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PubMed1997

Defects in glucuronate biosynthesis disrupt Wingless signaling in Drosophila.

In vitro experiments suggest that glycosaminoglycans (GAGs) and the proteins to which they are attached (proteoglycans) are important for modulating growth factor signaling. However, in vivo evidence to support this view has been lacking, in part because mutations that disrupt the production of GAG polymers and the core proteins have not been available. Here we describe the identification and characterization of Drosophila mutants in the suppenkasper (ska) gene. The ska gene encodes UDP-glucose dehydrogenase which produces glucuronic acid, an essential component for the synthesis of heparan and chondroitin sulfate. ska mutants fail to put heparan side chains on proteoglycans such as Syndecan. Surprisingly, mutant embryos produced by germ-line clones of this general metabolic gene exhibit embryonic cuticle phenotypes strikingly similar to those that result from loss-of-function mutations in genes of the Wingless (Wg) signaling pathway. Zygotic loss of ska leads to reduced growth of imaginal discs and pattern defects similar to wg mutants. In addition, genetic interactions of ska with wg and dishevelled mutants are observed. These data demonstrate the importance of proteoglycans and GAGs in Wg signaling in vivo and suggest that Wnt-like growth factors may be particularly sensitive to perturbations of GAG biosynthesis.

Amino Acid Sequence↗

PubMed2026

A guide to selecting high-performing antibodies for Syntenin-1 (O00560) for use in western blot, immunoprecipitation, and immunofluorescence.

Syntenin-1 is the Syndecan-binding protein 1 and a PDZ domain-containing adaptor protein that regulates diverse cellular processes through its interactions with transmembrane receptors, cytoskeletal components, and signaling molecules. Here we have characterized twelve Syntenin-1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Syntenins↗

PubMed2005

Decreasing the metastatic potential in cancers--targeting the heparan sulfate proteoglycans.

The heterogeneity of proteoglycans (PG)s contributes to their functional diversity. Many functions depend on their ability to bind and modulate the activity of components of the extracellular matrix (ECM). The ability of PGs to interact with other molecules, such as growth factors, is largely determined by the fine structure of the glycosaminoglycan (GAG) chains. Tumorigenesis is associated with changes in the PG synthesis. Heparan sulfate (HS) PGs are involved in several aspects of cancer biology including tumor progression, angiogenesis, and metastasis. PGs can have both tumor promoting and tumor suppressing activities depending on the protein core, the GAG attached, molecules they associate with, localization, the tumor subtype, stages, and degree of tumor differentiation. Perlecan is an angiogenic factor involved in tumor invasiveness. The C-terminal domain V of perlecan, named endorepellin, has however been shown to inhibit angiogenesis. Another angiogenic factor is endostatin, the COOH-terminal domain of the part-time PG collagen XVIII. Glypicans and syndecans may promote local cancer cell growth in some cancer tissues, but inhibit tissue invasion and metastasis in others. The GAG hyaluronan (HA) promotes cancer growth by providing a loose matrix for migrating tumor cells and mediates adhesion of cancer cells. HSPG degrading enzymes like heparanase, heparitinase, and other enzymes such as hyaluronidase and MMP are also important in tumor metastasis. Several different treatment strategies that target PGs have been developed. They have the potential to be effective in reducing tumor growth and inhibit the formation of metastases. PGs are also valuable tumor markers in several cancers.

Angiogenesis Inhibitors↗

PubMed1999

Fatty acids modulate the composition of extracellular matrix in cultured human arterial smooth muscle cells by altering the expression of genes for proteoglycan core proteins.

In diabetes-associated microangiopathies and atherosclerosis, there are alterations of the extracellular matrix (ECM) in the intima of small and large arteries. High levels of circulating nonesterified fatty acids (NEFAs) are present in insulin resistance and type 2 diabetes. High concentrations of NEFAs might alter the basement membrane composition of endothelial cells. In arteries, smooth muscle cells (SMCs) are the major producers of proteoglycans and glycoproteins in the intima, and this is the site of lipoprotein deposition and modification, key events in atherogenesis. We found that exposure of human arterial SMCs to 100-300 micromol/albumin-bound linoleic acid lowered their proliferation rate and altered cell morphology. SMCs expressed 2-10 times more mRNA for the core proteins of the proteoglycans versican, decorin, and syndecan 4 compared with control cells. There was no change in expression of fibronectin and perlecan. The decorin glycosaminoglycan chains increased in size after exposure to linoleic acid. The ECM produced by cells grown in the presence of linoleic acid bound 125I-labeled LDL more tightly than that of control cells. Darglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, neutralized the NEFA-mediated induction of the decorin gene. This suggests that some of the NEFA effects are mediated by PPAR-gamma. These actions of NEFAs, if present in vivo, could contribute to changes of the matrix of the arterial intima associated with micro- and macroangiopathies.

Aggrecans↗

PubMed2005

[Development and congenital malformations of the skeleton].

The article presents current knowledge on skeleton development, which is initiated by mesenchymal cells condensation. The mesenchymal to epithelial transformation, vascular regression, cartilage formation and ossification are involved in normal skeletogenesis. All the processes are influenced by various genes and regulatory molecules, e.g., N-cadherin, vascular endothelial growth factor, transforming growth factor-beta, syndecan-3 and others. The chondrogenesis depends on the Sox genes expression - Sox5, Sox6 and especially Sox9, which continued action produce persistent cartilage. The essential role in osteogenesis is played by the transcription factor Runx2 that directly activates expression of genes coding typical bone proteins like collagen type I, osteocalcin, osteopontin and osteonectin. Development of fibrous, cartilaginous and synovial joints, bone maturation and remodeling, and mechanisms leading to skeleton dysmorphogenesis are also discussed.

Antigens, CD↗

PubMed1992

Proteoglycans: many forms and many functions.

Proteoglycans are produced by most eukaryotic cells and are versatile components of pericellular and extracellular matrices. They belong to many different protein families. Their functions vary from the physical effects of the proteoglycan aggrecan, which binds with link protein to hyaluronan to form multimolecular aggregates in cartilage; to the intercalated membrane protein CD44 that has a proteoglycan form and is a receptor and a cell-binding site for hyaluronan; to heparan sulfate proteoglycans of the syndecan and other families that provide matrix binding sites and cell-surface receptors for growth factors such as fibroblast growth factor (FGF). One feature that recurs in proteoglycan biology is that their structure is open to extensive modulation during cellular expression. Examples of protein changes are known, but a major source of structural variation is in the glycosaminoglycan chains. The number of chains and their length can vary, as well as their pattern of sulfation. This may result in the switching of different chain types with different properties, e.g., chondroitin sulfate and heparan sulfate, and it may also result in the selective expression of sulfated chain sequences that have specific functions. The control of glycosaminoglycan structure is not well understood, but it does appear to be used to change the properties of proteoglycans to suit different biological needs. Proteoglycan forms of proteins are thus important modifiers of the organization of the pericellular and extracellular matrices and modulators of the processes that occur there.

Aggrecans↗

PubMed1993

Potential markers (enzymes, proteoglycans) for human liver tumors.

In the present work the activities of GGT and G-6-Pase and the content of Cyt P-450 were determined in surgically removed liver specimens (16 hepatocellular carcinomas, 8 focal nodular hyperplasias and 4 adenomas). The activities were compared to the surrounding seemingly normal liver tissue. In the adenomas neither of the enzymes studied showed alterations, characteristic for hepatocarcinogenesis. Four out of 8 FNHs had the enzyme pattern that was found in experimental liver carcinogenesis. Liver carcinoma specimens proved to be heterogenous. Neither elevated GGT nor reduced G-6-Pase activity was consistent in these samples although the average of G-6-Pase activity decreased to 50 percent. Cytochrome P-450 was significantly reduced in the majority of cases, showing the best agreement with the tendency observed in experimental models. As an other approach, the qualitative and quantitative alterations of proteoglycans (PG) were analized in the same tumor samples. The amount of sugar components of PGs the glycosaminoglycans (GAG) increased by many times in liver tumors. Carcinoma samples were characterized by about twentyfold increase in chondroitin sulfate content, compared to normal liver. The enhancement of GAGs is partly the consequence of a selective alteration in PG expression. The amount of perlecan and decorin was found to be increased, while syndecan disappeared from liver carcinomas. These data suggest that malignant transformation in liver is accompanied by specific alteration in the content, composition and structure of PGs. Presumably, these changes have significance in tumor progression and have also the potential to be used as markers for liver tumors.

Adenoma↗

PubMed1999

Coordinated modulation of the fibroblast growth factor dual receptor mechanism during transformation from human colon adenoma to carcinoma.

Basic fibroblast growth factor (bFGF) is dependent on heparan sulphate for its ability to activate the cell surface signal transducing receptor. We have investigated the FGF dual receptor mechanism in a novel model of the transformation from human colon adenoma to carcinoma in vitro. Reverse transcription-polymerase chain reaction showed that mRNA for FGF receptors 1 and 2 were expressed in both the adenoma and carcinoma cells whereas immunocytochemistry showed that the expression of the FGF R1 was reduced significantly in the carcinoma cells. We have reported previously that the composition and sequence of human colon adenoma and carcinoma heparan sulphate (HS) differ in a defined and specific manner. The functional significance of these changes was assessed by affinity co-electrophoresis, which showed that the affinity of adenoma HS for bFGF was 10-fold greater than that of the carcinoma HS (Kd 220 nM vs. 2493 nM, respectively). In addition, Northern studies of the expression of syndecan 1 and 4 mRNA showed that proteoglycan core protein expression was reduced significantly in the carcinoma cells. These findings were associated with a reduced biological response to bFGF in the carcinoma cells that could be partially reversed by the addition of exogenous heparin, suggesting that both the proteoglycan and signal transducing receptor control the cells' response to bFGF.

Adenoma↗

PubMed1997

FGF-2 converts mature oligodendrocytes to a novel phenotype.

Fibroblast growth factor (FGF)-2 differentially regulates oligodendrocyte progenitor proliferation and differentiation in culture, and modulates gene expression of its own receptors, in a developmental and receptor type-specific manner (Bansal et al., 1996a,b). Three FGF receptors (types 1, 2, 3) are expressed in postmitotic, terminally differentiating oligodendrocytes. Exposure of such cells to FGF-2 results in: (a) the down-regulation of myelin-specific gene expression (e.g., ceramide galactosyltransferase, 2',3'-cyclic nucleotide 3'-phosphohydrolase, myelin basic protein, proteolipid protein), (b) dramatic increases in the length of cellular processes in a time- and dose-dependent manner, (c) re-entrance into the cell cycle without accompanying mitosis, and (d) the alteration of the expression of both low- and high-affinity FGF receptors. Compared to oligodendrocyte progenitors, the differentiated oligodendrocytes treated with FGF-2 incorporate BrdU at a slower rates, exhibit different patterns of both FGF high- and low-affinity (syndecans) receptors, and are morphologically very different. In addition, they do not re-express the progenitor markers A2B5, NG2 or PDGFalpha receptor. Therefore, although the FGF-treated cells lose their differentiated OL/myelin markers, they do not revert to progenitors and clearly represent a different, apparently novel, phenotype both morphologically and biochemically, which we have termed NOLs. These data indicate that terminally differentiated oligodendrocytes retain the plasticity to reprogram their differentiation fate under the influence of environmental factors. The possible significance of this response to FGF relative to normal and pathological physiology is discussed. In particular, on the basis of these data we predict the appearance of cells in and around multiple sclerosis plaques with the phenotype O4+, NG2-, A2B5-, O1-, MBP-.

Animals↗

PubMed1996

Specific involvement of glypican in thrombin adhesive properties.

We have previously demonstrated that thrombin possesses an active yet cryptic Arg-Gly-Asp (RGD) site which upon exposure induces endothelial cell (EC) adhesion via alpha nu beta 3 integrin [Bar-Shavit et al. (1991): J Cell Biol 112:335]. This was achieved in the presence of cell surface-associated heparan sulfate proteoglycans (HSPG) and exceedingly low concentrations of plasmin [Bar-Shavit et al. (1993): J Cell Biol 123:1279]. A portion of the cell surface-associated HSPG (glypican) is anchored via a covalently linked glycosyl-phosphatidylinositol (PI) residue, which can be released by treatment with glycosyl-PI-specific phospholipase C (PI-PLC). We report here that exposure of either bovine aortic EC, smooth muscle cells (SMC), or wild-type CHO cells to PI-PLC released HSPG involved in the conversion of thrombin to an adhesive molecule. The adhesion-promoting activity of the released HSPG was abolished following treatment with heparinase but not chondroitinase ABC. Incubation of thrombin with heparan sulfate-deficient CHO cells or cells that were pretreated with PI-PLC failed to induce its conversion to an adhesive molecule, indicating that glypican was playing a major role in this conversion. Moreover, affinity-purified glypican, but not syndecan or fibroglycan, elicited efficient conversion of plasmin-treated thrombin into an adhesive molecule. Antibodies raised against the RGD site in thrombin failed to interact with native thrombin, prothrombin, or the RGD site in other adhesive proteins such as vitronectin, fibrinogen, or fibronectin. Anti-thrombin-RGD antibodies which blocked the adhesion-promoting activity of thrombin were also capable of recognizing thrombin that was first incubated with a suboptimal concentration of plasm in in the presence of PI-PLC-released HSPG. Heparin, heparan sulfate, and PI-PLC-released HSPG had no effect on other cellular properties of thrombin such as receptor binding and growth-promoting activity. Altogether we have demonstrated that the heparin binding domain in thrombin plays a specific role in promoting thrombin adhesive properties and that membrane-associated glypican is likely to be the major physiological inducer of this property.

Animals↗

PubMed1996

Shedding of heparan sulfate proteoglycan by stimulated endothelial cells: evidence for proteolysis of cell-surface molecules.

Activation of endothelial cells by cytokines and endotoxin causes procoagulant and pro-inflammatory changes over a period of hours. We postulated that the same functional state might be achieved more rapidly by changes in the metabolism of heparan sulfate, which supports many of the normal functions of endothelial cells. We previously found that binding of anti-endothelial cell antibodies and activation of complement on endothelial cells causes the rapid shedding of endothelial cell heparan sulfate. Here we report the biochemical mechanism responsible for the release of the heparan sulfate. Stimulation of endothelial cells by anti-endothelial cell antibodies and complement resulted in the release of 35S-heparan sulfate proteoglycan and partially degraded 35S-heparan sulfate chains. Degradation of the 35S-heparan sulfate chains was not necessary for release since heparin and suramin prevented cleavage of the heparan sulfate but did not inhibit release from stimulated endothelial cells. The 35S-heparan sulfate proteoglycan released from endothelial cells originated from the cell surface and had a core protein similar in size (70.5 kD) to syndecan-1. Release was due to proteolytic cleavage of the protein core by serine and/or cysteine proteinases since the release of heparan sulfate was inhibited 87% by antipain and 53% by leupeptin. Release of heparan sulfate coincided with a decrease of approximately 7 kD in the mass of the protein core and with a loss of hydrophobicity of the proteoglycan, consistent with the loss of the hydrophobic transmembrane domain. The cleavage and release of cell-surface 35S-heparan sulfate proteoglycan might be a novel mechanism by which endothelial cells may rapidly acquire the functional properties of activated endothelial cells.

Animals↗

PubMed1998

Disrupted expression of matrix genes in the growth plate of the mouse cartilage matrix deficiency (cmd) mutant.

Chondrodysplasia in the autosomal recessive cartilage matrix deficiency (cmd) mutant is caused by lack of the proteoglycan aggrecan arising from a mutation in the gene. Homozygous cmd/cmd mice are characterized by disorganisation of chondrocytes in the growth plate, disproportionate dwarfism, cleft palate, and perinatal lethality. We have studied the impact of the aggrecan deficiency on the expression of other matrix genes during the differentiation of chondrocytes in the growth plate of cmd/cmd 18.5 day fetuses. Compared with the wild-type, there are significant differences in the growth plates of cmd mutants in the combinations of co-expression of genes encoding the glycoprotein link protein, proteoglycan syndecan 3, collagens alpha 1 (X) [Col10a1], alpha 2(XI) [Col11a2], and the alternative transcripts of alpha 1 (II) [Col2a1 type IIA form], and alpha 1 (IX) [Col9a1 long and short forms]. The discordance of gene expression in cmd chondrocytes may be additional factors contributing to the disrupted cellular architecture of the growth plate resulting from the primary absence of aggrecan.

Aggrecans↗

PubMed2000

All for one and one for all: condensations and the initiation of skeletal development.

Condensation is the pivotal stage in the development of skeletal and other mesenchymal tissues. It occurs when a previously dispersed population of cells gathers together to differentiate into a single cell/tissue type such as cartilage, bone, muscle, tendon, kidney, and lung and is the earliest stage during organ formation when tissue-specific genes are upregulated. We present a synopsis of our current understanding of how condensations are initiated and grown, how their boundaries and sizes are set, how condensation ceases, and how overt differentiation begins. Extracellular matrix molecules, cell surface receptors and cell adhesion molecules, such as fibronectin, tenascin, syndecan, and N-CAM, initiate condensation formation and set condensation boundaries. Hox genes (Hoxd-11-13) and other transcription factors (CFKH-1, MFH-1, osf-2), modulate the proliferation of cells within condensations. Cell adhesion is ensured indirectly through Hox genes (Hoxa-2, Hoxd-13), and directly via cell adhesion molecules (N-CAM and N-cadherin). Subsequent growth of condensations is regulated by BMPs, which activate Pax-2, Hoxa-2 and Hoxd-11 among other genes. Growth of a condensation ceases when Noggin inhibits BMP signalling, setting the stage for transition to the next stage of skeletal development, namely overt cell differentiation. BioEssays 22:138-147, 2000.

Animals↗

PubMed2000

Mesenchymal-epithelial transition in the developing metanephric kidney: gene expression study by differential display.

The developing metanephric kidney is a convenient model to study molecular events associated with epithelial cell differentiation. To determine the genes involved in the defining event of this process, namely, the conversion of metanephric mesenchyme to the epithelium of the nephron, we applied differential display (DD) techniques. Explants of rat metanephric mesenchymes were induced to condense ex vivo with fibroblast growth factor 2 (FGF2) or to form tubules with FGF2 and conditioned medium (CM) from a cell line (RUB1) of ureteric bud, the renal inductive tissue. Three time points (6, 24, and 72 h) were chosen to track the dynamics of gene expression during morphogenesis. Seventy-two up- or down-regulated mRNAs were identified, including 36 novel sequences and those of cell cycle regulatory proteins (TGF-beta2, Cyclin D1, p57Kip2), transcription factors (beta-catenin, Sox11, DP1), signaling proteins (SH3-domain binding protein, G-protein-coupled receptor, Ser-Thr protein kinase), cell adhesion molecules (syndecan-4, integrin-beta1), and also gene33, H19, SM20, IGFBP5, MAMA receptor, lectin, keratin, beta-tubulin, calreticulin, GRP78, ERp72, MnSoD, thioredoxin, and others. Some have previously been associated with kidney development and serve as good controls for expected changes, while most have not been linked with kidney epithelial cell differentiation. Using thin sections of embryonic kidney and labeled antisense RNA probes, we applied RNA hybridization to confirm the results of DD and related the expression of these genes to specific cell lineages of the developing kidney. These results provide a window into the events that mediate this critical differentiation process and suggest that a limited number of interrelated events direct the epithelial conversion of metanephric mesenchyme. genesis 27:22-31, 2000. Published 2000 Wiley-Liss, Inc.

Animals↗

PubMed1992

Epithelial-mesenchymal interactions in murine organogenesis.

Reciprocal, sequential interactions between embryonic epithelial and their mesenchymal stroma guide the cytodifferentiation and organization of both components. These morphogenetic interactions and their consequences are examined in two model systems in vitro: the mouse metanephric blastema and the tooth rudiment. Experimental approaches include dissection and recombination of the interacting tissues, localization of molecular changes by immunohistology and in situ hybridization. An early response of the mesenchyme is increased proliferation of cells in the vicinity of the epithelial inductor and their subsequent aggregation (condensation). In the kidney model disruption of this aggregation or prevention of assembly of the programmed cells results in impaired cytodifferentiation. If the cells are allowed to reaggregate, a phenotype is expressed not unlike that seen in normal in vivo conditions. Our present interest is focused on the early metabolic events associated with the condensation phenomenon. The cell surface proteoglycan syndecan and the matrix glycoprotein tenascin are expressed in the condensed mesenchyme and may mediate cell-matrix interactions. The expression patterns of certain growth factors suggest functions in signal transduction.

Animals↗

PubMed2002

Comparison of the biologic effects of MA5 and B-B4 monoclonal antibody labeled with iodine-131 and bismuth-213 on multiple myeloma.

BACKGROUND: Using a specific monoclonal antibody (MAb), B-B4, coupled to bismuth-213 ((213)Bi) by a chelating agent (CITC-DTPA), the feasibility of alpha-radioimmunotherapy (RIT) for multiple myeloma (MM) has been demonstrated previously. METHODS: In this study, the two MAbs tested, MA5 and B-B4, target the epithelial antigens Muc-1 and syndecan-1, respectively, which are both expressed by MM cell lines. Antibody characterization was evaluated by flow cytometric analysis of normal and tumoral hematopoeitic cells of MM patients as well as immunohistochemical tests of normal, nonhematopoetic tissues. Radiobiologic effects were evaluated for (213)Bi- and iodine-131 ((131)I)--labeled antibodies. We assessed in vitro mortality (thymidine incorporation, MTT, and clonogenic assays) and cell cycle modifications with propidium iodide staining. These tests were performed on MM cell lines until 120 hours postirradiation at several time points, using radiolabeled antibody concentrations ranging from 0.5 to 20 nM and specific activities ranging from 240 to 1200 MBq/mg of MAb. RESULTS: MA5 stained all MM cells in only 50% of patients, whereas B-B4 recognized all MM cells in all patients. B-B4 principally showed hepatic, pulmonary, and duodenal staining, whereas MA5 marked renal and pulmonary tissues. RIT with (213)Bi-B-B4 induced specific mortality and G(2)/M phase cell cycle arrest, which depended on the concentrations and specific activity. For (213)Bi-MA5, this arrest appeared at concentrations above 10 nM, an amount fivefold higher than that required with B-B4. This difference was also found in thymidine incorporation assays. Furthermore, with (213)Bi-B-B4, the arrest at the G(2)/M phase appeared quickly, within 24 hours after irradiation, and affected up to 60% of the cells (for 20 nM of (213)Bi-B-B4 at 1,200 MBq/mg). Conversly, (131)I-B-B4 had a very limited effect on cell mortality and did not induce any cell cycle arrest. CONCLUSIONS: The results of this study show that B-B4 might be the more effective therapeutic antibody and suggest that alpha-RIT might be more suitable than beta-RIT for treating single-cell tumor models. Thus, these findings set the stage for the beginning of clinical trials using alpha-emitter--radiolabeled B-B4, with special attention paid to hepatic, pulmonary, and intestinal side effects.

Antibodies, Monoclonal↗

PubMed1992

Functional and molecular characterization of single, (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific, IgG1+ B cells from antibody-secreting and memory B cell pathways in the C57BL/6 immune response to NP.

We have used multiparameter flow cytometry to identify a population of IgG1+ IgM- antigen-specific B cells which emerges in spleens of C57BL/6 mice following immunization with the hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP). Characterization of the specificities of IgG1 antibodies produced by single, sorted IgG1+ NP+ cells in both Elispot assays and in microcultures containing lipopolysaccharide, interleukin (IL)-2, IL-4 and IL-5 indicates that the splenic IgG1+ NP+ B cell population includes both IgG1 anti-NP antibody-secreting cells and non-secreting, IgG1+ memory B cells. Each functionally discrete population of IgG1+ B cells expresses a distinctive surface phenotype defined by a wide range of B cell markers. In particular, antibody-secreting, IgG1+ cells were uniquely identified by co-expression of the matrix receptor, syndecan. The NP-specific B cell population emerging in the day 7 primary response was assessed for clonotypic diversity by amplification and direct sequencing of the rearranged V186.2 heavy chain variable region gene expressed by single, ex vivo IgG1+ NP+ lambda+ B cells. Memory B cell clones, distinguished by junctional diversity, carried either no mutation or a single mutation within rearranged V186.2, suggesting isolation of these cells at the onset of the hypermutation mechanism. This novel approach, therefore, allows the direct and unambiguous identification and characterization of individual B cell clonotypes during their initial selection and activation in antibody responses in vivo.

Animals↗

PubMed1996

The phenotype and fate of the antibody-forming cells of the splenic foci.

The first product of the humoral response to antigen is low-affinity antibody, produced by extrafollicular foci of antibody-forming cells (AFC) in organs such as spleen and lymph node. These cells proliferate rapidly but then undergo an equally rapid decline, so that they are present in only small numbers 14 days after immunization. We have used 6-parameter flow cytometry to isolate and examine the characteristics of (4-hydroxy-5-nitrophenyl)acetyl-specific AFC, looking in particular for those markers that might differentiate them from cells of the intrafollicular (germinal center) arm of the T-dependent immune response. At day 7 of the primary response, most AFC were found to express surprisingly low levels of B220, high levels of syndecan, and retain significant levels of surface IgG1. We then used enzyme-linked immunospot assays to demonstrate that the rapid decline of these cells was not likely to be due to migration to organs such as the bone marrow. Their decline could, however, be explained by apoptosis in situ, which was demonstrated immunohistologically by nick-end labeling.

Animals↗