AIMTo design and evaluate the small peptide mimetic of anti-P-glycoprotein (P-gp) antibody (PHMA02).

METHODSFrom the three dementional structure analysis of computer modeling of PHMA02 CDR loops, a small peptide mimetic was designed and determined by flow cytometry.

RESULTSAnti-P-gp peptide mimetic functionally similar to PHMA02 was developed. The peptide mimetic competitively inhibits PHMA02 binding to P-gp and partially block the P-gp function as a drug efflux pump in K562/A02 cells.

CONCLUSIONSome special conformational properties of CDR loops of antibody might serve as lead structures for develop new biological peptide mimetics. Antibody-structure-based design would develop new drug in the future.

ATP-Binding Cassette, Sub-Family B, Member 1 ; chemistry ; immunology ; Antibodies, Monoclonal ; chemistry ; Binding, Competitive ; Complementarity Determining Regions ; chemistry ; Drug Design ; Drug Resistance, Multiple ; Humans ; K562 Cells ; Molecular Mimicry ; Peptides ; chemical synthesis ; chemistry ; metabolism ; Protein Conformation

OBJECTIVETo study the expression of TCR BV CDR3 family in fulminant hepatitis B (FHB) patients.

METHODSTotally 28 patients with fulminant hepatitis B (FHB) (FHB group), who were treated in our hospital from Oct. 2010. to Mar. 2012, and 20 healthy controls( HC group) were included in the study. PBMCs were isolated from anticogulated blood, and RT-PCR was used to detect the levels of TCR BV CDR3 family in the 2 groups.

RESULTSThe levels of DeltaCt1, DeltaCt12 and DeltaCt20 in FHB group were higher than those in HC group (P < 0.05); The levels of DeltaCt5, DeltaCt7, DeltaCt13, DeltaCt14, DeltaCt15, DeltaCt22, DeltaCt23 in FHB group were lower than those in HC group (P < 0.05).

CONCLUSIONSThe result indicates that cellular immunology is involved in the pathogenesis of the liver inflammation process of FHB.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Complementarity Determining Regions ; chemistry ; genetics ; immunology ; Female ; Hepatitis B ; genetics ; immunology ; virology ; Humans ; Male ; Middle Aged ; Receptors, Antigen, T-Cell ; chemistry ; genetics ; immunology ; Young Adult

The gene fingerprinting of T cell receptor beta varial region (TCRbetaV) was used to analyze the T cell clone variations in peripheral blood lymphocytes from a patient with chronic eosinophilic leukemia (CEL) and to find out the characters of CDR3 amino acid sequence related to CEL. RT-PCR was used to amplify betaV1-24 family genes and the gene fingerprinting was set up by denatured polyacrylamide sequence gel electrophoresis. The expanded band was cut and then sequenced. The results showed that one clonal expansion of T cells could be found in TCRbetaV13.2 family. Its nucleotide sequence indicated a monoclonality. The CDR3 amino acid sequence was SFSYEQY and the modif SFSY was special reserved sequence. The other TCRbetaV families remained unchanged. It is concluded that the monoclonal T cells in TCRbetaV13.2 may be related to CEL and it is conjectured that the monoclonal T cells proliferation is induced by CEL malignant cells.
Aged ; Amino Acid Sequence ; Chronic Disease ; Complementarity Determining Regions ; chemistry ; Genes, T-Cell Receptor beta ; Humans ; Hypereosinophilic Syndrome ; genetics ; immunology ; Lymphocyte Activation ; Male ; Molecular Sequence Data ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes ; immunology

OBJECTIVEThe complementarity determining region 3 (CDR3) of T cell receptor (TCR) is the place through which T cells connect to the antigen. The lengths and DNA sequences of CDR3s are different according to different T cell clones. This leads to a diverse TCR CDR3 repertoires which can reflects the functional status of T cells precisely. This study aimed at elucidating the abnormality of TCR beta chain variable region (BV) CDR3 repertoires of children with acute B lymphoblastic leukemia, the pathogenesis of leukemia associated with T cell dysfunction and the immuno-reconstruction of T cells after the chemotherapy.

METHODSTwelve children aged from 3 to 13 years (average 4.50 +/- 3.78 years) with acute B lymphoblastic leukemia before chemotherapy and 8 healthy control donors aged from 6 to 16 years (average 10.30 +/- 3.00 years) were enrolled. Four of 12 patients were studied for the second time 3 months after complete remission (CR). Reverse transcription-polymerase chain reaction (RT-PCR) and polyacrylamide sequencing gel electrophoresis were used to detect the diversity of TCR BV CDR3 repertoires of these children.

RESULTS(1) The expression of BV2 and BV3 in 12 children increased and BV17 and BV18 decreased before the chemotherapy as compared with controls (P < 0.05). There were 4 children with a lower level expression of BV21 before the chemotherapy, and much lower level expression was found after the remission. (2) Normally each lane contained eight to ten bands, which represented unique CDR3 sizes for a given TCR BV family. Bands differed in size by 3 nucleotides and generally form a Gaussian distribution. There were 14% TCR BV families with abnormal CDR3 length distribution in 12 patients before therapy, which was significantly higher than that of controls (5.5%, P < 0.05). Restricted CDR3 length distribution was observed in BV14, BV1, BV16, BV20, BV13.1, BV13.2 and BV6. Every abnormal BV family recovered to normal Gaussian distribution 3 months after CR.

CONCLUSIONT lymphocytes in children with acute B lymphoblastic leukemia revealed markedly skewed repertoires, which suggests the abnormality of T cell functions. Most abnormal T cell repertoires recovered to normal Gaussian distribution 3 months after the first CR, which suggests the immunological reconstitution of T cell repertoires.

Adolescent ; Burkitt Lymphoma ; genetics ; immunology ; Child ; Child, Preschool ; Complementarity Determining Regions ; genetics ; DNA, Complementary ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Female ; Humans ; Male ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.
Animals ; Antibody Diversity ; genetics ; Base Sequence ; Complementarity Determining Regions ; chemistry ; genetics ; DNA ; genetics ; DNA, Complementary ; genetics ; Drug Discovery ; methods ; Gene Library ; High-Throughput Nucleotide Sequencing ; methods ; Humans ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; methods

BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy. METHODS: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1x10(10) size, human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing. RESULTS: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1microgram of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15x10(-8) M to 1.75x10(-6) M. CONCLUSIONS: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.
Antibodies, Monoclonal/biosynthesis/genetics/*pharmacology ; Complementarity Determining Regions/chemistry ; Gene Products, pol/*antagonists & inhibitors/genetics/immunology ; Genetic Vectors ; Hepatitis B virus/enzymology/genetics ; Humans ; Peptide Library ; RNA-Directed DNA Polymerase/genetics/*immunology ; Recombinant Fusion Proteins/biosynthesis/genetics ; Reverse Transcriptase Inhibitors/chemistry/metabolism/*pharmacology

The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ~2% of the productive human TCRβ CDR3 sequences.
Complementarity Determining Regions ; genetics ; DNA Primers ; chemistry ; genetics ; Female ; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor ; genetics ; Gene Rearrangement, delta-Chain T-Cell Antigen Receptor ; genetics ; Genes, T-Cell Receptor beta ; genetics ; Genetic Variation ; High-Throughput Nucleotide Sequencing ; Humans ; Immunoglobulin Joining Region ; genetics ; Immunoglobulin Variable Region ; genetics ; Male ; Receptors, Antigen, T-Cell, alpha-beta ; genetics