Effects of shikonin on the development of ovarian follicles and female germline stem cells.

OBJECTIVE: To investigate the effects and potential mechanism of action of shikonin (SHK) on the development of ovarian follicles and female germline stem cells (FGSCs). METHODS: Female Kunming adult mice were administered SHK (0, 20 and 50 mg/kg) by oral gavage. Cultures of FGSCs were treated with SHK 32 µmol/l for 24 h. The ovarian index in mouse ovaries was calculated. Numbers of primordial, primary and atretic follicles were counted. Germline stem cell markers and apoptosis were examined. Levels of glutathione (GSH), superoxide dismutase (SOD) and reactive oxygen species (ROS) were measured. RESULTS: Both doses of SHK significantly decreased the ovarian index, the numbers of primordial follicles, primary follicles and antral follicles in mice. SHK significantly increased the numbers of atretic follicles and atretic corpora lutea. SHK promoted apoptosis in vivo and in vitro. SHK significantly decreased the levels of the germline stem cell markers. SHK significantly lowered GSH levels and the activity of SOD in the peripheral blood from mice, whereas SHK significantly elevated cellular ROS content in FGSCs. CONCLUSIONS: These current results suggested that follicular development and FGSCs were suppressed by SHK through the induction of apoptosis and oxidative stress might be involved in this pathological process.

Upregulation of CD4+CD25+ T lymphocyte by adenovirus-mediated gene transfer of CTLA4Ig fusion protein in experimental autoimmune myocarditis.

OBJECTIVE: To explore the effects of adenovirus vector-mediated gene transfer of CTLA4Ig fusion protein on CD4+CD25+ T cells in experimental autoimmune myocarditis (EAM). METHODS: EAM was induced by porcine cardiac myosin as previously described. Adenovirus vector-mediated CTLA4Ig gene was administrated intravenously in EAM rats on days 1, 4 and 7, with EGFP as control. On day 21, myocardium histopathology was examined and CD4+CD25+ T cells were isolated. Proliferation and suppression assays were used to evaluate the suppressive capacity of CD4+CD25+ T cells in vitro. Relative mRNA level of Foxp3 and TGF-beta was determined by quantitative real-time RT-PCR; expression of CTLA-4, B7-1 and B7-2 protein was compared with Western blot in CD4+CD25+ Tregs. RESULTS: Severe inflammatory lesions were observed in the hearts of EGFP-treated EAM rats and the untreated ones, while Ad-CMV-CTLA4Ig alleviated the myocarditis histologically. Adenovirus vector-mediated CTLA4Ig gene transfer up-regulated the proportion of CD4+CD25+ Tregs significantly. T cell proliferation was greatly inhibited in the CTLA4Ig group compared with the untreated and EGFP-treated groups in vitro. CTLA-4 and B7-2 proteins were down-regulated in the CTLA4Ig group, Foxp3 and TGF-beta mRNA was up-regulated significantly by CTLA4Ig treatment. CONCLUSIONS: Adenovirus vector-mediated CTLA4Ig gene transfer alleviated inflammation in EAM, one of the potential mechanisms is up-regulation of CD4+CD25+ Tregs.

Identification of differential gene expression between intestinal and diffuse gastric cancer using cDNA microarray.

To compare the gene expression profiling between intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC), cDNA microarray containing 7334 gene elements was performed on 12 paired IGC specimens/its' normal epithelial tissue and 11 paired DGC specimens/its' normal epithelial tissue. Twenty-seven genes were co-overexpressed in IGC and DGC. These overexpressed genes were related to transcription and translation, DNA replications and mitosis, calcium binding, apoptosis and mitochondria protein. Twelve genes were co-underexpressed in IGC and DGC. These underexpressed genes were associated with cell adhesion and migration, organelle movement and intracellular transport, and matrix metalloproteinase. A clustering dendrogram of IGC and DGC with 27 genes significantly differed between IGC and DGC. Nineteen genes were more overexpressed in DGC than in IGC, including annexin A1 (ANXA1), chemokine ligand 7 (CCL7), and chemokine ligand 8 (CCL8). Eight genes were more overexpressed in IGC than in DGC, including claudin 4 (CLDN4). The results of quantitative real-time PCR and immunohistochemical staining confirmed the microarray finding. The gene expression profiling between IGC and DGC suggested that they might have unique genetic pathways which share some of the same and some different genetic alterations.

Molecular signature of clinical severity in recovering patients with severe acute respiratory syndrome coronavirus (SARS-CoV).

BACKGROUND: Severe acute respiratory syndrome (SARS), a recent epidemic human disease, is caused by a novel coronavirus (SARS-CoV). First reported in Asia, SARS quickly spread worldwide through international travelling. As of July 2003, the World Health Organization reported a total of 8,437 people afflicted with SARS with a 9.6% mortality rate. Although immunopathological damages may account for the severity of respiratory distress, little is known about how the genome-wide gene expression of the host changes under the attack of SARS-CoV. RESULTS: Based on changes in gene expression of peripheral blood, we identified 52 signature genes that accurately discriminated acute SARS patients from non-SARS controls. While a general suppression of gene expression predominated in SARS-infected blood, several genes including those involved in innate immunity, such as defensins and eosinophil-derived neurotoxin, were upregulated. Instead of employing clustering methods, we ranked the severity of recovering SARS patients by generalized associate plots (GAP) according to the expression profiles of 52 signature genes. Through this method, we discovered a smooth transition pattern of severity from normal controls to acute SARS patients. The rank of SARS severity was significantly correlated with the recovery period (in days) and with the clinical pulmonary infection score. CONCLUSION: The use of the GAP approach has proved useful in analyzing the complexity and continuity of biological systems. The severity rank derived from the global expression profile of significantly regulated genes in patients may be useful for further elucidating the pathophysiology of their disease.

Expression of IL-37 contributes to the immunosuppressive property of human CD4+CD25+ regulatory T cells.

Interleukin-37 (IL-37) possesses the function of down-regulate systemic and local inflammation. It is unknown whether IL-37 is expressed in human regulatory T cells (Tregs) and its role in modulating the immune response of Tregs. In the present study, cell surface molecules and secretory cytokines were analyzed in order to determine the function of IL-37 in regulating inhibitory effect of human CD4(+)CD25(+)Tregs. Meanwhile, the effects of IL-37 on T cell differentiation and proliferation as co-culture of CD4(+)CD25(+)Treg/CD4(+)CD25(-)T cell were also investigated. It was showed that IL-37 was expressed in cytoplasm of CD4(+)CD25(+)Tregs, and the levels of IL-37 were gradually elevated with the enhanced activity of CD4(+)CD25(+)Tregs. Secretory cytokines such as transforming growth factor (TGF)-ß and interleukin (IL)-10, and expressions of cell surface molecules, including forkhead/winged helix transcription factor p3 (FOXP3) and cytotoxic T-lymphocyte associated antigen (CTLA)-4, were significantly decreased when IL-37 gene was silenced by siRNA. Furthermore, down-regulation of IL-37 expression in human CD4(+)CD25(+)Tregs obviously promoted proliferation of co-cultured T cell and differentiation, together with observably enhancement of IL-2 formation. These results demonstrated that IL-37 might manifest as a critical protein involving in immunosuppression of human CD4(+)CD25(+)Tregs.

Molecular characterization of adenocarcinoma and squamous carcinoma of the uterine cervix using microarray analysis of gene expression.

In an attempt to understand the molecular mechanisms for the different clinical features between adenocarcinoma/adenosquamous carcinoma (AC/ASC) and squamous carcinoma (SC) of the uterine cervix, we analyzed gene expression profiles of different histological subtypes of cervical cancer. Cancer specimens and the surrounding normal tissue counterparts were separately collected from cervical cancer patients undergoing type III radical hysterectomy. Paired total RNA (cancer and normal tissues) was isolated and analyzed with cDNA microarrays containing duplicate spots of 7 334 sequence-verified human cDNA clones. Selected differentially expressed genes specific for AC or SC were further verified using real-time quantitative polymerase chain reaction (RTQ-PCR) and immunohistochemistry. Genes, including CEACAM5, TACSTD1, S100P and MSLN were upregulated in AC. Contrarily, genes involved in epidermal differentiation complex such as S100A9 and ANXA8 were upregulated in SC. Cross-validation of the results using an independent but comparable group of patients with known long-term outcomes (n = 63, median follow-up 70.3 months; range, 4-208 months) showed that the correlation between the selected 6 differentially expressed genes and histology was highly significant. CEACAM5 (p < 0.0001) and TACSTD1 (p = 0.009) were significant prognostic factors by multivariate Cox proportional hazards regression analysis. The combination of cDNA microarray, RTQ-PCR and immunohistochemical results of this study showed that it is possible to define different gene profiles for AC and SC. Moreover, TACSTD1 expression may be a novel poor prognostic factor.

Establishment of cDNA microarray analysis at the Genomic Medicine Research Core Laboratory (GMRCL) of Chang Gung Memorial Hospital.

BACKGROUND: Advances in molecular and computational biology have led to the development of powerful, high-throughput methods for analysis of differential gene expression, which are opening up new opportunities in genomic medicine. DNA microarray technology has been enthusiastically integrated into basic biomedical research and will eventually become a molecular monitoring tool for various clinical courses. METHODS: As a core research facility of Chang Gung University (CGU) and Chang Gung Memorial Hospital (CGMH), the Genomic Medicine Research Core Laboratory (GMRCL) welcomes investigators from every discipline to employ DNA microarray technology in the quest for knowledge of genomic medicine. The first tasks for GMRCL are to optimize the standard operating procedures (SOP) for each instrument and to assure the quality of every procedure. RESULTS: During the first year after the establishment of the GMRCL at the CGMH, we tested and adopted procedures that were satisfactory for our purposes. These procedures included: replication of bacterial stocks, amplification of human DNA clones, annotation of each DNA clone, production of cDNA microarrays, validation of RNA quality and quantity, labeling of target specimens, competitive hybridization, scanning of slides, data analysis, and post-microarray validation of results. We present a summarization of the materials and procedures used at the GMRCL and discuss the reasons for using them. CONCLUSIONS: The information about the cDNA microarray analysis system at the GMRCL is compliant with the minimal information about a microarray experiment (MIAME) format. The information may be useful to both the investigators who are using this core facility and researchers at other institutes, who will establish their own in-house cDNA microarray systems.