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L Santos-Argumedo

Publications and source records attributed to L Santos-Argumedo.

At least 19 recordsLinked to original sources
PubMed2005

Production and characterization of a monoclonal antibody specific for NS3 protease and the ATPase region of Dengue-2 virus.

Dengue is considered a reemerging disease of worldwide distribution. The Dengue virus non-structural protein 3 (NS3) is known to possess ATPase, helicase, and protease activities that are a constitutive part of the replication complex of Dengue virus. In this report, we discuss the cloning, expressing, and purifying of the Dengue-2 NS3 protein, to immunize mice and then generate monoclonal antibodies (MAbs). Our results show the production of MAbs specific to NS3 protein of Dengue-2 virus, which by immunofluorescence recognize the native protein in experimentally infected endothelial cells (HMEC). Likewise, C6/36-infected lisates were used in Western blots, and observed the specific characteristic band that defines the NS3 protein. We conclude that these antibodies may be a useful tool, not only to study the replicative process of Dengue virus, but also to generate specific diagnostic tools for Dengue infection.

Adenosine Triphosphatases↗
PubMed2002

Expression and function of CD22, a B-cell restricted molecule.

In this work, we studied the expression and function of CD22 in murine B cells. CD22 has been previously characterized as an activation marker of mature B lymphocytes. However, we found that CD22 is expressed early during the ontogeny of B cells in the bone marrow and spleen, and was found on B cells isolated from all the different lymphoid compartments. We also found that B cells stimulated through the B-cell antigen receptor (BCR), CD38 and CD40, upregulated CD22 expression to maximal levels within 24 h after stimulation, but that the levels of CD22 declined at later times (48 and 72 h). CD22 is rapidly phosphorylated after BCR signal transduction, and is believed to downregulate B-cell activation. In this study, we did not detect CD22 phosphorylation in activated B cells after CD38 or CD40 cross-linking, even though CD22 was clearly phosphorylated in the BCR-stimulated B cells. Consistent with this, we found no evidence of physical association between CD38 or CD40 and CD22 in B cells. The lack of association or phosphorylation of CD22 induced by CD38 and CD40 cross-linking indicates that CD22 may not downregulate the activation induced by these two molecules.

ADP-ribosyl Cyclase↗
PubMed2001

Ontogeny, distribution and function of CD38-expressing B lymphocytes in mice.

Analysis of expression of CD38, CD45R (B220), IgM and IgD on splenic B lymphocytes from mice of different ages demonstrated CD38 on both immature (B220(+), BCR(-)) and mature (B220(+), BCR(+)) B lymphocytes. Similarly, CD38 is expressed as early as B220 on the surface of progenitor B cells in the bone marrow. In spite of expressing of CD38 and IgM, neonatal B cells, in contrast to the adult, failed to proliferate to either anti-CD38 or anti-IgM cross-linking when IL-4 was present. They did, however, respond to LPS and anti-CD40, and by 2 weeks of age they began to respond to anti-CD38 and anti-IgM, reaching adult B cell levels by 4 weeks. Although the distribution of CD38 on adult B cells from most different lymphoid compartments was broadly similar, significantly higher levels of CD38 were expressed on peritoneal B lymphocytes. A detailed analysis, using IgM / IgD ratio and staining with anti-CD5 confirmed that B1 lymphocytes were expressing a high level of CD38. Interestingly, both immature B cells and peritoneal B1 lymphocytes were unresponsive to anti-CD38. However, they were activated by LPS or anti-CD40.

ADP-ribosyl Cyclase↗
PubMed2001

Enforced and prolonged CD40 ligand expression triggers autoantibody production in vivo.

CD40, a glycoprotein expressed on B lymphocytes plays an important role in B cell development, growth and differentiation. The ligand for the CD40 is a 39-kDa glycoprotein (CD154) expressed on the surface of activated T lymphocytes and is essential for thymus-dependent humoral immunity. The expression of CD154 is tightly regulated and its transient expression reduces the chances of potentially deleterious bystander activation of B cells. Stimulation through CD40 has been studied in vitro by using antibodies against CD40, by membranes of activated T cells or lately, by CD154 transfected cells. In this work we have evaluated the outcome of CD40-CD40 ligand interaction in vitro and in vivo by using CD154-transfected L929 cells. In vitro assays showed that CD154-L929 cells can induce on B cells: IL-4-dependent proliferation, up-regulation of CD23, CD54 and class II molecules and can also rescue WEHI-231 B cell lymphoma from anti-IgM-induced apoptosis. Interestingly, in vivo assays revealed that when CD154-L929 cells were inoculated into the spleen, mice developed a strong but transient production of anti-erythrocyte autoantibodies. Through B lymphocyte activation with CD154-transfected L929 cells both in vitro and in vivo, our data reveal that enforced and prolonged expression of CD40 ligand overcomes the tightly regulated mechanisms of B cell activation, triggering the production of autoantibodies. This system might be used to evaluate the early steps of an autoimmune response and the role of CD40-CD154 in the induction of primary responses in vivo.

Animals↗
PubMed2000

CD45R, CD44 and MHC class II are signaling molecules for the cytoskeleton-dependent induction of dendrites and motility in activated B cells.

Anti-CD44 or anti-MHC II antibodies bound to tissue culture plates have previously been shown to induce a dramatic generation of dendritic processes in activated murine B cells. In this study, we demonstrate a similar generation of dendrites and cell motility in activated B cells through CD45R. The dynamic formation of dendritic processes and associated induction of cell motility were analyzed by video microscopy and were characterized by a rapid, and multidirectional emission of dendrites with retractile behavior. The addition of cytochalasin E totally blocked dendrites formation and motility induced through either CD45R, CD44 or MHC II, suggesting that the necessary cytoskeletal rearrangements require active polymerization of actin. Confocal microscopy showed an accumulation of F-actin in the dendrites, as long as cells were elongating. In contrast, G-actin was localized in the perinuclear area and also accumulated in sites where dendrites originated. Preincubation of B cells with staurosporine (a PKC inhibitor) or BAPTA-AM (a calcium chelator) prevented these morphological changes, indicating additionally a requirement for a PKC-calcium-dependent activity. Dendrite formation and cellular motility, therefore, seem to be two manifestations of the same phenomenon, and CD44, CD45R and MHC II appear to be signaling molecules for the observed cytoskeleton-dependent morphological changes.

Actins↗
PubMed1999

Cell surface expression of CD154 inhibits alloantibody responses: A mechanism for the prevention of autoimmune responses against activated T cells?

Binding of CD40 by CD154 expressed on activated T cells is a pivotal event in T cell help to B cells, macrophages, and other antigen-presenting cells. Expression of CD154 by MHC mismatched cells, in contrast to expectations, strongly suppressed alloantibody responses against the cells. This was caused by a failure of priming of antibody responses by the CD154 expressing cells. We hypothesize that this lack of response against CD154 expressing cells may represent a mechanism that has evolved to prevent autoantibody responses being generated against the CD154 antigen itself, as B cells expressing antibody reactive with CD154 would probably escape deletion on binding antigen in the bone marrow due to rescue by the simultaneous ligation of CD40.

Animals↗
PubMed1998

Arrest of B lymphocyte terminal differentiation by CD40 signaling: mechanism for lack of antibody-secreting cells in germinal centers.

Despite extensive research, the role of CD40 signaling in B cell terminal differentiation remains controversial. Here we show that CD40 engagement arrests B cell differentiation prior to plasma cell formation. This arrest is manifested at a molecular level as a reduction in mRNA levels of secretory immunoglobulin gene products such as mu(s) and J chain as well as the loss of the transcriptional regulator BLIMP-1. Furthermore, the inhibition of B cell differentiation by CD40 engagement could not be overcome by either mitogens or cytokines, but could be reversed by antibodies that interfere with the CD40/gp39 interaction. These data suggest that secretory immunoglobulin is not produced by B cells that are actively engaged by gp39-expressing T cells.

Animals↗
PubMed1997

CD44-stimulated dendrite formation ('spreading') in activated B cells.

A rat monoclonal antibody (mAb) (NIM-R8), insolubilized by binding to plastic plates, induced a rapid and extensive formation of dendrite processes ('spreading') in B lymphocytes activated by anti-IgM and interleukin-4 (IL-4) or anti-CD38 and IL-4. In contrast, resting B cells were unable to spread similarly on the NIM-R8-coated plates. The NIM-R8 antibody recognized a 90,000 MW surface glycoprotein (gp90) present on both B and T lymphocytes. The expression of this molecule was greatly increased after polyclonal (lipopolysaccharide, anti-IgM plus IL-4 or concanavalin A) activation. The NIM-R8 mAb with or without IL-2 or IL-4 was unable to induce proliferation of splenic lymphocytes. Following the demonstration that the NIM-R8 mAb recognizes the murine equivalent of human CD44, the induction of spreading of activated B lymphocytes was studied using a panel of mAb recognizing different epitopes of murine CD44. All of these different mAb induced similar spreading of activated B cells. The ligand-inducible spreading of activated B lymphocytes may be an important mechanism for providing an increased cell-surface area for cell-cell or cell-matrix interactions, and thus may be an important factor controlling the response of activated lymphocytes.

Animals↗
PubMed1996

Evolution of lymphocyte populations in armadillos (Dasypus novemcinctus) inoculated with M. leprae.

In human leprosy patients there are changes in the percentages of T and B lymphocytes in peripheral blood, and there is a correlation with the clinical characteristics or manifestations of the disease. These phenomena still require clarification regarding the triggering mechanism involved that may lead to one or the other clinical entities. Much has yet to be learned about the intricacies of whether the changes in subpopulations of T and B lymphocytes are a causative factor or an effect attributable to the microorganism itself. The armadillo is an excellent animal model to study how Mycobacterium leprae spread, turning into an established infection. The application of modifications in percentages of the subpopulations of B and T lymphocytes in armadillos may well lead to extrapolation of the results obtained in this animal model in an attempt to be able to manipulate the course of the disease in humans. The purpose of the study was to evaluate changes in the percentages of rosette-forming and sIgM+ mononuclear cells during a full year in groups of armadillos: five randomly chosen animals formed the control group and 11 armadillos were inoculated with M. leprae obtained from a human leproma at the onset of the 12-month period of the study. Of the 11 randomly selected armadillos that were inoculated, only five developed an active and disseminated infection. The percentage of rosette-forming cells did not show statistically significant variations during the first 6 months of the study. However, at months 8 and 12 a significant increment in this parameter was observed (p < 0.05) in the animals with active infection. In regard to the variations in the numbers of sIgM+ cells, significant changes occurred in the armadillos with active infection at month 2. However, results returned to normal and no changes were seen at later times. No significant changes occurred in the group of animals inoculated but not developing active infection compared with the other groups. The results are considered sufficiently interesting to encourage further study on the cell-mediated immune system of the armadillo and the changes that occur during the development and dissemination of an inoculated infection with M. leprae. Since this mammal is of great value as an effective animal model in the experimental research of M. leprae, there is an urgent need to obtain, as quickly as possible, a thorough understanding of the cellular branch of its immune system and, thereby, be in a position to extrapolate immune modulation to benefit human leprosy patients.

Animals↗
PubMed1995

CD38 unresponsiveness of xid B cells implicates Bruton's tyrosine kinase (btk) as a regular of CD38 induced signal transduction.

CD38 is a 42 kDa membrane-associated ectoenzyme expressed by a large proportion of human and mouse lymphocytes. Agonistic antibodies to CD38 induce a strong proliferative response in lymphocytes additionally co-stimulated with other growth co-factors such as IL-4, IL-2 plus accessory cells or sub-mitogenic doses of endotoxin. We show here that B lymphocytes from unstimulated X-linked immunodeficient (xid) mice are unresponsive to CD38 stimulation, both in terms of proliferative response and surface antigen modulation. This CD38 unresponsiveness is evident in the presence of excess quantities of, and normal responses to, the accessory growth co-stimulants required for this response. CD38 molecules expressed on xid B cells are normal in terms of expression levels, size and enzymatic activity, suggesting that CD38 unresponsiveness reflects a down-stream signaling defect. In light of the recent proposal that the xid gene encodes a tyrosine kinase called Bruton's tyrosine kinase (btk), these data suggest that btk is either an integral component or an indirect regulator of the CD38-induced signal transduction pathway.

ADP-ribosyl Cyclase↗
PubMed1995

CD38 expression on mouse T cells: CD38 defines functionally distinct subsets of alpha beta TCR+CD4-CD8- thymocytes.

We have examined CD38 expression on mouse lymphocytes using the rat mAb NIM-R5 and demonstrate that CD38 expression is restricted to approximately 8% of thymocytes. Although CD38 is absent from the majority of CD4+CD8- and CD4-CD8+ T cells, we detected a strong correlation between CD38 expression and alpha beta+CD4-CD8- T cells in the thymus, with nearly 80% of alpha beta TCR+CD4-CD8- thymocytes being CD38+. Using heat stable antigen (HSA) and CD38, we divided alpha beta+CD4-CD8- thymocytes into four subsets: HSA+CD38-, HSA-CD38hi, HSA-CD38low and HSA-CD38-. Two established characteristics of alpha beta TCR+CD4-CD8- cells, bias towards V beta 8.2 TCR expression and high levels of IL-4 production, were used to establish a possible relationship between the above thymocyte subsets. Our present data show that the HSA+CD38- subset is not biased towards V beta 8.2 TCR expression whereas the HSA-CD38- subset does show this bias (approximately 47%). Neither of these subsets make IL-4 upon CD3 mediated stimulation. In contrast, the CD38+ subsets are heavily biased toward V beta 8.2 expression and produce large amounts of IL-4 upon stimulation, particularly the CD38low cells. Taken together, these data suggest that these four subsets represent various stages of a possible differentiation pathway for alpha beta TCR+CD4-CD8- cells, with the HSA+CD38- subset being the most immature while the HSA-CD38low subset is the most functionally mature. These characteristics support the view that alpha beta TCR+CD4-CD8- T cells represent an independent lineage with a distinct, but as yet obscure, role in immunity.

ADP-ribosyl Cyclase↗
PubMed1995

Identification and purification of armadillo (Dasypus novemcinctus) immunoglobulins: preparation of specific antisera to evaluate the immune response in these animals.

In this work we describe the purification and characterization of armadillo immunoglobulins. The IgM was precipitated using low-strength ionic solution and further purified by filtration through Sephadex G-200. The IgG was obtained in pure form by precipitation of serum with ammonium sulfate and DEAE-cellulose ion exchange chromatography. The purity of these immunoglobulins was evaluated by polyacrylamide gel electrophoresis. The results showed 28-kDa light chains and 55-kDa and 70-kDa heavy chains for IgG and IgM, respectively. The rabbit antibodies against these molecules were used to prepare fluorescein (FITC) and peroxidase conjugates. The FITC conjugate was used to quantify IgM-bearing lymphocytes. An average of 17% of peripheral blood lymphocytes were sIgM+ from 14 healthy animals. Additionally, in the same animals we quantified lymphocytes with the capacity to form rosettes with sheep red-blood cells; the average for this marker was 10%. Also, the production of crossreacting antibodies to BCG was evaluated in healthy and Mycobacterium leprae-inoculated animals using the peroxidase conjugates. All animals with active infection recognized BCG antigens.

Animals↗
PubMed1995

Immune response of armadillos (Dasypus novemcinctus). I. Use of lectins to identify lymphocyte subpopulations and to evaluate cell proliferation.

Lectins have been used to study populations and discrete differentiation stages of lymphocytes. Likewise, lectins have been of practical importance in promoting mitogenic stimulation of lymphocytes in numerous species. In this research project, we took advantage of these tools in an attempt to identify specific subsets of peripheral blood lymphocytes obtained from healthy nine-banded armadillos (Dasypus novemcinctus). The same cell source served to evaluate mitogenic stimulation. Twelve FITC-labeled lectins were used; 5 (ConA, LcH, RCA, WGA and UEA) reacted with almost 100% of the lymphocytes and 7 (PNA, DBA, SBA, PCA, PHA-L, PWM and VVA) recognized variable percentages (< 100% of these cells). This latter group of lectins may be useful in the identification of armadillo lymphocyte subsets, or may correlate with discrete stages of differentiation of these cells. The same lectins served to evaluate mitogenic stimulation in an aliquot of the same peripheral blood mononuclear cells. Of the 12 lectins studied, 5 (ConA, PHA-L, PWM, DBA and SBA) had the capacity to induce mitogenic stimulation in the whole mixture of mononuclear cells, giving rise to variable degrees in the corresponding mitogenic index obtained for each of the 5 lectins. Those lectins that gave an indication of selective identification of lymphocytes, that is, the percentages at or below 75%, may prove useful in the evaluation of the immune response of healthy armadillos as well as the evolution of progression stages of lepromatous leprosy in armadillos inoculated with the same strain of Mycobacterium leprae that affects humans.

Animals↗
PubMed1994

Antibodies to murine CD40 protect normal and malignant B cells from induced growth arrest.

We have previously described the production of polyclonal anti-murine CD40 antibodies that specifically bind recombinant murine CD40 expressed on L cells and induce vigorous proliferation of normal murine B lymphocytes. The current study utilizes these antibodies to explore the distribution and function of CD40 in murine B cell development. Murine CD40 is expressed at high levels by normal splenic B cells and all Ig-positive B cell lymphomas tested to date. It is not expressed by the 70Z/3 pre-B cell line, BaF3 pre-B cell line, or by numerous T cell and myeloid cell lines. 70Z/3 pre-B cells can be induced to express CD40 by LPS stimulation of the cells. Stimulation of purified splenic B cells with anti-CD40 antibodies causes upregulation of class II MHC antigens, CD23, and ICAM-1 and results in extensive aggregation of the cells. Antibodies to murine CD40 are extremely effective at rescuing malignant and normal B cells from induced growth arrest. Anti-CD40 antibodies protect WEHI-231 and CH31 B lymphoma cells from growth arrest induced by soluble anti-IgM antibodies, TGF beta, or a combination of both stimulants. Similarly, anti-IgM preactivated normal splenic B cells which normally die rapidly from growth arrest after 1 or 2 days culture produce a vigorous proliferative response to subsequent stimulation with anti-CD40 antibodies plus IL-4. Interestingly, anti-CD40 antibodies provide little to no protection against B lymphoma growth arrest induced by immobilized anti-IgM antibodies. These data confirm and extend functional properties assigned previously to human CD40 and identify numerous defined murine model systems to explore the molecular basis of CD40-mediated protection from induced B cell growth arrest.

Animals↗
PubMed1994

Murine B-cell activation via CD38 and protein tyrosine phosphorylation.

CD38 has been implicated in the regulation of both proliferation and rescue from apoptosis of B cells. The signalling events associated with CD38-mediated activation of murine B cells are, as yet, not well defined but it is clear that ligation of CD38 by a mitogenic antibody, NIMR-5, induces a calcium influx in resting B cells. Interestingly, however, cross-linking of CD38 does not mobilize intracellular stores of calcium. We now provide a rationale for these findings by demonstrating that CD38 is not coupled to the generation of inositol phosphates in resting B cells. We do, however, show that CD38 ligation stimulates one, or more, protein tyrosine kinase activities which may play a central role in the transduction of CD38-mediated signals leading to B-cell activation.

ADP-ribosyl Cyclase↗
PubMed1993

Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38.

CD38 is a 42-kilodalton glycoprotein expressed extensively on B and T lymphocytes. CD38 exhibits a structural homology to Aplysia adenosine diphosphate (ADP)-ribosyl cyclase. This enzyme catalyzes the synthesis of cyclic ADP-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+) with calcium-mobilizing activity. A complementary DNA encoding the extracellular domain of murine CD38 was constructed and expressed, and the resultant recombinant soluble CD38 was purified to homogeneity. Soluble CD38 catalyzed the formation and hydrolysis of cADPR when added to NAD+. Purified cADPR augmented the proliferative response of activated murine B cells, potentially implicating the enzymatic activity of CD38 in lymphocyte function.

ADP-ribosyl Cyclase↗
PubMed1993

Expression cloning of a cDNA encoding a novel murine B cell activation marker. Homology to human CD38.

A rat mAb (NIM-R5) has recently been prepared against a novel murine B cell activation marker. We report here isolation of a cDNA (1-19) encoding the B cell-derived protein recognized by NIM-R5 antibody. This cDNA contains an open reading frame that encodes a polypeptide of 304 amino acids with a predicted molecular weight of 34,500. The existence of a 22-amino acid hydrophobic region located 23 amino acids from the amino terminal of the deduced protein, together with four potential N-linked glycosylation sites, characterize the deduced protein encoded by I-19 cDNA as a typical type II transmembrane glycoprotein. Although I-19 cDNA appears to encode a novel murine protein, its nucleotide sequence and deduced amino acid sequence show approximately 70% homology to the previously reported sequence of human CD38, suggesting that I-19 cDNA encodes either the mouse homologue of CD38 or a closely related protein. Northern blot analysis of the expression of this cDNA product in a variety of cell types, together with immunoprecipitation of the recombinant protein expressed in BaF3 cells, indicated that I-19 cDNA encodes not only the epitope recognized by NIM-R5 but also a protein that is indistinguishable biochemically and in terms of distribution from the murine B cell activation marker recognized by NIM-R5 antibody. Chromosomal mapping studies have localized this locus to the proximal region of mouse chromosome 5. We anticipate that the availability of probes for the murine B cell activation marker recognized by NIM-R5, and the recombinant protein itself, will greatly aid efforts to define the role of this molecule in murine B cell development.

ADP-ribosyl Cyclase↗