A Comprehensive Investigation of Stimulatory Agents on MAIT and Vα7.2+/CD161- T Cell Response and Effects of Immunomodulatory Drugs.

Mucosal-associated invariant T (MAIT) cells, a subset of Vα7.2+ T cells, are a crucial link between innate and adaptive immunity, responding to various stimuli through TCR-dependent and independent pathways. We investigated the responses of MAIT cells and Vα7.2+/CD161- T cells to different stimuli and evaluated the effects of Cyclosporin A (CsA) and Vitamin D3 (VitD). Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with various agents (PMA/Ionomycin, 5-OP-RU, 5-OP-RU/IL-12/IL-33) with or without CsA and VitD. Flow cytometric analysis assessed surface markers and intracellular cytokine production. Under steady-state conditions, MAIT cells displayed elevated expression of CCR6 and IL-13. They showed upregulated activation and exhaustion markers after activation, producing IFNγ, TNFα, and TNFα/GzB. CsA significantly inhibited MAIT cell activation and cytokine production. Conversely, Vα7.2+/CD161- T cells exhibited distinct responses, showing negligible responses to 5-OP-RU ligand but increased cytokine production upon PMA stimulation. Our study underscores the distinct nature of MAIT cells compared to Vα7.2+/CD161- T cells, which resemble conventional T cells. CsA emerges as a potent immunosuppressive agent, inhibiting proinflammatory cytokine production in MAIT cells. At the same time, VitD supports MAIT cell activation and IL-13 production, shedding light on potential therapeutic avenues for immune modulation.

Case report: Complex evaluation of coagulation, fibrinolysis and inflammatory cytokines in a SARS-CoV-2 infected pregnant woman with fetal loss.

Background: SARS-CoV-2 infection during pregnancy increases the risk of severe obstetrical complications. Detailed evaluation of COVID-19-associated coagulopathy in a pregnancy with stillbirth hasn't been described so far. Besides knowledge gaps in the pathomechanism leading to stillbirth in COVID-19 pregnancies, currently, no prognostic biomarker is available to identify pregnant patients who are at imminent risk of COVID-19-associated maternal and fetal complications, requiring immediate medical attention. Case: Here we report the case of a 28-year-old SARS-CoV-2 infected pregnant patient, admitted to our hospital at 28 weeks of gestation with intrauterine fetal loss. The presence of SARS-CoV-2 placentitis was confirmed by immunohistological evaluation of the placenta. She had only mild upper respiratory symptoms and her vital signs were within reference throughout labor and postpartum. The stillborn infant was delivered per vias naturales. Fibrinogen concentrate was administered before and after labor due to markedly decreased fibrinogen levels (1.49 g/l) at admission and excessive bleeding during and after delivery. Although coagulation screening tests were not alarming at admission, the balance of hemostasis was strikingly distorted in the patient. As compared to healthy age- and gestational age-matched pregnant controls, increased D-dimer, low FVIII activity, low FXIII level, marked hypocoagulability as demonstrated by the thrombin generation assay, together with shortened clot lysis and decreased levels of fibrinolytic proteins were observed. These alterations most likely have contributed to the increased bleeding observed during labor and in the early postpartum period. Interestingly, at the same time, only moderately altered inflammatory cytokine levels were found at admission. Serum ACE2 activity did not differ in the patient from that of age- and gestational age-matched healthy controls, suggesting that despite previous speculations in the literature, ACE2 may not be used as a potential biomarker for the prediction of COVID-19 placentitis and threatening fetal loss in SARS-CoV-2-infected pregnancies. Conclusions: Although based on this case report no prognostic biomarker could be identified for use in pregnant patients with imminent risk of fetal loss associated with COVID-19 placentitis, the above-described hemostasis alterations warrant awareness of postpartum hemorrhagic complications and could be helpful to identify patients requiring intensified medical attention.

Investigating Vα7.2+/CD161- T Cell and MAIT Cell Profiles Using Flow Cytometry in Healthy Subjects and Subjects with Atopic Dermatitis.

This study investigates the roles of mucosal-associated invariant T (MAIT) cells and Vα7.2+/CD161- T cells in skin diseases, focusing on atopic dermatitis. MAIT cells, crucial for bridging innate and adaptive immunity, were analyzed alongside Vα7.2+/CD161- T cells in peripheral blood samples from 14 atopic dermatitis patients and 10 healthy controls. Flow cytometry and machine learning algorithms were employed for a comprehensive analysis. The results indicate a significant decrease in MAIT cells and CD69 subsets in atopic dermatitis, coupled with elevated CD38 and polyfunctional MAIT cells producing TNFα and Granzyme B (TNFα+/GzB+). Vα7.2+/CD161- T cells in atopic dermatitis exhibited a decrease in CD8 and IFNγ-producing subsets but an increase in CD38 activated and IL-22-producing subsets. These results highlight the distinctive features of MAIT cells and Vα7.2+/CD161- T cells and their different roles in the pathogenesis of atopic dermatitis and provide insights into their potential roles in immune-mediated skin diseases.

Enhancing HLA-B27 antigen detection: Leveraging machine learning algorithms for flow cytometric analysis.

As the association of human leukocyte antigen B27 (HLA-B27) with spondylarthropathies is widely known, HLA-B27 antigen expression is frequently identified using flow cytometric or other techniques. Because of the possibility of cross-reaction with off target antigens, such as HLA-B7, each flow cytometric technique applies a "gray zone" reserved for equivocal findings. Our aim was to use machine learning (ML) methods to classify such equivocal data as positive or negative. Equivocal samples (n = 99) were selected from samples submitted to our institution for clinical evaluation by HLA-B27 antigen testing. Samples were analyzed by flow cytometry and polymerase chain reaction. Features of histograms generated by flow cytometry were used to train and validate ML methods for classification as logistic regression (LR), decision tree (DT), random forest (RF) and light gradient boost method (GBM). All evaluated ML algorithms performed well, with high accuracy, sensitivity, specificity, as well as negative and positive predictive values. Although, gradient boost approaches are proposed as high performance methods; nevertheless, their effectiveness may be lower for smaller sample sizes. On our relatively smaller sample set, the random forest algorithm performed best (AUC: 0.92), but there was no statistically significant difference between the ML algorithms used. AUC values for light GBM, DT, and LR were 0.88, 0.89, 0.89, respectively. Implementing these algorithms into the process of HLA-B27 testing can reduce the number of uncertain, false negative or false positive cases, especially in laboratories where no genetic testing is available.

Gender-dependent frequency of unconventional T cells in a healthy adult Caucasian population: A combinational study of invariant NKT cells, γδ T cells, and mucosa-associated invariant T cells.

This study tested the hypothesis of gender bias in frequency of unconventional T cells. Unconventional T cells exist as minor subsets of T cells in peripheral blood. Despite their low number, they play a crucial role in various immune-mediated diseases such as inflammation, autoimmunity, allergy, and cancer. Gender-based frequency of these cells altogether on large number of healthy individuals are unestablished creating hurdles to manifest association with various immune-mediated pathologic conditions. In this study, we used a multicolor flow cytometric panel to identify iNKT cells, γδ T cells, and MAIT cells altogether in the peripheral blood samples of 93 healthy adult males and 109 healthy adult females from the Caucasian population. The results revealed iNKT cell median value (% T cells) in females was higher: 0.114% ranging from 0.011 to 3.84%, than males: 0.076% (p value 0.0292), ranging from 0.007 to 0.816% and found to be negatively correlated with age in females (p value 0.0047). However, γδ T cell median value in males was higher: 2.52% ranging from 0.31 to 16.09%, than females: 1.79% (p value 0.0155), ranging from 0.078 to 12.49% and each gender was negatively correlated with age (male p value 0.0003 and female p value 0.0007). MAIT cell median values were 3.04% ranging from 0.11 to 10.75% in males and 2.67% ranging from 0.2 to 18.36% in females. MAIT cells did not show any statistically significant difference between genders and found to be negatively correlated with age (p value < 0.0001). Our results could be used for further gender-wise investigations of various pathologic conditions such as cancer and their prognosis, autoimmune diseases, allergies, and their pathogenicity.

Comparative Evaluation of Body Fluid Analysis by Sysmex XN Hematology Analyzers, CellaVision, Manual Microscopy and Multicolor Flow Cytometry.

OBJECTIVE: The aim of this study was to investigate the correlation between instrumental and morphological cell differential methods in body fluid (BF) samples. METHODS: Forty ascitic (AF) and forty cerebrospinal (CSF) fluid samples were measured with Sysmex XN1000 and XN2000 instruments in BF mode. Flow cytometry (FC) was carried out with FACS Canto II. From the centrifuged cytospin preparations mononuclear (MN%) and polymorphonuclear (PMN%) cell percentages were determined using optical microscopy (OM) and a digital cell morphology system CellaVision (CV). RESULTS: Both Passing-Bablok and Bland-Altman analysis showed strong correlation between the hematology analyzers for total cell count (TC), white blood cell count (WBC), MN%, and PMN% in BFs. With slightly inferior results, all other WBC differential methods showed acceptable correlation. Passing-Bablok regression analysis yielded a slope encompassing 1.0 in all method comparisons except for three scenarios in CSF. The bias calculated with Bland-Altman plots was comprised between -6.05% and 6.05%. Strong correlations were found when comparing XN1000, XN2000, and CV to OM and FC method with linear regression analysis (r values between 0.905 and 0.984). CONCLUSION: We found strong correlation between instrumental and manual morphological WBC differential methods when testing BF samples containing no tumor cells.

Age-dependent frequency of unconventional T cells in a healthy adult Caucasian population: a combinational study of invariant natural killer T cells, γδ T cells, and mucosa-associated invariant T cells.

Unconventional T cells show distinct and unique features during antigen recognition as well as other immune responses. Their decrease in frequency is associated with various autoimmune disorders, allergy, inflammation, and cancer. The landscape frequency of the unconventional T cells altogether (iNKT, γδ T, and MAIT) is largely unestablished leading to various challenges affecting diagnosis and research in this field. In this study, we have established the age group-wise frequency of iNKT, γδ T, and MAIT cells altogether on a total of 203 healthy adult samples of the Caucasian population. The results revealed that iNKT cells were 0.095%, γδ T cells were 2.175%, and MAIT cells were 2.99% of the total T cell population. γδ and MAIT cell frequency is higher in younger age groups than elderly; however, there is no statistically significant difference in the frequency of iNKT cells. Furthermore, γδ and MAIT cells were negatively correlating with age, supporting immunosenescence, unlike iNKT cells. Our finding could be used for further age-wise investigation of various pathological conditions such as cancer and their prognosis, autoimmune diseases and their pathogenicity.

Combined use of different antibody clones improves the efficiency of human leukocyte antigen B27 detection by flow cytometry.

BACKGROUND: Human leukocyte antigen B27 (HLA-B27) is an major histocompatibility complex Class I cell surface antigen that shows strong association with spondylarthropathies. Although polymerase chain reaction (PCR) is the gold standard method for HLA-B27 detection, monoclonal antibodies, and flow cytometric analysis is also frequently used. We aimed to compare the efficiency of two commercially available monoclonal antibody clones and the DuraClone kit that uses simultaneously these clones. METHODS: Blood samples drawn from 63 patients were analyzed by flow cytometry and PCR. For flow cytometry analysis ABCm3 and FD705 clones were used for flow cytometry as well as the DuraClone Reagent Kit. Results of flow cytometric analysis were confirmed by PCR. RESULTS: Numbers of false-positive and equivocal samples were high when ABCm3 or FD705 clones were used separately: 34 out of 63 and 27 out of 63, respectively. Simultaneous use of the two antibody clones and pre-selection of CD3+ T cells, in the DuraClone kit, significantly decreased the number of these samples. Using the DuraClone kit, only 11 out of 63 samples were inconclusive. Influence of HLA-B7 expression was detectable in some cases when ABCm3 and FD705 were used separately. CONCLUSIONS: Our results show that simultaneous use of HLA-B27 monoclonal antibodies and pre-selection of T cells significantly increased the specificity of the flow cytometric assay, however it did not reach the specificity of PCR. Nevertheless, based on our results, performing the PCR test exclusively in equivocal cases by DuraClone kit reduces the burden of PCR assays and the turn-around time.

T-cell Subset Profile in Kidney Recipients of Extended or Standard Donors.

INTRODUCTION: The usage of extended-criteria donors (ECD) became a routinely accepted manner in the last decade. ECD is a potential risk factor for antibody-mediated rejection. Analysis of lymphocyte subsets might be a complementary diagnostic toolkit because there is limited knowledge about this term. METHOD: Between May 12, 2016, and September 4, 2019, a total of 130 patients who had undergone kidney transplant were investigated. Patients were divided in ECD and standard criteria donor (SCD) groups. Blood samples were collected before the operation, then in the first week and after 30, 60, 180, and 365 days. Besides routine laboratory tests, multicolor flow cytometry was performed for lymphocyte subsets. RESULTS: ECD grafts were transplanted to older recipients. The number of CD4+ cells increased in the SCDs from the first week to until the end of first month, and then decreased. The number of CD4+ cells decreased from the beginning of the study until the end of first year to 66% of its original value in ECDs. At the first month, the number of CD19+ cells was higher in SCD compared with ECD cases; the number then decreased in both groups. T-regulatory cells had a drop at the first week that lasted until the first month. A bigger increase in SCD and a moderate increase in ECD group were then observed. The kinetics of CD19+ and CD19+ naive cells are similar in the ECD and SCD groups. In the SCD group, cell count decreased in both CD19+ (13%) and CD19+ naive (12%) between third and sixth month. The count of CD19+ cells decreased by 9%, but the count of CD19+ naive cells increased by 11% between the sixth month and first year. DISCUSSION: The prolonged postoperative uremic state caused by the poorer initial function, together with an aging immune system, explains the weaker immune response in ECD patients, which may be the cause of the decreased number of memory and regulatory T cells. Older patients with an ECD graft need a tailored, personalized, and less aggressive immunosuppressive treatment.

Evaluation of Sample Quality As Preanalytical Error in Flow Cytometry Analysis in Childhood Acute Lymphoblastic Leukemia.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is the most common cancer among children. The intensity of chemotherapy and further therapeutic decisions depend on several prognostic factors, including response to initial treatment by examining peripheral blood (PB), bone marrow (BM) and cerebrospinal fluid (CSF) samples at certain time points. (e.g. day 15 BM). Sample quality is crucial for the correct risk assessment. PATIENTS AND METHODS: We aimed to explore the rate of inadequate samples as a source of preanalytical error. We retrospectively analyzed flow cytometry results of BM (day 15 and day 33) and CSF samples from children with ALL in different cohorts focusing on PB contamination and viable cell ratio among nucleated cells. We also compared viable cell percentages in native and stabilized CSF samples. RESULTS: Due to PB contamination (erythroid precursors < 2%) 12.5% of day 15 and 14% of day 33 BM samples were inadequate for flow cytometry risk stratification. Significantly fewer CSF samples had to be considered inadequate for analysis (defined as viable cells < 30%) in the subgroup of stabilized samples compared to native samples. Four of the CSF samples from children with ALL had identifiable malignant cell population despite the low viable cell percentage. DISCUSSION: Poor sample quality can hamper risk stratification and further therapeutic decision in childhood ALL. Despite low viable cell count malignant cell populations may still be identified in a CSF sample, therefore establishing a certain cutoff point for viable cells is difficult.

The role of microRNAs in congenital heart disease.

Congenital heart diseases (CHDs) are the leading inherited cause of perinatal and infant mortality. CHD refers to structural anomalies of the heart and blood vessels that arise during cardiac development and represents a broad spectrum of malformations, including septal and valve defects, lesions affecting the outflow tract and ventricules. Advanced treatment strategies have greatly improved life expectancy and led to expanded population of adult patients with CHD. Thus, a better understanding of the pathogenesis and molecular mechanisms underlying CHDs is essential to improve the diagnosis and prognosis of patients. The etiology of CHD is largely unknown, genetic and environmental factors may contribute to the disease. In addition to the mutations affecting genomic DNA, epigenetic changes are being increasingly acknowledged as key factors in the development and progression of CHDs. The posttranscriptional regulation of gene expression by microRNAs (miRs) controls the highly complex multi-cell lineage process of cardiac tissue formation. In recent years, multiplex experimental models have provided evidence that changes in expression levels of miRs are associated with human cardiovascular disease, including CHD. The newly described correlations between miRs and heart development suggest the potential importance of miRs as diagnostic markers in human cardiovascular diseases. In the future, more intensive research is likely to be carried out to clarify their contribution to personalized management and treatment of CHD patients. In this paper, we discuss the current knowledge on the causative role of miRs in cardiac development and CHDs.

T Cell Subset Profile and Appearance of Donor-specific Antibodies in Primary and Retransplanted Kidney Recipients.

INTRODUCTION: Accelerated antibody-mediated rejection is a major challenge after kidney transplantation. While the clinical course, diagnosis, and treatment of cell-mediated acute rejection is agreed upon and has been successfully performed, the antibody-mediated rejection remains a problem. Biopsies cannot be repeated several times, are not always representative, and are refused by many patients. Analysis of T-cell subsets and donor-specific antibodies (DSAs) might be an additive diagnostic tool in the case of kidney transplantation. METHOD: Between 2015 and 2017, 50 kidney transplant patients were enrolled in the study. Patients were divided into 2 clinical groups: primary transplants and regrafted patients. Serum samples were collected right before the operation, then in 1 week; 30, 60, and 180 days; and yearly. Besides routine laboratory, multicolor flow cytometry was performed for T cell subsets, and Luminex Single Antigen Bead assay for the detection on donor-specific anti-HLA antibodies. Medical data were also fixed. RESULTS: The percentage of CD4+ and CD8+ cells (the CD4+/CD8+ rate) did not change much over time in either group. The percentage of CD19+ cells increased until week 1, then decreased back to its original level by day 180. CD56+/3-% was high in both groups and had no characteristic kinetics by the time. The CD4+ naïve absolute cell count increased in first-time transplants and did not decreased back to its original value until the end of year 1. This is in contrast to retransplants, where CD4+ naïve cell count rapidly dropped below its original value and remained low throughout the first year after transplantation. The CD8+ effector memory absolute cell-count was higher in first-time transplants compared to retransplanted patients in all time points. By the end of month 1, the CD19+ naïve absolute cell-count increased in first-time transplants to 170% of its original value; however, it remained or decreased in second transplants. By the end of the first year, the CD19+ naïve absolute cell count diminished to 70% in first-time transplants and 38% in second transplants. DSA was detected in 9 out of the 38 first-time transplants (23.7%) compared to 7 out of 12 (58.3%) in regrafted patients during the observational period (P = .001). It was typical for regrafted patients for DSAs to appear earlier after transplantation, and that more simultaneously different antibodies were detected against more antigens at the first time point compared to first-time transplants. DISCUSSION: The 2 groups were similar in demographics and there were no differences regarding the clinical course, complications, or output data. However, we found statistical differences regarding the dynamics of T cell subsets and DSAs. The parallel measurement of CD subsets and DSAs might be a sensitive and useful additive tool in diagnosing subclinical immunologic changes after transplantation.

Peripheral Lymphocyte Multidrug Resistance Activity as a Predictive Tool of Biological Therapeutic Response in Rheumatoid Arthritis.

OBJECTIVE: Multidrug resistance (MDR) transporters may be used as biomarkers to monitor disease progression in RA and as a predictive tool to establish responsiveness to biological therapy. In this multicenter clinical trial, we aimed to assess the predictive value of activity measurement of transporters MDR1, MD resistance protein (MRP)1, and breast cancer resistance protein (BCRP) for biological therapeutic response in RA before the initiation of biological therapy as well as 4 to 6 and 12 weeks after. METHODS: Peripheral blood samples were collected from 27 responders and 12 nonresponders to biological disease-modifying antirheumatic drugs (bDMARD) at the indicated timepoints as well as from 35 healthy controls. MDR activity factor (MAF) of MDR1, MRP1, and BCRP was measured in CD3+ and CD19+ cells using the Solvo MDQ Kit and cell surface staining by flow cytometry following peripheral blood mononuclear cells isolation. RESULTS: At the start of therapy, MAFC (composite MAF of MRP1 and MDR1) and MAFMDR values, and at 4 to 6 weeks of treatment, MAFC, MAFMRP, and MAFMDR values of CD3 cells were higher in nonresponders compared to responders. Receiver-operation characteristic curve analysis revealed that RA patients with MAFC values above 21.3 in CD3 cells at the start of bDMARD therapy are likely to be nonresponders. At 4 to 6 weeks of treatment, these also predict unfavorable response: MAFC values above 20.3, MAFMRP values above 6.0, and MAFMDR values above 13.9 in CD3 cells. CONCLUSION: Our results indicate that the determination of MAFC values in CD3 cells of patients with RA may be of predictive value prior to the initiation of biological therapy, to establish whether the patient will demonstrate sufficient therapeutic response.

Determination of Reference Values of MDR-ABC Transporter Activities in CD3+ Lymphocytes of Healthy Volunteers Using a Flow Cytometry Based Method.

BACKGROUND: MDR transporters are important biomarkers of drug resistance in cancer and in autoimmune conditions. We determined the MDR1, MRP1 and BCRP activity in CD3+ lymphocytes using a flow cytometry based method from 120 healthy volunteers in order to describe normal reference values of the activity of these transporters. The effects of gender and age were also determined. METHODS: The Solvo MDQ Kit™ was used for measurements. In this assay, fluorescent reporter substrates (Calcein-AM for MDR1 and MRP1 and mitoxantrone for BCRP, respectively) are trapped in the cytoplasm and pumped out by MDR proteins depending on the presence or absence of specific inhibitors (verapamil for MDR1 and MRP1, indomethacin for MRP1 and KO134 for BCRP, respectively), allowing for quantitative, standardized assessment. Cell surface staining was applied to select CD3+ cells. RESULTS: MAF values of MRP1 and BCRP are independent from age. MAFC and MAF of MDR1 show negative correlation with the age of the studied subjects (P = 0.003, r = -0.27 and P = 0.0001, r = -0.34, respectively). No difference was detected in any of the four MAF values between men and women. Gender does not affect the presence or lack of correlation between MAF values and age. CONCLUSIONS: The determination of the functional activity of MDR-ABC transporters is achievable using a flow cytometry based standardized method. Having established the normal range of MAF values on CD3+ lymphocytes of a healthy population, our results allow for the development of novel flow cytometry based diagnostic tools. © 2018 International Clinical Cytometry Society.

Myeloid but not plasmacytoid blood DCs possess Th1 polarizing and Th1/Th17 recruiting capacity in psoriasis.

Psoriasis is a common inflammatory skin disease and dendritic cells (DCs) play crucial role in the development of skin inflammation. Although the characteristics of skin DCs in psoriasis are well defined, less is known about their peripheral blood precursors. Our aim was to characterize the phenotypic features as well as the cytokine and chemokine production of CD1c+ myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in the blood samples of psoriatic patients. Blood DCs were isolated by using a magnetic separation kit, and their intracytoplasmic cytokine production and CD83/CD86 maturation/activation marker expression were investigated by 8-colour flow cytometry. In CD1c+ mDCs the intracellular productions of Th1, Th2, Th17, Th22 and Treg polarizing cytokines were examined simultaneously, whereas in pDCs the amounts of IFNα as well as IL-12, IL-23 and IL-6 were investigated. The chemokine production of both DC populations was investigated by flow-cytometry and ELISA. According to our results psoriatic CD1c+ mDCs were in a premature state since their CD83/CD86 maturation/activation marker expression, IL-12 cytokine, CXCL9 and CCL20 chemokine production was significantly higher compared to control cells. On the other hand, blood pDCs neither produced any of the investigated cytokines and chemokines nor expressed CD83/CD86 maturation/activation markers. Our results indicate that in psoriasis not only skin but also blood mDCs perform Th1 polarizing and Th1/Th17 recruiting capacity, while pDCs function only in the skin milieu.

The assessment of immune-regulatory effects of extracorporeal photopheresis in systemic sclerosis: a long-term follow-up study.

The therapeutic options in systemic sclerosis (SSc) are limited mainly to the management of complications, and decelerating fibrosis and preventing disease progression are still great challenges. Extracorporeal photopheresis (ECP) is one of the promising therapeutic strategies in SSc; nevertheless, there is no consensus on the ideal timing and frequency of treatment cycles. In the present study, we evaluated the long-term effects of consecutive ECP treatments, and the stability of clinical and laboratory improvements. We enrolled nine patients with diffuse cutaneous SSc and performed 12 ECP cycles (24 ECP treatments) per patient in total. ECP cycles were carried out once in every 6 weeks, and each cycle consisted of two procedures. Sixteen healthy individuals served as controls for laboratory assessment. Following the sixth ECP cycle, we observed further improvement in skin score, which was confirmed by high-resolution ultrasonography as well. After the second ECP cycle, values of Tr1 and CD4+CD25(bright) Treg cells increased; however, Tr1 cells remained under control values until the 10th cycle. Suppressor activity of CD4+CD25+ Treg cells improved, while percentages of Th17 cells decreased. At the end of 12-month follow-up, we did not observe significant deterioration in skin involvement; however, improvement in laboratory parameters diminished after 12 months. If the first six ECP cycles are effective, uninterrupted continuation of treatment should be considered, which may lead to the normalization of Tr1 cell values along with further clinical improvement. Our laboratory observations indicate that immunomodulatory effect of ECP treatments lasts for 1 year only.

Regulatory T-cell subsets with acquired functional impairment: important indicators of disease severity in atopic dermatitis.

Our aim was to assess whether the presence of highly active effector T cells in atopic dermatitis (AD) is associated with changes in the number and/or function of regulatory T cells (Tregs). Flow cytometry was utilised to determine the percentage of CD4+ CD25bright CD127-/low FOXP3+ and skin-homing CLA+ CD4+ CD25bright FOXP3+ Tregs in healthy controls and AD patients. The correlation between disease severity and Treg percentages was estimated. Treg suppressor activity and cell proliferation were measured after T-cell stimulation. Significantly increased percentages of Tregs were found in AD patients compared to healthy individuals, and significant correlation between the frequency of Tregs and disease severity was also detected. The otherwise normal suppressor activity of Tregs decreased in the presence of Staphylococcus enterotoxin B (SEB). In conclusion, the continuous presence of SEB can trigger an acquired functional impairment of Tregs in AD patients and the correlation between the increased frequency of Tregs and disease severity supports their important role in AD pathogenesis.

Evaluation of significance of lymphocyte subpopulations and non-specific serologic markers in B-cell non-Hodgkin's lymphoma patients.

The use of rituximab brought attention to the hosts' immune system and to the microenvironment in non-Hodgkin's lymphoma cases. Our aim was to identify prognostic factors that can be measured easily to indicate the current state of the patient's immune status and possible reaction against malignant cells. In the retrospective analysis (2000-2008), 66 patients diagnosed with B-cell non-Hodgkin's lymphomas were enrolled (40 women, 26 men; mean age: 51 years). White blood cells, lymphocytes, CD3 +; CD4 +; CD8 + T-cells, immunoglobulin types A; G; M, anti-cardiolipin antibody isotypes A; G; M; and levels of beta-2-microglobulin were measured before the initiation of the first cycle of chemotherapy, during and after 4-weeks treatment. As for CD 3+ T-lymphocytes, the absolute CD 3+ T -lymphocyte numbers were higher before (0.78 × 10(9)/L) versus during (0.27 × 10(9)/L) treatment, and increased percentages were detected in pre- (66.57 %) and post-treatment (75.32 %). Absolute numbers of CD 8+ T-lymphocyte levels showed reduction before (0.26 × 10(9)/L) versus during (0.10 × 10(9)/L) therapy, but were elevated after (0.28 × 10(9)/L) treatment, while increased percentage before (21.99 %) versus after (29.85 %), and during (24.56 %) versus after (29.85 %) therapy were seen. Average white blood cell numbers were increased before (9.71 × 10(9)/L) versus during (12.07 × 10(9)/L) treatment, while decreased numbers could be observed, after (5.47 × 10(9)/L) treatment. IgA levels were decreased before (2.51 g/L) versus after (1.63 g/L) therapy. IgG levels were higher before (12.25 g/L) vs. after (8.64 g/L) treatment. IgM levels were decreased before (1.76 g/L) and after (0.83 g/L) as well as before (1.76 g/L) versus during (0.73 g/L) treatment. Anti-cardiolipin antibody type A level were decreased before (2.76 U/ml) versus after (2.49 U/ml) treatment. Decreased level of beta-2-microglobulin could be observed before (2.91 mg/L) versus post (2.28 mg/L) chemotherapy. Findings may provide better insight into the effects of immuno-chemotherapy on the hosts' immune system.

The in vitro treatment with vitamin D3 is ineffective on the expression of PKC isoenzymes, but decreases further the impaired production of IL-2 in the T lymphocytes of SLE patients.

The objective of the study was to investigate the possibility whether the in vitro treatment with vitamin D3 can restore the impaired expression of protein kinase C (PKC) isoenzymes and IL-2 production in the lymphocytes of patients with systemic lupus erythematosus (SLE). Purified T lymphocytes from 14 patients with SLE and 13 healthy controls were cultured for 48 h in the presence and absence of 1 and 100 nM doses of vitamin D3. The expressions of various PKC isoenzymes were tested by Western blot analysis, and the amounts of various cytokines were detected by ELISA in the culture supernatants. Neither the low (1 nM) nor the high (100 nM) doses of vitamin D3 (1α,-25-dihydroxyvitamin) applied in vitro for 48 h were able to restore the decreased expression of PKC isoenzymes in the T cells of SLE patients. However, 100 nM of vitamin D3 significantly increased the release of IL-10, but suppressed the production of IL-2, IL-6, interferon γ and TNF α in the culture supernatants of both groups. As the low production of IL-2 is one of the main pathologic features of SLE, we recommend to avoid the use of high doses of vitamin D3 for treatment of lupus patients with vitamin D3 deficiency.

Changes in the number of CD8⁺ T lymphocytes in the peripheral blood of patients with various autoimmune diseases after autologous hematopoietic stem cell transplantations and their relations to the survival times.

The changes in the number of CD8⁺ T lymphocytes were studied before (0 day) and then 30 days after the autologous hematopoietic stem cell transplantations (AHSCT) in 14 therapy refractory patients with autoimmune diseases. The years of survival and the clinical states were also evaluated. The number of CD8⁺ T cells was determined by an hematologic automat and by flow cytometry. Longer than 5-year survival times were found in 6 cases, whereas there was no progression (improvement) in 2 cases, and 4 patients were lost. The increase in the number of CD8⁺ cytotoxic T cells was gradual in the first 2 months and reached the significantly highest values among all subtypes of lymphocytes. It was of a special interest that in all the 4 patients who died, the numbers of CD8⁺ T cells were less than 150/µl on the 30th day after AHSCT, whereas all the 10 patients with a higher cell number survived. These results suggest that the early monitoring of the number (not only the ratio) of regenerating CD8⁺ T cells in the peripheral blood can be a useful and quantitative laboratory measurement after AHSCT, and it has a significant relation also to the survival times of transplanted patients.