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

To examine the self-association of CD4 molecules and preliminary studies on its biological function by several indirect methods. A series of CD4 chimeras were generated including truncated CD4 lacking the short cytoplasmic tail, deleted mutantsD1/D2 devoid of D3 and D4 and D3/D4 devoid of D1 and D2 by PCR techniques, as well as another three CD4 chimeric genes by fused human Fas cytoplasmic death domain to the downstream of the above chimeras respectively. All these molecules were subcloned into pEGFP-N1, forming the corresponding expression vectors. After introducing into HEK293 cells, gene-modified cell morphological changes and target protein subcellular localization were observed and analyzed by a confocal microscopy. Moreover, stable 293/CD4 clones were obtained by transfecting the truncated CD4 recombinant plasmid into the HEK293 cell line and selected by G418. The fluorescene intensity and rosette formation of different clones was each analyzed by a confocal microscopy and cell adhesive assays. It's seen that CD4-Fas fusion gene could induce approximately 80% cell apoptosis of transfected HEK293 cells, compared to FKBP12-Fas is about 30% and CD4 gene only is 7%. Furthermore, both D1/D2-Fas and D3/D4 Fas chimeras could trigger nearly all transfected HEK293 cells to death. Cell adhesion assays showed that neither the D1/D2 nor D3/D4 chimeras when expression in HEK293 cells binds to MHC class II + Raji B cells. Interestedly, there were two type stable clones among 293/CD4. Fluorescence intensity analysis displayed that one' mean fluorescence intensity value is about twice of the other while cell-cell binding examination showed that the former is capable of forming rosette with Raji cells but the latter. All these results suggest that CD4 molecules most likely could exist as a dimer or even an oligomer on transfected HEK293 cell surface, which constitute a functional form for stable binding to MHC class II molecules.
Antigen-Presenting Cells ; immunology ; metabolism ; CD4 Antigens ; chemistry ; genetics ; metabolism ; CD4-Positive T-Lymphocytes ; immunology ; metabolism ; Cell Line ; Dimerization ; Fas Ligand Protein ; metabolism ; Histocompatibility Antigens Class II ; genetics ; immunology ; metabolism ; Humans ; Mutagenesis, Site-Directed ; Protein Binding ; genetics ; Protein Multimerization

Analysis of the T-cell receptor (TCR) repertoire of innate CD4+ T cells selected by major histocompatibility complex (MHC) class II-dependent thymocyte-thymocyte (T-T) interaction (T-T CD4+ T cells) is essential for predicting the characteristics of the antigens that bind to these T cells and for distinguishing T-T CD4+ T cells from other types of innate T cells. Using the TCRmini Tg mouse model, we show that the repertoire of TCRalpha chains in T-T CD4+ T cells was extremely diverse, in contrast to the repertoires previously described for other types of innate T cells. The TCRalpha chain sequences significantly overlapped between T-T CD4+ T cells and conventional CD4+ T cells in the thymus and spleen. However, the diversity of the TCRalpha repertoire of T-T CD4+ T cells seemed to be restricted compared with that of conventional CD4+ T cells. Interestingly, the frequency of the parental OT-II TCRalpha chains was significantly reduced in the process of T-T interaction. This diverse and shifted repertoire in T-T CD4+ T cells has biological relevance in terms of defense against diverse pathogens and a possible regulatory role during peripheral T-T interaction.
Amino Acid Sequence ; Animals ; Antigens, Surface/metabolism ; CD4-Positive T-Lymphocytes/cytology/*immunology/*metabolism ; Cell Communication ; Cell Differentiation/genetics/immunology ; Clonal Evolution ; Histocompatibility Antigens Class II/*immunology ; *Immunity, Innate ; Immunophenotyping ; Lymphocyte Count ; Mice ; Mice, Knockout ; Mice, Transgenic ; Peptide Fragments/chemistry ; Phenotype ; Receptors, Antigen, T-Cell/chemistry/*genetics/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/chemistry/genetics ; Spleen/cytology ; Thymocytes/cytology/immunology/metabolism

Complete HIV-1 env genes were amplified by nested PCR from uncultured peripheral blood mononuclear cells (PBMCs) DNA of 60 HIV-1 positive paid blood donors in Henan province, and the amplified full-length genes were sequenced. Twenty one full-length env genes were obtained, sequence analysis found that 15 of them had intact open reading frame (ORF). Fourteen sequences conformed to subtype B', their average genetic distance with the international reference sequence RL42 was 4.87% +/- 0.31%. One was subtype B, its genetic distance with the international reference sequence HXB2 was 5.43%. The amino acid sequences of these env genes were deduced according to their nucleotide sequences and extensive analysis and comparison of important structural motifs were performed. The results indicated that there was no drastic alteration in the number and position of potential N-linked glycosylation sites among these 15 sequences. And the residues involved in forming the CD4 binding site were highly conserved. Genotype prediction of coreceptor usage based on V3 sequence and net charge suggested that most samples use CCR5 coreceptor. GPGR motif at the tetrapeptide crown in the V3 loop was most common in these samples and it was detected in 40% sequences. The cleavage site of gp120/gp41 was highly conserved, so Gp160 precursor of all isolates would be efficiently cleaved into the Gp120 and Gp41 subunits. The known neutralizing antibody binding sites for 2G12, IgG1b12, 4E10 and 2F5 were also highly conserved, it is expected that most of these isolates will be sensitive to neutralization by these antibodies. Further study to elucidate the correlation of the env genotype to functionally relevant motifs is necessary and that will aid vaccine and novel drug design.
Base Sequence ; Blood Donors ; supply & distribution ; CD4 Antigens ; metabolism ; China ; Clinical Laboratory Techniques ; Conserved Sequence ; HIV Envelope Protein gp120 ; genetics ; HIV-1 ; genetics ; Humans ; Receptors, CCR5 ; chemistry ; genetics ; env Gene Products, Human Immunodeficiency Virus ; chemistry ; genetics

OBJECTIVETo explore the mechanisms of Chinese herbal medicine Sanqi Oral Liquid, composed of Astragalus membranaceus and Panpax notoginseng, in alleviating renal injury by observing its effect on the expressions of CD4(+), CD8(+) and CD68(+) cells in 5/6 nephrectomized rats with chronic renal failure.

METHODSA total of 102 SD rats were randomly divided into six groups: three treatment groups were administrated with high, medium and low dosage of Sanqi Oral Liquid respectively by gavage; a normal group, a 5/6 nephrectomized model group, and a group treated with coated aldehyde oxygenstarch were used as controls. Following oral administration of Sanqi Oral Liquid for 12 weeks, the general condition and renal pathological changes were observed, and the renal function, platelet count (PLT) and the expressions of CD4(+), CD8(+) and CD68(+) cells were determined for each group.

RESULTSThere were proliferation of mesangial matrix, renaltubularnecrosis and obvious tubulointerstitial fibrosis in the model group, and they were much milder in the treatment groups. Compared with the model group, the amounts of blood urea nitrogen (BUN), serum creatinine (Scr) and PLT in the treatment groups decreased (P<0.05 for all); and in the group administrated of medium dosage of Sanqi Oral Liquid, the expression of CD4(+) cells was up-regulated and those of CD8(+) and CD68(+) cells were down-regulated (P<0.05 for all), leading to an increased ratio of CD4(+)/CD8(+)(P<0.01).

CONCLUSIONSanqi Oral Liquid has a significant effect on regulating lymphocyte subsets, reducing the infiltration of macrophages in renal tissues and alleviating tubulointerstitial fibrosis, and this may be one of mechanisms of Sanqi Oral Liquid in delaying the progression of chronic kidney diseases.

Administration, Oral ; Animals ; Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Astragalus membranaceus ; chemistry ; CD4-Positive T-Lymphocytes ; drug effects ; pathology ; physiology ; CD8-Positive T-Lymphocytes ; drug effects ; pathology ; physiology ; Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Kidney Failure, Chronic ; drug therapy ; immunology ; pathology ; surgery ; Lymphocyte Count ; Male ; Nephrectomy ; Panax notoginseng ; chemistry ; Rats ; Rats, Sprague-Dawley ; Solutions

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