An in vitro investigation of telocytes-educated macrophages: morphology, heterocellular junctions, apoptosis and invasion analysis.

BACKGROUND: Telocytes (TCs), a recently discovered novel type of interstitial cells, were also found in a wide variety of human and mammalian reproductive organs/tissues, including uterus, oviduct and placenta. Previously, we demonstrated that TCs-conditioned media was capable of activating peritoneal macrophages (pMACs) through paracrine effects. This study investigates the hypothesis that direct interaction of TCs with pMACs will also play a significant role in immunoregulation of pMACs. METHODS: TCs and pMACs were derived from the uterus and intraperitoneal cavity of female BALB/c mice, respectively. TCs were identified by immunofluorescence and then co-cultured directly with pMACs for 24 h without added cytokines, to observe the in vitro biological behavior of pMACs. We used histochemical staining to study morphology and mitochondrial metabolism of pMACs, scanning electron microscopy to study heterocellular junctions, flow cytometry to investigate mitochondrial membrane potential (ΔΨm) and apoptosis, and transwell chambers to study invasion ability. Student-t test was used accordingly. RESULTS: Presently, TCs with typical structure and immunophenotype of double CD-34-positive/vimentin-positive were successfully isolated. pMACs co-cultured with TCs showed obviously morphological activation, with enhanced energy metabolism (P < 0.05). Meanwhile, direct physical cell-to-cell interaction promoted the development of heterocellular junctions between TCs and pMACs. Furthermore, TCs treatment markedly reduced the depletion of ΔΨm in co-cultured pMACs (all P < 0.05), and inhibited their apoptosis (P < 0.05). Functionally, pMACs co-cultured with TCs showed enhanced invasion ability (P < 0.05). CONCLUSIONS: Direct physical cell-to-cell interaction promoted the development of heterocellular junctions between TCs and pMACs, presumably responsible for the observed novel efficient way of pMACs activation via mitochondrial signaling pathway. TCs-educated pMACs might be a promising way to restore the defective immunosurveillance in endometriosis (EMs), led to the enhanced treatment efficacy of EMs in a simple and clinically feasible fashion.

In vitro morphology, viability and cytokine secretion of uterine telocyte-activated mouse peritoneal macrophages.

Telocytes (TCs), a distinct interstitial cell population, have been identified in the uterus, oviduct and placenta, with multiple proposed potential biological functions. Their unique structure allows them to form intercellular junctions with various immunocytes, both in normal and diseased tissues, suggesting a potential functional relationship with the local immune response. It has been hypothesized that through direct heterocellular junctions or indirect paracrine effects, TCs influence the activity of local immunocytes that are involved in the inflammatory process and in immune-mediated reproductive abnormalities. However, no reliable cytological evidence for this hypothesis is currently available. In this study, we cultured primary murine uterine TCs and collected TC conditioned media (TCM). Mouse peritoneal macrophages (pMACs) were co-cultured for 48 hrs with TCM or with DMEM/F12 or lipopolysaccharide (LPS) as negative and positive controls, respectively. Normal uterine TCs with a typical structure and a CD-34-positive/vimentin-positive/c-kit-negative immunophenotype were observed during culture. Morphologically, TCM-treated pMACs displayed an obvious activation/immunoresponse, in contrast to over-stimulation and cell death after LPS treatment and no sign of activation in the presence of DMEM/F12. Accordingly, a cell counting kit 8 (CCK-8) assay indicated significant activation of pMACs by TCM and LPS compared to DMEM/F12, thus supporting the marked morphological differences among these groups of cells. Furthermore, within a panel of macrophage-derived cytokines/enzymes, interleukin-6 (IL-6) and inducible nitric oxide synthase were significantly elevated in TCM-treated pMACs; tumour necrosis factor α, IL1-R1, and IL-10 were slightly, but significantly, up-regulated; and no changes were observed for transforming growth factor-ß1, IL-1ß, IL-23α and IL-18. Our results indicate that TCs are not simply innocent bystanders but are rather functional players in the activation of pMACs; they trigger and maintain the immune response, likely through indirect paracrine effects. Thus, we provide preliminary in vitro evidence of immunoregulatory and immunosurveillance roles for TCs.

Ultrastructure damage of oviduct telocytes in rat model of acute salpingitis.

Acute salpingitis (AS) is an inflammatory disease which causes severe damage to a subset of classically described cells lining in oviduct wall and contributes to interstitial fibrosis and fertility problems. Telocytes (TCs), a newly discovered peculiar type of stromal cells, have been identified in many organs, including oviduct, with proposed multiple potential bio-functions. However, with recent increasing reports regarding TCs alterations in disease-affected tissues, there is still lack of evidence about TCs involvement in AS-affected oviduct tissues and potential pathophysiological roles. We presently identified normal TCs by their characteristic ultrastructural features and immunophenotype. However, in AS-affected oviduct tissues, TCs displayed multiple ultrastructural damage both in cellular body and prolongations, with obvious loss of TCs and development of tissue fibrosis. Furthermore, TCs lose their interstitial 3-D network connected by homocellular or heterocellular junctions between TCs and adjacent cells. And especially, TCs connected to the activated immunocytes (mononuclear cells, eosinophils) and affected local immune state (repression or activation). Meanwhile, massive neutrophils infiltration and overproduced Inducible Nitric Oxide Synthase (iNOS), COX-2, suggested mechanism of inflammatory-induced TCs damage. Consequently, TCs damage might contribute to AS-induced structural and reproductive functional abnormalities of oviduct, probably via: (i) substances, energy and functional insufficiency, presumably, e.g. TC-specific genetic material profiles, ion channels, cytoskeletal elements, Tps dynamics, etc., (ii) impaired TCs-mediated multicellular signalling, such as homeostasis/angiogenesis, tissue repair/regeneration, neurotransmission, (iii) derangement of 3-D network and impaired mechanical support for TCs-mediated multicellular signals within the stromal compartment, consequently induced interstitial fibrosis, (iv) involvement in local inflammatory process/ immunoregulation and possibly immune-mediated early pregnancy failure.

Telocytes damage in endometriosis-affected rat oviduct and potential impact on fertility.

Women with endometriosis (EMs) have unexplained infertility. The recently identified telocytes (TCs) might participate in the maintenance of structural and functional integrity of oviduct tissue, but so far the involvement of TCs in EMs-affected oviduct tissue and potential impact on fertility capacity remain unknown. By an integrated technique of haematoxylin and eosin staining, in situ immunohistochemistry and double-labelled immunofluorescence staining and electron microscopy approach, TCs were studied in the autotransplantation Sprague-Dawley rat model of EMs-affected oviduct tissue and in sham control, respectively, together with determination of iNOS, COX-2, LPO and estradiol. TCs were found in perivascular connective tissue and smooth muscle bundles in sham oviduct, with typical ultrastructural features (a slender piriform/spindle/triangular cell body, and one or more extremely long prolongations, emerged from cell bodies and extend to various directions), and specific immunophenotype of CD34-positive/vimentin-positive/c-kit-negative. However, in EMs-affected oviduct tissue (grade III), extensive ultrastructural damage (degeneration, discontinue, dissolution and destruction), significant decrease or loss of TCs and interstitial fibrosis were observed, together with elevated level of iNOS, COX-2, LPO and estradiol, thus suggestive of inflammation and ischaemia-induced TCs damage. Based on TCs distribution and intercellular connections, we proposed that such damage might be involved in structural and functional abnormalities of oviduct, such as attenuated intercellular signalling and oviduct contractility, impaired immunoregulation and stem cell-mediated tissue repair, 3-D interstitial architectural derangement and tissue fibrosis. Therefore, TCs damage might provide a new explanation and potential target for EMs-induced tubal damage and fertility disorders.

MicroRNA-370 suppresses proliferation and promotes endometrioid ovarian cancer chemosensitivity to cDDP by negatively regulating ENG.

MicroRNAs (miRNAs) are a class of non-coding RNAs that post-transcriptionally inhibit gene expression. In this study, we discovered that microRNA-370 (miR-370) was down-regulated in endometrioid ovarian cancer cells. In IGROV1 and TOV112D endometrioid ovarian cancer cells, miR-370 suppressed cellular viability and colony formation. miR-370 also enhanced endometrioid ovarian cancer cell chemosensitivity to cDDP. Endoglin (ENG) was directly and negatively regulated by miR-370. In addition, hypermethylation was a potential mechanism of miR-370 epigenetic silencing. We conclude that miR-370 acts as a tumor suppressor in endometrioid ovarian cancer via ENG regulation.

Analysis of HGF, MACC1, C-met and apoptosis-related genes in cervical carcinoma mice.

To understand the underlying pharmacological basis and the molecular mechanism of Taxol in therapy of cervical carcinoma (CC) disease, we need to explore the effect of Taxol on CC-related genes and pro-apoptosis and anti-apoptosis genes expression. Immunohistochemistry, western blot and reverse transcription-polymerase chain reaction were applied to examine postive expression levels of Bcl-2, Bax and Caspase-3, HGF, MACC1, Caspase-3 and C-met proteins and MACC1 mRNA expression in tumour of CC mice. Results showed that treatment of Taxol could increase the inhibition rate of tumour growth, positive expression levels of Caspase-3, Bax and decrease positive expression levels of Bcl-2 and Bcl-2/Bax, expression levels of HGF, MACC1 and C-met proteins and MACC1 mRNA in tumour tissue of CC mice. It can be concluded that inhibitory activity of Taxol against tumour growth in CC mice is closely associated with its modulating positive expression of Bcl-2, Bax, Caspase-3, expression of HGF, MACC1, Caspase-3 and C-met proteins and MACC1 mRNA in tumour of CC mice. In conclusion, HGF, MACC1 and C-met genes involve into malignant cervical tumors occurrence, development and prognosis, and might become potential molecular target therapy site of cervical cancer. Taxol intervention may serve as a multi-targeted CC therapeutic capable of inducing selective cancer cell death.

Immunohistochemical alterations of cajal-like type of tubal interstitial cells in women with endometriosis and tubal ectopic pregnancy.

PURPOSE: The aim of the study was to observe alterations of pacemaker cells termed cajal-like type of tubal interstitial cells (t-ICC) in oviduct from early-stage EMs and tEP, discuss underlying mechanisms and potential role in tubal factor infertility (TFI). METHODS: Ten patients with early-stage EMs, 10 with unruptured tEP and 10 control subjects were included in this retrospective comparative study, received adnexectomy (salpingectomy) and/or hysterectomy. Paraffin-embedded full-thickness isthmic segment of oviduct specimens received immunohistochemistry with c-kit/CD117 antibody. Network distribution and area density of cells with features of t-ICC were analyzed. RESULTS: t-ICC was detected mainly in lamina propria and smooth muscle layers. t-ICC lost its network integrity, became less densely stained, sparse and almost invisible with relatively very rare connections in EMs and tEP, apparently differing in morphology of t-ICC from control, which demonstrated rich t-ICC immunostaining and intact network. Further quantitative analysis showed the area density of t-ICC decreased significantly in early-stage EMs and tEP compared with the control (73.9 ± 8.8 vs. 156 ± 18.3 mm(2); and 76 ± 7.4 vs. 156 ± 18.3 mm(2); both P < 0.001). CONCLUSIONS: We revealed that t-ICC underwent certain degree of cell damage, suggested that decreased expression of t-ICC network may be involved in early development of EMs and tEP, and might serve as an explanation for TFI in these patients.

[Association between serum bisphenol-A and recurrent spontaneous abortion: a 1:2 case-control study, China].

OBJECTIVE: This study was to investigate the association between serum Bisphenol-A (BPA) and unexplained recurrent spontaneous abortion (RSA). METHODS: A hospital-based 1:2 matched case-control study was conducted.Sixty-two patients with unexplained recurrent abortion were included and matched with 2 normal controls by factors as age (± 2 years), living in the same district and the same gestational age.The levels of BPA in serum for 62 cases and 108 controls were detected under high performance liquid chromatography after fluorescent derivatization. Levels of serum BPA in each case was compared with that in control of age, BMI, education levels, occupation, exposure for passive smoking. RESULTS: The values of serum BPA in cases and controls were (0.009 ± 0.002) and (0.004 ± 0.012) µg/ml, respectively. The levels of serum BPA in cases was significantly higher than in controls (Z = 3.506, P = 0.0005). After adjusted by age, BMI, education levels, occupation, passive smoking history and other factors, when compared to BPA below 0.004 µg/ml. The adjusted ORs were 4.39 (1.15 - 16.71) for BPA levels between 0.004 µg/ml and 0.012 µg/ml, and 4.95 (1.77 - 13.82) for BPA over 0.012 µg/ml. The risk of unexplained recurrent spontaneous abortion increased progressively with the growth of serum BPA levels (χ(2) = 9.179, trend test P = 0.0024). There were significant differences on BPA among controls that with histories of two, three or more abortions (the levels were 0.004, 0.008, 0.018 µg/ml, respectively, F = 8.92, P = 0.0002). CONCLUSION: High BPA level might be associated with unexplained recurrent spontaneous abortion.

[A correlative study on Bisphenol A and recurrent spontaneous abortion].

OBJECTIVE: This study was to investigate the association of Bisphenol A and unexplained recurrent spontaneous abortion. METHODS: A hospital-based 1:1 matched case-control study was conducted. Sixty patients with unexplained recurrent abortion were included. Each case was matched with one normal control by age (± 2 years), living district and the same gestational age. The levels of Bisphenol A in urine for 60 cases and 60 controls were detected using high performance liquid chromatography after fluorescent derivatization. The levels of urinary Bisphenol A in case was compared with that in control in education levels, occupation, smoking history. Data was analyzed by means of Wilcoxon-test, Student-Newman-Keuls after rank transform, univariate and multivariate conditional Logistic regression analysis. The software used was SAS 9.1.3. RESULTS: The values of urinary Bisphenol A in cases and controls were (0.10 ± 0.21) µg/ml, (0.03 ± 0.08) µg/ml, respectively. The level of urinary Bisphenol A in cases was significantly higher than that in controls (Z = 3.988, P < 0.0001). The urinary Bisphenol A levels in cases were significant higher than those in controls from senior middle school (the levels were 0.10, 0.06 µg/ml respectively, Z = 1.996, P = 0.0459), college (the levels were 0.14, 0.03 µg/ml respectively, Z = 2.586, P = 0.0097), workers or farmers (the levels were 0.08, 0.03 µg/ml respectively, Z = 2.265, P = 0.0235), businessmen (the levels were 0.10, 0.03 µg/ml respectively, Z = 2.544, P = 0.0109), and no passive smokers (the levels were 0.09, 0.03 µg/ml respectively, Z = 3.767, P = 0.0002). After adjustment by age, body mass index, marital status during pregnancy and other factors, compared to Bisphenol A below 0.06 µg/ml, the adjusted OR was 4.03 (1.67 - 9.74) for Bisphenol A levels between 0.06 µg/ml and 0.20 µg/ml, and was 5.46 (1.95 - 15.27) for Bisphenol A over 0.20 µg/ml. The risk of unexplained recurrent spontaneous abortion increased progressively with the growth of urinary Bisphenol A levels (χ(2) = 13.042, trend test P = 0.0003). There were significant differences on Bisphenol A among controls, two abortions, and three or more abortions (the levels were 0.03 µg/ml, 0.09 µg/ml, 0.21 µg/ml respectively, F = 9.04, P = 0.0002). CONCLUSION: Exposure to Bisphenol A may be associated with unexplained recurrent spontaneous abortion.

[Differences in the expression of inhibin receptors and activin receptors in normal human ovaries and their significance].

OBJECTIVE: To explore the differences in the expression of inhibin (INH) receptors and activin (ACT) receptors in the follicular/luteinic phase in normal human ovaries and their relationship with female endocrine hormone levels. METHODS: Real time PCR and immunohistochemistry were used to determine the expression of inhibin receptors (INHR) genes, activin receptors (ACTR) genes. Serum estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), INHB, ACTA levels were determined by a solid quantitative sandwich enzyme immunoassay technique (Sandwich ELISA) in 21 women during follicular phase and another 21 women during luteinic phase, the correlations between each gene and each hormone were analyzed. RESULTS: (1) ACT type I and II receptors genes (ACTR I A, ACTR I B, ACTR II A, ACTR II B) and INH receptor beta-glycan genes were expressed higher in the follicular phase than in the luteinic phase: ACTR I A (0.50 +/- 0.17 vs 0.36 +/- 0.18; P < 0.05), ACTR I B (0.050 +/- 0.019 vs 0.036 +/- 0.020; P < 0.05), ACTR II A (0.10 +/- 0.04 vs 0.07 +/- 0.04; P < 0.05), ACTR II B (0.28 +/- 0.10 vs 0.19 +/- 0.11; P < 0.05), beta-glycan (0.26 +/- 0.10 vs 0.17 +/- 0.09; P < 0.01). (2) The intensities of ACTR I A, ACTR II A, beta-glycan immunostaining in human normal ovaries in the follicular phase were significantly stronger compared to those in luteinic phase. In the follicular phase beta-glycan expression was positively correlated with serum E2, FSH, LH, INHB levels. The correlation coefficient was 0.53 (P < 0.05), 0.74 (P < 0.01), 0.85 (P < 0.01) and 0.76 (P < 0.01) respectively. CONCLUSION: In normal human ovary in the follicular phase INH and ACT bind their receptors and down-regulate or up-regulate FSH, thus influencing the follicular development.