(1/945) Patterns of A2A extracellular adenosine receptor expression in different functional subsets of human peripheral T cells. Flow cytometry studies with anti-A2A receptor monoclonal antibodies.
Signaling through A2A adenosine receptors (A2AR) regulates T lymphocyte expansion and modulates T cell receptor (TCR)-mediated effector functions in vitro. To understand the role of A2ARs in the regulation of immune response, we investigated the expression levels of this receptor in different functional lymphocyte subsets. Monoclonal anti-A2AR antibody was used to develop a flow cytometric assay to quantify the expression A2ARs on lymphocytes. We report that detectable levels of expression of A2ARs are much higher among T cells than B cells. More CD4(+) than CD8(+) T cells express A2ARs, but activation of T cells increases A2AR expression, predominantly in CD8(+) T cells. No significant differences were found in the proportion of A2AR+ cells between CD8(low) and CD8(high) T cells or between TCR/CD3(low) and TCR/CD3(high) T cells. Studies of T helper cell subsets (TH1 and TH2) reveal that lymphokine-producing cells are much more likely to express A2ARs than are cells that do not produce lymphokines. These results suggest that A2ARs are variably expressed on T cell subsets and may regulate cytokine production in activated T lymphocytes. (+info)
(2/945) Immunohistochemical analysis of arterial wall cellular infiltration in Buerger's disease (endarteritis obliterans).
PURPOSE: The diagnosis of Buerger's disease has depended on clinical symptoms and angiographic findings, whereas pathologic findings are considered to be of secondary importance. Arteries from patients with Buerger's tissue were analyzed histologically, including immunophenotyping of the infiltrating cells, to elucidate the nature of Buerger's disease as a vasculitis. METHODS: Thirty-three specimens from nine patients, in whom Buerger's disease was diagnosed on the basis of our clinical and angiographic criteria between 1980 and 1995 at Nagoya University Hospital, were studied. Immunohistochemical studies were performed on paraffin-embedded tissue with a labeled streptoavidin-biotin method. RESULTS: The general architecture of vessel walls was well preserved regardless of the stage of disease, and cell infiltration was observed mainly in the thrombus and the intima. Among infiltrating cells, CD3(+) T cells greatly outnumbered CD20(+) B cells. CD68(+) macrophages or S-100(+) dendritic cells were detected, especially in the intima during acute and subacute stages. All cases except one showed infiltration by the human leukocyte antigen-D region (HLA-DR) antigen-bearing macrophages and dendritic cells in the intima. Immunoglobulins G, A, and M (IgG, IgA, IgM) and complement factors 3d and 4c (C3d, C4c) were deposited along the internal elastic lamina. CONCLUSION: Buerger's disease is strictly an endarteritis that is introduced by T-cell mediated cellular immunity and by B-cell mediated humoral immunity associated with activation of macrophages or dendritic cells in the intima. (+info)
(3/945) Plasma cell development in synovial germinal centers in patients with rheumatoid and reactive arthritis.
Plasma cells are found surrounding the inflammatory infiltrates of macrophages, T, and B cells in the synovial tissue of patients with rheumatoid and reactive arthritis. This characteristic arrangement suggests that in the synovial tissue CD20+ B cells differentiate into plasma cells. To examine clonal relationships, we have used micromanipulation to separately isolate CD20+ B cells and plasma cells from single infiltrates. DNA was extracted, and from both populations the VH/VL gene repertoires was determined. The data show that in the inflamed synovial tissue activated B cells are clonally expanded. During proliferation in the network of follicular dendritic cells, V gene variants are generated by the hypermutation mechanism. Surprisingly, we do not find identical rearrangements between CD20+ B cells and plasma cells. Nevertheless, the finding of clonally related plasma cells within single infiltrates suggests that these cells underwent terminal differentiation in the synovial tissue. These results indicate that B cell differentiation in the synovial tissue is a dynamic process. Whereas CD20+ B cells may turnover rapidly, plasma cells may well be long lived and thus accumulate in the synovial tissue. The analysis of individual B cells recovered from synovial tissue opens a new way to determine the specificity of those cells that take part in the local immune reaction. This will provide new insights into the pathogenesis of chronic inflammatory diseases like rheumatoid or reactive arthritis. (+info)
(4/945) Histopathologic features and expression of Bcl-2 and p53 proteins in primary gastric lymphomas.
The aim of this study is to present a histopathologic and immunohistochemical analysis of primary gastric lymphomas which were reclassified according to the concept of mucosa associated lymphoid tissue (MALT). The resected specimens from 41 patients with primary gastric lymphoma were investigated retrospectively. Immunohistochemical study was done to analyze the immunophenotype and bcl-2 and p53 proteins expression. Twenty three of the cases had tumors mainly located in the antrum. Histologically, 12 were low grade and 20 were high grade B-cell lymphoma of MALT, 9 other B-cell nonHodgkin's lymphomas. Helicobacter pylori was identified in 72% of the cases. According to Musshoff's modification, most of the MALT lymphoma cases had stage I or II disease. There was significant difference between low and high grade cases, in respect to depth of invasion in gastric wall. Immunohistochemically, the neoplastic cells in all MALT lymphomas expressed B-cell phenotype. Bcl-2 protein was found to be expressed in 59% and p53 protein expression was detected in 72% of cases. Among the B-cell lymphoma of MALT, bcl-2 positivity decreased and p53 positivity increased significantly as the histological grade advanced. So, an inverse correlation was observed between the expression of bcl-2 and p53. In conclusion, most primary gastric lymphomas are low or high grade B-cell MALT lymphomas and appear to arise in MALT acquired as a reaction to Helicobacter pylori infection. Expression of bcl-2 and p53 in gastric lymphomas may be associated with transformation from low-grade to high-grade disease. (+info)
(5/945) Therapy of B-cell lymphoma with anti-CD20 antibodies can result in the loss of CD20 antigen expression.
Rituximab is a chimeric antibody with human gamma-1 and kappa constant regions and murine variable regions. It recognizes the CD20 antigen, a pan B-cell marker. Therapeutic trials in patients with B-cell non-Hodgkin's lymphoma (NHL) have shown significant efficacy with a primary response rate of 50%, and a secondary response rate of 44% after repeat treatments in prior responders. The selection for proliferating tumor cells that no longer express CD20 may compromise repeated treatment. We have identified a patient who developed a transformed NHL that lost CD20 protein expression after two courses of therapy with rituximab. In a pretreatment lymph node biopsy, 83% of B cells (as defined by CD19 and surface immunoglobulin) expressed surface CD20. A biopsy from the recurrent tumor after two courses of rituximab revealed a diffuse large cell NHL where 0% of B cells expressed CD20 with no evidence of bound rituximab. Cytoplasmic staining showed no CD20 protein. Sequencing of immunoglobulin heavy chain cDNA identified identical variable sequences in the initial and recurrent lymphomas, confirming the association between the two tumors. Literature and database review suggests that approximately 98% of diffuse large cell lymphomas express CD20, which suggests that these tumors rarely survive without CD20. This is the first identified case of loss of CD20 expression in a lymphoma that has relapsed after rituximab therapy, although several other cases have since been identified. Considering the significant number of patients treated with anti-CD20 antibodies, this may occur only rarely and is unlikely to preclude recurrent therapy with anti-CD20 antibodies in the majority of patients. However, because many patients have relapsed after anti-CD20 antibody therapy and have not been biopsied to identify clones with down-regulated CD20 antigen, we do not currently know the true frequency of this phenomenon. When possible, patients should undergo evaluation for CD20 expression before repeated courses of anti-CD20 therapy. (+info)
(6/945) Molecular analysis of single B cells from T-cell-rich B-cell lymphoma shows the derivation of the tumor cells from mutating germinal center B cells and exemplifies means by which immunoglobulin genes are modified in germinal center B cells.
T-cell-rich B-cell lymphoma (TCRBCL) belongs to the group of diffuse large cell lymphomas (DLL). It is characterized by a small number of tumor B cells among a major population of nonmalignant polyclonal T cells. To identify the developmental stage of the tumor progenitor cells, we micromanipulated the putative neoplastic large CD20(+) cells from TCRBCLs and amplified and sequenced immunoglobulin (Ig) V gene rearrangements from individual cells. In six cases, clonal Ig heavy, as well as light chain, gene rearrangements were amplified from the isolated B cells. All six cases harbored somatically mutated V gene rearrangements with an average mutation frequency of 15.5% for heavy (VH) and 5.9% for light (VL) chains and intraclonal diversity based on somatic mutation. These findings identify germinal center (GC) B cells as the precursors of the transformed B cells in TCRBCL. The study also exemplifies various means how Ig gene rearrangements can be modified by GC B cells or their malignant counterparts in TCRBCL: In one case, the tumor precursor may have switched from kappa to lambda light chain expression after acquiring a crippling mutation within the initially functional kappa light chain gene. In another case, the tumor cells harbor two in-frame VH gene rearrangements, one of which was rendered nonfunctional by somatic mutation. Either the tumor cell precursor entered the GC with two potentially functional in-frame rearrangements or the second VHDHJH rearrangement occurred in the GC after the initial in-frame rearrangement was inactivated by somatic mutation. Finally, in each of the six cases, at least one cell contained two (or more) copies of a clonal Ig gene rearrangement with sequence variations between these copies. The presence of sequence variants for V region genes within single B cells has so far not been observed in any other normal or transformed B lymphocyte. Fluorescence in situ hybridization (FISH) points to a generalized polyploidy of the tumor cells. (+info)
(7/945) Distribution of lymphocytes and adhesion molecules in human cervix and vagina.
Knowledge of the histological distribution of leucocytes and adhesion molecules in the human genital tract is scarce although local immunity in this region is important. Using immunohistochemical methods, we here describe the organization of CD3+, CD8+ and CD4+ T cells, CD19+ B cells, CD38+ plasma cells, major histocompatibility complex (MHC) class II+ antigen-presenting cells and CD14+ monocytes, as well as the expression of endothelial addressins in normal human ecto-cervical and vaginal mucosa. T cells were clustered in a distinct band beneath the epithelium and were also dispersed in the epithelium and the lamina propria, whereas CD38+ plasma cells were present only in the lamina propria. MHC class II+ cells were numerous in the lamina propria and in the epithelium, where they morphologically resembled dendritic cells. Lymphoid aggregates containing CD19+ and CD20+ B cells as well as CD3+, CD4+ and CD8+ cells were also found in the cervix. The mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was not expressed on the vascular endothelium in the cervical or vaginal mucosa. In contrast, intercellular adhesion molecule-1 (ICAM-1), vascular adhesion protein-1 (VAP-1) and P-selectin were expressed in all tissue samples, and vascular cell adhesion molecule-1 (VCAM-1) and E-selectin were found in four of seven samples. We conclude that the distribution of leucocytes and adhesion molecules is very similar in the ecto-cervical and the vaginal mucosa and that the regulation of lymphocyte homing to the genital tract is different from that seen in the intestine. Our results also clearly suggest that the leucocytes are not randomly scattered in the tissue but organized in a distinct pattern. (+info)
(8/945) Characterization of scFv-Ig constructs generated from the anti-CD20 mAb 1F5 using linker peptides of varying lengths.
The heavy (VH) and light (VL) chain variable regions of the murine anti-human CD20 mAb 1F5 were cloned, and four single-chain Ab (scFv) molecules were constructed using linker peptides of variable lengths to join the VH and VL domains. Three constructs were engineered using linker peptides of 15, 10, and 5 aa residues consisting of (GGGGS)3, (GGGGS)2, and (GGGGS)1 sequences, respectively, whereas the fourth was prepared by joining the VH and VL domains directly. Each construct was fused to a derivative of human IgG1 (hinge plus CH2 plus CH3) to facilitate purification using staphylococcal protein A. The aggregation and CD20 binding properties of these four 1F5 scFv-Ig derivatives produced were investigated. Both size-exclusion HPLC column analysis and Western blots of proteins subjected to nonreducing SDS-PAGE suggested that all four 1F5 scFv-Ig were monomeric with m.w. of approximately 55 kDa. The CD20 binding properties of the four 1F5 scFv-Ig were studied by ELISA and flow cytometry. The 1F5 scFv-Ig with the 5-aa linker (GS1) demonstrated significantly superior binding to CD20-expressing target cells, compared with the other scFv-Ig constructs. Scatchard analysis of the radiolabeled monovalent GS1 scFv-Ig revealed a binding avidity of 1.35 x 108 M-1 compared with an avidity of 7.56 x 108 M-1 for the native bivalent 1F5 Ab. These findings suggest that the GS1 scFv-Ig with a short linker peptide of approximately 5 aa is the best of the engineered constructs for future studies. (+info)