected individuals with various malignancies.

CD69 partially inhibits apoptosis and erythroid differentiation via CD24, and their knockdown increase imatinib sensitivity in BCR-ABL-positive cells.

Chronic myeloid leukemia (CML) is caused by a constitutively active BCR-ABL tyrosine kinase. Tyrosine kinase inhibitors (TKIs) imatinib and its derivatives represent a breakthrough for CML therapy, but the use of TKI alone is ineffective for many CML patients. CD69, an early activation marker of lymphocytes, participates in immune and inflammatory responses. Previous studies revealed that BCR-ABL upregulates CD69 expression; however, the role of CD69 in CML cells is unknown. Here, we demonstrate that BCR-ABL induced CD69 promoter activity and mRNA and protein expression via the NF-κB pathway. CD69 knockdown partially increased apoptosis and expression of erythroid differentiation markers, α-globin, ζ-globin, and glycophorin A, and increased imatinib sensitivity in K562 and KU812 CML cells. Gene microarray analysis and quantitative real-time PCR verified that CD24, an oncogenic gene, downregulated in K562 cells upon CD69 knockdown. CD69 overexpression increased, whereas CD69 knockdown inhibited CD24 promoter activity and mRNA and protein levels. CD24 knockdown also partially increased apoptosis, erythroid differentiation, and imatinib sensitivity in K562 cells, whereas its overexpression inhibited the effects of CD69 knockdown on apoptosis, erythroid differentiation, and imatinib sensitivity in K562 cells. Imatinib-induced apoptosis and erythroid differentiation were also inhibited by CD69 or CD24 overexpression in BCR-ABL-expressing CML cell lines and CD34+ cells. Taken together, CD24 is a downstream effector of CD69. CD69 and CD24 partially inhibit apoptosis and erythroid differentiation in CML cells; thus, they may be potential targets to increase imatinib sensitivity.

Generation of anti-porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis.

T cell-mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti-porcine CD69 mAb-producing mouse hybridomas, 01-14-22-51 (IgG2b-κ) and 01-22-44-102 (IgG2a-κ), both showing fine reactivity with phorbol 12-myristate 13-acetate (PMA) and ionomycin-stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti-interferon-γ mAb and anti-tumor necrosis factor-α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs.

THE STUDY OF THE LEVELS OF METALLOPROTEINASES, CYTOKINES AND LYMPHOCYTE ACTIVATION MARKERS IN SEMINAL PLASMA OF MEN, DEPENDING ON FERTILITY

The study of the concentration of metalloproteinases, pro- and anti-inflammatory cytokines, lymphocyte activation markers in seminal plasma of men with oligosymptomatic forms of chronic inflammation of the urogenital tract (CIUT), depending on the fertility rate are presented in the article. Quantitative characteristics in male sperm with different forms of pathospermia were studied for matrix metalloproteinases (MMP) -2 and MMP-9 and their inhibitors - tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2. It was shown, that during chronic inflammation of the urogenital tract of men are shifting levels of cytokine profile, reducing the concentration of metalloproteinase-2, chemokines - fractalkine and regulated by activation, expression and secretion of normal T-cells (RANTES), sharp increase in IL-8, MCP-1 and elevation of the CD25+ / CD95+, indicating that the disturbance of apoptosis of pathological forms generative cells and their accumulation in the sperm. The ratio of immunological indices IL-2/IL-4, IL-10/IL-12 was calculated and a significant increase in the IL-10/IL-12 index was noted in the group of individuals with elevated levels of hypercapitated form of sperm, and a reduced proportion of this ratio was observed in the microsomatic morphology of sperm. Prolonged inflammation in the genital area accompanied by depletion of the local immune system, resulting in the development of infertility. Immunocorrection therapy for men with CIUT should take into account the peculiarities of changes in local immunity and be differentiated depending on the prevalence of certain pathological forms of sperm and changes in the cytokine profile of the seminal plasma.

Effect of Priming of Multipotent Mesenchymal Stromal Cells with Interferon γ on Their Immunomodulating Properties.

Multipotent mesenchymal stromal cells (MSCs) are widely used for cell therapy, in particular for prophylaxis and treatment of graft-versus-host disease. Due to their immunomodulatory properties, MSCs affect the composition of lymphocyte subpopulations, which depends on the immunological state of the organism and can change in different diseases and during treatment. Administration of MSCs is not always effective. Treatment of MSCs with different cytokines (in particular IFN-γ) leads to enhancement of their immunomodulatory properties. The aim of this study was to investigate subpopulational alterations and activation markers in lymphocytes (activated and non-activated) after interaction with MSCs and MSCs pretreated with IFN-γ (γMSCs) in vitro. Lymphocytes were co-cultured with MSCs or γMSCs for 4 days. The proportion of CD4+ and CD8+ expressing CD25, CD38, CD69, HLA-DR, and PD-1 and distribution of memory and effector subsets were measured by flow cytometry after co-cultivation of lymphocytes with MSCs or γMSCs. The distribution of lymphocyte subpopulations changes during culturing. In non-activated lymphocytes cultured without MSCs, decrease in the proportion of naïve cells and increase in the number of effector cells was observed. That could be explained as activation of lymphocytes in the presence of serum in culturing medium. Co-culturing of lymphocytes with MSCs and γMSCs leads to retention of their non-activated state. Activation of lymphocytes with phytohemagglutinin increases the number of central memory cells and activates marker expression. Interaction with MSCs and γMSCs prevents activation of lymphocytes and keeps their naïve state. Priming with IFN-γ did not induce MSCs inhibitory effect on activation of lymphocytes.

Crucial role for CD69 in allergic inflammatory responses: CD69-Myl9 system in the pathogenesis of airway inflammation.

CD69 has been known as an early activation marker of lymphocytes; whereas, recent studies demonstrate that CD69 also has critical functions in immune responses. Early studies using human samples revealed the involvement of CD69 in various inflammatory diseases including asthma. Moreover, murine disease models using Cd69-/- mice and/or anti-CD69 antibody (Ab) treatment have revealed crucial roles for CD69 in inflammatory responses. However, it had not been clear how the CD69 molecule contributes to the pathogenesis of inflammatory diseases. We recently elucidated a novel mechanism, in which the interaction between CD69 and its ligands, myosin light chain 9, 12a and 12b (Myl9/12) play a critical role in the recruitment of activated T cells into the inflammatory lung. In this review, we first summarize CD69 function based on its structure and then introduce the evidence for the involvement of CD69 in human diseases and murine disease models. Then, we will describe how we discovered CD69 ligands, Myl9 and Myl12, and how the CD69-Myl9 system regulates airway inflammation. Finally, we will discuss possible therapeutic usages of the blocking Ab to the CD69-Myl9 system.

THE ANTIGEN-SPECIFIC CELL IN VITRO TESTS FOR POST-VACCINATION ANTIPLAGUE IMMUNITY FORMATION

The possibility of post-vaccination anti-plague immunity evaluation was researched using antigen-stimulated cells tests in vitro and cytometry analysis. The object of study — the blood samples of 17 people immunised by the live plague vaccine (Yersinia pestis EV) epicutaneously. Blood taking was carried out before vaccination and after immunisation on 7 and on 21 days, in 3 and in 6 months. Intensity antigen reactivity of lymphocytes was detected by cell tests in vitro, analysing markers of early (CD45+CD3+CD25+) and late (CD45+CD3+HLA-DR+) lymphocyte activation using flow cytometry. The complex of water-soluble Y. pestis antigens and allergen — pestin PP was tested as antigen. The high stimulating potential was defined of the water-soluble antigens Y. pestis complex. It is shown that coefficient of stimulation of relative level T- lymphocytes which express receptors for IL-2 was positive for all observation times after immunisation. The coefficient of stimulation had maximum values at 21 days (56.37%) and at 3 (47.41%) months. In identifying HLADR-positive lymphocytes before vaccination, the negative coefficient of stimulation was indicated on 7 and 21 days and the positive coefficient of stimulation was indicated at 3 and at 6 months. Analysis of intensity expression of early and late lymphocyte activation markers dynamics showed the possibility and prospect of application of cellular in vitro tests for the laboratory evaluation of specific reactivity of cellular immunity in both the early (7 days) and late (6 months) periods after vaccination. The results can be the basis for developing a new algorithm for assessment of immunological effectiveness of vaccination people against plague. It is the algorithm based on the identification of lymphocyte activation markers by antigen stimulation in conditions in vitro.

Low level laser (LLL) attenuate LPS-induced inflammatory responses in mesenchymal stem cells via the suppression of NF-κB signaling pathway in vitro.

BackgroundConsidering promising results in animal models and patients, therapeutic use of MSCs for immune disease is likely to undergo continued evaluation. Low-lever laser (LLL) has been widely applied to retard the inflammatory reaction. LLL treatment can potentially be applied in anti-inflammatory therapy followed by stem cell therapy.Aim of the studyThe purpose of this study was to investigate the effect of LLL (660 nm) on the inflammatory reaction induced by LPS in human adipose derived mesenchymal stem cells (hADSCs) and pertinent mechanism.Materials and methodsAnti-inflammatory activity of LLL was investigated by LPS-induced mesenchymal stem cells. The production and expression of pro-inflammatory cytokines were evaluated by ELISA kits and RT-qPCR. Nuclear translocation of NF-κB was indicated by immunofluorescent staining. Phosphorylation status of NF-κB p65 and IκBα were illustrated by western blot assay. ROS generation was measured with CM-H2DCFDA, and NO secretion was determined by DAF-FM. We studied surface expression of lymphocyte activation markers when Purified peripheral blood mononuclear cell (PBMC) were activated by phytohaemagglutinin (PHA) in the presence of 3 types of treated MSCs.ResultsLLL reduced the secretion of IL-1β, IL-6, IL8, ROS and NO in LPS treated MSCs. Immunofluorescent assay demonstrated the nuclear translocation decrease of NF-κB in LLL treated LPS induced MSCs. Western blot analysis also suggested that LLL suppressed NF-κB activation via regulating the phosphorylation of p65 and IκBα. MSC significantly reduced the expression of activation markers CD25 and CD69 on PHA-stimulated lymphocytes.ConclusionThe results indicate that LLL suppressed the activation of NF-κB signaling pathway in LPS treated MSCs through inhibiting phosphorylation of p65 and IκBα, which results in good anti-inflammatory effect. In addition, LLL attenuated activation-associated markers CD25 and CD69 in co-cultures of PBMC and 3 types of treated MSCs.

CD69: from activation marker to metabolic gatekeeper.

CD69 is a membrane-bound, type II C-lectin receptor. It is a classical early marker of lymphocyte activation due to its rapid appearance on the surface of the plasma membrane after stimulation. CD69 is expressed by several subsets of tissue resident immune cells, including resident memory T (TRM) cells and gamma delta (γδ) T cells, and is therefore considered a marker of tissue retention. Recent evidence has revealed that CD69 regulates some specific functions of selected T-cell subsets, determining the migration-retention ratio as well as the acquisition of effector or regulatory phenotypes. Specifically, CD69 regulates the differentiation of regulatory T (Treg) cells as well as the secretion of IFN-γ, IL-17, and IL-22. The identification of putative CD69 ligands, such as Galectin-1 (Gal-1), suggests that CD69-induced signaling can be regulated not only during cognate contacts between T cells and antigen-presenting cells in lymphoid organs, but also in the periphery, where cytokines and other metabolites control the final outcome of the immune response. Here, we will discuss new aspects of the molecular signaling mediated by CD69 and its involvement in the metabolic reprogramming regulating TH-effector lineages.

The environment as a driver of immune and endocrine responses in dolphins (Tursiops truncatus).

Immune and endocrine responses play a critical role in allowing animals to adjust to environmental perturbations. We measured immune and endocrine related markers in multiple samples from individuals from two managed-care care dolphin groups (n = 82 samples from 17 dolphins and single samples collected from two wild dolphin populations: Indian River Lagoon, (IRL) FL (n = 26); and Charleston, (CHS) SC (n = 19). The immune systems of wild dolphins were more upregulated than those of managed-care-dolphins as shown by higher concentrations of IgG and increases in lysozyme, NK cell function, pathogen antibody titers and leukocyte cytokine transcript levels. Collectively, managed-care care dolphins had significantly lower levels of transcripts encoding pro-inflammatory cytokine TNF, anti-viral MX1 and INFα and regulatory IL-10. IL-2Rα and CD69, markers of lymphocyte activation, were both lower in managed-care care dolphins. IL-4, a cytokine associated with TH2 activity, was lower in managed-care care dolphins compared to the free-ranging dolphins. Differences in immune parameters appear to reflect the environmental conditions under which these four dolphin populations live which vary widely in temperature, nutrition, veterinary care, pathogen/contaminant exposures, etc. Many of the differences found were consistent with reduced pathogenic antigenic stimulation in managed-care care dolphins compared to wild dolphins. Managed-care care dolphins had relatively low TH2 lymphocyte activity and fewer circulating eosinophils compared to wild dolphins. Both of these immunologic parameters are associated with exposure to helminth parasites which is uncommon in managed-care care dolphins. Less consistent trends were observed in a suite of hormones but significant differences were found for cortisol, ACTH, total T4, free T3, and epinephrine. While the underlying mechanisms are likely multiple and complex, the marked differences observed in the immune and endocrine systems of wild and managed-care care dolphins appear to be shaped by their environment.

Abstract 454: Myeloid CD98hc Deficiency Reduces Atherosclerotic Plaque Development via Impaired Proliferation of Macrophages

Macrophage accumulation is a key process affecting all stages of atherosclerosis. Whether these cells accumulate in plaque solely by recruitment of monocytes from circulation or by proliferation within the plaque is an important question that has garnered much interest in recent years. Originally identified as a lymphocyte activation marker, CD98hc (SLC3A2) is a transmembrane protein involved in cell proliferation and survival via integrin signaling and MAP kinase activation. We hypothesized that CD98hc deficiency in myeloid cells would have a protective effect on atherosclerosis development and plaque composition by limiting macrophage proliferation. For the studies described, we utilized mice with myeloid-specific deletion of the CD98hc ( CD98hc fl/fl LysMCre + ) to determine the effects of CD98hc deficiency on macrophage function in the context of atherosclerosis . We performed in vitro assays to investigate the role of CD98hc in the proliferation and survival of primary mouse bone marrow derived macrophages. Although we found no differences in the number of bone marrow cells isolated from control or CD98hc -/- animals, after differentiation with MCS-F for 7 days, the number of macrophages obtained from CD98hc -/- mice was approximately 80% lower (7.2 ± 2.2 x 10 6 vs. 42.4 ± 4.6 x 10 6 per mouse) compared to control mice. Proliferation assays in vitro using EdU revealed approximately 50% (15.4 ± 2.5% vs. 7.5±1.8%) reduced cell proliferation in CD98hc -/- macrophages compared to control cells that could not be rescued with the addition M-CSF. In a 6-week atherosclerosis study using Ldlr -/- CD98hc fl/fl LysMCre + mice, Oil-Red O staining of whole aortae as well as aortic sinus sections showed that atherosclerotic plaque development was reduced compared to Ldlr -/- CD98hc fl/fl LysMCre - control mice. Additionally, immunohistochemical staining of atherosclerotic tissues revealed a reduction in macrophage abundance and proliferation within the plaque of Ldlr -/- CD98hc fl/fl LysMCre + mice compared to control mice. These findings support an important role of CD98hc in macrophage proliferation within the plaque environment, and provide a novel target for reducing atherosclerosis.

Study of CD69 antigen expression and integrity of leukocyte cellular membrane in stored platelet concentrates following irradiation and treatment with Mirasol® PRT System.

Leukocytes in transfused blood components, particularly residual lymphocytes, have been shown to contribute to the occurrence of various adverse reactions. One of the most severe is transfusionassociated graft versus host disease (TA-GvHD) following transfusion of blood components contaminated with immunocompetent T lymphocytes. Irradiation is a routine method for protection against TA-GvHD. According to the literature, some pathogen reduction methods have also been proven effective for the inactivation of T lymphocytes, and so they may be considered as an alternative to irradiation. Comparison of CD69 antigen expression and the integrity of the leukocyte cellular membrane in stored platelet concentrates (PCs) following irradiation with the Gammacell 3000 Elan (Nordion Inc., Ottawa, Canada) and treatment with the Mirasol® Pathogen Reduction Technology (PRT) System (Terumo BCT, Lakewood, USA). The study included seven experiments. For each experiment we used 3 PCs, for Mirasol® PRT System treatment (M), for Gammacell 3000 Elan irradiation (R), and for the control (C). 7-amino-actinomycin D (7-AAD, Becton Dickinson, Franklin Lakes, USA) permeability was used to determine lymphocyte viability while CD69 antigen expression was the marker of lymphocyte activation. Analyses of 7-AAD and CD69 antigen expression were performed in a FACS Canto I flow cytometer (Becton Dickinson, USA). During 6 storage days, viable lymphocyte count decreased to 28% (p = 0.001) in the Mirasol® PRT System treated PCs and to 65% (p = 0.004) in the irradiated PCs. A statistically significant increase in CD69 expression in the irradiated PCs was observed; 1.3-fold on day 3 and 1.5-fold on day 6. In the Mirasol ® PRT System treated PCs, no statistically significant increase was observed. The in vitro results suggest that the Mirasol® PRT System is as effective as irradiation due to donor leukocyte inactivation capacity.

Intermittent and graded exercise effects on NK cell degranulation markers LAMP-1/LAMP-2 and CD8+CD38+ in chronic fatigue syndrome/myalgic encephalomyelitis.

There is substantial evidence of immune system dysfunction in chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) but little is understood of exercise training effects on lymphocyte function in this illness. This study investigated whether graded and intermittent exercise improved CD8+ lymphocyte activation and natural killer cell degranulation markers compared to no exercise. Twenty‐four chronic fatigue syndrome (CFS) patients (50.2 ± 10 year) were randomized to graded exercise (GE), intermittent exercise (IE) or usual care (UC) groups; a control group (CTL) of 18 matched sedentary non‐CFS/ME participants were included for immunological variable comparisons. Main outcome measures were pre‐ and postintervention expression of CD3+ CD8+ CD38+ and CD3− CD16+56+ CD107a+ (LAMP‐1) CD107b+ (LAMP‐2) and aerobic exercise capacity. The postintervention percentage of NK cells expressing LAMP‐1 and ‐2 was significantly higher in IE compared to UC, and higher in GE compared to UC and CTL. LAMP‐1 and LAMP‐2 expression (absolute numbers and percent positive) increased significantly pre‐to‐postintervention for both GE and IE. Preintervention, the absolute number of CD8+ CD38+ cells was significantly lower in CTL compared to UC and IE. There were no significant pre‐ to postintervention changes in CD8+ CD38+ expression for any group. Aerobic exercise capacity was significantly improved by GE and IE. Twelve weeks of GE and IE increased the expression of NK cell activation and degranulation markers, suggesting enhanced immunosurveillance. Low‐intensity exercise may also reduce CD8+ CD38+ expression, a marker of inflammation. Both GE and IE improved exercise capacity without worsening CFS/ME symptoms, and more robust trials of these exercise modalities are warranted.

Effects of Traumeel (Tr14) on recovery and inflammatory immune response after repeated bouts of exercise: a double-blind RCT.

The purpose of this double-blind, randomized, placebo-controlled clinical trial was to investigate the effects of the natural combination medicine Traumeel (Tr14) consisting of 14 diluted biological and mineral components on the inflammatory immune response and recovery up to 72h after repetitive bouts of bicycle tests. Antigen-stimulated IL-1ra and IL-6 were defined as primary outcome measures. Moreover, various immunological and serum muscle damage markers were investigated. The evaluation was performed using the score of the area under the curve with respect to increase (AUCi) for 24 and 72h after the second exercise test (EX2). The Tr14 group indicated a lower decrease of lymphocytes by tendency (p = 0.06) and a lower activation of lymphocyte activation markers (CD62L absolute: p = 0.04; CD69: p = 0.01 and CD69 absolute: p = 0.05) in the period 24h after EX2. In addition, the Tr14 group indicated a higher expression of antigen-stimulated CCL3 (p = 0.01), CCL4 (p = 0.07) and serum CCL2 (p = 0.05) in the period 24h after EX2. There was a tendentially lower decrease of monocytes (p = 0.09) and a lower expression of antigen-stimulated MMP-3 (p = 0.01) in the Tr14 group in the period 72h after EX2. However, antigen-stimulated IL-1ra and IL-6 showed no group differences. In line with the previous results, it was shown that Tr14 attenuates the adaptive immune response partially. Furthermore, the results indicate that Tr14 is able to stimulate the innate immune system via an increased production of pro-inflammatory chemokines. It is speculated that the higher expression of chemokines might play a role in the regeneration and recovery after exercise.

Сравнительная оценка уровня экспресси молекулярных маркеров активации лимфоцитов периферической крови у больных меланомой хориоидеи малых размеров и у здорових лиц

Сравнительная оценка уровня экспресси молекулярных маркеров активации лимфоцитов периферической крови у больных меланомой хориоидеи малых размеров и у здорових лиц