OBJECTIVE: To gain insights into structural characteristics of immunoglobulin kappa chain repertoire expressed in the inflammed synovium of rheumatoid arthritis (RA), we analyzed V kappa transcirpts expressed in the synovium of a patient with longstanding RA and compared to those expressed in the PBLs of RA and normal controls. METHODS: RT-PCR was done to amplify kappa chain transcripts from RA synovial lymphocytes and the cDNA sequences were compared to those from PBL of RA patient or normal control. RESULTS: Kappa chain repertoire from RA patient's synovial lymphocytes or PBL revealed increased somatic mutation and unusually long complementarity determining region (CDR) 3 compared to normal control. CONCLUSIONS: These changes in kappa chain repertoire in RA patient are suggesting that the antibody repertoire expressed in the synovium or PBL is unique and may be related with systemic dysregulation of B cell development
Arthritis, Rheumatoid ; Autoimmune Diseases* ; Complementarity Determining Regions ; DNA, Complementary ; Genes, Immunoglobulin* ; Humans ; Immunoglobulin kappa-Chains ; Immunoglobulins* ; Lymphocytes ; Synovial Membrane*

The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) antibody (trastuzumab) with artificial sequences of 7–18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)₂ of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition.
Antibodies ; Chromatin Immunoprecipitation ; Clone Cells ; Complementarity Determining Regions ; DNA-Directed RNA Polymerases* ; Exons* ; Peptides ; Phosphopeptides ; Phosphorylation* ; Phosphoserine ; Receptor, Epidermal Growth Factor ; RNA Polymerase II* ; RNA* ; Sensitivity and Specificity ; Serine

Phage display technology provides a powerful tool to screen a library for a binding molecule via an enrichment process. It has been adopted as a critical technology in the development of therapeutic antibodies. However, a major drawback of phage display technology is that because the degree of the enrichment cannot be controlled during the bio-panning process, it frequently results in a limited number of clones. In this study, we applied next-generation sequencing (NGS) to screen clones from a library and determine whether a greater number of clones can be identified using NGS than using conventional methods. Three chicken immune single-chain variable fragment (scFv) libraries were subjected to bio-panning on prostate-specific antigen (PSA). Phagemid DNA prepared from the original libraries as well as from the Escherichia coli pool after each round of bio-panning was analyzed using NGS, and the heavy chain complementarity-determining region 3 (HCDR3) sequences of the scFv clones were determined. Subsequently, through two-step linker PCR and cloning, the entire scFv gene was retrieved and analyzed for its reactivity to PSA in a phage enzyme immunoassay. After four rounds of bio-panning, the conventional colony screening method was performed for comparison. The scFv clones retrieved from NGS analysis included all clones identified by the conventional colony screening method as well as many additional clones. The enrichment of the HCDR3 sequence throughout the bio-panning process was a positive predictive factor for the selection of PSA-reactive scFv clones.
Antibodies ; Bacteriophages ; Chickens ; Clone Cells* ; Cloning, Organism ; Complementarity Determining Regions ; DNA ; Escherichia coli ; Immunoenzyme Techniques ; Mass Screening ; Methods ; Polymerase Chain Reaction ; Prostate-Specific Antigen ; Single-Chain Antibodies

OBJECTIVETo observe anti-cancer effects of Jianpi Jiedu Recipe (JJR) on liver cancer (LC) rats with Pi deficiency syndrome (PDS) and its relation with the third complementary-determining region gene spectratyping of TCRVβ-chain (TCRVβCDR3).

METHODSRats were divided into 8 groups according to random digit table, i.e., the blank control group (normal), the PDS group, the LC model group, the LC-PDS group, high, middle, and low dose JJR groups (75.00, 37.50, 18.75 g/kg, respectively by gastrogavage, once per day), the thymus pentapeptide group (5 mg/kg, intramuscular injection, twice per week), 8 in each group. Rats in the normal group were administered with physiological saline by gastrogavage once per day. PDS rat model was prepared by bitter-cold purgation. LC model was prepared by orthotopic transplantation method. Twenty gene subfamilies of TCRβCDR3 in the thymus, liver, and LC tissues were detected by Gene Scan.

RESULTSHigh and middle dose JJR could postpone the growth of LC volume (P < 0.05), with equivalent liver index and thymus index to those of the normal group (P > 0.05). In thymus and liver tissue of the normal group, the number of clones (20 and 19), gene fragment number (220 and 113), Quasi-Gaussian distribution ratio of TCRVβCDR3 gene repertoire (100.0% and 42.1%), and fragment fluorescence peak area (6,539 ± 2,325 and 1,238 ± 439) were at the highest level among the 8 groups. TCRVβCDR3 expressions in thymus and liver tissue of high and middle dose JJR groups were approximate to those of the normal group. They were in the middle of the thymus pentapeptide group, the PDS group, the LC model group, and poorest in the LC-PDS group. TCRVβCDR3 in liver tissue expressed the best in the thymus pentapeptide group.

CONCLUSIONJJR might inhibit the growth of LC cells, and its mechanism might be related to enhancing TCRVβCDR3 spectratype expression.

Animals ; Complementarity Determining Regions ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation, Neoplastic ; Genes, T-Cell Receptor beta ; Liver Neoplasms ; drug therapy ; genetics ; Random Allocation ; Rats

The cross reactivity of mimotopes of hepatitis C virus (HCV) hypervariable region 1 (HVR1) was investigated to obtain epitopes that have high cross reactivity. Five expression vectors encoding B cell mimotopes fused with Trx were constructed, and the mimotope proteins were purified. The cross reactivity of mimotope proteins with HCV positive sera was determined by ELISA. HCV pseudotype particles (HCVpp) were generated and applied to evaluate neutralization effects of the sera of BALB/c mice immuned with the mimotope proteins on infection of Huh7. 5 cells. Our data showed that the mimotope proteins (P1, P2, P5, P6, P8) could react to the HCV positive sera. The HCVpp infection inhibition of the sera of BALB/c mice immuned with P6 or P8 was detectable. These results suggest that the mimotopes may be valuable in the studies of anti-HCV infection and development of HCV vaccines.
Amino Acid Sequence ; Animals ; Complementarity Determining Regions ; immunology ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Hepacivirus ; immunology ; Humans ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data

OBJECTIVETo investigate the association of the mitochondrial DNA region np16181-16193 variations with type 2 diabetes mellitus (T2DM).

METHODSBlood samples of 199 unrelated T2DM patients and 205 normal controls were collected to detect the mitochondrial DNA region np16181-16193 variations by PCR and sequencing, and to analyze the association of the variations with the major clinical symptoms.

RESULTSThe mitochondrial DNA np16181-16193 region is a hypervariable area, with several polymorphisms. Four types of np16181-16193 region variations were found only in T2DM. The 1-hour postprandial blood glucose (P1BG) in the T2DM individuals with np16181-16193 region variations was significantly higher than those without variations (P<0.05), while there was no significant difference in other biochemical parameters (P>0.05).

CONCLUSIONThe mitochondrial DNA np16181-16193 variations could not be regarded as a risk factor for T2DM.

Adult ; Complementarity Determining Regions ; genetics ; DNA Mutational Analysis ; DNA, Mitochondrial ; analysis ; Diabetes Mellitus, Type 2 ; genetics ; Female ; Genetic Predisposition to Disease ; Genome, Mitochondrial ; genetics ; Humans ; Male ; Sequence Analysis, DNA

OBJECTIVETo analyze the drift of the complementarity-determining region 3 (CDR3) of T cell receptor beta (TCRbeta) chain variable region in T cells of healthy volunteers cultured with interleukin-2 (IL-2).

METHODST cells were isolated from the peripheral blood and cultured in vitro in the presence of IL-2. The non-specific killing effect of the cells was analyzed by LDH releasing assay, and the distribution of TCRbeta chain CDR3 in healthy volunteers by immunoscope spectratyping method to evaluate the clonality of the T cells.

RESULTSThe results showed Gaussian distribution of TCR Vbeta gene CDR3 in healthy volunteers. The T cell cultured with IL-2, however, displayed some anomalous and oligoclonal expansion in different TCR Vbeta families without killing effect against nasophargngal carcinoma cell line CNE2.

CONCLUSIONIL-2 may affect TCRbeta chain CDR3 distribution in T cells cultured in vitro.

Cells, Cultured ; Complementarity Determining Regions ; genetics ; Genetic Drift ; Humans ; Interleukin-2 ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; T-Lymphocytes ; immunology ; metabolism

BACKGROUND: This study characterized clonal IG heavy V-D-J (IGH) gene rearrangements in South Indian patients with precursor B-cell acute lymphoblastic leukemia (precursor B-ALL) and identified age-related predominance in VDJ rearrangements. METHODS: IGH rearrangements were studied in 50 precursor B-ALL cases (common ALL=37, pre-B ALL=10, pro-B ALL=3) by polymerase chain reaction (PCR) heteroduplex analysis. Twenty randomly selected clonal IGH rearrangement sequences were analyzed using the IMGT/V-QUEST tool. RESULTS: Clonal IGH rearrangements were detected in 41 (82%) precursor B-ALL cases. Among the IGHV1-IGHV7 subgroups, IGHV3 was used in 25 (50%) cases. Among the IGHD1-IGHD7 genes, IGHD2 and IGHD3 were used in 8 (40%) and 5 (25%) clones, respectively. Among the IGHJ1-IGHJ6 genes, IGHJ6 and IGHJ4 were used in 9 (45%) and 6 (30%) clones, respectively. In 6 out of 20 (30%) IGH rearranged sequences, CDR3 was in frame whereas 14 (70%) had rearranged sequences and CDR3 was out of frame. A somatic mutation in Vmut/Dmut/Jmut was detected in 14 of 20 IGH sequences. On average, Vmut/Dmut/Jmut were detected in 0.1 nt, 1.1 nt, and 0.2 nt, respectively. CONCLUSION: The IGHV3 gene was frequently used whereas lower frequencies of IGHV5 and IGHV6 and a higher frequency of IGHV4 were detected in children compared with young adults. The IGHD2 and IGHD3 genes were over-represented, and the IGHJ6 gene was predominantly used in precursor-B-ALL. However, the IGH gene rearrangements in precursor-B-ALL did not show any significant age-associated genotype pattern attributed to our population.
Child ; Clone Cells ; Complementarity Determining Regions* ; Gene Rearrangement* ; Genotype ; Heteroduplex Analysis ; Humans ; Immunoglobulins* ; Polymerase Chain Reaction ; Precursor Cell Lymphoblastic Leukemia-Lymphoma* ; Precursor Cells, B-Lymphoid* ; Young Adult

OBJECTIVETo observe the distribution of TCR V alpha gene repertoire and clonal expansion in peripheral blood mononuclear cells from 9 donors and 16 workers exposed to benzene.

METHODSComplementarity determining region 3 (CDR3) of TCR V alpha subfamily genes were amplified using RT-PCR. The PCR products were further analyzed by genescan to evaluate clonality of T cells.

RESULTSAlmost all of 29 V alpha subfamily could be detected in 9 donors. 1 approximately 11 V alpha subfamilies were identified in all but one of the workers studied. The most frequently expressed V alpha subfamily were V alpha 3, V alpha 12 and V alpha 19 (68.8%), V alpha 14 (56.3%), with a lower expression rate found in V alpha 5, V alpha 15, V alpha 16, V alpha 22, V alpha 23 and V alpha 24 (6.3%). Clonal expansion T cells in one or more V alpha subfamily were found in 12 out of all workers studied, including oligoclonal, oligoclonal trend and biclonal patterns. The frequency of clonal expansion T cells in V alpha 12, V alpha 14 and V alpha 19 subfamilies were higher than others.

CONCLUSIONSkewed distribution and clonal expansion of TCR V alpha subfamily T cells could be found in workers exposed to benzene. V alpha 12, V alpha 14 and V alpha 19 subfamilies may be highly sensitive to benzene exposed. This is the first report of clonal expansion TCR V alpha T cells in the benzene-exposed group. The bias pattern of TCR V alpha T cells may be due to the immune cytotoxicity from benzene. However, whether the oligoclonality in some V alpha subfamilies reflect the phenomenon of clone absence or may be a response clone to benzene-related impairment during exposed to benzene, remains an open question.

Adult ; Benzene ; poisoning ; Complementarity Determining Regions ; genetics ; Female ; Gene Expression Profiling ; Genes, T-Cell Receptor alpha ; genetics ; Humans ; Leukocytes, Mononuclear ; Male ; Middle Aged ; Occupational Diseases ; genetics ; Young Adult

BACKGROUNDIn general, it is very important to understand the state of T cell immune response against tumor cells in leukemia patients and it is especially critical to assess the T cell repertoire of untreated patients. As we know, few studies have dealt with the distribution of oligoclonal T cells in leukemia, so we investigated the distribution and clonality of TCR Vbeta repertoire of T cells in patients with chronic myelogenous leukemia (CML) in chronic phase.

METHODSThe complementarity determining region 3 (CDR3) of TCR Vbeta24 subfamily genes were amplified in peripheral blood mononuclear cells from 27 cases with CML using reverse transcription-polymerase chain reaction (RT-PCR). In order to observe the distribution of TCR Vbeta repertoire, the PCR products were further analyzed by genescan technique to evaluate clonality of the detectable TCR Vbeta T cells. The PCR products of the oligoclonal T cells from three cases were analyzed by direct sequencing to define the sequence of CDR3.

RESULTSThe expression pattern of TCR Vbeta repertoire in different individuals are different. Vbeta2-21 subfamilies could be detected in CML cases. The frequent usage Vbeta repertoire in CML was Vbeta1, Vbeta2 or Vbeta13. Most of the PCR products from 27 patients displayed polyclonality, while a part of the PCR products from 21 out of 27 samples displayed clonal expansion pattern. The clonal expanded T cells in CML could be found in Vbeta16 subfamilies. The frequent usage of Vbeta genes in clonal expansion was Vbeta3, Vbeta13 or Vbeta21. Multiple Vbeta clonal expansion was a general phenomenon in the same patient. The CDR3 sequence of Vbeta21 oligoclonal T cells from 3 cases showed some difference in splice regions and in the usage of J segments.

CONCLUSIONSThese results indicated that clonal expanded T cells could be found in patients with CML and were tendentious in Vbeta3, Vbeta13 and Vbeta21 subfamilies that may be related to the specific immune response for leukemia cell associated antigen.

Clone Cells ; Complementarity Determining Regions ; analysis ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; immunology ; Receptors, Antigen, T-Cell, alpha-beta ; analysis ; T-Lymphocytes ; immunology ; pathology