Biomedical subjects

R B Sim

Publications and source records attributed to R B Sim.

At least 37 records · Page 2Linked to original sources

PubMed2000

Contribution of C5-mediated mechanisms to host defence against Echinococcus granulosus hydatid infection.

The aim of this work was to investigate the contribution of complement C5-mediated mechanisms, with an emphasis on inflammation, to host defences against Echinococcus granulosus hydatid disease. Thus, we compared the systemic and local inflammatory responses induced by the parasite, and the outcome of infection, between congenic C5-sufficient (B10.D2 n/SnJ) and C5-deficient (B10.D2 o/SnJ) mice challenged with protoscoleces. Indirect evidence of in-vivo complement activation during the establishment phase was obtained; infection induced serum amyloid P and eosinophil responses which were dependent on C5. Early recruitment of polymorphonuclear cells was not dependent on the presence of C5. The higher capacity of C5-sufficient mice to recruit eosinophils was also observed during the cystic phase of infection, and mice recruiting more eosinophils developed lower parasite masses. Analysis of the outcome of infection after 8 months showed that C5-sufficient mice were more resistant to infection than C5-deficient mice in terms of individuals with no cysts; this trend was not statistically significant. In addition, C5-deficient mice developed higher numbers of large (> 5 mm in diameter) cysts and higher cyst weights than C5-sufficient mice indicating that C5-mediated mechanisms are detrimental for parasite growth. Taken together, our results suggest that complement, through C5-mediated effectors, contributes to host defences by both restricting the establishment of infection and controlling the growth of established cysts. This contribution may, at least partially, be associated with the ability of C5a to promote eosinophil infiltration.

Animals↗

PubMed2000

Lung surfactant protein A provides a route of entry for respiratory syncytial virus into host cells.

Lung surfactant protein A (SP-A) has a central role in host defense mediated by the interaction of surface carbohydrates of inhaled pathogens with the lectin domains of SP-A. Respiratory syncytial virus (RSV), the most important viral pathogen of neonates and infants, encodes a highly glycosylated attachment protein, G. Binding studies were performed with G-protein from RSV (human, A2 strain) and human SP-A. The effect of SP-A on the interaction between RSV and host cells was determined by two methods: an infectivity study with monolayers of Hep-2C cells and by interleukin-8 (IL-8) release from buffy coat (BC) cells. SP-A binds to RSV G-protein in a concentration-dependent manner that is inhibitable by both ethylenediamine tetraacetic acid (EDTA) and mannan, indicating that binding is through the carbohydrate recognition domain of the SP-A and a carbohydrate moiety of the G-protein. The level of RSV infection of Hep-2C cells increases with increasing concentrations of SP-A. The amount of IL-8 released by BC cells in the presence of RSV is increased with SP-A concentrations of 2.9 microg/mL or greater. Our results show that SP-A enhances the attachment of RSV and subsequent entry into host cells. The effect of SP-A on viral uptake by epithelial cells and macrophage may determine both innate and adaptive immune responses to RSV.

Bronchoalveolar Lavage Fluid↗

PubMed2000

Two edged role of mannose binding lectin in rheumatoid arthritis: a cross sectional study.

OBJECTIVE: We investigated whether polymorphisms in the gene of mannose binding lectin (MBL) may be associated with onset of rheumatoid arthritis (RA), and whether MBL in conjunction with aggregated agalactosyl IgG (IgG-G0) may be associated with clinical and paraclinical variables. METHODS: MBL genotypes and serum concentrations were measured by polymerase chain reaction and ELISA in 189 patients with established RA. Binding of purified MBL to IgG-G0 in serum was assessed and clinical and paraclinical variables were recorded. RESULTS: The median age at onset of RA in the 3 genotypes (normal: A/A, hetero: A/0, and homozygous: 0/0 for variant alleles) was 54.1 (n = 108), 47.0 (n = 68), and 38.4 years (n = 13), respectively (p = 0.01). The frequency of variant alleles in patients with onset below the median age (50.8 yrs) was 0.32, but was 0.17 in patients with onset above 50.8 years (p = 0.003) and 0.20 in 250 controls (p = 0.001). Stratification according to erosion score (no, small, large) revealed an increasing tendency among the different groups in binding of MBL to IgG-G0, increased Health Assessment Questionnaire score, and acute phase reactants in A/A individuals, while no difference was seen among carriers of variant alleles. This effect was most pronounced in those with late onset RA. CONCLUSION: Presence of MBL variant alleles was associated with early onset of RA. MBL deficiency may, therefore, accelerate the disease. However, in patients with late onset and advanced disease our results indicate that the A/A type may be associated with additional inflammation different from that seen in carriers of variant alleles.

Adult↗

PubMed2000

Expression of the proteinase specialized in bone resorption, cathepsin K, in granulomatous inflammation.

BACKGROUND: The cysteine proteinase cathepsin K has aroused intense interest as the main effector in the digestion of extracellular matrix during bone resorption by osteoclasts. The enzyme is not a housekeeping lysosomal hydrolase, but is instead expressed with striking specificity in osteoclasts. In this work, we present evidence for the association of cathepsin K with the granulomatous reaction. Granulomas are inflammatory tissue reactions against persistent pathogens or foreign bodies. We came across cathepsin K while working on Echinococcus granulosus, a persistent tissue-dwelling, cyst-forming parasite that elicits a granulomatous response. MATERIALS AND METHODS: The walls of hydatid cysts from infected cattle were solubilized. Strong proteolytic activity was detected in the extracts. The proteinase responsible was purified by anion exchange and gel filtration. The purified protein was subjected to N-terminal sequencing, and its identity further confirmed by Western blotting, with a cathepsin K-specific antibody. The same antibody was used to localize the proteinase in paraffin-embedded sections of the parasite and the local host response. RESULTS: A proteinase was purified to near homogeneity from hydatid cyst extracts. The enzyme was unequivocally identified as host cathepsin K. Both the proenzyme and the mature enzyme forms were found. Cathepsin K was then immunolocalized both to the parasite cyst wall and to the epithelioid and giant multinucleated cells of the host granulomatous response. CONCLUSIONS: In the granulomatous response to the hydatid cyst, cathepsin K is expressed by epithelioid and giant multinucleated cells. We propose that, by analogy with bone resorption, cathepsin K is secreted by the host in an attempt to digest the persistent foreign body. Both processes, bone resorption and granulomatous reactions, therefore tackle persistent extracellular material (the bone matrix or the foreign body), and utilize specialized cells of the monocytic lineage (osteoclasts or epithelioid/giant cells) secreting cathepsin K as an effector.

Amino Acid Sequence↗

PubMed1999

Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B.

Factor B is a five-domain 90 kDa serine protease proenzyme which is part of the human serum complement system. It binds to other complement proteins C3b and properdin, and is activated by the protease factor D. The fourth domain of factor B is homologous to the type A domain of von Willebrand Factor (vWF-A). A full-length human factor B cDNA clone was used to amplify the region encoding the vWF-A domain (amino acids 229-444 of factor B). A fusion protein expression system was then used to generate it in high yield in Escherichia coli, where thrombin cleavage was used to separate the vWF-A domain from its fusion protein partner. A second vWF-A domain with improved stability and solubility was created using a Cys(267)-->Ser mutation and a four-residue C-terminal extension of the first vWF-A domain. The recombinant domains were investigated by analytical gel filtration, sucrose density centrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consistent with known vWF-A crystal structures. This expression system and its characterization permitted the first investigation of the function of the isolated vWF-A domain. It was able to inhibit substantially the binding of (125)I-labelled factor B to immobilized C3b. This demonstrated both the presence of a C3b binding site in this portion of factor B and a ligand-binding property of the vWF-A domain. The site at which factor D cleaves factor B is close to the N-terminus of both recombinant vWF-A domains. Factor D was shown to cleave the vWF-A domain in the presence or absence of C3b, whereas the cleavage of intact factor B under the same conditions occurs only in the presence of C3b.

Amino Acid Sequence↗

PubMed1999

Identification of human complement Factor H as a ligand for L-selectin.

The selectin family of adhesion molecules (E-, P- and L-selectins) is involved in leukocyte recruitment to sites of inflammation and tissue damage. Recently it has been shown that L-selectin is involved not only in leukocyte tethering and rolling, but also plays an important role in leukocyte activation. For example, glycosylation-dependent cell-adhesion molecule 1 (GlyCAM-1), a known ligand for L-selectin, has been shown to enhance beta2-integrin function. GlyCAM-1 is a secreted protein and is present in mouse serum at a concentration of approx. 1.5 microg/ml. There is no obvious GlyCAM-1 homologue in man and, to date, L-selectin ligand(s) from human serum have not been characterized. Therefore we have used L-selectin affinity chromatography, followed by ion-exchange chromatography, to isolate specific ligand(s) for L-selectin. Using this procedure, we have isolated three major glycoproteins of apparent molecular masses 170 kDa, 70kDa and 50 kDa. The 170 kDa protein band was digested with trypsin and peptides were analysed by delayed extraction matrix-assisted laser desorption ionization MS and protein database searching. The 170 kDa protein was identified as the human complement protein Factor H. Human Factor H, isolated by a different method, was shown to bind specifically to L-selectin in the presence of CaCl2, and binding was inhibited by anti-L-selectin antibodies, fucoidan and lipopolysaccharide. Only a part of the purified Factor H preparation bound to immobilized L-selectin. The interaction of Factor H with leukocyte L-selectin was shown to induce the secretion of tumour necrosis factor-alpha (TNF-alpha). Pretreatment of Factor H with sialidase reduced both the binding of L-selectin to Factor H and the Factor H-induced L-selectin-mediated TNF-alpha secretion by leukocytes. Taken together, these results demonstrate that a post-translationally modified form of human plasma Factor H is a potential physiological ligand for L-selectin.

Blood Proteins↗

PubMed1999

In vitro analysis of complement-dependent HIV-1 cell infection using a model system.

Previous studies based on the use of human serum as a source of C have provided evidence for the C-dependent enhancement of cell infection by HIV-1. The present study was undertaken to distinguish C from other serum factors and to identify the proteins and the mechanisms involved in C-dependent cell infection by HIV-1. The classical C activation pathway was reconstituted from the proteins C1q, C1r, C1s, C4, C2, C3, factor H, and factor I; each were purified to homogeneity. A mixture of these proteins at physiological concentrations was shown to reproduce the ability of normal human serum to enhance the infection of MT2 cells by HIV-1 at low doses of virus. This enhancing effect was abolished when heat-inactivated serum and C2- or C3-depleted serum were used, and was restored upon addition of the corresponding purified proteins. A mixture of two synthetic peptides corresponding to positions 10-15 and 90-97 of human C receptor type 2 (CD21) as well as soluble CD4 both inhibited the C-dependent infection process. These data provide unambiguous evidence that HIV-1 triggers a direct activation of the classical C pathway in vitro and thereby facilitates the infection of MT2 cells at low doses of virus. These findings are consistent with a mechanism involving increased interaction between the virus opsonized by C3b-derived fragment(s) and the CD21 cell receptors and subsequent virus entry through CD4 receptors.

Acquired Immunodeficiency Syndrome↗

PubMed1999

A recombinant trimeric surfactant protein D carbohydrate recognition domain inhibits respiratory syncytial virus infection in vitro and in vivo.

The pulmonary collectin, lung surfactant protein D (SP-D), plays a role in host defense mediated by the interaction of surface carbohydrates of inhaled pathogens with the lectin domains of SP-D. Respiratory syncytial virus (RSV), the most important viral pathogen of neonates and infants, encodes a highly glycosylated attachment protein, G. Binding studies were performed with G protein from RSV (human, A2 strain) and both native and recombinant human SP-D. The effect of recombinant trimeric SP-D lectin domains (rSP-D) on the interaction between RSV and host cells was determined by two methods: an infectivity study with monolayers of Hep-2C cells and in vivo infections in BALB/c mice. These studies show that full-length and recombinant SP-D bind to RSV G protein in a concentration-dependent manner. Both EDTA and mannan inhibited binding of full-length SP-D. These results indicate that binding occurs via the carbohydrate recognition domain of the SP-D. The recombinant SP-D inhibited RSV infectivity in cell culture in a dose-dependent manner, giving 100% inhibition of replication. Intranasal administration of recombinant SP-D to RSV-infected mice inhibited replication of the virus in the lungs, reducing levels of lung virus by 80%. These results suggest that SP-D plays a major role in clearing RSV from the lungs.

Animals↗

PubMed1999

Control of host complement activation by the Echinococcus granulosus hydatid cyst.

Cystic hydatid disease is caused by the multicellular parasite Echinococcus granulosus. The hydatid cyst, being a long-lived, large, antigenic structure lodged in the host's internal organs, could potentially elicit major inflammatory responses. However, in practice, the cyst causes only minimal local inflammation. The complement system is a major pathway to immune-mediated inflammation. Recent results have shown that the host-exposed structure of the cyst, the hydatid cyst wall (HCW), fails to trigger the complement system strongly. We have carried out a wide survey for the mechanisms making the cyst wall relatively complement-inert. The results of those studies are summarised in this work, with emphasis on the most recently identified of the complement inhibitory mechanisms. This is based on a non-protein heat-stable, parasite inhibitor of the activation of host complement factor B.

Animals↗

PubMed1998

The first subcomponent of complement, C1q, triggers the production of IL-8, IL-6, and monocyte chemoattractant peptide-1 by human umbilical vein endothelial cells.

We and others have demonstrated previously the occurrence of cC1qR/CaR, a receptor for the collagen-like stalks of complement component C1q, on endothelial cells. In the present study we investigated whether binding of C1q to endothelial cells resulted in enhancement of cytokine or chemokine production. HUVEC produced 82 +/- 91 pg/ml of IL-8, 79 +/- 113 pg/ml of IL-6, and 503 +/- 221 pg/ml of monocyte chemoattractant peptide-1 (MCP-1) under basal conditions. Incubation with C1q resulted in a time- and dose-dependent up-regulation of IL-8 (1012 +/- 43 pg/ml), IL-6 (392 +/- 20 pg/ml), and MCP-1 (2450 +/- 101 pg/ml). This production is dependent on de novo protein synthesis, as demonstrated by the detection of specific mRNA after C1q stimulation, and inhibition of peptide production in the presence of cycloheximide. The production of all factors was inhibited (69 +/- 7%) by the collagenous fragments of C1q, while the C1q globular heads only induced 13 +/- 11% inhibition. When HUVEC were incubated with C1q in the presence of aggregated IgM, enhanced production of IL-8 (2500 +/- 422 pg/ml), IL-6 (997 +/- 21 pg/ml), and MCP-1 (5343 +/- 302 pg/ml) was found. Furthermore, F(ab')2 anti-calreticulin partially inhibited the production of IL-8, confirming at least the involvement of cC1qR/CaR. These experiments suggest that in an inflammatory response C1q not only is able to activate the complement pathway, but when presented in a proper fashion also might induce the production of factors that contribute to acute phase responses and recruitment of inflammatory cells.

Animals↗

PubMed1998

Induction of TNF-alpha release from human buffy coat cells by Pseudomonas aeruginosa is reduced by lung surfactant protein A.

Lipopolysaccharide (LPS) induction of TNF-alpha release is a central event in the pathophysiology of gram-negative bacterial septicaemia. Lung surfactant protein A (SP-A) mediates pathogen/host cell interactions. Binding of SP-A to Pseudomonas aeruginosa LPS and the effects of SP-A with LPS or whole bacteria on buffy coat cells were investigated. SP-A interacts with P. aeruginosa LPS in a concentration and calcium dependent manner, either through the lipid A portion of LPS or through another lectin/carbohydrate interaction. SP-A decreased TNF-alpha secretion induced by bacteria or LPS from buffy coat cells, in a concentration dependent manner.

Apoproteins↗

PubMed1998

Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope.

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Here we have investigated the resistance of EEV and IMV to neutralization by complement in the absence of immune antibodies. When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV resistance was not a result of EEV protein B5R, despite its similarity to proteins of the regulators of complement activation (RCA) family, or to any of the other EEV proteins tested (A34R, A36R, and A56R gene products). EEV was sensitive to complement when the virus was grown in one species and challenged with complement from a different species, suggesting that complement resistance might be mediated by host RCA incorporated into the EEV outer envelope. This hypothesis was confirmed by several observations: (i) immunoblot analysis revealed that cellular membrane proteins CD46, CD55, CD59, CD71, CD81, and major histocompatibility complex class I antigen were detected in purified EEV but not IMV; (ii) immunoelectron microscopy revealed cellular RCA on the surface of EEV retained on the cell surface; and (iii) EEV derived from rat cells expressing the human RCA CD55 or CD55 and CD59 were more resistant to human complement than EEV derived from control rat cells that expressed neither CD55 nor CD59. These data justify further analysis of the roles of these (and possible other) cellular proteins in EEV biology.

Animals↗

PubMed1998

Mannan-binding lectin in human serum, cerebrospinal fluid and brain tissue and its role in Alzheimer's disease.

Mannan-Binding lectin (MBL) is a serum lectin which can activate the classical complement pathway. Complement proteins of the classical pathway have been found in the brains of patients with Alzheimer's disease (AD) in association with AD brain pathology. To investigate the role for MBL in AD we have looked for its presence in the brain by immunohistochemistry and determined the levels of MBL in paired samples of cerebrospinal fluid and serum from AD patients and controls. MBL was detected in association with blood vessels in the brain tissue of both AD patients and control subjects. There was no apparent difference in the distribution of MBL in the brain tissue between the two groups. The mean concentration of MBL in the CSF was 44% lower in AD patients than in controls (AD 154 +/- 35 pg/ml, n = 19; non-AD 276 +/- 50 pg/ml, n = 15, p < 0.05). The levels of MBL in serum were not significantly different in the two groups. Thus, this study shows that MBL is associated with blood vessels in the brains of both AD and control subjects. Moreover, CSF levels of MBL appear to be lower in AD patients than in control subjects which may indicate a higher degree of MBL consumption connected with complement activation in the AD patients.

Aged↗

PubMed1998

Echinococcus granulosus: an intraperitoneal diffusion chamber model of secondary infection in mice.

The present work describes a new experimental model of secondary infection which allows, through the recovery of the parasite together with its local in vivo environment, examination of the local nonadaptive immune response of the infected host and the differentiation of the parasite from protoscoleces to cysts. In this model we administered protoscoleces within silicone diffusion chambers, previously implanted into the peritoneal cavities of mice. The process of designing the model involved, first, determination of the optimal time postimplantation to infect the mice and, second, evaluation of the parasite's ability to establish infection within the chambers. The optimal time for infection was considered to be after the inflammation caused by implantation of the chambers had subsided. Our results showed that by day 20 postsurgery, three parameters used as indications of inflammation (complement C3, serum amyloid P protein, and polymorphonuclear cells in the peritoneum and in the chamber contents) had reverted to their normal levels. In our study of parasite differentiation, we found that 2-3% of the total number of parasites inoculated into the chambers were recovered as viable cysts after 100 days. Throughout the infection period, the population of parasites recovered was heterogeneous; certain parasite morphologies that have not been described previously were observed. In conclusion, the use of intraperitoneal diffusion chambers offers a potential tool for investigating the in vivo differentiation process of secondary cysts of Echinococcus granulosus in mice and the early local interactions between host and parasite during this process.

Animals↗

PubMed1998

Human complement factor I: its expression by insect cells and its biochemical and structural characterisation.

Factor I is a five-domain plasma serine protease which is essential for the regulation of the complement system. In order to express this, the factor I coding sequence was cloned into a recombinant baculovirus system, which was used to infect Trichoplusia ni cells. Using the native factor I leader sequence, recombinant factor I (rFI) was secreted into the culture medium. Purified rFI was recognised by polyclonal antisera and by the factor I-specific monoclonal antibody MRC-OX21. SDS PAGE showed that rFI was processed into two chains with molecular weights of 48,000 and 36,000. Amino acid sequence analysis showed that the N-terminal sequences of the rFI chains were the same as those of serum-derived factor I (sFI), confirming that processing was correct. Since both molecular weights were less than those observed for sFI, this is attributed to the replacement of complex-type oligosaccharides by high mannose ones in rFI. C3(NH,) cleavage assays showed that rFI had 55% the activity of sFI. Circular dichroism and Fourier transform infrared spectroscopy showed that the protein folding of rFI and sFI were very similar. Both had a secondary structure low in alpha-helix and high in beta-sheet, as expected from crystal structure and multiple sequence alignment analyses. It is inferred that the reduced activity of rFI is attributable to its changed glycosylation. The availability of rFI and structures for the domains in factor I makes possible new approaches to determine the molecular basis of its interactions with factor H and C3b.

Animals↗

PubMed1998

Interaction of C1q and the collectins with the potential receptors calreticulin (cC1qR/collectin receptor) and megalin.

Several proteins have been identified as candidate cell-surface receptors for the complement protein C1q. Some of these also interact with the structurally-related collectin proteins. Previous descriptions of C1q-binding properties of cells, and information on the cellular distribution of candidate receptors suggest that there is more than one physiologically relevant receptor for C1q. Two such candidate receptors, cell-surface calreticulin (also referred to as cC1qR or collectin receptor) and megalin are discussed in this review.

Amino Acid Sequence↗

PubMed1998

Mechanism of action of anti-C1-inhibitor autoantibodies: prevention of the formation of stable C1s-C1-inh complexes.

BACKGROUND: Acquired C1-inhibitor (C1-inh) deficiency is usually associated with the presence of circulating C1-inh autoantibodies. These autoantibodies have been shown previously to bind to two synthetic peptides corresponding to C1-inh amino acid residues 438-449 (peptide 2) and 448-459 (peptide 3) but not to peptide 1 (residues 428-440). MATERIALS AND METHODS: Affinity-purified C1-inh autoantibodies from two patients with acquired C1-inh deficiency were studied for their effects on the inhibition of C1s activity by C1-inh using SDS-PAGE and hydrolysis of a synthetic ester. RESULTS: Functional studies confirmed that the anti-C1-inh autoantibodies abrogated C1-inh activity, and their maximum effect was produced when the concentrations of C1-inh and autoantibody were approximately equimolar. The autoantibodies prevent the formation of the C1s-C1-inh complex, but they do not dissociate the preformed complex, suggesting that the autoantibodies act prior to the formation of the enzyme-inhibitor complex. In the presence of autoantibodies, C1s cleaves C1-inh, and a stable covalent bond between C1s and C1-inh does not form. Peptides 2 and 3, but not peptide 1 inhibited autoantibody activity, thus C1-inh inhibitory activity for C1s was expressed fully. CONCLUSIONS: Our data indicate that the anti-C1-inh autoantibodies convert C1-inh to a substrate by preventing the formation of the stable covalent protease-serpin complex. The data also suggest a possible therapeutic use for peptides 2 and 3 or their derivatives in the management of patients with type II acquired angioedema (AAE).

Autoantibodies↗

PubMed1998

Human lung surfactant protein A exists in several different oligomeric states: oligomer size distribution varies between patient groups.

BACKGROUND: Lung surfactant protein A (SP-A) is a complex molecule composed of up to 18 polypeptide chains. In vivo, SP-A probably binds to a wide range of inhaled materials via the interaction of surface carbohydrates with the lectin domains of SP-A and mediates their interaction with cells as part of a natural defense system. Multiplicity of lectin domains gives high-affinity binding to carbohydrate-bearing surfaces. MATERIALS AND METHODS: Gel filtration analyses were performed on bronchoalveolar lavage (BAL) fluid samples from three patient groups: pulmonary alveolar proteinosis (n = 12), birch pollen allergy (n = 11), and healthy volunteers (n = 4). Sucrose density gradient centrifugation was employed to determine molecular weights of SP-A oligomers. SP-A was solubilized from the lipid phase to compare oligomeric state with that of water soluble SP-A. RESULTS: SP-A exists as fully assembled complexes with 18 polypeptide chains, but it is also consistently found in smaller oligomeric forms. This is true for both the water- and lipid-soluble fractions of SP-A. CONCLUSION: The three patient groups analyzed show a shift towards lower oligomeric forms of SP-A in the following sequence: healthy-pulmonary alveolar proteinosis-pollen allergy. Depolymerization would be expected to lead to loss of binding affinity for carbohydrate-rich surfaces, with loss or alteration of biological function. While there are many complex factors involved in the establishment of an allergy, it is possible that reduced participation of SP-A in clearing a potential allergen from the lungs could be an early step in the chain of events.

Allergens↗