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ABSTRACT: Puumala virus (PUUV) is the causative agent of nephropathia epidemica, a mild form of haemorrhagic fever with renal syndrome. PUUV is transmitted to humans via aerosolized excreta of the infected bank vole (Clethrionomys glareolus). Current methods for screening of the PUUV prevalence among bank vole populations are laborious, combining sampling in the field and subsequent analyses in the laboratory. In order to facilitate animal testing, a new serological immunochromatographic rapid test was developed. The test uses PUUV nucleocapsid protein as antigen, and it detects anti-PUUV IgG antibodies in rodents. With fresh and undiluted bank-vole blood samples (n = 105) the efficacy of the test was 100%, and with frozen and diluted samples (n = 78) the efficacy was 91%. The test was also shown to detect related hantavirus infections in Norway lemmings and sibling voles (n = 31) with 99% efficacy. The test provides an applicable tool for studying PUUV and related hantavirus infections in arvicoline rodents.
Epidemiology and Infection 07/2004; 132(3):549-53. · 2.84 Impact Factor
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ABSTRACT: Puumala virus (PUUV) is the causative agent of nephropathia epidemica, a mild form of haemorrhagic fever with renal syndrome. PUUV is transmitted to humans via aerosolized excreta of the infected bank vole (Clethrionomys
glareolus). Current methods for screening of the PUUV prevalence among bank vole populations are laborious, combining sampling in the field and subsequent analyses in the laboratory. In order to facilitate animal testing, a new serological immunochromatographic rapid test was developed. The test uses PUUV nucleocapsid protein as antigen, and it detects anti-PUUV IgG antibodies in rodents. With fresh and undiluted bank-vole blood samples (n=105) the efficacy of the test was 100%, and with frozen and diluted samples (n=78) the efficacy was 91%. The test was also shown to detect related hantavirus infections in Norway lemmings and sibling voles (n=31) with 99% efficacy. The test provides an applicable tool for studying PUUV and related hantavirus infections in arvicoline rodents.
Epidemiology and Infection 05/2004; 132(03):549 - 553. · 2.84 Impact Factor
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ABSTRACT: Hantaviruses are associated with two human diseases: haemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome (HPS). Puumala virus (PUUV), which is one of the Hantaviruses, is a causative agent of nephropathia epidemica (NE), a mild form of HFRS.
a new 5 min rapid test, POC PUUMALA (Erilab Ltd, Finland), for detecting IgM antibodies to PUUV was evaluated and compared with the commercially available Hantavirus (Puumala) IgM ELISA test (Progen, Germany). Discrepant test results between the two tests were confirmed by a mu-capture reference EIA.
Two hundred and thirty five serum samples, which had earlier been analyzed with the Progen IgM ELISA, were assayed with the POC PUUMALA rapid test. Five persons, without knowing the Progen IgM ELISA test results, interpreted independently the rapid test results. In addition, a panel of 48 serum samples was analyzed in parallel with the rapid test and the Progen IgM ELISA by one technician in daily routine diagnostics in a clinical microbiology laboratory.
the agreement between the results of the five interpreters was 95%, and the congruence of the results between individual readers and commercial ELISA test varied from 93 to 96%. Diagnostic efficacy of the rapid test varied between 98 and 99% compared with 96% of the Progen IgM ELISA. The POC PUUMALA rapid test showed higher or similar sensitivity compared with the Progen IgM ELISA, whereas both the tests had similar levels of specificity.
the analytical performance of the POC PUUMALA rapid test was found to be as good or even slightly better than the analytical performance of the Progen IgM ELISA. In addition, the rapid and straightforward procedure makes the POC PUUMALA a feasible tool for the diagnosis of the acute PUUV infection.
Journal of Clinical Virology 01/2002; 23(1-2):79-85. · 3.97 Impact Factor
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ABSTRACT: Intermolecular interactions of Tula hantavirus N (nucleocapsid) protein were detected in the yeast two-hybrid system, prompting further attempts to study this phenomenon. Using chemical cross-linking and immunoblotting it was shown that the N protein from purified virus and from infected cell lysates as well as recombinant protein produced in a baculovirus expression system are capable of forming dimers, trimers and multimers, thus confirming the capacity of the protein molecules to interact with each other. An ELISA format was developed in which molecules of the recombinant N protein were shown to associate non-covalently, via electrostatic interactions. Divalent cations (Ca(2+), Mn(2+), Mg(2+), Ba(2+)) enhanced the process 3- to 8-fold suggesting that adequate folding of the N protein is crucial for the association. Based on these data a model for hantavirus nucleocapsid assembly is proposed, in which N molecules first trimerize around the viral RNA molecule, and then the trimers gradually assemble forming longer multimers.
Journal of General Virology 09/2001; 82(Pt 8):1845-53. · 3.36 Impact Factor
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ABSTRACT: A new immunochromatographic rapid test, POC PUUMALA (Erilab Ltd., Kuopio, Finland), for detection of acute-phase Puumala virus (PUUV) infection was developed based on a highly purified baculovirus-expressed PUUV nucleocapsid protein antigen and lateral immunodiffusion techniques. After addition of sample (5 microl of serum, plasma, or fingertip blood) and buffer, PUUV-specific immunoglobulin M (IgM) antibodies, if present, together with the gold-conjugated anti-human IgM, formed a specific colored line in 5 min. The sensitivity and specificity of the test were evaluated with 200 serum samples and 30 fingertip blood samples. The reference method for the serum samples was a micro-capture enzyme immunoassay (EIA) for IgM and an immunofluorescence assay (IFA) for IgG antibodies. The analytical sensitivity and specificity of the rapid test were 100 and 99%, respectively, for unfrozen serum samples (n = 103; 12 PUUV IgM-positive samples). When freeze-thawed serum samples were used, the sensitivity and specificity were each 97.1% (n = 70; 35 PUUV IgM-positive samples). The specificity of the test was 96.2% for 27 serum samples with nonspecific IgM antibodies or rheumatoid factor (RF). The fingertip blood samples (n = 30) were negative, but they gave clear positive results when spiked with IgM-positive sera (n = 20). The results were in good agreement with the standard diagnostic methods. The rapid performance, the lack of need for refined laboratory equipment, and the high specificity with fresh serum and fingertip blood samples indicate that the developed POC PUUMALA rapid test is a useful tool for fast diagnosis of acute PUUV infection.
Journal of Clinical Microbiology 07/2001; 39(6):2146-50. · 4.15 Impact Factor
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ABSTRACT: Factor H (fH) restricts activation of the alternative pathway of complement at the level of C3, both in the fluid phase and on self-structures, but allows the activation to proceed on foreign structures. To study the interactions between fH and C3b we used surface plasmon resonance analysis (Biacore(R)) and eight recombinantly expressed fH constructs containing fragments of the 20 short consensus repeat domains (SCRs) of fH. We analyzed the binding of these constructs to C3b and its cleavage products C3c and C3d. Three binding sites for C3b were found on fH. Site 1 was localized to the five amino-terminal SCRs (SCR1-5), and its reciprocal binding site on C3b was found to be lost upon the cleavage of C3b to C3c and C3d. Site 2 on fH was localized by exclusion probably within or near SCRs 12-14 (fragment SCR8-20 bound to C3b, C3c, and C3d; SCR8-11 did not bind to C3b at all; and SCR15-20 bound only to the C3d part of C3b). Site 3 on fH for C3b was localized to the carboxyl-terminal SCRs 19-20, and its reciprocal binding site was mapped to the C3d part of C3b. In conclusion, we confirmed and mapped three binding sites on fH for C3b and demonstrated that the three binding sites on fH interact with distinct sites on C3b. Multiple reciprocal interactions between C3b and fH can provide a basis for the different reactivity of the alternative pathway with different target structures.
Journal of Biological Chemistry 10/2000; 275(36):27657-62. · 4.77 Impact Factor
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ABSTRACT: In the alternative pathway of complement (APC) factor H is the primary control factor involved in discrimination between potential pathogens. The APC deposits C3b on possible Ags, and the interaction with factor H determines whether the initial C3b activates the APC. Factor H is composed of a linear array of 20 homologous short consensus repeats (SCR) domains with many functional sites. Three of these sites are involved in binding C3b and regulating complement activation; others bind to sialic acid and/or heparin and are responsible for host recognition. Using site-directed mutations we have examined the contributions of each of these sites to target discrimination and to functional activities of factor H. Decay acceleration by SCR1-4 of C3/C5 convertases bound to nonactivators was strongly dependent on SCR domains 11-15 and 16-20. Loss of these regions caused a 97% loss of activity, with SCR16-20 being the most critical (>90% loss). On APC activators the pattern of site usage was different and unique on each. On yeast, deletion of the 10 C-terminal domains (SCR11-20) had no effect on specific activity. On rabbit erythrocytes, this deletion caused loss of 75% of the specific activity. An examination of binding affinity to C3b on the four cell types demonstrated that factor H exhibits a unique pattern of SCR involvement on each cell. The results reveal a complex molecular mechanism of discrimination between microbes and host in this ancient innate defense system and help explain the different rates and intensities of APC activation on different biological particles.
The Journal of Immunology 06/2000; 164(9):4742-51. · 5.79 Impact Factor
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ABSTRACT: The human factor H-related proteins FHR-3 and FHR4 are members of a family of proteins related to the complement factor H. Here, we report that the two proteins bind to the C3d region of complement C3b. The apparent K(A) values for the interactions of FHR-3 and FHR-4 with C3b are 7.5 x 10(6) M(-1) and 2.9 x 10(6) M(-1), respectively. Binding studies performed with C3b-coated pneumococci confirmed the results obtained with the biosensor system. A C-terminal construct of factor H showed similar binding characteristics. The interaction of FHR-3, but not of FHR4, with opsonised pneumococci was inhibited by heparin.
FEBS Letters 01/2000; 462(3):345-52. · 3.54 Impact Factor
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ABSTRACT: The factor H gene family provides a prime example of a multidomain multifunctional protein family whose individual members are defined by conserved common structural elements and display diverse but often overlapping functions. The six identified members of this protein family represent secreted plasma proteins that are primarily synthesized in the liver. Here, we summarize the current understanding of the function of these proteins and suggest a common role in complement control. Factor H is the best characterized member and acts as a complement regulator. The protein displays cofactor activity for factor I in the degradation of the central complement component C3b, acts as a decay accelerating factor for the C3 convertase, C3bBb and is a competitor for factor B binding to C3b. Factor H is a multifunctional protein and displays functions outside the complement system: it binds to the cellular integrin receptor (CD11b/CD18), interacts with cell surface glycosaminoglycans and also binds to the surface of certain pathogenic microorganisms. In addition, factor H has several binding sites for the C3 protein. The factor H-like protein 1 (FHL-1) or reconectin shares the complement regulatory functions with factor H and interacts with heparin. The protein displays cell spreading activity and binds to the N-terminus of the streptococcal M protein. The function of the factor H-related proteins (FHR-1 to FHR-4) is currently under investigation. These proteins are differently distributed. Three proteins (FHR-1, FHR-2 and FHR-4) are constituents of lipoproteins, while FHR-3 interacts with heparin. Binding to C3b and C3d has been demonstrated for FHR-3 and FHR-4 and the two proteins display a cofactor related activity.
Immunopharmacology 06/1999; 42(1-3):53-60.
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ABSTRACT: The authors examined the effect of four different kinds of contrast media (ionic/non-ionic, monomer/dimer) on the activation of the complement (C) system (haemolytic activity and anaphylatoxin generation) in vitro. In addition, the authors compared the effect of contrast media on inulin-mediated generation of the anaphylatoxin derivative C3a des Arg in sera from urticarial reactors and their non-reacting controls. It was observed that the incubation of commercial iohexol, ioxaglate, iodixanol and meglumin amidotriz solutions in normal human serum (NHS) resulted in a dose-dependent decrease in the haemolytic activity of the alternative C pathway. Contrary to expectations the contrast media did not activate C in NHS. Instead, inulin-induced generation of C3a des Arg was inhibited by all the four contrast media. The strongest inhibitor was ioxaglate, an ionic dimer. No significant difference between the urticarial reactors and non-reactors in the inhibition of C3a des Arg generation was observed. In analyzing the mechanism of C inhibition we found that the contrast media solutions, particularly the ionic ones, prevented formation of the alternative pathway C3 convertase, C3bBb, by inhibiting the binding of factor B to surface-associated C3b molecules. The results suggest that the previously observed decrease in haemolytic C titres by contrast media is due to direct suppression of C activity rather than activation-induced consumption.
Scandinavian Journal of Immunology 05/1997; 45(4):371-7. · 2.23 Impact Factor
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ABSTRACT: beta 2 integrin (CD11a,b,c/CD18)-mediated cell adhesion is required for many leukocyte functions. Under normal circumstances, the integrins are nonadhesive, and become adhesive for their cell surface ligands, the intercellular adhesion molecules (ICAMs), or soluble ligands such as fibrinogen and iC3b, when leukocytes are activated. Recently, we defined a peptide derived from ICAM-2, which specifically binds to purified CD11a/CD18. Furthermore, this peptide strongly induces T cell aggregation mainly mediated by CD11a/CD18-ICAM-1 interaction, and natural killer cell cytotoxicity. In the present study, we show that the same ICAM-2 peptide also avidly binds to purified CD11b/CD18, but not to CD11c/CD18. This binding can be blocked by the CD11b antibody OKM10. The peptide strongly stimulates CD11b/CD18-ICAM-1-mediated cell aggregations of the monocytic cell lines THP-1 and U937. The aggregations are energy and divalent cation-dependent. The ICAM-2 peptide also induces CD11b/CD18 and CD11c/CD18-mediated binding of THP-1 cells to fibrinogen and iC3b coated on plastic. These findings indicate that in addition to induction of CD11a/CD18-mediated cell adhesion, the ICAM-2 peptide may also serve as a "trigger" for high avidity ligand binding of other beta 2 integrins.
The Journal of Cell Biology 06/1995; 129(4):1143-53. · 10.26 Impact Factor
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ABSTRACT: Immunopathological evidence suggests that activation of the alternative pathway of complement (AP) is involved in membranoproliferative glomerulonephritis (MPGN) and in immunoglobulin A nephropathy. In this report we describe an AP dysfunction-associated factor that was isolated from the serum and urine of a patient with hypocomplementemic MPGN. Extensive glomerular deposits of C3, properdin, and of the terminal complement components were observed in the kidney of the patient. In her serum the AP hemolytic activity was virtually absent. When mixed with fresh normal serum, the patient's serum induced a 96% C3 conversion during a 30-min incubation at +37 degrees C. This activity was found to be due to a circulating factor that by immunochemical characterization proved to be a 46-kD monoclonal immunoglobulin lambda light (L) chain dimer (lambda L). Purified lambda L, but not control lambda or kappa L chains from patients with L chain disease, activated the AP in a dose- and ionic strength-dependent manner. Functionally, lambda L was differentiated from C3 nephritic factor (an autoantibody against the AP C3 convertase, C3bBb) by its inability to bind to and stabilize the C3bBb enzyme. Instead, lambda L was observed to interact directly with the AP control factor H. Thus, lambda L represents a novel type of immunoglobulin-related AP-activating factor with the capacity to initiate alternative complement pathway activation in the fluid phase.
Journal of Experimental Medicine 05/1992; 175(4):939-50. · 13.85 Impact Factor
