Biomedical subjects

P F Zipfel

Publications and source records attributed to P F Zipfel.

At least 19 recordsLinked to original sources

PubMed2006

Deletion of Lys224 in regulatory domain 4 of Factor H reveals a novel pathomechanism for dense deposit disease (MPGN II).

We report a novel pathomechanism for membranoproliferative glomerulonephritis type II (MPGN II) caused by a mutant Factor H protein expressed in the plasma. Genetic analyses of two patients revealed deletion of a single Lys residue (K224) located within the complement regulatory region in domain 4 of Factor H. This deletion resulted in defective complement control: mutant protein purified from the plasma of patients showed severely reduced cofactor and decay-accelerating activity, as well as reduced binding to the central complement component C3b. However, cell-binding activity of the mutant protein was normal and comparable to wild-type Factor H. The patients are daughters of consanguineous parents. As both patients but also their healthy mother were positive for C3 nephritic factor, the mutant Factor H protein is considered relevant for unrestricted activation of the disease-causing activation of the alternative complement pathway. Replacement of functional Factor H by fresh frozen plasma (10-15 ml/kg/14 days) was well tolerated, prevented so far disease progression in both patients, and is in the long run expected to preserve kidney function.

Amino Acid Sequence↗

PubMed2006

The C-terminus of complement regulator Factor H mediates target recognition: evidence for a compact conformation of the native protein.

The complement inhibitor Factor H has three distinct binding sites for C3b and for heparin, but in solution uses specifically the most C-terminal domain, i.e. short consensus repeats (SCR) 20 for ligand interaction. Two novel monoclonal antibodies (mABs C14 and C18) that bind to the most C-terminal domain SCR 20 completely blocked interaction of Factor H with the ligands C3b, C3d, heparin and binding to endothelial cells. In contrast, several mAbs that bind to the N-terminus and to the middle regions of the molecule showed no or minor inhibitory effects when assayed by enzyme-linked immunosorbent assay (ELISA) and ligand interaction assays. This paradox between a single functional binding site identified for native Factor H versus multiple interaction sites reported for deletion constructs is explained by a compact conformation of the fluid phase protein with one accessible binding site. On zymosan particles mAbs C14 and C18 blocked alternative pathway activation completely. Thus demonstrating that native Factor H makes the first and initial contact with the C terminus, which is followed by N terminally mediated complement regulation. These results are explained by a conformational hypothetical model: the native Factor H protein has a compact structure and only one binding site accessible. Upon the first contact the protein unfolds and exposes the additional binding sites. This model does explain how Factor H mediates recognition functions during complement control and the clustering of disease associated mutations in patients with haemolytic uraemic syndrome that have been reported in the C-terminal recognition domain of Factor H.

Antibodies, Monoclonal↗

PubMed2005

Variations in the complement regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated with membranoproliferative glomerulonephritis type II (dense deposit disease).

INTRODUCTION: Membranoproliferative glomerulonephritis type II or dense deposit disease (MPGN II/DDD) causes chronic renal dysfunction that progresses to end stage renal disease in about half of patients within 10 years of diagnosis. Deficiency of and mutations in the complement factor H (CFH) gene are associated with the development of MPGN II/DDD, suggesting that dysregulation of the alternative pathway of the complement cascade is important in disease pathophysiology. SUBJECTS: Patients with MPGN II/DDD were studied to determine whether specific allele variants of CFH and CFHR5 segregate preferentially with the MPGN II/DDD disease phenotype. The control group was compromised of 131 people in whom age related macular degeneration had been excluded. RESULTS: Allele frequencies of four single nucleotide polymorphisms in CFH and three in CFHR5 were significantly different between MPGN II/DDD patients and controls. CONCLUSION: We have identified specific allele variants of CFH and CFHR5 associated with the MPGN II/DDD disease phenotype. While our data can be interpreted to further implicate complement in the pathogenesis of MPGN II/DDD, these associations could also be unrelated to disease pathophysiology. Functional studies are required to resolve this question.

Biopsy↗

PubMed2005

Factor H binds to washed human platelets.

BACKGROUND: Factor H regulates the alternative pathway of complement. The protein has three heparin-binding sites, is synthesized primarily in the liver and copurifies from platelets with thrombospondin-1. Factor H mutations at the C-terminus are associated with atypical hemolytic uremic syndrome, a condition in which platelets are consumed. Objectives The aim of this study was to investigate if factor H interacts with platelets. METHODS: Binding of factor H, recombinant C- or N-terminus constructs and a C-terminus mutant to washed (plasma and complement-free) platelets was analyzed by flow cytometry. Binding of factor H and constructs to thrombospondin-1 was measured by surface plasmon resonance. RESULTS: Factor H bound to platelets in a dose-dependent manner. The major binding site was localized to the C-terminus. The interaction was partially blocked by heparin. Inhibition with anti-GPIIb/IIIa, or with fibrinogen, suggested that the platelet GPIIb/IIIa receptor is involved in factor H binding. Factor H binds to thrombospondin-1. Addition of thrombospondin-1 increased factor H binding to platelets. Factor H mutated at the C-terminus also bound to platelets, albeit to a significantly lesser degree. CONCLUSIONS: This study reports a novel property of factor H, i.e. binding to platelets, either directly via the GPIIb/IIIa receptor or indirectly via thrombospondin-1, in the absence of complement. Binding to platelets was mostly mediated by the C-terminal region of factor H and factor H mutated at the C-terminus exhibited reduced binding.

Binding Sites↗

PubMed2004

The hyphal and yeast forms of Candida albicans bind the complement regulator C4b-binding protein.

Candida albicans, an important pathogenic yeast, activates all three pathways of the complement system. To understand how this yeast evades the effects of the activated system, we have analyzed the binding of the classical pathway inhibitor C4b-binding protein (C4BP) by C. albicans. Purified native as well as recombinant C4BP bound dose dependently to the yeast and hyphal forms, as shown by multiple methods, such as confocal microscopy, flow cytometry, a novel enzyme-linked immunosorbent assay, absorption from human serum, and direct binding assays with purified proteins. A prominent binding site was identified at the tip of the germ tube, a structure that is considered important for tissue penetration and pathogenesis. The binding site in C4BP was localized to the two N-terminal complement control protein domains by using recombinant deletion constructs and site-specific monoclonal antibodies. As the alternative pathway inhibitors factor H and FHL-1 also bind to C. albicans, the binding of all three plasma proteins was compared. Simultaneous binding of the classical regulator C4BP and the alternative pathway regulator factor H was demonstrated by confocal microscopy. In addition, FHL-1 competed for binding with C4BP, suggesting that these two related complement regulators bind to the same structures on the yeast surface. The surface-attached C4BP maintains its complement regulatory activities and inactivates C4b. The surface-attached human C4BP serves multiple functions relevant for immune evasion and likely pathogenicity. It inhibits complement activation at the yeast surface and, in addition, mediates adhesion of C. albicans to host endothelial cells.

Binding Sites↗

PubMed2004

Attachment of the soluble complement regulator factor H to cell and tissue surfaces: relevance for pathology.

Complement is a central element of innate immunity and this vital defense system initiates and coordinates immediate immune reactions which attack and eliminate microbes, foreign particles and altered self cells. Newly generated activation products are extremely toxic and consequently, activation is highly restricted in terms of time and space. The initial activation of the alternative complement pathway occurs continuously and the early phase acts indiscriminatoryl and forms on any surface. However, the system discriminates between self and foreign, and therefore allows activation on foreign surfaces e.g. microbes, and restricts activation on host cells. Consequently, self cells and tissues are protected from the harmful activation products. This protection is mediated by specific regulators or inhibitors, which exist in the fluid phase and/or in membrane-bound forms. Here we review a novel mechanism, i.e. the attachment of the soluble complement regulator factor H to the surface of self cells. This attachment, which is demonstrated experimentally by means of immunofluorescense microscopy and by flow cytometry, increases the inhibitory potential at the cell surface and mediates protection by reducing the local formation of toxic inflammatory products. This attachment is highly relevant and has pathophysiological consequences in several human diseases, including Factor H-associated hemolytic uremic syndrome (FH-HUS), membrano-proliferative glomerulonephritis type II, recurrent microbial infections and chronic inflammation, e.g. rheumatoid arthritis and immune evasion of tumor cells. Defects of this safeguard activity have been recently understood in patients with FH-HUS. Point mutations in the Factor H gene occurring in the C-terminus of the protein result in impaired cell binding capacity of Factor H and, consequently, during an inflammatory insult endothelial cells are not properly protected and are damaged.

Animals↗

PubMed2003

CTLA-4 positive T cells in contrast to procalcitonin plasma levels discriminate between severe and uncomplicated Plasmodium falciparum malaria in Ghanaian children.

Procalcitonin (PCT) plasma levels and the fraction of CTLA-4-positive T cells are both elevated in acute Plasmodium falciparum malaria in human adults and the degree of elevation is positively correlated with other markers of disease severity, for example with parasitaemia. However, the clinical manifestations of malaria are strongly age-dependent and children from endemic areas carry the main disease burden. Therefore, we measured PCT plasma levels and CTLA-4 expression by T cells in four groups of children from the Ashanti Region in Ghana: asymptomatic children with or without parasitaemia, children with uncomplicated P. falciparum malaria and children with severe disease. PCT levels were highly elevated in both groups with acute malaria but they did not discriminate between uncomplicated and severe disease. In contrast, CTLA-4-expression by T cells was increased only in severe malaria. The fraction of CTLA-4 positive T cells in the blood of children with severe disease differed significantly from that in uncomplicated malaria, which was not elevated in spite of the high parasite loads observed in these children. This was unexpected, as in adults uncomplicated malaria is associated with a dramatic sixfold increase of the fraction of CTLA-4-positive T cells. The data from this study support the hypothesis that strong T cell activation as measured here by CTLA-4 expression is not just the by-product of a high parasite burden, but that it contributes to the pathogenesis of P. falciparum malaria.

Antigens, CD↗

PubMed2003

Release of endogenous anti-inflammatory complement regulators FHL-1 and factor H protects synovial fibroblasts during rheumatoid arthritis.

Rheumatoid arthritis is a chronic inflammatory disease of unknown aetiology predominantly affecting cells and tissues of synovial joints. Here we show that the two important complement regulators FHL-1 and factor H play a protective anti-inflammatory role in rheumatoid arthritis. Expression analyses at the mRNA- and protein level show in vitro expression and secretion of both regulators by synovial fibroblasts derived from patients with rheumatoid arthritis. Similarly the two regulators are synthesized in vivo in diseased synovial tissue, and in particular synovial lining cells express high levels of FHL-1. The anti-inflammatory role of these regulators in rheumatoid arthritis is highlighted by their induction with IFN-gamma and dexamethasone, whilst the pro-inflammatory cytokine TNF-alpha had no effect. Transient transfection experiments with various FHL-1/factor H promoter-luciferase reporter constructs into cells of distinct origin show independent cell and tissue specific promoter regulated transcription of these two regulators. The inducible expression, specifically of FHL-1 has physiological consequences. By binding directly to surfaces the released proteins protect cells from inflammatory damage and complement-mediated cell lysis. This study shows a novel protective and anti-inflammatory role of the two important complement regulators FHL-1 and factor H in rheumatoid arthritis and suggests a disease controlling role of the two proteins.

Arthritis, Rheumatoid↗

PubMed2003

Haemolytic uraemic syndrome and mutations of the factor H gene: a registry-based study of German speaking countries.

BACKGROUND: The aetiology of atypical haemolytic uraemic syndrome (aHUS) is, in contrast to classical, Shiga-like toxin induced HUS in children, largely unknown. Deficiency of human complement factor H and familial occurrence led to identification of the factor H gene (FH1) as the susceptibility gene, but the frequency and relevance of FH1 mutations are unknown. METHODS: We established a German registry for aHUS and analysed in all patients and 100 controls the complete FH1 gene by single strand confirmational polymorphism and DNA sequencing. In addition, complement C3 and factor H serum levels were assayed. Demographic data at onset of aHUS and follow up were compared for the mutation positive and negative groups. RESULTS: Of 111 patients with aHUS (68 female, 43 male, mean age 33 years) 14% had FH1 germline mutations, including two of eight patients with familial aHUS. For each of these eight patients, both parents were tested, and we were able to trace the mutation for five cases. In the other three cases (one with the mutation 3749 C/T, one with 3200 T/C, and one with 3566+1 G/A), we could not detect the mutation in either parent, although paternity was proven by genetic fingerprinting, suggesting that these subjects have new mutations. C3 was decreased in five mutation carriers but also in two non-carriers, and factor H was decreased in none of the carriers, but elevated in six carriers and 15 non-carriers. Clinical parameters including associated medications and diseases, and outcome of aHUS and of post-aHUS kidney transplantation were similar in the mutation positive and negative groups. CONCLUSION: FH1 germline mutations occur with considerable frequency in patients with aHUS. Hypocomplementaemia is not regularly associated with a germline mutation, and factor H serum levels can even be elevated. Screening for FH1 mutations contributes to the classification of aHUS.

Adult↗

PubMed2002

Secretion of soluble complement inhibitors factor H and factor H-like protein (FHL-1) by ovarian tumour cells.

We observed that the soluble complement regulators factor H and factor H-like protein were abundantly present in ascites samples as well as in primary tumours of patients with ovarian cancer. RT-PCR and immunoblotting analyses showed that the two complement inhibitors were constitutively produced by the ovarian tumour cell lines SK-OV-3 and Caov-3, but not PA-1 or SW626 cells. The amounts of factor H-like protein secreted were equal to those of factor H. This is exceptional, because e.g. in normal human serum the concentration of factor H-like protein is below 1/10th of that of factor H. In ascites samples the mean level of factor H-like protein (130+/-55 microg ml(-1)) was 5.5-fold higher than in normal human serum (24+/-3 microg ml(-1)). Ovarian tumour cells thus preferentially synthesise factor H-like protein, the alternatively spliced short variant of factor H. The tumour cells were found to bind both (125)I-labelled factor H and recombinant factor H-like protein to their surfaces. Surprisingly, the culture supernatants of all of the ovarian tumour cell lines studied, including those of PA-1 and SW626 that did not produce factor H/factor H-like protein, promoted factor I-mediated cleavage of C3b to inactive iC3b. Subsequently, the PA-1 and SW626 cell lines were found to secrete a soluble form of the membrane cofactor protein (CD46). Thus, our studies reveal two novel complement resistance mechanisms of ovarian tumour cells: (i) production of factor H-like protein and factor H and (ii) secretion of soluble membrane cofactor protein. Secretion of soluble complement inhibitors could protect ovarian tumour cells against humoral immune attack and pose an obstacle for therapy with monoclonal antibodies.

Ascitic Fluid↗

PubMed2002

Factor H family proteins: on complement, microbes and human diseases.

At present, the human Factor H protein family represents seven multidomain, multifunctional serum proteins. This group includes the complement and immune regulators Factor H, the Factor H-like protein 1 (FHL-1) and five Factor H-related proteins proteins (FHR-1, -2, -3, -4 and -5). Each is exclusively composed of individually folded protein domains, termed short consensus repeats (SCRs) or complement control modules. Structure-function analyses allowed the localization of the complement regulatory domain of Factor H and FHL-1 in the N-terminal region within SCRs 1-4. In addition, multiple binding sites for C3b, heparin and microbial surface proteins were localized in the N-terminus, within the middle region and also in the C-terminus of Factor H and FHL-1. Recent results show a central role for the C-terminus of Factor H, i.e. SCRs 19-20. These particular domains are conserved in all FHRs identified so far, include contact points for C3b, heparin and microbial surface proteins and represent a 'hot-spot' for gene mutations in patients that suffer from the Factor H-associated form of haemolytic uraemic syndrome.

Borrelia↗

PubMed2002

The yeast Candida albicans binds complement regulators factor H and FHL-1.

The human facultative pathogenic yeast Candida albicans causes mucocutaneous infections and is the major cause of opportunistic fungal infections in immunocompromised patients. C. albicans activates both the alternative and classical pathway of the complement system. The aim of this study was to assay whether C. albicans binds human complement regulators in order to control complement activation at its surface. We observed binding of two central complement regulators, factor H and FHL-1, from normal human serum to C. albicans by adsorption assays, immunostaining, and fluorescence-activated cell sorter (FACS) analyses. Specificity of acquisition was further confirmed in direct binding assays with purified proteins. The surface-attached regulators maintained their complement regulatory activities and mediated factor I-dependent cleavage of C3b. Adsorption assays with recombinant deletion mutant proteins were used to identify binding domains. Two binding sites were localized. One binding domain common to both factor H and FHL-1 is located in the N-terminal short consensus repeat domains (SCRs) 6 and 7, and the other one located in C-terminal SCRs 19 and 20 is unique to factor H. These data indicate that by surface acquisition of host complement regulators, the human pathogenic yeast C. albicans is able to regulate alternative complement activation at its surface and to inactivate toxic complement activation products.

Binding Sites↗

PubMed2001

Characterization of factor H-related cell membrane molecules expressed by human B lymphocytes and neutrophil granulocytes.

The human factor H protein family comprises six plasma glycoproteins. Earlier we described a membranal factor H-related (mFHR) molecule that is expressed by human B lymphoblastoid cell lines and exerts cofactor activity. In our present study we screened human blood cells for the presence of mFHR proteins and further characterized these molecules. By cytofluorimetry it is shown that the factor H-specific rabbit antiserum reacts strongly with B cells and neutrophil granulocytes, but not with T cells and monocytes. On B lymphocytes mFHR is shown to be down-regulated upon activation of the cells via sIg. In experiments studying which short consensus repeat (SCR) domains are part of the cell membrane proteins we found that antibodies raised against SCRs 1-4, 19-20 and FHR-3 bound to neutrophils but not to B cells. While mFHRs derived both from B cells and granulocytes are shown to bind heparin, their size and structure are different as revealed by Western blotting. A further characteristic of the granulocyte-derived mFHR is its sensitivity to the PI-specific PLCgamma enzyme. These data demonstrate the existence of new members of the FHR protein family, as two distinct, membranal forms are identified. Based on the differences, the B cell derived molecule is termed mFHR-1 and the neutrophil derived protein mFHR-2.

B-Lymphocytes↗

PubMed2001

Immune evasion of Borrelia burgdorferi by acquisition of human complement regulators FHL-1/reconectin and Factor H.

To understand immune evasion mechanisms of Borrelia burgdorferi we compared serum-resistant B. afzelii and serum-sensitive B. garinii isolates for their capacity toacquire human complement regulators. Here we demonstrate that the two borrelial genospecies show different binding of the two important human complement regulators, FHL-1/reconectin and Factor H. All serum-resistant B. afzelii isolates bound FHL-1/reconectin and also Factor H, and all analyzed serum-sensitive B. garinii isolates showed no or a significantly lower binding activity. Using recombinant deletion mutants, the binding domains were localized to the C terminus of FHL-1/reconectin to short consensus repeats 5-7. The borrelial binding proteins were located in the surface of the bacteria as demonstrated by immunofluorescence staining of intact, serum-exposed bacteria and by enrichment of outer membrane proteins. The surface-attached complement regulators maintained complement regulatory activity as demonstrated in a cofactor assay. By ligand blotting two different borrelial binding proteins were identified that were responsible for the surface attachment of FHL-1/reconectin and Factor H. These borrelial complement regulators acquiring surface proteins (CRASP) were further characterized as either CRASP-1, a 27.5-kDa molecule which preferentially binds FHL-1/reconectin and which was present in all serum-resistant borreliae, or CRASP-2, a 20/21-kDa protein which interacts preferentially with Factor H and the expression of which was more restricted, being detected in four of the six isolates analyzed. In summary, we describe a new immune evasion mechanism of B. burgdorferi, as these bacteria acquire human complement regulators to control complement activation on their surface and to prevent formation of toxic activation products.

Adsorption↗

PubMed2001

Hemolytic uremic syndrome: how do factor H mutants mediate endothelial damage?

Hemolytic uremic syndrome is characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. Data from recent genetic analyses reveal a clear association between the complement regulator factor H and the atypical form of this fatal human disease. The clustering of the identified mutations in the C-terminus of factor H identifies a "hot spot" that is central to the pathogenesis of the disease. What are the possible biological and functional consequences of the identified mutations for the disease process and mechanisms of disease progression?

Amino Acid Sequence↗

PubMed2001

Mechanism of complement resistance of pathogenic Borrelia burgdorferi isolates.

Borrelia burgdorferi, the causative agent of Lyme disease, differ in their susceptibility to normal human serum and are consequently classified as complement-resistant, complement-sensitive and intermediate complement-sensitive. Most isolates belonging to the genospecies B. afzelii are complement-resistant, while particularly B. garinii isolates were rapidly killed by complement. In general, isolates of the genospecies B. burgdorferi sensu stricto (s.s.) are intermediate complement-sensitive. Independent of the genospecies, all Borreliae were capable to activate the classical and/or the alternative pathway. Deposition of the activation products C3, C6, and TCC is much stronger by B. burgdorferi s.s. and B. garinii isolates than by B. afzelii isolates. The mechanism(s) on how Borreliae evade complement-mediated bacteriolysis has recently been described by showing that complement-resistant B. afzelii isolates but not the complement-sensitive B. garinii isolates absorb human complement regulators FHL-1/reconectin and factor H. Surface-attached FHL-1/reconectin maintains its complement regulatory activity and supports factor I-mediated C3b cleavage to iC3b. In complement-resistant Borreliae, two outer surface proteins, the 27.5 kDa (CRASP-1, complement regulator-acquiring surface protein 1) and the 20/21 kDa (CRASP-2), are responsible for the surface attachment of the two complement regulators. CRASP-1, which is present in complement-resistant Borreliae, binds preferentially FHL-1/reconectin while CRASP-2, which is restrictively expressed, binds preferentially factor H. Thus, complement-resistant Borreliae bind human complement regulators and control complement activation on their surface and prevent the formation of toxic activation products.

Bacterial Outer Membrane Proteins↗

PubMed2001

Complement factor H and hemolytic uremic syndrome.

Factor H is a 150 kDa single chain plasma glycoprotein that plays a pivotal role in the regulation of the alternative pathway of complement. Primary sequence analysis reveals a structural organization of this plasma protein, in 20 homologous units, called Short Consensus Repeats (SCRs), each about 60 amino acids long. Biochemical and genetic studies show an association between factor H deficiency and human diseases, including Systemic Lupus Erythematosus, susceptibility to pyogenic infection and a form of membranoproliferative glomerulonephropathy. More recently, factor H deficiency has also been associated with susceptibility to Hemolytic Uremic Syndrome (HUS), a disease consisting of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure, caused by platelet thrombi which mainly, but not exclusively, form in the microcirculation of the kidney. In this review, we summarize recent genetic and biochemical data, which indicate a critical role for factor H in the pathogenesis of HUS and suggest an important role of the most C-terminal domain, i.e. SCR 20, in the disease. In addition, we discuss the physiological consequences of these findings, as novel functional data show a particular essential role of SCR 20 of factor H as the central discriminatory and regulatory site of this multidomain, multifunctional plasma protein.

Complement Factor H↗

PubMed2001

Group A streptococcal phagocytosis resistance is independent of complement factor H and factor H-like protein 1 binding.

Factor H (FH) and factor H-like protein 1 (FHL-1) regulate complement activation through the alternative pathway. Several extracellular bacterial pathogens, prime targets for the complement system, bind FH and FHL-1, thereby acquiring a potential mechanism for minimizing complement deposition on their surface. For group A streptococci (GAS), surface-bound antiphagocytic M proteins mediate the interaction. To study the role of the FH-FHL-1 interaction for complement deposition and opsonophagocytosis of GAS, we first constructed a set of truncated M5 protein variants and expressed them on the surface of a homologous M-negative GAS strain. Binding experiments with the resulting strains demonstrated that the major FH-FHL-1 binding is located in a 42-amino-acid region within the N-terminal third of M5. Measurement of bacteria-bound complement factor C3 after incubation in plasma showed that the presence of this region had little impact upon complement deposition through the alternative pathway. Moreover, streptococci expressing M5 proteins lacking the major FH and FHL-1 binding sequence resisted phagocytosis in human blood as efficiently as bacteria expressing the wild-type protein. Consequently, the data suggest that the binding of the regulators of the alternative pathway is of limited importance for GAS phagocytosis resistance.

Antigens, Bacterial↗