Haocai Huang

Transforming growth factor-β1 modulates lipopolysaccharide-induced cytokine/chemokine production and inhibits nuclear factor-κB, extracellular signal-regulated kinases and p38 activation in dendritic cells in mice.

Tolerogenic dendritic cells (DCs) are crucial for peripheral tolerance mediated by a variety of cytokines, including transforming growth factor-β1 (TGF-β1). We have observed that TGF-β1-treated DCs (TGFβ-DCs) were resistant to the maturation stimulus of lipopolysaccharide (LPS) and that TGF-β1 down-regulated Toll-like receptor 4 (TLR4) expression on DCs. The purpose of this study was to analyze whether TGF-β1 affected the production of cytokines/chemokines and proteins in the TLR4 signal transduction pathway following LPS stimulation. We observed that TGF-β1 induced a significant increase in interleukin (IL)-10, impaired IL-12 secretion, and attenuated messenger RNA (mRNA) expression of chemokines CCL2, CCL3, and CXCL10 in DCs following LPS administration. We also noted that TGF-β1 suppressed LPS-induced activation of nuclear factor (NF)-κB, extracellular signal-related kinases (ERK)-1/2, and p38 in DCs. Taken together, our results identified the suppressive effects of TGF-β1 on TLR4 signal transduction, strengthening the notion that TGFβ-DCs are a unique type of tolerogenic DC exhibiting distinct characteristics.

Cytomegalovirus Infection in Mesenchymal Stem Cells and Their Activation Could Be Enhanced by Nuclear Factor-κB Inhibitor Pyrrolidinedithiocarbamate In Vitro

Because of the central role of the transcription factor nuclear factor (NF)-κB in cell survival and proliferation in many kinds of cancer cells, NF-κB inhibitors may have a potential role in cancer therapy. Currently, many NF-κB inhibitors are used for immunosuppression to treat hematologic malignancy patients after stem cell transplantation (SCT). Human cytomegalovirus (HCMV) infection is one of the most common complications following SCT. Some workers have reported that HCMV infection has a close relationship to NF-κB activation; however, the specific effects of NF-κB inhibitors, such as pyrrolidinedithiocarbamate (PDTC), on infection with and activation of CMV in mesenchymal stem cells (MSCs) remain unknown. In our study, we isolated MSCs from the bone marrows of healthy human donors for infection with 1 tcid(50) of HCMV with or without 1 μmol/L PDTC. After 48 hours of culture in dmem supplemented with 10% (volume per volume) fetal calf serum, we tested MSCs using reverse transcription-polymerase chain reaction (RT-PCR) assays to detect messenger RNA (mRNA) expression of HCMV immediate early (IE) gene and the GAPDH gene. Flow cytometry was used to detect HCMV pp65 antigen-positive cells and transmission electron microscopy (TEM) for intra cellular HCMV particles. We observed that the shape of the MSCs changed in response to infection by 1 TCID(50) of HCMV. MSCs infected by 1 TCID(50) of HCMV in combination with 1 μmol/L of PDTC changed their shapes more profoundly; almost all cells went from a thin elongated profile to a round, thick ball. In contrast, the shape of cells treated with PDTC alone or the HCMV mock-infected elements did not change. The RT-PCR assay showed that there was a bright band corresponding to HCMV IE mRNA in MSCs infected with 1 TCID(50) of HCMV in combination with 1 μmol/L of PDTC, as compared with cells infected by only 1 TCID(50) of HCMV. The HCMV mock-infected MSCs did not express HCMV IE mRNA. Using flow cytometry, we detected more HCMV pp65 antigen-positive cells among MSCs infected with 1 TCID(50) of HCMV in combination with 1 μmol/L of PDTC. HCMV particles were observed by TEM in the nucleus and cytoplasm of MSCs infected with HCMV. There were more HCMV particles in cells infected by HCMV in combination with PDTC. In conclusion, NF-κB activation may affect HCMV infection efficiency of MSCs. An NF-κB inhibitor increased the infection by activation of HCMV in MSCs, thus we should pay close attention to HCMV infection when we prescribe an NF-κB inhibitor in clinical settings.

Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

OBJECTIVE To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on beta-actin mRNA and microfilaments. METHODS HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, beta-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. RESULTS Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of beta-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. CONCLUSION CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of beta-actin mRNA and the microfilaments.

Novel gas-tight multi-sampler for discrete deep-sea water

The issues of how to quickly collect seawater samples and of how to make sure that those samples truly reflect the in-situ information on gas composition and concentration have therefore become a hot but difficult topic in the field of ocean technology. Most conventional seawater samplers only focus on collecting seawater itself, but take little consideration on gas preservation. A set of new oceanographic tools are presented for ocean resource exploration such as hydrothermal sulfide and gas hydrate, and for investigations on the processes and mechanisms of marine physical, chemical and biological evolutions. A gas-tight deep-sea water sampling system (GTWSS) is designed for the collection of deep-sea geochemical samples. This set of tools mainly consists of a conductivity temperature depth profiler (CTD), release devices and gas-tight deep-sea water samplers (GTWS). The GTWS is able to hold the gases in deep-sea water samples tightly, providing in-situ information on gas contents in the seawater samples and can be deployed on a routine wire-deployed CTD sampler for multi-layer discrete sampling of gas-tight seawater. Sea trials are performed successfully in 2008 and 2009, on a research vessel named HaiYang Si Hao in South China Sea, with the deepest trial depth 3 930 m. GTWSS is capable of quickly sampling 12 discrete gas-tight seawater samples (8.3 L per sample) during its single deployment. The head space method is employed to separate the gases from the seawater samples immediately after recovery of the seawater samples on the vessel. Field geochemical analysis is carried out by gaseous hydrocarbon sensors and an infrared gas analyzer. Results show that the concentrations of CH4 and CO2 in the seawater sampled by GTWSS are higher than those sampled by general non-gas-tight water samplers, thus confirming the gas tightness of GTWSS. Seawater samples can be collected quickly by using GTWSS, and GTWSS can keep the samples’ integrity quite well.