Heat-stable Antigen Research Articles

The heat-stable antigen can alter very late antigen 4-mediated adhesion.

The integrin very late antigen, (VLA-4) alpha 4 beta 1 and its counter receptor vascular cell adhesion molecule 1 (VCAM-1) are involved in B cell maturation and pre-B cell attachment to bone marrow stroma cells. We have analyzed whether heat-stable antigen (HSA), a marker for immature leukocytes, is involved in such cell adhesion phenomena. HSA is a glycolipid-anchored, highly glycosylated surface protein differentially expressed on cells during the maturation of both the hematopoietic and nervous systems. We found that pre-B cells lacking HSA (due to targeted disruption of both alleles) can still bind via VLA-4 to tumor necrosis factor alpha-stimulated endothelioma cells. This binding, however, cannot be blocked by an anti-VCAM-1 antibody. Restoration of HSA expression restores the inhibitable VCAM-1 binding. We also found that pre-B cells lacking HSA did not bind to the FN40 fragment of fibronectin but reexpression of HSA restored VLA-4-mediated binding to fibronectin. Thus, expression of HSA on pre-B cells modifies the binding specificity of VLA-4 for two known ligands.

A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [ 3H]thymidine incorporation assay

A one-step non-radioactive assay to determine the proliferation of murine lymphocytes, lymphoid tumor cells and hybridoma cells is described. This assay requires the addition of Alamar Blue dye to cell cultures and the degree of change in its color, which is reflective of the extent of cellular proliferation, can be determined by an ELISA plate reader. Alamar Blue must be added during the initial phase of cell culture. The pattern of concanavalin A (ConA) or anti-CD3 antibody-induced proliferative response of murine lymphocytes as assessed by Alamar Blue was similar to that of a [ 3H]thymidine assay. Similarly, the spontaneous proliferation curve of anti-CD3 antibody secreting cell line (YCD3-1), monocytic macrophage cell lines (PU5-1.8, P388D1, J774.A1) and myeloma cells (Sp2/0) as determined by Alamar Blue closely resembled that of the [ 3H]thymidine assay. The minimum detectable number of proliferating cells was comparable in Alamar Blue and [ 3H]thymidine assays. Since cell lysis/extraction and washing procedures are not involved in the Alamar Blue assay, this approach has several distinct advantages over currently available assays (eg. [ 3H]thymidine). First, it allows daily monitoring of proliferation without compromising the sterility of cultures. An indication of proliferation can be evaluated (spectrophotometrically or visually) as early as 24 h after ConA stimulation. Second, unlike previously reported assays, Alamar Blue permits further analysis of proliferating cells by other methods. Analysis of cells in culture with Alamar Blue for various surface antigens (CD44, CD45RB, CD4, heat stable antigen) by flow cytometry revealed that the fluorescent profile and relative percentage of cells in cultures with the Alamar Blue were comparable to those without this reagent. The salient advantages of Alamar Blue assay over the [ 3H]thymidine assay include: (i) non-radioactivity; (ii) simplicity; (iii) less costly; (iv) non-labor intensive; (v) rapidity of assessment of proliferation of large number of samples; (vi) non-toxicity; (vii) usefulness in determining the kinetics of cell growth of hybridomas; and (viii) non-interference of secretion of antibodies by a hybridoma cell line.

Heat-stable antigen is an important costimulatory molecule on epidermal Langerhans' cells.

Heat-stable antigen (HSA), expressed by activated B cells, has been described as a costimulatory molecule for CD4+ T cells. Because epidermal Langerhans cells (LC) are known to express HSA, we determined whether LC HSA also served as a costimulator of Th cells. We have confirmed that HSA is expressed by freshly prepared (fresh) and, to a lesser extent, short-term cultured (cultured) LC and we demonstrate that costimulatory effects of HSA are prominent on fresh and 1-day cultured LC, whereas 2- to 4-day cultured LC exhibit less HSA-costimulatory activity. The anti-HSA mAb 20C9 almost completely blocked the proliferative response of Th1 or lymph node T cells induced by fresh or cultured LC. 20C9 also specifically inhibited LC-dependent IL-2 production by Th1 cells. The inhibitory effect of 20C9 was not observed when Th2 cells were substituted for Th1 cells or peripheral lymph node T cells. Furthermore, Th1 cells rescued from cocultures of T cells and 20C9-treated, Ag-pulsed LC, (but not from control-treated cocultures) were anergic to restimulation with untreated Ag-pulsed LC. These data suggest that LC HSA is an important costimulatory molecule in Th1 cell-dependent cutaneous immune reactions.

Heat-stable antigen/CD24 on mouse T lymphocytes: evidence for a costimulatory function.

Heat-stable antigen (HSA)/mouse CD24 (formerly termed Nectadrin) is a membrane glycoprotein with an unusual structure consisting of a small protein core and extensive glycosylation. It is expressed by hematopoietic cells but not by mature T lymphocytes. HSA on accessory cells is an important costimulatory molecule required for the clonal expansion of T lymphocytes. HSA is also involved in cell-cell adhesion events and the isolated antigen has been shown to possess self-binding properties. In the present study we have re-investigated the role of HSA in T cell proliferation. We find that following stimulation of T lymphocytes with concanavalin A or of CD4+ T lymphocytes with a combination of anti-CD3/CD28 monoclonal antibodies (mAb) the HSA antigen is transiently expressed. The expression correlated with the appearance of CD25 and a CD2 activation epitope at the cell surface. Induction of HSA was also seen in vivo on V beta 8+ T lymphocytes in BALB/c mice that were injected with Staphylococcal enterotoxin B. Biosynthetic labeling and analysis of mRNA by reverse transcriptase-polymerase chain reaction showed that HSA was synthesized by activated T lymphocytes. A combination of anti-CD3 and mAb 79 to HSA was incapable of inducing proliferation of purified CD4+ T lymphocytes. However, the antibody strongly enhanced the response obtained with a combination of anti-CD3/CD28 mAb. The augmenting effect of the HSA-specific mAb was dose dependent. Since HSA is bound to the membrane via a glycosyl-phosphatidylinositol (GPI) anchor and GPI-anchored molecules have been implicated in lymphocyte activation, it is conceivable that HSA is not only a costimulatory molecule on accessory cells but is also a signaling molecule in T lymphocytes. The possibility of a homotypic HSA/HSA interaction between T lymphocytes and accessory cells is discussed.

Differential, LFA-1-Sensitive Effects of Antibodies to Nectadrin, the Heat-Stable Antigen, on B Lymphoblast Aggregation and Signal Transduction

Nectadrin, the heat-stable antigen (HSA), is a highly glycosylated GPI-linked glycoprotein that can undergo homophilic and heterophilic binding. In the present work we have examined short-term effects of nectadrin antibodies on splenic B lymphoblast aggregation and signal transduction. Monoclonal antibody 79 inhibited cell aggregation and induced an intracellular Ca ++ signal in the absence of cross-linking. Both these effects were perturbed in the presence of LFA-1 antibodies. Nectadrin antibody M1/69 and polyclonal nectadrin antibodies stimulated cell aggregation, did not induce a Ca ++ signal, and their effects were functionally independent of LFA-1. These results suggest that nectadrin may concomitantly mediate primary and activate secondary adhesion mechanisms whereby each of these processes may be related to a different signal transduction pathway.

Isolation and Developmental Expression of Retinoic-Acid-Induced Genes

The vitamin A derivative retinoic acid (RA) is involved in vertebrate anteroposterior axis formation and cellular differentiation and has been shown to modulate the expression of a number of genes implicated in the control of early embryonal development. Under defined culture conditions, all- trans-RA induces differentiation of P19 embryonal carcinoma cells into neural derivatives. In this report, we describe the isolation and partial characterization of 14 RA-regulated genes from P19 cells committed to differentiate along the neural pathway. In addition to the previously recognized genes encoding vimentin, heat stable antigen, neural cadherin, Hox-B2, F52/MacMARCKS, thymosin β4b, and the murine homolog of COUP-TF1, we identified 7 novel genes, 5 of which are predominantly expressed in developing neural tissues as shown by in situ hybridization. The results confirm the usefulness of the P19 system in the isolation and study of the regulation of developmentally expressed genes.

Selective expansion of activated Vδ4+ cells during experimental infection of mice with Leishmania major

Previous work from this laboratory has revealed that infection of mice with Leishmania major leads to an expansion of gamma delta+ T cells in the spleen. Further examination of the gamma delta+ T cells expanding in infected mice has shown that the majority of these cells in the spleen, lymph nodes, blood and liver expressed the V delta 4 gene segment. Cell cycle analysis, using propidium iodide incorporation, demonstrated that while only 1% of alpha beta+ T cells in the spleen were in either S + G2/M phase, up to 10% of the gamma delta+ T cells were in cycling phase 8 weeks after infection. Comparison of the state of activation of the two populations in different organs after infection, confirmed that gamma delta+ T cells are actively dividing in lymph nodes, liver and blood, but not in the thymus or among intraepithelial lymphocytes. Examination of the expression of different activation markers on the surface of gamma delta+ T cells in the spleen of both normal and chronically infected BALB/c mice by FACS analysis, revealed increased expression of LFA-1, CD25, CD44, 4F2, CD28 and the heat-stable antigen, whereas Thy-1 and CD5 decreased. Collectively, these results suggest an oligoclonal expansion and activation of gamma delta+ T cells in response to L. major infection.

Parallels in T lymphocyte development between lpr and normal mice

The unusual phenotype and function of lpr T cell subsets has considerably aided the determination of various stages in normal T lymphocyte development through the identification of parallel populations. This has included the description of memory T cells that express high levels of CD44. Lpr mature (CD4 + and CD8 +) T cells are all CD44 hi and produce large amounts of several cytokines, in contrast to normal murine peripheral T cells. However, the minor CD44 hi subset of normal memory T cells produces similarly high levels of cytokines. The expression of intermediate density surface T cell antigen receptor (TCR)-αβ by lpr CD4 -, CD8 - (CD4 -8 -) cells guided the identification of a minor subset of Heat Stable Antigen—normal CD4 -8 -thymocytes that also expresses TCR-αβ. The unresponsiveness of lpr CD4 -8 - T cells correlates closely with their absence of CD2 expression. This is paralleled in normal mice by CD2 - and CD2 + subsets of thymocytes and gut lymphocytes in which the proliferative capacity also segregates with CD2 expression. Finally, lpr CD4 -8 - T cells bear many similarities to anergic T cells, including high expression of p59 fyn, lack of IL-2 production, and recovery of function following induction of cell cycling in the presence of IL-2. The developmental block in lpr CD4 -8 - T cells has therefore provided considerable insight into normal T cell development and function.

Heat-stable antigen (CD24) as ligand for mouse P-selectin.

Heat-stable antigen (HSA)/CD24 is a cell surface molecule expressed by many cell types in the mouse. The molecule has an unusual structure because of its small protein core and extensive glycosylation. In order to study the functional role of the HSA-associated glycoconjugates we have isolated different forms of HSA. Using lectin analysis we provide evidence for extensive heterogeneity in carbohydrate composition and sialic acid linkage. Several HSA forms were recognized by mouse P-selectin-IgG but not E-selectin-IgG in ELISA. As expected, P-selectin-IgG also bound to L2/HNK-1-positive neural glycoproteins (L2-glycoproteins) and sulfatides but not to gangliosides and other control glycoproteins. The binding of P-selectin-IgG to L2-glycoproteins and HSA required bivalent cations. The reactivity to HSA was sensitive to sialidase treatment whereas the binding to L2-glycoproteins was not. Studies with alpha 2-6 sialytransferase indicated that alpha 2-6 linked sialic acid was not involved in the P-selectin binding to HSA. Surprisingly, an L2/HNK-1 specific antibody was found to cross-react with some HSA glycoforms and its binding correlated with P-selectin-IgG reactivity. L2/HNK-1-positive or L2/HNK-1-negative HSA glycoforms were also analyzed after coating to polystyrene beads. Only the L2/HNK-1-positive HSA coated beads were reactive with P-selectin-IgG and could bind to activated bend3 endothelioma cells expressing P-selectin whereas the L2/HNK-1-negative HSA beads did not. It is suggested that in its L2/HNK-1 modified form the HSA molecule on leukocytes could represent a ligand for P-selectin on endothelial cells or platelets.

Defective development of thymocytes overexpressing the costimulatory molecule, heat-stable antigen.

Heat-stable antigen (HSA) is a small, glycosyl phosphatidylinositol-anchored protein that can act as a costimulatory molecule for antigen-dependent activation of helper T cells. In addition to being expressed on antigen-presenting B cells, HSA is also expressed during the initial stages of T cell development in the thymus. HSA levels are very high on immature CD4-, CD8- double negative thymocytes, but are reduced on CD4+, CD8+ double positive cells undergoing selection in the thymus, and are entirely eliminated when these cells differentiate into immunologically competent CD4+ or CD8+ single positive T cells. To examine the potential roles of this molecule in T cell development and selection, we generated transgenic mice in which HSA was highly expressed on all classes of thymocytes. The consequence of deregulated HSA expression was a pronounced reduction in the numbers of double positive and single positive thymocytes, whereas the numbers of their double negative precursors were largely unaffected. These results demonstrate that downregulation of HSA expression at the double positive stage is a critical event in thymocyte development. The depletion of thymocytes resulting from HSA overexpression begins at the same time as the onset of negative selection, suggesting that HSA may provide signals that contribute to determining the efficiency of this process.

Induction of B cell costimulatory function by recombinant murine CD40 ligand.

T cell-dependent regulation of B cell growth and differentiation involves an interaction between CD40, a B cell surface molecule, and the CD40 ligand (CD40L) which is expressed on activated CD4+ T cells. In the current study, we show that recombinant membrane-bound murine CD40L induces B cells to express costimulatory function for the proliferation of CD4+ T cells. CD40L- or lipopolysaccharide (LPS)-activated, but not control-cultured B cells were strong costimulators of anti-CD3 or alloantigen-dependent T cell responses. The molecular interactions responsible for the increased costimulatory functions were examined by analyzing the activated B cells for changes in the expression of two costimulatory molecules, B7 and heat-stable antigen (HSA), as well as by the use of antagonists of B7 and HSA (CTLA4.Fc and 20C9, respectively). The expression of both B7 and HSA was enhanced on B cells activated with LPS. As observed in previous studies, the costimulatory activity of the LPS-activated B cells was dependent on both B7 and HSA and was completely inhibited in the presence of a combination of CTLA4.Fc and 20C9. In contrast, activation of B cells with CD40L induced the expression of B7 but did not enhance the expression of HSA. In addition the costimulatory activity of the CD40L-activated B cells was partially, but not completely, inhibited by the combination of CTLA4.Fc and 20C9. These results demonstrate that CD40L regulates costimulatory function of B cells in part by inducing the expression of B7 and suggest that CD40L-activated B cells express an additional costimulatory activity that is not associated with LPS-activated B cells.

Reduced life span of anergic self-reactive B cells in a double-transgenic model.

The life span of anergic self-reactive B cells was determined by 5-bromo-2'-deoxyuridine (BrdU) loading of tolerant double-transgenic (Dbl-Tg) mice produced by mating hen egg lysozyme (HEL)-transgenic mice with the corresponding immunoglobulin-transgenic (Ig-Tg) mice, the B cells of which express anti-HEL IgM and IgD. B cells from Dbl-Tg mice, despite being exposed to soluble antigen throughout their development, are not deleted, but persist in an anergic state. As a prelude to studying the life span of these anergic B cells, BrdU was administered to nontransgenic mice; B cells from the bone marrow, spleen, and lymph nodes displayed distinct kinetic profiles based on reciprocal expression of the B220 isoform of CD45 and heat-stable antigen (HSA). Thus, immature B220lo/HSAhi B cells incorporated BrdU rapidly suggesting recent generation from dividing precursors, whereas uptake by B cells expressing the mature B220hi/HSAlo phenotype was significantly slower, consistent with a longer life span. Such gating allowed analysis to be directed at the stable mature B cell population in transgenic mice. Comparison of BrdU uptake in Ig- and Dbl-Tg mice indicated that B cells from Dbl-Tg mice were renewed at a much higher rate (50% renewal times of 0.64 vs. 3.4 wk for total B cells, and 1.2 vs. 5.0 wk for mature B200hi/HSAlo cells from Dbl- and Ig-transgenic mice, respectively). This difference was even more marked when analysis in Dbl-Tg mice was restricted to HEL-binding cells, which had a 50% renewal time of 3-4 d compared with 4-5 wk for non-HEL-binding B cells. While the proportion of B cells in cell cycle, and the rate of entry of newly generated B cells into the spleen of Ig- and Dbl-Tg mice, were similar, B cell numbers were reduced in Dbl-Tg mice. It was therefore concluded that anergic B cells have a markedly decreased life span in the periphery. According to studies in radiation chimeras produced by reconstituting HEL-transgenic recipients expressing different serum levels of antigen with Ig-Tg bone marrow, the reduced life span of anergic B cells was associated with the anergic state per se, the serum concentration of HEL being important only in attaining the critical threshold necessary for tolerance induction. B cells rendered tolerant by exposure to soluble self-antigen therefore survive for a relatively short period in an anergic state once they have reached peripheral lymphoid tissue and fail to enter the long-lived compartment.(ABSTRACT TRUNCATED AT 400 WORDS)

Immortalized dendritic cell line fully competent in antigen presentation initiates primary T cell responses in vivo.

Dendritic cells (DC) can provide all the known costimulatory signals required for activation of unprimed T cells and are the most efficient and perhaps the critical antigen presenting cells in the induction of primary T cell-mediated immune responses. It is now shown that mouse cell lines with many of the features of DC can be generated using the MIB phi 2-N11 retroviral vector transducing a novel envAKR-mycMH2 fusion gene. The immortalized dendritic cell line (CB1) displays most of the morphologic, immunophenotypic, and functional attributes of DC, including constitutive expression of major histocompatibility complex (MHC) class II molecules, costimulatory molecules B7/BB1, heat stable antigen, intracellular adhesion molecule 1, and efficient antigen-presenting ability. Granulocyte/macrophage colony-stimulating factor (GM-CSF) proved to be effective in increasing MHC class II molecule expression and in enhancing presentation of native protein antigens. In comparison with macrophages, CB1 dendritic cells did not exhibit phagocytic and chemotactic activity in response to various stimuli and lipopolysaccharide activation was ineffective in inducing tumor necrosis factor alpha or interleukin 1 beta production. CB1 cells, pulsed with haptens in vitro and injected into naive mice were able to induce delayed-type hypersensitivity responses, further increased with pretreatment with GM-CSF, indicating that these cells may represent an immature, rather than a mature DC. The ability of CB1 to prime T cells in vivo could provide a tool to design novel immunization strategies.

Suppression of B lymphopoiesis during normal pregnancy.

We describe a dramatic reduction in numbers and activity of committed B lymphocyte precursors in the bone marrow of normal pregnant mice. Changes in cells responsive to IL-7 were evident as early as 6.5 d of pregnancy and values were < 10% of normal at parturition. B lineage precursors, identified by display of CD45R and absence of surface IgM, were also substantially depressed, and subpopulations representing different stages in the B lineage were assessed by three-color flow cytometry. Early pro-B cells are medium to large in size and have been previously characterized by low expression of the heat-stable antigen (HSA). This category of cells was not reduced, and in fact may have been slightly elevated, during pregnancy. In contrast, all subsequent populations of B lineage precursors, defined by patterns of expression of heat-stable and CD43 antigens, were substantially depressed. The immediate precursors of B cells (small pre-B cells) were identified by small size, expression of CD45R, absence of CD43, and lack of surface IgM. These were the most reduced of any phenotypically defined population in bone marrow. Numbers of newly formed B cells, characterized by the presence of sIgM, but not sIgD, were also diminished. However, B cells with a mature phenotype (sIgM+, sIgD+) were present in normal to somewhat elevated numbers. Mitogen-responsive B cells clonable in a semisolid agar assay were not significantly affected. A bromodeoxyuridine (BrdU) labeling technique was used to evaluate mitotic activity, which revealed an increased proportion of long-lived lymphocytes in the bone marrow of pregnant mice. These observations indicate that B lymphopoiesis is markedly downregulated during pregnancy and that all precursor populations beyond the early pro-B cell stage are affected. The pregnancy-related changes in bone marrow were selective for B lineage precursors, as cells expressing myeloid and erythroid markers were not reduced. In spleen, evidence was obtained for partial depletion of one subset of B cells. These cells, which have been reported to be recent immigrants from marrow, are characterized as having high levels of sIgM and HSA. Changes in other major B lymphocyte subsets in the spleen were less remarkable. When considered with results from the BrdU labeling procedure, the findings indicate that both production and export of lymphocytes from marrow may be substantially decreased. Numbers of B cell precursors were higher in postpartum animals whose litters were removed at birth, suggesting that lactation may prolong regeneration of lymphocyte production.(ABSTRACT TRUNCATED AT 400 WORDS)

The heat stable antigen (mouse CD24) gene is differentially regulated but has a housekeeping promoter.

Expression of the mouse heat stable antigen (HSA or mouse CD24) shows tissue-specific as well as developmental regulation. During the maturation of several hematopoietic lineages, HSA expression is generally high in immature precursor cells and low or absent in terminally differentiated cells. We present evidence suggesting that this regulation of the HSA gene (Cd24a) occurs at the transcriptional level. In addition, sequence and methylation analysis of the Cd24a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, HpaII tiny fragment (HTF) island, multiple putative SP1 and AP-2 consensus binding sites, and a TATA box. Functional analysis of a 0.6-kilobase DNA fragment containing these elements fused to the CAT reporter gene in transient transfection experiments showed activity in both HSA expressing and non-expressing cell lines with a strength similar to that of the herpes-simplex virus-thymidine kinase promoter. Large fragments from the flanking region of the Cd24a promoter did not influence the ubiquitous nature of this promoter. Finally, we mapped the Cd24a, Cd24b, and Cd24c genes to mouse chromosomes 10, 8, and 14 respectively.

Ontogenic development of murine fetal thymocytes is accelerated by 3,3′,4,4′-tetrachlorobiphenyl

Polychlorinated hydrocarbons such as biphenyls or dioxins interfere with cellular processes by gene induction via ligand-activated binding of the cytosolic Ah-receptor to specific DNA elements. The thymus is a target organ for these processes and immunosuppression a hallmark of polychlorinated hydrocarbon toxicity. Using flow cytometry we analysed the development of thymocytes in fetal thymus organ cultures (FTOC) exposed to tetrachlorobiphenyl (TCB) for up to one week. We show that exposure to TCB changes the normal developmental pathways of fetal thymocytes within days. Overall fewer thymocytes are found in TCB-treated cultures from day 4 on, and significantly more CD8 positive thymocytes are detectable. These cells express the T-cell receptor, but not heat-stable antigen or IL2-receptor, giving them a mature phenotype. Moreover, relatively more CD4/CD8 double-negative thymocytes express CD44, a molecule involved in lymphocyte-epithelial interaction. We suggest that, at least for the CD8 single-positive thymocyte population, maturation is accelerated, and this may be due to TCB interference with physiological thymocyte-epithelial interactions.

Peripheral B cell maturation. II. Heat-stable antigen(hi) splenic B cells are an immature developmental intermediate in the production of long-lived marrow-derived B cells.

HSAhi B cells comprise 5 to 10% of adult mouse splenic B cells and are phenotypically and functionally immature. To assess their origin and relationship to mature, heat-stable Ag (HSA)lo B cells, we determined HSA and surface IgD phenotype among splenic B cells throughout development, as well as during reconstitution of lethally irradiated adults given adult B-depleted bone marrow. In each case, HSAhi splenic B cells predominate during the earliest stages of B cell genesis. Furthermore, 5-bromo-2'-deoxyuridine labeling experiments indicate rapid turnover within both the marrow and peripheral HSAhi pools, and adoptive transfer studies show that peripheral HSAhi splenic B cells differentiate to HSAlo within 4 days. Finally, splenic HSAhi B cells reconstitute both primary and memory humoral responses. Together, these data indicate that splenic B cells in the HSAhi subset are an intermediate maturational stage in the adult periphery.

Serological grouping of indigenous Bradyrhizobium sp. (Arachis) isolated from various soils of Thailand

ELISA and antibody adsorption tests were applied to determine the minimal somatic antigen constitution of 243 strains of Bradyrhizobium sp. (Arachis) using 12 antisera. The 243 indigenous bradyrhizobial isolates were from 15 sites in four regions of Thailand. A total of 29 serogroups were identified. Most (80%) of the isolates tested had at least one heat-stable antigen in common with strain 280A, forming a so-called 280A serocluster. At 11 of 15 sites tested, 53 to 100% of the isolates fell into one or two predominant serogroups. The serological properties of the indigenous bradyrhizobia were not related to the cropping history of the cultivated fields from which they were isolated.

Identification and characterization of hematopoietic stem cells from the yolk sac of the early mouse embryo.

The yolk sac is the first site of hematopoiesis in the mammalian embryo. However, little is known about the initial stem cells in the yolk sac. We have isolated hematopoietic stem cells from early mouse embryonic yolk sac by using a sequential protocol of nonadherence to plastic, density gradient centrifugation, immunocytoadherence, and cell sorting. Isolated, nonadherent, density < 1.077-g/cm3, surface antigen AA4.1+, wheat germ agglutinin bright (WGAbright) cells give rise to multiple lineages, including T cells, B cells, and myeloid cells, as detected by using fetal thymus organ culture, S17 stromal feeder layers, or methylcellulose culture colony-forming cells, respectively. AA4.1+, WGAbright cells expressed high levels of heat-stable antigen (HSA) and CD45 (Ly-5) but did not significantly express major histocompatibility complex antigens, CD44, or Sca-1. Peak stem cell concentration is reached by day 11, before stem cells can be found in the liver, omentum, or thymus. In vivo long-term reconstitution of lethally irradiated mice was effected by as few as 720 AA4.1+, WGAbright yolk sac cells, but it required addition of a subset of bone marrow cells capable of providing immediate (short-term) radiation protection. Yolk sac donor-derived T cells, B cells, and macrophages were readily identified 6 months after transfer of yolk sac-derived stem cells. We suggest that, because of their cell surface phenotype as well as their capacity to differentiate in vitro and in vivo, the cells isolated from the mouse embryonic yolk sac may include the most primitive hematopoietic pluripotential stem cells yet identified.

K‐antigens in Porphyromonas gingivalis are associated with virulence

We investigated antigens in spreading and non-spreading Porphyromonas gingivalis strains. On the basis of differences in virulence in the mouse model, 8 strains were selected for antiserum production in rabbits. Hyperimmune sera were tested by double immunoprecipitation and immunoelectrophoresis. Besides a common antigen, differences in antigenic composition were observed in the thermolabile antigens between all strains tested. Two different heat-stable antigens were found after heating at 120 degrees C. One such antigen was detected after sonication of the pellet fraction of autoclaved P. gingivalis cells. This antigen cross-reacted with 6 of the 8 immune sera. This somatic antigen was almost neutrally charged and sensitive to sodium periodate treatment, suggestive of lipopolysaccharide. A second heat-stable antigen was detected in the supernatant of autoclaved strains of W83, W50, HG184 and A7A1-28. These non-somatic antigens were strain-specific, i.e., no cross-reactivity was found with heterologous hyperimmune sera. An exception was strain W50, which had a non-somatic heat-stable antigen which was recognized by W83 antiserum. These antigens were resistant to DNAse, RNAse and proteinase-K treatment but were degraded by sodium periodate. In immunoelectrophoresis, these antigens appeared to be negatively charged. These properties are characteristics of a K-antigen, which likely represent a thermostable carbohydrate capsule. The presence of K-antigen correlates very well with the serum resistance, the low chemiluminescence, the resistance to phagocytosis and the need for opsonization with specific antibodies for complement-mediated killing of virulent P. gingivalis strains.(ABSTRACT TRUNCATED AT 250 WORDS)