Immunodeficiency among children with recurrent invasive pneumococcal disease.

BACKGROUND: Recurrent invasive pneumococcal disease (rIPD) occurs mostly in children with an underlying disease, but some cases remain unexplained. Immunodeficiency has been described in children with rIPD, but the prevalence is unknown. We used a nationwide registry of all laboratory-confirmed cases of rIPD to identify cases of unexplained rIPD and examine them for immunodeficiency. METHODS: Cases of rIPD in children 0-15 years of age from 1980 to 2008 were identified. Children without an obvious underlying disease were screened for complement function, T-cell, B-cell, natural killer--cell counts and concentration of immunoglobulins. B-cell function was evaluated by measuring antibody response to polysaccharide-based pneumococcal vaccination and the extent of fraction of somatic hypermutation. Toll-Like receptor (TLR) signaling function and mutations in key TLR-signaling molecules were examined. RESULTS: In total, rIPD were observed in 54 children (68 cases of rIPD of 2192 IPD cases). Children with classical risk factors for IPD were excluded, and among the remaining 22 children, 15 were eligible for analysis. Of these 6 (40%) were complement C2-deficient. Impaired vaccination response was found in 6 children of whom 3 were C2 deficient. One patient had a severe TLR signaling dysfunction. No mutations in IRAK4, IKBKG or MYD88 were found. CONCLUSION: Of an unselected cohort of children with rIPD at least 11% were C2 deficient. Data suggest that screening for complement deficiencies and deficient antibody response to pneumococcal vaccines in patients with more than 1 episode of IPD is warranted.

Development, manufacturing and characterization of a highly purified, liquid immunoglobulin g preparation from human plasma.

BACKGROUND: The use of plasma-derived immunoglobulin G (IgG) is increasing, and the number of diseases, including immunodeficiencies, neurological diseases and autoimmune conditions, treated with intravenous IgG (IVIG) is expanding. Consequently, there is a great need for high-yield production processes for plasma-derived IgG. The aim of this work was to develop a high-yield process leading to a highly purified, liquid, ready-to-use IgG for intravenous use. METHODS: Plasma from healthy, voluntary, non-remunerated donors was fractionated by ethanol precipitation. IgG was extracted from fraction II + III using a phosphate/acetate buffer, pH 4, and purified by chromatography. RESULTS: Precipitation with 6% polyethylene glycol at pH 7 removed high molecular-weight contaminating proteins, aggregates and contaminating viruses. Ion exchange chromatography at pH 5.7 on serially connected anion and cation exchange columns allowed for elution of IgG from the cation exchange column in good yield and high purity. Further safety was achieved by solvent/detergent treatment and repeated ion exchange chromatography. The product consisted of essentially only IgG monomers and dimers, and had a high purity with very low levels of IgM and IgA. CONCLUSION: A process providing highly purified IVIG in good yield was developed.

Stability of HE4 and CA125 in blood samples from patients diagnosed with ovarian cancer.

OBJECTIVE: To investigate the influence of handling and storage on HE4 and CA125 serum and EDTA plasma levels to clarify any important consequences for a clinical setting. METHODS: Blood samples from 13 ovarian cancer (OC) patients were collected and allowed to clot or sediment for up to 72 hours at 4 °C or 20 °C, then processed into serum and EDTA plasma. Furthermore, the effects of up to eight repetitive cycles of freeze/thaw were investigated. HE4 and CA125 were analyzed using a Chemiluminescent Microparticle Immunoassay on the Architect i2000sr System. RESULTS: No significant effect of processing time for HE4 could be shown. HE4 EDTA plasma levels were insignificantly lower (3%) than serum levels (p = 0.41). Similarly, no significant effect of processing time for CA125 could be demonstrated. CA125 levels at 4 °C were significantly reduced compared to levels at 20 °C (p = 0.024). No significant difference between CA125 serum and plasma levels were found (p = 0.46). Serum and EDTA plasma samples were stable during the eight cycles of freezing and thawing (CA125: all p > 0.2; HE4: all p > 0.5). CONCLUSION: No systematic difference could be demonstrated for HE4. CA125 is not dependent on processing time, EDTA plasma or serum. Levels of CA125 are significantly reduced at 4 °C compared to levels at 20°C, but this difference was less than 6% and is not considered clinically relevant.

Non-specific binding in solid phase immunoassays for autoantibodies correlates with inflammation markers.

Enzyme-linked immunosorbent assay (ELISA) is a validated and sensitive method for detection of human autoantibodies, but may have problems with specificity. Non-specific binding is a well-known problem often observed in tests for autoantibodies, when sera are incubated on plastic surfaces, e.g. an ELISA plate. To understand the mechanisms underlying non-specific immunoglobulin deposition, we here analyse the phenomenon in detail and we propose means of reducing false positive test results caused by non-specific binding. The level of non-specific binding, in sera with suspected autoreactivity, was analysed in non-coated and autoantigen-coated ELISA wells and 4-32% of sera showed a high level of non-specific binding depending on the assay conditions and serum properties. Non-specifically binding sera were found to contain increased concentrations of IgG and other inflammatory mediators. Moreover, non-specific binding could be induced in serum by increasing the concentration of IgG and incubating the serum at 40 °C. This suggests that non-specific binding immunoglobulins can be formed during inflammation with high immunoglobulin levels and elevated temperature. We show that the level of non-specific binding correlates with the IgG concentration and therefore propose that non-specific binding may be interpreted as an informative finding indicative of elevated IgG and inflammation.

Aluminum hydroxide adjuvant differentially activates the three complement pathways with major involvement of the alternative pathway.

Al(OH)3 is the most common adjuvant in human vaccines, but its mode of action remains poorly understood. Complement involvement in the adjuvant properties of Al(OH)3 has been suggested in several reports together with a depot effect. It is here confirmed that Al(OH)3 treatment of serum depletes complement components and activates the complement system. We show that complement activation by Al(OH)3 involves the three major pathways by monitoring complement components in Al(OH)3-treated serum and in Al(OH)3-containing precipitates. Al(OH)3 activation of complement results in deposition of C3 cleavage products and membrane attack complex (MAC) and in generation of the anaphylatoxins C3a and C5a. Complement activation was time dependent and inhibited by chelation with EDTA but not EGTA+Mg(2+). We thus confirm that Al(OH)3 activates the complement system and show that the alternative pathway is of major importance.

Evaluation of HE4, CA125, risk of ovarian malignancy algorithm (ROMA) and risk of malignancy index (RMI) as diagnostic tools of epithelial ovarian cancer in patients with a pelvic mass.

OBJECTIVE: Diagnostic factors are needed to improve the currently used serum CA125 and risk of malignancy index (RMI) in differentiating ovarian cancer (OC) from other pelvic masses, thereby achieving precise and fast referral to a tertiary center and correct selection for further diagnostics. The aim was to evaluate serum Human Epididymis protein 4 (HE4) and the risk of ovarian malignancy algorithm (ROMA) for these purposes. METHODS: Serum from 1218 patients in the prospective ongoing pelvic mass study was collected prior to diagnosis. The HE4 and CA125 data were registered and evaluated separately and combined in ROMA and compared to RMI. RESULTS: 809 benign tumors, 79 borderline ovarian tumors, 252 OC (64 early and 188 late stage), 9 non-epithelial ovarian tumors and 69 non-ovarian cancers were evaluated. Differentiating between OC and benign disease the specificity was 62.2 (CA125), 63.2 (HE4), 76.5 (ROMA) and 81.5 (RMI) at a set sensitivity of 94.4 which corresponds to RMI=200. The areas under the curve (AUC) were 0.854 (CA125), 0.864 (HE4), 0,897 (ROMA) and 0.905 (RMI) for benign vs. early stage OC. For premenopausal benign vs. OC AUC were 0.925 (CA125), 0.905 (HE4), 0.909 (ROMA) and 0.945 (RMI). CONCLUSION: HE4 and ROMA helps differentiating OC from other pelvic masses, even in early stage OC. ROMA performs equally well as the ultrasound depending RMI and might be valuable as a first line biomarker for selecting high risk patients for referral to a tertiary center and further diagnostics. Further improvements of HE4 and ROMA in differentiating pelvic masses are still needed, especially regarding premenopausal women.

MASP interactions with plasma-derived MBL.

The interaction of mannan-binding lectin (MBL) with its associated serine proteases (MASPs) was investigated using recombinant (r) MBL, plasma-derived (pd) MBL, rMASP-3 and rMAp19. When mixed with MBL-deficient serum, rMBL and pdMBL associated with free MASP-2 to (re)gain complement-activating activity. MASPs already associated with pdMBL did not exchange with rMASP-3 or rMAp19, which bound to non-overlapping sites on MBL. Thus, rMASP-3 and rMAp19 bound to free available sites on rMBL and pdMBL. These results have important implications for the therapeutic use of MBL preparations.

High levels of immunoglobulin E and a continuous increase in immunoglobulin G and immunoglobulin M by age in children with newly diagnosed type 1 diabetes.

The incidence of type 1 diabetes (T1D) is increasing, either because of environmental factors accelerating onset of the disease or because of inducement of autoimmune diabetes in children who previously were at lower risk. High levels of immunoglobulin (Ig), specifically, IgM and IgA, and a low level of IgG were reported in adult patients; however no studies have analyzed the increasing incidence in relation to Ig levels. Our aim was to describe Ig in children newly diagnosed with diabetes and in their healthy siblings. Children with T1D expressed significantly lower IgG (p < 0.01) and higher IgA levels (p = 0.045), whereas no differences in IgE or IgM (p > 0.5) levels were found. Age-specific levels were unchanged over a 9-year period. In patients and siblings IgG, IgA and IgE increased by age (p < 0.001); which was in contrast to IgM (p > 0.05). The continued increase in IgG levels by age indicates that adult levels are reached later than in previously studied cohorts, thereby indicating a slower maturation of the immune system.

Investigating the humoral immune response in chronic venous leg ulcer patients colonised with Pseudomonas aeruginosa.

The ability to manage the bioburden in chronic wounds is most likely coupled to the humoral immune response of the patient. We analysed markers of systemic immune response in patients with chronic venous leg ulcers (CVLUs) colonised (no-systemic infection) with the opportunistic pathogen Pseudomonas aeruginosa. Sera from 44 clinically non infected patients with CVLUs were analysed for total IgM and IgG isotype 1-4, complement C3, mannose-binding lectin (MBL), interleukin (IL)-6, C-reactive protein (CRP) and specific anti-P. aeruginosa antibodies against exotoxin A, elastase and alkaline phosphatase. Concentrations of IL-6 versus CRP intercorrelated (ß = 2.43 95% CI (1.34-4.34)), but were independent of P. aeruginosa colonisation. MBL deficiency (MBL < 500 ng/ml) correlated to high serum levels of IgG(1) (P = 0.038) consistent with a compensatory mechanism, but not related to presence of P. aeruginosa in the ulcers. Twenty-four patients (54.5%) were culture positive for P. aeruginosa, also conferring significantly high serum levels of complement C3 (P = 0.014), but only two of these had positive titres for antibodies against exotoxin A. All patient sera were negative for antibodies against elastase and alkaline phosphatase. Fluorescent in situ hybridization analysis on randomly selected culture-positive patients could not establish unambiguous presence of P. aeruginosa biofilms in the ulcers. A multiple regression model showed P. aeruginosa and systemic CRP as significant factors in deterioration of ulcer healing rate.

Proteomic investigations of the ventriculo-lumbar gradient in human CSF.

Cerebrospinal fluid (CSF) is an ideal biological material in which to search for new biomarkers for improved diagnosis of neurological diseases. During a lumbar puncture between 5 and 15 mL of CSF are obtained. Previous studies have assessed the ventriculo-lumbar concentration gradient of a number of specific proteins. In the present study we took a proteomics approach to investigate the possible concentration gradient of a panel of proteins and peptides in the CSF of 16 patients with neurodegenerative diseases. Using two different mass spectrometry techniques, matrix assisted laser desorption ionization time of flight (MALDI-TOF) and surface enhanced laser desorption ionization time of flight (SELDI-TOF), we found that only one of the investigated proteins, apolipoprotein CI, was significantly decreased between the 1st and the 10th mL of CSF. Furthermore, we confirmed previous results showing a significant decrease in albumin concentration from the first to the last CSF aliquots. In conclusion, we found a significant gradient effect for only two of the measured proteins. However, a standardized procedure for CSF collection for diagnostic and research purposes is crucial to allow comparisons of results between patient groups and between laboratories. This is especially important since CSF is usually collected at several centres and variation in sampled CSF due to pre-analytical factors could complicate the interpretation of the results.

Safety pharmacology, toxicology and pharmacokinetic assessment of human Gc globulin (vitamin D binding protein).

Gc globulin is an important protein of the plasma actin-scavenger system. As such, it has been shown to bind free actin and prevent hypercoagulation and shock in patients with massive actin release resulting from severe tissue injuries. Treatment of such patients with Gc globulin could therefore potentially be life-saving. This article presents pre-clinical toxicology experiments conducted on purified plasma-derived human Gc globulin. The Gc globulin formulation was shown to be stable for at least 4 years with full retention of actin-binding capacity. In vitro studies did not reveal activation of the kallikrein system or the complement system and cellular studies showed no toxic effects on a variety of human cell lines. In vivo studies showed no acute toxic effects in mice, rats or guinea pigs upon intravenous infusion. A 14-day local tolerance study in rabbits showed no adverse effects, and 14-day toxicity studies in rats and horses did not show any unwanted reactions. In a 14-day toxicology study in beagle dogs, formation of antibodies was seen and in the end of the study period, three out of four dogs showed clinical immunological reactions, which could be ascribed to the formation of antibodies. The half-life, T, for human Gc globulin was 12 hr in rats, 16 hr in horses and 30 hr in dogs. The safety profile of plasma-derived Gc globulin is concluded to be consistent to that required for use in man.

Estimating glomerular filtration rate using the new CKD-EPI equation and other equations in patients with autosomal dominant polycystic kidney disease.

BACKGROUND: No studies have compared the performance of equations for estimating glomerular filtration rate (GFR) in patients with autosomal dominant polycystic kidney disease (ADPKD), where the declining GFR typically is followed for many years or even decades. This was the purpose of the present investigation. METHODS: 101 ADPKD patients with chronic kidney disease stages 1-5 were recruited and GFR was measured with the (51)Cr-EDTA clearance method, and estimated with the Modification of Diet in Renal Disease Study (MDRD) equation with 4 variables, the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation, the Cockcroft-Gault equation adjusted for body surface area and the MDRD equation with cystatin C. Performance was evaluated by mean bias, precision and accuracy. RESULTS: The MDRD equation with cystatin C had 97% of GFR estimates within 30% of measured GFR (accuracy). Both the CKD-EPI and Cockcroft-Gault equations had an accuracy of 90% whereas the MDRD equation had an accuracy of 83%. This difference of accuracy was especially marked with GFR >60 ml/min/1.73 m(2). CONCLUSION: For estimating GFR in ADPKD patients the MDRD equation with cystatin C incorporated had the best performance. The CKD-EPI or the Cockcroft-Gault equations showed better performance compared to the 4-variable MDRD equation.

Epidemiology of chronic wound patients and relation to serum levels of mannan-binding lectin.

The aim of this study was to describe the epidemiology of chronic wounds in a large cohort of patients from a tertiary hospital out-patient clinic, and examine the significance of serum mannan-binding lectin for the occurrence and clinical presentation of such wounds. The study comprised 489 consecutive patients with chronic foot and leg ulcers. A clinical classification of wound- aetiology was performed, and mannan-binding lectin was measured in the sera of patients and healthy controls. The patients presented with 639 wounds altogether; diabetic foot ulcers (309), venous leg ulcers (188), arterial ulcers (109), and vasculitis (33). The mannan-binding lectin levels of patients with venous leg ulcer, alone or in combination with other types of wounds, differed significantly from the control group, and the frequency of values < 100 ng/ml was significantly higher. In diabetic and arterial ulcer patients the frequency of values >or= 3000 ng/ml was significantly higher than that of the control group. This suggests a role for the innate immunity in the pathology of venous leg ulcers, and indicates different roles for mannan-binding lectin in the development of ulcers with different aetiologies; it further suggests that mannan-binding lectin substitution should be tested in a controlled clinical trial.

First trimester maternal serum pregnancy-specific beta-1-glycoprotein (SP1) as a marker of adverse pregnancy outcome.

OBJECTIVE: To establish the first trimester levels of pregnancy-specific beta-1-glycoprotein (SP1) in pregnancies with adverse outcome. Furthermore, to determine the screening performance for adverse outcome using SP1 alone and in combination with other first trimester markers including proMBP and PAPP-A. METHODS: A case-control study was conducted in a primary hospital setting. The SP1 concentration was measured in first trimester maternal serum in pregnancies with small-for-gestational age fetuses (SGA) (n = 150), spontaneous preterm delivery (n = 88), preeclampsia (n = 40) and in controls (n = 500). Concentrations were converted to multiples of the median (MoM) in controls and groups were compared using Mann-Whitney U-test. Logistic regression analysis was used to determine significant factors for predicting adverse pregnancy outcome. Screening performance was assessed using receiver operating characteristic (ROC) curves. RESULTS: The SP1 MoM median was significantly reduced in cases with SGA (0.76 MoM, p < 0.0005) and spontaneous preterm delivery (0.77 MoM, p < 0.0005) whereas no alteration was found in cases with preeclampsia (0.94 MoM, p = 0.723). A significant correlation (r = 0.217) between log(10)(SP1 MoM) and the birth weight percentile was found in the SGA group. Screening performance was only slightly improved when SP1 was combined with PAPP-A or proMBP. CONCLUSION: SP1 is a first trimester maternal serum marker of SGA and preterm delivery.

Mannose-binding lectin (MBL) substitution: recovery of opsonic function in vivo lags behind MBL serum levels.

Mannose-binding lectin (MBL) deficiency is often associated with an increased risk of infection or worse prognosis in immunocompromised patients. MBL substitution in these patients might diminish these risks. We therefore performed an open, uncontrolled safety and pharmacokinetic MBL-substitution study in 12 pediatric oncology patients with chemotherapy-induced neutropenia. Twice weekly MBL infusions with plasma-derived MBL yielded MBL trough levels >1.0 microg/ml. We tested whether MBL substitution in vivo increased MBL-dependent complement activation and opsonophagocytosis of zymosan in vitro. Upon MBL substitution, opsonophagocytosis by control neutrophils increased significantly (p < 0.001) but remained suboptimal, although repeated MBL infusions resulted in improvement over time. The MBL-dependent MBL-associated serine protease (MASP)-mediated complement C3 and C4 activation also showed a suboptimal increase. To explain these results, complement activation was studied in detail. We found that in the presence of normal MASP-2 blood levels, MASP-2 activity (p < 0.0001) was reduced as well as the alternative pathway of complement activation (p < 0.05). This MBL-substitution study demonstrates that plasma-derived MBL infusions increase MBL/MASP-mediated C3 and C4 activation and opsonophagocytosis, but that higher circulating levels of plasma-derived MBL are required to achieve MBL-mediated complement activation comparable to healthy controls. Other patient cohorts should be considered to demonstrate clinical efficacy in phase II/III MBL-substitution studies, because we found a suboptimal recovery of (in vitro) biological activity upon MBL substitution in our neutropenic pediatric oncology cohort.

Safety and pharmacokinetics of plasma-derived mannose-binding lectin (MBL) substitution in children with chemotherapy-induced neutropaenia.

Mannose-binding lectin (MBL)-deficient children with cancer may benefit from substitution of the innate immune protein MBL during chemotherapy-induced neutropaenia. We determined the safety and pharmacokinetics of MBL substitution in a phase II study in MBL-deficient children. Twelve MBL-deficient children with cancer (aged 0-12 years) received infusions of plasma-derived MBL once, or twice weekly during a chemotherapy-induced neutropaenic episode (range: 1-4 weeks). Four patients participated multiple times. Target levels of 1.0 microg/ml were considered therapeutic. In total, 65 MBL infusions were given. No MBL-related adverse reactions were observed, and the observed trough level was 1.06 microg/ml (range: 0.66-2.05 microg/ml). Pharmacokinetics were not related to age after correction for body weight. The half-life of MBL, for a child of 25 kg, was 36.4h (range: 23.7-66.6h). No anti-MBL antibodies were measured 4 weeks after each MBL course. Substitution therapy with MBL-SSI twice weekly was safe and resulted in trough levels considered protective.

Characterisation of the 1st SSI purified MBL standard.

BACKGROUND: Mannan-binding lectin (MBL) is of importance in innate immunity. MBL-deficiency, the most common immune defect, is significant in several clinical contexts. The request for MBL diagnostic is increasing, hence we developed a high-purity MBL standard assigned with a traceable value. METHODS AND RESULTS: The standard material was produced from human plasma; and the protein concentration determined by amino acid analysis after a preceding desalting. The standard value was assessed by two series of sub-sample analyses from nine vials by the grand mean: 235.7 microg protein/ml (range 191.1-280.3 microg/ml). The loss during desalting was 7% and the protein content 253.4 microg/ml after correction. After SDS/PAGE the MBL content was estimated by densitometric scanning. The MBL band (non-MBL bands being MASPs) comprised 76%. Therefore, the standard was assigned a value of 192.6 microg MBL/ml (range 156.0-229.2 microg/ml). A calibrated time-resolved immuno-flourescence assay was used for stability evaluation of the MBL standard, after transfer from -80 degrees C, showing stability for at least 10 days at 25 degrees C, 14 days at 5 degrees C, and 16 weeks at -20 degrees C. CONCLUSION: The 1st SSI purified MBL standard has been produced, and assigned the value 192.6 microg MBL/ml, traceable to an accurate realisation of the unit.

Ficolins and Mannose-Binding Lectin in Danish patients with sarcoidosis.

Mannose-Binding Lectin (MBL) is a prognostic marker in pulmonary diseases. Ficolins, sharing many structural and functional similarities with MBL, may also be involved in the pathogenesis of pulmonary diseases. The objectives of the study were to establish whether plasma concentrations of Ficolin-2, -3, and MBL in Danish patients with sarcoidosis and control persons differed and whether they were of prognostic significance. We retrospectively included 46 consecutive patients (26 male, 20 female) and 51 age- and sex-matched healthy control persons (28 male, 23 female). Information about the patients was obtained from their medical records. We measured plasma concentrations of Ficolin-2, -3, and MBL using ELISA. There was a significant difference in the patients' mean Ficolin-3 plasma level (14.9 microg/ml; +/-2SD: 6.7-23.1) compared with the control persons' (21.6 microg/ml; +/-2SD: 12.7-30.5). The difference was 6.7 microg/ml (95% CI: 5.0-8.4 microg/ml; p<0.001). In the patients, Ficolin-3 correlated inversely with the CD4(+)/CD8(+)-ratio (Spearman's Rho=-0.37; p=0.021; n=39). There were no significant differences in plasma concentrations of Ficolin-2 or MBL between the two groups. Ficolin-3 concentrations were lower in plasma from patients with sarcoidosis. This suggests a possible involvement of Ficolin-3 in the complex pathophysiology of sarcoidosis. However, we could not show the applicability of Ficolin plasma level measurement as a marker of disease activity or of prognostic significance in sarcoidosis.

The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases.

The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding was observed. Interaction of calreticulin with recombinant MBL was fully inhibited by recombinant MASP-2, MASP-3 and MAp19, but not by the MASP-2 D105G and MAp19 Y59A variants characterized by defective MBL binding ability. Furthermore, MBL point mutants with impaired MASP binding showed no interaction with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins. In conclusion, the potential MBL co-receptor calreticulin binds to MBL at the MASP binding site and the interaction may involve a conformational change in MBL.