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W Schwaeble

Publications and source records attributed to W Schwaeble.

At least 19 recordsLinked to original sources
PubMed2005

Functional MASP2 single nucleotide polymorphism plays no role in psoriasis.

BACKGROUND: Psoriasis is a heritable disease and genome-wide scans have implicated several loci of susceptibility. The gene for MASP-2, a protease involved in complement activation, is located within one of these loci on chromosome 1p. OBJECTIVES: To assess whether partial or total MASP-2 deficiency is a risk factor for developing psoriasis. METHODS: We screened a cohort of patients affected by plaque psoriasis and their parents by restriction fragment length polymorphism analyses. RESULTS: We detected a single nucleotide polymorphism that leads to an amino acid exchange, which results in dissociation of MASP-2 from a carbohydrate recognition complex. CONCLUSIONS: We show that this mutant allele is not associated with psoriasis. There was no favoured transmission from parents to affected offspring. The calculated allele frequency in this psoriasis group (Scottish and English) was 0.0326, and in the unaffected group 0.0379.

Alleles↗
PubMed2001

Recombinant expression of human mannan-binding lectin.

Mannan-binding lectin (MBL) constitutes an important part of the innate immune defence by effecting the deposition of complement on microbial surfaces. MBL deficiency is among the most common primary immunodeficiencies and is associated with recurrent infections and symptoms of poor immune complex clearance. Plasma-derived MBL has been used in reconstitution therapy but concerns over viral contamination and production capacity point to recombinant MBL (rMBL) as a future source of this protein for clinical use. Natural human MBL is an oligomer of up to 18 identical polypeptide chains. The synthesis of rMBL has been accomplished in several mammalian cell lines, however, the recombinant protein differed structurally from natural MBL. In this, study we compare rMBL produced in myeloma cells, Chinese hamster ovary (CHO) cells, human hepatocytes, and human embryonic kidney (HEK) cells. We report that rMBL structurally and functionally similar to natural MBL can be obtained through synthesis in the human embryonic kidney cells followed by selective carbohydrate affinity chromatography.

Carrier Proteins↗
PubMed2001

The human gene for mannan-binding lectin-associated serine protease-2 (MASP-2), the effector component of the lectin route of complement activation, is part of a tightly linked gene cluster on chromosome 1p36.2-3.

The proteases of the lectin pathway of complement activation, MASP-1 and MASP-2, are encoded by two separate genes. The MASP1 gene is located on chromosome 3q27, the MASP2 gene on chromosome 1p36.23-31. The genes for the classical complement activation pathway proteases, C1r and C1s, are linked on chromosome 12p13. We have shown that the MASP2 gene encodes two gene products, the 76 kDa MASP-2 serine protease and a plasma protein of 19 kDa, termed MAp19 or sMAP. Both gene products are components of the lectin pathway activation complex. We present the complete primary structure of the human MASP2 gene and the tight cluster that this locus forms with non-complement genes. A comparison of the MASP2 gene with the previously characterised C1s gene revealed identical positions of introns separating orthologous coding sequences, underlining the hypothesis that the C1s and MASP2 genes arose by exon shuffling from one ancestral gene.

Base Sequence↗
PubMed2001

Role of the classical pathway of complement activation in experimentally induced polymicrobial peritonitis.

The complement system and the natural antibody repertoire provide a critical first-line defense against infection. The binding of natural antibodies to microbial surfaces opsonizes invading microorganisms and activates complement via the classical pathway. Both defense systems cooperate within the innate immune response. We studied the role of the complement system in the host defense against experimental polymicrobial peritonitis using mice lacking either C1q or factor B and C2. The C1q-deficient mice lacked the classical pathway of complement activation. The factor B- and C2-deficient mice were known to lack the classical and alternative pathways, and we demonstrate here that these mice also lacked the lectin pathway of complement activation. Using inoculum doses adjusted to cause 42% mortality in the wild-type strain, none of the mice deficient in the three activation routes of complement (factor B and C2 deficient) survived (mortality of 100%). Mortality in mice deficient only in the classical pathway of complement activation (C1q deficient) was 83%. Application of further dilutions of the polymicrobial inoculum showed a dose-dependent decrease of mortality in wild-type controls, whereas no changes in mortality were observed in the two gene-targeted strains. These results demonstrate that the classical activation pathway is required for an effective antimicrobial immune defense in polymicrobial peritonitis and that, in the infection model used, the remaining antibody-independent complement activation routes (alternative and lectin pathways) provide a supporting line of defense to gain residual protection in classical pathway deficiency.

Animals↗
PubMed2001

cDNA cloning, chromosome assignment, and genomic structure of a human gene encoding a novel member of the RBM family.

We have cloned and characterised a novel human gene mapping to chromosome 20q11.2. A partial transcript was initially isolated from a human cDNA library transcribed from RNA of the colon carcinoma cell line T-84. In order to determine the full coding sequence of this novel mRNA, we isolated seven cDNA clones from a human cDNA library transcribed from RNA of the acute monocytic leukemia cell line THP1 by colony hybridization. On Northern blot analysis of four human cell lines, the cDNAs isolated hybridize with an abundantly expressed mRNA species of 3.5 kb. A full-length cDNA transcript of this novel mRNA has an open reading frame of 2,796 bp encoding a protein with a calculated molecular weight of 97 kDa. Two repetitive structural consensus motifs are contained within the deduced protein sequence, namely five distinct RNA binding motifs and two proline rich regions. The derived protein sequence also contains putative transmembrane domains. These structural motifs identify this novel protein as a member of an expanding protein family containing RNA binding motifs (RBM). As seen from recently completed sequence of the genomic area encoding this novel mRNA by the Sanger Centre Human Genome Project, the coding region of this gene, RBM12, is intronless.

Amino Acid Motifs↗
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↗
PubMed1999

Microfibril-associated protein 4 is present in lung washings and binds to the collagen region of lung surfactant protein D.

We have purified a glycoprotein from bovine lung washings using affinity chromatography on a maltose-affinity column. On SDS-polyacrylamide gel electrophoresis the protein showed a molecular mass of 36 kDa in the reduced state and 66 kDa in the unreduced state. On gel permeation chromatography the apparent molecular mass was 250 kDa. N-terminal sequencing showed homology to the human matrix protein microfibril-associated protein (hMFAP4), and the glycoprotein was designated bovine MFAP4 (bMFAP4). Lung surfactant protein D (SP-D) was also purified from lung washings, and calcium-dependent binding was demonstrated between bMFAP4 and SP-D. hMFAP4 was cloned, and recombinant hMFAP4 showed the same binding pattern to SP-D as bMFAP4. No binding was seen to recombinant SP-D composed of the neck region and carbohydrate recognition domain of SP-D, indicating that the interaction between MFAP4 and SP-D is mediated via the collagen region of SP-D. MFAP4 also showed calcium-dependent binding to mannan, which was partially inhibited by maltose. Our findings indicate that MFAP4 has two binding specificities, one for collagen and one for carbohydrate, and we suggest that MFAP4 may fix the collectins in the extracellular compartment during inflammation.

Amino Acid Sequence↗
PubMed1999

Expression and regulation by interferon-gamma of the membrane-bound complement regulators CD46 (MCP), CD55 (DAF) and CD59 in gastrointestinal tumours.

The membrane-bound complement inhibitors CD46 (membrane cofactor protein), CD55 (decay-accelerating factor) and CD59 (protectin) protect tumour cells against lysis by activated complement. In this study, a total of 14 (3 gastric, 3 colonic and 8 pancreatic) gastrointestinal tumour cell lines were examined for the expression of CD46, CD55 and CD59 with respect to the regulatory efficacy of interferon-gamma (IFN-gamma). The effects of IFN-gamma on mRNA and protein expression levels of CD46, CD55 and CD59 were evaluated by Northern blot hybridisation, RT-PCR, flow cytometry and immunostaining. In unstimulated cell lines, CD46 and CD59 transcripts were expressed at comparable levels, whereas the basal expression of CD55 mRNA was heterogeneous. The complement inhibitor proteins were detected in all cell lines using specific antibodies. Additional immunohistochemical stainings of gastrointestinal tissue specimens supported these findings. IFN-gamma evoked a weak induction of certain transcripts in a subset of the cell lines. Upregulation of protein expression was only observed in HT29 cells for CD55 and CD59 and was accompanied by a marked increase of the corresponding transcripts. We conclude that membrane-bound complement inhibitors are broadly expressed in gastrointestinal tumour cells and vary in their susceptibility to IFN-gamma. Thus, they may be involved in tumour escape mechanisms in gastric, pancreatic and colorectal cancer.

Antigens, CD↗
PubMed1998

Characterization of the murine gene of gC1qBP, a novel cell protein that binds the globular heads of C1q, vitronectin, high molecular weight kininogen and factor XII.

gC1qBP is a novel cell protein which was found to interact with the globular heads of C1q, high mol. wt kininogen, factor XII and the heparin-binding, multimeric form of vitronectin. The protein sequence shows no homology to any protein family. This paper describes the genomic organization of mouse gC1qBP and the characterization of its 5' flanking region. The mouse gene consists of six exons separated by five introns, and its total length is approximately 6kb. Exon 1 encodes the putative signal peptide, a long stretch of 70 amino acid residues, and the first four amino acid residues found in the mature gC1qBP. Exons 2-5 encode four very hydrophilic domains, whereas exon 6 encodes a neutral domain. The amino acid sequence responsible for binding to the heparin-binding, multimeric form of vitronectin is located in exon 2. A 1kb DNA fragment upstream of the first initiation codon was sequenced, which contained four potential TATA boxes, seven CAAT boxes, six SP1 sites and various putative transcription factor-binding elements, indicating that the promoter region is in close proximity to the first exon. The mouseC1qbp gene was mapped to chromosome 11, closely linked to D11Mit4 using genomic DNAs from a (C57BL/6J x Mus spretus)F1 x Mus spretus backcross.

Amino Acid Sequence↗
PubMed1997

Intracellular localization of the human receptor for the globular domains of C1q.

This study was performed to determine the localization of the recently described receptor for the globular domain of C1q, gC1qR. In contrast to previous reports, we were not able to detect significant surface expression of gC1qR on Raji cells, monocytes, neutrophils, human or rat mesangial cells, the endothelial cell line EA.hy 926, or HUVEC using FACS analysis. Only by using digoxigenin-conjugated Abs could some surface staining of gC1qR be observed on rat mesangial cells and neutrophils. However, after permeabilizing these cells with saponin, a strong positive intracellular staining for gC1qR was observed by FACS, fluorescence microscopy on coverslips, and confocal laser scanning microscopic analysis. By reflection contrast microscopy and electron microscopy on ultrathin sections of permeabilized Raji cells, it was shown that gC1qR is present in double membranous cytoplasmic vesicles located in the proximity of the plasma membrane. To determine whether certain conditions could induce surface expression of gC1qR, Raji cells were either stimulated with T cell growth factor, LPS, or driven to apoptosis by incubation with fenretinide or by serum depletion. None of the conditions resulted in significant surface expression of gC1qR. Our hypothesis that gC1qR is not a surface molecule but a soluble molecule that is secreted by cells is supported by the observation that gC1qR is found in significant concentrations in supernatants of several cultured cells and in normal human and rat sera. Our results suggest that the recently described gC1qR is not a cell surface receptor, but a soluble binding protein with affinity for the globular heads of C1q. Excreted gC1qR might act as a potential fluid phase regulator of complement activation.

Animals↗
PubMed1997

A second serine protease associated with mannan-binding lectin that activates complement.

The complement system comprises a complex array of enzymes and non-enzymatic proteins that is essential for the operation of the innate as well as the adaptive immune defence. The complement system can be activated in three ways: by the classical pathway which is initiated by antibody-antigen complexes, by the alternative pathway initiated by certain structures on microbial surfaces, and by an antibody-independent pathway that is initiated by the binding of mannan-binding lectin (MBL; first described as mannan-binding protein) to carbohydrates. MBL is structurally related to the complement C1 subcomponent, C1q, and seems to activate the complement system through an associated serine protease known as MASP (ref. 4) or p100 (ref. 5), which is similar to C1r and C1s of the classical pathway. MBL binds to specific carbohydrate structures found on the surface of a range of microorganisms, including bacteria, yeasts, parasitic protozoa and viruses, and exhibits antibacterial activity through killing mediated by the terminal, lytic complement components or by promoting phagocytosis. The level of MBL in plasma is genetically determined, and deficiency is associated with frequent infections in childhood, and possibly also in adults (for review, see ref. 6). We have now identified a new MBL-associated serine protease (MASP-2) which shows a striking homology with the previously reported MASP (MASP-1) and the two C1q-associated serine proteases C1r and C1s. Thus complement activation through MBL, like the classical pathway, involves two serine proteases and may antedate the development of the specific immune system of vertebrates.

Amino Acid Sequence↗
PubMed1997

Complement and complement deficiencies.

The complement system provides a first line of defense and mediates a large variety of cellular and humoral interactions within the immune response, including chemotaxis, phagocytosis, cell adhesion, and B-cell differentiation. The system involves more than 30 serum components and numerous cell surface regulators and receptors. Similar to the blood clotting system, complement activation is initiated through a series of complex activation cascades involving enzymatic cleavage. Three independent complement activation cascades, the classical, the alternative, and the lectin pathway, have been described. The liver is the main site of biosynthesis for most of the serum components of complement and diseases of the liver can lead to alterations of the normally stable plasma levels of complement. Deficiencies of single components can lead to a broad variety of secondary diseases, caused by either imbalanced activation or defects in the humoral or cellular response to microbial infections.

Complement C3a↗
PubMed1996

Synthesis of classical pathway complement components by chondrocytes.

Using immunohistochemical studies, C1q, C1s, C4 and C2 were detected in chondrocytes in normal human articular cartilage and macroscopically normal articular cartilage from the inferior surfaces of hip joints of patients with osteoarthritis. Using reverse-transcribed polymerase chain reaction (RT-PCR), mRNA for C1q, C1s, C4 and C2 was also detected in RNA extracted from articular cartilage. C1r, C3, C1-inhibitor, C4-binding protein and factor I were not detected by either technique. Articular chondrocytes cultured in vitro synthesized C1r, C1s, C4, C2, C3 and C1-inhibitor but not C1q, C4-binding protein or factor I, as assessed by enzyme-linked immunosorbent assay (ELISA) and Northern blot analysis. Thus cultured articular chondrocytes have a complement profile that is similar to that of cultured human fibroblasts rather than that of articular chondrocytes in vivo. Complement synthesis in cultured chondrocytes was modulated by the cytokines interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), showing that cytokines can probably regulate complement synthesis in intact cartilage. The possible roles of local synthesis of complement components by chondrocytes in matrix turnover and the regulation chondrocyte function are discussed.

Base Sequence↗
PubMed1995

Follicular dendritic cells, interdigitating cells, and cells of the monocyte-macrophage lineage are the C1q-producing sources in the spleen. Identification of specific cell types by in situ hybridization and immunohistochemical analysis.

In a mouse model, we have shown previously that macrophages are the principal source of complement C1q. Furthermore, spleen, heart, and brain were found to contain substantial levels of murine C1q-specific mRNA, whereas liver, kidney, lung, and small intestine contained only trace amounts of C1q-specific mRNA. This work addresses the identification of C1q-expressing spleen cells in the rat, using Northern blotting and in situ detection of rat C1q mRNA combined with immunohistochemical analysis. The complete sequence of mRNA encoding the B chain of rat C1q was established. The cloned cDNA was found to hybridize primarily with spleen-derived mRNA of 1.2 kb, and additionally with a novel mRNA species of 3 kb. In situ hybridization together with immunohistochemistry revealed most of the C1q-expressing cells to be located in the red pulp of the spleen, and to be mainly of the monocyte-macrophage lineage, as indicated by coexpression of ED-1, an established marker for this type of cell. In addition, C1q was expressed in S-100-positive but ED-1-negative cells, in germinal center follicular dendritic cells, and in some interdigitating dendritic cells of the periarteriolar lymphatic sheath (PALS). These results indicate that the spleen, containing the above APCs that are all involved to a major extent in the adaptive immune response and are all capable of synthesizing C1q that is involved intimately in the innate immune response, may provide the site at which the innate and adaptive immune systems merge.

Amino Acid Sequence↗
PubMed1995

Expression of C1q, a subcomponent of the rat complement system, is dramatically enhanced in brains of rats with either Borna disease or experimental allergic encephalomyelitis.

In situ hybridization, RT-PCR and Northern blot analysis as well immunohistochemistry were used to examine the expression of C1q, a subcomponent of the rat complement system, in brains of rats infected with Borna disease virus (BDV) and rats afflicted with experimental allergic encephalomyelitis (EAE) induced by the adoptive transfer of myelin basic protein specific T cells. C1q mRNA, which was not detected in normal brain, became clearly detectable using RT-PCR analysis by d14 post infection (p.i.) with BDV. Maximal levels of C1q mRNA were reached 21 days p.i. when inflammatory reactions in the brain were also at a peak. Similarly, C1q mRNA was elevated when the clinical symptoms of EAE became evident 5 days following cell transfer. C1q positive cells, as identified by immunohistology, were preferentially localized in grey and white matter of the hippocampus and basolateral cortex. The C1q positive cells resembled microglial cells in morphology. The correlation of C1q expression with the development of neurological disease as well as the dramatic increase of C1q within brain regions with inflammatory lesions suggest that local biosynthesis of C1q may play a role in the pathogenesis of Borna virus induced and autoimmune encephalomyelitis.

Animals↗
PubMed1994

Localization of kappa-opioid receptor mRNA in neuronal subpopulations of rat sensory ganglia and spinal cord.

A partial cDNA encoding a kappa-opioid receptor was isolated and used to generate specific 35S-labeled probes to investigate the gene expression of the kappa-opioid receptor in sensory, sympathetic and spinal neurons of the rat by in situ hybridization. A subpopulation of mainly small and medium-sized neurons within dorsal root and trigeminal ganglia expressed kappa-receptor mRNA, but no signal was detectable in the superior cervical ganglion. kappa-Receptor mRNA was distributed over neurons throughout the dorsal horn and in laminae VII/VIII. Highest concentrations of positive neurons were seen in laminae I/II, dorsal lamina X and in the lateral spinal nucleus. alpha-Motoneurons and glial cells were not labeled. This distribution of kappa-receptor mRNA indicates preferential functions of kappa-receptors in sensory signalling with particular importance to nociception.

Animals↗
PubMed1994

Expression of properdin in human monocytes.

Properdin is the only known positive regulator of the alternative pathway of complement activation. Northern blot analysis of cell lines derived from fibroblasts, B-cells, hepatoma cells, and cells of the monocyte-macrophage lineage revealed properdin expression only in the myelomonocytic cell line HL-60, in the monoblastic cell line U-937 and in the monocytic line Mono Mac 6. Culture of Mono Mac 6 cells for 24 h with phorbol 12-myristate 13-acetate, bacterial lipopolysaccharide and the cytokines interleukin-1 beta and tumour necrosis factor-alpha enhanced mRNA abundance, with the strongest effect (tenfold) being observed with the lipopolysaccharide. In contrast, recombinant interferon-gamma consistently halved properdin mRNA abundance. The same pattern was found for the secretion of properdin as detected by ELISA of Mono Mac 6 supernatants. The suppressive effect of interferon-gamma on properdin mRNA abundance was also demonstrated for primary blood monocytes. The data suggest that the expression and secretion of this complement regulatory protein by monocytes is differentially regulated by cytokines and link the immune response with alternative pathway activation.

Adult↗