Exogenous IFN-beta regulates the RANKL-c-Fos-IFN-beta signaling pathway in the collagen antibody-induced arthritis model.

BACKGROUND: Although a variety of drugs have been used to treat the symptoms of rheumatoid arthritis (RA), none of them are able to cure the disease. Interferon ß (IFN-ß) has pleiotropic effects on RA, but whether it can be used to treat RA remains globally controversial. Thus, in this study we tested the effects of IFN-ß on RA patients and on collagen antibody-induced arthritis (CAIA) model mice. METHODS: The cytokine and auto-antibody expression profiles in the serum and synovial fluid (SF) from RA patients were assessed using enzyme-linked immunosorbent assay (ELISA) and compared with the results from osteoarthritis (OA) patients. Exogenous IFN-ß was administered to RA patients and CAIA model mice, and the therapeutic effects were evaluated. Endogenous IFN-ß expression in the joint bones of CAIA model mice was evaluated by quantitative real-time PCR (qRT-PCR). The effects of exogenous IFN-ß on CAIA model mice were assessed using a clinical scoring system, hematoxylin eosin and safranin-O with fast green counterstain histology, molybdenum target X-ray, and tartrate-resistant acid phosphatase (TRAP) staining. The RANKL-RANK signaling pathway was analyzed using qRT-PCR. The RAW 264.7 cell line was differentiated into osteoclasts with RANKL stimulation and then treated with exogenous IFN-ß. RESULTS: The expression of inflammatory cytokines (IFN-γ, IL-17, MMP-3, and RANKL) and auto-antibodies (CII antibodies, RF-IgM, and anti-CCP/GPI) were significantly higher in RA compared with OA patients. After IFN-ß intervention, some clinical symptoms in RA patients were partially alleviated, and the expression of IFN-γ, IL-17, MMP-3, and OPG) returned to normal levels. In the CAIA model, the expression of endogenous IFN-ß in the joint bones was decreased. After IFN-ß administration, the arthritis scores were decreased; synovial inflammation, cartilage, and bone destruction were clearly attenuated; and the expression of c-Fos and NFATc1 were reduced, while RANKL and TRAF6 expression was unchanged. In addition, exogenous IFN-ß directly inhibited RANKL-induced osteoclastogenesis. CONCLUSIONS: Exogenous IFN-ß administration immunomodulates CAIA, may reduce joint inflammation and, perhaps more importantly, bone destruction by inhibiting the RANKL-c-Fos signaling pathway. Exogenous IFN-ß intervention should be selectively used on RA patients because it may only be useful for RA patients with low endogenous IFN-ß expression.

Knockdown of metallopanstimulin-1 inhibits NF-κB signaling at different levels: the role of apoptosis induction of gastric cancer cells.

The ribosomal protein S27 (metallopanstimulin-1, MPS-1) has been reported to be a multifunctional protein, with increased expression in a number of cancers. We reported previously that MPS-1 was highly expressed in human gastric cancer. Knockdown of MPS-1 led to spontaneous apoptosis and repressed proliferation of human gastric cancer cells in vitro and in vivo. However, how does MPS-1 regulate these processes is unclear. Here we performed microarray and pathway analyses to investigate possible pathways involved in MPS-1 knockdown-induced apoptosis in gastric cancer cells. Our results showed that knockdown of MPS-1 inhibited NF-κB activity by reducing phosphorylation of p65 at Ser536 and IκBα at Ser32, inhibiting NF-κB nuclear translocation, and down-regulating its DNA binding activity. Furthermore, data-mining the Gene-Regulatory-Network revealed that growth arrest DNA damage inducible gene 45ß (Gadd45ß), a direct NF-κB target gene, played a critical role in MPS-1 knockdown-induced apoptosis. Over-expression of Gadd45ß inhibited MPS-1 knockdown-induced apoptosis via inhibition of JNK phosphorylation. Taken together, these data revealed a novel pathway, the MPS-1/NF-κB/Gadd45ß signal pathway, played an important role in MPS-1 knockdown-induced apoptosis of gastric cancer cells. This study sheds new light on the role of MPS-1/NF-κB in apoptosis and the possible use of MPS-1 targeting strategy in the treatment of gastric cancer.

Helicobacter pylori induces promoter hypermethylation and downregulates gene expression of IRX1 transcription factor on human gastric mucosa.

BACKGROUND AND AIM: Gene silence of IRX1 tumor suppressor by promoter CpG methylation combined with loss of heterozygosity (LOH) has been identified in human gastric cancer. This study investigated the association between methylation of IRX1 and Helicobacter pylori infection in gastric mucosa tissues and cell line. METHODS: IRX1 methylation was studied by methylation specific polymerase chain reaction (MSP) and bisulfate sequencing polymerase chain reaction (BSP) methods in gastric mucosa tissues from H. pylori-positive chronic gastritis patients or H. pylori-negative chronic gastritis patients. Promoter activity, methylation status and gene expressing level of IRX1 were evaluated by persistent infecting H. pylori on human gastric cells GES-1 in vitro. Electron microscopy was used to observe the effect of H. pylori infection on GES-1 gastric mucosa cells. RESULTS: The methylation level of IRX1 promoter in H. pylori positive chronic gastritis and H. pylori negative chronic gastritis was 55.30%±13.17 versus 5.20%±6.31, respectively (P<0.01). H. pylori infection stimulated increased microvillus, and mucous secretion on GES-1 cells. Infection of H. pylori induced IRX1 promoter methylation and downregulation of the promoter activity as well as gene expression significantly. CONCLUSIONS: This study firstly demonstrated that H. pylori infection contributes to IRX1 promoter methylation on gastric mucosa.

Cell cycle dependent genes from the gastric cancer cell MKN45 can affect tumorigenesis.

BACKGROUND/AIMS: To investigate the cell cycle dependent genes involved in gastric tumorigenesis, possibly determining the relationship between the cell cycle and tumorigenesis. METHODOLOGY: MKN45 cells were collected every hour from Oh to 12h after release from G2/M and G1/S blocks. Nine samples (a-i), chosen at key times of the cell cycle, were prepared for RNA isolation and cDNA microarray analysis. RESULTS: In 2001 viable clones, 959 genes showed periodic variations during the cell cycle. Among 2001 genes that were clustered, a series of up-regulated genes were assigned to different cell cycle phases. Many periodically dependent genes in the cell cycle were ubiquitously expressed and participated in various cell physiological functions, such as transcription, translation, ubiquitination and signal transduction. These cell cycle dependent genes could affect cancer cell proliferation, apoptosis, activation of oncogenes and inactivation of tumor suppressor genes. CONCLUSIONS: We provided a comprehensive understanding of the gene expression profile involved in gastric cancer cell cycles and laid a foundation for further research on mechanisms of gastric tumorigenesis.

[Establishment of experimental angiogenic models with applications of quantitative digital image analysis].

OBJECTIVE: To establish experimental models for tumor neovascularization and to apply quantitative digital imaging analysis in the study. METHODS: An endothelial tube formation model was established by human umbilical vein endothelial cells (HUVECs). A vasculogenic mimicry model was established by SGC-7901 gastric cancer cell line. Fertilized eggs were used to establish a chorioallantoic membrane angiogenesis model. Using gene transfection experiment, IRX1 tumor suppressor gene was chosen as a therapeutic target. Image Pro Plus (IPP) analysis software was used for digital vascular images analysis with parameters including points, lines, angles and integral absorbance (IA) for the tubular formation or vasculogenic mimicry. RESULTS: Digital image analysis by IPP showed that HUVEC tubular formation was significantly inhibited in IRX1 transfectant, compared with controls. The tubular numbers in three groups were 12.80 +/- 3.83, 29.00 +/- 5.34 and 28.20 +/- 4.32 (P<0.01). The connection points of tubules in three groups were 13.20 +/- 2.59, 25.00 +/- 2.24 and 24.60 +/- 3.21 (P<0.01). The tubular lengths of three groups were (821.5 +/- 12.5), (930.9 +/- 13.5) and (948.4 +/- 18.1) microm (P=0.022). The IA values of PAS stain in three groups were 3606 +/- 363, 14 200 +/- 1251 and 15 043 +/- 1220 (P<0.01). In chick chorioallantoic membrane model, the angular numbers of tubules in three groups were 6.41 +/- 2.60, 10.27 +/- 2.65 and 9.18 +/- 1.99 (P<0.01). CONCLUSIONS: The endothelial tube formation model, vasculogenic mimicry model and chorioallantoic membrane angiogenesis model are useful for gene therapy and drug screening with targeting neoplastic vascularization. Professional image analysis software may greatly facilitate the quantitative analysis of tumor neovascularization.

[The characteristics of CT imaging and diagnosis of pulmonary cryptococcosis in 42 cases with non-acquired immune deficiency syndrome].

OBJECTIVE: To further elucidate the CT characteristics and diagnostic approaches to non-acquired immune deficiency syndrome patients with pulmonary cryptococcosis. METHODS: The histories of forty-two pulmonary cryptococcosis (PC) patients diagnosed in Zhongshan Hospital from 2003-2008 were collected and analyzed for demography data, underlying conditions, clinical symptoms, chest CT and diagnostic studies. RESULTS: None of the 42 PC patients had avian or its feces contacting history, and 71.4% (30/42) of them were immunocompetent. The most frequent CT lesions were multiple nodules (67.9%) with peripheral predominance (67.9%), and cavitations (50%) often presented within them. Masses/consolidation (31.4%) and patching lesions (2.9%) could exist occasionally. Positive detection rates of non-aggressive examinations including sputum, bronchoalveolar lavage fluid and bronchofibroscopy aspiration were 4.3%, 8.3% and 6.3% respectively, while those of aggressive approaches including transbronchial lung biopsy (TBLB), thin needle aspiration biopsy (TNAB) and pneumonectomy by surgery were 64.7%, 64.3% and 100% respectively. Non-aggressive serum cryptococcus antigen test was performed in 14 patients who had been diagnosed by histopathology or pathogen culture, and all of them were positive. CONCLUSION: Our study suggests that PC is common in immunocompetent population. Avian or its feces contacting is not so important as used opinion to PC differential diagnosis. CT characteristics of PC are diversiform and always change very slowly. Besides the most frequent multiple nodules with subpleural predominance, pulmonary lesions can present as masses, consolidation or patching. Aggressive techniques such as TBLB and TNAB are benefit to clinical diagnosis of PC, and non-aggressive serum cryptococcus antigen test may be promising for its early diagnosis as well as clinical course follow-up and therapeutic effect evaluation.

Aberrant expression and demethylation of gamma-synuclein in colorectal cancer, correlated with progression of the disease.

Recent evidence suggests that gamma-synuclein is abnormally expressed in a high percentage of tumor tissues of diversified cancer types, but rarely expressed in tumor-matched non-neoplastic adjacent tissues (NNAT). The molecular mechanism of CpG island demethylation may underlie aberrant gamma-synuclein expression. To fully understand the roles of aberrant gamma-synuclein expression and demethylation in the development of colorectal cancer (CRC), we examined the expression and methylation status of gamma-synuclein in 67 CRC samples, 30 NNAT samples, and five CRC cell lines as well. By using reverse transcription-polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry analyses, gamma-synuclein expression was detected in both HT-29 and HCT116 cells, and was much higher in CRC samples than in NNAT samples (P < 0.05). The demethylating agent, 5-aza-2 cent-deoxycytidine, can induce re-expression of gamma-synuclein in COLO205, LoVo, and SW480 cells. Unmethylated gamma-synuclein alleles were detected in HT-29, HCT116, and LoVo cells by nested methylation-specific PCR, and the demethylated status of gamma-synuclein was much higher in CRC samples than in NNAT samples by real-time quantitative methylation-specific PCR (P < 0.05). The results of genomic bisulfite DNA sequencing further confirmed that the aberrant gamma-synuclein expression in CRC was primarily attributed to the demethylation of CpG island. The protein expression and demethylation status of gamma-synuclein in 67 CRC samples correlated with clinical stage, lymph node involvement, and distant metastasis. These findings suggest an involvement of aberrant gamma-synuclein expression and demethylation in progression of CRC, especially in advanced stages.

Comparative proteomics analysis of human gastric cancer.

AIM: To isolate and identify differentially expressed proteins between cancer and normal tissues of gastric cancer by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). METHODS: Soluble fraction proteins of gastric cancer tissues and paired normal tissues were separated by 2-DE. The differentially expressed proteins were selected and identified by MALDI-TOF-MS and database search. RESULTS: 2-DE profiles with high resolution and reproducibility were obtained. Twenty-three protein spots were excised from sliver staining gel and digested in gel by trypsin, in which fifteen protein spots were identified successfully. Among the identified proteins, there were ten over-expressed and five under-expressed proteins in stomach cancer tissues compared with normal tissues. CONCLUSION: In this study, the well-resolved, reproducible 2-DE patterns of human gastric cancer tissue and paired normal tissue were established and optimized and certain differentially-expressed proteins were identified. The combined use of 2-DE and MS provides an effective approach to screen for potential tumor markers.

Effects of N(omega)-nitro-L-arginine methyl ester and aminoguanidine on lipopolysaccharide-induced airway hyperresponsiveness in guinea pigs.

BACKGROUND: The down-regulation of constitutive nitric oxide synthase (cNOS) and up-regulation of inducible nitric oxide synthase (iNOS) are associated with the allergen-provocated airway hyperresponsiveness (AHR). This study aimed to determine whether their alteration also plays an important role in the AHR induced by lipopolysaccharide (LPS). METHODS: Hartley male guinea pigs, weighing between 250 g and 350 g, were injected with LPS at a dose of 1 mg/kg every 24 hours for three days. A non-selective NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), or a selective inducible NOS inhibitor, aminoguanidine (AG), were used thirty minutes before each injection of LPS. Airway reactions, nitric oxide (NO) production and inflammatory changes were detected 24 hours after the last dose of LPS. RESULTS: AG significantly decreased the NO production in the bronchoalveolar lavage fluid (BALF) and sharply reduced the intensity of bronchoconstriction to histamine challenge. L-NAME also significantly decreased the NO production in the BALF, but had no effect on airway reactions or, perhaps, a tendency to enhance the intensity of AHR. CONCLUSIONS: The data suggest that inducible NOS contributes to the AHR induced by repetitive intraperitoneal LPS, and constitutive NOS was also involved.