Establishment and Comparison of Two Mouse Models of Celiac and Cervical Heterotopic Heart Transplantation / 中国实验动物学报

Objective Objective In order to better keep up with the development of transplantation immunobiology.we established and compared two types of mouse heterotopic heart transplantation,and hope to help further organ transplantation studies.Methods According to the surgical procedures of Ono's type and Chen's type of mouse model of heterotopic heart transplantation with some modification,we performed celiac and cervical heteropotic heart transplantation between iso-strains and hetero-strains,and compared the operation suecess rate,operation time,allografi survival time,and histopathology of those establishment methods.Results The success rates of mouse celiac and cervical heterotopic heart transplantation were 86.7% and 83.3%,respectively,with a non-significant difference(P>0.05) between the two methods of operation regarding the total operation time,survival time of the allografts,and histopathological findings.Conclusions Based on the mastery of microsurgical techniques,the two models of heterotopic mouse heart transplantation can be established equally,and either of them can be considered depending on the particular requirements of studies.

Different neurotropic pathogens elicit neurotoxic CCR9- or neurosupportive CXCR3-expressing microglia.

What mechanism that determines microglia accomplishing destructive or constructive role in CNS remains nebulous. We report here that intracranial priming and rechallenging with Toxoplasma gondii in mice elicit neurotoxic CCR9+ Irg1+ (immunoresponsive gene 1) microglia, which render resistance to apoptosis and produce a high level of TNF-alpha; priming and rechallenging with lymphocytic choriomeningitis virus elicit neurosupportive CXCR3+ Irg1- microglia, which are sensitive to apoptosis and produce a high level of IL-10 and TGF-beta. Administration of CCR9 and/or Irg1 small interfering RNA alters the frequency and functional profiles of neurotoxic CCR9+ Irg1+ and neurosupportive CXCR3+ Irg1- microglia in vivo. Moreover, by using a series of different neurotropic pathogens, including intracellular parasites, chronic virus, bacteria, toxic substances, and CNS injury to intracranially prime and subsequent rechallenge mice, the bi-directional elicitation of microglia has been confirmed as neurotoxic CCR9+ Irg1+ and neurosupportive CXCR3+ Irg1- cells in these mouse models. These data suggest that there exist two different types of microglia, providing with a novel insight into microglial involvement in neurodegenerative and neuroinflammatory pathogenesis such as Alzheimer's disease and AIDS dementia.

Establishment of Stable High Expression Cell Line with Green Fluorescent Protein and Resistance Genes / 华中科技大学学报（医学）（英德文版）

In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

Establishment of stable high expression cell line with green fluorescent protein and resistance genes / 华中科技大学学报（医学）（英德文版）

In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

V alpha 24-invariant NKT cells from patients with allergic asthma express CCR9 at high frequency and induce Th2 bias of CD3+ T cells upon CD226 engagement.

We have demonstrated that Valpha24(+)Vbeta11(+) invariant (Valpha24(+)i) NKT cells from patients with allergic asthma express CCR9 at high frequency. CCR9 ligand CCL25 induces chemotaxis of asthmatic Valpha24(+)i NKT cells but not the normal cells. A large number of CCR9-positive Valpha24(+)i NKT cells are found in asthmatic bronchi mucosa, where high levels of Th2 cytokines are detected. Asthmatic Valpha24(+)i NKT cells, themselves Th1 biased, induce CD3(+) T cells into an expression of Th2 cytokines (IL-4 and IL-13) in cell-cell contact manner in vitro. CD226 are overexpressed on asthmatic Valpha24(+)i NKT cells. CCL25/CCR9 ligation causes directly phosphorylation of CD226, indicating that CCL25/CCR9 signals can cross-talk with CD226 signals to activate Valpha24(+)i NKT cells. Prestimulation with immobilized CD226 mAb does not change ability of asthmatic Valpha24(+)i NKT cells to induce Th2-cytokine production, whereas soluble CD226 mAb or short hairpin RNA of CD226 inhibits Valpha24(+)i NKT cells to induce Th2-cytokine production by CD3(+) T cells, indicating that CD226 engagement is necessary for Valpha24(+)i NKT cells to induce Th2 bias of CD3(+) T cells. Our results are providing with direct evidence that aberration of CCR9 expression on asthmatic Valpha24(+)i NKT cells. CCL25 is first time shown promoting the recruitment of CCR9-expressing Valpha24(+)i NKT cells into the lung to promote other T cells to produce Th2 cytokines to establish and develop allergic asthma. Our findings provide evidence that abnormal asthmatic Valpha24(+)i NKT cells induce systemically and locally a Th2 bias in T cells that is at least partially critical for the pathogenesis of allergic asthma.

CD226 expression deficiency causes high sensitivity to apoptosis in NK T cells from patients with systemic lupus erythematosus.

Humans and mice with systemic lupus erythematosus (SLE) and related autoimmune diseases have reduced numbers of NK T cells. An association between NK T cell deficiency and autoimmune disease has been identified. However, the mechanisms for reduction of NK T cell number in patients with SLE are unknown. In the present study we report that NK T cells from active SLE patients are highly sensitive to anti-CD95-induced apoptosis compared with those from normal subjects and inactive SLE patients. CD226 expression is deficient on NK T cells from active SLE patients. The expression of one antiapoptotic member protein, survivin, is found to be selectively deficient in freshly isolated NK T cells from active SLE patients. CD226 preactivation significantly up-regulates survivin expression and activation, which can rescue active SLE NK T cells from anti-CD95-induced apoptosis. In transfected COS7 cells, we confirm that anti-CD95-mediated death signals are inhibited by activation of the CD226 pathway through stabilization of caspase-8 and caspase-3 and through activation of survivin. We therefore conclude that deficient expression of CD226 and survivin in NK T cells from active SLE is a molecular base of high sensitivity of the cells to anti-CD95-induced apoptosis. These observations offer a potential explanation for high apoptotic sensitivity of NK T cells from active SLE, and provide a new insight into the mechanism of reduction of NK T cell number in SLE and understanding the association between NK T cell deficiency and autoimmune diseases.

Studies on activity of NK cells in preeclampsia patients / 华中科技大学学报（医学）（英德文版）

The activity of the NK cells in patients with preeclampsia was studied to investigate the pathogenesis of preeclampsia. By using MTT and 51Cr releasing technique, the proliferation and killing ability of the NK cells in maternal and umbilical blood from preeclampsia patients (n = 18) and normal third trimester pregnant women (n = 18) were detected. The NK-92 cell line was as the positive control. The results showed that the NK cell counts of umbilical blood in preeclampsia patients and normal third trimester pregnant women were significantly greater than those of maternal blood (both P<0.05). Compared with that in normal third trimester pregnant women, the proliferative ability of the NK cells in preeclampsia patients was apparently increased (P<0.05). Compared with that in maternal blood, the proliferative ability of the NK cells in umbilical blood from both preeclampsia patients and normal third trimester pregnant women was dramatically increased. The killing ability of the NK cells in preeclampsia patients was significantly higher than that in normal third trimester pregnant women (P <0.05). It was suggested that both number and function of the NK cells in preeclampsia women were increased, and that in umbilical blood was greater than that in maternal blood, speculating that the function of the NK cells may affect the maintenance of the maternal and fetal immune tolerance during pregnancy.

Studies on activity of NK cells in preeclampsia patients / 华中科技大学学报（医学）（英德文版）

The activity of the NK cells in patients with preeclampsia was studied to investigate the pathogenesis of preeclampsia. By using MTT and 51Cr releasing technique, the proliferation and killing ability of the NK cells in maternal and umbilical blood from preeclampsia patients (n = 18) and normal third trimester pregnant women (n = 18) were detected. The NK-92 cell line was as the positive control. The results showed that the NK cell counts of umbilical blood in preeclampsia patients and normal third trimester pregnant women were significantly greater than those of maternal blood (both P<0.05). Compared with that in normal third trimester pregnant women, the proliferative ability of the NK cells in preeclampsia patients was apparently increased (P<0.05). Compared with that in maternal blood, the proliferative ability of the NK cells in umbilical blood from both preeclampsia patients and normal third trimester pregnant women was dramatically increased. The killing ability of the NK cells in preeclampsia patients was significantly higher than that in normal third trimester pregnant women (P <0.05). It was suggested that both number and function of the NK cells in preeclampsia women were increased, and that in umbilical blood was greater than that in maternal blood, speculating that the function of the NK cells may affect the maintenance of the maternal and fetal immune tolerance during pregnancy.

Peptide nucleic acid antisense prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into graft.

CXCR3, predominantly expressed on memory/activated T cells, is a receptor for both IFN-gamma-inducible protein 10/CXC chemokine ligand (CXCL)10 and monokine induced by IFN-gamma/CXCL9. It was reported that CXC chemokines IFN-gamma-inducible protein 10/CXCL10 and monokine induced by IFN-gamma/CXCL9 play a critical role in the allograft rejection. We report that CXCR3 is a dominant factor directing T cells into mouse skin allograft, and that peptide nucleic acid (PNA) CXCR3 antisense significantly prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into allografts in mice. We found that CXCR3 is highly up-regulated in spleen T cells and allografts from BALB/c recipients by day 7 of receiving transplantation, whereas CCR5 expression is moderately increased. We designed PNA CCR5 and PNA CXCR3 antisenses, and i.v. treated mice that received skin allograft transplantations. The PNA CXCR3 at a dosage of 10 mg/kg/day significantly prolonged mouse skin allograft survival (17.1 +/- 2.4 days) compared with physiological saline treatment (7.5 +/- 0.7 days), whereas PNA CCR5 (10 mg/kg/day) marginally prolonged skin allograft survival (10.7 +/- 1.1 days). The mechanism of prolongation of skin allograft survival is that PNA CXCR3 directly blocks the CXCR3 expression in T cells, which is responsible for directing T cells into skin allograft to induce acute rejection, without interfering with other functions of the T cells. These results were obtained at mRNA and protein levels by flow cytometry and real-time quantitative RT-PCR technique, and confirmed by chemotaxis, Northern and Western blot assays, and histological evaluation of skin grafts. The present study indicates the therapeutic potential of PNA CXCR3 to prevent acute transplantation rejection.

The inhibitory effects of an antisense u-PAR vector on invasion of highly invasive human prostate carcinoma PC-3M cell subclones / 华中科技大学学报（医学）（英德文版）

To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found that in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79% and 60%, respectively. Although the antisense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups was statistically significant (P < 0.01). The antisense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclones in vitro but also restrain the growth of those cell subclones in vivo.

Construction of antisense RNA expression plasmid for u-PAR and its transfection to highly invasive PC-3M cell subclones / 华中科技大学学报（医学）（英德文版）

To evaluate the specific inhibition of antisense u-PAR on the u-PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u-PAR obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the antisense u-PAR recombinant was transfected into highly invasive cell subclones. The u-PAR expression in neo-resistant cells was examined by RT-PCR and immunohistochemical assay. Compared to the control cells, the content of mRNA and protein of u-PAR in transfected cells decreased sharply, and the rate of inhibition was 53% and 73%, respectively, indicating that an antisense u-PAR might have played a specific inhibitory role in its expression in the cells, which may provide a good cell model for making further investigation of the inhibitory effects of the antisense u-PAR on invasion in highly invasive cell subclones of human prostate carcinoma.

Possible association of HLA-DRB1 gene with the autoantibody against myocardial mitochondria ADP/ATP carrier in dilated cardiomyopathy / 华中科技大学学报（医学）（英德文版）

To probe the genetic background and immunopathogenesis of dilated cardiomyopathy (DCM) 77 patients with DCM, HLA-DRB1 gene polymorphism were analyzed by using the polymerase chain reaction/sequence specific primer (PCR/SSP) technique and autoantibody against myocardial mitochondria ADP/ATP carrier were examined by using the Immunoblot analysis. The frequency of HLA-DRB1*0901 allele was significantly higher in DCM patients in which autoantibody against ADP/ATP carrier of myocardial mitochondria is positive in contrast with those in which the autoantibody is negative (25.46% vs 3.45%, P < 0.05), the relative risk (RR) being 9.56. The other frequencies of HLA-DRB1 alleles have no significant difference in the antibody positive group and negative group. It is possible that a subset of DCM patients may exist in which autoimmunity is associated with genetic factors.

Antitumor effects of the fibroblasts transfected TNF-alpha gene and its mutants / 华中科技大学学报（医学）（英德文版）

To compare the anti-tumor effects of transmembrane TNF-alpha (TM-TNF) and secreted TNF-alpha (S-TNF) in vivo, mouse fibroblasts NIH3T3 were transfected separately with three types of retrovirus containing wild type TNF-alpha (Wt-TNF), TM-TNF mutant (TM-TNFm), S-TNF mutant (S-TNFm). Southern blot, RT-PCR, FACS and bioassay were used to investigate TNF-alpha gene integration, expression and its biological activity. It was found that both fixed cells and supernatant of NIH3T3/Wt-TNF, the fixed cells of NIH3T3/TM-TNFm and the supernatant of NIH3T3/S-TNFm could express high level of TNF-alpha or its mutants and effectively kill H22 in vitro. The transfected NIH3T3 were separately injected into the mice at the sites of H22 tumor cell inoculation according to a ratio of 5:1 or 1:1 (effector/target cells, E/T) after the third day of H22 challenge, respectively. At the E/T = 5:1, the NIH3T3/TM-TNFm induced the highest tumor regression, while NIH3T3/S-TNFm exerted the strongest tumor depressing effect at the E/T = 1:1 in vivo. No obvious side effects were noted throughout the course of treatment. The results suggest that both TM-TNF and S-TNF could cause tumor regression. The anti-tumor effect of TM-TNF would be more powerful and safe than that of S-TNF at the proper E/T ratio.

Study of the effects of LPS on the TACE gene expression and its function / 华中科技大学学报（医学）（英德文版）

In order to investigate the effects of LPS on the TACE gene transcription and expression and its regulating effect on the TM-TNF secretion, in vitro studies were carried out on HL-60 cells stimulated by LPS. TACE, TNF-alpha mRNA levels were detected by Dot-Elisa and the distribution of membrane molecules determined by flow cytometry assay and indirect immunofluorescence. The results showed that: (1) TACE was detected in or on HL-60 cells and it is predominantly localized on cell surface and to a perinuclear compartment. (2) LPS induced a time dependent increasement of TNF-alpha mRNA and enhanced TNF conversion with decreasing distribution of TNF in cell surface and increasing secretion of TNF protein. Such conversion could be inhibited by TACE ODN. (3) LPS also induced time-dependently increased expression of TACE gene and activation of its function. On the other hand, TACE protein in cell lysate and on cell surface was decreased. It was suggested that TACE molecular structure might change following its mediating membrane-anchored molecular secretion.

Molecular cloning and sequencing of the cDNA of the soluble human leukocyte antigen-G1 / 免疫学杂志

Objective　To construct a recombinant plasmid pcDNA3-sHLA-G1 expressing soluble HLA-G1. Methods　 Total cell RNA was extracted from the cell line Jeg-3 and the cDNA was amplified by RT-PCR； The cDNA fragment was inserted into the eukaryotic expressing vector pcDNA3 and the recombinant plasmid was identified by restriction endonucleases digestion and sequencing. Results　 After restriction endonucleases treatment and sequencing, it was confirmed that the pcDNA3-sHLA-G1 had been constructed successfully. Conclusion　 In this study, the recombinant plasmid pcDNA3-sHLA-G1 had been constructed successfully.

Effect of transfection of tumor necrosis factor ? gene and its mutants on tumorigenicity of H22 tumor cells in vivo / 中国病理生理杂志

AIM: To compare the tumorigenecity of H22 cells transfected with TNF-? gene and its mutants (secreted TNF-? mutant, S-TNFm, transmembrane TNF-? mutant, TM-TNFm and wild type of TNF-?, Wt-TNF) in vivo . METHODS: Three kinds of mouse liver cancer cell line H22 expressing TNF-? and its two mutants were mixed with untransfected H22 at different effector/target ratio separately. The growth of tumor was examined after injection of 2.5?10 5 (100 ?L) mixed H22 tumor cells into mice. The lymphocyte infiltration in the site of tumor and the expression of Fas on tumor cells were detected by immunohistochemistry. RESULTS: The tumorigenecity of H22 cells transfected with TNF-? gene and its mutants was significantly weakened ( P

The effect of secreted and transmembrane stem cell factor on biological activities of mast cells / 中国免疫学杂志

Objective:To study the effects of secreted stem cell factor(S SCF) and transmembrane SCF(TM SCF) on the biological functions of mast cells,including proliferation,release of histamine and secretion of cytokine to explore the biological characteristics of the two forms of SCF.Methods:MTT,fluorescence technique,in situ hybridization were used to detect the several biological functions of mast cells,such as proliferation,release of histamine and the transcription of TNF ? mRNA.Results:It was shown that S SCF could significantly promote the proliferation of mast cells,release of histamine and TNF ? mRNA transcription,while TM SCF had only a weaker positive effect on histamine relase and no any effect on proliferation and TNF ? mRNA transcription.Conclusion:The biological actions of TM SCF on mast cells are different from that of S SCF.

The Inhibitory Effects of an Antisense u-PAR Vector on Invasion by Highly Invasive Human Prostate Carcinoma PC-3M Cell Subclones / 中国肿瘤生物治疗杂志

Objective: To observe the inhibiting effects of an antisense u-PAR vector on invasion by highly invasive PC-3M cell subclones. Methods: The effects of an antisense vector on invasion by highly invasive PC-3M cell subclones were observed and compared in vitro by monolayer invasion assay and soft agar clone. Then, both a quantitative RT-PCR and zymography were used to exam the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones. Furthermore, the tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. Results: It is found that the speed of growth in vitro was slowing down by highly invasive PC-3M cell subclones transfected with the antisense u-PAR, and the ability of anchorage-independent growth of those cell subclones was also decreasing sharply,and the inhibiting rate was 79% and 60%, respectively. Although the antisense u-PAR didn′t change MMP-9 gene transcription, but they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups reached statistical significance ( P