Vitamin D3 Controls TLR4- and TLR2-Mediated Inflammatory Responses of Endometrial Cells.

OBJECTIVES: Vitamin D has potent immunoregulatory features and modulates innate and adaptive immune responses. There is a significant association between intrauterine infection-associated inflammatory responses and pregnancy complications such as abortion and preterm labor. Here, we investigated how 1,25 (OH)2 D3 could modulate inflammatory responses of endometrial cells. DESIGN: This is an in vitro experimental study. Endometrial stromal cells (ESCs) and whole endometrial cells (WECs) were collected from 15 apparently normal women, and the immunomodulatory effects of 1,25 (OH)2 D3 on lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-treated ESCs and WECs were investigated. Participants/Materials, Setting, and Methods: Women with no history of abortion, infertility, endometriosis, or sign of vaginal infection were enrolled in this study. Endometrial samples were collected by gynecologists using a Pipelle pipette in the proliferative phase of the menstrual cycle. WECs and ESCs were collected and treated with either LPS or LTA. The levels of IL-6, IL-8, and TNF-α in culture supernatants were quantified using the ELISA technique. TLR2, TLR4, and MyD88 expressions were assessed by RT-qPCR. TLR4 expression at the protein level was studied by the Western blot technique. RESULTS: 1,25 Dihydroxycholecalciferol (1,25 (OH)2 D3) significantly reduced TNF-α production in LPS-activated ESCs and TNF-α and IL-6 production by LTA-stimulated WECs. In contrast, 1,25 (OH)2 D3 pretreatment increased the production of IL-8 by LPS- and LTA-stimulated endometrial cells. 1,25 (OH)2 D3 pretreatment markedly reduced LPS-induced TLR4 protein expression by ESCs. LPS treatment of ESCs significantly induced MyD88 gene expression. This effect was reversed when these cells were pretreated with 1,25 (OH)2 D3 before stimulation with LPS. LIMITATIONS: Because of the small size of samples, doing experiments all together on some samples was not feasible. Confirmation of the results obtained here needs well-designed in vivo studies. CONCLUSIONS: 1,25 (OH)2 D3 is an immunomodulatory molecule essential for maintaining endometrial immune homeostasis by controlling potentially harmful inflammatory responses associated with female reproductive tract infections.

Association Study of Single Nucleotide Polymorphisms of Endoplasmic Reticulum Aminopeptidase 1 and 2 Genes in Iranian Women with Preeclampsia.

BACKGROUND: Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and 2) are involved in blood pressure regulation and single nucleotide polymorphisms (SNPs) of these genes have been linked to preeclampsia. This study intended to assess the association of ERAP1 and 2 genes polymorphism with Iranian preeclamptic women. METHODS: In this case-control study, 148 preeclamptic and 133 pregnant women were selected from the Kosar Hospital, Qazvin, Iran, during 2013-2015. In order to genotype the subjects for rs28096, rs30187, rs26653, rs3734016, rs34750 and rs2549782, rs17408150 for ERAP1 and 2 genes, respectively, Real-Time PCR allelic discrimination approach was exploited. RESULTS: Neither allelic nor genotype frequencies of all seven polymorphisms were significantly different between two groups. Though, ACGACTT and GTCAGGA haplotypes were related with decreased (P=0.0079, OR=0.559, 95% CI: 0.363-0.861 and P=0.02, OR=0.417, 95% CI: 0.194-0.896, respectively), but ACGACGT and GTGACTT haplotypes were associated with an increased (P=0.00082, OR=3.657, 95% CI: 1.630-8.206 and P=0.02, OR=2.401, 95% CI: 1.119-5.151, respectively) risk of preeclampsia. Moreover, some positions were detected to be in linkage disequilibrium. CONCLUSION: Ongoing investigation resulted differently from before performed studies considering the role of ERAP1 and ERAP2 gene polymorphisms in predisposing women to preeclampsia, emphasizing on the genetic structure differences among various racial populations.

Lipopolysaccharide- and Lipoteichoic Acid-mediated Pro-inflammatory Cytokine Production and Modulation of TLR2, TLR4 and MyD88 Expression in Human Endometrial Cells.

BACKGROUND: Toll-like receptor (TLR)-mediated inflammatory processes are supposed to be involved in pathophysiology of spontaneous abortion and preterm labor. Here, we investigated functional responses of human endometrial stromal cells (ESCs) and whole endometrial cells (WECs) to lipopolysaccharide (LPS) and lipoteichoic acid (LTA). METHODS: Endometrial tissues were obtained from 15 cycling women who underwent laparoscopic tubal ligation. Modulation of TLR2, TLR4 and MyD88 expression and production of pro-inflammatory cytokines by WECs and ESCs in response to LPS and LTA were assessed. RESULTS: WECs and ESCs expressed significant levels of TLR4 and MyD88 transcripts but, unlike WECs, ESCs failed to express TLR2 gene. Regardless of positive results of Western blotting, ESCs did not express TLR4 at their surface as judged by flow cytometry. Immunofluorescent staining revealed intracellular localization of TLR4 with predominant perinuclear pattern. LPS stimulation marginally increased TLR4 gene expression in both cell types, whereas such treatment significantly upregulated MyD88 gene expression after 8 hr (p < 0.05). At the protein level, however, LPS activation significantly increased TLR4 expression by ESCs (p < 0.05). LTA stimulation of WECs was accompanied with non-significant increase of TLR2 and MyD88 transcripts. LPS and LTA stimulation of WECs caused significant production of IL-6 and IL-8 in a dose-dependent manner (p < 0.05). Similarly, ESCs produced significant amounts of IL-6, IL-8 and also TNF-α in response to LPS activation (p < 0.05). CONCLUSION: Our results provided further evidence of initiation of inflammatory processes following endometrial TLR activation by bacterial components which could potentially be harmful to developing fetus.

Upregulation of CD200 is associated with Foxp3+ regulatory T cell expansion and disease progression in acute myeloid leukemia.

Immunosuppression in acute myeloid leukemia (AML) is an important mechanism of tumor escape. CD200, as an immunosuppressive molecule, is overexpressed in some hematological malignancies and it has also been shown to be an independent prognostic factor in AML. In the current study, simultaneous CD200 expression and Foxp3(+) regulatory T cell levels were investigated in Iranian patients with AML by flow cytometry. We also assessed the effect of CD200-CD200R blockade on Th1 and T-reg cytokine production and T cell proliferation in autologous AML- and monocyte-DC mixed lymphocyte reactions (MLRs). ELISA assay was performed to detect IL-2, IL-12, IFN-γ, IL-10, and TGF-ß production in MLR supernatants. Expression of Foxp3, IL-10, and TGF-ß mRNAs in MLRs were detected by real-time PCR. Our results demonstrated significant overexpression of CD200 (P = 0.001) in association with higher frequencies of Foxp3(+) T cells in AML patients (r = 0.8, P < 0.001). Blocking of CD200-CD200R interaction demonstrated a significant decrease in TGF-ß and IL-10 expression in AML-DC MLRs and a significant increase in IL-12 and IFN-γ expression in monocyte-DC MLRs. Elevated T cell levels with lower Foxp3 intensity was also shown in CD200-CD200R-blocked MLRs. Expression of IL-10 mRNA declined significantly only in AML-DC MLRs where CD200-CD200R interaction was blocked and the same result was observed for TGF-ß and Foxp3 mRNA in both AML- and monocyte-DC MLRs. These data present a significant role for CD200 in suppressing anti-tumor immune response through stimulation of regulatory mechanisms in AML patients and suggest that CD200 may have a prognostic value in this malignancy and its blockade may be used as a target for AML immunotherapy.

Serum levels of tumor necrosis factor-α, interleukin-15 and interleukin-10 in patients with pre-eclampsia in comparison with normotensive pregnant women.

BACKGROUND: Pre-eclampsia (PE) is a pregnancy associated disorder characterized by hyper-tension and proteinuria. The first 2 stages of PE cause dysfunction in uteroplacental perfusion and oxidative stress while the third stage of PE is due to the release of inflammatory and angiogenic factors, which could lead to maternal endothelial damage and systemic inflammatory response. In this study, we compared the serum levels of tumor necrosis factor-α (TNF-α), interleukin-15 (IL-15), and interleukin-10 (IL-10) in PE and normotensive women. MATERIALS AND METHODS: Serum samples of 84 pregnant women (44 PE and 40 normotensive) were evaluated for TNF-α, IL-15 and IL-10 by sandwich ELISA assay. RESULTS: The women with PE showed significantly higher serum levels of TNF-α and IL-15 (P = 0.001 and 0.01 respectively) in comparison with normotensive pregnant women. Conversely, the serum levels of IL-10 in normotensive women were significantly higher compared to PE patients (P = 0.01). CONCLUSION: The results of this study demonstrated that inflammatory T helper 1-type responses are increased in PE women compared to normotensive pregnant women.

Erythropoietin levels in aqueous humor of patients with glaucoma.

PURPOSE: To compare the aqueous concentration of erythropoietin (EPO) in eyes with primary open-angle (POAG), pseudoexfoliative (PXFG), and neovascular (NVG) glaucoma with age-matched eyes with cataracts, and to correlate its concentration with other factors including age, gender, intraocular pressure (IOP), type of glaucoma, and severity of glaucoma. METHODS: In this prospective non-randomized comparative study, a total of 26 eyes with cataracts (control group) and 92 glaucomatous eyes (POAG, 40 eyes; PXFG, 26 eyes; NVG, 26 eyes) were enrolled. Aqueous samples (0.1 to 0.2 ml) were obtained during phacoemulsification, trabeculectomy, phacotrabeculectomy, or Ahmed valve glaucoma implants. The aqueous concentration of EPO was measured using an enzyme-linked immunosorbent assay. RESULTS: The mean±SEM aqueous level of EPO was statistically significantly higher in eyes with glaucoma (56.7±9.3 mIU/ml) compared to the control group (0.8±0.51 mIU/ml; p<0.001). Eyes with NVG had the highest aqueous level of EPO. Aqueous EPO concentrations remained considerably elevated even in eyes with controlled IOP in all three types of glaucoma. Eyes with PXFG displayed the greatest change in aqueous EPO concentration proportionate to the IOP level. In simple regression analysis, IOP, mean deviation, and the type of glaucoma were the factors that had a statistically significantly positive correlation with the aqueous level of EPO (p=0.011 and <0.001, respectively). Only the type of glaucoma remained statistically significant in the multiple regression analysis (adjusted R(2)=0.278). CONCLUSIONS: Compared to the control group, the aqueous humor EPO concentration is increased in eyes with POAG, PXFG, and NVG, both with and without controlled IOP. The aqueous level of EPO was more proportionate to the level of IOP in eyes with PXFG compared to eyes with POAG and NVG.

Effect of 1,25(OH)2 vitamin D3 on cytokine production by endometrial cells of women with repeated implantation failure.

Repeated implantation failure (RIF) is a worldwide health problem that imposes a great deal of cost on patients and health care system. Vitamin D(3) has been proposed to have positive impact on the process of implantation. The present study was performed to compare the effect of 1,25-dihydroxy vitamin D(3) (1,25(OH)(2)D(3)) on cytokine production by endometrial cells of women with RIF and healthy fertile controls. Whole endometrial cells (WECs) and endometrial stromal cells (ESCs) from RIF and normal fertile women were treated with 1,25(OH)(2)D(3). The levels of IL-10, TGF-ß, IFNγ, Il-6, IL-8 and IL-17 in culture supernatants were assayed by ELISA. Also, ability of the cells from both groups to produce 1,25(OH)(2)D(3) was evaluated and compared. 1,25(OH)(2)D(3) down-regulated cytokine production in WECs from both groups except for IL-8 which was upraised. Similar trends were also observed in ESCs except up-regulation of TGF-ß in RIF group. Endometrial cells of both groups had comparable capacity to produce 1,25(OH)(2)D(3). Based on the minimal differential immunoregulatory effect of vitamin D(3) on endometrial cells from RIF and control women, it may be suggested that circulating levels of maternal vitamin D(3) be the subject of further investigation.

Cytokine profile in the endometrium of normal fertile and women with repeated implantation failure.

BACKGROUND: Repeated Implantation Failure (RIF) is one of the most intricate obstacles in assisted reproduction. The cytokine and chemokine composition of uterine cavity seem to play important roles in the implantation process. OBJECTIVE: To compare the cytokine profile in the endometrium of normal fertile women and those with repeated implantation failure. METHODS: After enzymatic digestion of endometrial tissues, whole endometrial cells and endometrial stromal cells from RIF and normal fertile women were cultivated and stimulated for cytokine secretion. The levels of IL-10, TGF-ß, IFN-γ, IL-6, IL-8 and IL-17 in culture supernatants of the two groups were assayed by ELISA and compared together. RESULTS: Endometrial stromal cells and whole endometrial cells of normal fertile women produced higher levels of IL-6, IL-8 and TGF-ß compared to RIF group, although this difference was statistically significant only in endometrial stromal cells (p=0.005, 0.002 and 0.001, respectively). In addition, endometrial stromal cells of normal fertile women produced lower levels of IL-10 in comparison with RIF group (p=0.005). CONCLUSION: Disturbances in cytokine production at the feto-maternal interface could be a cause of implantation failure. A pro-inflammatory cytokine milieu seems to be pivotal for successful implantation.

Analysis of switched memory B cells in patients with IgA deficiency.

BACKGROUND: Selective IgA deficiency (SIGAD) is the most common primary antibody deficiency, characterized by significant decreased serum levels of IgA in the presence of normal IgG and IgM. Despite several investigations into the nature of the disease, the exact pathophysiology of SIGAD is still unknown. METHODS: In this study, switched memory B cells (CD19+/CD27+/IgD- cell population) of 28 patients with SIGAD and 28 matched healthy controls were investigated using flow cytometry. RESULTS: The percentage of switched memory B cells in all healthy controls was more than 0.4%. In SIGAD patients, who were classified as group I, the percentage of switched memory B cells was less than 0.4% (0.34 ± 0.06) in 7 patients (25%). The remaining 21 patients were designated as group II (1.74 ± 0.12%). The mean concentration of IgG in group I was significantly lower than in group II (1,014 ± 278 vs. 1,388 ± 406 mg/dl, p = 0.028). Comparison of clinical features between the 2 groups revealed that episodes of pneumonia during the course of disease were significantly higher in group I than in group II (p = 0.002). Autoimmune diseases in group I (57.1%) were also significantly higher (p = 0.01) than in group II (23.8%). The prevalence of bronchiectasis was 57% in group I, while only 1 patient (4.7%) in group II developed bronchiectasis (p = 0.006). Specific antibody deficiency in group I was documented in 5 patients and in group II in 4 patients (p = 0.01). CONCLUSIONS: The classification of SIGAD patients by assessment of switched memory B cells could help physicians with the clinical prognosis for these patients, whereas the patients with reduced switched memory B cells are prone to severe phenotypes.

Association of tumor necrosis factor-alpha and interleukin-10 gene polymorphisms in Iranian patients with pre-eclampsia.

PROBLEM: Considering that certain cytokines may change during pre-eclampsia (PE), because of functional polymorphisms in their genes, our purpose was to determine the association between tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) gene polymorphisms and development of PE. METHOD OF STUDY: The genetic polymorphisms of TNF-alpha and IL-10 was studied by polymerase chain reaction-sequence specific primers in the DNA of peripheral blood cell from 160 patients with PE and 100 healthy pregnant women. RESULTS: We found a significant difference between TNF-alpha A allele (-308) and G allele (-238) in PE patients compared with those of the control groups. A significantly higher C/C genotype frequency of IL-10 (-592 and -819) was observed in the PE patients than in the control groups. In addition, the frequencies of three common IL-10 haplotypes (GCC, ACC, and ATA) did not show any significant difference between the study groups. CONCLUSION: These findings would support the concept of contribution of TNF-alpha and IL-10 gene polymorphisms in the pathogenesis of PE in our population.

Increased natural killer cell cytotoxicity and IL-2 production in recurrent spontaneous abortion.

PROBLEM: To determine whether natural killer (NK) cells cytotoxicity in peripheral blood is altered in patients with a history of recurrent spontaneous abortion (RSA); also, if there is any correlation between cytokine production and NK cytotoxicity. METHOD OF STUDY: In this case-control study, 21 patients with RSA within 24 hr of the last abortion (group I), and 32 pregnants with no history of abortion (group II) were surveyed. NK cell cytotoxicity was evaluated by flow cytometry, and IL-2, IL-10, transforming growth factor beta1 were measured in cell culture supernatant by ELISA method. RESULTS: Group I showed higher NK cytotoxicity than group II at all of effector to target (E:T) ratios (P < or = 0.045). The correlation between production of IL-2 and NK cytotoxicity was positively significant (R = 0.350, P = 0.001). Group I had significantly higher levels of IL-2 than group II (P = 0.001). In group II, the production of IL-10 by peripheral blood mononuclear cells was higher than group I (P = 0.002). CONCLUSION: Increased NK cell cytotoxicity and high level of IL-2 may be considered as a risk factor for RSA.

Serum cytokines profiles in Iranian patients with preeclampsia.

BACKGROUND: Preeclampsia is a major cause of mortality and morbidity and is also a leading cause of preterm birth and intrauterine growth retardation. Several studies have reported abnormal levels of cytokines in women with preeclampsia. OBJECTIVES: To detect serum levels of various cytokines in pregnant women with and without preeclampsia in the third trimester of pregnancy. METHODS: Thirty patients with preeclampsia and thirty normal pregnant women were enrolled in the study. Blood samples were taken and serum levels of IFN gamma, IL-12p70, IL-18, IL-15, IL-4 and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Preeclamptic women had significantly increased levels of circulating IL-12p70 (p < 0.05), IL-18 (p < 0.001), IL-4 (p < 0.001), IL-15 (p < 0.05) and IFN gamma (p < 0.001). By contrast, circulating levels of IL-10 were not significantly different between the two groups. CONCLUSIONS: The present study supports the hypothesis of altered immune response in preeclampsia and suggests that dysregulation of cytokine expression occurs in preeclampsia with increased levels of IFN gamma, IL-12p70, IL-15, IL-18 and IL-4.