Child, Preschool ; Epithelioid Cells ; Heart Atria ; pathology ; Heart Neoplasms ; Humans ; Perivascular Epithelioid Cell Neoplasms

OBJECTIVETo study the retentive force and deformation of acetal resin clasp.

METHODS40 premolars and 40 molars were cast respectively. Undercut of 0.25 mm or 0.50 mm depth were measured for each with undercut gage. According to the type of abutment and the depth of undercut, the specimens were divided into 4 groups: Premolars with 0.25 mm undercut, premolars with 0.50 mm undercut, molars with 0.25 mm undercut and molars with 0.50 mm undercut, 20 specimens each group. 10 three-arm clasps with resin and Co-Cr alloy were fabricated in each group, respectively. The clasps were set into the corresponding abutments and soaked in distilled water. The retentive force of the clasps when 0, 720, 1440, 2160, 2880, 3600, 4320 consecutive times of setting in and removing out from the abutments were measured. The distance between the tips of retentive arm and resistant arm after 0 and 4320 cycles were recorded.

RESULTS1) The mean retentive force of resin clasps (1.69 N) was significantly lower than that of Co-Cr clasps (5.87 N) (P<0.01). With the same factors, the retentive force of resin clasps were significantly less than that of Co-Cr clasps (P<0.01). The retentive force of molar clasps were significantly lower than that of premolar models (P<0.01). The retentive force of 0.25 mm undercut clasps were significantly lower than that of 0.50 mm undercut clasps (P<0.01). With increasing time of the cycles, the retentive force of Co-Cr clasps significantly reduced (P<0.01), but the retentive force of resin clasps didn't change significantly (P>0.05). 2) After 4320 times, the distance between the tips of retentive arm and resistant arm of Co-Cr clasps increased significantly (P<0.05), but the distance between the tips of resin clasps didn't change significantly (P>0.05).

CONCLUSIONThe retentive force and deformation of the resin clasp are significantly lower than those of Co-Cr clasp.

Bicuspid ; Chromium Alloys ; Dental Clasps ; Denture Retention ; Humans ; In Vitro Techniques

OBJECTIVETo induce anti-B-cell acute lymphoblastic leukemia (B-ALL) cytotoxic T lymphocyte response against immunoglobulin heavy chain frame-derived nonapeptide.

METHODSThe peptide, QLVQSGAEV, containing IgHV1 frame region 3rd-11th amino acids (IgHV1(3-11)), was synthesized. IgHV1(3-11)-T2 binding tests were performed. HLA-A * 0201-positive normal peripheral blood mononuclear cells (PBMNC) were stimulated by IgHV1(3-11)-loaded antigen presenting cells three times at weekly intervals. HLA-A * 0201/IgHV1(3-11) tetramers were used to detect the proliferation of IgHV1(3-11)-specific CD8(+) T cells in the culture. Seven IgHV gene families of B-ALL patients were respectively amplified by PCR and the PCR products were sequenced to select IgHV1 and IgHV3 family monoallelic functional rearrangements. Among them, HLA-A * 0201 positive individuals were subsequently identified. Cytotoxicity of IgHV1(3-11)-specific CD8(+) T cells against HLA-A * 0201-positive IgHV1/IgHV3 family B-ALL cells was measured by MTT assay.

RESULTSThe synthesized IgHV1(3-11) up-regulated HLA-A * 0201 expression on T2 cell surface by 1.63-folds. The percentage of IgHV1(3-11)-specific CD8(+) T cells in HLA-A * 0201-positive normal PBMNC was increased from 1.64% after second stimulation to 82.57% after third stimulation. At effector: target ratio of 20:1, the killing rate of IgHV1(3-11)-specific CD8(+) T cells against IgHV1 family B-ALL cells was 18.24%, being 1.8-folds as that against IgHV3 family B-ALL cells (P = 0.01).

CONCLUSIONCytolytic T lymphocytes generated in vitro against immunoglobulin heavy chain frame-derived nonapeptides can kill B-ALL cells expressing these peptides.

Cells, Cultured ; Humans ; Immunoglobulin Heavy Chains ; immunology ; pharmacology ; Leukemia, B-Cell ; immunology ; pathology ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology

OBJECTIVETo investigate the origin of childhood B-cell acute lymphoblastic leukemia (B-ALL) and its epitopes recognized by cytotoxic T lymphocytes (CTL) in immunoglobulin heavy chain variable region (IgHV).

METHODSSeven IgHV gene families were respectively amplified by PCR and directly sequenced in 108 childhood ALL. The amino acid sequences were deducted from sequenced nucleotides. Bioinformatics was applied to analyses of recombination patterns, somatic mutations and germline gene segments usage, and to prediction of epitopes recognized by CTL.

RESULTSIgHV gene rearrangements were identified in 66% of the cases, including 37 (52.1%) monoallelic rearrangements, 26 (36.6%) biallelic rearrangements and 8 (11.3%) oligoclonal rearrangements. Among the obtained 40 B-ALL IgHV gene sequences, 8 (20.0%) were in frame rearrangements without stop codons. V(H3) (11/40), V(H4) (11/40) and V(H1) (8/40) amounted to 75% rearranged V(H) families. V(H)(4-59) and V(H)(4-34) were the most frequently rearranged V(H)(4) family gene segments. Usage of D2 and D3 families was most prominent (35.9% and 28.2%, respectively). Increased frequency of D7-27 (15.4%) was found as compared to that of normal peripheral B lymphocytes (P = 0.02). J(H)(6) was found in 47.5% rearrangements followed by J(H)(4) (27.5%). 8/40 (20.0%) DJ(H) junctions lacked N nucleotides, which was higher than that reported for normal peripheral B lymphocytes (P = 0.02). 17.5% B-ALL IgHV contained scattered replacement mutations with replacement (R) to silent (S) substitution ratio (R/S ratio)

CONCLUSIONB-ALL originated from progenitor or precursor B lymphocytes. B-ALL IgHV genes are of germline characteristics. Potential T cell epitopes were derived from framework regions 1 and 3 of immunoglobulin heavy chain in B-ALL.

Adolescent ; Child ; Child, Preschool ; Female ; Gene Rearrangement ; Genes, Immunoglobulin ; HLA-A Antigens ; genetics ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Infant ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology

Child ; Child, Preschool ; Female ; Hand, Foot and Mouth Disease ; complications ; Humans ; Infant ; Male ; Nail Diseases ; etiology

Primary systemic light chain amyloidosis or immunoglobulin light-chain amyloidosis (AL) is the most common type of systemic amyloidosis.AL is a proteotoxic clonal plasma cell disease, a hematological malignancy, characterised by overproduction of immunoglobulin light chains that form characteristic abnormally folded and aggregated, insoluble fibrillar deposits in various organs, including kidneys, heart, liver, and autonomic and peripheral nerves, etc, these processes lead to organ dysfunction and death. Systemic amyloidosis have various types with different causes, thereby its clinical diagnosis and treatment are more difficult. Recent developments on studies that have significantly aided the management of patients with AL include diagnostic techniques for definitive typing of amyloid deposits by using flow cytometry and immunophenotype analysis. These methods can detect abnormalities of bone marrow plasma cell clones, such as CD38(+), CD138(+), CD56(+), CD19(-) in AL patients. The monitoring abnormal plasma cells with immunoglobulin light chain restriction and abnormal plasma cell phenotypic characteristics contributes to the early diagnosis of AL and detection of minimal residual disease after treatment, which greatly improved AL treatment and prognosis. In this review the diagnosis and typing, clinical characteristics, flow cytometry, immunophenotyping of bone marrow cells, immunoglobulin light chain restriction and phenotypic characteristics of abnormal plasma cells of AL are briefly summarized.
Amyloidosis ; diagnosis ; Bone Marrow Cells ; cytology ; Humans ; Immunoglobulin Light-chain Amyloidosis ; Plasma Cells ; cytology

Adolescent ; Child ; Collagen Type I ; metabolism ; Extracellular Matrix ; metabolism ; Female ; Fibrillin-1 ; Fibrillins ; Heart Defects, Congenital ; metabolism ; pathology ; Humans ; Male ; Microfilament Proteins ; metabolism

OBJECTIVETo clone IgHV genes from childhood B-ALL cells and identify CTL epitopes deduced from IgHV gene.

METHODSSeven IgHV gene families were respectively amplified by PCR and directly sequenced for 37 childhood B-ALL cases. Bioinformatics were applied for analyzing characteristics of sequences available and predicting HLA-A*0201 molecule-binding nonapeptides derived from IgHV. The predicted nonapeptide QLVQSGAEV was synthesized and its binding affinity to T2 cells determined. CD8+ T cells from a healthy HLA-A*0201+ donor peripheral blood were stimulated repeatedly with QLVQSGAEV-loaded antigen presenting cells.

RESULTSIgHV gene rearrangements were identified in 37 B-ALL. Forty IgHV gene sequences available preferentially utilized V(H)4-59 and V(H)4-34 gene segments. Increased frequency (15.4%) of D7-27 in B-ALL IgHV was found compared to that reported for adult PBLs (P = 0.02); 20.0% DJ(H) junctions in B-ALL lacked non-encoding nucleotides, a frequency higher than that reported for adult PBLs (P = 0.02). 17.5% B-ALL IgHV contained < 2% replacement mutations. Forty B-ALL IgHV sequences acquired 12 high HLA-A*0201-binding nonapeptides, 10 (83.0%) peptides were located in frame region (FR)1 and 3. The synthesized peptide QLVQSGAEV up-regulated HLA-A*0201 expression 1.63 fold on the surface of T2 cells. The frequency of QLVQSGAEV-specific CD8+ T cells in a healthy HLA-A*0201+ donor peripheral blood increased from 1.6% and 82.6% after two-round and 3-round stimulations, respectively.

CONCLUSIONIgHV genes in childhood B-ALL are of germline characteristics. Their heavy chain framework regions contain HLA-A*0201-binding nonapeptides. These peptides are capable of inducing specific CD8+ T cells to activate and proliferate.

Adolescent ; Burkitt Lymphoma ; genetics ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Child ; Child, Preschool ; Epitopes, T-Lymphocyte ; immunology ; Gene Rearrangement ; HLA-A Antigens ; immunology ; metabolism ; HLA-A2 Antigen ; Humans ; Immunoglobulin Heavy Chains ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Infant ; Oligopeptides ; immunology

BACKGROUNDImmunoglobulin heavy chain variable region (IgHV) is a well-characterized tumor antigen for B-cell malignancies. It can function as a target for T cell-mediated immune response. Clinical trials of IgHV protein vaccines against lymphoma have demonstrated induction of tumor-specific cytotoxic T lymphocyte (CTL) responses. However, complementary determining regions-based individual vaccines have disadvantages for wide clinical application. Although a recent study demonstrated that immunogenic peptides are derived from framework regions (FR) shared among patients with B-cell lymphoma, how to choose the appropriate peptides for each patient is still unsolved. The aim of this study was to investigate whether immunoglobulin heavy chain FR-derived peptides shared in each IgHV family are potential CTL epitopes presented by B-cell acute lymphoblastic leukemia (B-ALL). Such CTL epitopes might be beneficial to shifting vaccination strategies against B-ALL from individual specificity to family specificity.

METHODSSeven IgHV gene families were amplified respectively by PCR and sequenced directly from 71 childhood B-ALL cases. Bioinformatics was applied in analyzing characteristics of sequences available and predicting HLA-A*0201-restricted CTL epitopes for each IgHV family. An antigen-specific T cell expansion system was used to generate peptide-specific CTLs. The cytotoxicity of CTLs against B-ALL cells was assessed in the lactate dehydrogenase release assay.

RESULTSComplete IgHV rearrangements were identified in all of the 71 B-ALL cases. All of 40 sequences available showed > or = 98% homology with the nearest germline IgHV genes, indicating IgHV genes in B-ALL of germline nature. Twelve nonapeptides of high HLA-A*0201-binding scores were obtained from 26 productive IgHV protein sequences. Ten (83%) of the peptides were located in FR1 and FR3 shared among the corresponding IgHV family. CTLs specific for the peptide QLVQSGAEV located in FR1 (3 - 11) shared among the IgHV1 family could be successfully generated from peripheral blood mononuclear cells of two HLA-A*0201 + healthy donors in vitro and were capable of killing HLA-matched B-ALL cell clones belonging to the IgHV1 family.

CONCLUSIONAnti-B-ALL CTLs against immunoglobulin heavy chain FR-derived peptides have family-specific cytotoxicity.

Amino Acid Sequence ; Burkitt Lymphoma ; genetics ; immunology ; Epitopes, T-Lymphocyte ; genetics ; immunology ; Genes, Immunoglobulin Heavy Chain ; genetics ; Humans ; Immunoglobulin Heavy Chains ; chemistry ; genetics ; immunology ; Immunoglobulin Variable Region ; chemistry ; genetics ; immunology ; Molecular Sequence Data ; Oligopeptides ; immunology ; Polymerase Chain Reaction ; Protein Binding ; T-Lymphocytes, Cytotoxic ; immunology

The Cre recombinase from bacteriophage P1 can recognize specific DNA sequences, cleave DNA at specific target sites, and then ligate it to the cleaved DNA of a second site. In this study, cre gene was cloned into the pGEM-T Easy vector via PCR procedure. Then the cre gene was inserted into an expression vector pET-29a and expressed in E. coli BL21 (DE3). A 38 kD soluble protein was expressed and named CRE. CRE was purified by DEAE-52 chromatography. Bioassay of the partially purified product showed that CRE can cleave the plasmid pGLGFP which contains two loxP site with the same direction.
Chromatography, DEAE-Cellulose ; Escherichia coli ; genetics ; Gene Expression Regulation, Enzymologic ; Green Fluorescent Proteins ; Integrases ; genetics ; metabolism ; Luminescent Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; isolation & purification ; metabolism ; Viral Proteins ; genetics ; metabolism