Effects of 15-lipoxygenase overexpressing adipose tissue mesenchymal stem cells on the Th17 / Treg plasticity.

15-lipoxygenase (15-LOX) is a critical enzyme that allows the direction of arachidonic acid metabolism to change from inflammation into the resolution. This study aims to reveal how the immunomodulation properties of mesenchymal stem cells (MSC) alter by the 15-LOX overexpression. For this purpose, peripheral blood mononuclear cells (PBMCs) isolated from seven healthy volunteers, and both MSCs and 15-LOX overexpressing MSCs (15-LOXMSCs) were co-cultured at different cell ratios (1/1, 1/5 and 1/10). Alterations of CD4+Tbet+, CD4+Gata3+, CD4+RoRC2+, and CD4+FoxP3+ lymphocyte frequencies were detected by flow cytometry, and IFN-γ, IL-4, IL-6, IL-10, IL-17a, TGF-ß and LXA4 levels of medium supernatants were measured by ELISA method. According to our findings, MSC and 15-LOXMSCs have a suppressive effect on PHA activated PBMCs. However, as the ratio of PBMCs increased, the effects of 15-LOXMSCs increased significantly, while the effects of MSCs decreased. The most notable effect of the 15-LOX modification was the significant reduction in IL-6, IL-10 and IL-17a expression and the accompanying increase in TGF-ß and LXA4 levels. We also observed a similar situation between CD4+RoRC2+ and CD4+FoxP3+ cell frequencies. These data suggested that the effects of MSCs on the balance of Th17 / Treg could change by the 15-LOX overexpression, and this might be in favor of the Treg cells.

Low LXA4, RvD1 and RvE1 levels may be an indicator of the development of hypertension.

Vascular structure and integrity are at the forefront of blood pressure regulation. However, there are many factors that affect the responses of the vessels. One of these is the inflammatory processes associated with high cholesterol and its modification. 15-lipoxygenase (15-LOX) is the critical enzyme in cholesterol oxidation, but this enzyme is also responsible for the synthesis of specialized proresoving lipid mediators (SPMs) called Lipoxin (Lxs) and Resolvin (Rvs). In this study, we determined serum LXA4, RvD1 and RvE1 levels in newly diagnosed hypertension (HT) and normotension (NT) cases. We evaluated how the presence of hypercholesterolemia (HC) in the follow-up changes the levels of these SPMs. We found that the three SPMs we measured decreased significantly in the presence of HC. In addition, we found a negative and significant correlation with systolic blood pressure and total cholesterol levels for the three SPMs. In conclusion, HT and HC are independent risk factors for cardiovascular death. However, the presence of HC may be an important factor for the development of HT. Increasing cholesterol levels may cause 15-LOX to shift towards LDL oxidation, thus leading to inflammation. This situation may negatively affect the vascular functions in the regulation of blood pressure. Serum LXA4, RvD1 and RvE1 measurements may provide clues that represent a shift of 15-LOX enzyme activity towards cholesterol.

Age-related changes in the immunomodulatory effects of human dental pulp derived mesenchymal stem cells on the CD4+ T cell subsets.

Mesenchymal stem cells (MSCs) are powerful immunomodulatory cells. The effects of the aging on these abilities of MSCs have not been adequately clarified. In this study, alterations in immunomodulatory abilities of MSCs caused by aging were investigated. For this, dental pulp (DP) MSCs and peripheral blood mononuclear cells (PBMCs) of elderly and young donors were co-cultured age-matched and cross. We detected that the effects of DP-MSCs on Th1 and Th2 cells and their specific cytokines IFN-γ and IL-4 are not affected by aging. However, we observed that young and elderly DP-MSCs have different effects on Th17 and Treg cells. Th17 frequencies of young and elderly PBMCs were significantly increased only by young DP-MSCs, in contrast, Treg frequencies were significantly increased by elderly DP-MSCs. IL-6, IL-17a and HGF levels of both young and elderly PBMCs showed a significant increase only by young DP-MSCs, but TGF-ß levels were significantly increased only by elderly DP-MSCs. The oral cavity is home to a rich microflora. The interactions of dental tissues with this microflora can lead them to acquire different epigenetic modifications. Aging can affect the microflora composition of the oral cavity and change this process in different directions. According to our findings, DP-MSCs are effective cells in the regulation of CD4+ T cells, and their effects on Th1 and Th2 cells were not affected by aging. However, pleiotropic molecules IL-6 and HGF expressions, which are important in dental and bone tissue regeneration, decreased significantly in elderly DP-MSCs. This situation may have indirectly made a difference in the modulation effects of young and elderly DP-MSCs on the Th17 and Treg cells.

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.

Low levels of pro-resolving lipid mediators lipoxin-A4, resolvin-D1 and resolvin-E1 in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a disease in which joint inflammation is at the forefront but the whole body is affected, and prevention of inflammation is the main treatment approach. Lipoxins (LXs) and resolvins (Rvs) are critical molecules in the resolution of inflammation. In this study, we aimed to investigate the role of LXs and Rvs in the RA pathogenesis. To this end, we measured the LXA 4, RvD 1, RvE 1 levels, and inflammatory cytokines and chemokines IL-6, IL-8, IL-10, IL-17a, IL-22 and MCP-1 in patients with RA and healthy individuals. We found that the LXA4, RvD1, RvE1 levels of the active RA cases were significantly lower than in remission RA and healthy individuals, but the levels of inflammatory cytokines and chemokines were significantly higher. The decreases in LXs and Rvs were independent of disease activity, suggesting that there might be an impairment of LX and Rvs synthesis or catabolism in patients with RA.

The investigation of immunomodulatory effects of adipose tissue mesenchymal stem cell educated macrophages on the CD4 T cells.

Mesenchymal stem cells (MSCs) are strong immunomodulatory cells investigated in numerous clinical studies on fatal pathologies, such as graft versus host disease and autoimmune diseases; e.g., systemic lupus erythematosus, Crohn's disease, and ulcerative colitis. Macrophages are one of the critical cells linking the innate and adaptive immune system, and it has been shown that MSCs can differentiate between pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype of macrophages. However, it has not yet been fully clarified whether these differentiated macrophages are functional. In this study, we compared the immunomodulatory effects on the CD4 T cells of M1, M2a and M2c macrophages with the macrophages that directly and indirectly cultured with MSCs. We analyzed the changes in CD14, CD64, CD80, CD163 and CD200R expression to evaluate macrophage phenotypes, and the changes in CD4, IFN-g, IL-4, IL-17a and FoxP3 expression to evaluate T helper subsets using the FACS method. The changes in IL-1b, IL-4, IL-10, IL-12p70, IL-17a and IFN-g in the media supernatants were analyzed using the Luminex method. We also performed WST-1 and Caspase-3 ELISA analyses to observe the proliferation and apoptosis status of the T cells. MSCs were found to differentiate macrophages into a distinctive phenotype, which was close to the M2c phenotype, but was not considered as an M2c cell due to the low expression of CD163, a characteristic marker for M2c. While MEM-D, MEM-ID and MSCs showed similar inhibitory effects on the Th2 and Th17 cells, the most significant increase in Treg cell frequencies was seen in MEM-D cells. Macrophages can alter their phenotypes and functions according to the stimuli from the environment. The fact that macrophages educated with MSCs suppressed the production of all the cytokines we evaluated even after the removal of MSCs suggests that these cells may be differentiated by MSCs into a suppressive macrophage subgroup. However, the Treg cell activation caused by direct interactions between MSCs and macrophage cells may be the most prominent observation of this study compared to previous work. As a result, according to our data, the interactions between MSCs and macrophages may lead to differentiation of macrophage cells into an immunosuppressive phenotype, and these macrophages may suppress the T lymphocyte subgroups at least as effectively as MSCs. However, our data obtained from in vitro experiments should be supported by future in vivo studies.

A comparison of cancer stem cell markers and nonclassical major histocompatibility complex antigens in colorectal tumor and noncancerous tissues.

Colorectal carcinoma (CRC) is one of the most fatal types of cancer in both women and men, and, unfortunately, patients are often diagnosed at an advanced stage. Cancer stem cells (CSCs) are associated with poor prognosis, metastasis, and recurrence, as well as chemotherapy and radiotherapy resistance. Therefore, different treatment alternatives are needed to facilitate the elimination of CSCs. One such approach is immunotherapy; however, tumor cells can evade immune cells by alteration of the expression patterns of human leukocyte antigens (HLA). In this study, we immunohistochemically investigated the expression patterns of CSC-specific markers CD44, CD133, Nanog, and Oct3/4, and immunosuppressive molecules HLA-G and -E in advanced CRC tumor tissues and noncancerous colon biopsies. We found significantly increased CD44, Nanog, Oct3/4, HLA-G, and HLA-E expression in the CRC tumor tissues compared with the noncancerous colon biopsies. These findings suggest that some tumor cells may be CSC-like and that the increased expression of HLA-G and HLA-E may be considered as an immune-evasive adaptation. Therefore, the nonclassical major histocompatibility complex class Ib antigens HLA-G and HLA-E may be potential targets in the elimination of CRC-CSCs. However, more detailed studies are required to support our findings.

The paracrine immunomodulatory interactions between the human dental pulp derived mesenchymal stem cells and CD4 T cell subsets.

Mesenchymal stem cells (MSCs) have strong immunomodulatory properties, however these properties may show some differences according to the tissue type of their isolate. In this study we investigated the paracrine interactions between human DP derived MSCs (hDP-MSCs) and the CD4+ T helper cell subsets to establish their immunomodulatory mechanisms. We found that the CD4+-Tbet+ (Th1) and CD4+-Gata3+ (Th2) cells were suppressed by the hDP-MSCs, but the CD4+-Stat3+ (Th17) and CD4+-CD25+-FoxP3+ (Treg) cells were stimulated. The expressions of T cell specific cytokines interferon gamma (IFN-g), interleukin (IL)-4 and IL-17a decreased, but IL-10 and transforming growth factor beta-1 (TGF-b1) increased with the hDP-MSCs. The expressions of indoleamine-pyrrole 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), soluble human leukocyte antigen G (sHLA-G) derived from hDP-MSCs slightly increased, but hepatocyte growth factor (HGF) significantly increased in the co-culture groups. According to our findings, the hDP-MSCs can suppress the Th1 and Th2 subsets but stimulate the Th17 and Treg subsets. The Stat3 expression of Th17 cells may have been stimulated by the HGF, and thus the pro-inflammatory Th17 cells may have altered into the immunosuppressive regulatory Th17 cells. Further prospective studies are needed to confirm our findings.

The association of HLA-G and immune markers in recurrent miscarriages.

OBJECTIVE: To determine role of human leukocyte antigen (HLA)-G, CD8, CD16, CD56, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α for recurrent miscarriages in feto-maternal interface. METHOD: Chorion and decidua samples were obtained from 11 women with unwanted pregnancies (healthy pregnancy, HP) and 10 women with missed abortion diagnosis after at least two pregnancy losses (recurrent miscarriage, RM). In addition, endometrial tissues were obtained from 10 non-pregnant women (NonP). The expressions of markers were evaluated using the Western blot analysis. The values obtained between different groups were compared. RESULTS: The highest protein expression of CD56 was found in the HP compared to NonP and RM. Meanwhile, the lowest protein expression of CD16 was observed in the NonP compared to HP and RM. The HLA-G expression exhibited the highest level in HP; however, there was no statistically significant difference between groups. CD8 and IFNγ expressions were lowest in the NonP group; however, TNF-α was highest in the RM group. CONCLUSIONS: The CD56 expression of uterine NK cells may be an indicator of a HP. However, not statistically significant, the increased expression of CD16, CD8, and also significantly increased expression of TNF may be associated with the predominant cytotoxic activity in the maternal immune system in patients with RM. Although there was no change in the expression of HLA-G, this finding may mean that the maternal immune system is unresponsive to HLA-G-mediated immunosuppressive signals originating from the fetus in these cases.

Prognostic value of serum pregnancy-associated plasma protein A level at the initial ED presentation in elderly patients with CAP.

OBJECTIVE: This study aims to compare serum pregnancy-associated plasma protein A (PAPP-A) levels in surviving and nonsurviving elderly patients with community-acquired pneumonia (CAP), investigating whether PAPP-A is correlated with CAP prediction scores and whether PAPP-A can successfully predict 28-day mortality rates in elderly patients. METHODS: This prospective, observational, single-center, cross-sectional study was conducted at the emergency department (ED) of Celal Bayar University Hospital in Manisa, Turkey, between January and September 2014. All patients underwent follow-up evaluations 28 days after admission. The end point was defined as all-cause mortality. RESULTS: A total of 100 elderly patients (mean age, 77.3 ± 7.6 years [range, 65-94 years]); 60% men) with CAP were enrolled in this study. All-cause mortality at the 28-day follow-up evaluation was 22%. Admission PAPP-A levels were significantly higher in nonsurvivors compared with 28-day survivors (10.3 ± 4.5 vs 3.8 ± 2.6 ng/mL, P < .001). A significant and positive correlation between admission PAPP-A levels and pneumonia severity index; confusion, oxygen saturation, respiratory rate, blood pressure, and age 75 years or older; and confusion, urea, respiratory rate, blood pressure, and age older than 65 years scores was found (r = .440, P < .001; r = .395, P < .001; and r = .359, P < .001, respectively). Moreover, we determined that the optimal PAPP-A cutoff for predicting 28-day mortality at the time of ED admission was 5.1 ng/mL, with 77.3% sensitivity and 77.9% specificity. CONCLUSIONS: Serum PAPP-A level is valuable for predicting mortality and the severity of the disease among elderly patients with CAP at ED admission. Thus, PAPP-A might play a further role in the clinical assessment of the severity of CAP.

The efficacy of complete blood count parameters in the diagnosis of tubal ectopic pregnancy.

OBJECTIVE: Ectopic pregnancy (EP) is the major cause of maternal morbidity and is responsible for maternal mortality in the first trimester. In order to reduce undesirable results, it is necessary to find rapid and accurate, non-surgical diagnostic tests for ER The goal of the study was to investigate the differences in complete blood count parameters between tubal EPs and healthy pregnancies in be used in the diagnosis of ectopic pregnancy. STUDY DESIGN: White blood cell (WBC), neutrophil, monocyte, lymphocyte, platelet (PLT) counts, mean PLT volume (MPV) and PLT distribution width (PDW) levels in the complete blood count samples have been obtained from subjects with diagnosed tubal EP (n=78; study group) and women with healthy intrauterine gestations (n=79; control group). Statistical comparisons between groups were performed using the t test. RESULTS: PDW levels were found to be significantly higher in the control group than EP (p<0.001). However no differences between the study and control groups with regard to PLT and MPV levels were observed. WBC levels were found to be significantly higher in the EP group as compared to controls (p<0.001). When leukocyte differentials were compared, monocyte counts in the EP group were significantly higher than in controls (p=0.005). No statistically significant differences in neutrophil and lymphocyte values were observed in either group. CONCLUSION: PDW as an indicator of PLT activation is lower in tubal EP than intrauterine pregnancy so, possibly endometrial invasion in the intrauterine pregnancy needs more PLT activation. Monocyte counts are higher in tubal EP, indicating that monocyte activation in the pathophysiology of EP could be effective in the formation of tubal motility and microenvironment regulation.

The Val762Ala polymorphism in the poly(ADP-ribose) polymerase-1 gene is not associated with susceptibility in Turkish rheumatoid arthritis patients.

The findings of the studies on poly(ADP-ribose) polymerase-1 (PARP-1) have suggested that the enzyme inactivation provides significant protection against systemic or tissue inflammation in animal models. It has also shown that the single-nucleotide polymorphism (Val762Ala) of the PARP-1 causes about 40% decrease of enzyme activity. The aim of this study was to analyze the association of the PARP-1 Val762Ala polymorphism in Turkish patients with rheumatoid arthritis. A total of 128 RA patients and 165 normal controls from the same geographic region were studied and polymerase chain reaction (PCR)-based restriction analysis was used to identify Val762Ala polymorphism of the PARP-1. Association analyses were performed using chi (2) tests. Our results indicated that the distribution of the PARP-1 genotypes and alleles did not differ significantly among subjects with or without RA (P > 0.05). The results of the study indicate that, for our Turkish sample, the V762A polymorphism of the PARP-1 may not be involved in susceptibility to RA, implying that the polymorphism may not function as a candidate gene marker for screening RA patients.