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R P da Silva

Publications and source records attributed to R P da Silva.

At least 19 recordsLinked to original sources
PubMed2001

Endogenous ligands of carbohydrate recognition domains of the mannose receptor in murine macrophages, endothelial cells and secretory cells; potential relevance to inflammation and immunity.

The macrophage mannose receptor (MR) has an established role in the phagocytosis of a wide range of microbes, and also functions in viral endocytosis, and clearance of a number of endogenous glycoproteins from the circulation. Its broad ligand specificity is mediated by tandemly linked carbohydrate recognition domains (CRDs). Recent studies suggest that binding or internalization of both natural and synthetic ligands of MR CRDs may modulate macrophage (MPhi ) function, for example to increase cidal capacity or cytokine synthesis. To identify endogenous ligands in the normal mouse we used an Fc-fusion protein (CRD4-7Fc) bearing four of the CRDs of MR. CRD4-7Fc recognized endocytic compartments of cultured MPhi, consistent with lysosomal enzymes being major ligands of MR. CRD4-7Fc also recognized MPhi and some endothelial cells in tissues, and intensely labeled secretory cells of the exocrine pancreas, salivary gland and thyroid. Strongly MR-positive interstitial cells were found in close proximity to the ligand-rich secretory cells, suggesting a role for MR in uptake of secretory glycoproteins, including thyroglobulin which was identified as a novel ligand in vitro. Endocytosis of these ligands by MR may have implications for tissue homeostasis and immunity, including antigen presentation, in secretory organs.

Animals↗
PubMed1999

Type 1 and type 2 cytokine regulation of macrophage endocytosis: differential activation by IL-4/IL-13 as opposed to IFN-gamma or IL-10.

Cytokine regulation of endocytic activity in primary human macrophages was studied to define ultrastructural changes and mechanisms of pinocytic regulation associated with cytokines secreted by activated T cells. The effects of IFN-gamma (type 1) and IL-4/IL-13 and IL-10 (type 2) cytokines on fluid phase and mannose receptor-mediated endocytosis were assessed by horseradish peroxidase and colloidal gold-BSA uptake and computer-assisted morphometric analysis. IL-4 and IL-13 enhanced fluid phase pinocytosis and mannose receptor-mediated uptake by activation of phosphatidylinositol 3-kinase. Inhibition of actin assembly showed that both cytokines exerted actin-dependent and -independent effects. Ultrastructurally, IL-4 and IL-13 increased tubular vesicle formation underneath the plasma membrane and at pericentriolar sites, concurrent with decreased particle sorting to lysosomes. By contrast, IL-10 or IFN-gamma decreased both fluid phase pinocytosis and mannose receptor-mediated uptake. IFN-gamma stimulated increased particle sorting to perinuclear lysosomes, while IL-10 decreased this activity. In summary, our data document differential effects on macrophage endocytic functions by type 1 or type 2 cytokines associated with induction and effector pathways in immunity.

Actins↗
PubMed1999

Phagocytosis stimulates alternative glycosylation of macrosialin (mouse CD68), a macrophage-specific endosomal protein.

Macrosialin (mouse CD68), a macrophage-specific member of the lysosomal-associated membrane protein family, displays N-linked glycosylation and a heavily sialylated, mucin-like domain. We show that phagocytosis of zymosan by inflammatory peritoneal macrophages potently alters glycan processing of macrosialin in vitro. The phagocytic glycoform is not induced by other forms of endocytosis and depends on particle internalization. Zymosan uptake does not influence macrosialin protein synthesis, but increases the specific incorporation of D-[2-3H]mannose, D-[6-3H]galactose, N-acetyl-D-[1-3H]glucosamine and L-[5,6-3H]fucose by 2-15-fold. The phagocytic glycoform displays increased binding of agglutinins from peanut, Amaranthus caudatus and Galanthus nivalis, whereas binding of the sialic-acid-specific Maakia amurensis agglutinin is slightly reduced. Digestion by N-Glycanase abolishes the incorporation of [3H]mannose label and Galanthus nivalis agglutinin binding activity, but preserves the incorporation of galactose and N-acetylglucosamine and specific lectin binding. We also show that phagocytosis increases the complexity and length of O-linked chains. The data presented highlight the importance of differential glycosylation in the biology of macrosialin, phagosomes and macrophages in general.

Animals↗
PubMed1999

Class A scavenger receptors and the phagocytosis of apoptotic cells.

Apoptotic cells are rarely seen in situ because of rapid clearance by phagocytes. A number of receptors have been implicated in the recognition and ingestion of dying cells. Class A scavenger receptors (SRs) are multi-domained membrane glycoproteins that can endocytose modified lipoproteins and bind a wide range of ligands. There is growing evidence that they contribute to several biological processes. We present data that suggest class A SRs are involved in the phagocytosis of apoptotic cells.

Animals↗
PubMed1999

Rapid recruitment of late endosomes and lysosomes in mouse macrophages ingesting Candida albicans.

Candida albicans is an important opportunistic pathogen, whose interaction with cells of the immune system, in particular macrophages (MO), is poorly understood. In order to learn more about the nature of the infectious mechanism, internalisation of Candida albicans was studied in mouse MO by confocal immunofluorescence and electron microscopy in comparison with latex beads of similar size, which were coated with mannosyl-lipoarabinomannan (ManLAM) to target the MO mannose receptor (MR). Uptake of Candida yeasts had characteristics of phagocytosis, required intact actin filaments, and depended on the activity of protein kinase C (PKC). Candida phagosomes rapidly attracted lysosome-associated membrane protein (Lamp)-rich vacuoles, indicative of fusion with late endosomes and lysosomes. Rapid recruitment of late endosomes and lysosomes could be observed regardless of heat-inactivation or serum-opsonisation of Candida, but did not follow binding of the mannosylated-beads to MO, which suggest that this phenotype is not MR-specific. The yeasts developed germ tubes within phagolysosomes, distended their membranes and escaped, destroying the non-activated MO. The filamentous form of Candida could penetrate intact MO even when phagocytosis was blocked, and also attracted Lamp-rich organelles. Inhibition of lysosomal acidification and associated lysosomal fusion reduced germ tube formation of Candida within the phagolysosomes. These data suggest that rapid recruitment of late endocytic/lysosomal compartments by internalizing C. albicans favours survival and virulence of this pathogen.

Acids↗
PubMed1998

Lack of p56lck expression correlates with CD4 endocytosis in primary lymphoid and myeloid cells.

In cell lines the endocytic properties of CD4 are regulated through its association with the src-family tyrosine kinase p56lck. In lymphoid cell lines expressing p56lck, CD4 is restricted to the cell surface and undergoes only limited internalization. Phosphorylation of the cytoplasmic domain of CD4 causes p56lck to dissociate and activates an endocytosis signal leading to the internalization of CD4 through clathrin-coated pits. In p56lck-negative transfected cell lines CD4 is constitutively internalized, but internalization is inhibited when p56lck is expressed in these cells. We now demonstrate that these endocytic properties of CD4 determined in transfected cell lines hold true for CD4 naturally expressed on myeloid cell lines (HL-60 and U937), as well as on primary lymphocytes, monocytes and macrophages isolated from human blood. CD4 showed limited internalization on p56lck-positive lymphocytes, but was rapidly internalized in p56lck-negative monocytes and macrophages. Surprisingly, rapid internalization of CD4 was seen with the lymphocytes from one unidentified donor. In these cells we failed to detect p56lck expression by Western blotting.

Animals↗
PubMed1998

Recognizing death: the phagocytosis of apoptotic cells.

Although apoptotic cell death is widespread, dying cells are rarely seen in situ because of their rapid clearance by neighbouring phagocytes. Phagocytic recognition of apoptotic cells is less well understood than the death programme itself, but an increasing number of recent studies are highlighting its importance. This review discusses the nature of the receptors that have been implicated in apoptotic cell phagocytosis, the mechanisms of uptake and the immunological consequences of apoptotic cell ingestion.

Animals↗
PubMed1996

Macrophage membrane molecules: markers of tissue differentiation and heterogeneity.

Monoclonal antibodies directed against murine macrophage differentiation antigens provide tools to characterize novel glycoproteins, their expression by different macrophage subpopulations in situ and regulation by cytokines and other agents in vitro. Studies are in progress to determine ligands for these molecules, and to establish possible functions. We summarize current knowledge of the following molecules: F4/80, a glycoprotein with homology to the G-protein linked transmembrane 7 hormone receptor family; macrosialin, a member of the lysosomal-associated membrane protein [lamp] family with a macrophage-specific mucin-like extracellular domain also present in its human homologue, CD68; sialoadhesin, a sialic acid binding lectin with multiple Ig superfamily domains; mannosyl receptor, a lectin-like molecule with multiple C-type lectin domains; type 3 complement receptor, a beta 2 integrin involved in cell migration and adhesion; scavenger receptor, a transmembrane homotrimer with a collagenous domain.

Animals↗
PubMed1993

Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family.

Macrosialin is a heavily glycosylated transmembrane protein of 87-115 kDa, highly and specifically expressed by mouse tissue macrophages, and to a lesser extent by dendritic cells. We have isolated cDNA clones encoding macrosialin from a thioglycollate-elicited peritoneal macrophage cDNA library by transient expression in COS cells and panning with the anti-macrosialin monoclonal antibody FA/11. A single 1.3-kilobase macrosialin transcript was detected in both untreated and phorbol 12-myristate 13-acetate-stimulated RAW cells. The cDNA sequence predicts a type I integral membrane protein of 326 residues with a heavily glycosylated extracellular domain of 306 residues containing nine potential N-linked glycosylation sites and numerous potential O-linked glycosylation sites. The extracellular domain consists of two distinct regions, separated by an extended 12 residue proline-rich hinge; a membrane-distal mucin-like domain of 89 residues containing short peptide repeats and consisting of 44% serine and threonine residues; and a membrane proximal domain of 170 residues, which has significant sequence homology to a family of lysosomal associated glycoproteins known as the lamp-1 group. Macrosialin is the murine homologue of the human macrophage glycoprotein CD68 (72% identity, 80% similarity). Both proteins are preferentially expressed by macrophages and share the same bipartite structure having a mucin-like domain and a domain common to the lamp family. Macrosialin and CD68 are the first examples of a lamp family protein with a restricted cell-type-specific expression. They may have evolved from the lamps to carry out specialized functions in dedicated phagocytic cells.

Amino Acid Sequence↗
PubMed1991

[Clinical aspects and development of dilated cardiomyopathy in infants and children].

PURPOSE: The evaluation of the clinical aspects of the dilated cardiomyopathy (DCM) in infants and children regarding, mainly, to the evolution and prognostic of this disease. PATIENTS AND METHODS: 38 patients divided in two groups: A) 22 infants till 22 (11.60 +/- 6.50) months of age, 15 female, and B) 15 children of 2 to 12 years of age (5.23 +/- 3.13) the majority males (10). A retrospective study was carried out based on the data from the patients's records. It was performed, in all the cases, a clinical, electrocardiographic, echocardiographic (M module and two dimensional echocardiography) and radiologic evaluation. RESULTS: The dyspnea on exercise (included sucking) was the predominant symptom in 15 (65.22%) patients of the A group and 10 (66.67%) of the B group followed by perilabial cyanosis in 7 (30.43%) and 6 (40%) patients, respectively. In the A group the clinical diagnostic hypothesis was inspecific myocarditis (IM) in 12 (52.17%), endocardial fibroelastosis (EFE), in 8 (34.79%), and "idiopathic" dilated cardiomyopathy (IDCM) in 3 (13.04%). In the B group to the diagnostic conclusion of myocarditis was made in 10 patients (66.67%)--5 of them IM--EFE in 3 (20%); and IDCM in 2 (13.33%). The average time of evolution was 5.48 months in the A group and 18.56 in the B group. In the A group the evolution was excellent in 3 (3.04%), good in 10 (43.46%), stable in 2 (8.70%) and bad in 1 (4.35%). In the B group, excellent in 8 (53.33%), good in 2 (20%) stable in 1 (6.67%). No bad evolution in this group. There was a decrease in the A group (4.34%); 6 patients in this group (26.09%) and 3 (20%) of the B group interrupt the follow-up. CONCLUSION: 1) The prognosis of infants with DCM including those with the diagnostic hypothesis of EFE seems to be less adverse than it could be supposed to be; 2) the prognostic in children with the diagnosis of DCM established above 2 years of age seems to be good; 3) the differential clinical diagnosis between EFE and IM is difficult and with no accuracy; 4) it is possible that the IM could be more prevalent in infants till 6 months of age than we suppose it was.

Cardiomyopathy, Dilated↗
PubMed1989

Cell surface nanoanatomy of Leishmania major as revealed by fracture-flip. A surface meshwork of 44 nm fusiform filaments identifies infective developmental stage promastigotes.

Fracture-flip (Anderson-Forsman and Pinto da Silva, J. Cell Sci. 90, 531-541; 1988) was used to reveal the nanoanatomy of the surface of Leishmania major promastigotes. Over the cell surface of infective metacyclic promastigotes we identify a meshwork of 44 nm long, fusiform filaments. These filaments are not seen in noninfective stages of the parasite. Replica-staining immunocytochemistry with monoclonal antibody against infective metacyclic lipophosphoglycan shows a uniform distribution of protein A-colloidal gold complexes over the cell surface. Thin sections show that acquisition of the high molecular weight lipophosphoglycan is reflected in a thicker glycocalyx. Conventional freeze-fracture shows that in infective metacyclic promastigotes there is a reversal of the partition of intramembrane particles--an additional morphological marker for the infective developmental stage. We hypothesize that the fusiform filaments represent metacyclic developmental lipophosphoglycan.

Animals↗
PubMed1988

Complement binding by two developmental stages of Leishmania major promastigotes varying in expression of a surface lipophosphoglycan.

The binding of complement by two developmentally distinct stages of Leishmania major has been studied. Noninfective log phase growth (LOG) promastigotes (serum sensitive) activate complement with deposition of covalently bound C3b onto the surface of the parasite. Infective, peanut agglutinin (PNA-) metacyclic stage promastigotes (serum resistant) also bear mainly C3b after incubation in serum, but a major portion of deposited C3 is present as a 110 X 10(3) mol wt C3 fragment. Whereas deposition of C3b on LOG promastigotes is mediated through the alternative pathway. PNA- parasites are unable to activate the alternative pathway in nonimmune serum. C3 is released from the parasite surface by proteolytic cleavage, at a rate which is nearly threefold greater for LOG than for PNA- promastigotes. Immunoprecipitation experiments show that the developmentally regulated lipophosphoglycan is a major C3 acceptor on both LOG and PNA- parasites. These experiments, which are the first to compare the form and processing of complement on infective and noninfective promastigotes of Leishmania, provide a framework for further definition of the differential C3 receptor-dependent uptake and survival of these parasites within mononuclear phagocytes.

Animals↗
PubMed1987

The generation of infective stage Leishmania major promastigotes is associated with the cell-surface expression and release of a developmentally regulated glycolipid.

A monoclonal antibody, 3F12, was generated which reacted specifically against infective or metacyclic stage Leishmania major promastigotes, but not with noninfective promastigotes obtained from log phase cultures. The antibody recognized a cell surface and released molecule that could be metabolically labeled with [14C]glucose, [3H]mannose, [3H]galactose, and [3H]palmitic acid, but not with [35S]methionine or [3H]leucine. The molecule was the major species surface-labeled by [3H]sodium borohydride after periodate treatment. The glycolipid appeared to be shed primarily as free carbohydrate because 70% of the released material partitioned in the aqueous fraction after phase separation in TX-114. The molecule could be distinguished from the L. major glycolipid which has already been extensively described because its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was of higher relative m.w. However, a close relationship between the two molecules was indicated by the finding that another monoclonal antibody, WIC-79.3, recognized both forms of the glycolipid; one produced and released only by log phase promastigotes, and one produced and released only by metacyclic promastigotes. The loss of agglutination with peanut agglutinin which has been shown to accompany metacyclogenesis was found to be caused by the loss of expression of the log form of the glycolipid which in most cases appeared to be the result of the developmental modification of this molecule. A survey of a number of virulent and avirulent. L. major strains and clones reinforced an absolute association between the ability of these promastigotes to initiate infection in BALB/c mice and their expression and release of the 3F12-binding, developmentally regulated form of the glycolipid. Not only does this glycolipid serve as the first well defined molecular marker for infective stage metacyclic promastigotes, but its unique structure is very likely to contribute to the adaptive changes that allow these parasites to survive within the vertebrate host.

Animals↗