BACKGROUND: We developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay. METHODS: We selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples. RESULTS: CV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein alpha-isothyocyanate (FITC)dim+CD4-FITCbright and with CD19-FITCdim+CD3-FITCbright showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-Thelper cells, and Thelper cells; r2=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITCdim+CD3-FITCbright and CD8-PEdim+CD4-PEbright, and MFC, in the 23 patient samples (B cells, T cells, Tcytotoxic cells, and Thelper cells; r2> or =0.98, 0.99, 0.99, and 0.99, respectively; P<0.05). CONCLUSIONS: The multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.
Antibodies, Monoclonal/chemistry/*immunology ; Antigens, CD19/chemistry/metabolism ; Antigens, CD3/chemistry/metabolism ; Antigens, CD4/chemistry/metabolism ; Antigens, CD8/chemistry/metabolism ; B-Lymphocyte Subsets/immunology/metabolism ; Color ; Flow Cytometry/*methods ; Fluorescein-5-isothiocyanate/*chemistry ; Humans ; T-Lymphocyte Subsets/immunology/metabolism

OBJECTIVETo define the mechanism of acute hepatitis in non-human primates after liver directed gene therapy.

METHODSDifferences in immune response exhibited by 8 rhesus monkeys receiving adenovirus (Ad) or lipofectamine-mediated gene transfer by various routes, the time course, and the nature of the specific immune responses to both adenoviral vectors and transgene products were studied using HE staining (H&E) and immunohistochemical staining.

RESULTSThe monkeys developed mild to moderate acute hepatitis 1 to 3 weeks after intravenous or intrabiliary injection of first generation replication-defective adenoviruses carrying the Escherichia coli lacZ gene. This was accompanied by adenovirus-mediated T-cell proliferation and neutralizing antibodies to the adenovirus. Increased numbers of CD3(+), CD4(+) and CD8(+) T-lymphocytes were detected in the diseased livers, while B-lymphocytes were absent. Hepatocytes demonstrated increased expression of beta 2-microglobulins (beta 2-MG) and HLA-DR antigens in the plasma membranes. The development of acute hepatitis and the accompanying immune abnormalities were delayed in immunosuppressed monkeys until after the discontinuation of immunosuppressive therapy. The monkeys infused with Ad. CMVluc showed more significant and longer durations of hepatitis than the monkeys infused with adenoviruses carrying the lacZ gene. Lipofectamine-mediated gene transfer was inefficient. There was neither lacZ expression nor significant immune response in the liver of monkeys infused with lipofectamine via the portal vein or the common bile duct.

CONCLUSIONImmune response to the hepatocytes in liver directed gene therapy is MHC class I restricted and T-cell mediated. Both adenoviral vectors and foreign genes are related to the liver damage. Mild to moderate hepatic inflammation seen with the E-1 deleted vector is reversible. Immunosuppression regimens may prolong transgene expression and delay the development of acute adenoviral hepatitis.

Acute Disease ; Adenoviridae ; genetics ; Adenoviridae Infections ; genetics ; Animals ; CD3 Complex ; analysis ; CD4 Antigens ; analysis ; CD8 Antigens ; analysis ; DNA, Recombinant ; administration & dosage ; genetics ; Gene Transfer Techniques ; HLA-DR Antigens ; analysis ; Hepatitis, Animal ; genetics ; virology ; Immunohistochemistry ; Liver ; chemistry ; metabolism ; pathology ; Macaca mulatta ; beta 2-Microglobulin ; analysis

The study investigated the effects of red clover extract (RCE) on mouse T macrophages and lymphocytes in vitro. The cell toxic effect of RCE was estimated by MTT assay. Multiple-fluorescence staining plus flow cytometry were used to detect the effect of RCE on CD69/CD25/CD71 expression of mouse T lymphocytes stimulated by Con A; CFDA-SE staining plus flow cytometry were used to analyze the effect of RCE on proliferation of T lymphocytes activated by Con A; The effect of RCE on nitric oxide (NO) secretion of mouse macrophages stimulated by lipopolysaccharide (LPS) for 24 h was assayed by Griess reagent system. We found that RCE had potent anti-inflammatory effects on mice. RCE had little cell toxic effect on mouse lymphocytes and macrophages. RCE strongly inhibited the excessive production of inflammatory mediators (NO, CD69, CD25, CD71), in a dose-dependent manner, like cyclosporine A injection. RCE could inhibit proliferation of CD3+ T lymphocytes. These data suggested that RCE might exhibit anti-inflammatory effect by inhibiting the activation and proliferation of mouse lymphocytes and the NO secretion of mouse macrophages.
Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; CD3 Complex ; analysis ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Lectins, C-Type ; Lymphocyte Activation ; drug effects ; Macrophages ; cytology ; secretion ; Male ; Mice ; Mice, Inbred BALB C ; Nitric Oxide ; secretion ; Plants, Medicinal ; chemistry ; Receptors, Transferrin ; metabolism ; T-Lymphocytes ; cytology ; drug effects ; metabolism ; Trifolium ; chemistry

OBJECTIVETo evaluate the therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis (EAE).

METHODSEAE was induced in the low-sensitive strain C57BL/6 mice with intraperitoneal injection of myelin basic protein (MBP) to simulate the human disease multiple sclerosis, followed by intramuscular injection of cationic liposome carrying the plasmid DNA SRalpha-mMIP-1alpha-DT390 in the leg muscle to elicit resistance to EAE development. The mice were then examined daily for clinical signs of EAE by an observer blind to the treatment protocol. For immunohistochemistry the mice were anesthetized and perfused with sterile PBS and paraformaldehyde, and the cerebrum, cerebellum, medulla and spinal cord were removed for preparation of serial sections. The mononuclear cells (MNCs) from the EAE mouse spleens were prepared for three-color flow cytometry analysis of the surface markers with appropriate antibodies following the BD Pharmingen cytokine staining protocol.

RESULTSEAE model was successfully established by active MBP immunization in C57BL/6 mice. Administration of the immunotoxin mMIP-1alpha-DT390 significantly delayed the disease onset and lowered the mean clinical score for EAE as compared with the control mice. Immunohistochemistry demonstrated much less CCR5(+) infiltrating cells in the central nervous system in mMIP-1alpha-DT390-treated mice than in the control. The treatment also eliminated reactive T cells in the periphery blood without affecting the number of B cells.

CONCLUSIONThe immunotoxin mMIP-1alpha-DT390 can attenuate the disease activity of EAE in mice, suggesting its potential use in the treatment of other autoimmune disorders.

Animals ; Antigens, CD19 ; analysis ; B-Lymphocytes ; cytology ; metabolism ; CD3 Complex ; analysis ; Chemokine CCL3 ; genetics ; metabolism ; Diphtheria Toxin ; genetics ; metabolism ; Disease Models, Animal ; Encephalomyelitis, Autoimmune, Experimental ; drug therapy ; Female ; Flow Cytometry ; Immunoglobulin Fragments ; genetics ; metabolism ; Immunohistochemistry ; Immunologic Factors ; therapeutic use ; Immunotoxins ; therapeutic use ; Meninges ; chemistry ; pathology ; Mice ; Mice, Inbred C57BL ; Multiple Sclerosis ; drug therapy ; NIH 3T3 Cells ; Receptors, CCR5 ; analysis ; Recombinant Fusion Proteins ; genetics ; metabolism ; therapeutic use ; T-Lymphocytes ; cytology ; metabolism

The detrimental effects of environmental pollutants on the health of the individual are generally accepted, although the mechanisms of these effects remain to be incompletely understood. In the present study, we examined the effects of B[a]P, 2-BP, phenol and TCDD on proinflammatory cytokine gene expression in mice spleen cells which were stimulated with anti-CD3. 10-9M TCDD increased IFN gammar and TNF alpha gene expression, but suppressed IL-1 gene expression. 10-6M phenol inhibited IL-1, IL-6 and TNF alpha gene expression, and 10-6M of 2-BP downregulated TNF alpha gene expression. However, 10-6M of B[a]P did not influence on IL-1, IL-6, IFN gammar and TNF alpha gene expression. These findings suggest that TCDD may impair the immune functions of mice by enhancing proinflammatory cytokines production, whereas phenol and 2-BP may impair the functions by inhibiting the production of these cytokines.
Animals ; Antigens, CD3/immunology ; Apoptosis/drug effects ; Benzo(a)pyrene/*toxicity ; Cells, Cultured ; Cytokines/*biosynthesis/genetics ; Environmental Pollutants/*toxicity ; Gene Expression/drug effects ; Hydrocarbons, Brominated/*toxicity ; Male ; Mice ; Mice, Inbred C3H ; Phenol/*toxicity ; RNA/chemistry/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Spleen/*drug effects/metabolism ; Tetrachlorodibenzodioxin/*toxicity