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Biomedical subjects

R B Sim

Publications and source records attributed to R B Sim.

At least 19 recordsLinked to original sources
PubMed2007

Severe fibrosis in hepatitis C virus-infected patients is associated with increased activity of the mannan-binding lectin (MBL)/MBL-associated serine protease 1 (MASP-1) complex.

Mannan-binding lectin (MBL) binds microorganisms via interactions with glycans on the target surface. Bound MBL subsequently activates MBL-associated serine protease proenzymes (MASPs). A role for MBL in hepatitis C virus (HCV) infection had been indicated by previous studies examining MBL levels and polymorphisms in relation to disease progression and response to treatment. We undertook this study to investigate a possible relationship between disease progression and functional MBL/MASP-1 complex activity. A functional assay for MBL/MASP-1 complex activity was employed to examine serum samples from patients with chronic HCV infection, non-HCV liver disease and healthy controls. Intrapatient consistency of MBL/MASP-1 complex activity levels was assessed in sequential samples from a subgroup of patients. Median values of MBL/MASP-1 complex activity were higher in sera from patients with liver disease compared with healthy controls. MBL/MASP-1 complex activity levels correlate with severity of fibrosis after adjusting for confounding factors (P = 0.003). MBL/MASP-1 complex activity was associated more significantly with fibrosis than was MBL concentration. The potential role of MBL/MASP-1 complex activity in disease progression is worthy of further study to investigate possible mechanistic links.

Adolescent↗
PubMed2005

[Concentration and protein composition of circulating immune complexes in the blood of patients with schizophrenia and subjects with positive familial history of disease].

A comparative study of blood serum concentrations and pathogenic properties of circulating immune complexes (CIC), along with identification of their protein composition, was conducted in 45 patients with schizophrenia, 15 their healthy relatives and 39 normal controls. In patients and their relatives, mean concentration of small CIC was within the normal range, while concentrations of giant, large and middle CIC were higher than those of the controls (p<0.001). Over 80% of schizophrenic patients and their relatives had pathogenic immune complexes in the circulation. Clinical and immunologic analysis of patients with schizophrenia revealed a correlation between the illness duration and CIC concentration for all sizes. Smokers had significantly lower levels of small CIC comparing to non-smokers. Determination of CIC composition in patients and relatives revealed a presence of specific proteins in the immune complexes with molecular weights of 36 and 25 kDa. The results suggest genetic determination of autoimmune processes in schizophrenia.

Adolescent↗
PubMed2004

Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA.

Primary defence against invading microorganisms depends on a functional innate immune system and the complement system plays a major role in such immunity. Deficiencies in one of the components of the complement system can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus (SLE) and renal disease. Screening for complement deficiencies in the classical or alternative complement pathways has mainly been performed by haemolytic assays. Here, we describe a simple ELISA-based format for the evaluation of three pathways of complement activation. The assays are based on specific coatings for each pathway in combination with specific buffer systems. We have standardized these assays and defined cut off values to detect complement deficiencies at the different levels of the complement system. The results demonstrate the value of these ELISA-based procedures for the functional assessment of complement deficiencies in clinical practice. The assay is now available commercially in kit form.

Complement Activation↗
PubMed2004

Proteases of the complement system.

The complement system is a group of about 35 soluble and cell-surface proteins which interact to recognize, opsonize and clear or kill invading micro-organisms or altered host cells (e.g. apoptotic or necrotic cells). Complement is a major part of the innate immune system. Recognition proteins such as C1q, MBL (mannan-binding lectin) and ficolins bind to targets via charge or sugar arrays. Binding causes activation of a series of serine protease proenzymes, such as C1r, C1s and MASP2 (MBL-associated serine protease 2), which in turn activate the atypical serine proteases factor B and C2, which then activate the major opsonin of the system, C3. Activated C3 binds covalently to targets, and is recognized by receptors on phagocytic cells. Two of the complement proteases, factors D and I, circulate not as proenzymes, but in activated form, and they have no natural inhibitors; their substrates are transient protein complexes (e.g. C3bB and C3bH) which form during complement activation. Factor B and C2 also have no natural inhibitor; they are active only when proteolytically cleaved and bound in an unstable, short-lived complex with C3b or C4b. C1r, C1s and the MASPs, in contrast, are regulated more conventionally by the natural serpin, C1-inhibitor. Complement proteases in general have very narrow specificity, and low substrate turnover with both natural and synthetic substrates. Excessive activation of complement is inflammatory, and causes tissue damage (e.g. in rheumatoid arthritis, or in ischaemia/reperfusion injury). Substances that regulate complement activation are likely to be useful in the regulation of inflammation. Complement activation might potentially be controlled at many different steps. Much attention has been focused on controlling the formation or activity of the protease complexes C3bBb and C4b2a (containing activated factor B and C2 respectively), as these generate the inflammatory peptides C3a and C5a.

Complement Activation↗
PubMed2003

Collectins and their role in lung immunity.

The collectins are a small family of secreted glycoproteins that contain C-type lectin domains and collagenous regions. They have an important function in innate immunity, recognizing and binding to microorganisms via sugar arrays on the microbial surface. Their function is to enhance adhesion and phagocytosis of microorganisms by agglutination and opsonization. In the lung, two members of the collectin family, surfactant proteins A and D, are major protein constituents of surfactant. Another collectin, mannan-binding lectin, is also present in the upper airways and buccal cavity and may protect against respiratory infections. Recent work has shown that collectins have roles in resistance to allergy and in the control of apoptosis and clearance of apoptotic macrophage in the lung.

Cell Adhesion↗
PubMed2003

Biochemistry and genetics of mannan-binding lectin (MBL).

Mannose- or mannan-binding lectin (MBL) is a member of the collectin protein family, which includes lung surfactant proteins SP-A and SP-D. Each member consists of similar or identical polypeptide chains with a region of collagen-like sequence followed by a C-type lectin domain. The polypeptides associate in threes to form a subunit containing a collagen-like helix, with three clustered lectin domains. These subunits associate into larger structures, usually with 12-18 polypeptides. The collectins bind to patterns of neutral sugars on surfaces (e.g. of micro-organisms) and mediate effector functions associated with killing/phagocytosis. MBL is the only collectin which activates complement. It resembles in quaternary structure the complement protein C1q, which recognizes targets via charge clusters. Binding of MBL to a surface activates MBL-associated serine proteases (MASPs) attached to MBL, and MASP-2 activates complement proteins C4 and C2. The MASPs are homologous to the C1q-associated proteases, C1r and C1s. MBL therefore activates complement by a mechanism very similar to C1q, and engages the opsonic activity of complement to clear micro-organisms. The serum concentration of MBL is very variable in humans. The variability is largely associated with mutations leading to amino acid substitutions in the collagen-like region which decrease MBL assembly and stability. Many studies demonstrate that MBL deficiency is associated with susceptibility to a range of infectious and inflammatory diseases.

Complement Pathway, Mannose-Binding Lectin↗
PubMed2002

Evaluation and clinical interest of mannan binding lectin function in human plasma.

The mannan binding lectin (MBL) plays a major role in innate immunity through its ability to activate complement upon binding to carbohydrate arrays on the surface of various microorganisms. The question of a possible association of the MBL structural gene polymorphism and the oligomeric state of MBL was poorly documented. For these reasons, it appears difficult to evaluate MBL in blood patients on the only basis of protein contents, even in combination with MBL genotyping. This study reports a method to calculate a specific activity for circulating MBL, that relies on: (i) the availability of purified MBL; and (ii) a simplified MBL activity assay based on complement activation. The three-step MBL purification from human plasma reported here is characterized by a highly purified MBL, that occurs in two different oligomeric forms. The results on the specific activity of these forms show that the higher oligomeric forms of MBL have the ability to induce C4 cleavage more efficiently than the corresponding lower oligomers. The usefulness of this approach is illustrated by its potential interest in the biological exploration of certain pathology, for example in the follow-up of chronic hepatitis C. Further investigation is needed to establish whether MBL specific activity (MBLsa) is correlated to the polymorphic state of the molecule. The relative simplicity of the test described here allows better investigation on the relationship between MBL biological activity and its genotype.

Animals↗
PubMed2001

Complement C4bC2 complex formation: an investigation by surface plasmon resonance.

Complex formation between the human complement proteins C4b and C2 was investigated by surface plasmon resonance. C4b was immobilised and C2 was used in the fluid phase to measure interaction at different ionic strengths (30-830 mM NaCl) and in the absence and presence of MgCl2. Maximum binding was observed at 30 mM NaCl, and was negligible above 300 mM NaCl. Binding was not greatly influenced by variation in Mg(2+) in the range of 2.5-15 mM. C4bC2 affinity (Kd) was determined by steady-state analysis to be 7.2x10(-8) M in physiological conditions (10 mM Hepes, 2.5 mM MgCl2, 0.75 mM CaCl2 and 140 mM NaCl, pH 7.4). For C4(H2O)C2 complex formation, a Kd of 4.0x10(-8) M was calculated. As far as detected by the applied method, complex formation does not involve conformational changes of one of the binding partners. Consistent with previous reports, C4bC2 binding takes place as a multiple-site binding event in the presence of Mg2+. C4bC2 complex formation in 10 mM Hepes, 2.5 mM EDTA and 140 mM NaCl (pH 7.4) was also observed and the interaction showed characteristics of a single-site binding event. Kd was 1.5x10(-8) M. Complement factor B (FB) was also tested for its binding to immobilised C4b. Weak interaction was observed at FB concentrations in the physiological range (500-1000 nM). Kd was 1.2x10(-6) M, indicating possible cross-reactivity between classical and alternative pathways of the activation of the complement system.

Complement C4↗
PubMed2001

Assessment of in vivo complement activation on the Echinococcus granulosus hydatid cyst wall.

The larval stage of the parasite Echinococcus granulosus causes hydatid disease. The hydatid cyst is potentially capable of activating host complement, since it is a large, persistent, carbohydrate-rich structure, coated with host immunoglobulins, and localized in the host's internal organs. Nonetheless, in vitro studies have suggested that the cyst surface, the hydatid cyst wall (HCW), is a poor complement activator. In this study, we assessed the occurrence of in vivo complement activation on the hydatid cyst by measuring the levels of two complement activation products, C3d and complexes bearing a C9 activation neoepitope (TCC/MAC), in extracts from HCW of human origin. Low amounts of C3d and TCC/MAC were found in HCW in comparison with their levels in normal human plasma and activated human sera, suggesting that in vivo complement activation on HCW is efficiently down-regulated. This regulation may contribute to limit host inflammation which has been observed to correlate with parasite degeneration and death.

Animals↗
PubMed2000

Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2.

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.

Carrier Proteins↗
PubMed2000

Complement C1q is dramatically up-regulated in brain microglia in response to transient global cerebral ischemia.

Recent evidence suggests that the pathophysiology of neurodegenerative and inflammatory neurological diseases has a neuroimmunological component involving complement, an innate humoral immune defense system. The present study demonstrates the effects of experimentally induced global ischemia on the biosynthesis of C1q, the recognition subcomponent of the classical complement activation pathway, in the CNS. Using semiquantitative in situ hybridization, immunohistochemistry, and confocal laser scanning microscopy, a dramatic and widespread increase of C1q biosynthesis in rat brain microglia (but not in astrocytes or neurons) within 24 h after the ischemic insult was observed. A marked increase of C1q functional activity in cerebrospinal fluid taken 1, 24, and 72 h after the ischemic insult was determined by C1q-dependent hemolytic assay. In the light of the well-established role of complement and complement activation products in the initiation and maintenance of inflammation, the ischemia-induced increase of cerebral C1q biosynthesis and of C1q functional activity in the cerebrospinal fluid implies that the proinflammatory activities of locally produced complement are likely to contribute to the pathophysiology of cerebral ischemia. Pharmacological modulation of complement activation in the brain may be a therapeutic target in the treatment of stroke.

Animals↗
PubMed2000

A Schistosoma protein, Sh-TOR, is a novel inhibitor of complement which binds human C2.

Human complement regulatory (also called inhibitory) proteins control misdirected attack of complement against autologous cells. Trypanosome and schistosome parasites which survive in the host vascular system also possess regulators of human complement. We have shown Sh-TOR, a protein with three predicted transmembrane domains, located on the Schistosoma parasite surface, to be a novel complement regulatory receptor. The N-terminal extracellular domain, Sh-TOR-ed1, binds the complement protein C2 from human serum and specifically interacts with the C2a fragment. As a result Sh-TOR-ed1 pre-incubated with C2 inhibits classical pathway (CP)-mediated haemolysis of sheep erythrocytes in a dose-dependent manner. In CP-mediated complement activation, C2 normally binds to C4b to form the CP C3 convertase and Sh-TOR-ed1 has short regions of sequence identity with a segment of human C4b. We propose the more appropriate name for TOR of CRIT (complement C2 receptor inhibitory trispanning).

Amino Acid Sequence↗
PubMed2000

How Echinococcus granulosus deals with complement.

Here, Ana Mar a Ferreira and colleagues discuss the interplay between the larval stages of Echinococcus granulosus and an important effector arm of immunity: the host complement system. During early infection, the parasite activates complement, and hence complement-dependent inflammatory responses. However, on differentiation into the hydatid cyst, the parasite exposes to the host a structure - the cyst wall - that does not activate complement strongly. Mechanisms inhibiting complement activation on the cyst wall have been elucidated, contributing to the understanding of how this large, persistent, tissue-dwelling pathogen controls the inflammatory response.

Animals↗
PubMed2000

Collectins and innate immunity in the lung.

Evidence from both in vitro and in vivo studies suggests that the collectins are important elements in host innate immune defences against infectious agents. Study of the collectins in specific disease settings now raises the prospects of developing therapies exploiting these mechanisms of innate immunity.

Animals↗
PubMed2000

Serine proteases of the complement system.

The complement system in blood plasma is a major mediator of innate immune defence. The function of complement is to recognize, then opsonize or lyse, particulate materials, including bacteria, yeasts and other microrganisms, host cell debris and altered host cells. Recognition occurs by binding of complement proteins to charge or saccharide arrays. After recognition, a series of serine proteases is activated, culminating in the assembly of complex unstable proteases called C3/C5 convertases. These activate the complement protein C3, which acts as an opsonin. The complement serine proteases include the closely related C1r, C1s, MASPs 1-3 (80-90 kDa), C2 and Factor B (100 kDa), Factor D (25 kDa) and Factor I (85 kDa). Each of these has unusually restricted specificity and low enzymic activity. The C1r, C1s and MASP group occur as proenzymes. When activated, they are regulated, like many plasma serine proteases, by a serpin, C1-inhibitor. C2 and Factor B, however, have complex multiple regulation by a group of complement proteins called the Regulation of Complement Activation (or RCA) proteins, whereas Factors I and D appear to have no natural inhibitors. Advances in structure determination and protein-protein interaction properties are leading to a more detailed understanding of the complement-system proteases, and are indicating possible new routes for potential therapeutic control of complement.

Animals↗
PubMed2000

Humoral autoreactivity directed against surfactant protein-A (SP-A) in rheumatoid arthritis synovial fluids.

SP-A is found principally in the lung, and has been associated with lamellar bodies also found in the synovial joint. Both SP-A and C1q contain collagen-like regions, and SP-A and C1q have some structural similarities, both having a globular head region and a collagen-like tail. Here we are able to show that (i) autoreactivity to SP-A, as expressed by IgG and IgM autoantibodies, is present in synovial fluid (SF) isolated from patients with rheumatoid arthritis (RA); (ii) in absorption experiments only a limited degree of cross-reactivity between autoantibodies reactive with C1q and SP-A is observed; (iii) there is no cross-reactivity between autoantibodies reactive with type II collagen (CII) and those reactive with SP-A or C1q; (iv) autoantibodies react with polymeric (dimers and larger) SP-A, but not with monomeric SP-A subunits, indicating that a degree of quaternary structure is required for antibody binding. Unlike CII, which not accessible in the normal joint, both SP-A and C1q are available within the SF in patients with RA and may therefore provide antigens driving an autoimmune response directed against collagen-like structures.

Animals↗