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

This study was aimed to investigate the normalization of serum free light chain ratio (sPLCR) after treatment of multiple myeloma (MM) and its influence on the prognosis of MM patients. The clinical data of 42 patients with MM were analyzed retrospectively from January 2009 to November 2013 in out department. According to sPLCR consecutive normalization for more 4 weeks or not after treatment, the patients were classified in patients with mormalized sPLCR and patients with abnormalized sPLCR, then the influence of traditional prognostic factors of MM on sPLCR and effect of sPLCR on overall survival (OS) time of MM patients were analyzed. The results showed that the influence of age, ISS stage displayed statistical difference between sFLCR normalization group and abnormalization group, the age ≥ 65 years and ISS stage III negatively impacted on sFLCR normalization (P < 0.05). The response rates of patients with normalized sFLCR were as follows: CR - 60%, VGPR - 38.89%; PR - 28.57%; 17 patients (40.48%) with sFLCR normalization showed superior OS, as compared with patients with sPLCR abnormalization (P < 0.01). It is concluded that the sFLCR normalization is the independent prognostic factor for MM, suggesting that the MM patients with sPLCR normalization after treatment have superior prognosis.
Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Humans ; Immunoglobulin Light Chains ; immunology ; Multiple Myeloma ; diagnosis ; drug therapy ; immunology ; Prognosis ; Retrospective Studies

No abstract available.
Asymptomatic Diseases ; Biological Markers/analysis ; Biopsy ; Crystallization ; Fluorescent Antibody Technique ; Humans ; Immunoglobulin Light Chains/*analysis ; Kidney Diseases/*diagnosis/immunology/pathology ; Kidney Tubules, Proximal/*immunology/ultrastructure ; Male ; Microscopy, Electron ; Proteinuria/*diagnosis/immunology/pathology

BACKGROUNDPrimary systemic light chain amyloidosis (AL) is a rare plasma cell disease, our purpose was to analyze the immunophenotypic characteristics of the plasma cells in bone marrow in AL patients, and explore whether the detection of abnormal plasma cell clones in bone marrow by flow cytometry (FCM) could be used as an important indicator of AL diagnosis.

METHODSFresh bone marrow samples were collected from 51 AL, 21 multiple myeloma (MM), and 5 Waldenström's macroglobulinemia (WM) patients. The immunophenotype of bone marrow cells were analyzed and compared by FCM using a panel of antibodies including CD45, CD38, CD138, CD117, CD56, and CD19.

RESULTSIn AL, light chain restriction could be identified in 31 cases (60.9%), in which the λ light chain restriction was found in 24 cases (77.4%). In MM, κ light chain restriction was found in 13 cases (61.9%), and λ light chain restriction in eight cases. CD45 on abnormal plasma cells was negative to weakly positive in both AL and MM, but was positive to strongly positive in WM. In the bone marrow plasma cells of the 51 AL, 78.4% were CD56+, 68.6% were CD117+, and 88.2% were CD19-. While in the 21 MM cases, 66.7% were CD56+, 38.1% were CD117+, and 90.4% were CD19-. The plasmacytoid lymphocytes in the five WM patients were CD19+ and CD56-, CD117-.

CONCLUSIONDetection of abnormal plasma cell clones in bone marrow by FCM is valuable for the diagnosis of AL.

Adult ; Aged ; Aged, 80 and over ; Amyloidosis ; immunology ; metabolism ; CD56 Antigen ; metabolism ; Female ; Flow Cytometry ; Humans ; Immunoglobulin Light-chain Amyloidosis ; Immunoglobulin lambda-Chains ; metabolism ; Immunophenotyping ; Leukocyte Common Antigens ; metabolism ; Male ; Middle Aged ; Multiple Myeloma ; immunology ; metabolism ; Proto-Oncogene Proteins c-kit ; metabolism ; Waldenstrom Macroglobulinemia ; immunology ; metabolism

Immunophenotype is critical for diagnosing common B-cell acute lymphoblastic leukemia (common ALL) and detecting minimal residual disease. We developed a protocol to explore the immunophenotypic profiles of common ALL based on the expression levels of the antigens associated with B lymphoid development, including IL-7Rα (CD127), cytoplasmic CD79a (cCD79a), CD19, VpreB (CD179a), and sIgM, which are successive and essential for progression of B cells along their developmental pathway. Analysis of the immunophenotypes of 48 common ALL cases showed that the immunophenotypic patterns were highly heterogeneous, with the leukemic cell population differing from case to case. Through the comprehensive analysis of immunophenotypic patterns, the profiles of patient-specific composite leukemia cell populations could provide detailed information helpful for the diagnosis, therapeutic monitoring, and individualized therapies for common ALL.
Adult ; Antigens, CD19 ; metabolism ; B-Lymphocytes ; immunology ; metabolism ; CD79 Antigens ; metabolism ; Female ; Humans ; Immunoglobulin Light Chains, Surrogate ; metabolism ; Immunophenotyping ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; pathology ; Receptors, Interleukin-7 ; metabolism

To prepare large naive phage antibody library, the host bacteria with high transformation efficiency is used in the Cre-LoxP recombination system. The variable regions of immunoglobulin light and heavy genes were amplified from lymphocytes collected from adult peripheral blood and newborn cord blood. The genes were spliced to form the single-chain variable fragments (scFv) by overlap PCR, cloned into pDAN5a vector and then transformed into XL2-blue MRF' with the Hte gene. Compared with XL1-blue strain, the size of the primary library was increased by 3.9 times. The primary library infected Cre recombinase-expressing bacteria, and the genes between phagemids created many new VH/VL combinations. The library was calculated to have a diversity of 1.7 x 10(11) and validated by the selection of antibodies against six different protein antigens. This library provides the basis for further selection of antibody-based drugs. It is the first time to report that XL2-blue MRF' can be used to improve the diversity of the library in the recombination system.
Adult ; Escherichia coli ; genetics ; immunology ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Infant, Newborn ; Integrases ; metabolism ; Lymphocytes ; immunology ; Peptide Library ; Recombination, Genetic ; genetics ; Single-Chain Antibodies ; genetics ; metabolism ; Transformation, Genetic

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.
Amino Acid Sequence ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; Antibody Affinity ; Asthma ; blood ; Base Sequence ; DNA, Complementary ; metabolism ; Escherichia coli ; metabolism ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Lymphocytes ; chemistry ; Peptide Library ; RNA, Messenger ; isolation & purification ; Recombination, Genetic ; genetics ; Ribosomes ; chemistry ; genetics ; immunology ; Single-Chain Antibodies ; genetics ; isolation & purification ; Transformation, Genetic

Small molecule antibodies are naturally existed and well functioned but not structurally related to the conventional antibodies. They are only composed of heavy protein chains or light chains, much smaller than common antibody. The first small molecule antibody, called Nanobody was engineered from heavy-chain antibodies found in camelids. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies called Vnar fragments can be obtained. In addition, free light chain (FLC) antibodies in human bodies are being developed as therapeutic and diagnostic agents. Comparing to intact antibodies, common advantages of small molecule antibodies are with better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. This article reviews the structural characteristics and mechanism of action of the Nanobody, IgNAR and FLC.
Animals ; Camelids, New World ; immunology ; Humans ; Immunoglobulin Light Chains ; chemistry ; immunology ; Receptors, Antigen ; immunology ; Sharks ; immunology ; Single-Chain Antibodies ; chemistry ; immunology ; therapeutic use ; Single-Domain Antibodies ; chemistry ; immunology ; therapeutic use

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

Structure of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody was verified by the application of high-performance liquid chromatography-mass spectrometry (HPLC-MS)and N-terminal sequencer. Molecular masses, N-terminal sequences and peptide maps of the antibody treated with different reagents and enzymes were measured. Results indicate that the amino acid sequences of light and heavy chains and 10 disulfide bonds were consistent with theoretical structure. By comparison of molecular masses and peptide maps for the fully glycosylated and deglycosylated samples, the N-linked glycosylation site was identified. The method is simple, rapid, precise, and could be referred to the quality control and structure determination of other IgG1 products.
Amino Acid Sequence ; Antibodies, Monoclonal ; chemistry ; Antigens, CD20 ; immunology ; Chromatography, High Pressure Liquid ; Glycosylation ; Immunoglobulin G ; chemistry ; immunology ; Immunoglobulin Heavy Chains ; chemistry ; Immunoglobulin Light Chains ; chemistry ; Mass Spectrometry ; Molecular Weight ; Peptide Mapping ; Recombinant Proteins ; chemistry ; Trypsin ; chemistry