METTL3 regulates the proliferation, metastasis and EMT progression of bladder cancer through P3H4.

Bladder cancer, the most common malignant tumor in the urinary system, exhibits significantly up-regulated expression of P3H4, which is associated with pathological factors. The objective of this study was to elucidate the underlying mechanism of P3H4 in bladder cancer. Initially, we analyzed P3H4 gene expression using the TCGA database and evaluated P3H4 levels in clinical samples and various bladder cell lines. P3H4 was found to be markedly overexpressed in bladder cancer samples. Subsequently, bladder cancer cells were transfected with shRNA targeting P3H4 (sh-P3H4), sh-METTL3, and P3H4 overexpression vectors (P3H4 OE). Viability, migration, and invasion of bladder cancer cells were assessed using CCK-8, wound healing, and transwell assays. Western blot analysis was performed to determine the levels of EMT-associated proteins, while RNA stability assays determined the half-life of P3H4. Knockdown of P3H4 resulted in inhibition of bladder cancer cell proliferation, migration, invasion, and EMT progression. Mechanistically, METTL3 was found to regulate the mRNA stability of P3H4 in bladder cancer. Moreover, overexpression of P3H4 reversed the inhibitory effects of METTL3 knockdown on bladder cancer cell behaviors. Stable cell lines were established by infecting EJ cells with lentiviral vectors containing sh-METTL3 or P3H4 OE. These cells were then implanted into the skin of BALB/c nude mice, and IHC analysis was used to analyze the expression levels of EMT-associated proteins. In vivo studies demonstrated that inhibition of METTL3 suppressed bladder cancer growth and EMT through P3H4. In conclusion, our findings suggest that METTL3 regulates the proliferation, metastasis, and EMT progression of bladder cancer through P3H4, highlighting its potential as a therapeutic target.

Efficient Delivery of P3H4 siRNA and Chlorin e6 by cRGDfK-Installed Polyarginine Nanoparticles for Tumor-Targeting Therapy of Bladder Cancer.

PURPOSE: Prolyl 3-hydroxylase family member 4 (P3H4) is a potent prognostic oncogene in bladder cancer (BC), and the inhibition of P3H4 suppresses BC tumor growth. This study aimed to evaluate the efficiency of P3H4 inhibition for BC tumor therapy via tumor-targeting nanoparticles. METHODS AND RESULTS: A linear polyarginine peptide (R9) was synthesized, azide-modified, and then assembled with cyclic pentapeptide cRGDfK. Chlorin e6 (ce6)-conjugated CH3-R9-RGD nanoparticles were prepared for the delivery of siP3H4 into T24 cells in vitro and BC tumors in vivo. Dynamic light scattering analysis identified that the optimum CH3-R9-RGD@siP3H4 molar ratio was 30/1. CH3-R9-RGD@ce6/siP3H4 nanocomposites decreased P3H4 expression and cell proliferation and promoted reactive oxygen species production, apoptosis, and calreticulin exposure in T24 cells in vitro. In vivo experiments showed that CH3-R9-RGD@ce6/siP3H4 nanocomposites caused pathological changes, suppressed BC tumor growth, promoted caspase 3 expression, and enhanced calreticulin exposure in tumor cells. CONCLUSIONS: The tumor-targeting CH3-R9-RGD nanocomposites encapsulating siP3H4 and ce6 might be an alternative therapeutic strategy or intravesical instillation chemotherapy for BC.

CDH1 overexpression predicts bladder cancer from early stage and inversely correlates with immune infiltration.

BACKGROUND: Bladder cancer (BC) seriously endangers public health, but effective biomarkers for BC diagnosis, particularly in the early stage, are still lacking. Identification of reliable biomarkers associated with early-stage BC is of great importance to early treatment and an improved outcome. METHODS: Differentially expressed genes (DEGs) were identified using four publicly available early-stage BC gene-expression profiles. Protein-protein interaction (PPI) and survival analysis for hub genes was evaluated. The correlation between methylation of genes and prognosis was evaluated using the MethSurv database. Co-expressed genes were explored using Cancer Cell Line Encyclopedia database and the corresponding expression were assessed in vitro. The competing endogenous RNA network and the immune cell infiltration in BC were generated using data of The Cancer Genome Atlas. RESULTS: Ten hub genes of the 213 integrated DEGs were identified, including CDH1, IGFBP3, PPARG, SDC1, EPCAM, ACTA2, COL3A1, TPM1, ACTC1, and ACTN1. CDH1 appeared to increase from tumor initiation stage and negatively correlated with methylation. Six methylated sites in CDH1 indicated a good prognosis and one site indicated an aberrant prognosis. High CDH1 expression was negatively correlated with infiltrations by most immune cells, such as plasmacytoid dendritic cells (pDCs), regulatory T cells, macrophages, neutrophils, DCs, and natural killer cells. CDH1 was highly positively correlated with EPCAM and appeared to be directly regulated by miR-383. CONCLUSIONS: The identified oncogenic alterations provide theoretical support for the development of novel biomarkers to advance early-stage BC diagnosis and personalized therapy.

Melatonin decreases androgen-sensitive prostate cancer growth by suppressing SENP1 expression.

BACKGROUND: Melatonin is a hormone naturally produced by the pineal gland in the brain. In addition to modulating circadian rhythms, it has pleiotropic biological effects including antioxidant, immunomodulatory, and anti-cancer effects. Herein, we report that melatonin has the ability to decrease the growth and metastasis of androgen-dependent prostate cancer. METHODS: To evaluate the anti-cancer effect of melatonin on androgen-sensitive prostate cancer in vitro or in vivo, the effects of cell proliferation, apoptosis, migration and invasion were analyzed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, flow cytometry, Transwell assay, and immunohistochemistry (IHC), respectively. Next, the interaction between androgen receptor (AR) and SUMO specific protease 1 (SENP1) was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting, and confirmed by luciferase reporter assay. Furthermore, the Small Ubiquitin-like Modifier (SUMO) proteins are a group of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. (SUMOylation) of histone deacetylases 1 (HDAC1) was measured by proximity ligation assay (PLA). RESULTS: The treatment of melatonin cripples the transcriptional activity of AR, which is essential for the growth of the androgen-dependent prostate cancer cell, LNCaP. The lower activity of AR was dependent on melatonin induced SUMOylation of HDAC1, which has been established as a key factor for the transcriptional activity of AR. Mechanistically, the effect of melatonin on AR was due to the decreased SENP1 protein level and the subsequent increased HDAC1 SUMOylation level. The overexpression of SENP1 abrogated the anti-cancer ability of melatonin on LNCaP cells. CONCLUSIONS: These findings indicate that melatonin is a suppressor of androgen-dependent prostate cancer tumorigenesis.

P3H4 and PLOD1 expression associates with poor prognosis in bladder cancer.

PURPOSE: The prolyl 3-hydroxylase family member 4 gene (P3H4) is involved in the development of human cancers. The association of P3H4 with bladder cancer (BC) prognosis is unclear. This study aimed to analyze the association of P3H4 with BC prognosis. METHODS: RNA-Seq data were downloaded from The Cancer Genome Atlas project and BC microarray datasets (GSE13507, GSE31684, and GSE32548) were downloaded from the Gene Expression Omnibus database. We analyzed the differences in P3H4 expression levels between BC tumors and non-tumor tissues and between samples with different clinical information. The association of P3H4 and P3H4-related genes with BC prognosis and the possibility of using P3H4 expression as a prognostic biomarker in BC patients were also analyzed. RevMan was used to perform the meta-analysis. RESULTS: P3H4 was upregulated in BC tissues compared with the adjacent non-tumor tissues (p = 4.06e-08). Univariate Cox regression analysis and meta-analysis showed that high P3H4 expression level contributed to a poor BC prognosis (Hazard ratio, HR = 1.348, 95% CI 1.140-1.594, p = 4.89e-04; meta-analysis: HR = 1.45, 95% CI 1.10-1.91; p = 9.00e-03). Among the genes related to P3H4, the PLOD1 gene was closely associated with P3H4 expression (r = 0.620, p = 2.49e-44). Also, a meta-analysis showed that PLOD1 expression was associated with a poor prognosis in BC patients (HR = 1.77, 95% CI 1.31-2.38; p = 2.00e-04). CONCLUSIONS: The P3H4 and PLOD1 genes might be used as reliable prognostic biomarkers for BC.

MAP30 Inhibits Bladder Cancer Cell Migration and Invasion In Vitro Through Suppressing Akt Pathway and the Epithelial/Mesenchymal Transition Process.

The antitumor activity of Momordica anti-human immunodeficiency virus protein of 30 kDa (MAP30) has been proved. However, the role of MAP30 on tumor metastasis has not yet been identified. For this purpose, we investigated this effect and underlying mechanism of MAP30 in bladder cancer (BC). Here, we reported that MAP30 significantly inhibited the cell proliferation and clone formation of 5637 and T24 cells in vitro by promoting apoptosis and cell cycle arrest. We also found MAP30 inhibited cell migration and invasion by suppressing the epithelial/mesenchymal transition (EMT) process. Moreover, the Affymetrix GeneChip assay revealed that MAP30 significantly changed gene expression profile in T24 cells, especially the genes in cell cycle regulation pathways. After the Ingenuity Pathway Analysis, we predicted that NUPR1 was the most important upstream regulator. Subsequently, we verified that the AKT and EMT signaling pathways were inhibited by MAP30 treatment in T24 cells. In conclusion, MAP30 treatment inhibited the progression of human BC, especially cell migration and invasion through suppressing AKT pathways.

Clinical analysis of uterine torsion and fibroids in full-term pregnancy: A case report and review of the literature.

This study was performed to describe the clinical features, risk factors, and treatment methods of uterine torsion in pregnancy. The most common symptoms are abdominal pain, fetal heart rate changes, and failure of cervical dilatation and are often accompanied by complete or partial placental abruption. Preoperative diagnosis is challenging even with the use of ultrasound. Uterine torsion in the third trimester is correlated with the presence of multiple uterine fibroids. The causes of gravid uterine torsion vary and the clinical manifestations are nonspecific. Early diagnosis and improved detection approaches are the keys to treatment of patients with uterine torsion. However, the preoperative diagnosis remains difficult and the diagnosis is often made during cesarean section.

Knockdown of P3H4 inhibits proliferation and invasion of bladder cancer.

The prolyl 3-hydroxylase family member 4 (P3H4) (alias SC65) is implicated in a variety of physiological and pathological processes. However, little is known about the role of P3H4 in tumors. This study aimed to investigate the role of P3H4 in bladder cancer (BC) and the regulatory mechanisms that influence its expression. P3H4 was highly expressed in BC tissues. Knockdown of P3H4 inhibited BC cell proliferation, cell cycle, migration and invasion in vitro, and inhibited BC growth in vivo. We also found that ETV4 bound directly to the promoter region of P3H4 and activated its transcription. Furthermore, overexpression of ETV4 rescued the inhibition of proliferation and invasion induced by PH4 silencing. ETV4 was significantly overexpressed in the BC tissues. In conclusion, P3H4 functioned an oncogene role in BC progression, and ETV4 bound directly to the P3H4 promoter region to regulate its transcription.

Effect of Deubiquitinase Ovarian Tumor Domain-Containing Protein 5 (OTUD5) on Radiosensitivity of Cervical Cancer by Regulating the Ubiquitination of Akt and its Mechanism.

BACKGROUND The aim of this study was to investigate the role of deubiquitinase [ovarian tumor domain-containing protein 5 (OTUD5)] in regulating Akt ubiquitination and its effect on the radiosensitivity of cervical cancer. MATERIAL AND METHODS Cervical cancer C33A cells were cultured, and then 2 groups of cells (overexpressed cells and silenced cells) were established by overexpressing and silencing OTUD5 gene. Next, quantitative polymerase chain reaction (qPCR) was employed to detect the expression level of OTUD5 in cells in each group. Co-immunoprecipitation and Western blot (WB) analysis were applied to measure the expression level of phosphorylated protein kinase B (Akt) and the level of ubiquitination. The sensitivity of cells to radiotherapy in each group was detected via clone-forming efficiency assay. After that, Statistical Product and Service Solutions (SPSS) 17.0 software was employed for analyses. The t test, one-way analysis of variance (ANOVA), and p test were used. P<0.05 suggested that a difference was statistically significant. RESULTS The levels of phosphorylated Akt and ubiquitination in OTUD5-overexpressed C33A cells were lower than those in the OTUD5-silenced group and control group. The sensitivity of OTUD5-overexpressed C33A cells to radiotherapy was higher than that of the OTUD5-silenced group and control group. CONCLUSIONS OTUD5 affects the radiosensitivity of cervical cancer through the regulation of Akt deubiquitination.

Effects of disodium cantharidinate on dendritic cells of patients with bladder carcinoma.

The present study explored the effects of disodium cantharidinate (DC) on the peripheral blood-derived dendritic cells of patients with bladder carcinoma. The peripheral blood mononuclear cells from the 15 cases of urinary bladder carcinoma of middle and advanced stage were separated, and dendritic cells were prepared. The morphological changes of dendritic cells were observed. Flow cytometry was used to detect the expression levels of CD1a and CD83 on dendritic cell surface. MTT assay was utilized to measure the proliferation ability of allogeneic lymphocyte stimulated by DC. Annexin V-FITC/propidium iodide (PI) double staining flow cytometry method was carried out to detect cell apoptosis after treatment with DC. The changes in caspase-3 and PARP expression levels were investigated by western blot method. The high-dose DC resulted in a significant increase in the expressions of dendritic cell phenotyptic molecules CDla and CD83 as compared to control group. In addition, the proliferation index of allogenic lymphocyte stimulated by DC was significantly higher than that of control group. Moreover, MTT assay showed significant inhibition of the growth of BIU-87 cells. After 24 h of DC treatment, double staining flow cytometry confirmed the ability of DC to induce cell apoptosis. Further, western blot method showed a significant elevation of caspase-3 and PARP protein expression after DC treatment. In conclusion, DC treatment could induce dendritic cell maturation of patient with carcinoma of urinary bladder and promote its functional changes. Furthermore, DC was able to inhibit the proliferation of cell BIU-87 and also has the ability to induce cell apoptosis.

The antitumor effect of the novel cancer-specific adenovirus Ad-VEGFR on bladder cancer in vitro and in vivo.

Bladder cancer is one of the most common cancers. Approaches that block tumor angiogenesis are a new therapeutic strategy for locally advanced or metastatic BC. VEGF/VEGFR signaling has been obviously and negatively correlated with the progression and invasion of cancer. In this study, we constructed the recombinant adenovirus vAd-VEGFR-3 to investigate its antitumor effector in vitro/vivo. First, we used the recombinant adenovirus vAd-VEGFR-3 to infect bladder cancer cells and then collected the cell culture supernatant to treat human umbilical vein endothelial cells (HUVECs). The proliferation, migration and apoptosis of HUVECs were respectively detected by MTT, transwell and Annexin V-FITC/PI double staining. In addition, mouse bladder mucosa was injured by trypsin, and the orthotopic transplantation model of human bladder cancer was successfully constructed to clarify the anti-tumor effect of Ad-VEGFR in vivo. The results showed that Ad-VEGFR could inhibit the cancer's proliferation and migration, while promoting the apoptosis of HUVECs in vitro. Moreover, Ad-VEGFR could significantly promote the apoptosis of bladder cancer cells and then prevent tumor growth in vivo. In addition, it also down-regulated the expression levels of CD31, an endothelial cell marker which is closely related to the angiogenesis. Taken together, it suggests that the infection of adenovirus-carrying VEGFR in bladder cancer cells may inhibit blood vessel formation and prevent tumor progression.

Stability analysis on the radioactive iodine-labelled prostate cancer-specific recombinant oncolytic adenovirus.

The aim of the present study was to construct the 125I-replication-selective oncolytic adenovirus (RSOAds)-human telomerase reverse transcriptase (hTERT)/prostate specific antigen (PSA) nuclide-oncolytic virus marker by labelling the hTERT/PSA double-regulation replicative oncolytic adenovirus with 125I nuclide, and investigate the influence of viral markers under various reaction conditions on labelling efficiency. N-bromosuccinimide (NBS) was used as the oxidizer for 125I labelling, and the best conditions for labelling were identified through the reactions between oncolytic adenovirus at various concentrations and NBS. Dosage of 125I, reaction duration, pH values and reaction volume were respectively evaluated to determine their effects on the labelling efficiency of 125I-RSOAds-hTERT/PSA nuclide-oncolytic adenovirus markers. Purified nuclide-oncolytic adenovirus markers were isolated by gel-filtration chromatography; paper chromatography was performed to assay the radiochemical purity of 125I-RSOAds-hTERT/PSA markers at various time points. Radiochemical purity of 125I-RSOAds-hTERT/PSA was >95%, and could be maintained at 4°C for 7 days. The best reaction conditions were set as follows: 0.5 µl of 125I (~0.2 m Ci, 7.4 MBq); 25 qg of NBS; 100 µl of 8×109 VP/ml 125I-RSOAds-hTERT/PSA virus solution; 30 min of reaction duration; pH 7.5; 120 µl of PBS. Labelling hTERT/PSA double-regulation replicative oncolytic adenovirus with 125I was identified to be available, and the radiochemical purity of acquired virus markers could be maintained under specific conditions.

Application of pirarubicin photosensitizer fluorescence cystoscopy in early detection of bladder cancer.

The aim of this study was to investigate the value of pirarubicin (THP) photosensitizer fluorescence cystoscopy for the early diagnosis of bladder cancer. From January 2012 to June 2015, 25 patients with painless gross hematuria, were injected with THP 15 min prior to surgery and subsequently underwent fluorescence cystoscopy examination. Locations of tumors were recorded and biopsies were performed (total of 109 biopsies) under white and fluorescence light guidance. Biopsies were conducted at the THP-positive and -negative areas under white light and THP-positive areas under fluorescence light. Positive rate of bladder tumor tissue at the THP-positive areas detected under fluorescence light was 92.86%. The sensitivity and specificity were 100 and 96.15%, respectively. The positive rate of tumor tissue at the THP-positive areas detected under white light was 70.97%. The sensitivity and specificity were 100 and 84.74%, respectively. Fifty biopsies taken at the THP-negative areas under white light were found to be non-urothelial carcinoma tumor lesions. Thus, applying (THP) photosensitizer fluorescence cystoscopy for early-period bladder cancer diagnosis is safe, effective and practical. It showed a high degree of specificity and a low rate of false positives. It was a convenient visual diagnostic method for the early detection of non-muscular invasive bladder cancer.

Tumor necrosis factor-related apoptosis-inducing ligand inhibits proliferation and induces apoptosis of prostate and bladder cancer cells.

The effect of recombinant and purified tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the proliferation and apoptosis of PC-3 prostate cancer cells and the 5637 bladder cancer cells were investigated. We used a cell proliferation assay and flow cytometry to measure the proliferation and apoptosis of cancer cells after 24-h incubation of PC-3 and 5637 cells with different concentrations of TRAIL. PC-3 cell proliferation rate significantly decreased when TRAIL was used at concentrations of 20, 40, 80 and 160 ng/ml compared with the untreated group. In the 5637 cells, the proliferation rate significantly decreased when TRAIL was used at concentrations of 5, 10, 20 and 40 ng/ml compared with the untreated group. The flow cytometry results also confirmed that the apoptosis rate of both cancer cell lines increased with TRAIL protein concentration. In conclusion, recombinant and purified TRAIL has anticancer activity by inhibiting proliferation and promoting apoptosis of prostate and bladder cancer cells.

ImmunoCyt test compared to cytology in the diagnosis of bladder cancer: A meta-analysis.

The aim of the present study was to evaluate the diagnostic value of the ImmunoCyt test compared with urine cytology in detecting bladder cancer. A systematic literature search was performed to locate all publications reporting on the diagnostic accuracy of the ImmunoCyt test for bladder cancer. Data were extracted from 2×2 tables or calculated from reported accuracy data. Collected data were meta-analyzed for sensitivity, specificity, positive likelihood ratio (LR), negative LR, diagnostic odds ratio (DOR), and summary receiver operator characteristic (sROC) curve analysis. We applied the Meta-DiSc 1.4 and STATA 13.0 software to the meta-analysis. Seven separate studies consisting of 1,602 patients with bladder cancer were considered in the meta-analysis. We found that the ImmunoCyt test had a higher sensitivity than the urine cytology test [0.725, 95% confidence interval (CI) 0.683-0.765 vs. 0.566, 95% CI, 0.521-0.611], but the specificity, positive LR, negative LR, DOR, area under the curve (AUC) and Q index of the ImmunoCyt test were lower compared with the urine cytology test. In addition, the pooled sensitivity, specificity, positive LR, negative LR, DOR, AUC, and Q index of the combined method (combination of ImmunoCyt and cytology) were 0.833, 0.644, 2.804, 0.228, 13.50, 0.8554 and 0.7863, respectively. The results of the Eggers test showed no publication bias (P>0.05). In conclusion, specificity, positive LR, negative LR, DOR, the AUC, and the Q index of the urine cytology test may be superior to the ImmunoCyt test, but the ImmunoCyt test has greater sensitivity than the urine cytology test. Use of ImmunoCyt and cytology in combination has the potential to improve the sensitivity and promises to be an alternative in the detection of bladder cancer.

A Mouse Model of Hypospadias Induced by Estradiol Benzoate.

We wished to establish a mouse model of hypospadias using injections of estradiol benzoate for investigating the molecular mechanisms of hypospadias. Fifty timed pregnant mice were randomly divided into five study groups: A, B, C, D, and E. These groups were injected subcutaneously with estradiol benzoate mixed with sesame oil at, respectively, the doses of 0, 0.1, 0.5, 2.5, or 12.5 mg kg(-1) days(-1) from gestation day (GD) 12 to GD 16. The pups' mortality was recorded on the day of delivery. Urethras and positions of testes were examined on postnatal day 28. The numbers of live pups were significantly lower in the study groups D and E compared to study group A (p < 0.01). Hypospadias was seen in groups C (3.3 %; 1/30), D (18.2 %; 4/22), and E (21.4 %; 3/14), while cryptorchidism was observed in groups C (10 %; 3/30), D (31.8 %; 7/22), and E (57.1 %; 8/14) on postnatal day 28. The experimental model of hypospadias induced by estradiol benzoate in the group D (2.5 mg kg(-1) days(-1)) was more reliable considering high mortality of the study group E. The dose of estradiol benzoate used in the group D is suitable for establishing mouse model of hypospadias.

Anti-tumor function of double-promoter regulated adenovirus carrying SEA gene, in the treatment of bladder cancer.

To construct an adenovirus carrying SEA gene and regulated by telomerase reverse transcriptase (hTERT) and hypoxia-inducible factor (HIF) promoters and investigate its anti-tumor function in vitro, as well as its role in lymphocyte production. hTERT and HIF genes were cloned into adenovirus E1A and E1B shuttle plasmids. The control vector for SEA gene expression is under the regulation of CMV and SV40 promoters. Double regulation was obtained through homologous recombination. The positive clones of replicable adenovirus H2-SEA-Ad were selected by plaque assay. The adenovirus was purified, titrated, and DNA was verified by PCR. The obtained virus was used to infect EJ bladder tumor cells and the SEA Mrna, and protein expression was measured by RT-PCR, western blot, and immunofluorescence microscopy, respectively. Co-culture of lymphocytes and tumor cells was observed dynamically under microscope. The construction of shuttle plasmid p315-CSS-SEA was confirmed by PCR and DNA sequencing. Insertion of superantigen SEA gene in adenovirus (H2-SEA-Ad.SEA) was obtained by homologous recombination. In lymphocytes and tumor cell co-culture, the number of viable tumor cells in test groups was significantly lower than that in control group after 12, 24, and 48 h of treatment. Production of interleukin-2, interleukin-4, and tumor necrosis factor were higher in test groups than in control group. Expression of SEA gene in bladder tumor cells by adenoviral vector caused reduced tumor cell proliferation, as well as stimulation of inflammatory cytokine productions in co-cultures with lymphocytes.

[Establishment of a mouse model of hypospadias induced by estradiol benzoate].

OBJECTIVE: To establish a mouse model of hypospadias induced by benzoate estradiol to further the studies on the molecular mechanisms of hypospadias. METHODS: A total of pregnant mice were randomly divided into 5 groups, Group A, B, C, D and E, and injected subcutaneously (sc) with estradiol benzoate at the dose of 0, 0.2, 1, 5 and 25 mg x kg(-1) d(-1) respectively from the 12th to the 16th gestational day. The mortality of the newborn mice was recorded and the male neonates of 2 pregnant mice from each group were anatomized to observe the testis position and prostate agenesis on the delivery day. Examinations were made for urethra and cryptorchidism on the 28th postnatal day. RESULTS: The death rates of the neonates in Group A, B, C, D and E were 21.6%, 21.5%, 41.4%, 56. 6% and 75.0%, respectively. Hypospadias was detected in Group C (3.3%, 1/30), D (20.0%, 4/20) and E (23.0%, 3/13), with significant difference between Group D and A (P < 0.05) and E and A (P < 0.05), but not between Group D and E (P > 0.05). Cryptorchidism was found in Group C (6.6%, 2/30) , D (30.0%, 6/20) and E (61.6%, 8/13), with significant difference between Group D and A (P < 0.05) and E and A (P < 0.05) , but not between Group D and E (P >0.05). CONCLUSION: Exposure of pregnant mice to large dose of estradiol benzoate can induce hypospadias and cryptorchidism in their neonates. And the right dose of estradiol benzoate for the establishment of the mouse model of hypospadias should be 5 mg x kg(-1) x d(-1).

Transplantation tolerance mediated by regulatory T cells in mice.

BACKGROUND: With potent suppressive effect on responder T cells, CD(4)(+)CD(25)(+) regulatory T (Treg) cells have become the focus of attention only recently and they may play an important role in transplantation tolerance. However, the mechanism of action is not clear. This study was designed to assess the possibility of using CD(4)(+)CD(25)(+) Treg cells to induce transplantation tolerance and to investigate their mechanism of action. METHODS: CD(4)(+)CD(25)(+) Treg cells were isolated using magnetic cell separation techniques. Mixed lymphocyte reactions were used to assess the ability of Treg cells to suppress effector T cells. Before skin transplantation, various numbers of CD(4)(+)CD(25)(+) Treg cells, which have been induced using complex skin antigens from the donor, were injected into the host mice either intraperitoneally [0.5 x 10(5), 1 x 10(5), 2 x 10(5), 3 x 10(5), 4 x 10(5), or 5 x 10(5)] or by injection through the tail vein [5 x 10(3), 1 x 10(4), 2 x 10(4), 5 x 10(4), 1 x 10(5), 2 x 10(5)]. Skin grafts from two different donor types were used to assess whether the induced Treg cells were antigen-specific. The survival time of the allografts were observed. Single photon emission computed tomography was also used to determine the distribution of Treg cells before and after transplantation. RESULTS: Treg cells have suppressive effect on mixed lymphocyte reactions. Grafts survived longer in mice receiving CD(4)(+)CD(25)(+) Treg cell injections than in control mice. There was a significant difference between groups receiving intraperitoneal injection of either 2 x 10(5) or 3 x 10(5) CD(4)(+)CD(25)(+) Treg cells and the control group (P < 0.05, respectively). Better results were achieved when Treg cells were injected via the tail vein than when injected intraperitoneally. The transplantation tolerance induced by CD(4)(+)CD(25)(+) Treg cells was donor-specific. Analysis of the localization of Treg cells revealed that Treg cells mainly migrated from the liver to the allografts and the spleen. CONCLUSIONS: CD(4)(+)CD(25)(+)Treg cells can induce donor-specific transplantation tolerance. Cell-to-cell contact may be the primary mechanism by which Treg cells act on effector T cells.

[A mouse model of hypospadias induced by flutamide].

OBJECTIVE: To establish a mouse model of hypospadias induced by flutamide to further studying molecular mechanisms of hypospadias etiology. METHODS: Eighty timed pregnant ICR mice were randomly divided into four groups. Flutamide was injected subcutaneously (s.c.) with mixture sesame oil at 0 (Group A), 25 (Group B), 50 (Group C), 100 (Group D) mg.kg-1.d-1 from GD (gestation days) 12 to 16, respectively. The fetuses of two pregnants from each group were anatomized to observe the position of testes and the development of prostates on the day of delivery. Urethras and the position of testes were examined on postnatal day 28. RESULTS: Hypospadias was seen in Group A (0), B (44.2%), C (92.7%) and D (100%), and cryptorchidism in Group A (0), B (4.8%), C (23.2%) and D (32.4%), respectively. Flutamide caused 100% incidence of prostate agenesis in Group C and D and 19.2% in Group B, and 100% incidence of female-like anogenital distance in Group B, C, and D. CONCLUSION: The experimental model of hypospadias induced by flutamide is steadier and more suitable for popularization.