Mediastinal lymph node removal modulates natural killer cell exhaustion in patients with non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death globally. In this study, the effect of complete removal of mediastinal lymph nodes by video-assisted mediastinoscopic lymphadenectomy (VAMLA) on natural killer (NK) cell phenotype and functions in patients with NSCLC was evaluated. The study included 21 NSCLC patients (cIA-IVA) undergoing VAMLA staging and 33 healthy controls. Mononuclear cells were isolated from peripheral blood of all participants and mediastinal lymph nodes of the patients. NK cells were analyzed by flow cytometry to define NK subsets, expressions of PD-1, CTLA-4, activating/inhibitory receptors, granzyme A, and CD107a. The plasma levels of soluble PD-1, PDL-1, and CTLA-4 were measured by ELISA. Mediastinal lymph nodes of NSCLC patients had increased ratios of exhausted NK cells, increased expression of PD-1 and IL-10, and impaired cytotoxicity. Mediastinal lymph nodes removal increased CD56dimCD16bright cytotoxic effector phenotype and reduced exhausted NK cells. PD-1+ NK cells were significantly more abundant in patients' blood, and VAMLA significantly reduced their ratio as well. The ratio of IL-10 secreting regulatory NK cells was also reduced after VAMLA. Blood NK cells had increased cytotoxic functions and spontaneous IFN-γ secretion, and these NK cell functions were also recovered by VAMLA. Mediastinal lymph node removal reversed NK cell exhaustion, reduced regulatory NK cells, and improved antitumoral functions of NK cells. Tumor-draining lymph nodes may contribute to tumor evasion from antitumoral immune responses. The role of their removal needs to be further studied both to better understand this mechanism and as a potential immunotherapeutic approach.

The role of cytokines and T-bet, GATA3, ROR-γt, and FOXP3 transcription factors of T cell subsets in the natural clinical progression of Type 1 Diabetes.

Th cells play an important role in pathogenesis of type 1 diabetes (T1D). Peripheral blood mononuclear cells were isolated from peripheral blood samples from newly diagnosed (ND), 1-year (1YD), and 5-year T1D (5YD) patients (n:8 of each group), 8 healthy controls (HC), and cultured for 24 h under unstimulated (US) and stimulated conditions. Cell ratios of Th1, Th2, Th17, Treg, and intracellular levels of IFN-γ, TNF-α, IL-10, TGF-ß, IL-5, IL-13, IL-17, and IL-21 cytokines were evaluated using the flow cytometry. mRNA expressions of transcription factors T-bet, GATA3, ROR-γt, and FOXP3 of these cells were determined by real-time PCR. Reduced CD4+CD25high cell ratios were detected in ND. CD4+CD25high cells were found to be reduced in ND and 1YD compared to HC under IL-2-stimulated conditions. Intracellular IFN-γ and TNF-α levels were low in all patients under US and IL-12-stimulated conditions. IL-17A and IL-21 were found to be high in patients with IL-6-stimulated conditions. Expressions of IL-10 and TGF-ß have been observed to be reduced in patients. Th1/Th2, Th17/Treg, and Th1/Treg ratios were higher in patient groups. FOXP3 and GATA3 mRNA expressions were found to be low in patients, while RORγt and T-bet mRNA levels were higher than HC. Th1, Th17, and Treg cells and their cytokines have been shown to be associated with type 1 diabetes.

Ozone therapy restores immune dysfunction in refractory idiopathic granulomatous mastitis as a novel potential therapeutic approach.

Immunological dysfunction has been suggested to play a major role in the pathogenesis of idiopathic granulomatous mastitis (IGM). We recently showed that ozone therapy was effective in patients with steroid-resistant IGM. This study assessed alterations in intracellular cytokine expression patterns in different T-lymphocyte subsets after ozone therapy in refractory IGM. Peripheral blood T lymphocyte subsets (CD8+ , CD4+ , CD4+ CD25+ CD127- ) were analyzed via flow-cytometry for intracellular cytokine expressions IFN-γ, TNF-α, IL-10, and TGF-ß before and after completion of 4-month systemic ozone therapy. Ozone therapy significantly increased the CD4+ IFN-γ+ (p = 0.032), CD4+ TNF-α+ (p = 0.028), and the CD8+ TNF-α+ (p = 0.012) T cells. In contrast, significant decreases in CD4+ IL-10+ (p = 0.047) and CD8+ IL-10+ T cells (p = 0.022) and CD4+ CD25+ CD127-//low Treg cells secreting TGF-ß (p = 0.005) were found after ozone therapy. When patients were analyzed according to the response to ozone therapy, patients with a complete remission were more likely to have increased CD3- CD16+ CD56+ natural killer cells (p = 0.0027) and decreased CD19+ B lymphocytes (p = 0.046) following ozone therapy. Our results suggest that ozone therapy stimulated a T-helper-1 response associated with IFN-γ production and downregulation of TGF-ß expression in CD4+ CD25+ CD127- Treg cells. These alterations in the immune system following ozone therapy can improve wound healing and restore immune dysfunction in patients with refractory IGM.

Mediastinal lymph node removal ameliorates cytotoxic T-lymphocyte functions in patients with non-small cell lung cancer.

INTRODUCTION: Mediastinal lymph node (MLN) removal by video-assisted mediastinoscopic lymphadenectomy (VAMLA) for preoperative cancer staging was reported to be associated with increased survival. The aim of this study was to evaluate the immunologic effects of complete MLN removal by VAMLA on cytotoxic T lymphocyte (CTL) phenotype and function. METHODS: Seventeen patients with non-small cell lung cancer (NSCLC) (stage cT1-4N0-3M0-1A) and 20 healthy participants were included in this study. Blood samples were collected before and 4 weeks after the procedure. Lymphocytes were isolated from the removed MLNs. CTL phenotypes and functions were evaluated by flow cytometry. Plasma levels of soluble programmed cell death protein 1 (sPD-1), soluble programmed cell death protein 1 ligand, and soluble CTL antigen 4 (sCTLA-4) were measured with enzyme-linked immunosorbent assay. RESULTS: The ratio of the immunosenescent CTLs (CD3+CD8+CD28-) was increased in peripheral blood and MLNs of the patients with NSCLC compared to controls (p = 0.037), and MLN removal did not change this ratio. PD-1 and CTL antigen 4 expressions were significantly reduced in peripheral blood CTLs after MLN removal by VAMLA (p = 0.01 and p = 0.01, respectively). Granzyme A expression was significantly reduced in the peripheral blood CTLs of the patients compared to controls (p = 0.006) and MLN removal by VAMLA significantly improved Granzyme A expression in CTLs (p = 0.003). Plasma concentrations of sPD-1 and sCTLA-4 remained unchanged after VAMLA. CONCLUSION: CTLs in the MLNs and peripheral blood of the patients with NSCLC had an immunosenescent phenotype, increased immune checkpoint receptor expression, and impaired cytotoxicity. MLN removal by VAMLA improved these phenotypic and functional characteristics of CTLs. These changes may explain the potential contribution of VAMLA to improved survival.

High co-expression of immune checkpoint receptors PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT on tumor-infiltrating lymphocytes in early-stage breast cancer.

High expression of immune checkpoint receptors (ICRs) in the tumor microenvironment regulates the anti-tumor response. In this study, the differential expressions of ICRs on tumor-infiltrating lymphocytes (TILs) in patients with early-stage breast cancer were investigated.The study included 32 patients who underwent surgery with a diagnosis of early-stage breast cancer between September 2018 and March 2020. TIL isolation was performed using a MACS tumor separation device and tumor separation kit. PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT expression of cytotoxic T and natural killer (NK) cells on TILs and peripheral blood lymphocytes (PBLs) were determined by flow cytometry.Patients with a high Ki-67 index, high TIL density, and HER-2 positivity were more likely to have increased CD16+CD56dim NK cells on TILs. Patients with T2 tumors were more likely to have increased expression of PD-1, LAG-3, and TIGIT on tumor-infiltrating CD8+ cytotoxic T cells than those with T1 tumors. PD-1, CTLA-4, TIGIT, LAG-3, and TIM-3 expression of CD8+ T and CD16-CD56bright NK cells in TILs showed significant positive correlations with each other. PD1+CD8+, TIGIT+CD16+, and CTLA-4+CD56+ cells in PBLs and TILs were found to be negatively correlated, whereas only TIM-3+ expression of CD8+ T and CD16+CD56dim cells in PBLs and TILs showed positive correlations.Our results suggest that CD16+CD56dim NK cells on TILs may play a major role in the immune response against HER2-positive or highly proliferating breast tumors in patients with early-stage breast cancer. Furthermore, various ICRs were found to be highly co-expressed with each other on TILs, including PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT. These receptors may synergistically suppress the response to the tumor, which may trigger immune escape mechanisms in the early stage of carcinogenesis. However, ICR expressions other than TIM3 on PBLs were not found to accompany their counterparts on TILs.

Immune modulation as a consequence of SARS-CoV-2 infection.

Erroneous immune responses in COVID-19 could have detrimental effects, which makes investigation of immune network underlying COVID-19 pathogenesis a requisite. This study aimed to investigate COVID-19 related alterations within the frame of innate and adaptive immunity. Thirty-four patients clinically diagnosed with mild, moderate and severe COVID-19 disease were enrolled in this study. Decreased ILC1 and increased ILC2 subsets were detected in mild and moderate patients compared to healthy controls. NK cell subsets and cytotoxic capacity of NK cells were decreased in severe patients. Moreover, CD3+ T cells were reduced in severe patients and a negative correlation was found between CD3+ T cells and D-dimer levels. Likewise, moderate and severe patients showed diminished CD3+CD8+ T cells. Unlike T and NK cells, plasmablast and plasma cells were elevated in patients and IgG and IgA levels were particularly increased in severe patients. Severe patients also showed elevated serum levels of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-8, reduced intracellular IFN-γ and increased intracellular IL-10 levels. Our findings emphasize that SARS-CoV-2 infection significantly alters immune responses and innate and acquired immunity are differentially modulated in line with the clinical severity of the disease. Elevation of IL-10 levels in NK cells and reduction of CD3+ and CD8+ T cells in severe patients might be considered as a protective response against the harmful effect of cytokine storm seen in COVID-19.

The relationship between serum A proliferation-inducing ligand and B-cell activating factor levels with disease activity and organ involvement in systemic lupus erythematosus.

OBJECTIVES: We aim to investigate the association between serum B-cell activating factor (BAFF) and A proliferation-inducing ligand (APRIL) levels with disease activity and clinical findings in SLE patients. METHODS: Seventy-nine patients with SLE and 27 healthy controls were included into the study. Serum BAFF and APRIL levels were measured by using ELISA. In 19 patients with active disease at the time of the assessment, BAFF/APRIL levels were reassessed after 6 months of follow-up and disease activity was evaluated by using SLEDAI-2K. The relationship between renal histopathology index scores and lupus nephritis (LN) classes with serum BAFF/APRIL levels was examined in 16 patients who had recent renal involvement and underwent biopsy during the study. RESULTS: Although both BAFF/APRIL levels were higher in patients with SLE compared to the control group (p < 0.001), no correlation was found between BAFF/APRIL levels and SLEDAI scores. Serum BAFF levels were higher in patients with renal disease activity (p = 0.01), and there was a significant correlation between APRIL levels and proteinuria (r = 0.42, p = 0.02). A weak inverse correlation was observed between BAFF and C3 levels (r = 0.25, p = 0.02). No correlation was found between BAFF/APRIL levels and renal SLEDAI scores, renal histopathology, activity, and chronicity index scores. In the active disease group after treatment, there was no significant change in serum BAFF levels, but a significant increase in serum APRIL levels was observed. CONCLUSION: These results suggest that both cytokines are involved in the pathogenesis of SLE and that serum BAFF can be valuable as a biomarker in SLE especially in patients with renal activity.

Extremely low frequency electromagnetic fields exposure during the prenatal and postnatal periods alters pro-inflammatory cytokines levels by gender.

Maternal exposure to the excessive electromagnetic fields is considered harmful to infants and associated with several health problems in life, such as neurological or immune diseases. In this present study we aimed to investigate the potential effects of extremely low-frequency electromagnetic field (ELF-EMF) exposure during the gestational and lactational period of dams on immune system parameters. The development of white blood cells (WBC), lymphocyte subpopulations (CD4+ T cells, CD8+ T cells, Natural Killer (NK) cells, and B cells) and production of T cell related cytokines were explored in the offsprings. Significant changes were found in WBC and lymphocyte counts. Although no changes in lymphocyte subunits were observed among groups, CD4+ cells were significantly increased in the female group exposed to ELF-EMF. Also, IL-17A and IFN-γ levels increased in plasma and spleen. The mean IL-4 level and the expression level of the IL-4 gene were not changed, in the experimental groups. But the expression of the IL-17A gene was also upregulated, which supports cytokine quantification analyses. In conclusion, ELF-EMF exposure in the prenatal and postnatal period increases the level of IL-17A in the spleen and blood of young female rats, and it upregulates IL-17 gene expression in the spleen, resulting in CD4+ cell proliferation and inflammation.

COVID-19 immunotherapy: Treatment based on the immune cell-mediated approaches.

Multiple efforts are currently underway to control and treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19) worldwide. Despite all efforts, the virus that emerged in Wuhan city has rapidly spread globally and led to a public health emergency of international concern (PHEIC) due to the lack of approved antiviral therapy. Nevertheless, SARS-CoV-2 has had a significant influence on the evolution of cellular therapeutic approaches. Adoptive immune cell therapy is innovative and offers either promising prophylactic or therapy for patients with moderate-to-severe COVID-19. This approach is aimed at developing safety and providing secure and effective therapy in combination with standard therapy for all COVID-19 infected individuals. Based on the effective results of previous studies on both inflammatory and autoimmune diseases, various immune cell therapies against COVID-19 have been reviewed and discussed. It must be considered that the application of cell therapy for treatment and to eliminate infected respiratory cells could result in excessive inflammation, so this treatment must be used in combination with other treatments, despite its many beneficial efforts.

Inhaled corticosteroids' effects on biomarkers in exhaled breath condensate and blood in patients newly diagnosed with asthma who smoke.

OBJECTIVE: Exposure to cigarette smoke complicates the treatment and management of asthma through a variety of inflammatory effects. This study aimed to investigate the differences between newly diagnosed cases of asthma in smokers and nonsmokers in terms of localized and systemic biomarkers following treatment with inhaled corticosteroids (ICS) or ICS in combination with a long-acting ß2 agonist (LABA). METHODS: Specimens of exhaled breath condensate (EBC) from newly diagnosed patients with asthma were used to quantify inflammation in the airways, while blood samples were used to assess systemic inflammation. In both samples, the levels of IL-6, LTB4, LTD4, and 8-isoprostane were measured and these were repeated after 3 months of treatment with ICS or ICS + LABA. RESULTS: Of the 20 patients, 10 (50%) were nonsmokers with asthma (NSA) and 10 (50%) smokers with asthma (SA). There was no statistically significant difference in the blood or EBC levels of IL-6, LTB4, LTD4, or 8-isoprostane between the groups prior to treatment. Only the decrease in 8-isoprostane level in the EBC samples was found to be significantly greater in the NSA group after treatment (for smokers, the change was 2.91 ± 23.22, while for nonsmokers it was -22.72 ± 33.12, p = 0.022). Post-treatment asthma control was significantly better in the NSA group (p = 0.033). CONCLUSION: Monitoring the alterations in 8-isoprostane levels in EBC in patients with asthma who smoke may be helpful in deciding on therapeutic management and switching treatments. Asthma control was better in nonsmokers than in smokers.

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

PD-1, CTLA-4, LAG-3, and TIGIT: The roles of immune checkpoint receptors on the regulation of human NK cell phenotype and functions.

The roles of immune checkpoint receptors were defined in many cancers and autoimmune diseases, while there is limited information on their functional roles in the NK cells of healthy individuals. Immune checkpoint receptor expression of NK cell subsets and their association with NK cell functions (cytotoxic capacity and cytokine production) in healthy population were investigated. PD-1, CTLA-4, LAG-3 and TIGIT expression of peripheral blood NK cells, cytokine levels (TNF-α, IFN-γ, IL-10) and cytotoxic functions (granzyme A, perforin, CD107a; with/without K562 target cell stimulation) were evaluated by flow cytometry. CD56dimCD16dim NK cells had the highest expression of TIGIT, while CD56dimCD16- NK cells had highest expression of PD-1, CTLA-4 and LAG-3. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had increased amount of IL-10 however, reduced IFN-γ and TNF-α levels. Cytotoxic granule expressions (perforin and granzyme A) were reduced in PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells. However, TIGIT expression did not alter perforin and granzyme A expressions. Degranulation capacity was reduced in three groups of NK cells (PD-1+ or LAG-3+ or TIGIT+). TIGIT+ NK cells responded strongly to target cell stimulation, while NK cells in the other groups (PD-1+ or CTLA-4+ or LAG-3+) were resistant. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had a regulatory phenotype, impaired cytotoxic functions, and response to target cell stimulation. In contrast, TIGIT+ NK cells had strong baseline cytotoxic activity that further increased in response to target cell stimulation.

Functions of NK and iNKT cells in pediatric and adult CVID, ataxia telangiectasia and agammaglobulinemia patients.

Primary immune deficiencies (PID) are known to be more than 400 genetic defects caused by the impairment in development and/or functions of the immune system. Common Variable Immunodeficiency (CVID), Ataxia Telangiectasia (AT) and Agammaglobulinemia (AG) are examples of the most common immunodeficiency syndrome. Natural killer (NK) cells are a component of innate immune system and play a major role in the host-rejection of both tumors and virally infected cells. iNKT cells have a role in autoimmune and infectious diseases and controlling of tumor rejection. In this study, NK and iNKT cells and their functions, and intracellular cytokine amount are aimed to determine in patients that suffer CVID, AT and AG. NKp30, NKp46, NKG2D, perforin and granzyme mRNA expression levels were analyzed using RT-PCR. Receptors, cytokine amount of NK cell subset and iNKT were analyzed by flow cytometry. Decreased CD3+ T and elevated NK cell subset in pediatric AT were found. Expression of NKp44 was decreased in adult AG, but not in pediatric patients. Low NKp44 expression in CD3-CD16+CD56dim NK cell subset was found in pediatric AT patients. High HLA-DR, perforin and granzyme expression were found in CD3-CD16+CD56dim NK cell subset of pediatric CVID and AT patients. Alteration of the number of NK subsets, NK receptor expression and cytokine production were observed in pediatric patients compared to healthy subjects.

Differential Expression of Novel Immune Checkpoint Receptors on Tumor Infiltrating Lymphocytes in Patients with Locally Advanced Breast Cancer after Neoadjuvant Chemotherapy.

Immune checkpoint receptors (ICRs) were recently found to modulate the anti-tumoral immune response. This study aimed to determine the clinical and pathological associations of ICRs expression on tumor-infiltrating lymphocytes (TILs) in patients with locally advanced breast cancer (LABC) treated with neoadjuvant chemotherapy (NAC). Expressions of ICRs including PD-1, LAG-3, TIM-3, TIGIT, and CTLA-4 on CD8+ T lymphocytes and Natural Killer (NK) cells on TILs were analyzed by flow cytometry. Patients <50 years were more likely to express CTLA-4 on CD8+ T lymphocytes compared to those ≥50 years (p=0.004). In addition, patients with ypT3-4 tumors were more likely to have increased LAG-3 expression on CD16-CD56bright NK cells (p=0.042) and PD-1 (p=0.014) and CTLA-4 (p=0.018) expressions on CD8+ T cells in regard to those with ypT1-T2, respectively. Contrarily, PD-1 expression on CD16-CD56bright NK cells was found to be decreased in patients with ypN+ compared to those with ypN- (p=0.022). Furthermore, patients with HER2+ tumors were more likely to have increased TIM-3 expression on CD8+ T cells (p=0.043), whereas patients with a better response to NAC were more likely to express TIGIT on CD8+ T (p=0.014) and CD16-CD56bright NK cells (p=0.003), respectively. The new generation ICRs, TIM-3, LAG-3, and TIGIT are highly expressed in LABC following NAC in patients with poor prognostic factors. Therefore, new evolving therapies using inhibitory mAbs directed to TIM-3, LAG-3, and TIGIT could be also be considered in locally advanced breast cancers expressing these ICRs.

Notch4 signaling limits regulatory T-cell-mediated tissue repair and promotes severe lung inflammation in viral infections.

A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections.

The Role of Natural Killer Cells in Autoimmune Diseases.

Natural killer (NK) cells, the large granular lymphocytes differentiated from the common lymphoid progenitors, were discovered in early 1970's. They are members of innate immunity and were initially defined by their strong cytotoxicity against virus-infected cells and by their important effector functions in anti-tumoral immune responses. Nowadays, NK cells are classified among the recently discovered innate lymphoid cell subsets and have capacity to influence both innate and adaptive immune responses. Therefore, they can be considered as innate immune cells that stands between the innate and adaptive arms of immunity. NK cells don't express T or B cell receptors and are recognized by absence of CD3. There are two major subgroups of NK cells according to their differential expression of CD16 and CD56. While CD16+CD56dim subset is best-known by their cytotoxic functions, CD16-CD56bright NK cell subset produces a bunch of cytokines comparable to CD4+ T helper cell subsets. Another subset of NK cells with production of interleukin (IL)-10 was named as NK regulatory cells, which has suppressive properties and could take part in immune-regulatory responses. Activation of NK cells is determined by a delicate balance of cell-surface receptors that have either activating or inhibitory properties. On the other hand, a variety of cytokines including IL-2, IL-12, IL-15, and IL-18 influence NK cell activity. NK-derived cytokines and their cytotoxic functions through induction of apoptosis take part in regulation of the immune responses and could contribute to the pathogenesis of many immune mediated diseases including ankylosing spondylitis, Behçet's disease, multiple sclerosis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus and type-1 diabetes. Dysregulation of NK cells in autoimmune disorders may occur through multiple mechanisms. Thanks to the rapid developments in biotechnology, progressive research in immunology enables better characterization of cells and their delicate roles in the complex network of immunity. As NK cells stand in between innate and adaptive arms of immunity and "bridge" them, their contribution in inflammation and immune regulation deserves intense investigations. Better understanding of NK-cell biology and their contribution in both exacerbation and regulation of inflammatory disorders is a requisite for possible utilization of these multi-faceted cells in novel therapeutic interventions.

Pseudomonas aeruginosa colonization in cystic fibrosis: Impact on neutrophil functions and cytokine secretion capacity.

BACKGROUND: Chronic colonization with Pseudomonas (P.) aeruginosa worsens the prognosis of cystic fibrosis (CF) patients. This study aims to analyze the functional properties of neutrophils in CF patients with P. aeruginosa colonization. METHODS: Patients with CF (n = 16) were grouped by positivity of P. aeruginosa in sputum culture, as positive (P.+) or negative (P.-), then compared with age and sex matched healthy controls (n = 8). Adhesion molecules, apoptotic index, intracellular CAP-18, interleukin 8 (IL-8), and tumor necrosis factor α (TNF-α) levels of neutrophils, following P. aeruginosa and lipopolysaccharides (LPS) stimulation, were analyzed by flow cytometry. IL-1ß, IL-6, TNF-α, and IL-17 plasma levels were determined by Luminex. RESULTS: Patients with CF had increased phagocytosis of Escherichia coli and P. aeruginosa, upregulated oxidative burst and chemotaxis. Increased neutrophil apoptosis was noted in CF patients. In unstimulated conditions, higher levels of CD16+ TNF-α+ and CD16+ IL-8+ neutrophils were determined, whereas bacteria and LPS stimulation significantly decreased secretion of CAP-18 from CD16+ neutrophils of CF patients. Plasma levels of IL-1ß, TNF-α and IL-17 in P.+ patients were higher than in P.- group. CONCLUSION: Our findings confirm inadequate neutrophil defense towards pathogens in CF. A significant difference in migration, phagocytosis, oxidative burst, percentage of IL-8 producing neutrophils, IL-1ß, TNF-α, and IL-17 secretions were noted among CF patients according to their colonization status, which might induce a further destructive effect on airways, resulting in an unfavorable prognosis for children with CF who also have colonization.

Mesenchymal Stem Cells: a Potential Treatment Approach for Refractory Chronic Spontaneous Urticaria.

The etiopathogenesis of chronic spontaneous urticaria (CSU) is not fully elucidated, and almost 30-40% of patients are resistant to treatments; therefore, there is still a need for the development of new and effective treatments. This study aimed to develop experimental cellular therapy for CSU patients resistant to current treatment options. Autologous adipose tissue mesenchymal stem cells (MSC) were administered to 10 refractory CSU patients who were then followed up for six months. The efficacy of treatment was evaluated according to the weekly urticaria activity scores (UAS7) and drug use scores (DUS7). To observe the effect of treatment on immune cells, CD4+ T cell subsets were analyzed by flow cytometry, and the serum IFN-γ, TNF-α, IL2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17a, IL-21, IL-22, TGF-ß1, PGE2, IDO and anti-FcεRI levels were measured using the Luminex and ELISA methods. The values obtained were compared with 10 control refractory CSU patients and five healthy controls. We found that the T cell subsets and inflammatory molecules were not affected by MSC treatment during the follow-up period. In control patients, a significant decrease was detected only at the Th2 subset, TGF-ß1, PGE2, IDO and anti-FcεRI levels on the 14th day of treatment. The UAS7 and DUS7 values of the MSC-treated patients significantly decreased during the follow-up period, but in control patients, a significant but temporary decrease was seen. According to our findings, unlike conventional treatment, MSC therapy resulted in longer and more effective recovery. Our data indicate that MSCs may be an alternative and effective approach for treatment-resistant CSU patients. Graphical Abstract.

Expression of Akt1 and p-Akt1 in peripheral T cell subsets of multiple sclerosis patients.

Multiple sclerosis is an autoimmune disorder induced by the infiltration of autoreactive immune cells into the central nervous system. Akt/PKB signaling pathway is crucially involved in T cell development and survival. We aimed to determine whether Akt1 expression levels of regulatory T (Treg) cells are altered in MS and are associated with disease activity. Relapsing-remitting multiple sclerosis (RR-MS, n = 17) patients and healthy individuals (n = 20) were enrolled. Peripheral blood mononuclear cells were isolated and anti-CD3, -CD4, -CD8, -CD25, -CD127 monoclonal antibodies were used to identify the T cell subsets. After stimulation with phorbol myristate acetate/ionomycin, the Akt1 and phosphorylated-Akt1 (p-Akt1) levels of T cell subsets were detected with intracellular staining using flow cytometry. Total Akt1 and p-Akt1 expression levels were found to be suppressed in CD4+ T cell and Treg populations of RR-MS patients. Progression indices were positively correlated with Akt1 expression levels of Tregs indicating that the Akt pathway might partake in the progression of multiple sclerosis. Flow cytometry may effectively be used for the evaluation of the Akt pathway activity. Our findings suggest that the magnitude of suppression of the Akt pathway might serve as a biomarker for the prognosis of multiple sclerosis.