Rudi Steffensen

An assay for the mannan-binding lectin pathway of complement activation.

The mannan-binding lectin (MBL) pathway of complement activation has been established as the third pathway of complement activation. MBL is a carbohydrate-binding serum protein, which circulates in complex with serine proteases known as mannan-binding lectin associated serine proteases (MASPs). When bound to microorganisms, the MBL complex activates the complement components C4 and C2, thereby generating the C3 convertase and leading to opsonisation by the deposition of C4b and C3b fragments. This C4/C2 cleaving activity is shared with the C1 complex of the classical pathway of complement activation. Therefore, in a generally applicable complement activation assay specific for the MBL pathway, the activity of the classical pathway must be inhibited. This can be accomplished by exploiting the finding that high ionic strength buffers inhibit the binding of C1q to immune complexes and disrupt the C1 complex, whereas the carbohydrate-binding activity of MBL and the integrity of the MBL complex is maintained under hypertonic conditions. In the assay described here, the specific C4b-depositing capacity of the MBL pathway was determined by incubating serum diluted in buffer containing 1 M NaCl in mannan-coated microtiter wells before the addition of purified C4. The interassay coefficient of variation in the ELISA version was 7.3%. As expected no activity was found in MBL-deficient serum. When 100 normal serum samples were analysed we found that the MBL level correlated with the amount of C4b deposited on the mannan-coated surface. However, we also found a threefold variation in C4b-depositing capacity between individuals with similar MBL concentrations. The assay permits for the determination of MBL complex activity in serum and plasma samples and may thus be used to evaluate the clinical implications of complement activation via this pathway.

MAp44, a Human Protein Associated with Pattern Recognition Molecules of the Complement System and Regulating the Lectin Pathway of Complement Activation

Essential effector functions of innate immunity are mediated by complement activation initiated by soluble pattern recognition molecules: mannan-binding lectin (MBL) and the ficolins. We present a novel, phylogenetically conserved protein, MAp44, which is found in human serum at 1.4 μg/ml in Ca2+-dependent complexes with the soluble pattern recognition molecules. The affinity for MBL is in the nanomolar range (KD = 0.6 nM) as determined by surface plasmon resonance. The first eight exons of the gene for MAp44 encode four domains shared with MBL-associated serine protease (MASP)-1 and MASP-3 (CUB1-EGF-CUB2-CCP1), and a ninth exon encodes C-terminal 17 aa unique to MAp44. mRNA profiling in human tissues shows high expression in the heart. MAp44 competes with MASP-2 for binding to MBL and ficolins, resulting in inhibition of complement activation. Our results add a novel mechanism to those known to control the innate immune system.

Report of the first nationally implemented clinical routine screening for fetal RHD in D- pregnant women to ascertain the requirement for antenatal RhD prophylaxis

BACKGROUND: A combination of antenatal and postnatal RhD prophylaxis is more effective in reducing D immunization in pregnancy than postnatal RhD prophylaxis alone. Based on the result from antenatal screening for the fetal RHD gene, antenatal RhD prophylaxis in Denmark is given only to those D− women who carry a D+ fetus. We present an evaluation of the first national clinical application of antenatal RHD screening.

Polymorphisms in innate immunity genes predispose to bacteremia and death in the medical intensive care unit

Objective:Critically ill patients are at risk of sepsis, organ failure, and death. Studying the impact of genetic determinants may improve our understanding of the pathophysiology and allow identification of patients who would benefit from specific treatments. Our aim was to study the influence of single nucleotide polymorphisms in selected genes involved in innate immunity on the development of bacteremia or risk of death in patients admitted to a medical intensive care unit. Design, Setting, and Patients:DNA was available from 774 medical intensive care unit patients. We selected 31 single nucleotide polymorphisms in 14 genes involved in host innate immune defense. Serum levels of MASP2 and chemotactic capacity, phagocytosis, and killing capacity of monocytes at admission were quantified. Univariate Kaplan-Meier estimates with log-rank analysis and multivariate logistic regression were performed. Bootstrap resampling technique and ten-fold cross-validation were used to assess replication stability, prognostic importance of the variables, and repeatability of the final regression model. Main Results:Patients with at least one NOD2 variant were shown to have a reduced phagocytosis by monocytes (p = 0.03) and a higher risk of bacteremia than wild-type patients (p = 0.02). The NOD2/TLR4 combination was associated with bacteremia using survival analyses (time to bacteremia development, log-rank p < 0.0001), univariate regression (p = 0.0003), and multivariate regression analysis (odds ratio [OR] 4.26, 95% confidence interval [CI] 1.85–9.81; p = 0.0006). Similarly, the same combination was associated with hospital mortality using survival analysis (log-rank p = 0.03), univariate regression (p = 0.02), and multivariate regression analysis (OR 2.27, 95% CI 1.09–4.74; p = 0.03). Also variants in the MASP2 gene were significantly associated with hospital mortality (survival analysis log-rank-p = 0.003; univariate regression p = 0.02; multivariate regression analysis OR 2.35, 95% CI 1.38–3.99; p = 0.002). Conclusions:Functional polymorphisms in genes involved in innate immunity predispose to severe infections and death, and may become part of a risk model, allowing identification of patients at risk, who could benefit from early introduction of specific preventive or therapeutic interventions.

Multifactorial Etiology of Recurrent Miscarriage and Its Scientific and Clinical Implications

A considerable proportion of recurrent miscarriage (RM) cases are caused by recurrent chromosomally abnormal conceptions. However, in younger patients and patients with multiple miscarriages, maternal causes seem to dominate. No single biomarker with a high predictive value of maternally caused RM has been identified. Non-genetic biomarkers in RM may not reflect conditions in the pregnant uterus and we rarely know whether they are causes or consequences of miscarriage. Studies of genetic biomarkers are probably the best way to reveal the pathophysiological mechanisms behind RM. Epidemiological and genetic studies suggest that RM due to maternal causes has a multifactorial background. The risk of RM in each patient is probably determined by the interaction of many genetic variants and environmental factors but only few of these have so far been identified. The genetic biomarkers for RM can probably be classified into three groups: (1) variants associated with excessive inflammatory responses and autoimmunity; (2) variants of importance for insulin and androgen sensitivity and turn-over, and (3) variants associated with thrombophilia. Identification of these markers will require whole genome association studies comprising thousands of individuals. Acknowledgement of the multifactorial background for RM has important implications for the management of patients in clinical practice.

Evaluation of Histo-Blood Group ABO Genotyping in a Danish Population: Frequency of a Novel O Allele Defined as O2

Traditional blood group ABO serology is based on immunoreactivity with the carbohydrate determinants A, B and H antigens. Recent advances at the DNA level of the ABO genes have provided a molecular genetic model for the ABO polymorphism. This genetic model has to date only been tested on a limited basis. The present study was initiated to evaluate the universality of the proposed genetic model on a larger group of serologically defined ABO phenotypes. Three hundred healthy Danish blood donors were analysed (A: 50, B: 50, AB: 50, O: 150) by PCR amplification followed by diagnostic restriction enzyme cutting. In all cases A, B, and AB at least one allele of correctly predicted status was found. However, in O phenotype individuals, 11 out of 150 carried one allele discordant to the proposed genetic model. This novel O allele (3.7% allele frequency) was further characterized by diagnostic restriction enzyme analysis in two positions divergent between A and B alleles and by DNA sequencing of the two major exons. The novel O allele is termed O2 as it typed as B in nucleotide position 526 and as A in positions 703, 796, and 803, in contrast to the most predominant O allele termed O1, which types as A in all 4 positions. The structural defect in the O2 allele appears to be an additional substitution at nucleotide position 802. The results clearly demonstrate that with the addition of the two distinctly different O alleles, O1, O2, the previously proposed molecular genetic basis of the ABO polymorphism is quite valid. More importantly the determined characteristics of these two O alleles have practical implications in ABO genotyping, because it establishes within the limits of the number of samples tested that ABO genotypes can be assessed directly by non‐allele specific PCR amplification and restriction enzyme analysis.

Activation of the complement system in human nonalcoholic fatty liver disease

Activation of the innate immune system plays a major role in nonalcoholic fatty liver disease (NAFLD). The complement system is an important component of innate immunity that recognizes danger signals such as tissue injury. We aimed to determine whether activation of the complement system occurs in NAFLD, to identify initiating pathways, and to assess the relation between complement activation, NAFLD severity, apoptosis, and inflammatory parameters. Liver biopsies of 43 obese subjects with various degrees of NAFLD and of 10 healthy controls were analyzed for deposition of complement factors C1q, mannose‐binding lectin (MBL), C4d, activated C3, and membrane attack complex (MAC)‐associated C9. Furthermore, hepatic neutrophil infiltration, apoptosis, and pro‐inflammatory cytokine expression were quantified. Whereas complement activation was undetectable in the liver of healthy subjects, 74% of the NAFLD patients showed hepatic deposition of activated C3 and C4d. C1q as well as MBL accumulation was found in most activated C3‐positive patients. Strikingly, 50% of activated C3‐positive patients also displayed MAC‐associated C9 deposition. Deposition of complement factors was predominantly seen around hepatocytes with macrovesicular steatosis. Subjects showing accumulation of activated C3 displayed increased numbers of apoptotic cells. Importantly, hepatic neutrophil infiltration as well as interleukin (IL)‐8 and IL‐6 expression was significantly higher in patients showing activated C3 deposition, whereas patients with C9 deposition additionally had increased IL‐1β expression. Moreover, nonalcoholic steatohepatitis (NASH) was more prevalent in patients showing hepatic C9 or activated C3 deposition. Conclusion: There is widespread activation of the complement system in NAFLD, which is associated with disease severity. This may have important implications for the pathogenesis and progression of NAFLD given the function of complement factors in clearance of apoptotic cells, hepatic fibrosis, and liver regeneration. (HEPATOLOGY 2009.)

Deficiency of mannan-binding lectin associated serine protease-2 due to missense polymorphisms.

Mannan-binding lectin (MBL) and ficolins distinguish between self, non-self and altered-self by recognizing patterns of ligands on the surface of microorganisms or aberrant cells. When this happens MBL-associated serine protease-2 (MASP-2) is activated and cleaves complement factors to start inflammatory actions. We examined human populations for MASP-2 levels, MASP-2 function and for the presence of mutations in coding exons of MASP2. The MASP-2 levels were lowest in Africans from Zambia (median, 196 ng/ml) followed by Hong Kong Chinese (262 ng/ml), Brazilian Amerindians (290 ng/ml) and Danish Caucasians (416 ng/ml). In the Chinese population, we uncovered a novel four amino-acid tandem duplication (p.156_159dupCHNH) associated with low levels of MASP-2. The frequency of this mutation as well as the SNPs p.R99C, p.R118C, p.D120G, p.P126L and p.V377A were analyzed. The p.156_159dupCHNH was only found in Chinese (gene frequency 0.26%) and p.D120G was found only in Caucasians and Inuits from West-Greenland. The p.P126L and p.R99Q were present in Africans and Amerindians only, except for p.R99Q in one Caucasian. The MASP-2 levels were reduced in individuals with p.V377A present. The MASP-2 present in individuals homozygous for p.377A or p.99Q had a normal enzyme activity whereas MASP-2 in individuals homozygous for p.126L was non-functional.

FcγRIIIB polymorphism: evidence that NA1/NA2 and SH are located in two closely linked loci and that the SH allele is linked to the NA1 allele in the Danish population

BACKGROUND: The neutrophil‐specific antigens NA1, NA2, and SH are well‐recognized allotypic forms of FcγRIIIB. Individuals carrying all three FcγRIIIB genes were recently described.

Effects of infectious mononucleosis and HLA-DRB1*15 in multiple sclerosis

Background Both human leukocyte antigen (HLA)-DRB1*15 and Epstein-Barr virus infection presenting as infectious mononucleosis (IM) are recognized as risk factors for multiple sclerosis (MS). However, their combined effect and possible interaction on MS risk is not known. Objective To assess the association between HLA-DRB1*15 and risk of MS in persons with and without IM. Methods We compared the prevalence of DRB1*15 in MS patients with (n = 76) and without (n = 1,836) IM with the corresponding distributions in blood donors with (n = 62) and without (n = 484) IM histories. This allowed us to estimate the relative risk of MS associated with DRB1*15 in the presence and absence, respectively, of previous IM. We then estimated the interaction between DRB1*15 and IM as the ratio of the two individual odds ratios. Results In IM-naïve individuals, DRB1*15 carried a 2.4-fold (95% confidence interval [CI], 2.0–3.0) increased MS risk. In contrast, among persons with IM history, DRB1*15 was associated with a 7.0-fold (95% CI, 3.3–15.4) increased MS risk. Thus, the MS risk conferred by HLA-DRB1*15 was 2.9 (95% CI, 1.3–6.5)-fold stronger in the presence than in the absence of IM. Combined with previous results, this result indicates that DRB1*15-positive persons with a history of IM may be at a 10.0-fold (95% CI, 6.0–17.9) increased risk of MS compared with persons who are DRB1*15 and IM-naïve. Conclusion DRB1*15 and IM may act in synergy causing MS.