Michael Osthoff

Mannose-Binding Lectin Deficiency is Associated with Smaller Infarction Size and Favorable Outcome in Ischemic Stroke Patients

Background The Mannose-binding lectin (MBL) pathway of complement plays a pivotal role in the pathogenesis of ischemia/reperfusion (I/R) injury after experimental ischemic stroke. As comparable data in human ischemic stroke are limited, we investigated in more detail the association of MBL deficiency with infarction volume and functional outcome in a large cohort of patients receiving intravenous thrombolysis or conservative treatment. Methodology/Principal Findings In a post hoc analysis of a prospective cohort study, admission MBL concentrations were determined in 353 consecutive patients with an acute ischemic stroke of whom 287 and 66 patients received conservative and thrombolytic treatment, respectively. Stroke severity, infarction volume, and functional outcome were studied in relation to MBL concentrations at presentation to the emergency department. MBL levels on admission were not influenced by the time from symptom onset to presentation (p = 0.53). In the conservative treatment group patients with mild strokes at presentation, small infarction volumes or favorable outcomes after three months demonstrated 1.5 to 2.6-fold lower median MBL levels (p = 0.025, p = 0.0027 and p = 0.046, respectively) compared to patients with more severe strokes. Moreover, MBL deficient patients (<100 ng/ml) were subject to a considerably decreased risk of an unfavorable outcome three months after ischemic stroke (adjusted odds ratio 0.38, p<0.05) and showed smaller lesion volumes (mean size 0.6 vs. 18.4 ml, p = 0.0025). In contrast, no association of MBL concentration with infarction volume or functional outcome was found in the thrombolysis group. However, the small sample size limits the significance of this observation. Conclusions MBL deficiency is associated with smaller cerebral infarcts and favorable outcome in patients receiving conservative treatment. Our data suggest an important role of the lectin pathway in the pathophysiology of cerebral I/R injury and might pave the way for new therapeutic interventions.

Autoantibodies against Complement C1q Specifically Target C1q Bound on Early Apoptotic Cells

Autoantibodies against complement C1q (anti-C1q) are frequently found in patients with systemic lupus erythematosus (SLE). They strongly correlate with the occurrence of severe lupus nephritis, suggesting a pathogenic role in SLE. Because anti-C1q are known to recognize a neoepitope on bound C1q, but not on fluid-phase C1q, the aim of this study was to clarify the origin of anti-C1q by determining the mechanism that renders C1q antigenic. We investigated anti-C1q from serum and purified total IgG of patients with SLE and hypocomplementemic urticarial vasculitis as well as two monoclonal human anti-C1q Fab from a SLE patient generated by phage display. Binding characteristics, such as their ability to recognize C1q bound on different classes of Igs, on immune complexes, and on cells undergoing apoptosis, were analyzed. Interestingly, anti-C1q did not bind to C1q bound on Igs or immune complexes. Neither did we observe specific binding of anti-C1q to C1q bound on late apoptotic/necrotic cells when compared with binding in the absence of C1q. However, as shown by FACS analysis and confocal microscopy, anti-C1q specifically targeted C1q bound on early apoptotic cells. Anti-C1q were found to specifically target C1q bound on cells undergoing apoptosis. Our observations suggest that early apoptotic cells are a major target of the autoimmune response in SLE and provide a direct link between human SLE, apoptosis, and C1q.

Urinary tract infections due to extended-spectrum beta-lactamase-producing Gram-negative bacteria: identification of risk factors and outcome predictors in an Australian tertiary referral hospital.

OBJECTIVES Extended-spectrum beta-lactamase-expressing Gram-negative bacilli (ESBL-GNB) now commonly cause community-acquired infections, including urinary tract infections (UTI), and represent a challenge for practitioners in choosing empirical antibiotics. The aim of this study was to describe the epidemiology and clinical characteristics of UTIs/bacteriuria due to ESBL-GNB in Australia. METHODS At a single-site tertiary referral hospital, 100 cases with UTIs/bacteriuria due to ESBL-GNB were matched to 100 cases where UTIs/bacteriuria were caused by organisms matching the ESBL bacterial species that had routine susceptibility to antibiotics. Potential risk factors for ESBL-GNB UTI/bacteriuria and differences in clinical outcomes were identified. RESULTS Length of admission prior to positive sample (odds ratio (OR) 1.3, p = 0.03, per week), exposure to antibiotics (OR 5.7, p < 0.001), return from overseas travel (OR 6.5, p = 0.002), and nursing home residency (OR 4.2, p = 0.03) were identified as risk factors associated with ESBL-GNB UTI/bacteriuria in the multivariate analysis. In addition, ESBL-GNB-infected cases subsequently had a longer inpatient stay (median 6 vs. 2 days, p = 0.002) and were admitted to the intensive care unit more frequently (28/100 vs. 8/100, p < 0.001). CONCLUSIONS Our results emphasize the need for culture of a mid-stream urine specimen prior to commencing antibacterials, especially in patients with the risk factors identified herein associated with ESBL-GNB UTI/bacteriuria.

Identification of a major linear C1q epitope allows detection of systemic lupus erythematosus anti-C1q antibodies by a specific peptide-based enzyme-linked immunosorbent assay

OBJECTIVE Autoantibodies against C1q strongly correlate with the occurrence of severe nephritis in patients with systemic lupus erythematosus (SLE). We undertook this study to determine whether identification of the C1q epitope(s) recognized by these autoantibodies might lead to a better diagnostic assay and help elucidate the putative role of C1q and anti-C1q in SLE. METHODS SLE patient-derived anti-C1q Fab were used in a microarray-based peptide scan to identify the peptide sequence recognized by anti-C1q. Anti-C1q Fab binding to the target peptide was further analyzed using real-time interaction measurements (surface plasmon resonance) and peptide-based enzyme-linked immunosorbent assays (ELISAs). RESULTS A peptide scan of the collagen-like region of C1q identified 2 regions, 1 on the A chain and 1 on the B chain, that were the targets of the anti-C1q Fab. Binding was confirmed by surface plasmon resonance and showed nanomolar affinity. The A chain-derived peptide could specifically be detected in a peptide-based ELISA by SLE patient sera. Competition experiments suggested that this peptide represented one of the major linear epitopes of C1q that is the target of anti-C1q in SLE. Serum antibodies from most SLE patients but not from healthy individuals specifically bound to this epitope. Binding to the peptide correlated with binding of the same sera to native C1q but was found to be more sensitive for the detection of lupus nephritis. CONCLUSION We identified a major linear epitope of C1q that is the target of anti-C1q in SLE. The ELISA using this peptide was more specific and more sensitive than a conventional anti-C1q assay for the detection of active nephritis in SLE patients.

Mannose-binding lectin levels and major infections in a cohort of very long-term survivors after allogeneic stem cell transplantation

Background Life-threatening infections are a major cause of death after allogeneic stem cell transplantation. Complement Mannose-binding lectin is a key component of innate immunity. Functional deficiency of mannose-binding lectin due to genetic polymorphism is frequent. Previous reports showed conflicting results with respect to the influence of functional mannose-binding lectin deficiency on infectious risk after allogeneic stem cell transplantation. The aim of this study was to clarify the impact of low mannose-binding lectin levels on infectious risk in a unique cohort of very long-term survivors after stem cell transplantation. Design and Methods Incidence of major infections was evaluable in 43 out of 44 very long-term survivors (over ten years) and studied retrospectively in relation to mannose-binding lectin serum concentrations. Results Recipients with mannose-binding lectin levels below 1,000 ng/mL were at increased risk to suffer from one or more major infections (P=0.002) during entire follow up. Infectious susceptibility was increased after neutrophil recovery, particularly until 24 months (Hazard Ratio 3.4) with sustained effects afterwards (Hazard Ratio 2.9). Mannose-binding lectin serum concentrations below 1,000 ng/mL were independently associated with major infections after neutrophil recovery (P=0.009). In subgroup analyses occurrence of severe herpes virus infections in particular was associated with significantly lower mannose-binding lectin levels (P=0.02). Conclusions Our findings indicate that low mannose-binding lectin levels may predict markedly increased susceptibility to severe infections with sustained effects even late after allogeneic stem cell transplantation. Determinations of mannose-binding lectin status should therefore be included into pre-transplantation risk assessment.

If there is an evolutionary selection pressure for the high frequency of MBL2 polymorphisms, what is it?

Either immune selection or stochastic processes may have influenced the frequency of highly polymorphic genes such as mannose‐binding lectin 2 (MBL2). This pattern recognition receptor of the innate immune system recognizes and binds to pathogenic microorganisms and apoptotic cells leading to lectin pathway complement killing or clearance. In almost all of a large number of studies in different ethnic groups worldwide there is 20–25% carriage of low MBL2 haplotypes, with 8–10% of each population having no MBL detectable in the blood. The source of this high variability of MBL2 remains cryptic. It arises from six main snps in the prompter and exon regions of the gene that assort into seven common haplotypes under linkage disequilibrium. While global studies of MBL2 show that it is not under immune selection pressure, these results are not the same when the same population genetic tools are used on large national studies. Other analyses point to the silenced MBL1 pseudogene and development of promoter polymorphisms in humans as evidence of selection pressure favouring low‐producing haplotypes. While these analyses cannot be reconciled readily, there are two processes by which MBL heterozygosity could have been advantageous in an evolutionary sense; protection against adverse effects of various infectious diseases and lethal manifestations of atherosclerosis – a disease that now seems to have a more ancient history than assumed previously. Ultimately, consideration of the context for possible future therapeutic manipulation of MBL means that this can proceed independently of resolution of the evolutionary forces that have shaped MBL2 polymorphism.

Procalcitonin as a diagnostic marker for sepsis

We appreciate the comments of Michael Osthoff and Damon Eisen about a lack of diagnostic accuracy of procalcitonin in our metaanalysis, the need for serial testing, and their suggested treatment decisions. However, previous metaanalyses on the diagnostic accuracy of procalcitonin measurements in patients with severe sepsis in intensive-care units were limited by selected populations, and were biased by the choice of gold standard for the definition of sepsis. We noted a mean sensitivity of 0·77 (95% CI 0·72–0·81) and specifi city of 0·79 (0·74–0·84) and an area under the receiver operating characteristic curve of 0·85 (0·81–0·88). By contrast, Tang and colleagues reported a mean sensitivity and specificity of 0·71 (0·67–0·76), and an area under the curve of 0·78 (0·73–0·83). We agreed that these results are far from ideal, and concluded that results must be interpreted carefully for clinical and microbiological assessment and have to be re-evaluated accordingly. However, in the daily practice of an intensive-care unit, accuracy of clinical confi rmation of the diagnosis alone is probably much worse. We also agree that the time course is more relevant than are the initial procalcitonin concentrations in patients in intensive-care units, and that higher cutoff ranges than are used in other settings should be considered. However, we strongly argue against any biomarker-guided approach to initiate treatment in sepsis, in situations in which, among others, broad-spectrum antibiotic treatment has to be given immediately. Treatment decisions and efficacy analyses were not one of the objectives of our Article. However, we investigated the effect of procalcitonin-guided treatment compared with standard care in another meta-analysis of seven studies with 1075 patients with severe sepsis. Findings showed that procalcitonin concentrations might be helpful to guide antimicrobial treatment through a reduction in duration of antimicrobial treatment without an obvious increase in mortality. Importantly, treatment algorithms and cutoffs differ substantially and should be clarified in future studies before recommendations are made. We also thank Gerta Rucker and Martin Schumacher for their interest in our work and for their thoughtful reanalysis of our data. We agree that the handling of diff erent cutoff values is a difficult problem in diagnostic meta-analyses. This issue is due to the fact that often only one pair of sensitivities and specificities are given for a particular study. Thus, the approach of Rucker and Schumacher helps to deal with this problem and is facilitated by the additional assumption that the authors of the individual studies report a result on the basis of an optimum cutoff value. More specifi cally, the overall summary receiver operating characteristic curve in this setting is obtained as a weighted sum of study-specific receiver operating characteristic curves in the logit space. However, we are not entirely convinced that these results are better than are those obtained with the bivariate generalised linear mixed model that we used. This assertion is based on two arguments. First, the approach of Rucker and Schumacher is a fi xed-eff ects model that ignores heterogeneity between studies. Our analysis revealed substantial heterogeneity between studies, which should be taken into account. Second, the proposed method works in logit space. Simulation studies have shown that an exact binomial generalised linear mixed model has better performance compared with logit transformed data. Thus, a comparison of methods by simulation studies would be interesting and seems necessary. We are not entirely convinced that the observed lower values of sensitivity and specifi city lead to a strong argument against the use of procalcitonin as a diagnostic marker for sepsis. Furthermore, we would like to prompt authors of diagnostic studies to report sensitivities and specifi cities for more than one cutoff value, since this knowledge would allow a better consideration of cutoff values.

Significance of Mannose-Binding Lectin Deficiency and Nucleotide-Binding Oligomerization Domain 2 Polymorphisms in Staphylococcus aureus Bloodstream Infections: A Case-Control Study

Background Pathways coordinated by innate pattern recognition receptors like mannose-binding lectin (MBL) and nucleotide-binding oligomerization domain 2 (NOD2) are among the first immune responses to Staphylococcus aureus (S. aureus) bloodstream infections (BSI) in animal models, but human data are limited. Here, we investigated the role of MBL deficiency and NOD2 mutations in the predisposition to and severity of S. aureus BSI. Patients and Methods A matched case-control study was undertaken involving 70 patients with S. aureus BSI and 70 age- and sex-matched hospitalized controls. MBL levels, MBL2 and NOD2 polymorphisms were analyzed. Results After adjusting for potential confounders, MBL deficiency (<0.5 µg/ml) was found less frequently in cases than controls (26 vs. 41%, OR 0.4, 95% confidence interval (CI) 0.20-0.95, p=0.04) as were low producing MBL genotypes (11 vs. 23%, OR 0.2, 95% CI 0.08-0.75, p=0.01), whereas NOD2 polymorphisms were similarly distributed. Cases with NOD2 polymorphisms had less organ dysfunction as shown by a lower SOFA score (median 2.5 vs. 4.5, p=0.02), whereas only severe MBL deficiency (<0.1 µg/ml) was associated with life-threatening S. aureus BSI (OR 5.6, 95% CI 1.25-24.85, p=0.02). Conclusions Contrary to animal model data, our study suggests MBL deficiency may confer protection against acquiring S. aureus BSI. NOD2 mutations were less frequently associated with multi-organ dysfunction. Further human studies of the innate immune response in S. aureus BSI are needed to identify suitable host targets in sepsis treatment.

Impact of mannose-binding lectin deficiency on radiocontrast-induced renal dysfunction: a post-hoc analysis of a multicenter randomized controlled trial

BackgroundLocal renal ischemia is regarded as an important factor in the development of contrast-induced nephropathy (CIN). Mannose-binding lectin (MBL) is involved in the tissue damage during experimental ischemia/reperfusion injury of the kidneys. The aim of the present study was to investigate the association of MBL deficiency with radiocontrast-induced renal dysfunction in a large prospective cohort.Methods246 patients with advanced non–dialysis-dependent renal dysfunction who underwent radiographic contrast procedures were included in the study. Baseline serum MBL levels were analyzed according to the occurrence of a creatinine-based (increase of ≥0.5 mg/dL or ≥25% within 48 hours) or cystatin C-based (increase of ≥10% within 24 hours) CIN.ResultsThe incidence of creatinine-based and cystatin C-based CIN was 6.5% and 24%, respectively. MBL levels were not associated with the occurrence of creatinine-based CIN. However, patients that experienced a cystatin C increase of ≥10% showed significantly higher MBL levels than patients with a rise of <10% (median 2885 (IQR 1193–4471) vs. 1997 (IQR 439–3504)ng/mL, p = 0.01). In logistic regression analysis MBL deficiency (MBL levels≤500 ng/ml) was identified as an inverse predictor of a cystatin C increase ≥10% (OR 0.34, 95% CI 0.15-0.8, p = 0.01).ConclusionMBL deficiency was associated with a reduced radiocontrast-induced renal dysfunction as reflected by the course of cystatin C. Our findings support a possible role of MBL in the pathogenesis of CIN.

Mannose-binding lectin-the forgotten molecule?

To the Editor: We read with great interest the excellent review on the role of immunity and inflammation in acute ischemic stroke by Iadecola and Anrather1. The authors highlight the role of complement in the acute and regenerative phase of ischemic stroke among several important components of the immune system cited. Moreover, they appropriately emphasize caution against overly translating results from animal studies to human trials, as no single animal model can truly replicate the symptoms and outcomes in people after stroke in all their complexity. However, many of the targets mentioned in the review have been shown to influence cerebral ischemia-reperfusion injury only in animal models, whereas human data are scarce. Additionally, their therapeutic potential cannot be readily explored, as effective drugs against these targets have not been developed in the majority of cases. In this context, there is a protein of the complement cascade, mannose-binding lectin (MBL), that is not mentioned but whose important influence on cerebral ischemia-reperfusion injury has been shown in both experimental and human studies. MBL is a recognition protein of the lectin pathway that shares a high degree of structural similarity with C1q, the initiating molecule of the classical pathway of complement activation. Interestingly, uninflamed tissues, which lack MBL, have numerous mannose receptor–positive macrophages, whereas circulating granulocytes or monocytes do not express mannose receptor. Evidence suggests that the circulating MBL protein is the functional serum equivalent of the tissue macrophage mannose receptor2. Hence, the ability to recognize mannose-rich ligands in peripheral blood is conferred to the hepatically derived MBL2. Similarly to C1q and the mannose receptor, MBL has been implicated in host defense3 and the removal of apoptotic cells4. And, notably, almost a third of the population worldwide suffers from functional MBL deficiency, sometimes referred to as the most frequent human immunodeficiency5. In the brain, mannose receptor–expressing macrophages and microglia are mainly located at the blood-brain–cerebrospinal fluid interfaces6, and the majority of C1q is synthesized by microglia and macrophages entering the central nervous system (CNS)7. In contrast, MBL has been detected only in association with endothelial cells of cerebral blood vessels8. MBL has a profound influence on microglial cells acting as a sensor of danger in the CNS that is distinct from C1q. Whereas C1q acts like a proinflammatory trigger for microglia, MBL impinges on the activation state of microglia by inducing proliferation and phagocytosis without eliciting strong inflammatory responses9. Hence, MBL may influence the inflammatory response after brain injury by regulating multiple pathways in the neurovascular unit10, including microglia and the complement and coagulation systems11. Although MBL delays the development of atherosclerotic lesions12, several experimental and human studies suggest that MBL and the lectin complement pathway may be crucial to augmenting ischemiareperfusion injury in various organs, such as the heart and the gut13–17. Recently, two mouse studies of cerebral ischemia showed that MBLdeficient mice have diminished C3 deposition and neutrophil influx into the affected brain region, which resulted in protection from stroke (as assessed 48 h after a transient 2-h ischemia)18,19. In contrast to the experimental data in mice and other animal models nicely summarized by Iadecola and Anrather1, the role of MBL in people with stroke has already been investigated in two human studies. The first one found a significant association of MBL deficiency with favorable outcome 3 months after acute stroke in a mixed population of 135 individuals with ischemic and hemorrhagic stroke19. The second, larger study (n = 297) showed that functional MBL deficiency is associated with smaller cerebral infarcts (as evaluated on magnetic resonance imaging) and improved outcome in individuals with ischemic stroke not eligible for treatment with thrombolytic agents, suggesting a crucial role of MBL in human acute ischemic stroke20. Regarding translation into effective therapies, a powerful recombinant inhibitor of the lectin pathway—that additionally blocks C1 of the classical pathway and, to a lesser extent, the alternative pathway and the kinin system21—already exists and has been recently approved for hereditary angioedema (rhC1INH, Ruconest, Pharming). This transient multiple-action, multiple-target inhibitor showed very promising results in a mouse model of transient cerebral ischemia when administered up to 18 h after ischemic stroke18. In addition, transient blockage of MBL-associated serine protease-2 might represent an alternative approach22. Iadecola and Anrather emphasize the potential risk of infections when modulating innate or adaptive immunity to lessen ischemiareperfusion injury1. In fact, infections are the most prevalent and relevant complications after stroke23. Although the human studies on MBL and stroke19,20 did not primarily assess the contribution of MBL deficiency to infection susceptibility, there was no evident increase of infectious complications in MBL-deficient individuals (ref. 19 and M.O., M. Katan (University Hospital Basel), F. Fluri (University Hospital Basel), P. Schuetz (Harvard School of Public Health), R. Bingisser (University Hospital Basel) et al., unpublished data), suggesting the potential use of rhC1INH in human cerebral ischemiareperfusion injury in future trials. The lectin pathway, and MBL in particular, seems to be an essential component of the inflammatory process leading to additional postischemic damage after stroke in mice and humans. Interventional studies to explore the effectiveness of blocking MBL or the lectin pathway are clearly warranted.