Application of immunoglobulin gene rearrangement-derived real-time quantitative polymerase chain reaction in monitoring minimal residual disease of B-cell lymphoblastic leukemia / 白血病·淋巴瘤

Objective To establish a real-time quantitative polymerase chain reaction (qPCR) assay for B-cell lymphoblastic leukemia according to individualized and specific immunoglobulin gene rearrangements in leukemia cells, and to use it for the monitoring of minimal residual disease (MRD) of B-cell lymphocytic leukemia. Methods The immunoglobulin gene rearrangements of bone marrow samples from 15 cases of B-cell lymphoblastic leukemia were analyzed with a validated European BIOMED-2 system, and the individualized and specific qPCR-based quantification of leukemic immunoglobulin gene rearrangements was established. Results Unique and specific gene rearrangements of immunoglobulin light and heavy chains were identified in 14 cases and Ig-qPCR based on these gene rearrangements had a sensitivity of 10-5 and high specificity which met the international criteria in 10 patients. Leukemia MRD quantification with immunoglobulin gene rearrangement-based qPCR was similar as compared with other MRD detection methods. Conclusion Immunoglobulin gene rearrangement-based leukemia MRD quantification is feasible, sensitive, specific, precise and much valuable for clinical decision of treatments in B-cell lymphoblastic leukemia.

Comparison of class switch recombination assays for immunoglobulin synthesis

The second step of immunoglobulin gene alteration consists of somatic hypermutation and class switch recombination. 80th are regulated by activation-induced cytidine deaminase (AID). Methods: Study on possible application of class switch recombination assays for immunoglobulin gene alteration via AID. Cell based assays using AID B lymphocyte and NIH3T3 cell carrying switch substrate; gene transfer using retrovirus system; FACS analysis; PCR and ELISA. Results: DNA sequencing for S region and gamma1CT are the most sensitive and accurate assays. However, gamma1CT assay seemed to be more reliable and applicable. Others are accurate assays but less applicable. Conclusion: gamma1CT determination is the best class switch recombination assay for immunoglobulin gene alteration via AID.

A Case of Idiopathic Lymphocytoma Cutis

The pseudolymphoma of the skin has the architectural and cytological features of a neoplastic proliferation of lymphoid tissue but pursue a benign course. Cutaneous B cell pseudolymphoma (CBPL) shares many histopathologic and clinical features with cutaneous B cell lymphoma (CBCL). Therefore, the differentiation between CBPL and CBCL is often very difficult, but it is important because each of them has a different therapeutic consequence. Recently, immunoglobulin gene rearrangement is considered as a reliable technique for differentiation of CBPL with CBCL. We herein report a case of idiopathic lymphocytoma cuffs, showing a typical nodular infiltrate of lymphocytes that formed a follicular germinal center resembling reactive lymph nodes with numerous tingible bodies, and that revealed a polyclonality in the immunoglobulin gene rearrangement.

Isolation of Mouse Ig Heavy and Light Chain Genomic DNA Clones, and Construction of Gene Knockout Vector for the Generation of Humanized Xenomouse

BACKGROUND: Monoclonal antibodies (mAb) of rodent origin are produced with ease by hybridoma fusion technique, and have been successfully used as therapeutic reagents for humans after humanization by genetic engineering. However, utilization of these antibodies for therapeutic purpose has been limited by the fact that they act as immunogens in human body causing undesired side effects. So far, there have been several attempts to produce human mAbs for effective in vivo diagnostic or therapeutic reagents including the use of humanized xenomouse that is generated by mating knockout mice which lost Ig heavy and light chain genes by homologous recombination and transgenic mice having both human Ig heavy and light gene loci in their genome. METHODS: Genomic DNA fragments of mouse Ig heavy and light chain were obtained from a mouse brain lamda genomic library by PCR screening and cloned into a targeting vector with ultimate goal of generating Ig knockout mouse. RESULTS: Through PCR screening of the genomic library, three heavy chain and three light chain Ig gene fragments were identified, and restriction map of one of the heavy chain gene fragments was determined. Then heavy chain Ig gene fragments were subcloned into a targeting vector. The resulting construct was introduced into embryonic stem cells. Antibiotic selection of transfected cells is under the progress. CONCLUSION: Generation of xenomouse is particularly important in medical biotechnology. However, this goal is not easily achieved due to the technical difficulties as well as huge financial expenses. Although we are in the early stage of a long-term project, our results, at least, partially contribute the successful generation of humanized xenomouse in Korea.

Cutaneous B-Cell Pseudolymphoma: Report of Two Cases

Cutaneous pseudolymphoma (CPL) has a microscopic appearance that resembles that of cutaneous lymphoma, but shows a clinically benign course. The differential diagnosis of CPL with cutaneous lymphoma is very important because clinical outcomes of them are quite different. We herein describe two cases of B-cell pseudolymphoma, which were difficult to differentiate from cutaneous B-cell lymphlma. All of two cases, Polymerase chain reaction of immunoglobulin heavy chain gene rearrangement showed polyclonal pattern.

Repertoire Antibody Library Constructed from Human Peripheral Blood Lymphocytes with in vitro Immunization with Colorectal Carcinoma-Associated Antigen / 中国肿瘤生物治疗杂志

A Strategy was established for construction of repertoire antibody library with affinity chromatography purifying antigen, antigen immunizing human lymphocytes, RT-PCR and phage display technology. The colorectal carcinoma-associated antigen CA-Hb3 was purified with affinity column and analysed with SDS-PAGE and Western-blot, then applied for immunizing peripheral blood lymphocytes (PBL) in vitro. The PBL were isolated from ten patients with colorectal carcinoma and cultured with interleukin 2 and pokeweed mitogen, lymphoblast-like cells occurred and colonies formed after immunization. Three VH-CHl(?) and five VL-CL(?,?) genes were amplified from their total RNA and mRNA with RT-PCR. Three VH (?) and 8 VL (?,?) genes were reamplified and randomly combined to construct 24 single-chain variable fragments (ScFv) genes through (Gly_(4)Ser)_(3) linker. ScFv genes digested with Sfi I were cloned into fUSE 5 vector and transformed into MC1061 with electroporation. Repertoire antibody library was obtained with 10~(6) tetracycline-resistant colonies, in which the percentage of ScFv inserts was 85 % . This strategy might be used for humanizing mouse-original monoclonal antibody.

Primary Cutaneous B Cell Lymphoma

We report a case of B-cell lymphoma primarily involving the skin in a 12-year-old boy. The histopathologic findings were compatible with those of small lymphocytic type of non-Hodgkin's lymphoma. A cutaneous lesion was the sole manifestation of his disease without any other organ involvement. Immunophenotypic studies and immunoglobulin gene rearrangement with Southern blot analysis determined its lineages and monoclonality with result of B-cell lineage neoplasm, i. d. CD20⁺, C1323⁺, CD35⁻ and rearranged band on JH probe. We treated him with surgical excision and CVP regimen of chemotherapy (cyclophosphamide, vincristine, prednisolone). There is no recurrence or metastasis during the last six months.

Immunoglobulin Gene Repertoire In Rheumatoid Synovium As A Model For Autoimmune Disease / 대한류마티스학회지

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

Alteration of configuration of immunoglobulin and T cell receptor genes in early stage of children acute leukemia / 中国免疫学杂志

Phenotypic markers of leukemic cells from 29 children with acute leukemia were examined. Of these cases, six were negative for myeloperoxidase and did not react with lineage-associated or mature lineage-associated monoclonal antibodies. Then, we analyzed the configuration of both immunoglobulin (heavy chain and kappa chain) and T-cell receptor (?, ?, ?) genes in these six cases. All cases had rearrangement of IgH were suggestive of B-lymphoid origin of these leukemic cells. Two cases without CD10 antigen had no rearrangement of kappa chain, one with retention of the germline configuration of TCR ?, ?. ?, the other with retention of the germline of TCR ? gene. In the cases with CD10 antigen, two cases showed kappa chain deletion, the alteration of TCR genes (rearrangements or deletions )was shown to occur frequently. These findings may implicate that the gene rearrange ments forming functional IgH gene in leukemic cells with CD10 antigen, induce the alteration of IgL and TCR genes.