OBJECTIVEEscape from the body's immune response is a basic characteristic of lung cancer, and indoleamine-2,3-dioxygenase (IDO) plays a key role in mediating immune escape of non-small-cell lung cancer, which leads to recurrence and metastasis. Feiji Recipe, a compound Chinese herbal medicine, has the effect of stabilizing lesions and prolonging survival in patients with lung cancer. The purpose of this study was to investigate the mechanisms underlying the anticancer properties of Feiji Recipe.

METHODSAn orthotopic transplant model of mouse Lewis lung cancer, with stable expression of IDO gene, was established in C57BL/6 mice. Optical imaging was used to observe the effects of Feiji Recipe in the treatment of lung cancer in vivo. The effects of Feiji Recipe on the proliferation of mouse Lewis lung cancer cell line 2LL, 2LL-enhanced green fluorescent protein (2LL-EGFP) and 2LL-EGFP-IDO were investigated, and the apoptosis of T-cells was examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide using flow cytometry. Chemical composition of Feiji Recipe was validated by high-performance liquid chromatography.

RESULTSCompared to the control group, the survival of animals treated with Feiji Recipe was significantly prolonged (P = 0.0074), and the IDO protein level decreased (P = 0.0072); moreover, the percentages of CD4CD25 T-cells and Foxp3 T-cells were significantly decreased (P < 0.05). The molecular mechanism of Feiji Recipe against lung cancer may relate to the regulation of immune cells, such as T-cells and regulatory T-cells.

CONCLUSIONThe molecular mechanism of Feiji Recipe in treatment of lung cancer is to restore the function of T-cells in the cancer microenvironment through interfering with the IDO pathway.

Animals ; Apoptosis ; drug effects ; Carcinoma, Lewis Lung ; drug therapy ; enzymology ; immunology ; physiopathology ; Cell Proliferation ; drug effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Growth Inhibitors ; administration & dosage ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; genetics ; immunology ; Lung Neoplasms ; drug therapy ; enzymology ; immunology ; physiopathology ; Male ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; drug effects ; immunology

OBJECTIVEWe used an animal model of collagen-induced arthritis (CIA) to study changes and roles of tyrosine hydroxylase (TH) expressed by CD4+ T cell subsets, and then explore the relationship between CD4+ T cell subset-derived catecholamines and inflammatory responses in CIA.

METHODSThirty-six male DBA/1 mice were randomly divided into control group, CIA model group (day 35) and CIA model group (day 55) (n = 12). CIA model was induced by type II collagen (CII) in DBA/1 mice. On the 35th and 55th day following primary immunization, the joints of the mice were observed for clinical score of swelling and the level of anti-CII IgG antibody in serum was examined. Expression of specific transcription factors and cytokines of Th1, Th17, Th2 and Treg cells and TH in mesenteric lymph nodes was measured by means of Western blot. The changes of TH expressed by CD4+ T cell subsets in mesenteric lymph nodes were determined by flow cytometry.

RESULTSClinical score and anti-CII antibody level increased in CIA compared with that in intact mice. Specific transcription factors and cytokines expressed by Th1 and Th17 cells were upregulated and cytokines expressed by Th2 and Treg cells were downregulated in mesenteric lymph nodes in CIA mice. Expression of TH was upregulated and the increased expression of TH in CD4+ T cells was attributed to Th1 and Th17 cells in mesenteric lymph nodes of CIA.

CONCLUSIONThe increase in catecholamines from CD4+ T cell subsets in mesenteric lymph nodes of CIA may be related to inflammatory alleviation in CIA progression.

Animals ; Arthritis, Experimental ; immunology ; CD4-Positive T-Lymphocytes ; enzymology ; Collagen Type II ; adverse effects ; Cytokines ; metabolism ; Disease Models, Animal ; Lymph Nodes ; immunology ; Male ; Mice ; Mice, Inbred DBA ; T-Lymphocyte Subsets ; immunology ; Transcription Factors ; metabolism ; Tyrosine 3-Monooxygenase ; metabolism

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.
Acute-Phase Proteins/metabolism ; Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; Antigens, CD14/metabolism ; Bone Marrow Cells/cytology/drug effects ; CD8-Positive T-Lymphocytes/*immunology ; Carrier Proteins/metabolism ; Cell Differentiation/drug effects ; Cell Nucleus/drug effects/metabolism ; Cell Proliferation/drug effects ; Cytokines/biosynthesis ; Dendritic Cells/cytology/drug effects/enzymology/*immunology ; Enzyme Activation/drug effects ; Lymphocyte Activation/*drug effects ; Membrane Glycoproteins/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinases/metabolism ; Myeloid Differentiation Factor 88/metabolism ; NF-kappa B/metabolism ; Neoplasms/immunology/*pathology ; Pectins/*pharmacology ; Phenotype ; Protein Transport/drug effects ; Receptors, Chemokine/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes, Cytotoxic/cytology/drug effects ; Toll-Like Receptor 4/*agonists/metabolism

M1-type macrophages are capable of inducing lysis in various types of cancer cells, but the mechanism of action is unclear. It has been noted that an "unknown protein" produced together with protease by activated macrophages is responsible for this action. Activated M1 macrophages have been recently reported to produce family 18 chitinases, all of which have been named chitotriosidase. Our experiments have demonstrated that family 18 chitinases work together with proteases and can damage various cancer cells both in vitro and in vivo. Thus, in this article, we suggest that the 50-kDa chitotriosidase is the reported "unknown protein". In addition, we discuss how to properly stimulate activated M1 macrophages to produce 50-kDa chitotriosidases and proteases for destroying cancer cells. Because family 19 chitinase has recently been reported to kill cancer cells, we also discuss the possibility of directly using human family 18 chitotriosidase and the humanized plant family 19 chitinase for cancer treatment.
Animals ; Antineoplastic Agents, Alkylating ; pharmacology ; Chitinases ; metabolism ; Cyclophosphamide ; pharmacology ; Hexosaminidases ; metabolism ; Humans ; Immunosuppressive Agents ; pharmacology ; Macrophage Activation ; immunology ; Macrophages ; classification ; enzymology ; immunology ; pathology ; Neoplasms ; immunology ; pathology ; Peptide Hydrolases ; metabolism ; T-Lymphocytes, Regulatory ; metabolism ; Th1 Cells ; metabolism ; Th2 Cells ; metabolism

BACKGROUNDOverwhelming evidences on chronic myeloid leukemia (CML) indicate that patients harbor quiescent CML stem cells that are responsible for blast crisis. While the hematopoietic stem cell (HSC) origin of CML was first suggested over 30 years ago, recently CML-initiating cells beyond HSCs are also being investigated.

METHODSWe have previously isolated fetal liver kinase-1-positive (Flk1(+)) cells carrying the BCR/ABL fusion gene from the bone marrow of Ph(+) patients with hemangioblast property. In this study, we isolated CML patient-derived Flk1(+)CD31(-)CD34(-) mesenchymal stem cells (MSCs) and detected their biological characteristics and immunological regulation using fluorescence in situ hybridization (FISH) analysis, fluorescence activated cell sorting (FACS), enzyme-linked immunoadsorbent assay, mixed lymphocyte reaction assays; then we compared these characters with those of the healthy donors.

RESULTSCML patient-derived Flk1(+)CD31(-)CD34(-) MSCs had normal morphology, phenotype and karyotype while appeared impaired in immuno-modulatory function. The capacity of patient Flk1(+)CD31(-)CD34(-) MSCs to inhibit T lymphocyte activation and proliferation was impaired in vitro.

CONCLUSIONSCML patient-derived MSCs have impaired immuno-modulatory functions, suggesting that the dysregulation of hematopoiesis and immune response may originate from MSCs rather than hematopoietic stem cells (HSCs). MSCs might be a potential target for developing efficacious treatment for CML.

Adolescent ; Adult ; Antigens, CD34 ; genetics ; metabolism ; Apoptosis ; drug effects ; Blotting, Western ; Cell Cycle ; drug effects ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Humans ; Immunomodulation ; In Situ Hybridization, Fluorescence ; Karyotype ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; enzymology ; immunology ; metabolism ; Male ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Mesenchymal Stromal Cells ; cytology ; immunology ; Middle Aged ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; T-Lymphocytes ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism ; Young Adult

OBJECTIVETo study the effect of Extractum trametes robiniophila murr on cardiac allograft rejection in mice.

METHODSAll abdominal heterotopic heart transplantation models were divided into three groups as follows: (A) Extractum trametes robiniophila murr group. (B) Rejection group. (C) Isograft group. In each group, mean survival times (MST) of transplanted hearts and their pathologic histological changes at postoperative fifth day were observed. With fluoroimmunoassay, granzyme B and CD8(+) expressions were examined.

RESULTSThe MST of heart allografts in group A were (6.38 +/- 0.69) d, significantly shorter than that of group B [(8.31 +/- 0.59) d] (P < 0.01). In group A, acute rejection was present in advance; transplanted hearts were seriously damaged into acute rejection pathological grade 3, and CD8(+) T lymphocytes infiltrated diffusely and the expression of granzyme B increased significantly as compared with other groups.

CONCLUSIONSExclusive application of Extractum trametes robiniophila murr can promote the acute rejection of graft in early phase of postoperation, and the mechanism may be the promoted proliferation and infiltration of CD8(+) T lymphocytes and the increased expression of granzyme B.

Animals ; CD8-Positive T-Lymphocytes ; immunology ; Drugs, Chinese Herbal ; adverse effects ; Female ; Graft Rejection ; chemically induced ; Granzymes ; metabolism ; Heart Transplantation ; Male ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Myocardium ; enzymology ; immunology ; Postoperative Care

Germ-line mutations in BRCA2 predispose to early-onset cancer. Homozygous mutant mouse, which has Brca2 truncated in exon 11 exhibit paradoxic occurrence of growth retardation and development of thymic lymphomas. However, due to its large embryonic lethality, cohort studies on the thymic lymphomas were not feasible. With the aid of Cre-loxP system, we demonstrate here that thymus-specific disruption of Brca2 allele without crossing it to p53-mutant background leads to the development of thymic lymphomas. Varying from 16 weeks to 66 weeks after birth, 25% of mice disrupted of Brca2 in the thymus died of thymic lymphomas, whereas previous report did not observe lymphomagenesis using similar Cre-loxP system. Future analysis of thymic lymphomas from these mice presented here will provide information on the cooperative mutations that are required for the BRCA2-associated pathogenesis of cancer.
Animals ; BRCA2 Protein/deficiency/*genetics ; CD4-CD8 Ratio ; Cell Separation ; Flow Cytometry ; Integrases/*genetics/immunology ; Lymphoma/*genetics/immunology/metabolism/pathology ; Mice ; Mice, Knockout ; Organ Specificity ; *Sequence Deletion ; T-Lymphocytes/enzymology/*immunology ; Thymus Gland/immunology/metabolism/pathology ; Thymus Neoplasms/*genetics/immunology/metabolism/pathology ; Tumor Suppressor Protein p53/deficiency/genetics/immunology

OBJECTIVETo study the anti-tumor immunotherapeutic effect induced by the suicidalcancer vaccine FC/TK, and to evaluate the safety of this vaccine.

METHODSThe suicidal cancer vaccine, named FC/TK, was prepared by fusion of suicide gene (HSVI,-TK gene) -modified ovarian carcinoma NuTu-19 cells with rat bone marrow-derived dendritic cells (DCs). The morphology of FC/TK was evaluated by scanning electron microscopy. The stimulatory effect of FC/TK on T cells was determined by T cell proliferation assay. In immunotherapeutic studies in vivo, Fischer344 rats were injected subcutaneously with NuTu-19 cells, followed by treatment of FC/TK on days 7 and 14, compared to controls treated with irradiated FC/TK, FC or PBS, respectively. Tumor incidence and volume were measured in 90 days after challenge. To determine the killing effect of FC/TK in vivo, TUNEL assays were applied to detect apoptotic cell death in spleen of vaccinated rats with prodrug ganciclovir administration.

RESULTSFC/TK cells were of irregular shape with surface membrane processes. Compared to the control groups, FC/TK significantly promoted T cell proliferation (P <0.01). The rats vaccinated with FC/TK and FC significantly inhibited the tumor growth compared to rats vaccinated with irradiated FC/TK (P <0.05) or with PBS ( P <0.01). The immunotherapeutic effect induced by FC/TK was similar to that using FC. Fluorescence microscopy showed that fluorescein-stained FC/TK cells migrated into spleen also showed to be TUNEL-positive, suggesting that the FC/TK cells were killed by ganciclovir in vivo.

CONCLUSIONOur data indicate that suicidal cancer vaccine is an effective and safe therapy for ovarian carcinoma and may serve as a broadly applicable approach for other cancer vaccines in the future.

Animals ; Apoptosis ; drug effects ; Cancer Vaccines ; immunology ; Cell Fusion ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dendritic Cells ; cytology ; immunology ; Female ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Herpesvirus 1, Human ; enzymology ; genetics ; Immunotherapy ; methods ; Microscopy, Electron, Scanning ; Microscopy, Fluorescence ; Neoplasms, Experimental ; enzymology ; pathology ; therapy ; Ovarian Neoplasms ; enzymology ; pathology ; therapy ; Rats ; Rats, Inbred F344 ; Survival Analysis ; T-Lymphocytes ; drug effects ; metabolism ; pathology ; Thymidine Kinase ; genetics ; metabolism ; Transfection

OBJECTIVETo study the effect of dendritic cells (DC) stimulated with K562 cell lysate in inducing specific cytotoxic T lymphocytes (CTL) against K562 cells in vitro.

METHODSThe DCs were derived from healthy human peripheral blood monocytes in the presence of granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4 and tumor necrosis factor (TNF) alpha. The T cells were stimulated by DCs loaded with freeze-thawed K562 cells and T-cell cytotoxicities were measured by lactate dehydrogenase (LDH) assay.

RESULTSThe DCs could be successfully obtained from peripheral blood monocyte after the culture. Mixed lymphocyte reactions induced by the antigen-loaded DC were much stronger than those induced by human peripheral blood monocytes (P<0.05). At the effector to target ratio of 10:1 and 20:1, cytotoxicities against K562 cells by CTL derived from culture with the antigen-loaded DCs were the strongest (P<0.05).

CONCLUSIONCTL derived from DCs pulsed with K562 cell lysate show effective and specific cytotoxicity against K562 cells.

Antigens ; immunology ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; drug effects ; immunology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; K562 Cells ; L-Lactate Dehydrogenase ; metabolism ; Lymphocyte Activation ; immunology ; Lymphocyte Culture Test, Mixed ; T-Lymphocytes, Cytotoxic ; enzymology ; immunology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVEAcute idiopathic thrombocytopenic purpura (AITP) is a common autoimmune disease in children. Thrombocytes decrease extremely in serious patients, its pathogenesis involves abnormal activation of T lymphocytes and T cell-dependent production of autoantibody. The aim of the present study was to investigate changes of protein kinase C (PKC) activity in peripheral blood T lymphocytes in children with AITP and the relationships between PKC activity and T lymphocytes activation and thrombocytopenia.

METHODSPeripheral blood specimens were collected from children with acute ITP (n = 35) and healthy children (n = 30), and T lymphocytes were isolated and purified by using T cells Segregation Enrichment Column. PKC activity was detected by using PepTag Assay, a non-radioactive detection method. The reaction mixture, in a final volume of 25 microl, consisted of 5 microl reaction buffer, PepTag C1 5 microl (0.4 microg/microl), PKC activator solution (DG) 5 microl, peptide protection solution 1 microl and sample 9 microl. Phosphorylation reaction was allowed to continue for 30 minutes, then 25 microl reaction mixture was subjected to electrophoresis on a 0.8% agarose gel at 100 V for about 20 minutes. After electrophoresis, the PepTag C1 peptides which were phosphorylated and non-phosphorylated were separated, phosphorylated PepTag C1 peptide with negative electricity migrated toward the anode (+), but nonphosphorylated PepTag C1 peptide with positive electricity migrated toward cathode (-), the gel was photographed. Electrophoresis bands on anode represented PKC activity and were analyzed quantitatively. FasL, which is T cell activation marker, was determined by flow cytometer and platelet was counted by cell counting meter.

RESULTSCompared with healthy children, children with AITP had significantly higher PKC total activity [(0.97 +/- 0.21) nmol/(min.ml) vs. (0.55 +/- 0.13) nmol/(min.ml), (P < 0.05)]. Expression of FasL on T cell subpopulation in children with AITP was significantly higher [Th FasL: (32.7 +/- 3.4) vs. (14.7 +/- 4.2); Tc FasL: (17.3 +/- 9.7) vs. (11.6 +/- 8.5)%, (P < 0.05)]. Besides, relationships between the changes of PKC activity, Th FasL and Tc FasL had positive correlation (r(1) = 0.68, r(2) = 0.53, P < 0.05). However, PKC activity and platelet count had a significantly negative correlation (r = -0.75, P < 0.05).

CONCLUSIONIncreased PKC activity was seen in children with AITP, which can cause damage to thrombocytes and reduction of thrombocytes. PKC signal transduction pathway might play an important role in the immunopathogenesis of AITP.

Acute Disease ; Case-Control Studies ; Cell Separation ; Child ; Electrophoresis ; Flow Cytometry ; Humans ; Lymphocyte Activation ; Platelet Count ; Protein Kinase C ; metabolism ; Purpura, Thrombocytopenic, Idiopathic ; enzymology ; immunology ; T-Lymphocytes ; enzymology