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Michael Kirschfink

Publications and source records attributed to Michael Kirschfink.

At least 19 recordsLinked to original sources
PubMed2007

Regioselectively modified sulfated cellulose as prospective drug for treatment of malaria tropica.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to placental chondroitin-4-sulfate (CSA) has been linked to the severe disease outcome of pregnancy-associated malaria. Consequently, sulfated polysaccharides with inhibitory capacity may be considered for therapeutic strategies as anti-adhesive drugs. During in vitro screening a regioselectively modified cellulose sulfate (CS10) was selected as prime candidate for further investigations because it was able to inhibit adhesion to CSA expressed on CHO cells and placental tissue, to de-adhere already bound infected erythrocytes, and to bind to infected erythrocytes. Similar to the undersulfated placental CSA preferred by placental-binding infected erythrocytes, CS10 is characterized by a clustered sulfate pattern along the polymer chain. In further evaluation of its effects on P. falciparum interactions with host erythrocytes, we now show that CS10 inhibits the in vitro asexual growth of parasites in erythrocytes. Furthermore, we show that CS10 interferes with C1 of the classical complement pathway but not with MBL of the lectin pathway. In order to gain insights into the possible interactions of CS10 with known parasite receptors at the molecular level, we designed 3D-structures of characteristic stretches of CS10. CS10 fragments with clustered sulfate groups showed complex patterns of hydrophobic and hydrophilic patches most likely suitable for interactions with protein binding partners. The significance of CS10 interactions with the complement system as well as its anti-malarial effect for prospective drug application are discussed.

Amino Acids↗
PubMed2006

MPGN II--genetically determined by defective complement regulation?

MPGN II is a rare disease which is characterized by complement containing deposits within the GBM. The disease is characterized by functional impairment of the GBM causing progressive loss of renal function eventually resulting in end stage renal disease. It now becomes evident that in addition to C3NeF, which inhibits the inactivation of the alternative C3 convertase C3bBb, different genetically determined factors are also involved in the pathogenesis of MPGN II. These factors though different from C3NeF also result in defective complement regulation acting either through separate pathways or synergistically with C3NeF. Following the finding of MPGN II in Factor H deficient animals, patients with MPGN II were identified presenting with an activated complement system caused by Factor H deficiency. Factor H gene mutations result in a lack of plasma Factor H or in a functional defect of Factor H protein. Loss of Factor H function can also be caused by inactivating Factor H autoantibodies, C3 mutations preventing interaction between C3 and Factor H, or autoantibodies against C3. Identification of patients with MPGN II caused by defective complement control may allow treatment by replacement of the missing factor via plasma infusion, thus possibly preventing or at least delaying disease progress.

Animals↗
PubMed2006

Schistosoma japonicum and S. mansoni cercariae: different effects of protein in medium, of mechanical stress, and of an intact complement system on in vitro transformation to schistosomula.

The cercariae of Schistosoma japonicum were subjected in vitro to treatments known for Schistosoma mansoni to generate schistosomula-like organisms. As a technical prerequisite to pipette or to otherwise handle the sticky cercariae of S. japonicum, the addition of protein to water or medium was found to abolish the stickiness of cercariae of this species. Shearing forces exerted in vitro by syringe (22 G) passage are known since long to fully transform S. mansoni cercariae, but this treatment was found to be much less efficient with S. japonicum. Thus, even with very narrow needles (27 G), complete transformation of cercariae was not obtained with S. japonicum. Complement, provided by fresh human serum, is also well known to induce rapid transformation of S. mansoni cercariae with subsequent killing of the schistosomula. This treatment of S. japonicum cercariae induced degeneration of the tails and strongly promoted the transformation to schistosomula-like organisms, but at a much slower pace. These effects were absent from sera either heat-inactivated or depleted of factor B or of complement component C8, but were restored after adding the purified respective complement components. The schistosomula-like organisms of S. japonicum were not susceptible to lysis after 1 day of in vitro culture in the presence of 50% fresh human serum, although both cercariae and schistosomula of S. mansoni were killed under these conditions. In conclusion, the dynamics of in vitro transformation of S. japonicum cercariae differ significantly from those of S. mansoni, and complement has a major transformation-promoting activity.

Animals↗
PubMed2006

Strategies of therapeutic complement inhibition.

The involvement of complement in the pathogenesis of a great number of partly life threatening diseases defines the importance to develop inhibitors which specifically interfere with its deleterious action. Endogenous soluble complement-inhibitors, antibodies or low molecular weight antagonists, either blocking key proteins of the cascade reaction or neutralizing the action of the complement-derived anaphylatoxins have successfully been tested in various animal models over the past years. Promising results consequently led to first clinical trials. This review is focused on different approaches for the development of inhibitors, on their site of action in the cascade, on possible indications for complement inhibition based on experimental animal data, and on potential side effects of such treatment.

Anaphylatoxins↗
PubMed2006

Functional characterization of BbCRASP-2, a distinct outer membrane protein of Borrelia burgdorferi that binds host complement regulators factor H and FHL-1.

Borrelia burgdorferi, the aetiological agent of Lyme disease, employs sophisticated means to survive in diverse mammalian hosts. Recent studies demonstrated that acquisition of complement regulators factor H and factor H-like protein-1 (FHL-1) allows spirochetes to resist complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. In this study we have identified and characterized one of those B. burgdorferi proteins, named BbCRASP-2. BbCRASP-2 is distinct from the four previously identified factor H/FHL-1-binding CRASPs of B. burgdorferi strains. The single copy of the gene encoding BbCRASP-2, cspZ, is located on the linear plasmid lp28-3. BbCRASP-2 is highly divergent from the factor H/FHL-1-binding protein BbCRASP-1 and from members of the factor H-binding Erp (OspE/F-related) protein family. Peptide mapping analysis revealed that the factor H/FHL-1 binding site is discontinuous and it was found that C-terminal truncations abrogate factor H and FHL-1 binding. The predominant BbCRASP-2 binding site of both host complement regulators was mapped to the short consensus repeat 7 (SCR 7). Factor H and FHL-1 bound to BbCRASP-2 maintain cofactor activity for factor I-mediated C3b inactivation and accelerate the decay of the C3 convertase. Expression of BbCRASP-2 in serum-sensitive B. burgdorferi mutant B313 increased resistance to complement-mediated lysis. The characterization of BbCRASP-2 now provides a complete picture of the three diverse complement regulator-binding protein families of B. burgdorferi yielding new insights into the pathogenesis of Lyme disease.

Amino Acid Sequence↗
PubMed2006

Interaction of complement and leukocytes in severe acute pancreatitis: potential for therapeutic intervention.

In acute pancreatitis, local as well as systemic organ complications are mediated by the activation of various inflammatory cascades. The role of complement in this setting is unclear. The aim of the present study was to determine the level of complement activation in experimental pancreatitis, to evaluate the interaction of complement and leukocyte-endothelium activation, and to assess the effects of complement inhibition by soluble complement receptor 1 (sCR1) in this setting. Necrotizing pancreatitis was induced in Wistar rats by the combination of intravenous cerulein and retrograde infusion of glycodeoxycholic acid into the biliopancreatic duct; edematous pancreatitis was induced by intravenous cerulein only. In control animals, a sham operation (midline laparotomy) was performed. Complement activation, leukocyte sequestration, and pancreatic as well as pulmonary injury were assessed in the presence/absence of sCR1. Increased levels of C3a were found in necrotizing but not in edematous pancreatitis. When complement activation in necrotizing pancreatitis was blocked by sCR1, levels of C3a and total hemolytic activity (CH50) were decreased. Leukocyte-endothelial interaction, as assessed by intravital microscopy, and pancreatic as well as pulmonary organ injury (wet-to-dry weight ratio, MPO activity, and histology) were ameliorated by sCR1. As a result of the present study, necrotizing but not edematous pancreatitis is characterized by significant and early complement activation. Based on the interaction of complement and leukocytes, complement inhibition by sCR1 may be a valuable option in the treatment of leukocyte-associated organ injury in severe pancreatitis.

Animals↗
PubMed2006

Hereditary angioedema.

Hereditary angioedema (HAE) is a rare autosomal dominant disorder caused by a C1-inhibitor deficiency. It is characterized by potentially life-threatening recurrent episodes of angioedema of the skin and mucosa. Several recent studies have further elucidated the immunology of HAE implicating bradykinin, the key mediator of the contact system. This article reviews the pathophysiology, subtypes, and clinical features of HAE. Therapeutic approaches for various clinical situations (emergency and prophylactic regimens) are also discussed.

Aminocaproates↗
PubMed2005

Linking C5 deficiency to an exonic splicing enhancer mutation.

As an important component of the innate immune system, complement provides the initial response to prevent infections by pathogenic microorganisms. Patients with dysfunction of C5 display a propensity for severe recurrent infections. In this study, we present a patient with C5 deficiency demonstrated by immunochemical and functional analyses. Direct sequencing of all C5 exons displayed no mutation of obvious functional significance, except for an A to G transition in exon 10 predicting an exchange from lysine to arginine. This sequence alteration was present in only one allele of family members with a reduced serum C5 concentration and in both alleles of the patient with almost complete C5 deficiency, suggesting that this alteration may be producing the phenotype. Recent findings indicate that distinct nucleotide sequences, termed exonic splicing enhancers (ESEs), influence the splicing process. cDNA from all family members harboring the mutated allele showed skipping of exon 10, which resulted in a premature STOP codon, explaining the lack of C5 in the propositus. Sequence analysis of the mutated region revealed the substitution to be located within an ESE, as predicted by the RESCUE-ESE program. The altered ESE sequence is located close to the 5' splicing site and also lowers the predicted strength of the splice site itself. This apparently inconsequential sequence alteration represents a noncanonical splicing mutation altering an ESE. Our finding sheds a new light on the role of putative silent/conservative mutations in disease-associated genes.

Alternative Splicing↗
PubMed2005

Membranoproliferative glomerulonephritis type II (dense deposit disease): an update.

Membranoproliferative glomerulonephritis type II (MPGN II) is a rare disease characterized by the deposition of abnormal electron-dense material within the glomerular basement membrane of the kidney and often within Bruch's membrane in the eye. The diagnosis is made in most patients between the ages of 5 and 15 yr, and within 10 yr, approximately half progress to end-stage renal disease, occasionally with the late comorbidity of visual impairment. The pathophysiologic basis of MPGN II is associated with the uncontrolled systemic activation of the alternative pathway (AP) of the complement cascade. In most patients, loss of complement regulation is caused by C3 nephritic factor, an autoantibody directed against the C3 convertase of the AP, but in some patients, mutations in the factor H gene have been identified. For the latter patients, plasma replacement therapy prevents renal failure, but for the majority of patients, there is no proven effective treatment. The disease recurs in virtually all renal allografts, and a high percentage of these ultimately fail. The development of molecular diagnostic tools and new therapies directed at controlling the AP of the complement cascade either locally in the kidney or at the systemic level may lead to effective treatments for MPGN II.

Child↗
PubMed2005

Successful plasma therapy for atypical hemolytic uremic syndrome caused by factor H deficiency owing to a novel mutation in the complement cofactor protein domain 15.

Quantitative or functional deficiency of complement factor H results in uncontrolled complement activation. This leads to thrombotic microangiopathy and finally causes renal failure (atypical hemolytic uremic syndrome [aHUS]). By regular analysis of factor H in patients with aHUS, the authors found a complete factor H deficiency in an infant in whom aHUS developed at 8 months of age. Factor H was quantified by enzyme-linked immunosorbent assay and further analyzed by Western blot using a factor H-specific antibody. Complement activation was determined by measuring total hemolytic activity of the classical (CH50) and alternative (APH50) pathways, C3 and C3d. The sequence of factor H gene was determined. Serial factor H measurements after fresh frozen plasma infusion allowed calculation of a factor H half-life. Factor H was absent in plasma (<1 mug/mL), and the complement system was highly activated (CH50, APH50, C3 decreased; C3d increased). Genetic analysis identified a novel homozygous factor H mutation (T2770A; Y899Stop) in CCP domain 15, most likely causing defective protein secretion. Time course measurements of factor H after plasma infusion established a factor H half-life of about 6 days. By repetitive plasma infusions (20 mL/kg over about 2 to 3 hours) the authors were able to interrupt the vicious circle of thrombotic microangiopathy in a factor H-deficient patient with aHUS. Based on the measured factor H half-life of about 6 days, regular plasma infusions in 2-week intervals were given, which prevented further aHUS episodes and stopped the decline of kidney function.

Complement Factor H↗
PubMed2005

Is immunity in diabetic patients influencing the susceptibility to infections? Immunoglobulins, complement and phagocytic function in children and adolescents with type 1 diabetes mellitus.

OBJECTIVE: Previous reports suggest an increased susceptibility of diabetes patients to infections, but little information is available on possible underlying immunologic dysfunctions. The aim of this study was to evaluate humoral factors in pediatric patients with type 1 diabetes mellitus. METHODS: There were 66 diabetic patients (39 males:27 females; 5-17 yr) classified into two groups according to levels of glycohemoglobin (limit 9%): Group C - controlled (n = 33) and Group UC - uncontrolled (n = 33). We evaluated five patients in C and six in UC who reported previous infections. Immunologic analysis included measurement of plasma concentrations of immunoglobulins (Ig), C3, and C4 levels (turbidimetry); functional hemolytic assays for complement evaluation (CPH for classical and APH for alternative pathways), quantification of C4 isotypes C4A and C4B (ELISA), phagocytosis assays, measurement of bactericidal activity against Staphylococcus aureus, as well as tests of fungicidal capacity for Candida albicans. RESULTS: The UC Group had higher mean age, received higher insulin doses, and had higher concentrations of glycohemoglobin than the C Group. No significant differences in duration of the disease or nutritional conditions were detected between the groups. Lower IgA values in C (10/33) and lower IgG levels in UC (23/33) were detected, and there were inverse relationship with HbA1c values. Analysis of CPH, APH, C3, and C4 showed normal levels in both groups and no statistical correlation with the HbA1c. However, 9/33 children of the UC Group had decreased C3 values. C4B levels were below the normal range in 8/20 and correlated with higher HbA1c. Both phagocytic assays for S. aureus and Candida albicans were within normal limits. CONCLUSIONS: Low IgG concentrations and to some degree reduction in C4B levels were related to impaired metabolic control. No strong link between the immunological alterations was found in diabetic patients and the occurrence of infections.

Adolescent↗
PubMed2005

Impact of inverse plasma filtration on complement activation in porcine blood.

During extracorporeal circulation, activation of the complement system as a bioincompatibility reaction upon contact with artificial surfaces is well known. This may induce adverse effects for the patient as well as for the bioreactor when using a hybrid liver support system. At present, the achievable clearance rates in hybrid liver support systems are restricted by the limitations of blood flow through the capillaries of the plasma filter. In order to enhance clearance rates it is desirable to develop a high-flow plasma separation unit in which the activation of the complement system is as low as possible. Conventional plasma filters operating in normal and in inverse (blood flow around the capillaries and the capillary lumen serving as plasma compartment) modes were investigated applying an in vitro circuit (n = 4 in each group). For this purpose heparinized porcine blood was used. Complement activation was measured as generation of the soluble membrane attack complex, SC5b-9. With a normal flow mode the maximum achievable filtration rates varied from 25 to 40 mL/min, whereas the maximal filtration rates with an inverse flow mode varied from 40 to 100 mL/min. While these values were significantly higher than those with regularly operating plasma filters, there was no significant difference between normal and inverse mode in the production of SC5b-9. We conclude that the outer surface of the investigated conventional plasma filtration membranes is suitable for an inverse mode and does not lead to an excessive activation of the complement system upon dynamic blood contact. Thus, the inversion of the blood and the plasma compartments may be a possible way forward in the development of a high-flow plasma separation filter.

Animals↗
PubMed2004

[Therapeutic approach of hereditary angioedema].

PURPOSE: Hereditary Angioedema was first described by William Osler in 1888 and it is caused by a hereditary or acquired deficiency of C1 esterase inhibitor (C1-INH). Treatment is indicated for acute attacks or prophylaxis of angioedema which occur in the subcutaneous tissue respiratory or gastrointestinal tracts. Treatment includes attenuated androgens, inhibitors of kininogen or plasminogen, like tranexamic acid or e-aminocaproic acid and the administration of C1-INH concentrate. We describe the peculiarities of the treatment chosen for 10 patients (4 families) with HAE and their evolution. METHODS: Ten patients (1-38 years old) with HAE were diagnosed by clinical history and laboratory evaluation. The following tests were performed for the complement system: C1-INH, C4 and C3 levels and hemolytic assay (CH50 and APH50) for the classic and alternative pathways. Treatment was initiated considering severity of symptoms, age, gender and therapeutic response of the patient. RESULTS: Clinical evaluation showed: 4/10 patients with recurrent subcutaneous edema; 3/10 with previous laryngeal edema and 3/10 with sporadic symptoms. Different severity of symptoms was verified in the same family. The laboratory evaluation detected: low C1-INH levels (10/10); low serum C4 level (8/10); undetectable CH50 (3/10) and low CH50 levels (6/10); low APH50 levels (2/10). Six out of ten patients did not receive any specific treatment and 2 of them had high risk of asphyxia. One adolescent had been controlled with e-aminocaproic acid, one child had been changed from danazol to tranexamic acid, a 30 year old female patient had received oxandrolone and a 38 year old man had been treated with danazol. CONCLUSIONS: Although HAE is caused by the same defect and affects members of the same family, various approaches have been taken to treat these patients. We observed different alternatives of prophylactic therapy for HAE, of which some did not require drug therapy.

Adolescent↗
PubMed2004

Crystallization and preliminary crystallographic analysis of BbCRASP-1, a complement regulator-acquiring surface protein of Borrelia burgdorferi.

Borrelia burgdorferi is the causative agent of Lyme disease. Serum-resistant strains of the pathogen are able to reduce the host's immune response to infection by recruiting fluid-phase complement regulators from the serum. B. burgdorferi complement regulator-acquiring surface protein-1 (BbCRASP-1) binds factor H and factor-H-like protein-1 to the bacterial surface, where they actively down-regulate complement response. Crystals of native and selenomethionine-substituted BbCRASP-1 have been obtained and a native data set to 2.7 A as well as selenomethionine MAD data to 3.2 A resolution have been collected. The selenium substructure has been solved and initial phases have been refined to 3.0 A by density-modification methods. Model building and refinement are under way.

Bacterial Proteins↗
PubMed2004

C1-inhibitor reduces hepatic leukocyte-endothelial interaction and the expression of VCAM-1 in LPS-induced sepsis in the rat.

INTRODUCTION: Increased leukocyte-endothelial interaction (LEI) leading to hepatic microperfusion disorders is proposed as major contributor for hepatic failure during sepsis. Recently it has been demonstrated that complement inhibition by C1-inhibitor (C1-INH) is an effective treatment against microcirculatory disturbances in various diseases. The purpose of this study was to investigate the influence of C1-INH on microcirculation and LEI in the liver in a rat model of sepsis. MATERIALS AND METHODS: Rats received lipopolysaccharides (LPS) from Escherichia coli intravenously. Controls received Ringer solution only. Ninety minutes after LPS infusion some animals were treated with C1-INH intravenously (LPS + C1-INH). Others (LPS + SC) and controls (Ringer + SC) received sodium chloride (SC). Hepatic LEI and mean erythrocyte velocity (MEV) were quantified by intravital microscopy (IVM) 90 min after LPS or Ringer infusion (0) and 30, 60, 90 and 120 min following treatment. VCAM-1 m-RNA in hepatic tissue, C3a, TNF-alpha and hepatic enzyme liberation in blood was analysed. RESULTS: Leukocyte sticking to the endothelial wall in postsinusoidal venules was significantly reduced in the LPS + C1-INH vs. the LPS + SC group 30, 60, 90 and 120 min after treatment. VCAM-1 m-RNA expression in the hepatic tissue was markedly and C3a levels in plasma were significantly reduced in the LPS + C1-INH vs. the LPS + SC group. No differences in TNF-alpha levels were detected between these two groups. MEV was improved in the LPS + C1-INH vs. the LPS + SC group. CONCLUSIONS: Our results indicate that even upon delayed treatment hepatic adhesion molecule expression and LEI can be reduced by C1-INH. The multifunctional regulator may reduce hepatic microcirculatory disturbances during sepsis under clinical conditions.

Animals↗
PubMed2004

Immunological characterization of the complement regulator factor H-binding CRASP and Erp proteins of Borrelia burgdorferi.

Complement activation plays an important role in the elimination of invading microorganisms. Borrelia (B.) burgdorferi sensu lato the etiological agent of Lyme borreliosis, can resist complement-mediated killing. The mechanism of complement resistance of B. burgdorferi sensu stricto apparently depends on the expression of several outer surface proteins described as CRASPs (complement regulator-acquiring surface proteins). These borrelial surface proteins are able to bind components of the complement regulatory system, factor H and/or factor H-like protein 1 (FHL-1), two crucial fluid-phase negative regulators of the alternative pathway of complement. It was previously demonstrated that one CRASP is encoded by a member of the erp gene family. The purpose of the study was to use a set of monoclonal antibodies (mAb) and polyclonal antisera to characterize the relatedness of factor H-binding CRASP and Erp proteins among several B. burgdorferi sensu stricto and B. afzelii strains. Based on the observed cross-reactivities between B. burgdorferi sensu stricto strains LW2 and PKa-1, it is concluded that BbCRASP-3 is similar to ErpP, BbCRASP-4 is structurally related to ErpC, and BbCRASP-5 is similar to ErpA. The BaCRASP-2 and BaCRASP-4 proteins of B. afzelii strain EB1 reacted with both anti-ErpA and anti-ErpP antibodies whereas BaCRASP-5 of B. afzelii strain FEM1-D15 exclusively reacted with BbCRASP-3/ErpP specific antibodies. Together, these data indicate that most of the factor H-binding CRASPs are members of the Erp protein family, which represents a polymorphic class of proteins with similar or identical immunological reactivities.

Antigens, Bacterial↗
PubMed2003

Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins.

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i). binds the host complement regulators factor H and FHL-1 with high affinity, (ii). is the key molecule of the complement resistance of spirochetes, and (iii). is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease.

Amino Acid Sequence↗