冠心病与红细胞免疫T淋巴细胞亚群的关系及治疗观察

冠心病的发病机理与影响因素,目前人们已作了诸多探讨,近年来人们发现冠心病的致病因素除与血管性因素相关外,与免疫因素也有着密切的内在联系。为此,我们作了目前人们普遍关注的红细胞免疫,T淋巴细胞亚群与冠心病之间的相关性研究,并根据多年临床经验自拟养心Ⅰ号及舒心Ⅰ号分别治疗冠心病虚实证型,得出了可喜的初步结论,报道如下。1 一般资料共收集经确诊的冠心病患者21例(诊断标准参照1979年国际冠心病诊断标准)。其中男性患者9例,女性12例;实证11例;虚证10例(中医分型参照1986年版中医内科学)。年龄最大71岁,最小34岁。

IgA肾病红细胞免疫功能的观察

作者测定了２８例ＩｇＡ肾病患者及１６例血缘亲属红细胞免疫功能。结果发现，红细胞Ｃ３ｂ受体活性均明显低于正常组，红细胞表面免疫复合物与正常组无明显差异。２５例ＩｇＡ肾病患者及１５例血缘亲属血清中存在红细胞免疫粘附促进因子，其活性与正常组无明显差异。２４例ｌｇＡ肾病患者及１５例血缘亲属血清中存在红细胞免疫粘附抑制因子，且ＩｇＡ肾病患者该因子活性明显高于正常组。提示，红细胞免疫功能改变可能参与ＩｇＡ肾病的发病。

Morphological Changes of Hemocytes of Musca domestica Larva in vitro Infected by Escherichia coli

[Objective] The research aimed to observe the effects of Escherichia coli infection on the morphology of hemocytes of the 3rd stage larva of Musca domestica in vitro and understand the hemocytes types that take part in the cell immunity of Musca domestica larval.[Method] The hemcytes of the 3rd stage larva of Musca domestica were cultured in vitro and the hemocyte morphology was observed about 2,4,6,8 h after culture in vitro.After Escherichia coli were injected into the hemocytes of the 3rd stage larva of Musca domestica in vitro,the morphology changes of hemocytes were observed at different time after infection.[Result] The hemocytes of of the 3rd stage larva of Musca domestica was divided into five types about 2 h after hemoculture.The hemocytes partly adherence was seen about 6 h after hemoculture.The vacuolation and morpholysis was found in plasmatocytes after being infected by E.coli and a great quantity bacterium were gathered around granulocyte,but the morphology changes of hemocytes were not found in the prohemocyte,shprulocyte and oenocytoid.[Conclusion] The plasmatocyte and granulocyte were primary participants of the cell immunity of Musca domestica larval,but the prohemocyte,sphrulocyte and oenocytoid do not participate in the cell immune reactions.

Cell Cycle Kinetic Analysis in the Cortical Regions of the Lentil Primary Root During Germination

Cell cycle kinetic activity in the cortical cells of the lentil (Lens culinaris Me-die. cv. Verte du Puy) primary root during germination was examined both temporally and spatially. Immunohistochemical and cytological evidence indicated that DNA replication and cell division started in the cortical cells of tire lentil primary root after around 13 and 17 h of imbibition, respectively. The first cells in DNA synthesis and the First mitotic figures all appeared in the cortical cells about I mm front the root-cap junction, but these divided cells had synthesized their DNA during the maturity of seed instead of during germination. The kinetic pattern of activity of the first cell cycle showed that these cells were not activated synchronously, but re-entered the cell cycle in turn depending on their places in the root tip, However, the adjacent cells partially synchronously proceeded their cell cycle.

The Expression of RECK mRNA and Protein in Esophageal Squamous Cell Carcinoma and Its Clinical Significance

OBJECTIVE To investigate the expression of RECK mRNA and protein in esophageal squamous cell carcinoma （ESCC） and to examine its relationship with the clinicopathologic features. METHODS The expression of RECK mRNA and protein in 62 cases of ESCC, 31 of paraneoplastic atypical hyperplasia （PAH） and 62 normal esophageal mucous membrane specimens was examined, using RT-PCR and immunohistochemistry. RESULTS During canceration of the ESCC, the mRNA of RECK increased sequentially from ESCC tissue to PAH and normal mucous membranes. Values were 1.052±0.078, 1.274±0.235 and 1.306±0.121, respectively, with a significant difference among different groups （F=49.936, P〈0.05）. There was a statistically significant difference in the relative amount of the RECK mRNA among the ESCC tissues at various levels of differentiation, depth of infiltration, and different types of lymph node metastasis （F=5.081, F=26.084, U=24.011, P〈0.05）. In the ESCC tissue and PAH, the positive rates of RECK protein expressions were lower compared to the normal mucosa tissue, i.e. 59.7% （37/62）, 71.0% （22/31） and 85.5% （53/62）, respectively. There was a significant difference among the inter-group comparisons （Х^2=10.331, P〈0.01）. In ESCC, there was a close correlation between the RECK protein expression and the degree of cancer differentiation, and the depth of invasion and the types of ESCC lymph node metastasis （P〈 0.05）. CONCLUSION The decrease in expression of both RECK mRNA and protein in ESCC suggest that these low expressions may relate to ESCC development. Examination of RECK mRNA and protein expression may develop into one of the molecular indices for early ESCC diagnosis and prognosis.

Optimization of Parameters of Exogenous Gene Mediated by Liposome to Transfect Yak Mammary Epithelial Cells in Vitro

[Objective] The aim of this study was to optimize conditions of exogenous gene mediated by liposome to transfect yak mammary epithelial cells in Vitro.[Method] Yak mammary epithelial cells were isolated and cultivated in Vitro by the methods of collagenase digestion and tissue adhesion.The expression of cytokeratin in yak mammary epithelial cell was detected by immunocytochemistry technique.With green fluorescence protein as the report gene,yak mammary epithelial cells were transfected with exogenous gene m...

Fixed-Tumor Vaccine: A Practical Formulation with Cytokine-Microspheres for Protective and Therapeutic Antitumor Immunity

Objective: To study the protective and therapeutic antitumor immunity against hepatocellular carcinoma (HCC) with the fixed-tumor vaccine.Methods: A tumor vaccine consisting of fixed tumor cells or fixed tumor fragments combined with sustained-releasers of cytokines and a non-toxic adjuvant was developed. C57BL/6J mice were immunized intra-dermally with the vaccine on day 0 and 7, followed by intrahepatic challenge with live Hepa 1–6 cells.Results: All of 15 nonimmunized control mice developed the hepatoma. Protection of mice immunized with fixed Hepa 1–6 cells and both of IL-2/GM-CSF microspheres or further mixed with TiterMax Gold reached 80% and 87%, respectively. Mass growth of the established tumors, vaccinated twice at 5 mm in diameter, the tumor of control animals continued to grow. However, 7–10 days after the second injection of the tumor vaccine, the tumor growth was suppressed in 9 of 10 mice and then markedly reduced. Complete tumor regression was observed in 60% (6/10) of mice. Splenocytes from the control mice were not able to lyse target Hepa 1–6 cells and other tumor cells. In contrast splenocytes from the vaccinated mice exhibited a 41% lytic activity against the Hepa 1–6 cells tested at an effector/target (E/T) ratio of 5, whereas they did not exhibited such activity against the melanoma cells (B16-F1), Lewis lung carcinoma cells (LLC), renal carcinoma cells (Renca), and bladder carcinoma cells (MBT-2). The cytotoxic activity was inhibited by the treatment with anti-CD3, anti-CD8, and anti-MHC-class I monoclonal antibodies but not with anti-CD4 and anti-MHC-class II antibodies. In the Phase-I clinical trial, vaccination of HCC patients with the autologous vaccine is a well-tolerated treatment and induces fixed tumor fragment-specific immunity.Conclusion: Fixed HCC vaccination elicited protective and therapeutic antitumor immunity against HCC. The tumor vaccine elicited antigen specific CTL response lysis of the target HCC was mediated by the typical MHC-class I restricted CD8+ T cells. Key words cancer vaccine - cytotoxic T lymphocyte - immunotherapy - hepatoma

Effect of New Immunosuppressant-Rapamycin on One-way Mixed Lymphocyte Culture

Objective: Observing human to mouse one-way mixed lymphocyte culture(xMLC) and the effect of new immunosuppressant-Rapamycin on XMLC. Methods: Mouse splenic lymphocyte were collected and treated by mitomycin as activating cell; Human Peripheral blood lymphocytes(hPBL)were separated and gathered as reacting cell; Mouse splenic lymphocyte and hPBL wee mixed to incubate for 1 week; The researchers designed control、RPM groups,and experiment(drugs)grup have different concentration. Results: HPBL in the experiment groups (mixed mouse lymphocyte)proliferated obviously,the amount of3H-TdR in corporation increased evidently(P<0 05,The mean percentage of CD 4, CD 8, LgG, LgM positive cells rose markedly. HPBL in the experiment groups less proliferated,the amount of 3H-TdR incorporation declined,RPM's ic50(50%inhibition concentration)approximately in 1.5 nmol/L; the mean percentage of CD 4, CD 8, IgG, IgM positive cells fell obviously. Conclusion: The human to mouse one-way MLC has obvious lymphocyte proliferation. New immunosuppressants-Rapamycin have powerful effete on XMLC.

Cellular immune function and liver damage in post hepatitic cirrhosis

AIM To study the cellular immune function in patients with post hepatitic cirrhosis (PHC) and its relation with different liver damages.

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

Setting up the Model of Xeno-graft Verse Host Disease

Objective: To observe human to mouse one way mixed lymphocyte(MLC); And to set up the xeno-grats verse host disease Xeno-graft host disease(XGVHD) model,probing its immunologic mechamism.Methods: Mouse splenic lymphocyte were collected in asepsis and treated by mitomycin as activating cell. Human Peripheral blood lymphocytes (hPBL)were separated and gathered as reacting cell; Mouse splenic lymphocyte and hPBL were mixed to incubate for a week. Destroying recipient (mouse) immune system by total body irradiation (TBI) and intraperitoneal injecting CTX、MTX; Separating and collecting hPBL; Injecting hPBL to mouse by caudal vein. Results; ①HPBL in the experiment groups(mixed mouse lymphocyte) proliferated obviously, the amount of 3H-TdR in corporation increased evidently(P<0.05); The mean percentage of CD 4、CD 8、IgG 、IgM positive cells rose markedly. ②Experiment groups,the hPBL were found in the spleen and kidney tissue, fas protein expressing, we occasionally discovered and apoptosis cells.Conclusion: The human to mouse one-way MLC has obvious lymphocyte proliferation. By these means,we succeed in inducing XGVHD and setting up a XGVHD model.

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

Immunosuppressive properties of cloned bone marrow mesenchymal stem cells

Mesenchymal stem cells （MSCs）, derived from adult tissues, are multipotent progenitor cells, which hold great promise for regenerative medicine. Recent studies have shown that MSCs are immunosuppressive in vivo and in vitro in both animals and humans. However, the mechanisms that govern these immune modulatory functions of MSCs remain largely elusive. Some studies with bulk populations of MSCs indicated that soluble factors such as PGE2 and TGFβ are important, while others support a role for cell-cell contact. In this study, we intended to clarify these issues by examining immunosuppressive effects of cloned MSCs. We derived MSC clones from mouse bone marrow and showed that the majority of these clones were able to differentiate into adipocytes and osteoblast-like cells. Importantly, cells from these clones exhibited strong inhibitory effects on TCR activation-induced T cell proliferation in vitro, and injection of a small number of these cells promoted the survival of allogeneic skin grafts in mice. Conditioned medium from MSC cultures showed some inhibitory effect on anti-CD3 induced lymphocyte proliferation independent of PGE2 and TGFβ. In comparison, direct co-culture of MSCs with stimulated lymphocytes resulted in much stronger immunosuppressive effect. Interestingly, the suppression was bi-directional, as MSC proliferation was also reduced in the presence of lymphocytes. Taking together, our findings with cloned MSCs demonstrate that these cells exert their immunosuppressive effects through both soluble factor（s） and cell-cell contact, and that lymphocytes and MSCs are mutually inhibitory on their respective proliferation.

A critical role of IFNγ in priming MSC-mediated suppression of T cell proliferation through up-regulation of BT-H1

Bone-marrow-derived mesenchymal stem cells （MSCs） have been shown to possess immunosuppressive properties, e.g., by inhibiting T cell proliferation. Activated T cells can also enhance the immunosuppression ability of MSCs. The precise mechanisms underlying MSC-mediated immunosuppression remain largely undefined, although both cell-cell contact and soluble factors have been implicated; nor is it clear how the immunosuppressive property of MSCs is modulated by T cells. Using MSCs isolated from mouse bone marrow, we show here that interferon gamma （IFNγ）, a well-known proinflammatory cytokine produced by activated T cells, plays an important role in priming the immunosuppressive property of MSCs. Mechanistically, IFNγ acts directly on MSCs and leads to up-regulation of B7-H1, an inhibitory surface molecule in these stem cells. MSCs primed by activated T cells derived from IFNγ-/- mouse exhibited dramatically reduced ability to suppress T cell proliferation, a defect that can be rescued by supplying exogenous IFNγ. Moreover, siRNA-mediated knockdown of B7-H1 in MSCs abolished immunosuppression by these cells. Taken together, our results suggest that IFNγ plays a critical role in triggering the immunosuppresion by MSCs through upregulating B7-H1 in these cells, and provide evidence supporting the cell-cell contact mechanism in MSC-mediated immunosuppression.

Heat-shocked tumor cell lysate-pulsed dendritic cells induce effective anti-tumor immune response in vivo

AIM. To study whether heat-shocked tumor cells could enhance the effect of tumor cell lysate-pulsed dendritic cells （DCs） in evoking anti-tumor immune response in vivo. METHODS： Mouse undifferentiated colon cancer cells （CT-26） were heated at 42℃ for 1 h and then frozenthawed. The bone marrow-derived DCs pulsed with heatshocked CT-26 cell lysate （HSCT-26 DCs） were recruited to immunize syngeneic naive BALB/c mice. The cytotoxic activity of tumor specific cytotoxic T lymphocytes （CTLs） in mouse spleen was evaluated by IFN-enzyme-linked immunospot （ELISpot） and LDH release assay. The immunoprophylactic effects induced by HSCT-26 DCs in mouse colon cancer model were compared to those induced by single CT-26 cell lysate-pulsed DCs （CT-26 DCs） on tumor volume, peritoneal metastasis and survival time of the mice. RESULTS： Heat-treated CT-26 cells showed a higher hsp70 protein expression. Heat-shocked CT-26 cell lysate pulsing elevated the co-stimulatory and MHC-Ⅱ molecule expression of bone marrow-derived DCs as well as interleukin-12 p70 secretion. The IFN-y secreting CTLs induced by HSCT-26 DCs were significantly more than those induced by CT-26 DCs （P=0.002）. The former CTLs＇ specific cytotoxic activity was higher than the latter CTLs＇ at a serial E/T ratio of 10：1, 20：1, and 40：1. Mouse colon cancer model showed that the tumor volume of HSCT-26 DC vaccination group was smaller than that of CT-26 DC vaccination group on tumor volume though there was no statistical difference between them （24 mm^3 vs 8 mm^3, P=0.480）. The median survival time of mice immunized with HSCT-26 DCs was longer than that of those immunized with CT-26 DCs （57 d vs 43 d, P = 0.0384）. CONCLUSION： Heat-shocked tumor cell lysate-pulsed DCs can evoke anti-tumor immune response in vivo effectively and serve as a novel DC-based tumor vaccine.

The role of apoptosis in the development and function of T lymphocytes

Apoptosis plays an essential role in T cell biology. Thymocytes expressing nonfunctional or autoreactive TCRs are eliminated by apoptosis during development. Apoptosis also leads to the deletion of expanded effector T cells during immune responses. The dysregulation of apoptosis in the immune system results in autoimmunity, tumorogenesis and immunodeficiency. Two major pathways lead to apoptosis: the intrinsic cell death pathway controlled by Bcl-2 family members and the extrinsic cell death pathway controlled by death receptor signaling. These two pathways work to- gether to regulate T lymphocyte development and function.

Role of serum interleukin-18 as a prognostic factor in patients with hepatocellular carcinoma

AIM:To determine whether serum interleukin-18 (IL-18) levels correlated with clinicopathologic features and prognosis in patients with hepatocellular carcinoma (HCC). METHODS:Serum IL-18,IL-6 and IL-12 levels were measured by enzyme-linked immunosorbent assay (ELISA) from 70 patients with HCC and 10 healthy controls. RESULTS:Serum IL-18,IL-6 and IL-12 levels of patients with HCC were significantly higher that those of the controls. The levels of IL-18 correlated significantly with the presence of venous invasion and advanced tumor stages classified by Okuda's criteria. Patients with high serum IL-18 levels (≥ 105 pg/mL) had a poorer survival than those with low serum IL-18 levels (< 105 pg/mL) (4 and 11 mo,respectively,P = 0.015). Multivariate analyses showed that serum IL-18 level,but not IL-6 and IL-12 levels,was a significant and independent prognostic factor of survival. CONCLUSION:These findings demonstrate that serum IL-8 may a useful biological marker of tumor invasiveness and an independent prognostic factor of survival for patients with HCC. Thus,the detailed mechanisms of IL-18 involving in tumor progression should be further investigated.

Mesenchymal stem cells： a new strategy for immunosuppression and tissue repair

Mesenchymal stem cells （MSCs） have great potential for treating various diseases, especially those related to tissue damage involving immune reactions. Various studies have demonstrated that MSCs are strongly immunosuppressive in vitro and in vivo. Our recent studies have shown that un-stimulated MSCs are indeed incapable of immunosuppression; they become potently immunosuppressive upon stimulation with the supernatant of activated lymphocytes, or with combinations of IFN-γ, with TNF-α, IL-1α or IL-1β. This observation revealed that under certain circumstances, inflammatory cytokines can actually become immunosuppressive. We showed that there is a species variation in the mechanisms of MSC-mediated immunosuppression： immunosuppression by cytokine-primed mouse MSCs is mediated by nitric oxide （NO）, whereas immunosuppression by cytokine-primed human MSCs is executed through indoleamine 2, 3-dioxygenase （IDO）. Additionally, upon stimulation with the inflammatory cytokines, both mouse and human MSCs secrete several leukocyte chemokines that apparently serve to attract immune cells into the proximity with MSCs, where NO or IDO is predicted to be most active. Therefore, immunosuppression by inflammatory cytokine-stimulated MSCs occurs via the concerted action of chemokines and immune-inhibitory NO or IDO produced by MSCs. Thus, our results provide novel information about the mechanisms of MSC-mediated immunosuppression and for better application of MSCs in treating tissue injuries induced by immune responses.

Lymphoepitelioma-like hepatocellular carcinoma: A case report and a review of the literature

Lymphoepitelioma is a particular form of undifferentiat-ed carcinoma, characterized by a prominent lymphoid stroma, originally described in the nasopharynx. Lym-phoid stroma-rich carcinomas arising in other organs have been termed lymphoepithelioma-like carcinoma (LELC). In the liver, primary LELCs are very rare, and the majority has been identified as cholangiocarcino-mas. Here a rare case of lymphoepithelioma-like hepa-tocellular carcinoma (HCC) is described. A 47-year old woman presented with abdominal pain. Ultrasonogra-phy revealed a liver nodule, 2.2 cm in diameter, local-ized in the right lobe, adjacent to the gallbladder. Viral markers for hepatic B virus (HBV), hepatic C virus (HCV) and Epstein-Barr virus (EBV) were negative. The nod-ule was hypoechogenic. The patient underwent sur-gery, with resection of the nodule. Histology showed hepatocellular carcinoma, characterized by a promi-nent lymphoid infiltrate. At immunocytochemistry, tumor cells were reactive for Hep Par1 and glypican 3. Immunophenotyping of tumor infiltrating lymphocytes evidenced the predominance of CD8+ cytotoxic sup-pressor T cells. The postoperative clinical outcome was favorable and the patient was recurrence-free 15 mo after resection. This case, to the best of our knowl-edge, is the first reported non EBV and non cirrhosis-associated lymphoepithelioma-like hepatocellular carci-noma. The association between the lack of EBV infec-tion, the absence of cirrhosis, a "cytotoxic profile" of the inflammatory infiltrate and a good prognosis could identify a variant of lymphoepithelioma-like HCC with a favorable clinical outcome.

Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells

AIM： To analyze the ability of nine different potentially probiotic bacteria to induce maturation and cytokine production in human monocyte-derived dendritic cells （moDCs）. METHODS： Cytokine production and maturation of moDCs in response to bacterial stimulation was analyzed with enzyme-linked immunosorbent assay （ELISA） and flow cytometric analysis （FACS）, respectively. The kinetics of mRNA expression of cytokine genes was determined by Northern blotting. The involvement of different signaling pathways in cytokine gene expression was studied using specific pharmacological signaling inhibitors. RESULTS： All studied bacteria induced the maturation of moDCs in a dose-dependent manner. More detailed analysis with S. thermophilus THS, B. breve Bb99, and L. lactis subsp, cremoris ARH74 indicated that these bacteria induced the expression of moDC maturation markers HLA class Ⅱ and CD86 as efficiently as pathogenic bacteria. However, these bacteria differed in their ability to induce moDC cytokine gene expression. S. thermophilus induced the expression of pro-inflammatory （TNF-α, IL-12, IL-6, and CCL20） and Thl type （IL-12 and IFN-γ） cytokines, while B. breve and L. lactis were also potent inducers of antiinflammatory IL-10. Mitogen-activated protein kinase （MAPK） p38, phosphatidylinositol 3 （PI3） kinase, and nuclear factor-kappa B （NF-κB） signaling pathways were shown to be involved in bacteria-induced cytokine production. CONCLUSION： Our results indicate that potentially probiotic bacteria are able to induce moDC maturation, but their ability to induce cytokine gene expression varies significantly from one bacterial strain to another,