The lectin complement pathway serine proteases (MASPs) represent a possible crossroad between the coagulation and complement systems in thromboinflammation.

UNLABELLED: ESSENTIALS: The lectin pathway's MASP-1/2 activates coagulation factors but the trigger of the activation is unknown. MASP-1/2 activation was assessed by quantifying complexes between MASPs and antithrombin/C1-inhibitor. Activated platelets and fibrin were demonstrated to activate MASP-1 and MASP-2 both in vitro and in vivo. These findings may represent a crossroad between the complement and the coagulation systems. BACKGROUND: The activated forms of the complement lectin pathway (LP) proteases MASP-1 and MASP-2 are able to cleave the coagulation factors prothrombin, fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor in vitro. In vivo studies also show that MASP-1 is involved in thrombogenesis. OBJECTIVES: To clarify the not yet identified mechanisms involved in triggering activation of the LP during thrombotic reactions. METHODS: Novel sandwich-ELISAs for detection of complexes between MASP-1 or MASP-2 and the serpins C1 inhibitor (C1-INH) or antithrombin (AT), were used to specifically detect and quantify the activated forms of MASP-1 and MASP-2. RESULTS: Activated platelets were shown by flow cytometry to bind Ficolin-1, -2 and -3 but not MBL, which was associated with activation of MASP-1 and MASP-2. We also demonstrated that fibrin and the plasmin-generated fibrin fragment DD in plasma, bind and activate MASP-1 and MASP-2. As demonstrated by the ELISA and SDS-PAGE/Western blotting, the fibrin-associated activation was reflected in a specific inactivation by AT during clotting without the assistance of heparin. In all other cases the MASPs were, as previously reported, inactivated by C1-INH. In systemic lupus erythematosus patients with thrombotic disease and in polytrauma patients, the levels of activated MASP-1 and MASP-2 in complex with both AT and C1-INH were associated with markers of thrombotic disease and contact/coagulation system activation. CONCLUSIONS: MASP-1 and MASP-2 are activated during blood clotting. This activation is triggered by activated platelets and by the generation of fibrin during thrombotic reactions in vitro and in vivo, and may represent a novel activation/amplification mechanism in thromboinflammation.

MBL1 genotypes in wild boar populations from Sweden, Austria, the Czech Republic, and Japan.

The single nucleotide polymorphism (SNP) G949T in the mannose-binding lectin ( MBL ) 1 gene has been associated with low MBL-A concentration in serum and detected at different frequencies in various European pig populations. However, the origin of this SNP is not known. Part of the MBL1 gene was sequenced in 12 wild boar/Large White crossbred pigs from the second backcross (BC 2 ) generation in a family material originating from two wild boar x Large White intercrosses. Also, MBL-A serum concentration was measured in the entire BC 2 generation (n = 45). Furthermore, the genotypes of 68 wild boars from Sweden, Austria, the Czech Republic, and Japan were determined in regard to five previously described SNPs in MBL1 . The T allele of G949T was present among the BC 2 animals. MBL-A serum concentration in the BC 2 animals showed a bimodal distribution, with one-third of the animals at levels between 0.7 and 1.6 µg mL(-1) and the remaining pigs at levels around 13 µg mL(-1) . There was a co-variation between the presence of the T allele and low MBL-A concentration in serum. The genotyping of the wild boars revealed differences between populations. The T allele of G949T was not detected in the Austrian and Japanese samples and is thus unlikely to be an original feature of wild boars. In contrast, it was present at high frequency (0.35) among the Swedish wild boars, probably representing a founder effect. Five MBL1 haplotypes were resolved. Only two of these were present among the Japanese wild boars compared to four in each of the European populations. This difference may reflect differences in selection pressure and population history.

Complement activation products in liquid stored plasma and C3a kinetics after transfusion of autologous plasma.

BACKGROUND AND OBJECTIVES: Keeping a small stock of liquid plasma readily available for transfusion is common practise in Sweden. We report data on complement activation markers in plasma components during storage in the liquid state and the kinetics of C3a-(desArg) after transfusion of autologous plasma with high content of C3a-(desArg). MATERIAL AND METHODS: Plasma components were prepared by apheresis or from whole blood. C3 fragments (C3a-(desArg), C3d,g, iC3), and soluble terminal complement complex (sC5b-9) were investigated. C3a-(desArg) kinetics was investigated in regular apheresis donors. RESULTS: Apheresis plasma prepared by membrane centrifugation had significantly higher level of C3a-(desArg), C3d,g and sC5b-9 from day 0 and low iC3, than plasma prepared by other methods. By storage day 7, C3a-(desArg)-levels were above the reference value in 88% of all components. After re-infusion of autologous plasma with high C3a-(desArg) content, there were rapid a(1) and a(2)-distribution followed by a slower b-elimination phase. CONCLUSION: Plasma components prepared by different methods and stored in the liquid phase differ significantly in the amount and timing of complement activation. C3a-(desArg) present in plasma is rapidly eliminated after transfusion. Autologous plasma could be used to study complement kinetics in different clinical situations.

Extensive polymorphism in the porcine Toll-like receptor 10 gene.

The great importance of the Toll-like receptors (TLRs) in innate immunity is well established, but one family member--TLR10--remains elusive. TLR10 is expressed in various tissues in several species, but its ligand is not known and its function is still poorly understood. The open reading frame of TLR10 was sequenced in 15 wild boars, representing three populations, and in 15 unrelated domestic pigs of Hampshire, Landrace and Large White origin. Amino acid positions corresponding to detected nonsynonymous single nucleotide polymorphisms (SNPs) were analysed in the crystal structures determined for the human TLR1-TLR2-lipopeptide complex and the human TLR10 Toll/Interleukin 1 receptor (TIR) dimer. SNP occurrence in wild boars and domestic pigs was compared, and haplotypes for the TLR10 gene and the TLR6-1-10 gene cluster were reconstructed. Despite the limited number of animals sequenced in the present study (N = 30), a larger number of SNPs were found in TLR10 than recently reported for TLR1, TLR6 and TLR2. Thirty-three SNPs were detected, of which 20 were nonsynonymous. The relative frequency of nonsynonymous (d(N) ) and synonymous (d(S) ) SNPs between wild boars and domestic pigs was higher in TLR10 than recently reported for TLR1, TLR6 and TLR2. However, the polymorphism reported in the present study seems to leave the function of the TLR10 molecule unaffected. Furthermore, no nonsynonymous SNPs were detected in the part of the gene corresponding to the hinge region of the receptor, probably reflecting rigorously acting functional constraint. The total number of SNPs and the number of nonsynonymous SNPs were significantly lower (P < 0.05) in the wild boars than in the domestic pigs, and fewer TLR10 haplotypes were present in the wild boars. The majority of the TLR6-1-10 haplotypes were specific for either wild boars or domestic pigs, probably reflecting differences in microbial environment and population history.

The burden of hepatitis C in Sweden: a national study of inpatient care.

The spread of hepatitis C virus (HCV) in Sweden in the 1970s indicated that serious liver complications (SLC) would increase in the 2000s. The aim of this study was to analyse the burden of HCV-associated inpatient care in Sweden, to demonstrate the changes over time and to compare the findings with a noninfected population. The HCV-cohort (n: 43,000) was identified from the national surveillance database 1990-2006, and then linked to national registers to produce an age-, sex-, and region-matched noninfected comparison population (n: 215,000) and to obtain information on demographics, cancers, inpatient care and prescriptions. Cox regression was used to estimate the likelihood (hazard ratios) for admission to hospital in the HCV compared with the noninfected cohort. The hazard ratios were 4.03 (95% CI: 3.98-4.08) for all care, 77.52 (71.02-84.60) for liver-related care and 40.74 (30.58-54.27) for liver cancer care. The admission rate in the HCV-cohort compared with the noninfected cohort, the rate ratio (age- and sex-adjusted) for all inpatient care was 5.91 (95% CI: 5.87-5.94), and the rate ratio for liver-related care was 70.05 (66.06-74.28). In the HCV-cohort, 45% of all episodes were for psychiatric, mostly drug-related, care. Inpatient care for SLC increased in the 2000s. To conclude, drug-related care was common in the HCV-infected cohort, the demand for liver-related care was very high, and SLC increased notably in the 2000s, indicating that the burden of inpatient care from serious liver disease in HCV-infected individuals in Sweden is an increasing problem.

European Antibiotic Awareness Day, 2008 - the first Europe-wide public information campaign on prudent antibiotic use: methods and survey of activities in participating countries.

Antibiotic resistance is a major European and global public health problem and is, for a large part, driven by misuse of antibiotics. Hence, reducing unnecessary antibiotic use, particularly for the treatment of certain respiratory tract infections where they are not needed, is a public health priority. The success of national awareness campaigns to educate the public and primary care prescribers about appropriate antibiotic use in Belgium and France stimulated a European initiative coordinated by the European Centre for Disease Prevention and Control (ECDC), and named European Antibiotic Awareness Day (EAAD), to take place each year on 18 November. Specific campaign materials, including key messages, logos, slogans and a media toolkit, were developed and made available for use in European countries. The focus of the first EAAD campaign was about not taking antibiotics for viral infections such as colds and flu. A post-campaign survey was conducted in January 2009. Thirty-two European countries participated in the first EAAD, producing information materials and implementing activities to mark EAAD. Media coverage peaked on 18 and 19 November. At EU level, EAAD was launched at a scientific meeting in the European Parliament, Strasbourg. The event received EU political engagement through support from the EU Commissioner for Health, the Slovenian and French EU Presidencies, and Members of the European Parliament. Critical factors that led to the success of the first EAAD were good cooperation and process for building the campaign, strong political and stakeholder support and development of campaign materials based on scientific evidence. Countries indicated wide support for another EAAD in 2009. For this purpose, ECDC is developing several TV spots as well as a second set of EAAD campaign materials targeting primary care prescribers.

Validity of routine surveillance data: a case study on Swedish notifications of methicillin-resistant Staphylococcus aureus.

Surveillance of communicable diseases is a public health corner stone. Routine notification data on communicable diseases are used as a basis for public health action as well as for policy making. While there are agreed standards for evaluating the performance of surveillance systems, it is rarely possible to analyse the validity of the data entered into these systems. In this study we compared data on all Swedish cases of methicillin-resistant Staphylococcus aureus (MRSA) routinely notified between 2000 and 2003 with follow-up information collected for each of these cases as part of a public health project. The variables Reason for testing (clinical sample, contact tracing, screening of risk group), Clinical presentation (disease, colonisation), Transmission setting (healthcare-acquired, community-acquired), Country of acquisition (Sweden, abroad) and Risk-occupation (yes, no) were analysed for sensitivity, positive predictive value and completeness of answers. The sensitivity varied between 23% and 83%, the positive predictive values were generally higher (55% to 97%), while missing answers varied from 11% to 59%. The proportion of community-acquired cases was markedly higher when excluding either cases of MRSA colonisation or cases found through public health-initiated activities (contact tracing or screening of risk groups). We conclude that the quality of routine surveillance data may be inadequate for in-depth epidemiological analyses. This should be taken into account when interpreting routine surveillance figures. Whether or not the case definition includes cases of MRSA colonisation may have a significant impact on population-wide estimates of MRSA occurrence.

Complement activation triggered by chondroitin sulfate released by thrombin receptor-activated platelets.

BACKGROUND: Chondroitin sulfate (CS) is a glycosaminoglycan released by activated platelets. OBJECTIVE: Here we test the hypothesis that CS released by activated platelets can trigger complement activation in the fluid phase. METHODS AND RESULTS: Thrombin receptor-activating peptide (TRAP)-6 was used to activate platelets in platelet-rich plasma and blood, anticoagulated with the thrombin inhibitor lepirudin. TRAP activation induced fluid-phase complement activation, as reflected by the generation of C3a and sC5b-9, which could be attenuated by the C3 inhibitor compstatin. Chondroitinase ABC treatment of supernatants from activated platelets totally inhibited the activation, indicating that platelet-derived CS had initiated the complement activation. Furthermore, addition of purified CS to plasma strongly triggered complement activation. C1q was identified as the recognition molecule, as it bound directly to CS, and CS-triggered complement activation could be restored in C1q-depleted serum by adding purified C1q. TRAP activation of whole blood increased the expression of CD11b on leukocytes and generation of leukocyte-platelet complexes. It was demonstrated that these leukocyte functions were dependent on C3 activation and signaling via C5a, as this expression could be inhibited by compstatin and by a C5aR antagonist. CONCLUSIONS: We conclude that platelets trigger complement activation in the fluid phase by releasing CS, which leads to inflammatory signals mediated by C5a.

Unexpected effects of donor gender on the storage of liquid plasma.

BACKGROUND: Swedish regulations in effect since 2006 allow the storage of plasma for transfusion up to 14 days at 2-6 degrees C and for 3 years at < or = -30 degrees C. In this study, the quality of currently used plasma components was investigated. MATERIALS AND METHODS: Plasma components, prepared from whole blood or by apheresis, either leucocyte depleted or not leucocyte depleted, were stored at 2-6 degrees C as liquid plasma or as thawed fresh-frozen plasma; 31% were from female donors. Concentration, function and activation markers of the plasma coagulation systems were investigated during storage for up to 42 days. RESULTS: Cold-induced contact activation was the dominant storage lesion, occurring earlier and at higher frequency in plasma from females. Increased kallikrein-like activity led to changes in activated partial thromboplastin time, prothrombin time, protein C and C1 inhibitor (C1INH). C1INH function dropped to 53% on Day 14 in cold-activated plasma components. CONCLUSION: Contact activation may be triggered before Day 14, especially in plasma from females, and may progress as a result of the consumption of C1INH. The data suggest that lack of cold-induced contact activation may be an important quality criterion. To achieve this, plasma from male donors could be selected for transfusion and the storage time limited to 7 days.

SmiNet-2: Description of an internet-based surveillance system for communicable diseases in Sweden.

Electronic systems for communicable diseases surveillance enhance quality by simplifying reporting, improving completeness, and increasing timeliness. In this article we outline the ideas and technologies behind SmiNet-2, a new comprehensive regional/national system for communicable disease surveillance in Sweden. The system allows for reporting from physicians (web form) and laboratories (direct from lab data system) over the internet. Using a unique personal identification number, SmiNet-2 automatically merges clinical and laboratory notifications to case records. Privileged users, at national and county level, work against a common central server containing all notifications and case records. In addition, SmiNet-2 has separate county servers with tools for outbreak investigations, contact tracing and case management. SmiNet-2 was first used in September 2004. Individual counties receive up to 90% of all notifications electronically. In its first year, SmiNet-2 received 54 980 clinical notifications and 32,765 laboratory notifications, which generated 58,891 case records. Since most clinicians in Sweden have easy access to the internet, a general web-based reporting has been feasible, and it is anticipated that within a few years all reporting to SmiNet-2 will be over the internet. In this context, some of the major advantages of SmiNet-2 when compared with other systems are timeliness in the dataflow (up to national level), the full integration of clinical and laboratory notifications, and the capability to handle more than 50 diseases with tailor-made notification forms within one single system.

An evaluation and comparison of three commonly used statistical models for automatic detection of outbreaks in epidemiological data of communicable diseases.

We evaluated three established statistical models for automated 'early warnings' of disease outbreaks; counted data Poisson CuSums (used in New Zealand), the England and Wales model (used in England and Wales) and SPOTv2 (used in Australia). In the evaluation we used national Swedish notification data from 1992 to 2003 on campylobacteriosis, hepatitis A and tularemia. The average sensitivity and positive predictive value for CuSums were 71 and 53%, for the England and Wales model 87 and 82% and for SPOTv2 95 and 49% respectively. The England and Wales model and the SPOTv2 model were superior to CuSums in our setting. Although, it was more difficult to rank the former two, we recommend the SPOTv2 model over the England and Wales model, mainly because of a better sensitivity. However, the impact of previous outbreaks on baseline levels was less in the England and Wales model. The CuSums model did not adjust for previous outbreaks.

A novel application of multi-wavelength TIRF spectroscopy for real time monitoring of antithrombin interactions with immobilized heparin.

Real time interactions of antithrombin (AT) with Corline Heparin Surfaces (CHS) with one and two layers of heparin conjugate have been examined using a multi-wavelength TIRF spectroscopy technique with continuous flow. Fluorescently labeled AT, adsorbed from citrated human blood plasma, showed significantly higher signals on CHS compared to the cationic surface used to attach the heparin conjugate. The AT binding to CHS was very stable, also after exposure to soluble heparin at a concentration of 1.5 IU/mL. Only a few percent of the bound AT were displaced from the surfaces by AT present in plasma after long-term exposure to plasma. In contrast, larger amounts of the freshly added AT had adsorbed to the surfaces, especially to the surface with two layers of heparin conjugate, indicating the presence of unsaturated AT binding sites. The amount of AT bound to the different surfaces was quantified after elution using an enzyme immunoassay (EIA). Characteristic emission spectra of proteins and fluorophores of labeled proteins, obtained at the surfaces after a long-term exposure to plasma, confirmed their presence at the surfaces. The multi-wavelength TIRF technique proved to be a useful tool when combined with other techniques to study the time course of interactions of fluorescently labeled proteins with biomaterials, even in a complex environment such as plasma.

SmiNet-2: Description of an internet-based surveillance system for communicable diseases in Sweden.

Electronic systems for communicable diseases surveillance enhance quality by simplifying reporting, improving completeness, and increasing timeliness. In this article we outline the ideas and technologies behind SmiNet-2, a new comprehensive regional/national system for communicable disease surveillance in Sweden. The system allows for reporting from physicians (web form) and laboratories (direct from lab data system) over the internet. Using a unique personal identification number, SmiNet-2 automatically merges clinical and laboratory notifications to case records. Privileged users, at national and county level, work against a common central server containing all notifications and case records. In addition, SmiNet-2 has separate county servers with tools for outbreak investigations, contact tracing and case management. SmiNet-2 was first used in September 2004. Individual counties receive up to 90% of all notifications electronically. In its first year, SmiNet-2 received 54 980 clinical notifications and 32 765 laboratory notifications, which generated 58 891 case records. Since most clinicians in Sweden have easy access to the internet, a general web-based reporting has been feasible, and it is anticipated that within a few years all reporting to SmiNet-2 will be over the internet. In this context, some of the major advantages of SmiNet-2 when compared with other systems are timeliness in the dataflow (up to national level), the full integration of clinical and laboratory notifications, and the capability to handle more than 50 diseases with tailor-made notification forms within one single system.

Sensitivity of the Swedish statutory surveillance system for communicable diseases 1998-2002, assessed by the capture-recapture method.

To assess the sensitivity of the Swedish surveillance system, four notifiable communicable diseases in Sweden were examined during 1998-2002 with the two-sources capture-recapture method, based on parallel clinical and laboratory notifications. The sensitivity (proportion of diagnosed diseases actually being notified) was highest for salmonellosis (99.9%), followed by meningococcal infection (98.7%), and tularaemia (98.5%). For penicillin-resistant pneumococci, introduced as a notifiable disease in 1996, the overall sensitivity was 93.4%--increasing from 86.5% in 1998 to 98.5% in 2002. The system benefited from parallel reporting, with a sensitivity of clinical and laboratory notifications alone (all diseases combined) of 91.6% and 95.9% respectively. The sensitivity of both clinical and laboratory notifications was markedly higher in counties using the national electronic reporting system, SmiNet. Thus, sensitivity was higher for diseases with a long tradition of reporting, and there is a run-in period after a new disease becomes notifiable.