Cell Subpopulations In Patients Research Articles

Effects of Reishimmune-S, a Fungal Immunomodulatory Peptide Supplement, on the Quality of Life and Circulating Natural Killer Cell Profiles of Patients With Early Breast Cancer Receiving Adjuvant Endocrine Therapy.

To evaluate the effects of Reishimmune-S, a fungal immunomodulatory peptide, on the quality of life (QoL) and natural killer (NK) cell subpopulations in patients receiving adjuvant endocrine therapy (ET) for breast cancer (BC). Patients who received adjuvant ET for stage I-III hormone receptor-positive BC without active infection were enrolled in this prospective pilot study. Reishimmune-S was administered sublingually daily for 6 months. QoL scores, circulating immune cell levels, including lymphocyte/NK cell subpopulations, and plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured at baseline and every 4 weeks. Data were analyzed using linear mixed-effect regression models. Nineteen participants were included in the analyses. One patient with underlying asthma did not complete the study owing to the occurrence of skin rashes 15 days after the initiation of Reishimmune-S. No other adverse events were reported. Reishimmune-S supplementation significantly improved the cognitive function at 3 months and significantly decreased the fatigue and insomnia levels at 3 and 6 months, respectively. There was no significant change in the global health/QoL score between baseline and week 4 of treatment. The proportion of CD19+ lymphocytes was significantly higher at 3 and 6 months, and that of NKG2A+ and NKp30+ NK cells was significantly lower at 6 months than at baseline. In addition, fatigue positively correlated with the proportion of NKp30+ NK cells (β ± standard error: 24.48 ± 8.75, P = .007 in the mixed-effect model). Short-term supplementation with Reishimmune-S affected the circulating immune cell composition and exerted positive effects on cognitive function, fatigue, and insomnia in patients with BC undergoing adjuvant ET, providing a potential approach for the management of treatment-related adverse reactions in this patient population.

Expansion of distinct peripheral blood myeloid cell subpopulations in patients with rheumatoid arthritis-associated interstitial lung disease

ObjectivesInterstitial lung disease (ILD) is associated with significant mortality in rheumatoid arthritis (RA) patients with key cellular players remaining largely unknown. This study aimed to characterize inflammatory and myeloid derived suppressor cell (MDSC) subpopulations in RA-ILD as compared to RA, idiopathic pulmonary fibrosis (IPF) without autoimmunity, and controls. MethodsPeripheral blood was collected from patients with RA, RA-ILD, IPF, and controls (N = 60, 15/cohort). Myeloid cell subpopulations were identified phenotypically by flow cytometry using the following markers:CD45,CD3,CD19,CD56,CD11b,HLA-DR,CD14,CD16, CD15,CD125,CD33. Functionality of subsets were identified with intracellular arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS) expression. ResultsThere was increased intermediate (CD14++CD16+) and nonclassical (CD14+/-CD16++) and decreased classical (CD14++CD16-) monocytes in RA, RA-ILD, and IPF vs. control. Intermediate monocytes were higher and classical monocytes were lower in RA-ILD vs. RA but not IPF. Monocytic (m)MDSCs were higher in RA-ILD vs. control and RA but not IPF. Granulocytic (g)MDSCs did not significantly differ. In contrast, neutrophils were increased in IPF and RA-ILD patients with elevated expression of Arg-1 sharing similar dimensional clustering pattern. Eosinophils were increased in RA-ILD vs. controls, RA and IPF. Across cohorts, iNOS was decreased in intermediate/nonclassical monocytes but increased in mMDSCs vs. classical monocytes. In RA-ILD, iNOS positive mMDSCs were increased versus classic monocytes. ConclusionsMyeloid cell subpopulations are significantly modulated in RA-ILD patients with expansion of CD16+ monocytes, mMDSCs, and neutrophils, a phenotypic profile more aligned with IPF than other RA patients. Eosinophil expansion was unique to RA-ILD, potentially facilitating disease pathogenesis and providing a future therapeutic target.

Exploring the evolution of T cell function and diversity across different stages of non-small cell lung cancer.

The immune system plays a key role in detecting and fighting cancerous tumors. T cells are a crucial component in both natural and therapeutic cancer immunoediting responses, but it is unclear if they are the primary agents of these processes. In this study, patients with lung lesions detected by CT scan were selected, and their peripheral blood samples were analyzed for T cell population and serum cytokines/chemokines. T cell subtypes (CD3, CD4, CD8, CD27, CD28, CD45, CD45RA, CD57, CCR7, and PD1) and serum cytokines/chemokines (IL-2, IL-6, IL-10, IFN-γ, TGF-β, TNFα, CXCL1, CXCL9, and CXCL12) were measured by flow cytometry and analysis before surgical resection or other cancer treatments. The frequency of T cell subpopulations in patients with lung cancer (n = 111) corresponded to those seen in patients with T cell exhaustion. As lung cancer progressed, the proportion of effector memory T cells decreased, while the proportion of naive T cells, PD-1, CD57+, CD28+CD27+, CD45RA+, and CD3+CD4+CCR7 increased. Circulating CD8+PD1+ T cells were positively correlated with intra-tumoral PD-L1 expression. Concurrently, serum levels of IL-2, TGF-β, and CXCL9 decreased, while IL-6, IL-10, IFN-γ, and CXCL12 increased during the progression of lung cancer. In conclusion, T cell dysfunction is associated with cancer progression, particularly in advanced-stage lung cancer, and cancer immunoediting will provide early-stage cancer detection and further therapeutic strategies.

Distribution characteristics of circulating B cell subpopulations in patients with chronic kidney disease

This study compared the levels of circulating B cell subpopulations in patients with different stages of chronic kidney disease (CKD), investigated the effects of haemodialysis (HD) on the B cell-related immune spectrum in patients with end-stage renal disease, and evaluated the link between renal function and immune homeostasis. Overall, 197 patients with CKD (158 non-dialysis patients with CKD stages I–V and 39 end-stage patients undergoing maintenance HD) and 77 healthy controls were included. Compared to healthy controls, patients with CKD stages I–II showed no significant differences except for the proportion of transitional B cells; patients with CKD stage V showed a significant decrease in the proportions of transitional B cells and CD5+ B cells and a significant increase in double-negative (DN) B cells. Compared with early-stage patients with CKD, the absolute count of various B cell subpopulations in advanced-stage patients with CKD showed a significant decrease. The distribution of circulating B cell subpopulations in patients with CKD was significantly altered and was associated with CKD progression. Furthermore, the proportion of DN B cells and CD5+ B cells was inconsistent pre- and post-HD. This in-depth study of the immune status of patients with CKD may have important clinical value.

The Characterization of Cardiac Explants Reveals Unique Fibrosis Patterns and a Predominance of CD8+ T Cell Subpopulations in Patients with Chronic Chagas Cardiomyopathy.

The present study aimed to characterize the histopathological findings and the phenotype of inflammatory cells in the myocardial tissue of patients with end-stage heart failure (ESHF) secondary to CCC in comparison with ESHF secondary to non-Chagas cardiomyopathies (NCC). A total of 32 explanted hearts were collected from transplanted patients between 2014 and 2017. Of these, 21 were classified as CCC and 11 as other NCC. A macroscopic analysis followed by a microscopic analysis were performed. Finally, the phenotypes of the inflammatory infiltrates were characterized using flow cytometry. Microscopic analysis revealed more extensive fibrotic involvement in patients with CCC, with more frequent foci of fibrosis, collagen deposits, and degeneration of myocardial fibers, in addition to identifying foci of inflammatory infiltrate of greater magnitude. Finally, cell phenotyping identified more memory T cells, mainly CD8+CD45RO+ T cells, and fewer transitioning T cells (CD45RA+/CD45RO+) in patients with CCC compared with the NCC group. CCC represents a unique form of myocardial involvement characterized by abundant inflammatory infiltrates, severe interstitial fibrosis, extensive collagen deposits, and marked cardiomyocyte degeneration. The structural myocardial changes observed in late-stage Chagas cardiomyopathy appear to be closely related to the presence of cardiac fibrosis and the colocalization of collagen fibers and inflammatory cells, a finding that serves as a basis for the generation of new hypotheses aimed at better understanding the role of inflammation and fibrogenesis in the progression of CCC. Finally, the predominance of memory T cells in CCC compared with NCC hearts highlights the critical role of the parasite-specific lymphocytic response in the course of the infection.

Imbalance of circulating innate lymphoid cell subpopulations in patients with chronic kidney disease

Innate lymphoid cells (ILCs) are a newly identified heterogeneous family of innate immune cells. We conducted this study to investigate the frequency of circulating ILC subsets in various chronic kidney diseases (CKD). In DN, the proportion of total ILCs and certain ILC subgroups increased significantly. Positive correlations between proportion of total ILCs, ILC1s and body mass index, glycated hemoglobin were observed in DN. In LN, a significantly increased proportion of ILC1s was found in parallel with a reduced proportion of ILC2s. The proportions of total ILCs and ILC1s were correlated with WBC count and the level of C3. In all enrolled patients, the proportion of total ILCs and ILC1s was significantly correlated with the levels of ACR and GFR. In the present study, the proportion of circulating ILC subsets increased significantly in various types of CKD and correlated with clinico-pathological features, which suggests a possible role for ILCs in CKD.

Expression analysis of miRNAs from the DLK1-DIO3 locus in CD4+ and CD14+ cells in patients with relapsing-remitting multiple sclerosis

To analyze the expression of several previously unstudied miRNAs from the DLK1-DIO3 locus in peripheral blood mononuclear cells (PBMC) and in CD14+ and CD4+ cell subpopulations in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy individuals. MiRNA expression analysis was performed in PBMC, CD14+, and CD4+ cells of 14 patients who did not take immunomodulatory drugs, and 14 individuals without autoimmune and inflammatory diseases by reverse transcription followed by real-time PCR. Expression levels of 10 miRNAs selected based on different criteria were significantly higher in PBMC in men with RRMS compared to healthy men; in women, their expression did not change in a similar comparison. No mass unidirectional changes in the expression of these miRNAs in RRMS men were observed in CD14+ and CD4+ cells. High consistency in the expression of miRNA pairs was also not found. The analysis of PBMC showed the involvement of 10 more miRNAs encoded by genes from the DLK1-DIO3 locus in the development of RRMS. The main observed effect of the increase in the expression levels of these miRNAs in men with RRMS compared with healthy men is not achieved due to the contribution of CD14+ and CD4+ cells. This opens up possibilities for studying the involvement of miRNAs in other PBMC subpopulations in the pathological processes in RRMS.

Decreased of BAFF-R expression and B cells maturation in patients with hepatitis B virus-related hepatocellular carcinoma.

BACKGROUNDRecent evidence has indicated the role of B cells and B cell-activating factor (BAFF) in the development of hepatocellular carcinoma (HCC).AIMTo characterize circulating BAFF receptor expression and B cell subpopulations in patients with hepatitis B virus (HBV)-related HCC.METHODSPeripheral blood samples collected from 41 patients with chronic HBV infection (25 patients without HCC and 16 patients with HCC) and 9 healthy controls were assessed for BAFF receptors [BAFF-R(B cell-activating factor receptor), transmembrane activator and cyclophilin ligand interactor, B-cell maturation antigen] and B cell subpopulations by multicolor flow cytometry.RESULTSThe frequency of BAFF-R expressing B cells to total B cells was significantly lower in patients with HCC (3.39% ± 2.12%) compared with the non-HCC group (5.37% ± 1.90%) and healthy controls (6.23% ± 2.32%), whereas there was no difference in transmembrane activator and cyclophilin ligand interactor and B-cell maturation antigen. The frequencies of CD27+IgD+ memory B cells, CD27+IgD- class-switched memory B cells and plasmablasts were significantly lower in the patients with HCC compared to patients without HCC (1.23 ± 1.17 vs 3.09 ± 1.55, P = 0.001, 0.60 ± 0.44 vs 1.69 ± 0.86, P < 0.0001 and 0.16 ± 0.12 vs 0.37 ± 0.30, P = 0.014, respectively). However, the ratio of naïve and transitional B cell did not differ significantly between the three groups. In addition, decreased BAFF-R expression on B cells was significantly correlated with large tumor size and advanced tumor stage.CONCLUSIONOur data demonstrated BAFF-R expression was reduced in B cells that involved with the frequencies of B cells maturation in patients with HCC. The depletion of BAFF-R might play an important role in the development of HCC in patients with chronic HBV infection.

Imbalance of Circulating Innate Lymphoid Cell Subpopulations in Patients With Septic Shock.

Background: Septic shock, a major cause of death in critical care, is the clinical translation of a cytokine storm in response to infection. It can be complicated by sepsis-induced immunosuppression, exemplified by blood lymphopenia, an excess of circulating Treg lymphocytes, and decreased HLA-DR expression on circulating monocytes. Such immunosuppression is associated with secondary infections, and higher mortality. The effect of these biological modifications on circulating innate lymphoid cells (ILCs) has been little studied.Methods: We prospectively enrolled patients with septic shock (Sepsis-3 definition) in the intensive care unit (ICU) of Timone CHU Hospital. ICU controls (trauma, cardiac arrest, neurological dysfunction) were recruited at the same time (NCT03297203). We performed immunophenotyping of adaptive lymphocytes (CD3+ T cells, CD19+ B cells, CD4+CD25+FoxP3+ Treg lymphocytes), ILCs (CD3−CD56+ NK cells and helper ILCs – ILC1, ILC2, and ILC3), and monocytes by flow cytometry on fresh blood samples collected between 24 and 72 h after admission.Results: We investigated adaptive and innate circulating lymphoid cells in the peripheral blood of 18 patients in septic shock, 15 ICU controls, and 30 healthy subjects. As expected, the peripheral blood lymphocytes of all ICU patients showed lymphopenia, which was not specific to sepsis, whereas those of the healthy volunteers did not. Circulating CD3+ T cells and CD3−CD56+ NK cells were mainly concerned. There was a tendency toward fewer Treg lymphocytes and lower HLA-DR expression on monocytes in ICU patients with sepsis. Although the ILC1 count was higher in septic patients than healthy subjects, ILC2, and ILC3 counts were lower in both ICU groups. However, ILC3s within the total ILCs were overrepresented in patients with septic shock. The depression of immune responses has been correlated with the occurrence of secondary infections. We did not find any differences in ILC distribution according to this criterion.Conclusion: All ICU patients exhibit lymphopenia, regardless of the nature (septic or sterile) of the initial medical condition. Specific distribution of circulating ILCs, with an excess of ILC1, and a lack of ILC3, may characterize septic shock during the first 3 days of the disease.

The effect of short-term intensive insulin therapy on circulating T cell subpopulations in patients with newly diagnosed type 2 diabetes mellitus

AimsTo evaluate the effect of short-term intensive insulin therapy on circulating T cell subpopulations in patients with newly diagnosed type 2 diabetes mellitus (T2DM). MethodsA total of 113 patients with T2DM and 28 normal subjects were enrolled. Demographic parameters and biochemical markers were collected at baseline, and flow cytometry was applied to determine the proportion of T cell subpopulations in participants. Then the patients underwent continuous subcutaneous insulin injection (CSII) treatment with euglycemia for 2 weeks, and the T cell subpopulations were measured again after CSII treatment. ResultsCompared with normal subjects, the proportion of Th1 cells and the ratio of Th1/Th2 increased, the proportion of Treg cells decreased in patients with T2DM (p < 0.05 for all). The ratio of Th1/Th2 was positively correlated with glycosylated hemoglobin A1c (HbA1c) and negatively correlated with high density lipoprotein cholesterol (HDL-C). Furthermore, there were negative associations between the proportion of Treg cells and fasting plasma glucose, HbA1c, triglyceride, low density lipoprotein cholesterol, and positive association between the proportion of Treg cells and HDL-C. After CSII treatment, the proportion of Th1 cells and the ratio of Th1/Th2 decreased (p < 0.05 for both), the proportion of Treg cells increased in patients with T2DM (p < 0.05). ConclusionsShort-term intensive insulin therapy could modulate circulating T cell subpopulations in patients with T2DM, which might alleviate inflammatory responses caused by hyperglycemia.This study was registered with ChiCTR-OPN-17010405.

Relapsing-remitting multiple sclerosis: clinical and immunological aspects of the pathology on the example Sema4D and CD72

To explore the expression of Sema4D, CD72 receptor and a role of Sema4D-CD72 signal in the control of immunocompetent cell function in remitting-relapsing multiple sclerosis (RRMS). Fifty-two patients with RRMS diagnosis according to 2010 revised McDonald's criteria were studied. The control group included 24 healthy people. A flow cytometry method was used to measure the expression of semaphorin Sema4D by T-lymphocytes of peripheral blood, expression of CD72 receptor by B-lymphocytes, percentage of cells containing pro- and anti-inflammatory cytokines. The level of soluble Sema4D (sSema4D) was evaluated by ELISA. The level of Sema4D expression on T-lymphocytes (Mean Fluorescence Intensity - MFI) prevailed in cell subpopulations in patients with RRMS compared with the control group. Characteristics of membrane and sSema4D correlate with clinical presentations of the autoimmune disease. An increase in sSema4D level during cell cultivation was identified in RRMS patients. The results show the involvement of Sema4D in the hyperactivation of B-cell-mediated immunity through CD72 receptor and induction of proinflammatory cytokine synthesis. RRMS is associated with elevated expression of Sema4D in the immune system. Membrane and sSema4D involved in the pathological process in RRMS. The authors suggest several mechanisms of the involvement of semaphorin and its receptor in the pathogenesis of RRMS: the direct damage of nervous tissues by sSema4D penetrated through the blood brain barrier disrupted in RRMS or by membrane Sema4D due to the infiltration of the central nervous system by T-lymphocytes and hyperactivation of B-cell-mediated immunity.

Dissection of a circulating CD3+ CD20+ T cell subpopulation in patients with psoriasis.

CD3+ CD20+ T cells are a population of CD3+ T cells that express CD20 and identified in healthy donors and autoimmune diseases. However, the nature and role of these cells in patients with psoriasis remain unclear. In this study, we aimed to investigate the level, phenotype, functional and clinical relevance of CD3+ CD20+ T cells in the peripheral blood of patients with psoriasis. We found that a small subset of CD3+ T cells expressed CD20 molecule in the peripheral blood of patients with psoriasis, and their levels were similar to those in healthy donors. Circulating CD3+ CD20+ Tcells in patients with psoriasis were enriched in CD4+ cells and displayed an activated effector phenotype, as these cells contained fewer CD45RA+ -naive and CCR7+ cells with increased activity than those of CD3+ T cells lacking CD20. In addition, compared with healthy donors, circulating CD3+ CD20+ T cells in patients with psoriasis produced more cytokines, interleukin (IL)-17A, tumour necrosis factor (TNF)-α and IL-21, but not IL-4 and IFN-γ. Furthermore, a significantly positive correlation was found between the levels of IL-17A, TNF-α and IL-21-production CD3+ CD20+ T cells with Psoriasis Area and Severity Index scores. Our findings suggest that CD3+ CD20+ T cells may play a role in the pathogenesis of psoriasis.

Impact and Modulations of Peripheral and Edaphic B Cell Subpopulations in Chronic Rhinosinusitis With Nasal Polyposis.

Objectives The pathophysiological mechanisms of chronic rhinosinusitis with nasal polyposis (CRSwNP) still are discussed controversially. Regulatory B cells (Breg) are responsible for the suppression of T cell activity: deficiencies for Breg have been demonstrated to contribute to autoimmune disorders, e.g., systemic lupus erythematosus. In order to evaluate the influence of B cell subpopulations, especially Breg, on the etiology of this disease, the aim of this study was to characterize subpopulations of peripheral and edaphic B cells in CRSwNP.Methods Polypoid tissue and blood samples were collected from 10 patients undergoing paranasal sinus surgery and lymphocytes were analyzed by multicolor flow cytometry.Results There was a significantly lower frequency of B cells in nasal polyps compared to peripheral blood mononuclear cells (PBMC) in patients with CRSwNP. Mature resting B cells were the main population within B cells in PBMC, and memory B cells in nasal polyps. Remarkably, Breg and mature B cells significantly decreased in nasal polyps compared to PBMC. Memory B cells significantly increased and represented the main subpopulation in nasal polyps in patients with CRSwNP.Conclusion In this study a detailed contemporary characterization of B cell subpopulations in patients with CRSwNP is presented. The influence of edaphic B cells could play a key role in the maintenance of this chronic infectious disease.

Dynamic variations of the peripheral blood immune cell subpopulation in patients with critical H7N9 swine-origin influenza A virus infection: A retrospective small-scale study.

H7N9 influenza is a recently emerging infection with a high mortality rate. The aim of the present study was to investigate dynamic fluctuations of peripheral blood immune cell subgroups in patients with critical H7N9 infection. Flow cytometry was used to assess the cells in whole blood samples from 9 cases. With regard to the innate immune system, in the majority of patients, the natural killer (NK) cell counts were similar to those of monocytes, which demonstrated a gradual increase in the progression period and an early increase followed by a reduction during recovery. B cells exhibited a reduction during progression and were further decreased during recovery. The CD4+T cells of all patients decreased during progression, and further decreased during recovery. By contrast, CD8+T cells increased in the majority of patients in the progression stage, and underwent an initial reduction followed by a gradual increase during recovery. However, CD8+ programmed death (PD)-1+T cell and T helper (Th) 1 cell frequencies demonstrated a moderate increase in all patients during the progression stage, and regulatory T cell (Treg) frequencies tended to be reduced during progression and increased during recovery. Notably, this preliminary data also showed that the frequencies of B cells, Th2 cells and Th17 cells in the progression period were higher than those in the recovery period. The frequencies of monocytes, CD4+T cell, CD8+T cell, CD4+PD-1+T cells and CD8+PD-1+T cells in the progression period were lower than those during recovery. In conclusion, different levels of peripheral blood immune cell subgroups during the pathogenesis of H7N9 infection may be associated with elimination of the virus and immune damage.

Low baseline levels of NK cells may predict a positive response to ipilimumab in melanoma therapy.

The introduction of immune checkpoint blockade (ICB) has been a breakthrough in the therapy of metastatic melanoma. The influence of ICB on T-cell populations has been studied extensively, but little is known about the effect on NK cells. In this study, we analysed the relative and absolute amounts of NK cells and of the subpopulations of CD56dim and CD56bright NK cells among the peripheral blood mononuclear cells (PBMCs) of 32 patients with metastatic melanoma before and under treatment with ipilimumab or pembrolizumab by flow cytometry. In 15 (47%) patients, an abnormal low amount of NK cells was found at baseline. Analysis of the subpopulations showed also low or normal baseline levels for CD56dim NK cells, whereas the baseline levels of CD56bright NK cells were either normal or abnormally high. The relative and absolute amounts of NK cells and of CD56dim and CD56bright NK cell subpopulations in patients with a normal baseline did not change under treatment. However, patients with a low baseline of NK cells and CD56dim NK cells showed a significant increase in these immune cell subsets, but the amounts remained to be lower than the normal baseline. The amount of CD56bright NK cells was unaffected by treatment. The baseline levels of NK cells were correlated with the number of metastatic organs. Their proportion increased, whereas the expression of NKG2D decreased significantly when more than one organ was affected by metastases. Low baseline levels of NK cells and CD56dim NK cells as well as normal baseline levels of CD56bright NK cells correlated significantly with a positive response to ipilimumab but not to pembrolizumab. Survival curves of patients with low amounts of CD56dim NK cells treated with ipilimumab showed a trend to longer survival. Normal baseline levels of CD56bright NK cells were significantly correlated with longer survival as compared to patients with high baseline levels. In conclusion, analysis of the amounts of total NK cells and of CD56dim and CD56bright NK cells subpopulations at baseline may help to predict the outcome of treatment with ipilimumab.

Peripheral T Cell Subpopulation in Patients with Hepatocellular Carcinoma: Relation to Ablation Therapy

Background: Ablation therapy with advances in its techniques becomes widely used in patients with hepatocellular carcinoma. Ablative techniques can induce tumor cell death and stimulate many immunological responses. These responses can be evaluated through assessment of peripheral immune cells changes in systemic circulation. Aims: To investigate changes in the peripheral immune cells presented in CD4, CD8 and CD4/CD8 ratio after HCC ablation by different procedures and their relations to ablation result. Subjects and Methods: This study investigated 73 HCC patients admitted to Department of Tropical Medicine, Mansoura University Hospital, Egypt. The patients were stratified into three groups according to ablative technique used. Radiofrequency ablation was performed for 24 cases, microwave ablation for 24 and transarterial chemoembolization for 25 cases. After history taking, clinical examination, basic investigations, triphasic abdominal computerized tomography before and 4 weeks after the treatment, HCC patients were selected according to EASL guideline. Lymphocyte subset assay using flow cytometry 1 day before, and 4 weeks post ablation was done. The patients subdivided into successful and unsuccessful subgroup according to the result of ablation by CT. Results: In patients treated with Radiofrequency ablation, significant increase in CD4 count and CD4/CD8 ratio after treatment (P<0.001), while CD8+ cells count significantly decreased (P<0.002). In HCC patients treated with microwave ablation, CD4+ count and CD4/CD8 ratio significantly increased after treatment (P<0.001,<0.007), without significant differences in CD8+ cells count. After transarterial chemoembolization, CD4+ cells count and CD4/CD8 ratio significantly decreased (P<0.001) with significant increase of CD8+ cells (P<0.001). Changes in CD4, CD8, and CD4/CD8 ratio remained comparable to that occurred in both successfully ablated and cases with residual tumor. Conclusion: Various ablation procedures of HCC are associated with significant changes in peripheral T cell subpopulation. These changes mostly were due to the ablation of tumor cells but these changes cannot predict the success of ablation or recurrence of previously ablated one.

THE TIME COURSE OF CHANGES IN B LYMPHOCYTE SUBPOPULATIONS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS DURING THERAPY WITH BIOLOGICAL AGENTS

Objective: to study the baseline level and time course of changes in peripheral blood B cell subpopulations in patients with systemic lupus erythematosus (SLE) versus the clinical manifestations of the disease during therapy with rituximab (RTM) and belimumab (BLM). Subjects and methods. 49 patients with a documented diagnosis of SLE and high and moderate SLEDAI-2K values were divided into three groups: 1) 40 patients received RTM, 2) 5 patients took BLM; 3) 4 patients had combination therapy (dual anti-B-cell therapy) with RTM + BLM. Peripheral blood B lymphocyte subpopulations were measured by multicolor flow cytometry using a panel of monoclonal antibodies against surface membrane markers of B lymphocytes; the patients underwent clinical immunological examination before therapy with biological agents and every 3 months during a year. Results and discussion. Patients with high SLE activity (SLEDAI-2K 18±5) and involvement of vital organs had generally higher levels of double negative memory cells (r = 0.52; p < 0.001) with relatively low counts of naive B cells (r=-0.54; p < 0.0007) than those without severe organ pathology (SLEDAI-2K 8±2). RTM therapy at 3 month of follow-up resulted in a noticeable decrease in the number of naive and double negative memory B cells and to a lesser extent in that of non-switched and switched memory B cell. A rapid recovery of the count of memory B cells and plasmablasts after a RTM cycle at 6 months after therapy initiation should be evaluated as a predictor of early exacerbation of SLE. There was a reduction in the clinical and laboratory signs of SLE activity, as well as inhibition of naive B cells, plasma cells, and plasmablasts in all the 5 patients receiving BLM at various stages of follow-up. Dual anti-B cell therapy most effectively reduced disease activity at 3 months, prolonged the achieved remission, contributed to the achievement and maintenance of a low B lymphocyte level at later stages of control, to the further decline in the number of plasmablasts and plasma cells, and prevented the synthesis of autoantibodies. Conclusion. Assessment of the time course of changes in peripheral blood B lymphocyte subpopulations provides new opportunities to identify impaired function and regulation of cellular immunity in patients with SLE, it may be a useful diagnostic parameter to monitor an autoimmune disease and a valuable tool to assess and predict a response.

The effect of dipeptidyl peptidase-IV inhibition on circulating T cell subpopulations in patients with type 2 diabetes mellitus

AimTo assess intraindividually the effects of DPP-IV inhibition on the subpopulations of immune cells in type 2 diabetes mellitus (DM2) patients during the course of treatment with sitagliptin. MethodsIn this open label non-randomized observational study with a control group DM2 patients were examined before the initiation of the DPP-IV inhibitor administration (sitagliptin 100mg once daily) and then after 4weeks and 12months. Inhibition of the blood plasma DPP-IV enzymatic activity was determined by a chromogenic assay, the immunophenotyping of the blood cell subpopulations was performed using flow cytometry and blood plasma cytokine concentrations were quantified using an array-based multiplex ELISA. All parameters were evaluated in relation to the entry values in individual patients. ResultsThe blood plasma DPP-IV enzymatic activity was effectively inhibited during the sitagliptin treatment. A significant decrease of the proportion of Treg cells (to 86±31% (median±SD) of entry values, p=0.001) and an increase of Th1 cells (to 120±103% (median±SD) of entry values, p=0.004) were observed after 4weeks but not after one year of the sitagliptin treatment. No changes were observed in the ratio of CD4+/CD8+ cells, in the quantity of NK and Th2 cells and blood plasma cytokine levels. ConclusionsSitagliptin treatment may cause temporary changes of the proportion of lymphocyte subpopulations in patients with DM2. The consequent deregulation of the immune system should be considered as a possible cause of the eventual side effects of long term DPP-IV inhibition.

Detection of circulating tumor cell subpopulations in patients with head and neck squamous cell carcinoma (HNSCC).

BackgroundSince image based diagnostic tools fail to detect early metastasis in head and neck squamous cell carcinoma (HNSCC) it is crucial to develop minimal invasive diagnostic methods. A promising approach is to identify and characterize circulating tumor cells (CTC) in the peripheral blood of HNSCC patients. In this pilot study, we assessed which non-hematopoietic cell types are identifiable and whether their numbers differ in pre- and postoperative blood samples.Methods20 ml citrated peripheral blood was taken from 10 HNSCC patients before and after curative resection. CTC were enriched using density gradient centrifugation. CTC presence was verified by multi-immunofluorescence staining against cytokeratin (CK; epithelial), N-cadherin (mesenchymal); CD133 (stem-cell), CD45 (hematopoietic) and DAPI (nucleus). Individual cell type profiles were analyzed.ResultsWe were able to detect cells with epithelial properties like CK+/N-cadherin−/CD45− and CK+/CD133−/CD45− as well as cells with mesenchymal features such as N-cadherin+/CK−/CD45− and cells with both characteristics like N-cadherin+/CK+/CD45−. We also observed cells showing stem cell-like features like CD133+/CK−/CD45− and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45−. The number of CK positive cells (p = 0.002), N-cadherin positive cells (p = 0.002) and CD133 positive cells (p = 0.01) decreased significantly after resection. Kaplan-Meier test showed that the survival was significantly shorter when N-cadherin+ cells were present after resection (p = 0.04; 474 vs. 235 days; [HR] = 3.1).ConclusionsThis is - to the best of our knowledge- the first pilot study identifying different CTC populations in peripheral blood of HNSCC patients and showing that these individual cell type profiles may have distinct clinical implications.

Predominance of IL‐5‐secreting drug‐specific T cells in subgroups of patients with drug‐induced delayed‐type hypersensitivity reaction

Methods We sought to characterize drug-specific T cell subpopulations in patients with delayed-type DHRs to beta-lactam antibiotics by enzyme-linked immunospot (ELISPOT) assay, a sensitive technique to detect antigen-specific cytokine-producing cells. Peripheral blood was drawn from eight patients who had experienced maculopapular exanthema as a clinical manifestation of a DHR against beta-lactam antibiotics (penicillin, amoxicillin or cefuroxim), which was confirmed by skin patch-test. The occurrence and frequencies of drug-specific IL-5-, IFNgamma- and IL-10-producing T cells were analyzed by ELISPOT assay and compared to ten healthy controls without history of delayed-type DHRs. Results and conclusion As expected, ELISPOT analysis of peripheral blood mononuclear cells (PBMC) revealed increases in drug-specific IFN-gamma-producing T cells. However, in a subgroup of patients with delayed-type DHRs, augmented numbers of drug-specific IL-5-secreting T cells were observed, regardless which drug was taken. Of note, IL-10 positive T cells were not decreased in drug-allergic patients when compared with healthy controls. Our results show that DHRs are characterized by distinct patterns of cytokine-secreting lymphocytes which resemble either Th1, Th2 or a mixed type of Th cells. Thus, determining both IFN-gamma- and IL-5-positive (but not IL-10-producing) cells by ELISPOT assay increases the sensitivity in detecting drug-specific T cells. This may help diagnosing drug-allergy in patients with suspected DHRs negative by other conventional allergy tests.