Biomedical subjects

Yanzheng Zhang

Publications and source records attributed to Yanzheng Zhang.

5 recordsLinked to original sources

PubMed2006

MICA antigens stimulate T cell proliferation and cell-mediated cytotoxicity.

In previous experiments we have found that transplant recipients had specific antibodies against MICA. In the present study, we measured T cell proliferation, cytokine production, and cytotoxicity to investigate whether immunization with MICA can produce a specific cellular immune response. BALB/c mice were immunized with recombinant MICA (rMICA). Lymphoid cell suspensions obtained after immunization were used to measure T cell proliferation. We detected a robust proliferative response in MICA-stimulated cultures as determined by [3H]thymidine uptake. Using carboxyfluorescein diacetate succinimidyl ester (CFSE) to measure proliferation, we found that in MICA-stimulated cultures, 21% of the CD3+ T cells were CD4+ and CFSE-low and 3% of the T cells were proliferating CD8+ T cells. Among CFSE-low CD4+ spleen cells, 25% secreted IL-4 and only 2% produced IFN-gamma, suggesting a predominant Th2-type response. Blocking of MHC class I or class II molecules with monoclonal antibodies resulted in prominent inhibition of CD8+ or CD4+ T cell proliferation, respectively. In addition, we found that blocking the NKG2D receptor did not cause inhibition of the T cell response. MICA-stimulated CD8+ T lymphocytes exerted cytotoxicity against a BALB/c monocyte cell line (RAW 267.4) primed with soluble rMICA. Our results suggested that MICA-activated T cells may have a role in a cellular component of transplant rejection.

Animals↗

PubMed2003

Multiplex single nucleotide extension: a robust and high throughput method for HLA-A locus typing.

This report describes a typing method that can identify all known human leukocyte antigen A (HLA-A) alleles by determining nucleotides present at polymorphic sites using single nucleotide extension. Allele specific primers are bound to capture oligonucleotides which allows for a multiplex approach during single nucleotide extension (SNE) reactions. Eleven group-specific polymerase chain reaction amplifications were performed to obtain the templates to be analyzed with sets of primers designed to investigate the polymorphisms. Extension of biotin-labeled ddNTPs onto allele-specific primers was catalyzed by a DNA polymerase and each primer was hybridized to a specific capture oligonucleotide covalently bound to a bead. After staining with streptavidin-PE, incorporated fluorescence was determined with a flow cytometer. Fluorescence intensities were interpreted by computer and the nucleotide sequence was translated into HLA-A genotypes. Group-specific amplification reactions and primer sets for SNE were validated with 42 reference samples of known HLA-A alleles. In addition, 296 samples from three populations (N. A. Caucasian, African-American, Terena S. A. Indian) were analyzed and results compared to previous typing by SSOP. Reproducibility between repeated typings was 100% and ambiguities were quite rare. The method has been found to be accurate, relatively simple to perform and fast. It is our method of choice for high resolution clinical HLA-A typing.

Alleles↗

PubMed2003

Study of MICA alleles in 201 African Americans by multiplexed single nucleotide extension (MSNE) typing.

We have developed a method for major histocompatibility complex class I chain-related gene A (MICA) genotyping using multiplexed single nucleotide extension (MSNE) and flow cytometric analysis of an array of fluorescent microspheres. This technique employs a polymerase chain reaction-derived target DNA containing all the polymorphic sites of MICA, synthetic complementary primers, biotinylated dideoxynucleotide triphosphate, fluorescent reporter molecules (streptavidin-phycoerythrin), and thermophilic DNA polymerase. Genomic DNA was amplified by MICA locus-specific primers and the MSNE reactions were carried out in the presence of 30 MSNE primers used to assay polymorphisms in exons 2, 3, and 4 of the MICA genes. Thirty-two previously typed cell lines were used as reference material. The MICA gene frequencies among 201 African-American unrelated donors were determined. Of 51 previously known alleles, 18 were observed in African-Americans, compared to 16 that were found in North American Caucasians and 9 in South American Indians, suggesting a more diversified allelic distribution in African-Americans. MICA*00201 and MICA*00801 were the two most frequent alleles in African-Americans. We observed a high degree of linkage disequilibrium between certain alleles of MICA and of human leukocyte antigen-B in the African-American population. The methodology described here offers a powerful new approach to DNA typing of the MICA alleles.

Black or African American↗

PubMed2002

MICA polymorphism in South American Indians.

We have studied the MICA alleles of 196 unrelated subjects from three South American Indian tribes (Toba, Wichi and Terena). They are members of isolated tribes located in the Gran Chaco area in northeastern Argentina and in Mato Grosso do Sul in South Central Brazil. Of 55 previously known alleles, nine were observed in South American Indians, compared with 16 that were found in North American Caucasians, suggesting a more restricted allelic distribution of MICA in these tribes. In South American Indians, MICA*00201 was the most frequent allele, with a gene frequency of 33% in Toba, 47% in Wichi and 44% in Terena. MICA*00201, MICA*027 (external domain sequence like MICA*008/TM allele A5) and MICA*010 accounted for more than 90% of all the MICA genes in South American Indians. In North American Caucasians, MICA*00801 (*008/A5.1) accounted for 42% of the genes and was the most common allele. We observed a high degree of linkage disequilibrium between certain alleles of MICA and of HLA-B in the South American Indian populations. Phylogenetic trees constructed using gene frequencies of the transmembrane short tandem repeats in the populations reported here, and in other populations taken from published reports, suggest that South American Indians are more closely related to Asians than to Europeans.

Adult↗

PubMed2002

MICA is a target for complement-dependent cytotoxicity with mouse monoclonal antibodies and human alloantibodies.

The highly polymorphic major histocompatibility class I related chain A (MICA) gene encodes glycoproteins that have been shown to be expressed in epithelial cells, endothelial cells, keratinocytes, monocytes, and tumor cells. In previous experiments, we have studied MICA antigens using rabbit sera obtained by immunization with MICA peptides. We also found that several transplant recipients had specific antibodies against MICA in an ELISA assay with recombinant of MICA (r-MICA). In the present work we produced monoclonal antibodies by immunization of mice with recombinant MICA*008. Based on the different patterns of reactivity observed in ELISA, Western blot, and flow cytometry, mAbs 1.9C2, 2.4F5, 1.7AD, and 2.3D4 only reacted with denatured MICA and mAb 1.7A8 and 3.2H3 reacted also with native MICA as illustrated by flow cytometry with live cells. These monoclonal antibodies were postulated to bind to different sites of the MICA molecule. In order to investigate whether MICA expressed on the cell surface is able to mediate cell killing, antibody absorption, flow cytometry and complement-dependent cytotoxicity (CDC) were performed. We found that mouse monoclonal antibody 3.2H3 was able to kill 70% of HeLa cells. Absorption of a patient serum with pooled human platelets to remove antibodies against class I HLA resulted in a small shift of fluorescence and reduced killing from 100% to 70-75%. Absorption with the platelets and r-MICA produced a remarkable reduction in fluorescence staining and virtually reduced complement-dependent killing to the level of the negative controls. The results suggested that MICA alloantigens may be more immunogenic than could have been previously suspected.

Animals↗