Jakob T. Bay

Low mannose-binding lectin serum levels are associated with reduced kidney graft survival

Activation of the complement system is initiated by the alternative, the classical, or the lectin pathway. As the complement system is involved in the pathophysiology of graft rejection after kidney transplantation, we investigated the possible role of mannose-binding lectin in kidney transplantation and the influence of human leukocyte antigen (HLA) immunization on this process. In a prospective study of 544 kidney transplant patients over a follow-up period of 5 years, low serum levels of this lectin at the time of transplantation were found to be significantly associated with decreased 5-year death-censored graft survival (hazard ratio 1.68). Subanalysis showed that this association was confined to non-HLA-immunized patients (hazard ratio 1.93). The strongest association was seen in non-HLA-immunized patients receiving a kidney from a deceased donor (hazard ratio 2.93). No significant association with mannose-binding lectin levels and graft survival were found in HLA-immunized patients. Variant MBL2 genotypes causing low mannose-binding lectin serum concentrations showed the same association pattern. Our findings demonstrate a clear protective role of mannose-binding lectin and thus innate immunity in maintaining kidney graft survival, but these are probably overruled by HLA immunization.

Ficolin-2 reveals different analytical and biological properties dependent on different sample handling procedures.

Ficolin-2 (L-ficolin) is a germ line encoded pattern recognition molecule circulating in the blood, and functions as a recognition molecule in the lectin complement pathway. However, consistent and reliable measurements of Ficolin-2 concentration and activity have been difficult to achieve. After recurrent observations of deviations in Ficolin-2 properties between different blood sample procedures, we decided to investigate this closer. Blood samples from ten healthy donors were collected in various serum and plasma tubes and Ficolin-2 properties were evaluated by different ELISA setups. We found that serum prepared from tubes containing the clot activator silica used as a standard technique in many routine laboratories held a significantly lower concentration of Ficolin-2 as compared to the other sample types. Furthermore, Ficolin-2 binding and complement activation potential in this type of serum was impaired when using an acetylated compound as matrix. On the other hand, Ficolin-2 in serum made without clot activator and in plasma irrespective of additive used, had the same concentration and was capable of initiating the lectin pathway measured as C4 and C3 deposition on the ligand. No Ficolin-2 mediated formation of the terminal complement complex was observed under the applied assay conditions. In conclusion, our results show that Ficolin-2 is a promiscuous molecule and that care should be taken during sampling, handling and matrix chosen for measurement of Ficolin-2 levels and activity.

Pre-transplant levels of ficolin-3 are associated with kidney graft survival

Ficolin-3 is an initiator of the lectin complement pathway. The complement system is a mediator of the pathophysiology of graft rejection in kidney transplantation, but the role of ficolin-3 in this process is unknown. Using a prospective study design, 527 kidney transplanted patients were included. 97 blood donors served as controls. Ficolin-3, C4 and C3 were measured in pre-transplant as well as in control serum samples. In controls, deposition of ficolin-3, C4, C3 and the terminal complement complex (TCC) was measured in an assay based on acetylated albumin as matrix. The ficolin-3 levels correlated with the serum levels of C4 and C3. The serum levels of ficolin-3 correlated with the deposition of ficolin-3, C4, C3 and TCC. Survival analyses showed that high pre-transplant serum levels of ficolin-3 were associated with decreased graft survival. These results suggest an important role of ficolin-3 in the pathophysiology of kidney graft rejection.

Low C4 gene copy numbers are associated with superior graft survival in patients transplanted with a deceased donor kidney

Complement C4 is a central component of the classical and the lectin pathways of the complement system. The C4 protein exists as two isotypes C4A and C4B encoded by the C4A and C4B genes, both of which are found with varying copy numbers. Deposition of C4 has been implicated in kidney graft rejection, but a relationship between graft survival and serum C4 concentration as well as C4 genetic variation has not been established. We evaluated this using a prospective study design of 676 kidney transplant patients and 211 healthy individuals as controls. Increasing C4 gene copy numbers significantly correlated with the C4 serum concentration in both patients and controls. Patients with less than four total copies of C4 genes transplanted with a deceased donor kidney experienced a superior 5-year graft survival (hazard ratio 0.46, 95% confidence interval: 0.25-0.84). No significant association was observed in patients transplanted with a living donor. Thus, low C4 copy numbers are associated with increased kidney graft survival in patients receiving a kidney from a deceased donor. Hence, the degree of ischemia may influence the clinical impact of complement.

High levels of mannose-binding lectin are associated with lower pulse wave velocity in uraemic patients

BackgroundUraemia is associated with a highly increased risk of cardiovascular disease. Mannose-binding lectin (MBL) has been shown to be involved in cardiovascular pathophysiology and a protective effect of MBL is suggested. The purpose of the present study was to evaluate a potential impact of MBL on vascular parameters in uraemic patients.MethodsA cohort of 98 patients with end stage renal disease (ESRD) awaiting kidney transplantation had pulse wave velocity (PWV) and augmentation index (AIX) examined by tonometry and endothelial dependent flow-mediated (FMD) and endothelial independent nitroglycerin-induced (NID) dilatory capacities of the brachial artery measured by ultrasound. An oral glucose tolerance test (OGTT) was performed and serum levels of MBL were measured using Luminex xMAP bead array technology.ResultsThe cohort was divided in two groups according to MBL-concentration below or above the median concentration. These groups were comparable regarding age, BMI, and duration of ESRD. PWV was significantly lower in the group with high MBL levels compared to the group with low MBL levels and trends toward better AIX and higher insulin sensitivity (ISI) was also seen in the group with high MBL levels. No difference was seen in FMD and NID.ConclusionsHigh levels of MBL are associated with lower PWV and the use of antihypertensive drugs in a cohort of patients with ESRD awaiting kidney transplantation suggesting a beneficial role of high levels of MBL on arterial stiffness in uraemia.

A rapid, accurate and robust particle-based assay for the simultaneous screening of plasma samples for the presence of five different anti-cytokine autoantibodies

PURPOSE To establish and validate a rapid, cost-effective and accurate screening assay for the simultaneous testing of human naturally occurring anti-cytokine autoantibodies (c-aAb) targeting interleukin-1α (IL-1α), interleukin-6 (IL-6), interleukin-10 (IL-10), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon α (IFNα). Because the c-aAbs can be transferred to patients through blood transfusion, the assay was used to assess c-aAb levels in a cohort of patients who were receiving blood transfusions and subsequently presented with or without febrile reactions. MATERIALS AND METHODS The microsphere-based Luminex platform was used. Recombinant forms of human IL-1α, IL-6, IL-10, GM-CSF, and IFNα were gently coupled to MAG-PLEX beads. Plasma IgG binding was measured with phycoerythrin (PE)-labeled secondary antibodies. Previously confirmed c-aAb positive and negative donor plasma samples and pooled normal immunoglobulin preparations were used to validate the assay. Plasma samples from 98 transfusion recipients, half of whom presented with febrile reactions, were tested by the assay. RESULTS The assay detected specific and saturable immunoglobulin G (IgG) binding to each of the tested cytokines in previously confirmed c-aAb positive plasmas and in preparations of pooled normal immunoglobulin. Confirmed c-aAb negative plasmas gave no saturable binding. The detection limit of the cytokine autoantibodies was estimated to be between 1 pM and 10 pM. The recovery of confirmed cytokine autoantibodies quantities in the negative plasma samples ranged between 80% and 125%. The analytical intra- and inter-assay variations were 4% and 11%, respectively. Varying c-aAb levels were detectable in the transfusion recipients. There was no difference in c-aAb frequency between the patients with or without febrile transfusion reactions. The c-aAb level before and after the blood transfusions varied only slightly and in an irregular manner. CONCLUSION This assay simultaneously detected up to five different c-aAbs in pooled human IgG and in plasma from individual blood donors, and it was deemed suitable for larger screenings. Based on confirmed antibody binding characteristics and the resultant reactivity in this multiplex assay, a classification of the c-aAb levels was suggested. The screening results of the recipients who received blood transfusions indicate that more studies are needed to clarify the role of antibodies, if any, in transfusion medicine and in high-dose immunoglobulin treatment.

Novel CFI mutation in a patient with leukocytoclastic vasculitis may redefine the clinical spectrum of Complement Factor I deficiency.

Factor I is an important regulator of the complement system. Lack of Factor I causes uncontrolled activation of the complement system leading to consumption of C3. Complete deficiency of Factor I is a rare condition and only around 40 cases has been reported in the literature. The clinical presentation of Factor I deficiency varies and includes severe recurrent bacterial infections, glomerulonephritis and autoimmune diseases. The patient, a 28-years old woman with consanguineous parents, presented with recurrent leukocytoclastic vasculitis in the lower extremities with no associated systemic involvement, and without increased infection tendency. Initial testing showed low C3 concentration and a detailed complement evaluation absence of complement Factor I. Sequencing revealed a homozygous missense mutation in exon 2 of the CFI gene (SCV000221312). Even though the clinical symptoms of CFI mutations vary among patients sole association with leukocytoclastic vasculitis redefines the clinical spectrum of complete Factor I deficiency.