OBJECTIVERNA interference was applied to knockdown the Dhcr7 gene in mouse embryonic palatal shelves to facilitate understanding of the function of Dhcr7 gene variants in the fusion of palatal shelves.

METHODSThe pAdTrack-CMV-siDhcr7 was constructed using the specific siRNA sequence of Dhcr7 from C57BL/6J mouse. The pAdTrack-CMV- siDhcr7 of positive clones was reconstructed in vitro, and the recombinant adenovirus pAdEasy-1-siDhcr7 of kanamycin resistance was screened. The adenovirus vector DNA was then prepared for transfecting the embryonic palatal shelves. Thirty pairs of embryonic palatal shelves at 13.5 d gestational age were harvested and then randomly divided into the following three groups: normal control group (n = 10), which included palatal shelves inculture medium without cholesterol; blank adenovirus control group (n = 10), which included palatal shelves in culture medium without cholesterol and blank adenovirus; and experimental group (n = 10), which included palatal shelves in culture medium without cholesterol and adenovirus encoding Dhcr7 siRNA. At 48 h after in vitro cultivation, the mRNA and protein of the palatal shelves were obtained for scanning electron microscopy (SEM), reverse transcription polymerase chain reaction (RT-PCR), and Western blot analyses.

RESULTSSEM showed that the palatal shelves of the normal control and blank adenovirus control groups fused and formed continuous palates, whereas those of the experimental group was almost undeveloped but exhibited large gaps between the two palatal shelves. RT-PCR and Western blot analyses showed that the mRNA and protein of Dhcr7 in the experimental group decreased compared with those in the normal control group with a significant difference (P < 0.05).

CONCLUSIONResults indicate that Dhcr7 gene silencing affects the fusion of palatal shelves. Thus, Dhcr7 gene may serve a function in the normal development of palates.

Animals ; Cleft Palate ; Gene Silencing ; Mice ; Mice, Inbred C57BL ; Microscopy, Electron, Scanning ; Organ Culture Techniques ; Oxidoreductases Acting on CH-CH Group Donors ; Palate ; growth & development ; RNA, Messenger

Hepatitis E ; immunology ; prevention & control ; Humans ; Viral Hepatitis Vaccines

Objective The 4-and 16-hydroxylated metabolites of estrogens have been implicated in carcinogenesis,whereas its 2-hydroxylated metabolites have been shown to have antiangiogenic effects.We aimed to examine whether the polymorphisms of catechol-O-methyltransferase(COMT)involved in the estrogen metabolism are associated with endometrial cancer risk.Methods Polymerase chain reaction- restrictive fragment length polymorphism(PCR-RFLP)analysis was used to study the variant allele frequency distributions of COMT Val158Met genetic polymorphism in a population based case-control study with 132 endometrial cancer cases and 110 controls.Odds ratios(OR)and 95% confidence intervals(CI) were estimated by unconditional logistic regression after adjustment for known or suspected risk factors for endometrial cancer.Results The most frequent genotype was COMT~(Val/Val)(47.2%,52/110)in control group and COMT~(Mal/Met)(58.3%,77/132)in endometrial cancer group.The difference between the two groups was of statistical significance(P

OBJECTIVETo investigate the clinical safety and effects of auto-dendritic cells pulsed with HLA-A201-binding peptides prostate-specific antigen (PSA) , prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP) in the treatment of hormone-refractory metastatic prostate cancer (HRPC).

METHODSSixteen HRPC patients with positive HLA-A201 were enrolled and their monocytes isolated and induced into dendritic cells with the combination of rhGM-CSF and rhIL4. The patients were inoculated subcutaneously near the inguinal region with auto-DCs pulsed with peptides PSA (KLQCVDLHV) , PSMA (ALDVYNGL L) and PAP (LLHETDSAV) every 2 weeks for 4 times, and the immunological and clinical responses were examined within 1 -2 weeks after the final vaccination.

RESULTSVaccination of dendritic cells was well tolerated and no toxicity was observed. The cytokine levels in the serum such as IL-2, IL-12 and IFN-gamma were significantly increased after the vaccination (P < 0.01). The delayed type hyper- sensitivity (DTH) test was positive in 4 of the patients (4/11), the percentage of antigen-special IFN-gamma+ CD8+ T increased in 5 (5/11), the level of the tumor marker PSA decreased in 6 (6/16) , hydrops abdominis reduced in 1 (1/16), and the size of the cervical lymph node lessened in 1 (1/16). Three patients showed partial remission (PR), 7 stability of the disease (SD), and the other 6 progression of the disease (PD).

CONCLUSIONAuto-DC vaccines loaded with PSA, PSMA and PAP peptides, capable of eliciting specific immune responses in HRPC patients, is a safe and effective option for the treatment of advanced HRPC.

Acid Phosphatase ; Aged ; Antigens, Surface ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Cancer Vaccines ; immunology ; Cytokines ; blood ; Dendritic Cells ; immunology ; Glutamate Carboxypeptidase II ; immunology ; HLA-A2 Antigen ; immunology ; Humans ; Male ; Middle Aged ; Prostate-Specific Antigen ; immunology ; Prostatic Neoplasms ; therapy ; Protein Tyrosine Phosphatases ; immunology ; Treatment Outcome

Objective To explore the clinical value of second polar body (Pb2) exclusion monitoring by timelapse in predicting the fertilization and embryo development efficiency for intracytoplasmic sperm injection ( ICSI). Methods A retrospective research was performed on 278 patients treated with ICSI, the clinical data and Time-lapse monitoring embryo culture data were collected and analyzed, to explore the exclusion of Pb2 after ICSI and the relationship between the specific exclusion time and the outcome of fertilization and embryo development. Results The average time of Pb2 exclusion after ICSI was ( 3. 03 ± 1. 21) hours; The fertilization rate, 2 pronucleus(PN) fertilization rate and 5 days ( D5) blastocyst formation rate in the Pb2 exclusion group were significantly higher than those in the without Pb2 exclusion group (99.95% vs f. 75%, P < 0 . 001; 97.18% vs 0.66%, P< 0.001; 60.50% vs 16.67%, P < 0 . 0 5 ) ; The 2PN fertilization rate in Pb2 exclusion time >3-4 hours group was significantly higher than that in 0-2 hours group and >5 hours group (98.80% vs 9 3 . 8 1 % , P<0. 05; 98.80% vs 95.40%, P<0. 0 5 ) ; The exclusion time of Pb2 was significantly correlated with the average number of blastomeres in D3 embryos (P<0. OOf). The D5 blastocyst formation rate of 3-4 hours group was significantly lower than that of > 2-3 hours group ( 56. 23% vs 67. 23%, P < 0. 05 ) , > 4-5 hours group was significantly lower than 0-2 hours group and >2-3 hours group ( 46. 6f % vs 62. 30% , P<0. 05; 46. 6f % vs 67. 23% , P< 0. 05) , and D5 blastocyst formation rate of >5 hours group was 7. f 4 % , which were significantly lower than that of the other four groups (P<0. 05). The fonnation rate of D5 high-quality blastocysts in 3-4 hours group was significantly lower than 0-2 hours group and > 2-3 hours group ( 9. 92% vs 16. 39% , P<0. 05; 9. 92% vs 20. 72% , P<0. 05) , and D5 highquality blastocysts formation rate in > 4-5 hours group was significantly lower than that in > 2-3 hours group (11. 02% vs 20.72%, P<0. 05). Conclusion Monitoring Pb2 exclusion by Time-lapse can accurately predict fertilization outcome. The time of Pb2 exclusion is significantly correlated with embiyo development potential. It is a valuable morphological index to predict fertilization and embiyo development outcome in ICSI.

BACKGROUNDThe CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of proteins linking chemokines and TM4SF. Different members exhibit diverse biological functions. In this study, the effect of intracellular CMTM2 on regulating human immunodeficiency virus type-1 (HIV-1) transcription was evaluated.

METHODSThe effects of CMTM2 on regulating full-length HIV-1 provirus and the HIV-1 long terminal repeat (LTR)-directed transcription were assessed by luciferase assay. Transcription factor assays, using the luciferase reporter plasmids of AP-1, CRE, and NF-κB were conducted to explore the signaling pathway(s) that may be regulated by CMTM2. The potential relationship between CMTM2 and the transcription factor AP-1 was further analyzed by Western blotting analyses to investigate the effect of CMTM2 on PMA-induced ERK1/2 phosphorylation.

RESULTSThe results from the current study revealed that CMTM2 acts as a negative regulator of HIV-1 transcription. CMTM2 exerted a suppressive action on both full-length HIV-1 provirus and HIV-1 LTR-directed transcription. Transcription factor assays showed that CMTM2 selectively inhibited basal AP-1 and CREB activity. Co-expression of HIV-1 Tat, a potent AP-1 and CREB activator, can not reverse CMTM2-mediated AP-1 and CREB inhibition, suggesting a potent and specific effect of CMTM2 on negatively regulating these two signaling pathways.

CONCLUSIONIntracellular CMTM2 can negatively regulate HIV-1 transcription, at least in part, by targeting the AP-1 and CREB pathways. Exploring the mechanisms further may lead to new ways to control HIV-1 replication.

Chemokines ; physiology ; Cyclic AMP Response Element-Binding Protein ; antagonists & inhibitors ; HIV Long Terminal Repeat ; HIV-1 ; genetics ; Humans ; Intracellular Space ; metabolism ; Jurkat Cells ; MARVEL Domain-Containing Proteins ; Tetradecanoylphorbol Acetate ; pharmacology ; Transcription Factor AP-1 ; antagonists & inhibitors ; Transcription, Genetic ; U937 Cells

The study was aimed to investigate the influence of penicillin and streptomycin on proliferation, apoptosis and extracellular secretion (ECS) produced from human umbilical cord derived mesenchymal stem cells (MSC). MSC were isolated from umbilical cord tissue, then the immunotyping, multipotent differentiation and proliferation of these cells were assayed by cytometry, cytochemistry and MTT respectively. The expressions of ECS and apoptosis-related genes (bcl-2, bax) were detected by quantitative RT-PCR. The results showed that the phenotype of these cells matched with the characteristics of MSC. Penicillin and streptomycin of low concentrations promoted MSC proliferation, with the most effective concentration of 100 U/ml. Expressions of ECS cultured in addition of penicillin and streptomycin were down-regulated. Furthermore, apoptosis-related factor (bcl-2/bax) expression levels in low concentrations penicillin and streptomycin groups were higher than that in the control group. It is concluded that low concentrations penicillin and streptomycin can promote the proliferation and reduce the apoptotic rate, but high dose can inhibit the ECS component expression of MSC.
Cell Differentiation ; Cells, Cultured ; Extracellular Matrix ; secretion ; Flow Cytometry ; Gene Expression Regulation ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; secretion ; Penicillins ; pharmacology ; Streptomycin ; pharmacology ; Umbilical Cord ; cytology

Objective To evaluated intra-articular injection of TNF-α inhibitors into the sacroiliac joint as an effective and viable alternative. Methods Sixteen patients with documented ankylosing spondylitis (AS), without steroids or disease modifying anti-rheumatic drugs (DMARDs) were performed CT-guided intra-articular injections of etanercept (TNF-α antagonist) at week 0, 4 and 8 (25 mg per dose). Similarly, 20 patients with AS in the control group received systemic etanercept therapy at a dose of 50 mg per week for 8 weeks. All patients were followed up clinically and evaluated periodically. Pathological features of sacroiliitis were observed with light microscopy and immunohistochemistry. Expression of cytokines in joint biopsy samples was estimated by RT-PCR. Image changes of sacroiliitis were observed by SPECT/CT and MRI. Ttest, t'tesr and χ2 Fisher's test were selected. Results All the 16 patients who received intra-articular etanercept, the mean value of radiological nuclide decrease of the SIJ ROI (region of interest) in the SPECT improved significantly after 8 weeks treatment [(1.38±0.16 vs 1.45±0.14) P<0.05] . Bone marrow edema and fat deposition in MRI were relieved significantly after 8 weeks (P<0.05). In 8 patients the expression of TNF-α and TGF-β mRNA in joint tissue decreased significantly after 8 weeks [(0.89±0.06, 0.84±0.05) vs (l.08± 0.19, 1.13±0.33) (P<0.05)]. The occurrence of gynonitis, enthesitis, chondritis, subehondral bony plate destruction, bone marrow inflammation and inflammatory cell index also decreased significantly (P<0.05). Participants given intra-articular injection showed significant clinical improvement after 8 weeks and 12 weeks treatment(P<0.01 ) in BASDAI score [(32±13) mm]. Conclusion This study has shown that intra-articular injection of etanercept in SIJ can improve joint function and quality of life. It has a satisfactory safety profile and is cost effective. This mode of treatment is most beneficial in local arthropathy of recent onset and in those patients who do not tolerate systemic etanercept therapy.

OBJECTIVETo observe the effect of Compound Zhajin Granule (CZG) on Toll-like re-ceptor 4 (TLR4) signaling pathway in high-fructose corn syrup induced NASH mice.

METHODSThirty 6-week-old male C3H mice were divided into the high fat and high fructose (HFHFr) group (n = 20) and the control group (n = 10) according to body weight. Mice in the HFHFr group ate high fat diet and drank 20% fructose water, while those in the control group ate common diet and drank common water. After 8 weeks mice in the HFHFr group were divided into two group according to body weight, the HFHFr group and the CZG group, 10 in each group. Mice in the CZG group were fed with high fat forage and 20% fructose water, and administered with 50 mL/kg 12. 8% CZG (prepared by hawthorn, Radix Curcumae, Alisma Orientale, Fritillaria Thunbergii, Silybum Marianum, peach seed in the ratio of 3:1.5:1.5:2:1.5:2:1) by gastrogavage. Mice in the HFHFr group were fed in the same way and daily administered with equal volume of distilled water by gastrogavage. Sixteen weeks later all mice were sacrificed. Body weight, liver wet weight, liver function, and lipid metabolism were detected. Pathological changes of liver tissues were assessed by HE staining, oil red O staining, and Masson staining. Expressions of TLR4, myeloid differentiation factor 88 (MyD88), tumor necrosis factor-alpha (TNF-α) were detected using immunohistochemical staining and real-time fluorescent quantitative PCR.

RESULTSBody weight, alanine aminotransferase (ALT), aspartate aminotransferase (AST) were obviously lower in the CZG group than in the HFHFr group (P < 0.05); oil red O stained area and density were decreased more in the CZG group than in the control group. HE staining showed ballooning inflammation was reduced more in the CZG group than in the HFHFr group. Masson staining was negative. Positive rates of TLR4 and MyD88 and mRNA expressions were significantly lower in the CZG group than in the HFHFr group (all P < 0.05).

CONCLUSIONCZG could significantly inhibit TLR4 signaling pathway of liver in NASH mice.

Alanine Transaminase ; metabolism ; Animals ; Aspartate Aminotransferases ; metabolism ; Diet, High-Fat ; Drugs, Chinese Herbal ; pharmacology ; Fructose ; administration & dosage ; adverse effects ; Inflammation ; Lipid Metabolism ; Male ; Mice ; Mice, Inbred C3H ; Myeloid Differentiation Factor 88 ; metabolism ; Non-alcoholic Fatty Liver Disease ; drug therapy ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism