Characteristics of TCRαβ+CD4-CD8- thymocytes in fetal thymic organ culture

TCRαβ+CD4-CD8- (TCR+ DN) thymocytes at different developmental periods, i.e. after either 9 or 18 days of culture in the fetal thymic organ culture (FTOC) system, were characterized in the properties of phenotype, proliferation, differentiation and apoptosis. The results showed that anti-CD3 mAb significantly promoted proliferation of TCRαβ+ DN cells generated after 18 days of culture in FTOC, whereas the cells generated after 9 days of culture responded to anti-CD3 mAb by proliferation weakly. IL-7 efficiently induced TCRαβ+ DN cells at day 9 of FTOC to differentiate into TCRαβ+CD4+/CD8+ SP cells without detectable transitional stage of TCRαβ+CD4+CD8+ (DP) cells. In contrast, fewer TCRαβ+ DN cells generated after 18 days of FTOC were induced to differentiate into SP cells. The thymic stromal cell line MTEC5 cells synergized with IL-7 to promote the differentiation of TCRαβ+ DN cells. In addition, TCRαβ+ DN cells were shown to be less susceptible to apoptosis compared with the other major thymocyte subsets. Taken together, these data have provided insight into the characteristics of TCRαβ+ DN thymocytes.     

CD8<Superscript>+</Superscript> T cell response mediates the therapeutic effects of oncolytic adenovirus in an immunocompetent mouse model

The role of anti-tumor immune responses in oncolytic adenoviral therapy has not been well studied due to lack of efficacious tu- mor model in immunocompetent mice.Here,we evaluated the contributions of immune components to the therapeutic effects of oncolytic adenoviruse in an immunocompetent murine tumor model permissive for infection and replication of adenovirus.We found that CD8+T cells were critical mediator for antitumor efficacy by oncolytic adenovirus.Intratumoral viral therapy induced intensive infiltration of CD8+T cells in tumor,increased tumor-specific IFN-?(interferon-?)production and CTL(cytotoxic T lymphocyte)activity of lymphocytes,and generated a long-term tumor-specific immune memory.Boosting CD8+T cell responses by agonistic anti-4-1BB(cluster differentiation 137,CD137)antibody showed synergistic anticancer effects with oncolytic viro- therapy.Our results provide insight into antitumor mechanisms of oncolytic adenovirus in addition to their direct oncolytic effect.     

Two tyrosines in CD3ε-ITAM are required to induce T lymphocyte apoptosis

CD3ε of T cell antigen receptor complex (TCR/CD3) plays an important role in the resembling of the complex and activation signaling through its conservative immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic tail. Previous study showed that a chimera molecule, consisting of the extracellular-transmembrane domain of human CD8α fused to the cytoplasmic domain of CD3ε, induced apoptosis of T lymphocytes, indicating that apoptotic signals were transduced through the CD3ε- ITAM. To elineate involvement of the two tyrosines in apoptotic signaling pathway, cDNAs with mutations at Y170F, Y181F and Y170F/Y181F in CD8-ε-ITAM were made by point mutation and PCR, and then cloned into pcDNA3 eukaryotic expression vectors. Stable expression cell lines were established after transfection of the expression vectors into CD8+- Jurkat T lymphocytes. Stimulation of these cell lines with anti-CD8 monoclonal antibody showed that only the cells with expression of wild type chimera CD8-ε died by apoptosis, but not those cells with expressions of mutated CD8-ε chimera, indicating that the two tyrosines in CD3ε-ITAM were required for the apoptotic signal transduction in T lymphocytes.     

Use of flow cytometry to investigate the cytokine response pattern in infants with respiratory syncytial virus infection and bronchiolitis

Objective:To investigate the cytokine response pattern(IL-4/IFN-γ)in infants with RSV infections and bronchiolitis during the acute phase,Methods:Four-color flow cytometry was used to measure intracellular IL-4 and IFN-γ expressions in peripheral blood CD3 and CD* lymphocytes from RSV-infected and bronchiolitis infants.Serum IL-4 and INF-γ levels were also determined.Results:RSV-infected and bronchiolitis infants showed no statistical differences from not-RSV-infected or pneumonia infants and control in the frequency of IL-4 and INF-γ levels were also determined.Results;RSV-infected and bronchiolitis infants showed no statistical differences from not-RSV-infected or pneumonia infants and control in the frequency of IL-4 and IFN-γ expressions in CD3 CD8- lymphocytes,showed no obvious Th1/Th2 imbalance,while IFN-γ was expressed much more frequently in CD3 CD* lymphocytes.Systematically,RSV-infected and bronchiolitis infants showed moch lower levels of serum IL-4 and IL-4/INF-γ ratios and much higher serum IFN-γ levels than control.Howerver,there were no statistical differences in the above three indices between RSV-infected and not-RSV infected infants or between bronchiolitis and pneumonia infants,except that bronchiolitis infants had a higher level of serum INF-γ than pneumonia infants statistically .Conclusions:There is no type-2 cytokine response predominance in the acute thase of RSV infection and bronchilitis.IL-4 productioin is suppressed and IFN-γ production upregulated,the latter being most prominent in bronchiolitis infants.     

Differentiation of bone mar-row derived Thy-1~+β_2M~- cells into hepatocytes induced by coculture with transgenic CFSCs

Studies of transplantation in vivo indicted thatbone marrow derived stem cells had a potential to differenti-ate into mature hepatocytes. However, there are lots ofdoubts and uncertainties in the influencing factors and con-trol agents of effectively inducing stem cell differentiation invitro, the efficiency of stem cells‘ differentiation into hepato-cytes and differentiated cells‘ life-span and functional state,etc. In this study, rat bone marrow derived Thy-l^ β2M^- cells(BDTCs) were induced to differentiate into hepatocytes byco-culturing with CFSC/HGF feeder layers which expressedhHGF efficiently and stably. RT-PCR and immunofluores-cent texts proved induced BDTCs expressed infant and adulthepatocyte specific genes. Further more, these cells displayedfunctions of indocyanine green (ICG) uptake, ammoniummetabolism and albumin production. It was shown thatgrowth factors together with hepatic nonparenchyma cellsprovided a feasible microenvironment for differentiation ofbone marrow stem cells into hepatocytes. The studies notonly provided a significant biological model for going deepinto the mechanism of stem cell plasticity, but also offered atheoretical and technical foundation of gene and stem cellengineering-based regenerative medicine for end-stage liverdiseases.     

Effect of dehydroepiandrosterone on insulin action and development of insulin-induced resistance in C2C12 muscle cells

Dehydroepiandrosterone (DHEA), a precursor of androgens and estrogens, has been demonstrated to have effect of preventing insulin resistance and development of diabetes mellitus. Administration of testosterone appears to induce a marked insulin resistance. How these two hormones affect insulin resistance through regulation of sensitivity of tissues to insulin deserves further studies. Here, the effects of DHEA and testosterone on response to insulin in C2C12 muscle cells are analyzed. After 24 h of DHEA (10-6 mol/L) treatment, C2C12 cells showed an increased insulin- stimulated glucose uptake and enhanced activities of glycogen synthase (GS), phosphofructokinase (PFK) and pyruvate dehydrogenase (PDH), whereas testosterone gave the opposite effects. Incubation of C2C12 cells with high-dose insulin (5×10-7 mol/L) for 24 hours decreased their sensitivity to insulin and led to a state of resistance as assessed on insulin-stimulated glucose uptake and activities of GS, PFK and PDH. Addition of DHEA to insulin-resistant C2C12 cells could reverse the response of these cells to high-dose insulin, but testosterone could further impair insulin sensitivity in insulin-resistant C2C12 cells. These results suggest that the two hormones may influence the development or inhibition of insulin-resistance in type 2 diabetes through regulating glucose uptake, glycogenesis and glycolysis to some extent.     

Observation of fiber ultrastructure of Ligon lintless mutant in upland cotton during fiber elongation

Lintless mutant is a super-short fiber mutanl in upland cotton only 4-8 mm in fiber length and also named Ligon cotton controlled by one dominant gene Li1. Fiber ultrastructure of the mutant (Li1) and its its wild (li1) in siti and in vitro was observed under an electron microscope to understand its cytological characteristics during the fiber cell elongation. The resulls showed that the mutant fiber in situ had thinner cytoplasm, more small vacuoles, less mitochondria, Golgi apparatus and endoplasmic reticula, and there were more starch granules which were free or packed in the amyloplast beside the cell wall than thai of wild type. It was indicated that scarcity of functional organelles and disability of transformation from starch to sugar might be associated with the tact that the mutant fiber cell was aborted too early to elongate into normal length. Mutant ovule in some media containing GA3 could produce a kind of huge callus that grew faster than normal ovules. The callus was covered with many white, loose, and semitransparent fiber-like cells that apt lo get off from ovule. These fiber-like cells were multicellular fibers generated by cell division and had black dots just like pigment glands in the stem and leaf of cotton. There were lots of micro-tubes beside cytoplasm membrane uf the multiecllular fiber, which were thought to be primary preparation for second wall deposition of multicellular fiber. It was indicated that GA3 might induce the expression of gene(s) that kept inactive in the field condition and then stimulate the original fiber cell in vitro to undergo divisionagain.     

Differentiation of bone mar-row derived Thy-1~+β_2M~- cells into hepatocytes induced by coculture with transgenic CFSCs

~~Differentiation of bone mar-row derived Thy-1~+β_2M~- cells into hepatocytes induced by coculture with transgenic CFSCs~~     

The seasonal variations of δ~(18)O , Cl~- , Na~+ , NO_3~- and Ca~(2+) in the snow and firn recovered from Princess Elizabeth Land, Antarctica

Snow and firn samples recovered from two snow pits (2.5 and 4.5m deep) and one 50-m firn core along the route of the 1996/1997 Chinese First Antarctic Inland Traverse Expedition in Princess Elizabeth Land, East Antarctica, have been measured for chemical composition and oxygen isotope ratio. In the two snow pits, the variations of NO3- are partly in phase with that of δ18O, while the variations of Cl~ and Na+ are in inverse phase with that of δ18O. The variations of CI- , Na+ , NO3- and δ18O show obvious seasonal variations and annual stratag-raphy. However, with the depth increasing, the seasonal variations of δ18O are gradually smoothed below 3 m (corresponding to about 10-year mass accumulation) in depth while the seasonal variations of Cl- , Na+ and NO3- are kept fairly well in the whole profile of the 50-m firn core (corresponding to about 250-year mass accumulation). The results provide a useful tool for dating the snow stratum in this region. On the contrary, no obvious seasonal variations of Ca2 + are found in the profiles.     

Evaluation of Novel 3D Architectures Based on Knitting Technologies for Engineering Biological Tissues

Textile-based technologies are considered as potential routes for the production of 3D porous architectures for tissue engineering( TE) applications. We describe the use of two polymers,namely polybutylene succinate( PBS) and silk fibroin(SF) to produce fiber-based finely tuned porous architectures by weft and warp knittings. The obtained knitted constructs are described in terms of their morphology, mechanical properties,swelling ability,degradation behaviour,and cytotoxicity. Each type of polymer fibers allows for the processing of a very reproducible intra-architectural scaffold geometry,with distinct characteristics in terms of the surface physicochemistry,mechanical performance,and degradation capability,which has an impact on the resulting cell behaviour at the surface of the respective biotextiles. Preliminary cytotoxicity screening shows that both materials can support cell adhesion and proliferation. Furthermore, different surface modifications were performed( acid /alkaline treatment, UV radiation,and plasma) for modulating cell behavior. An increase of cell-material interactions were observed,indicating the important role of materials surface in the first hours of culturing. Human adipose-derived stem cells( hASCs) became an emerging possibility for regenerative medicine and tissue replacement therapies. The potential of the recently developed silk-based biotextile structures to promote hASCs adhesion,proliferation,and differentiation is also evaluated. The obtained results validate the developed constructs as viable matrices for TE applications. Given the processing efficacy and versatility of the knitting technology, and the interesting structural and surface properties of the proposed polymer fibers,it is foreseen that our developed systems can be attractive for the functional engineering of tissues such as bone,skin,ligaments or cartilage and also for develop more complex systems for further industrialization of TE products.