PubMed Health⌕ Search

Biomedical subjects

Delin Zhu

Publications and source records attributed to Delin Zhu.

10 recordsLinked to original sources
PubMed2006

Deglycosylation to obtain stable and homogeneous Pichia pastoris-expressed N-A1 domains of carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is a seven domain membrane glycoprotein widely used as a tumour marker for adenocarcinomas and as a target for antibody-directed therapies. Structural models have proposed that the first two domains of CEA (the N terminal and adjoining A1 domains) bind MFE-23, a single chain Fv antibody in experimental clinical use. We aimed to produce recombinant N-A1 to test this hypothesis. The N-A1 domains were expressed as soluble protein with a C-terminal hexahistidine tag (His6-tag) in the yeast Pichia pastoris. His6-tagged N-A1 was captured from the supernatant by batch purification with copper-loaded Streamline Chelating, an immobilised metal affinity chromatography (IMAC) matrix usually utilised in expanded bed techniques. Purified N-A1 was heterogeneous with a molecular weight range from 38 to 188 kDa. Deglycosylation with endoglycosidase H (Endo H) resulted in three discrete molecular weight forms of N-A1, one partially mannosylated, one fully Endo H-digested and one fully Endo H-digested but lacking the His6-tag. These were separated by concanavalin A chromatography followed by HiTrap IMAC. The procedure resulted in single-band-purity, mannose-free N-A1. The binding interaction of MFE-23 to N-A1 was analysed by surface plasmon resonance. The affinity constants retrieved were KD = 4.49 x 10(-9)M for the P. pastoris expressed, native N-A1, and 5.33 x 10(-9) M for the Endo H-treated N-A1. To our knowledge this is the first time that two consecutive domains of CEA have been stably expressed and purified from P. pastoris. This work confirms that the CEA epitope recognised by MFE-23 resides in N-A1.

Adenocarcinoma↗
PubMed2004

Deregulated expression of the Myc cellular oncogene drives development of mouse "Burkitt-like" lymphomas from naive B cells.

Chromosomal translocations juxtaposing immunoglobulin (Ig) and MYC genes are the hallmarks of human Burkitt lymphoma (BL), with deregulated MYC expression being a critical factor in pathogenesis. By inserting an intact mouse Myc gene into the mouse genome, proximal to the Ig enhancer Emu, the effect of a precise mimic of the major t(8;14) translocation of human endemic BL (eBL) could be investigated. Knock-in mice developed IgM-positive B-cell tumors, with most being typical of eBL by histology and immunophenotype, including expression of the germinal center (GC)-associated protein, BCL6. Unlike eBL, however, analysis of Ig V(H) sequences revealed no significant level of somatic mutation. Thus, constitutive expression of Myc in the knock-in mice is apparently able to induce "Burkitt-like" lymphomas before antigen stimulation and formation of a GC. In contrast, human eBL development occurs in a GC or post-GC site with a likely contribution to pathogenesis from Epstein-Barr virus (EBV) and other epigenetic factors.

Animals↗
PubMed2004

DNA vaccines to attack cancer.

Delivery of antigens by injection of the encoding DNA allows access to multiple antigen-presenting pathways. Knowledge of immunological processes can therefore be used to modify construct design to induce selected effector functions. Expression can be directed to specific intracellular sites, and additional genes can be fused or codelivered to amplify responses. Therapeutic vaccination against cancer adds a requirement to overcome tolerance and to activate a weakened immune repertoire. Induction of CD4(+) T helper cells is critical for both antibody and T cell effector responses. To activate immunity against tumor antigens, we fused the tumor-derived sequences to genes encoding microbial proteins. This strategy engages T helper cells from the large antimicrobial repertoire for linked help for inducing antibody against cell-surface tumor antigens. The principle of linked T cell help also holds for induction of epitope-specific antitumor CD8(+) T cells, but the microbial sequence has to be minimized to avoid competition with tumor antigens. Epitope-specific DNA vaccination leads to powerful antitumor attack and can activate immunity from a profoundly tolerized repertoire. Vaccine designs validated in preclinical models are now in clinical trial with immune responses detected against both tumor antigens and fused microbial antigens. DNA priming is highly efficient, but boosting may benefit from increased antigen expression. Physical methods including electroporation provide increased expression without introducing additional competing antigens. A wide range of cancers can be targeted, and objective assays of response will determine efficacy.

Animals↗
PubMed2004

Tumor vaccines.

Explore the source record for details and available documents.

Animals↗
PubMed2004

DNA fusion gene vaccines against cancer: from the laboratory to the clinic.

Vaccination against target antigens expressed by cancer cells has now become a realistic goal. DNA vaccines provide a direct link between identification of genetic markers in tumors and vaccine formulation. Simplicity of manufacture facilitates construction of vaccines against disease subsets or even for individual patients. To engage an immune system that exists to fight pathogens, we have developed fusion gene vaccines encoding tumor antigens fused to pathogen-derived sequences. This strategy activates high levels of T-cell help, the key to induction and maintenance of effective immunity. We have dissected the immunogenic tetanus toxin to obtain specific sequences able to activate antibody, CD4+, or CD8+ T cells to attack selected fused tumor antigens. Principles established in preclinical models are now being tested in patients. So far, objective immune responses against idiotypic antigen of neoplastic B cells have been observed in patients with B-cell malignancies and in normal transplant donors. These responses provide a platform for testing physical methods to improve DNA delivery and strategies to boost responses. For cancer, demands are high, because vaccines have to activate powerful immunity against weak antigens, often in a setting of immune damage or tolerance. Vaccination strategies against cancer and against microbes are sharing knowledge and technology for mutual benefit.

Antigens, Neoplasm↗
PubMed2003

Incidence of potential glycosylation sites in immunoglobulin variable regions distinguishes between subsets of Burkitt's lymphoma and mucosa-associated lymphoid tissue lymphoma.

Recently, a high incidence of novel N-glycosylation sites introduced by somatic mutation was observed in the immunoglobulin variable region genes of follicular lymphoma. As these are positively selected and are uncommon in normal B cells, they may have a role in tumour growth and behaviour. Sites are not characteristic of chronic lymphocytic leukaemia or myeloma, but are detectable in approximately 50% of diffuse large cell lymphomas. Another feature of the variable region genes of certain lymphomas is ongoing somatic mutation. To determine whether glycosylation is associated with this phenomenon, we analysed variable region gene sequences of Burkitt's lymphoma (BL) and mucosa-associated lymphoid tissue (MALT) lymphoma. Novel sites were common in endemic BL (82%) and in 4/5 patients with Iranian BL. However, sporadic BL had a lower incidence (43%). Patients with MALT lymphoma had a low frequency (9%) of novel sites, comparable to normal B cells. These findings distinguish glycosylation sites from ongoing mutation and may reflect different environmental influences on these tumours.

Binding Sites↗
PubMed2002

V(H) gene analysis of splenic marginal zone lymphomas reveals diversity in mutational status and initiation of somatic mutation in vivo.

Tumors of the splenic marginal zone can present in spleen or blood. The maturational status of the neoplastic B cells from each site appears heterogeneous, with either unmutated or mutated variable-region heavy chain (V(H)) genes. To determine an influence of tissue location, we assessed matched blood and splenic tumor cells from 4 patients and found them identical. However, one patient with unmutated V(H) genes in blood and spleen developed a clonally related diffuse large B-cell lymphoma in the chest wall. Strikingly, this subclone had undergone significant somatic mutation, with clear intraclonal heterogeneity. To our knowledge, this is the first case of a B-cell tumor showing initiation of somatic mutation in vivo. The finding emphasizes that the tissue microenvironment can influence tumor cell behavior and possibly affect disease progression. Importantly, because several replacement mutations were located within or close to the complementarity-determining regions (CDRs), it raises the question of a role for antigen in driving tumor growth.

Amino Acid Sequence↗
PubMed2002

Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma.

Most patients with follicular lymphoma (FL) have somatically mutated V genes with intraclonal variation, consistent with location in the germinal center site. Using our own and published sequences, we have investigated the frequency of potential N-glycosylation sites introduced into functional V(H) genes as a consequence of somatic mutation. FL cells were compared with normal memory B cells or plasma cells matched for similar levels of mutation. Strikingly, novel sites were detected in 55 of 70 (79%) patients with FL, compared to 7 of 75 (9%) in the normal B-cell population (P <.001). Diffuse large B-cell lymphoma (DLCL) showed an intermediate frequency (13 of 32 [41%] patients). Myeloma and the mutated subset of chronic lymphocytic leukemia showed frequencies similar to those of normal cells in 5 of 64 (8%) patients and 5 of 40 (13%) patients, respectively. In 3 of 3 random patients with FL, immunoglobulin was expressed as recombinant single-chain Fv in Pichia pastoris, and glycosylation was demonstrated. These findings indicate that N-glycosylation of the variable region may be common in FL and in a subset of DLCL. Most novel sites are located in the complementarity-determining regions. V(H) sequences of nonfunctional V(H) genes contained few sites, arguing for positive selection in FL. One possibility is that the added carbohydrate in the variable region contributes to interaction with elements in the germinal center environment. This common feature of FL may be critical for tumor behavior.

Amino Acid Sequence↗
PubMed2002

DNA gene fusion vaccines against cancer.

The ability of DNA vaccination to induce effective immune responses has been shown in a range of preclinical disease models. However, the potency of DNA vaccines must be further improved for their use in patients. DNA fusion vaccine strategies, whereby target antigens are genetically linked to immuno-enhancing molecules, are currently being explored. The ease of DNA manipulation has allowed incorporation of a wide variety of molecules able to promote antigen uptake, processing and presentation by professional antigen-presenting cells, to provide critical CD4 T-cell help and to activate more effective immune effector pathways. These strategies are particularly important for cancer vaccines to increase their immunogenicity and to overcome tolerance.

Animals↗