In order to assess changes in the activity of immunecompetency present in Crassostrea gigas infected with Marteilioides chungmuensis (Protozoa), the total hemocyte counts (THC), hemocyte populations, hemocyte viability, and phagocytosis rate were measured in oysters using flow cytometry. THC were increased significantly in oysters infected with M. chungmuensis relative to the healthy appearing oysters (HAO) (P<0.05). Among the total hemocyte composition, granulocyte levels were significantly increased in infected oysters as compared with HAO (P<0.05). In addition, the hyalinocyte was reduced significantly (P<0.05). The hemocyte viability did not differ between infected oysters and HAO. However, the phagocytosis rate was significantly higher in infected oysters relative to HAO (P<0.05). The measurement of alterations in the activity of immunecompetency in oysters, which was conducted via flow cytometry in this study, might be a useful biomarker of the defense system for evaluating the effects of ovarian parasites of C. gigas.
Animals ; Cell Count ; Cell Survival ; Cercozoa/*immunology/*pathogenicity ; Crassostrea/*immunology ; Flow Cytometry ; Granulocytes/immunology ; Hemocytes/immunology ; Phagocytosis

Sertoli cells (SC) are known to contain immunoprotective properties, which allow them to survive as allografts without the use of immunosuppressive drugs. Experiments were designed to determine which factors are related to prolonged survival of allogeneic SC. Balb/c derived Sertoli (TM4) and colon cancer (CT-26) cell lines were implanted beneath the kidney capsule of non-immunosuppressed C57BL/6 mice and compared their survival as allografts. Compared to TM4 graft, which survived more than 7 days after transplantation, CT-26 showed massive infiltration of polymorphonuclear cells, necrosis and enlargement of draining lymph nodes. Cultured cell lines showed no differences in their expression patterns of FasL, TGF beta1, clusterin and two complement regulatory proteins (CRP, i.e., membrane cofactor protein, MCP; decay accelerating factor, DAF), but protectin (CD59), another member of CRP was expressed only on TM4. These results suggest that CD59 and unknown factors may contribute to the prolonged survival of SC in non-immunoprivileged sites.
Transplantation, Homologous/immunology ; Transforming Growth Factor beta1/*immunology ; Sertoli Cells/*immunology/*transplantation ; Mice, Inbred C57BL ; Mice ; Male ; Graft Survival/*immunology ; Female ; Fas Ligand Protein/*immunology ; Complement System Proteins/*immunology ; Clusterin/*immunology ; Cells, Cultured ; Cell Survival ; Animals

The understanding of main mechanisms that determine the ability of immune privilege related to Sertoli cells (SCs) will provide clues for promoting a local tolerogenic environment. In this study, we evaluated the property of humoral and cellular immune response modulation provided by porcine SCs. Porcine SCs were resistant to human antibody and complement-mediated formation of the membrane attack complex (38.41+/-2.77% vs. 55.02+/-5.44%, p=0.027) and cell lysis (42.95+/-1.75% vs. 87.99 +/-2.25%, p<0.001) compared to immortalized aortic endothelial cells, suggesting that porcine SCs are able to escape cellular lysis associated with complement activation by producing one or more immunoprotective factors that may be capable of inhibiting membrane attack complex formation. On the other hand, porcine SCs and their culture supernatant suppressed the up-regulation of CD40 expression (p<0.05) on DCs in the presence of LPS stimulation. These novel findings, as we know, suggest that immune modulatory effects of porcine SCs in the presence of other antigen can be obtained from the first step of antigen presentation. These might open optimistic perspectives for the use of porcine SCs in tolerance induction eliminating the need for chronic immunosuppressive drugs.
Animals ; Antibodies, Heterophile/immunology ; Antibody Formation/*immunology ; Antigens, CD40/immunology ; Aorta/cytology ; Cell Line, Transformed ; Cell Survival/immunology ; Complement Membrane Attack Complex/immunology ; Complement System Proteins/immunology ; Dendritic Cells/cytology/immunology ; Endothelial Cells/cytology/immunology ; Epitopes/immunology ; Humans ; Immune Tolerance/*immunology ; Immunity, Cellular/*immunology ; Male ; Mice ; Mice, Inbred C57BL ; Sertoli Cells/cytology/*immunology ; Swine ; *Tissue Engineering ; Transplantation, Heterologous

To investigate the biological properties of cryopreserved dendritic cell (DC) derived from K562 cell line, thus to provide a simple, quick and efficient preservative method of DC for infusion of DC to patients with leukemia after complete remission, fresh DC induced from K562 cell line (K562-DC) was frozen in -196 degrees C liquid nitrogen and -80 degrees C mechanical freezer by method of steps (RPMI 1640 with 10% DMSO, 20% FCS as cryopreservatives), and thawed in different time, respectively. Survivals of cryopreserved and fresh K562-DC, expression of surface antigens, stimulating index (SI) and cytotoxic eliminating rate were detected. The results of fresh induced cells were compared with that of cryopreserved ones. The results showed that before and after frozen in liquid nitrogen, the morphological characteristics of K562-DC had no distinct change; and both their expression rates of surface molecular and capacity to stimulate allogeneic lymphocyte had no statistic significance (P > 0.05). In addition, there were no differences in terms of viability, stimulatory capacity and cytotoxicity of K562-DC from two ways for less than one month (P > 0.05), but there were differences when frozen for more than one month (P < 0.05). It is concluded that there is no significant difference when frozen less than one month between liquid nitrogen and -80 degrees C freezer; but when time is more than one month, K562-DC frozen in -196 degrees C liquid nitrogen is better than that in -80 degrees C freezer.
Antigens, Surface ; immunology ; Cell Shape ; Cell Survival ; Cells, Cultured ; Cryopreservation ; methods ; Dendritic Cells ; immunology ; pathology ; Humans ; K562 Cells ; T-Lymphocytes, Cytotoxic ; immunology ; pathology ; Time Factors

This study sought to shed light on the killing effect of peripheral blood mononuclear cells (PBMCs) irradiated by gamma ray at a dose of 1 Gy on cultured human gastric tumor cell line MKN-28. The radiation dose rate of 17 Gy/min was used. The groups in the experiment were MKN-28 cell control group, PBMCs control group, MKN-28 tumor cells with irradiated or non-irradiated PBMCs co-cultured groups. Radiation dosage was one Gray, acridine orange/ethidium bromide (AO/EB) staining was used for observation of the killing effects of PBMCs on tumor cells in different period. Cells were harvested 240 h later and observed by transmission electron microscopy. The result showed the living period of irradiated PBMCs was shorter than that of non-irradiated PBMCs. In the irradiated and non-irradiated groups,a few PBMCs were still alive after being cultured for 240 h, but the cell volume was larger than that of lymphocytes. These cells were identified as monocytes (95%) or DCs (5%) by transmission electron microscopy. The co-culture of irradiated PBMCs and MKN-28 cells showed that tumor cells were eliminated after 96 h. As compared with the non-irradiated goup, the irradiated PBMCs had more potent ability for killing tumor. The results demonstrate that 1 Gy gamma irridiation can improve the killing effect of PBMCs on the tumor cells, and that 1 Gy gamma irritation can also induce shorter living period of lymphocytes in PBMCs cultured in vitro, but such irritation has little effect on the living period of monocytes and DCs in PBMCs.
Cell Survival ; Coculture Techniques ; Gamma Rays ; Humans ; Leukocytes, Mononuclear ; cytology ; immunology ; radiation effects ; Stomach Neoplasms ; immunology ; pathology ; Tumor Cells, Cultured

OBJECTIVETo investigate the mechanism of autologous mesenchymal stem cells (MSCs) in prolonging the survival of dogs receiving living donor liver transplantation.

METHODSCanine models of allogenic living donor liver transplantation was established in 14 beagle dogs by non-venous by-pass method, and in 7 of the recipients, autologous MSCs labeled by BrdU was infused into the portal vein, with the other 7 dogs as the control. The survival time of the two groups of the dogs was observed after the operation. The liver function (AST and ALT levels), liver pathologies and the differentiation of the transplanted cells were also evaluated postoperatively.

RESULTSCompared with the control group, the dogs receiving MSC transplantation showed significantly increased median survival time (P<0.001) with lowered levels of AST and ALT (P<0.01). The two groups exhibited similar graft rejection after the operation. In dogs with MSC transplantation, the BrdU-labeled MSCs differentiated into liver-like cells in the liver and secreted albumin.

CONCLUSIONAutologous MSCs infusion through the portal vein during allogenic living donor liver transplantation can prolong the survival of the recipient dogs. The stem cells transplanted can differentiate into mature liver-like cells and secrete albumin in the hepatic tissue.

Animals ; Dogs ; Graft Survival ; Immune Tolerance ; immunology ; Liver Transplantation ; immunology ; Living Donors ; Male ; Mesenchymal Stem Cell Transplantation ; Random Allocation

As a part of our ongoing search for a safe and efficient anti-tumor vaccine, we attempted to determine whether the molecular nature of certain tumor antigens would influence immune responses against tumor cells. As compared with freeze-thawed or formaldehyde-fixed tumor antigens, heat-denatured tumor antigens elicited profound anti-tumor immune responses and greatly inhibited the growth of live tumor cells. The heat-denatured tumor antigens induced a substantial increase in the anti-tumor CTL response in the absence of any adjuvant material. This response appears to be initiated by strong activation of the antigen-presenting cells, which may recognize heat-denatured protein antigens. Upon recognition of the heat-denatured tumor antigens, macrophages and dendritic cells were found to acutely upregulate the expression of co-stimulatory molecules such as B7.2, as well as the secretion of inflammatory cytokines such as IL-12 and TNF-alpha. The results of this study indicate that heat-denatured tumor extracts might elicit protective anti-tumor adaptive immune responses and also raise the possibility that a safe and efficient adjuvant-free tumor vaccine might be developed in conjunction with a dendritic cell-based tumor vaccine.
Adjuvants, Immunologic ; Animals ; Antibodies, Neoplasm/immunology ; Antibody Specificity/immunology ; Antigens, Neoplasm/immunology ; Cancer Vaccines/*immunology ; Cell Line, Tumor ; Cell Proliferation ; Cytokines/biosynthesis ; Cytotoxicity, Immunologic/immunology ; Dendritic Cells/immunology ; *Hot Temperature ; Immunity, Cellular/immunology ; Immunization ; Immunologic Memory/immunology ; Macrophages, Peritoneal/immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Neoplasms/*immunology/*pathology ; Survival Analysis ; T-Lymphocytes, Cytotoxic/immunology

This study investigated whether elevated host immune capacity can inhibit T. gondii infection. For this purpose, we used silk protein extracted from Bombyx mori cocoons as a natural supplement to augment immune capacity. After silk protein administration to BALB/c mice for 6 weeks, ratios of T lymphocytes (CD4+ and CD8+ T-cells) and splenocyte proliferative capacities in response to Con A or T. gondii lysate antigen (TLA) were increased. Of various cytokines, which regulate immune systems, Th1 cytokines, such as IFN-gamma, IL-2, and IL-12, were obviously increased in splenocyte primary cell cultures. Furthermore, the survival of T. gondii (RH strain)-infected mice increased from 2 days to 5 or more days. In a state of immunosuppression induced by methylprednisolone acetate, silk protein-administered mice were resistant to reduction in T-lymphocyte (CD4+ and CD8+ T-cells) numbers and the splenocyte proliferative capacity induced by Con A or TLA with a statistical significance. Taken together, our results suggest that silk protein augments immune capacity in mice and the increased cellular immunity by silk protein administration increases host protection against acute T. gondii infection.
Animals ; Bombyx/*chemistry ; CD4-CD8 Ratio ; Cell Proliferation ; Cells, Cultured ; Cytokines/secretion ; Insect Proteins/*immunology ; Leukocytes, Mononuclear/immunology ; Male ; Mice ; Mice, Inbred BALB C ; Silk/immunology ; Spleen/immunology ; Survival Analysis ; Toxoplasma/*immunology/pathogenicity ; Toxoplasmosis, Animal/immunology/*prevention & control

OBJECTIVETo explore in vitro expansion of CD34+CD59+ cells from patients with PNH, and compare the capabilities of survival, proliferation and expansion between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control.

METHODSCD34+CD59+ cells from patients with PNH and CD34+ cells from normal control were selected from the bone marrow mononuclear cells by means of two-step sorting method with immunomagnetic microbead-flow cytometry, then underwent in vitro expansion for two weeks and semi-solid culture in vitro before and after expansion.

RESULTS(1) CD34+CD59+ cells from patients with PNH can be expanded effectively in vitro, and the biggest expansion of CD34+CD59+ cells was about 23.49 fold on the 7th day. (2) There were some similar characteristics between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control, such as: the best combination of hematopoietic factors for in vitro expansion was SCF+ IL-3 + IL-6 + FL + Tpo + Epo, and the seventh day was the most suitable in course of 4-14 days for in vitro expansion, and after in vitro expansion, the cells remained CD59 positive and strong capability of performing colony-forming. (3) CD34+ cells from normal control had better proliferation, expansion and stronger potential to survive than CD34+CD59+ cells from patients with PNH.

CONCLUSIONS(1) In vitro expansion of CD34+CD59+ cells from patients with PNH can be performed. The present study showed the possibility of performing ABMT or APBSCT clinically for patients with PNH. (2) There were some similar characteristics between CD34+CD59+ cells from patients with PNH and CD34+ cells from normal control, but the latter had better proliferation, expansion and stronger potential to survive than the former. CD34+CD59+ cells from patients with PNH were not completely normal cells.

Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; immunology ; CD59 Antigens ; analysis ; Cell Differentiation ; Cell Division ; Cell Survival ; Cells, Cultured ; Hemoglobinuria, Paroxysmal ; immunology ; pathology ; Humans ; Immunophenotyping

This study discusses the effects of Shenlian extracts (SL) on M1 macrophages in atherosclerosis. The MTT assay was used to detect the growth inhibition rates of RAW264.7 cells. RAW264.7 cells were stimulated with murine interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS) to induce M1 macrophages. The different concentrations of SL extracts (high-dose 50 mg x L(-1), moderate-dose 25 mg x L(-1), low-dose 12.5 mg x L(-1)) were added. The CD86 of M1 macrophages in cell membrane was measured by flow cytometry. The mRNA expression of iNOS and TNF-alpha gene was detected by reverse transcription PCR (RT-PCR). And the supernatants were collected, the content of IL-6 and TNF-alpha were detected with ELISA kits. The results of this experiment show that the expression of the cell membrane molecule CD86, iNOS and TNF-alpha gene, the content of IL-6 and TNF-alpha was obviously increased in M1 macrophages by IFN-gamma and LPS. The different doses of SL extract could reduce the expression of the above indicators. The above experimental results demonstrate that IFN-gamma combined LPS can induce RAW264.7 cell to type into M1 macrophages, and SL extracts can inhibit M1 macrophages.
Animals ; Cell Line ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Interferon-gamma ; genetics ; immunology ; Interleukin-6 ; genetics ; immunology ; Macrophages ; cytology ; drug effects ; immunology ; Mice ; Tumor Necrosis Factor-alpha ; genetics ; immunology