OBJECTIVETo study the rearrangement of immunoglobulin (Ig) heavy chain variable region (V(H)) genes in human neonates with different gestational ages (GA).

METHODSPeripheral blood from the neonates with GA of 27 weeks (4 cases), 28-32 weeks (9 cases), 33-36 weeks (12 cases), and 37-42 weeks (13 cases) was collected. RT-PCR was used to amplify the Ig V(H) gene, and the PCR products were separated by electrophoresis and analyzed using 6% denaturing PAGE gel.

RESULTSAll Ig V(H) family genes had several rearranged genes in each GA group, and the neonates with different GA showed no significant difference in the median molecular weight for each rearranged Ig V(H) family gene.

CONCLUSIONThe neonates with GA of 27-42 weeks exhibit diversity in Ig V(H) gene rearrangement, and for the same Ig V(H) family, the median length of the arranged Ig V(H) genes is independent of the gestational age.

Gene Rearrangement ; Genes, Immunoglobulin Heavy Chain ; Gestational Age ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Infant, Newborn ; Multigene Family ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo investigate the overrepresentation of specific gene segments of immunoglobulin heavy chain variable region (IgVH) among unmutated and mutated chronic lymphocytic leukemia (CLL) patients and its prognostic implication.

METHODSMultiplex PCR was used to identify the expression of IgVH segment and its mutation status in CLL.

RESULTSAnalyses were successfully performed in 80 of 85 samples. Marked skewed IgVH families were disclosed. The most commonly used VH was VH3 (40.0%), followed by VH4 (30.0%), VHI (13.8%), VH2 (10.0%) and VH5, VH7 (2.5%). Fifty-six patients (70.0%) had mutated VH, 24 (30.0%) unmutated VH. Nine cases (11.3%) were with 100% germline sequence. Fifteen cases (15/24, 62.5%) in VH4, 29 (29/32, 90.7%) in VH3, and 4 (4/11, 36.3%) in VH1 had mutated VH. The most frequently used IgVH gene was VH4-39 (13.8%), and VH4-34 (8.8%). J4 (36/66, 54.5%) and D3 (25/66, 37.8%) were the most frequently used in J and D genes. The progression-free survival (PFS) was 82 and 17 months (P = 0.000), and the overall survival (OS) was 90 and 41 months (P = 0.009), respectively, for mutated and unmutated cases. Recurrent CDR3 sequences were found in our patients and 2 patients with VH1-69 had CDR3 sequences highly similar to those reported in literature.

CONCLUSIONThere is difference in IgVH gene segment usage and mutational status in different area CLL patients. Recurrent CDR3 sequences were found in specific IgVH gene segments, which highlights the importance of immunoglobulin mediated stimulation in the development of CLL.

Adult ; Aged ; Aged, 80 and over ; DNA Mutational Analysis ; Female ; Gene Rearrangement, B-Lymphocyte, Heavy Chain ; Genes, Immunoglobulin ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; immunology ; pathology ; Male ; Middle Aged ; Mutation

BACKGROUND: The clonality of lymphoid infiltrates determined by polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) or T cell receptor (TCR) genes is not only useful in confirming the diagnosis of malignant lymphoma but also in establishing the lineage of a clonal lymphoid proliferation. We analyzed the efficiency of PCR analyses for IgH and TCRgenes that have been routinely applied for the diagnosis of malignant lymphoma in our laboratory. METHODS: Paraffin sections of 200 cases were analyzed by seminested PCR. Primers were FRIIIA-LJH/VLJH consensus primer for IgH gene and V-J consensus primer for TCR gene. The cases showing negative results by PCR for TCR gene were further analyzed by multiplex V family primers with heteroduplex analysis. RESULTS: PCR approach for IgH gene allowed detection of clonality in 100% of cases with false positive rate of 0.3% and false negative rate of 0%. The combination of PCR for TCR consensus primers with multiplex V family primers allowed detection of clonality in 91% of cases with false positive rate of 0.6% and false negative rate of 10.3%. CONCLUSIONS:Combined analysis of IgH and TCR gene rearragnements by the PCR technique followed by heteroduplex analysis can be a useful diagnostic adjunct to determine the clonality of various lymphoproliferative diseases with high sensitivity. But clinical, morphological and immunophenotypical correlation should be considered to reach the final diagnosis due to a few false positive cases.
Consensus ; Diagnosis ; Gene Rearrangement* ; Genes, T-Cell Receptor ; Heteroduplex Analysis ; Humans ; Immunoglobulin Heavy Chains* ; Immunoglobulins* ; Lymphoma ; Paraffin ; Polymerase Chain Reaction ; Receptors, Antigen, T-Cell* ; T-Lymphocytes*

BACKGROUND: Immunoglobulin heavy chain (IgH) gene rearrangement has been known to be a useful marker for determining the clonality as well as detecting minimal residual disease in B cell malignancies. This study was performed to establish single polymerase chain reaction (PCR) methods for the detection of IgH gene rearrangements in formalin-fixed, paraffin-embedded tissue of patients with B cell lymphoma and determine the type of JH segments used. METHODS: We obtained formalin-fixed, paraffin-embedded tissue sections of 44 patients diagnosed with B cell lymphoma at Ajou University Hospital from January 2005 to January 2007 and reviewed medical records retrospectively. After the extraction of DNA, PCR was performed using VH3 and JHPST primers to detect the third complementarity determining region (CDR3) gene of IgH. Sequence analysis of the PCR products was also done in 23 patients. RESULTS: The CDR3 gene rearrangements were detected in 26 (59%) out of 44 patients with B cell lymphoma. Sequence analysis of the amplified CDR3 gene was successful in 16 (70%) of 23 patients. JH3, JH4, JH5, and JH6 segments were used for CDR3 gene rearrangements in 3 (25%), 4 (33%), 1 (8%), and 4 (33%) patients with diffuse large B cell lymphoma, respectively. CONCLUSION: Although there are some limitations due to a low sensitivity less than 60%, single PCR using consensus primers could be an effective tool for the detection of CDR3 gene rearrangements in routine laboratory settings. Furthermore, sequence analysis of the CDR3 PCR products will provide basic information necessary for further studies.
Complementarity Determining Regions ; Consensus ; DNA ; Formaldehyde* ; Gene Rearrangement* ; Humans ; Immunoglobulin Heavy Chains* ; Immunoglobulins* ; Lymphoma, B-Cell* ; Medical Records ; Neoplasm, Residual ; Polymerase Chain Reaction* ; Retrospective Studies ; Sequence Analysis

BACKGROUND/AIMS: The gastrointestinal (GI) tract often becomes involved in patients with systemic amyloidosis. As few GI amyloidosis data have been reported, we describe the clinical features and outcomes of patients with pathologically proven GI amyloidosis. METHODS: We identified 155 patients diagnosed with systemic amyloidosis between April 1995 and April 2013. Twenty-four patients (15.5%) were diagnosed with GI amyloidosis using associated symptoms, and the diagnoses were confirmed by direct biopsy. RESULTS: Among the 24 patients, 20 (83.3%) had amyloidosis light chain (AL), three (12.5%) had amyloid A, and one (4.2%) had transthyretin-related type amyloidosis. Their median age was 57 years (range, 37 to 72), and 10 patients were female (41.7%). The most common symptoms of GI amyloidosis were diarrhea (11 patients, 45.8%), followed by anorexia (nine patients, 37.5%), weight loss, and nausea and/or vomiting (seven patients, 29.2%). The histologically confirmed GI tract site in AL amyloidosis was the stomach in 11 patients (55.0%), the colon in nine (45.0%), the rectum in seven (35.0%), and the small bowel in one (5.0%). Patients with GI involvement had a greater frequency of organ involvement (p = 0.014). Median overall survival (OS) in patients with GI involvement was shorter (7.95 months; range, 0.3 to 40.54) than in those without GI involvement (15.84 months; range, 0.0 to 114.53; p = 0.069) in a univariate analysis. A multivariate analysis of prognostic factors for AL amyloidosis revealed that GI involvement was not a significant predictor of OS (p = 0.447). CONCLUSIONS: The prognosis of patients with AL amyloidosis and GI involvement was poorer than those without GI involvement, and they presented with more organ involvement and more advanced disease than those without organ involvement.
Adult ; Aged ; Amyloid Neuropathies, Familial/*diagnosis/immunology/mortality/pathology/therapy ; Biomarkers/analysis ; Biopsy ; Female ; Gastrointestinal Diseases/*diagnosis/immunology/mortality/pathology/therapy ; Gastrointestinal Tract/immunology/*pathology ; Humans ; Immunoglobulin Heavy Chains/analysis ; Immunoglobulin Light Chains/analysis ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Multivariate Analysis ; Predictive Value of Tests ; Prognosis ; Proportional Hazards Models ; Republic of Korea ; Retrospective Studies ; Risk Factors ; Serum Amyloid A Protein/analysis ; Time Factors

OBJECTIVETo develop a recombinant single domain antibody against hTERT, human telomerase catalytic subunit.

METHODSA previously prepared His-tagged hTERT fusion protein was used as the antigen, and the variable regions in heavy chain (VH) of immunized mice were RT-PCR amplified and cloned into the pCANTAB 5E, a phagemid vector. By transfection, the display library of mouse VH was developed. The candidate clones were selected by affinity panning, and soluble VH were obtained after expression in E. coli, HB2151. The resultant single VH antibodies were characterized on their binding potentials by western blotting.

RESULTSAn about 350 bp VH fragment was amplified from spleen cells of mice immunized by His-tagged hTERT and expressed by phage displayed as VH library. The size of the library was 8 x 10(4). After three rounds of affinity panning, 4 independent clones were chosen and consequently expressed as soluble single domain antibodies (Mr = 16 000). In Western blot analysis, the single domain antibody from 2 of 4 clones proved to react with the His-tagged hTERT fusion protein (Mr = 167 000) without dependence of His-tags and also detect the native hTERT (Mr = 127 000) extracted from the human HeLa cancer cell line. DNA sequencing showed both of the single domain antibodies were encoded by the heavy chain variable region of the mouse.

CONCLUSIONSThe single domain antibodies developed were hTERT recognizable and hTERT specific, thus providing a basis for application of recombinant single domain antibody in inhibition of telomerase activity and anticancer therapy.

Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; genetics ; immunology ; Base Sequence ; Cloning, Molecular ; Complementarity Determining Regions ; genetics ; DNA-Binding Proteins ; HeLa Cells ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Mice ; Molecular Sequence Data ; Sequence Analysis, DNA ; Telomerase ; immunology

BACKGROUND: For patients with multiple myeloma (MM), different strategies are used to detect chromosomal abnormalities (CA). There have been a few studies that have directly compared FISH with conventional cytogenetics (CC) for the detection of CA. In this study, we employed a combined approach of metaphase cytogenetics and interphase FISH to investigate the genetic basis for the great heterogeneity observed in the clinical behavior of 28 MM patients. METHODS: Cytogenetic analysis was performed via traditional metaphase karyotype analysis. The FISH studies were done using DNA probes to detect translocations involving the immunoglobulin heavy chain gene (IGH) at 14q32 and deletions of 17p13.1 and 13q14. RESULTS: CA were detected by CC in 16 patients (57.1%) and by FISH in 14 patients (50.0%) of the 28 patients we studied. 14q32 abnormalities and deletion abnormalities of 13q14 and 17p13.1 were detected by CC in five patients (17.9%), three patients (10.7%) and no patients (0%), respectively and these were detected by FISH in 12 (42.8%), four (14.3%) and five (17.8%), respectively, of the 28 patients we studied. The median follow-up timefor the patients was 23.85 months (range: 0.3~58.13 months). On the univariate and multivariate analyses, none of the abnormalities detected by cytogenetics and interphase FISH affected survival. CONCLUSION: On comparing the cytogenetics and interphase FISH results, we can suggest that both studies should be an essential part of the workup for the diagnosis of patients with MM. Also, both studies may complement each other to predict the prognosis.
Chromosome Aberrations ; Complement System Proteins ; Cytogenetic Analysis ; Cytogenetics ; DNA Probes ; Fluorescence ; Follow-Up Studies ; Humans ; Immunoglobulin Heavy Chains ; In Situ Hybridization ; Interphase ; Karyotype ; Metaphase ; Multiple Myeloma ; Multivariate Analysis ; Population Characteristics ; Prognosis

OBJECTIVETo acquire the genes of light chain variable region (VL) and heavy chain variable region (VH) of a novel clone ZCH-7-2F9(2F9) of anti-hCD14 for construction of anti-hCD14 single chain antibody(ScFv).

METHODSFrom the mouse hybridoma cell line 2F9 and its fusion partner murine myeloma cell line NS-1, total RNA was prepared. The VL and VH genes were amplified by RT-PCR with family specific primer pairs, respectively. The PCR products were cloned into pGEM(sound recording copyright sign)-T Easy vectors, then transfected into DH5alpha and the positive recombinants were identified and purified. After sequencing with automatic DNA sequencer the sequences were analyzed online.

RESULTSVL gene of the new clone of CD14 monoclonal antibody (McAb) 2F9 consisted of 321 bps encoding a peptide of 107 amino acid residues, and VH gene of the 2F9 antibody contained 360 bps encoding a peptide of 120 amino acid residues. According to IMMUNOGENETICS online analysis by IMGT/V-QUEST, the VL and VH genes belonged to mouse IGKV and mouse IGHV subgroups, respectively. On the position 23/88 of the light chain and 22/96 of the heavy chain genes there were cysteines, which play the key role in forming disulfo-bond in each chain. Both VL and VH chains had definitely 4 frame regions (FR) and 3 complementary determinant regions (CDR).

CONCLUSIONThe treatment of CML consisting of myeloablative Allo-SCT combined with Gleevec before and after transplantation is an effective and safe method for CML.

Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; genetics ; immunology ; Base Sequence ; Cloning, Molecular ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Lipopolysaccharide Receptors ; immunology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Polymerase Chain Reaction ; Sequence Analysis, DNA

BACKGROUNDThe established clinical staging systems (Rai/Binet) of chronic lymphocytic leukemia (CLL) cannot accurately predict the appropriate treatment of patients in the earlier stages. In the past two decades, several prognostic factors have been identified to predict the outcome of patients with CLL, but only a few studies investigated more markers together. To predict the time to first treatment (TTFT) in patients of early stages, we evaluated the prognostic role of conventional markers as well as cytogenetic abnormalities and combined them together in a new prognostic scoring system, the CLL prognostic index (CLL-PI).

METHODSTaking advantage of a population of 406 untreated Chinese patients with CLL at early and advanced stage of disease, we identified the strongest prognostic markers of TTFT and, subsequently, in a cohort of 173 patients who had complete data for all 3 variables, we integrated the data of traditional staging system, cytogenetic aberrations, and mutational status of immunoglobulin heavy chain variable region (IGHV) in CLL-PI. The median follow-up time was 45 months and the end point was TTFT.

RESULTSThe median TTFT was 38 months and the 5-year overall survival was 80%. According to univariate analysis, patients of advanced Rai stages (P < 0.001) or with 11q- (P = 0.002), 17p- (P < 0.001), unmutated IGHV (P < 0.001), negative 13q- (P = 0.007) and elevated lactate dehydrogenase levels (P = 0.001) tended to have a significantly shorter TTFT. And subsequently, based on multivariate Cox regression analysis, three independent factors for TTFT were identified: advanced clinical stage (P = 0.002), 17p- (P = 0.050) and unmutated IGHV (P = 0.049). Applying weighted grading of these independent factors, a CLL-PI was constructed based on regression parameters, which could categorize four different risk groups (low risk [score 0], intermediate low [score 1], intermediate high [score 2] and high risk [score 3-6]) with significantly different TTFT (median TTFT of not reached (NR), 65.0 months, 36.0 months and 19.0 months, respectively, P < 0.001).

CONCLUSIONSThis study developed a weighted, integrated CLL-PI prognostic system of CLL patients which combines the critical genetic prognostic markers with traditional clinical stage. This novel modified PI system could be used to discriminate among groups and may help predict the TTFT and prognosis of patients with CLL.

Adult ; Aged ; Aged, 80 and over ; China ; Chromosome Aberrations ; Chromosomes, Human, Pair 17 ; genetics ; DNA Mutational Analysis ; Female ; Humans ; Immunoglobulin Heavy Chains ; genetics ; metabolism ; In Situ Hybridization, Fluorescence ; Leukemia, Lymphocytic, Chronic, B-Cell ; diagnosis ; genetics ; metabolism ; Male ; Middle Aged ; Mutation ; Prognosis

OBJECTIVESTo construct and to express a human-mouse chimeric antibody against Aβ peptide involved in Alzheimer disease by genetic antibody engineering with reducing of its human anti-mouse antibody response.

METHODSTotal RNA was extracted from a murine hybridoma cell line that secreted anti-Aβ monoclonal antibody. The entire gene coding heavy and light chains were amplified using RT-PCR and analyzed by Genebank Blast. The chimeric antibody gene was acquired by variable region gene of the monoclonal antibody with constant region gene of human IgG, in which point mutations were incluced by recombinant PCR technology, respectively. The eukaryotic expression vectors established by cloning chimeric antibody genes of the heavy and light chains into 3.1 were co-transfected into COS-7 cells. The expressed products were analyzed using ELISA and immunohistochemistry subsequently.

RESULTSGenebank Blast analysis showed that the entire cloned antibody genes were in accordance with the murine antibody genes. DNA sequencing confirmed that the expression vectors of chimeric antibody were constructed successfully after splicing the variable region and constant region sequences. By co-transfecting COS-7 cells, a chimeric antibody was produced and collected in the culture medium. The antibody was humanized and bound Aβ specifically by ELISA and immunohistochemistry evaluations.

CONCLUSIONSExpression vector of chimeric antibody against Aβ was constructed successfully and expressed in the eukaryotic cells. It provides a solid base for developing diagnostic and therapeutic methods for Alzheimer's disease in clinic and paves a way for a further humanization in the future.

Alzheimer Disease ; immunology ; metabolism ; Amyloid beta-Peptides ; immunology ; metabolism ; Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; COS Cells ; Cell Line ; Cercopithecus aethiops ; Genetic Vectors ; Humans ; Hybridomas ; cytology ; immunology ; Immunoglobulin Heavy Chains ; genetics ; metabolism ; Immunoglobulin Light Chains ; genetics ; metabolism ; Mice ; Point Mutation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Sequence Analysis, DNA ; Transfection