BackgroundSmad4,a key intracellular mediator in transforming growth factor-β (TGF-β)signaling,plays a critical role in the normal development of many tissues/organs.However,its functional role in the development of lacrimal gland has rarely been reported.ObjectiveThe aim of this study was to investigate the role that Smad4 may play in the development of lacrimal glands using Smad4 conditional knockout (CKO) mice( C57BL/6 mouse line),MethodsSmad4 in lacrimal glands,as well as in the lens,cornea and ectoderm of the eyelids,was conditionally inactivated by the Pax6 promoter-driven Cre transgenic mice and Smad4 conditional gene mice,LacZ reporter was used to visualize the developing lacrimal gland by X-gal staining,and standard histological approaches were used to reveal morphological changes.Six or more mice or embryos in each group were used for comparisons at the same stage.ResultsLacZ staining showed that E15.0,Smad4 CKO mice could still develop primary lacrimal bud,but much shorter than the wild-type ones.At E16.5,the primary lacrimal bud in wild-type mice began branching,but no branching was found in Smad4 CKO mice except that the primary lacimal bud became blunt at the tip.At E18.0,although Smad4 CKO mice develop some acini,the branching and size and number of acini were obviously less than ones in Smad4 wild-type mice.Based on histological findings,lacrimal glands in Smad4 CKO mice developed slowly,and the size was considerably smaller,and the numbers of lobes as well as the numbers of acini were much fewer than those of Smad4 wild-type mice lacrimal glands at various stages.Pigment and adipose tissue were also observed within the lacrimal glands starting from P7 in Smad4 CKO mice and increased with age growing.Lacrimal glands in mutant adult mice were eventually replaced by adipose tissue and accumulation of pigments.Conclusions These results support the notion that Smad4 is essential for the normal development and maintenance of the mouse LG and may be involved in the metabolism of pigment and adipose tissue in LG.

The mitogen activated protein kinases-extracellular signal regulated kinases (MAPK-ERK) pathway is involved in regulation of multiple cellular processes including the cell cycle.In the present study using a Huh7 cell line Con1 with an HCV replicon,we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein expression and might influence IFN-α signalling.Epithelial growth factor (EGF) was able to stimulate ERK activation and decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A protein amounts in Con1 cells.It could be further demonstrated that the inhibition of the MAPK-ERK pathway facilitated the translation directed by the HCV internal ribosome entry site.Consistently,a U0126 treatment enhanced activity of the HCV reporter replicon in transient transfection assays.Thus,the MAPK-ERK pathway plays an important role in the regulation of HCV gene expression and replication.In addition,cyclin-dependent kinases (CDKs) downstream of ERK may also be involved in the modulation of HCV replication since roscovitine,an inhibitor of CDKs had a similar effect to that of U0126.Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA levels.Further,the replication of HCV replicon in Conl cells was inhibited by IFN-α.The inhibitory effect of IFN-α could be partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs.It could be shown that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.

The woodchuck model is an excellent animal model to study hepadnaviral infection. The new progresses in this model made possible to examine the T-cell mediated immune responses in acute and chronic hepadnaviral infection. Recently, a new assay for cytotoxic T-cells based on detection of CD107 was established for the woodchuck model. In addition, new immunotherapeutic approaches based on combination of potent antiviral treatment and DNA-protein vaccines were proven to be useful for treatment of chronic hepatitis B.

In more recent times, health-related quality of life (HRQOL) measurements have formed an important part of assessing the quality of routine care in general practice. For a measure to have clinical usefulness it must not only be valid, appropriate, reliable, responsive, and capable of being interpreted, but it must also be simple, fast to complete, easy to score, and provide useful clinical data. The Two-step method of choosing appropriate measures is introduced. Then through comparison of generic instruments with disease-specific instruments, we can conclude that sometimes a combination of generic and disease-specific HRQOL measures may be more appropriate for monitoring changes in a patient's health status due to an intervention.
Family Practice ; Humans ; Outcome Assessment (Health Care) ; Quality Assurance, Health Care ; Quality of Life

OBJECTIVETo define the mechanism of acute hepatitis in non-human primates after liver directed gene therapy.

METHODSDifferences in immune response exhibited by 8 rhesus monkeys receiving adenovirus (Ad) or lipofectamine-mediated gene transfer by various routes, the time course, and the nature of the specific immune responses to both adenoviral vectors and transgene products were studied using HE staining (H&E) and immunohistochemical staining.

RESULTSThe monkeys developed mild to moderate acute hepatitis 1 to 3 weeks after intravenous or intrabiliary injection of first generation replication-defective adenoviruses carrying the Escherichia coli lacZ gene. This was accompanied by adenovirus-mediated T-cell proliferation and neutralizing antibodies to the adenovirus. Increased numbers of CD3(+), CD4(+) and CD8(+) T-lymphocytes were detected in the diseased livers, while B-lymphocytes were absent. Hepatocytes demonstrated increased expression of beta 2-microglobulins (beta 2-MG) and HLA-DR antigens in the plasma membranes. The development of acute hepatitis and the accompanying immune abnormalities were delayed in immunosuppressed monkeys until after the discontinuation of immunosuppressive therapy. The monkeys infused with Ad. CMVluc showed more significant and longer durations of hepatitis than the monkeys infused with adenoviruses carrying the lacZ gene. Lipofectamine-mediated gene transfer was inefficient. There was neither lacZ expression nor significant immune response in the liver of monkeys infused with lipofectamine via the portal vein or the common bile duct.

CONCLUSIONImmune response to the hepatocytes in liver directed gene therapy is MHC class I restricted and T-cell mediated. Both adenoviral vectors and foreign genes are related to the liver damage. Mild to moderate hepatic inflammation seen with the E-1 deleted vector is reversible. Immunosuppression regimens may prolong transgene expression and delay the development of acute adenoviral hepatitis.

Acute Disease ; Adenoviridae ; genetics ; Adenoviridae Infections ; genetics ; Animals ; CD3 Complex ; analysis ; CD4 Antigens ; analysis ; CD8 Antigens ; analysis ; DNA, Recombinant ; administration & dosage ; genetics ; Gene Transfer Techniques ; HLA-DR Antigens ; analysis ; Hepatitis, Animal ; genetics ; virology ; Immunohistochemistry ; Liver ; chemistry ; metabolism ; pathology ; Macaca mulatta ; beta 2-Microglobulin ; analysis

OBJECTIVETo investigate the expression profile of genes which are involved in IFN antiviral activity and IFN signal transduction pathway in Hep G2 and HepG2.2.15 cells.

METHODSGenes of interest were selected from the UniGene database (http://www.ncbi.nlm.gov/UniGene/Hs.Home.html). The 5'IMAGE clones with 0.5-0.8 kb length were chosen and ordered from RZPD company. The cDNA inserts were amplified by PCR and then were spotted onto the Hybond-N+ membranes. The membranes were denatured and neutralized for Macroarray analysis. HepG2.2.15 and Hep G2 cells were treated without or with IFN-alpha for 6 h, and the total cellular RNA was isolated using Trizol Reagent. Radio-labelled cDNA was generated from 20 microgram of RNA by reverse transcription using 360 units of reverse transcriptase in the presence of 30 microCi of alpha-32P dCTP. Hybridization was performed between 32P-labelled cDNA and membrane arrays. The membranes were then scanned, and the intensity of autoradiographic spots was quantitated by Cyclone Storage Phosphor System. The images were subsequently analysed by the OptiQuant Imager Analysis Software and converted into digital data.

RESULTSThe authors found that just partially IFN-inducible genes were expressed in Hep G2 and HepG2.2.15 cells, and the majority of IFN-inducible genes was lowly responsive or non-responsive to IFN-a treatment. Some interferon-stimulated genes (ISGs) were inhibited or blocked, especially in HepG2.2.15 cells. Interestingly, the authors found that the IFN signal transduction pathway (Jak-STAT) was intact and unimpaired in HepG2.2.15 cells.

CONCLUSIONDifferential gene expression profiles in response to IFN were found between Hep G2 and HepG2.2.15 cells.

Antiviral Agents ; pharmacology ; Carcinoma, Hepatocellular ; genetics ; pathology ; virology ; Cell Line, Tumor ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; drug effects ; Hepatitis B virus ; drug effects ; Humans ; Interferons ; pharmacology ; Liver Neoplasms ; genetics ; pathology ; virology ; Oligonucleotide Array Sequence Analysis ; methods ; RNA, Viral ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUNDTo construct a DC-Ad-Ki-ras(V12) vaccine and investigate the anti-tumor activities of lung cancer dendritic cell vaccine modified by mutant Ki-ras gene in vitro.

METHODSKi-ras(V12) cDNA was transfected into cultured bone marrow-derived DC with the recombinant adenovirus [(Ad-Ki-ras(V12)] containing human mutant Ki-ras gene. Anti-tumor activity of the vaccine was studied in vitro by flow cytometry, PCR, MLR and cytotoxicity assay.

RESULTS(1) The DC vaccine was confirmed not only to express Ki-ras(V12) gene, but also to remarkably stimulate lymphocyte proliferation and improve CTL activity. (2) The DC vaccine modified by mutant Ki-ras gene could induce specifical CTL activity of immunized mice against Lewis lung carcinoma that could express Ki-ras(V12) gene, but not to B16.

CONCLUSIONSThe DC vaccine modified by mutant Ki-ras gene can induce obvious anti-tumor activities against Lewis lung carcinoma that can express Ki-ras(V12) gene.

BACKGROUND AND PURPOSE: Interventions to reduce the risk for cerebrovascular events (CVE; stroke and transient ischemic attack [TIA]) after radiotherapy (RT) for head and neck cancer (HNCA) are needed. Among broad populations, statins reduce CVEs; however, whether statins reduce CVEs after RT for HNCA is unclear. Therefore, we aimed to test whether incidental statin use at the time of RT is associated with a lower rate of CVEs after RT for HNCA. METHODS: From an institutional database we identified all consecutive subjects treated with neck RT from 2002 to 2012 for HNCA. Data collection and event adjudication was performed by blinded teams. The primary outcome was a composite of ischemic stroke and TIA. The secondary outcome was ischemic stroke. The association between statin use and events was determined using Cox proportional hazard models after adjustment for traditional and RT-specific risk factors. RESULTS: The final cohort consisted of 1,011 patients (59±13 years, 30% female, 44% hypertension) with 288 (28%) on statins. Over a median follow-up of 3.4 years (interquartile range, 0.1 to 14) there were 102 CVEs (89 ischemic strokes and 13 TIAs) with 17 in statin users versus 85 in nonstatins users. In a multivariable model containing known predictors of CVE, statins were associated with a reduction in the combination of stroke and TIA (hazard ratio [HR], 0.4; 95% confidence interval [CI], 0.2 to 0.8; P=0.01) and ischemic stroke alone (HR, 0.4; 95% CI, 0.2 to 0.8; P=0.01). CONCLUSIONS: Incidental statin use at the time of RT for HNCA is associated with a lower risk of stroke or TIA.
Cohort Studies ; Data Collection ; Female ; Follow-Up Studies ; Head and Neck Neoplasms ; Head ; Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; Ischemic Attack, Transient ; Neck ; Proportional Hazards Models ; Radiotherapy ; Risk Factors ; Stroke

[Objective] To evaluate lipid -lowering efficacy and safety of RYR Cholestin , or Monascus purpureus (Red Yeast) Rice, in Americans with moderate hypercholesterolemia. [Methods] This study was an open-label, self-control, and multi-center clinical trial.A total of 187 subjects were entered into this trial (serum LDL-Cholesterol 3.50~4.92 mmol/L,total cholesterol 5.18~7.25 mmol/L, male:female=116∶71) , of whom 162 completed the study .Subjects were placed on the NCEP Step I Diet throughout the study and RYR Cholestin (2.4 g/day) was administered for 8 weeks following initial 4-week diet control . [ Results] Being on the diet alone for 4 weeks resulted in no significant changes in serum lipids .RYR Cholestin treatment for 8 weeks reduced serum total cholesterol , LDL-Cholesterol and triglycerides by 16 .6%, 24 .0%, and 25 .2%, respectively , and increased HDL-Cholesterol by 14 .3%( all P<0 .001 ) .There were 97 .5% of patients having ≥10% improvement in at least one of lipid risk factors, and 79.0%having ≥20% improvement.Discontinuation of RYR Cholestin intervention for 14 d led to a return of serum lipids to baseline of pre -study .And 29 possible product -related mild adverse re-actions were reported . [ Conclusion] RYR Cholestin is well tolerated and effective in reducing total and LDL-Cholesterol, and triglycerides, as well as in increasing HDL-Cholesterol in hypercholesterolemic patients, but those indicators return to the beginning baseline when the treatment is discontinued .

OBJECTIVES: Spiral ganglion neurons (SGNs) include potential endogenous progenitor populations for the regeneration of the peripheral auditory system. However, whether these populations are present in adult mice is largely unknown. We examined the presence and characteristics of SGN-neural stem cells (NSCs) in mice as a function of age. METHODS: The expression of Nestin and Ki67 was examined in sequentially dissected cochlear modiolar tissues from mice of different ages (from postnatal day to 24 weeks) and the sphere-forming populations from the SGNs were isolated and differentiated into different cell types. RESULTS: There were significant decreases in Nestin and Ki67 double-positive mitotic progenitor cells in vivo with increasing mouse age. The SGNs formed spheres exhibiting self-renewing activity and multipotent capacity, which were seen in NSCs and were capable of differentiating into neuron and glial cell types. The SGN spheres derived from mice at an early age (postnatal day or 2 weeks) contained more mitotic stem cells than those from mice at a late age. CONCLUSION: Our findings showed the presence of self-renewing and proliferative subtypes of SGN-NSCs which might serve as a promising source for the regeneration of auditory neurons even in adult mice.
Adult ; Animals ; Cochlea ; Deafness ; Hearing Loss ; Humans ; In Vitro Techniques* ; Mice* ; Nestin ; Neural Stem Cells* ; Neuroglia ; Neurons ; Regeneration ; Spiral Ganglion* ; Stem Cells