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.

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.

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.

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.

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.

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.

Follicular helper T cells may play an important role in the severity of primary Sjögren's syndrome.

The aim of this study was to investigate the possible role of follicular helper T (TFH) cells in the pathogenesis of primary Sjögren's syndrome (pSS) by analyzing immune-competent cells and serological markers with special emphasis on clinical symptoms. We enrolled 50 pSS patients and 16 healthy individuals in the study. Patients had elevated ratio of peripheral TFH cells, however, when dividing patients into two groups defined by the presence of extraglandular manifestations (EGMs), only patients with EGMs differed from controls significantly. Moreover, TFH cell percentages correlated positively with both activated T cell and Tr1 cell values. On the contrary, TFH cell percentages showed negative correlation with both IgM and IgG memory B cell proportions. Elevated TFH percent\ages were observed in the anti-SSA/SSB positive patients, and also in patients with higher IL-12, IL-21 levels and focus score values. Increased TFH cell proportions seem to have an important role in disease development.

Immunomodulatory effects of extracorporeal photochemotherapy in systemic sclerosis.

The aim of this study was to evaluate the clinical and immunomodulatory effects of extracorporeal photochemotherapy (ECP) in systemic sclerosis (SSc). We enrolled 16 patients with diffuse cutaneous SSc, who received 12 ECP treatments in total. After ECP treatments, the dermal thickness reduced and the mobility of joints improved. Internal organ involvement did not deteriorate. The percentages and numbers of peripheral Th17 cells decreased, the values of Tr1 and Treg cells increased, and the suppressor capacity of Treg cells improved. Interestingly, we found a positive correlation between the reduction of IL-17 levels and skin thickness measured by ultrasound. Moreover, levels of CCL2 and TGF-beta decreased, while the concentration of IL-1Ra, IL-10 and HGF elevated during the therapy. ECP treatments contribute to the restoration of disproportional autoimmune responses and attenuate fibrotic processes, thus decelerate the disease progression. Accordingly, ECP can be a useful element of novel treatment modalities proposed for SSc.

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.

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.

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.

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.

The immunoregulatory role of vitamins A, D and E in patients with primary Sjogren's syndrome.

OBJECTIVE: The aim of the present study was to investigate the immunomodulating role of fat-soluble vitamins in 25 patients with primary SS (pSS) and 15 healthy individuals. METHODS: Plasma levels of vitamins A, D and E were determined by HPLC. Peripheral NK, NK T cells, T-cell subsets, B cells, IL-10 producing Tr1 cells, CD4(+)CD25(+) Treg cells and Th17 were determined by flow cytometry. Various Th1- and Th2-soluble cytokines were assessed by ELISA, whereas intracytoplasmic cytokines (IFN-gamma, IL-4, -10 and -17) were measured by flow cytometry. Correlation was assessed between vitamin levels and immunological and clinical parameters. RESULTS: Vitamin A levels did not differ between patients and controls, yet in patients with extraglandular manifestations (EGMs) a significant decrease in vitamin A levels was apparent compared with pSS patients without EGMs (P = 0.005). Vitamin E levels were increased in patients compared with controls (P = 0.004), whereas vitamin D levels were similar in pSS and control subjects. In patients, vitamin A showed a positive correlation with both NK cell (P = 0.038) and Th17 cell (P = 0.025), and a negative correlation with Schirmer's test values (P = 0.035). Positive correlation was found between vitamin E and NK cells (P = 0.043), Th1 cells (P = 0.049) and the Th1/Th2 ratio (P = 0.043). In the control group, we found correlation between vitamin E and serum IL-10 levels (P = 0.003). CONCLUSIONS: Our data suggest that fat-soluble vitamins may be important in immunoregulatory processes in patients with pSS.

Progressive divergent shifts in natural and induced T-regulatory cells signify the transition from undifferentiated to definitive connective tissue disease.

The objectives of the study is to determine clinical signs and distribution of peripheral T-cell subsets, B cells and T regulatory cells in patients with undifferentiated connective tissue disease (UCTD) and during the development toward well-established connective tissue diseases (CTD). The methods include 46 patients with UCTD were followed and investigated for differentiation into defined CTDs for 2 years. Cell subsets were determined on the basis of cell surface markers, intracellular cytokine production by flow cytometry and serum cytokine levels by ELISA. The results are as follows: 45.6% of UCTD patients developed into a defined CTD. The number and percentage of activated T cells, memory T cells and NKT cells were increased in patients compared with controls. In addition, in patients with UCTD, the percentage of CD4+/IFN gamma+ T(h)1 was significantly higher compared with controls and further increased in patients that developed CTDs. The percentage and absolute number of CD4+CD25+Foxp3+ regulatory T cells (Tregs) were diminished in UCTD patients compared with healthy controls, while the number of CD4+/IL-10+ Tregs increased. The conclusions are Overproduction of IFN gamma and the decrease of natural (Foxp3+) Tregs seem to be characteristic features of UCTD patients. The increased IL-10 production of CD4+ T cells might be a compensatory suppressive mechanism; however, it is probably not able to balance the overproduction of IFN gamma and the observed decrease of Foxp3+ Tregs. The shift toward T(h)1 with increased IFN gamma production in patients with UCTD combined with the degree of immunoregulatory disturbances characterized by the progressive divergent shifts in natural and induced T-regulatory cell populations signify the transition from undifferentiated to definitive CTD.

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.

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.

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.