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1.
Gynecol Obstet Invest ; 86(1-2): 139-148, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33540416

RESUMO

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.


Assuntos
Calcitriol/farmacologia , Endométrio/imunologia , Inflamação/prevenção & controle , Receptor 2 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/efeitos dos fármacos , Adulto , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Fatores Imunológicos/farmacologia , Lipopolissacarídeos/farmacologia , Fator 88 de Diferenciação Mieloide/efeitos dos fármacos , Fator 88 de Diferenciação Mieloide/genética , Gravidez , Células Estromais/efeitos dos fármacos , Células Estromais/fisiologia , Ácidos Teicoicos/farmacologia , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologia
2.
PLoS One ; 11(4): e0153684, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27101408

RESUMO

Kisspeptins (KPs) are major regulators of trophoblast and cancer invasion. Thus far, limited and conflicting data are available on KP-mediated modulation of breast cancer (BC) metastasis; mostly based on synthetic KP-10, the most active fragment of KP. Here, we report for the first time comprehensive functional effects of term placental KPs on proliferation, adhesion, Matrigel invasion, motility, MMP activity and pro-inflammatory cytokine production in MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). KPs were expressed at high level by term placental syncytiotrophoblasts and released in soluble form. Placental explant conditioned medium containing KPs (CM) significantly reduced proliferation of both cell types compared to CM without (w/o) KP (CM-w/o KP) in a dose- and time-dependent manner. In MDA-MB-231 cells, placental KPs significantly reduced adhesive properties, while increased MMP9 and MMP2 activity and stimulated invasion. Increased invasiveness of MDA-MB-231 cells after CM treatment was inhibited by KP receptor antagonist, P-234. CM significantly reduced motility of MCF-7 cells at all time points (2-30 hr), while it stimulated motility of MDA-MB-231 cells. These effects were reversed by P-234. Co-treatment with selective ER modulators, Tamoxifen and Raloxifene, inhibited the effect of CM on motility of MCF-7 cells. The level of IL-6 in supernatant of MCF-7 cells treated with CM was higher compared to those treated with CM-w/o KP. Both cell types produced more IL-8 after treatment with CM compared to those treated with CM-w/o KP. Taken together, our observations suggest that placental KPs differentially modulate vital parameters of estrogen receptor-positive and -negative BC cells possibly through modulation of pro-inflammatory cytokine production.


Assuntos
Apoptose , Neoplasias da Mama/patologia , Movimento Celular , Kisspeptinas/metabolismo , Placenta/metabolismo , Receptores de Estrogênio/metabolismo , Adulto , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Feminino , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Kisspeptinas/genética , Gravidez , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Cicatrização , Adulto Jovem
3.
J Reprod Infertil ; 16(2): 72-81, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25927023

RESUMO

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.

4.
Cancer Cell Int ; 14: 54, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24966802

RESUMO

BACKGROUND: Many types of tumors are organized in a hierarchy of heterogeneous cell populations with different molecular signature. Such heterogeneity may be associated with different responsiveness to microenvironment stimuli. In the present study, the effects of lipopolysaccharide (LPS) and lipoteichoic acid (LTA), as well-known mediators of inflammation, on cancerous behavior of three prostate tumor cells, LNCaP, PC3 and DU145, were investigated. METHODS: Expression of TLR1-10, CD14 and MyD88 transcripts was investigated by RT-PCR. Protein expression of TLR2 and 4 was scrutinized by flow cytometry, immunofluorescent staining and Western blotting. Experiments were set up to assess the effects of LPS and LTA at different concentrations and times on cell proliferation, extracellular matrix invasion, adhesion and cytokine production. RESULTS: We showed that prostate cancer cell lines differentially express TLR1-10, MyD88 and CD14 transcripts. DU145 failed to express TLR4 gene. Positively-identified TLR2 protein in all prostate cancer cells and TLR4 protein in PC3 and LNCaP by Western blotting was not accompanied by cell surface expression, as judged by flow cytometry. Immunofluorescent staining clearly demonstrated predominantly perinuclear localization of TLR2 and TLR4. LTA activation of all prostate cancer cells significantly increased cell proliferation. Regardless of lacking TLR4, DU145 cells proliferated in response to LPS treatment. While LPS caused increased invasiveness of LNCaP, invasive capacity of PC3 was significantly reduced after LPS or LTA stimulation. Stimulation of all prostate tumor cells with LTA was associated with increased cell adhesion and IL-8 production. IL-6 production, however, was differentially regulated by LPS stimulation in prostate tumor cells. CONCLUSION: The data shows that cancer cells originated from the same histologically origin exhibit heterogeneous response to the same TLR ligand. Therefore, a thorough and comprehensive judgment on how and to what extent a particular cancer is affected by TLR agonist could not be inferred by studying an individual cell line.

5.
Avicenna J Med Biotechnol ; 1(1): 41-6, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23407674

RESUMO

Prevalence of abortion is higher in women with autoimmune thyroid disease. In the majority of cases, however, no abnormality of thyroid function is detected despite the high levels of antithyroid antibodies. The direct influence of such harmful autoantibodies in female reproductive organs may serve a role in pregnancy loss. In this study, expression of thyroglobulin in the reproductive tissues of cycling mice has been evaluated. Stages of estrous cycle were determined by cellular morphology and ratio of epithelial cells to leukocytes in vaginal smear of Balb/C mice. At each phase, the mice were sacrificed and their uterus, ovary and fallopian tubes were removed. Expression of thyroglobulin-specific transcript in endometrium was investigated by two sets of primers using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, expression of thyroglobulin in reproductive tissues was assessed by immunohistochemistry and dot blot analysis. The results showed that thyroglobulin mRNA is not expressed in endometrial tissue of Balb/C mice at any stage of estrous cycle. Immunohistochemical analysis also confirmed that thyroglobulin or its cross reactive-antigens are not expressed at the protein level in the female reproductive organs. The results showed that thyroglobulin was not expressed in the reproductive organs of female mice. It is plausible that antithyroglobulin antibodies could interact with newly-generated antigens during placentation and pregnancy.

6.
Pak J Biol Sci ; 10(22): 4015-21, 2007 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19090273

RESUMO

Water supply and Cooling Tower Water (CTW) are among the most common sources of Legionella pneumophila (LP) contamination. A nonradio active method is described to detect LP in industrial CTW samples. DNA was purified and amplified by nested -PCR with amplimers specific for the 16s rRNA gene of LP. The 5' end biotinylated oligomer probe was immobilized on sterptavidin B coated microtiter plates. The nested-PCR product was labeled with digoxigenin and then hybridized with 5'-biotinylated probes. The amplification products were detected by using proxidase-labled anti dioxygenin antibody in a colorimetric reaction. The assay detected LP present in 1 L of 5 CTW samples examined. All of the samples were Legionella positive in both culture and PCR-ELISA methods. The PCR-ELISA assay appears to exhibit high specificity and is a more rapid technique in comparison with bacterial culture method. Thus could prove suitable for use in the routine examination of industrial CTW contamination.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Legionella pneumophila/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Purificação da Água/métodos , Biotinilação , Primers do DNA/química , Eletroforese em Gel de Ágar , Legionella pneumophila/genética , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/química , Microbiologia da Água , Abastecimento de Água
7.
Pak J Biol Sci ; 10(24): 4490-4, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19093517

RESUMO

This study was designed to estimate the prevalence of Chlamydia infection in women attending Obstetrics and Gynaecology clinics in Tehran, during May 2003 to October 2003. Women attending Obstetrics and Gynaecology clinics aged 15-42 were recruited by Sequential Random Sampling. Those who had not passed urine in the last hour were eligible. Informed consent was obtained and a questionnaire completed after being interviewed by a midwife. First void urine was collected and after DNA extraction from urine specimen, PCR tests were performed; urine DNA samples were tested by strand displacement amplification (SDA) for Chlamydia confirmation. 12.6% (133/1052) tested positive for Chlamydia by PCR. Of these PCR positive samples, 86 were available for re-testing by SDA and 67 were positive giving a correlation between the tests of 78%. This gave an overall true prevalence of 6.4% which is however, underestimated. No statistical differences were seen between patient age groups, details of personal and reproductive history and combined PCR and SDA positivity for C. trachomatis. A 12.6% prevalence of Chlamydia trachomatis was found by PCR testing which is cost effective to screen and treat. Despite limitations in re-testing PCR-positive samples by SDA, a 78% correlation between tests confirms a high prevalence of C. trachomatis. Non-invasive screening of women was therefore a success in this group of patients. As this was the first time that more sensitive molecular methods were used for detection of C. trachomatis, prevalence in such a big sample size, the results are considerable. However, we suggest further such testing.


Assuntos
Infecções por Chlamydia/epidemiologia , Chlamydia trachomatis/isolamento & purificação , Doenças dos Genitais Femininos/microbiologia , Adolescente , Adulto , Chlamydia trachomatis/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Emprego , Feminino , Doenças dos Genitais Femininos/epidemiologia , Humanos , Irã (Geográfico)/epidemiologia , Casamento , Plasmídeos , Reação em Cadeia da Polimerase , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Complicações Infecciosas na Gravidez/microbiologia , Prevalência , Comportamento Sexual , Inquéritos e Questionários , Adulto Jovem
8.
Ann Hematol ; 85(4): 268-71, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16450127

RESUMO

Factor V Leiden (FVL) G1691A, methylenetetrahydrofolate reductase (MTHFR) C677T, and factor II (FII) G20210A mutations are three important causes of thrombophilia, the condition that might be related to infertility and recurrent spontaneous abortion (RSA). In this study we evaluated the presence of these three mutations in 36 female patients with unexplained infertility, 65 female patients with unexplained RSA, and 62 healthy fertile women as control group. DNA was extracted from peripheral blood samples and PCR-RFLP was performed for the molecular diagnosis of each mutation. In addition, activated protein C resistance (APC-R) was also evaluated. The frequencies of FVL, MTHFR, and FII mutations (heterozygous and homozygous) in the control group were 0.0%, 38.7%, and 3.2%, respectively. The frequency of FVL mutation in patients with infertility (30.6%) or RSA (20.0%) was significantly higher than that of the control group. A significantly higher MTHFR mutation rate was also observed in patients with RSA (63.1%) as compared to controls. However, the mutation rate of MTHFR in patients with infertility (50.0%) was not statistically different from that in controls. No significant difference was observed in the frequencies of FII mutations between the patients and controls. Decreased levels of APC-R were observed in 25.0% of infertile patients and 18.9% of patients with RSA. In conclusion, our results show a skew towards higher mutation frequencies of FVL and MTHFR in patients that may necessitate detection of such mutations in these Iranian patients.


Assuntos
Aborto Habitual/genética , Infertilidade/genética , Trombofilia/genética , Aborto Habitual/epidemiologia , Comorbidade , Fator V/genética , Feminino , Frequência do Gene , Genótipo , Heterozigoto , Homozigoto , Humanos , Infertilidade/epidemiologia , Irã (Geográfico)/epidemiologia , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Mutação , Reação em Cadeia da Polimerase/métodos , Protrombina/genética , Sensibilidade e Especificidade , Trombofilia/epidemiologia
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