Structural immunology and crystallography help immunologists see the immune system in action: how T and NK cells touch their ligands.

Host immune system is an important and sophisticated system, maintaining the balance of host response to "foreign" antigens and ignorance to the normal-self. To fulfill this achievement the system manipulates a cell-cell interaction through appropriate interactions between cell-surface receptors and cell-surface ligands, or cell-secreted soluble effector molecules to their ligands/receptors/counter-receptors on the cell surface, triggering further downstream signaling for response effects. T cells and NK cells are important components of the immune system for defending the infections and malignancies and maintaining the proper response against over-reaction to the host. Receptors on the surface of T cells and NK cells include a number of important protein molecules, for example, T cell receptor (TCR), co-receptor CD8 or CD4, co-stimulator CD28, CTLA4, KIR, CD94/NKG2, LILR (ILT/LIR/CD85), Ly49, and so forth. These receptor molecules interact with their ligands on the target cells, including major histocompatibility complex (MHC) (or human leukocyte antigen, HLA), CD80, CD86, and so forth. Detailed understanding of these receptor-ligand pair interactions is crucial for our full knowledge of the immune system, ultimately for us to manipulate the T cell and NK cell functions. Accumulations of the receptor-ligand complex crystal structures in the recent years have provided us a unique angel to see how the immune cells interacting with their partner cells. In this review, we discussed binding specificity, plasticity, and flexibility of the T cell and NK cell receptor/ligand interaction, fitting the structural data with their functions. Structural immunology indeed helps us see how T and NK cells "touch" their target cells in our immune system.

Modification and identification of a vector for making a large phage antibody library.

BACKGROUND: The large phage antibody library is used to obtain high-affinity human antibody, and the Loxp/cre site-specific recombination system is a potential method for constructing a large phage antibody library. In the present study, a phage antibody library vector pDF was reconstructed to construct diabody more quickly and conveniently without injury to homologous recombination and the expression function of the vector and thus to integrate construction of the large phage antibody library with the preparation of diabodies. METHODS: scFv was obtained by overlap polymerase chain reaction (PCR) amplification with the newly designed VL and VH extension primers. loxp511 was flanked by VL and VH and the endonuclease ACC III encoding sequences were introduced on both sides of loxp511. scFv was cloned into the vector pDF to obtain the vector pDscFv. The vector expression function was identified and the feasibility of diabody preparation was evaluated. A large phage antibody library was constructed in pDscFv. Several antigens were used to screen the antibody library and the quality of the antibody library was evaluated. RESULTS: The phage antibody library expression vector pDscFv was successfully constructed and confirmed to express functional scFv. The large phage antibody library constructed using this vector was of high diversity. Screening of the library on 6 antigens confirmed the generation of specific antibodies to these antigens. Two antibodies were subjected to enzymatic digestion and were prepared into diabody with functional expression. CONCLUSIONS: The reconstructed vector pDscFv retains its recombination capability and expression function and can be used to construct large phage antibody libraries. It can be used as a convenient and quick method for preparing diabodies after simple enzymatic digestion, which facilitates clinical trials and application of antibody therapy.

BPI700-Fc gamma1(700) chimeric gene expression and its protective effect in a mice model of the lethal E. coli infection.

BACKGROUND: Infections caused by gram-negative bacteria (GNB) often lead to high mortality in common clinical settings. The effect of traditional antibiotic therapy is hindered by drug-resistant bacteria and unneutralizable endotoxin. Few effective methods can protect high risk patients from bacterial infection. This study explored the protection of adeno-associated virus 2 (AAV2)-bacteriacidal permeability increasing protein 700 (BPI(700))-fragment crystallizable gamma one 700 (Fc gamma1(700)) chimeric gene transferred mice against the minimal lethal dose (MLD) of E. coli and application of gene therapy for bacterial infection. METHODS: After AAV2-BPI(700)-Fc gamma1(700) virus transfection, dot blotting and Western blotting were used to detect the target gene products in Chinese hamster ovary-K1 cells (CHO-K1cells). Reverse transcription-polymerase chain reaction and immunohistochemical assay were carried out to show the target gene expression in mice. Modified BPI-enzyme linked immunosorbent assay was used to identify the target gene products in murine serum. The protection of BPI(700)-Fc gamma1(700) gene transferred mice was examined by survival rate after MLD E. coli challenge. Colony forming unit (CFU) count, limulus amebocyte lysate kit and cytokine kit were used to quantify the bacteria, the level of endotoxin, and proinflammatory cytokine. RESULTS: BPI(1-199)-Fc gamma1 protein was identified in the CHO-K1 cell culture supernatant, injected muscles and serum of the gene transferred mice. After MLD E. coli challenge, the survival rate of AAV2-BPI(700)-Fc gamma1(700) gene transferred mice (36.7%) was significantly higher than that of AAV2-enhanced green fluorescent protein (AAV2-EGFP) gene transferred mice (3.3%) and PBS control mice (5.6%). The survival rate of AAV2-BPI(700)-Fc gamma1(700) gene transferred mice treated with cefuroxime sodium was 65.0%. The bacterium number in main viscera, the levels of endotoxin and proinflammatory cytokine (tumor necrosis factor-alpha and interleukin-1beta) in serum of the AAV2-BPI(700)-Fc gamma1(700) gene transferred mice were markedly lower than that of PBS control mice (P < 0.01). CONCLUSIONS: AAV2-BPI(700)-Fc gamma1(700) gene transferred mice can resist MLD E. coli infection through expressing BPI(1-199)-Fc gamma1 protein. Our findings suggested that AAV2 mediated BPI(700)-Fc gamma1(700) gene delivery could be used for protection and treatment of clinical GNB infection in high-risk individuals.

[Synthesis of dominant epitopes on N-terminal part of BPI and preparation of corresponding antisera].

AIM: To synthesize B cell dominant epitopes on N-terminal part of bactericidal/permeability increasing protein (BPI) and prepare the corresponding antisera. METHODS: The antigenicity, hydrophilicity, flexibility, surface probability and secondary structure of N-terminal amino acids 1-199 on BPI were predicted by bioinformatics applications. Two antigen peptides TA/IK were designed and synthesized on the basis of the above analysis. Then the TA/IK were respectively conjugated to keyhole limpet hemocyanin (KLH) and injected into rabbits to prepare corresponding antisera. Indirect ELISA was performed to analyze the antigenicity of TA/IK and to test the titer of the antisera. And Western blot was used to identify the specificity of the antisera. RESULTS: (1) Two B cell epitope-based peptides TA/IK were successfully synthesized; (2) the peptides could bind to commercial polyclonal antibody, anti-BPI(55); (3) titers of the antisera against TA/IK were up to 1:51,200, 1:25,600, respectively; (4) Western blot analysis revealed that these antisera could specifically react with the standard sample of BPI(55). CONCLUSION: The two synthetic antigen peptides TA/IK are indeed dominant epitopes of BPI N-terminal part, and the corresponding antisera are competent for detecting and identifying the N-terminal fragments of BPI.