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.

Functionality of natural killer cells in obese asthma phenotypes.

BACKGROUND: Obesity-associated asthma (OA) is a difficult to treat asthma phenotype due to its severity and poor response to inhaled steroids. Early-onset allergic (EoOA) and late-onset non-allergic (LoOA) OA are suggested subtypes of this phenotype. Natural Killer (NK) cells are key elements of innate immunity involved in cytotoxicity and immune regulation, with uncertain role in OA pathogenesis. METHODS: Early-onset allergic and LoOA patients together with obese non-asthmatic (ONA) controls have been enrolled in the study. Peripheral blood samples have been collected for analysis. Percentages of total NK cells, CD3- CD56dim and CD3- CD56bright NK cell subsets, cytotoxic activity, intracellular interferon-γ, interleukin (IL)-10, IL-13, IL-17 secretion and activatory receptors (NKG2D, NKp46i and NKp44) have been investigated by flow cytometry. The effect of IL-12 and IL-23 stimulation on NK cells and intracellular cytokines in different groups have also been analysed and compared with unstimulated conditions. RESULTS: Results of ONA (n = 5, age 42 ± 8), EoOA (n = 5, age 42 ± 10) and LoOA (n = 8, age 46 ± 8) patients have analysed. Body Mass Index has been found to be negatively correlated with CD69 (p = .022, r = -0.534). NKG2D receptor has been significantly low in CD56dim cells of asthma population (p = .046). NKp44 receptor expression has increased after IL-12 stimulation in EoOA and control group (p = .02). Intracellular IL-10 content has increased in LoOA and control subjects (p = .018, p = .03) but not in the EoOA group. Intracellular IL-17 level has found be higher in allergic OA group. LoOA patients showed a decreased NK cytotoxicity compared with the early-onset asthma group (p = .05). CONCLUSION: Our study suggests an impaired NK receptor expression, activation and reduced cytotoxicity in OA patients together with variances between different subtypes of this phenotype. This data would be beneficial for tailoring a more personalized treatment strategy combatting steroid resistance and frequent exacerbations in this group of patients.

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.

Effects of different pulp-capping materials on cell death signaling pathways of lipoteichoic acid-stimulated human dental pulp stem cells.

PURPOSE: This study aimed to evaluate the response of dental pulp stem cells (DPSCs) cultured with and without lipoteichoic acid (LTA) to different pulp-capping materials. METHODS: The cells were cultured and seeded in 6-well plates and exposed to 1% LTA solution. Dycal, ProRoot MTA and Biodentine materials were applied on cells and all groups were evaluated by cell proliferation, viability, cell cycle and cell death signaling pathways for 24 and 72 h. RESULTS: LTA + Dycal treatment significantly inhibited the proliferation of DPSCs and increased the apoptosis rate of cells more than the other groups at 72 h. Compared to other groups, LTA + Dycal treatment significantly increased the levels of Caspase-3 and AKT and decreased the levels of p-AKT. CONCLUSIONS: The results of this study revealed that all tested materials caused apoptosis in DPSCs via an extrinsic apoptotic pathway. The DPSCs showed an early apoptosis response to the Dycal and a late apoptosis response to the ProRoot MTA and Biodentine treatments. LTA led autophagy and inhibited the proliferation of DPSCs. ProRoot MTA and Biodentin eliminated the LTA's bioactivity with higher efficiency than Dycal.

CD133+ cells are associated with ADIPOCYTOKINES and endothelial dysfunction in hemodialysis patients.

BACKGROUND: Hemodialysis (HD) patients have increased risk of cardiovascular disease (CVD). Impaired stem cell health and adipocytokine metabolism may play important roles in the complex pathophysiological mechanisms of CVD in this patient population. We aimed to investigate the relationships between CD133+ cell counts, adipocytokines and parameters of endothelial dysfunction and atherosclerosis in HD patients. METHODS: In 58 chronic HD patients (male/female:28/30, mean age:58 ± 14 years), serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), leptin, adiponectin and resistin were measured by ELISA. Left ventricular mass index (LVMI), carotid intima-media thickness (CIMT), flow-mediated dilatation (FMD) of the brachial artery were measured. CD133+ cells were counted by flow cytometry (BD FACSCalibur-BD Bioscience,CA). RESULTS: CD133+ cell counts were inversely associated with FMD (r = -0.39, p = 0.007) and positively correlated with serum resistin (r = 0.45, p < 0.001) and serum TNF-α (r = 0.31, p = 0.02). Serum leptin levels were higher in high CD133 group compared to low CD133 group [32.37(12.74-72.29) vs 15.50(5.38-37.12)ng/mL, p = 0.03]. Serum leptin levels were correlated with TNF-α(r = 0.35, p = 0.009). Serum adiponectin levels were negatively correlated with serum leptin (r = -0.28, p = 0.03). Serum resistin levels were associated with TNF-α (r = 0.54, p < 0.001) and leptin (r = 0.29, p = 0.03). Serum IL-6 levels were significantly associated with LVMI (r = 0.31, p = 0.03). Serum IL-6 levels were significantly higher in patients with carotid plaque compared to patients without plaque [12.75(9.91-28.68) vs 8.27(5.97-14.04) pg/mL, p = 0.02]. In multiple linear regression analysis to determine the factors predicting LogFMD; dialysis vintage, LVMI and LogCD133+ cell counts were included as independent variables(R = 0.57, adjusted R-square = 0.27, p = 0.001). CD133+ cell count and LVMI were found to significantly predict FMD (p = 0.03 and p = 0.04 respectively). CONCLUSION: CD133+ cells were associated with inflammation and endothelial dysfunction in HD patients. Serum leptin, resistin and TNF-α levels were positively related to CD133+ cell count. Impaired regulation of undifferentiated stem cells and adipocytokines might contribute to endothelial dysfunction in HD patients.

Risk factors for persistence of asthma in children: 10-year follow-up.

OBJECTIVE: Risk factors related to the outcome of childhood asthma are not yet well established. We aimed to investigate the long-term outcome for children with asthma to determine the risk factors in predicting persistence of disease. METHODS: Sixty-two children with asthma were evaluated retrospectively at the end of a 10-year follow-up. Patients were asked to complete a questionnaire requesting clinical information, and underwent physical examination, skin prick testing, a pulmonary function test and bronchial provocation testing. Immunologic parameters evaluated were allergen-specific IgE and IgG4 levels, and allergen-induced generation of CD4(+)CD25(+) cells. RESULTS: Mean age at final assessment was 15.9 ± 3.6 years, and duration of follow-up was 10.30 ± 1.27 years. Fifty percent of patients outgrew their asthma during the 10-year follow-up period. All the non-atopic patients outgrew their disease during the study period, whereas 67% of atopic patients did not. We identified two risk factors independently related to the persistence of symptoms: presence of bronchial hyper-responsiveness and presence of rhinitis. Atopic children who were in remission demonstrated significantly higher allergen-induced CD4(+)CD25(+) T cells compared to healthy controls. CONCLUSIONS: Atopy, presence of rhinitis, positive and presence of bronchial hyper-reactivity are important risk factors for the persistence of asthma in children. Allergen-induced CD4(+)CD25(+) T cells were higher in the atopic children who outgrew their disease, implicating an immunological mechanism of asthma remission in children.

Decrease in endothelial progenitor cells associated with inflammation, but not with endothelial dysfunction in chronic hemodialysis patients.

INTRODUCTION: Endothelial progenitor cells (EPC), bone marrow derived cells, are considered to have a pivotal role in maintaining the integrity and repair of the endothelium. Endothelial dysfunction, atherosclerosis and inflammation are implicated for increased CV mortality in uremia. In this study, we aimed to investigate the possible association of EPC with inflammation, endothelial dysfunction and atherosclerosis in chronic hemodialysis (HD) patients. PATIENTS AND METHODS: 67 HD patients (male/female: 30/37, mean age: 58 ± 15 years) and 22 healthy controls (male/female: 13/9; mean age: 48 ± 8 years) were included. EPC were cultivated in the fibronectin-covered culture dishes and counted. Also EPC markers were studied by flow cytometry using anti-CD34, anti-CD133 and anti-vascular endothelial growth factor receptor 2 (VEGFR-2) antibodies. Serum levels of IL-6, TNF-α, intercellular cell adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM) and asymmetric dimethyl-arginine (ADMA) were measured by ELISA method. Endothelial function was investigated by measuring flow-mediated dilatation (FMD) of the brachial artery. Carotid intima-media thickness (CIMT) and ratio (CIMR) were also examined. RESULTS: EPC number was decreased in HD patients when compared to controls (63.7 ± 8.9 vs. 101.5 ± 19.6/ high power field, p < 0.001). Also CD34+ cell count was significantly lower in the HD group (2.26 ± 3.52 vs. 6.03 ± 4.73%, p < 0.0001). EPC number was significantly inversely correlated with serum TNF-α levels in HD patients(r: -0.453, p < 0.001) and also in the control group (r = -0.509, p = 0.044). There was an inverse association between VEGFR-2+/CD34+cell count and serum IL-6 levels (r: -0.364, p = 0.006) in HD patients. However, EPC count was not related to FMD and CIMT/CIMR. In HD patients, there was a positive correlation between serum IL-6 levels with CIMT (r = 0.358, p = 0.01) and CIMR was positively correlated with serum ICAM (r = 0.430, p = 0.002). CONCLUSION: EPC number was decreased in uremia and was associated with inflammation. TNF-α might have specific inhibitory actions on EPC in both HD patients and healthy controls. No relationship was present between EPC and endothelial dysfunction/atherosclerosis.

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.

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 modulatory action of C-Vx substance on the immune system in COVID-19.

The modulatory effect of C-Vx, a novel therapeutic agent, on the immune system of COVID-19 patients was investigated. The functions of T and NK cells of COVID-19 patients with different disease severity were evaluated by flow cytometry in response to C-Vx stimulation. The levels of pro- and anti-inflammatory cytokines were detected by multiplex assay in supernatants after cell culture with C-Vx. Bradykinin, IRF3, and IFN-α levels were also measured by ELISA in the presence or absence of C-Vx stimulation. As a result, increased CD107a expression was observed on NK cells in response to C-Vx addition. The proliferation of T cell subsets was increased by C-Vx, decreasing by disease severity. IL-4 and IL-10 levels were elevated while IFN-γ and IL-17 levels were reduced in T cells following C-Vx stimulation. However, the levels of pro-inflammatory IL-1ß, IL-6, IL-8, IFN-γ and GM-CSF were significantly increased upon C-Vx stimulation. IFN-α levels tended to increase after incubation with C-Vx. These findings support an immunomodulatory action of C-Vx on the immune system of patients with a mild and moderate phase of COVID-19.

Decreased therapeutic effects of noscapine combined with imatinib mesylate on human glioblastoma in vitro and the effect of midkine.

BACKGROUND: Glioblastoma (GBM) develops resistance to the advances in chemotherapy leading to poor prognosis and life quality. Consequently, new treatment modalities are needed. Our aims were to investigate the effects of combined noscapine (NOS) and imatinib mesylate (IM) on human GBM in vitro and the role of midkine (MK) in this new combination treatment. METHODS: Monolayer and spheroid cultures of T98G human GBM cell line were used to evaluate the effects of IM (10 µM), Nos (10 µM) and their combination on cell proliferation and apoptotic indexes, cell cycle, the levels of antiapoptotic MK, MRP-1, p170, PFGFR-α, EGFR, bcl-2 proteins, apoptotic caspase-3 levels, morphology (SEM) and ultrastructure (TEM) for 72 hrs. Results were statistically analyzed using the Student's t-test. RESULTS: The combination group induced highest decrease in cell proliferation and apoptotic indexes, caspase-3 levels, MRP-1 and PDGFR-α levels. The decrease in p170 levels were lower than IM but higher that NOS. The highest increases were in EGFR, MK, bcl-2 and cAMP levels in the combination group. The G0+G1 cell cycle arrest at the end of 72nd hr was the lowest in the combination group. Apoptotic appearence was observed rarely both in the morphologic and ultrastructural evaluation of the combination group. In addition, autophagic vacuoles which were frequently observed in the IM group were observed rarely. CONCLUSIONS: The combination of Nos with IM showed antagonist effect in T98G human GBM cells in vitro. This antagonist effect was correlated highly with MK levels. The effects of NOS on MRP-1, MK and receptor tyrosine kinase levels were firstly demonstrated in our report. In addition, we proposed that MK is one of the modulator in the switch of autophagy to cell death or survival/resistance.

Differences in lymphocyte subpopulation count and function in cord, maternal and adult blood.

OBJECTIVE: Phenotypical characterization and functional activity of lymphocytes and natural killer (NK) cells in cord blood (CB) were investigated, and maternal peripheral blood (MPB) values were compared to those of adult peripheral blood (APB) (control). METHODS: To determine cytotoxic activity target cells (K562) were labeled with carboxyfluorescein diacetate (CFDA) or fluorescein isothiocyanate (FITC), and propidium iodide (PI) was used to label dead cells. Cell surface expression in CB, APB, and MPB cells were analyzed using flow cytometry. RESULTS: CB and MPB mononuclear cells had similar CD45, CD34, CD4, and surface molecule for T helper cell expression, but had low-level expression of total T-lymphocyte surface molecules CD3 and CD8. CD19 and HLA-DR expression was higher in CB than in MPB. The same high-level of expression for CD19 and HLA-DR was observed in APB, as compared to MPB. All other cell surface expressions were similar in APB and MPB samples. NK (CD16+ and CD56+) cells in CB was similar to that in MPB and APB, and the level of inhibitory KIR receptors in NK cells was higher in venous CB than in MPB and APB. The only difference between MPB and APB was that the CD158a level was higher in MPB. No difference was observed in NK cells in CB and MPB, in terms of cytotoxicity. CONCLUSION: The present results show that there was numerical and proportional variability of lymphocytes and their subgroups in CB and APB, but no cytological difference.

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.

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.

Enhancement of vinorelbine-induced cytotoxicity and apoptosis by clomipramine and lithium chloride in human neuroblastoma cancer cell line SH-SY5Y.

The aim of this work is to investigate whether clomipramine (CIM) and lithium chloride (LiCl) potentiate the cytotoxicity of vinorelbine (VNR) on SH-SY5Y human neuroblastoma cells in vitro and whether midkine (MK) can be a resistance factor for these treatments. Four groups of experiments were performed for 96 h using both monolayer and spheroid cultures of SH-SY5Y cells: (1) control group, (2) singly applied VNR, CIM, and LiCl, (3) VNR with CIM, and (4) VNR with LiCl. Their effects on monolayer and spheroid cultures were determined by evaluating cell proliferation, bromodeoxyuridine labeling index (BrdU-LI), apoptosis, cyclic adenosine monophosphate (cAMP) and midkine levels, colony-forming efficiency, spheroid size, and ultrastructure. In comparison with the control group, single and combination drug treatments significantly reduced the proliferation index (PI) for 96 h. The most potent reduction of PI was observed with VNR in combination with CIM and LiCl for all time intervals. VNR with CIM and LiCl seemed to be ineffective in reducing BrdU-LI of both monolayer cell and spheroid cultures, spheroid size, and cAMP level. VNR with LiCl increased apoptosis at 24 h, however VNR with CIM increased apoptosis at 96 h. VNR was the most potent drug in inhibiting colony-forming efficiency. The combination of VNR with CIM was the most potent in reducing midkine levels among all groups. Interestingly, the combination of VNR with LiCl led to both nuclear membrane breakdown and disappearance of the cellular membranes inside the spheroids. Both CIM and LiCl seemed to potentiate VNR-induced cytotoxicity, and MK was not a resistance factor for VNR, LiCl, and CIM.

Cytotoxic effects of different self-adhesive resin cements: Cell viability and induction of apoptosis.

PURPOSE: The effects of four different self-adhesive resin cement materials on cell viability and apoptosis after direct and indirect exposure were evaluated using different cell culture techniques. MATERIALS AND METHODS: Self-adhesive cements were applied to NIH/3T3 mouse fibroblasts by the extract test method, cell culture inserts, and dentin barrier test method. After exposure periods of 24 h and 72 h, the cytotoxicity of these self-adhesive materials was evaluated using the MTT assay (viability) and the Annexin-V-FITC/PI staining (apoptosis). RESULTS: The lowest cell viability was found in cells exposed to BeautiCem SA for 24 h in the extract test method. Cell viability was reduced to 70.6% compared to negative controls. After the 72 h exposure period, viability rate of cell cultures exposed to BeautiCem SA decreased more than 2- fold (29.5%) while cells exposed to RelyX U200 showed the highest viability rate of 71.4%. In the dentin barrier test method, BeautiCem SA induced the highest number of cells in apoptosis after a 24 h exposure (4.1%). Panavia SA Cement Plus was the material that caused the lowest number of cells in apoptosis (1.5%). CONCLUSION: The used self-adhesive cements have showed different cytotoxic effects based on the evaluation method. As exposure time increased, the materials showed more cytotoxic and apoptotic effects. BeautiCem SA caused significantly more severe cytotoxic and apoptotic effects than other cements tested. Moreover, cements other than BeautiCem SA have caused necrotic cell death rather than apoptotic cell death.

Relationship between CD107a expression and cytotoxic activity.

NK cells play important roles in innate immunity against tumors and infections of the host. Studies show that CD107a (LAMP-1) may be a marker for degranulation of NK and activated CD8+ T cells. In our study, the relationship between the expression of CD107a, cytokine secretion and cytotoxic activity in CD56+ NK, CD8+ T cells and lymphocytes has been determined after various stimuli. Effector cells from PBMCs of healthy subjects were isolated and K562 cell line was used as target of cytotoxicity. IL-2 stimulation resulted in a significant increase of CD107a expression in CD56+ NK, CD8+ T cells and lymphocytes. Increased expression of CD107a after IL-2 stimulation of NK cells was parallel to the increase of cytotoxicity. Our results suggest that CD107a expression may be a sensitive marker for the cytotoxic activity determination.

Interleukin-10 is increased in successful drug desensitization regardless of the hypersensitivity reaction type.

BACKGROUND: Little is known about the mechanism of desensitization in hypersensitivity drug reactions. OBJECTIVE: The aim of this study was to evaluate the effects of drug desensitization on some cytokine levels in patients desensitized for drug hypersensitivity reactions. METHODS: Patients with a hypersensitivity reaction to any drug for whom desensitization was planned with the culprit drug, patients who could tolerate the same drugs and healthy subjects who were not exposed to these drugs were enrolled. Bead-based Milliplex MAP multiplex technology was used to determine interleukin (IL)-4, IL-5, interferon-γ and IL-10 levels in the sera of the subjects as a baseline and 24 hours after desensitization had been completed in the patients. RESULTS: A total of 26 patients (16 female [61.5%]; mean age 48.46 ± 15.97 years old), 10 control patients (5 female [50%]; mean age 47.4 ± 15.4 years old) and 5 healthy subjects (3 female [60%]; mean age 34.2 ± 5.6 years old) were enrolled. Four of the 26 patients did not tolerate the procedure and were grouped as the 'unsuccessful desensitization group' whereas 22 patients successfully completed the procedure and formed the 'successful desensitization group.' Baseline cytokine levels in the 3 groups were not statistically different. Postdesensitization IL-10 levels in the successful desensitization group were significantly higher than their initial levels (p = 0.005) whereas none of the cytokine levels significantly changed in the unsuccessful desensitization group. The rise in IL-10 levels was greater in chemotherapeutic desensitizations when compared to other drugs (p = 0.006). CONCLUSION: Successful desensitization independent of the hypersensitivity reaction type seems to be related to the increase of IL-10.

The role of atopy in the pathogenesis of bleomycin pulmonary toxicity.

INTRODUCTION: Bleomycin pulmonary toxicity (BPT) is a potentially life-threatening consequence of bleomycin usage in patients. An overproduction of epithelium-derived cytokines, habitually linked to allergic inflammation, has been recently revealed in experimental models of BPT. METHODS: We assessed retrospectively our cohort of patients with Hodgkin Lymphoma treated with bleomycin between 2014 and 2016 for their demographic, clinical features, including BPT development, atopy status and risk factors for BPT. Then they were invited for allergy testing and blood sample collection. The samples were stimulated with different stimuli (Bleomycin, IL-33, TSLP) for 24 h on cell culture. The culture supernatants were analysed for TGF-ß, Galectin3, Arginin, Amphiregulin, Eotaxin, IFNγ, TNFα, IL1ß, 4, 5, 6, 10, 13, 17, MIP-1α, and bleomycin hydrolase (BLH) levels. RESULTS: The cohort consisted of 51 patients showed that atopy was the only significant risk factor for BPT occurrence (OR: 7.2, p = 0.007). Fourteen subjects were included for blood analysis. The analysis of supernatants at the unstimulated condition revealed that BLH and Amphiregulin were significantly lower in patients who had BPT than controls. The BLH cut-off that best identified a history of BPT was 175.31 (Sensitivity: 62.5%, specificity: 100%). Following the stimulation, BLH reduced compared to the unstimulated condition and the difference between groups remained significant (p < 0.05). CONCLUSION: Our study is the first to report that low levels of bleomycin hydrolase in allergic individuals may be predisposing to a possible pathway of fibrosis.

Potentiation of cytotoxicity by combination of imatinib and chlorimipramine in glioma.

Rat C6 glioma is a chemo-resistant experimental brain tumor that is difficult to treat with various drug combinations. Previous studies suggested that imatinib mesylate (Gleevec) is effective in pre-clinical trials for glioblastoma. Also, chlorimipramine (Anafranil) is an anti-depressant drug in use in the clinic and shown to have anti-neoplastic activity. We hypothesized that treatment of resistant C6 glioma with combination of imatinib and chlorimipramine may potentiate cytotoxicity and reverse resistance. C6 glioma was examined both as monolayer and as spheroid cultures. Several experimental designs were examined all of which showed synergistic activity albeit at different time kinetics. Combination treatment resulted in inhibition of cell growth and enhanced cell death as determined by dye exclusion. Further, the combination treatment resulted in significant induction of apoptosis as determined by Annexin V-FITC and PI. Also, there was inhibition of DNA synthesis and cAMP. Altogether, these findings supported the anti-proliferative and cytotoxic effects of the combination treatment. Morphological studies were also performed using transmission and scanning electron microscopy. Significant synergistic apoptosis was detected by the combination treatment in both the monolayers and spheroid cultures. There was also a synergistic effect in autophagy by the combination. Several altered morphological features were noted by both the individual compound and enhanced by the combination treatment. The present findings support our hypothesis and demonstrate the potentiation of cytotoxicity by the combination of imatinib and chlorimipramine in C6 glioma. Further, the findings suggest the potential clinical application of the combination in the treatment of drug-resistant glioma.