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Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, originally discovered for its eponymous effect and now known for pleiotropic biologic properties in immunology and oncology. Circulating MIF levels are elevated in several types of human cancer including prostate cancer. MIF is released presumably by both stromal and tumor cells and enhances malignant growth and metastasis by diverse mechanisms, such as stimulating tumor cell proliferation, suppressing apoptotic death, facilitating invasion of the extracellular matrix, and promoting angiogenesis. Recently described fully human anti-MIF antibodies were tested in vitro and in vivo for their ability to influence growth rate and invasion of the human PC3 prostate cancer cell line. In vitro, the selected candidate antibodies BaxG03, BaxB01, and BaxM159 reduced cell growth and viability by inhibiting MIF-induced phosphorylation of the central kinases p44/42 mitogen-activated protein kinase [extracellular signal–regulated kinase-1 and -2 (ERK1/2)] and protein kinase B (AKT). Incubation of cells in the presence of the antibodies also promoted activation of caspase-3/7. The antibodies furthermore inhibited MIF-promoted invasion and chemotaxis as transmigration through Matrigel along a MIF gradient was impaired. In vivo, pharmacokinetic parameters (half-life, volume of distribution, and bioavailability) of the antibodies were determined and a proof-of-concept was obtained in a PC3-xenograft mouse model. Treatment with human anti-MIF antibodies blunted xenograft tumor growth in a dose-dependent manner. We therefore conclude that the anti-MIF antibodies described neutralize some of the key tumor-promoting activities of MIF and thus limit tumor growth in vivo. Mol Cancer Ther; 12(7); 1223–34. ©2013 AACR.

PDF file - 247K, Suppl. Fig. 1. Pharmacokinetics of BaxG03 after intravenous and intraperitoneal injection; Suppl. Fig. 2. Trough plasma concentrations of BaxG03 after 28 days of administration; Suppl. Fig. 3. Dose-dependent inhibition of xenograft outgrowth by BaxB01 and BaxM159.

Precise applying of PPP (Plant Protection Products) in orchards and vineyards requires new kinds of sprayer technologies and new methods of sensor data evaluation. In this paper a selective electrical driven sprayer, carried by the autonomous robotic platform elWObot, is introduced. A 3D-Simulation environment and the framework ROS (Robot Operating System) helps developing and testing the interaction between the sprayer and the robot. The calculated leaf wall area (LWA) and the distance from the sprayer to the leaves in the spray region, control the flow-rate and the air-assist of eight adjustable sprayers individually. First field trials showed that the adaption of the software from the simulation to the hardware worked as expected.

Background This prospective, open-label, non-comparative, multicentre, long-term phase IV study is examining the efficacy and safety of somatropin [recombinant human growth hormone (rhGH)] in short children born small for gestational age (SGA) and its impact on the incidence of diabetes. This report is the first interim analysis of patients who have completed 1 year of treatment. Methods A total of 278 pre-pubertal patients were enrolled. Key eligibility criteria included height standard deviation score (HSDS)

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, originally discovered for its eponymous effect and now known for pleiotropic biologic properties in immunology and oncology. Circulating MIF levels are elevated in several types of human cancer including prostate cancer. MIF is released presumably by both stromal and tumor cells and enhances malignant growth and metastasis by diverse mechanisms, such as stimulating tumor cell proliferation, suppressing apoptotic death, facilitating invasion of the extracellular matrix, and promoting angiogenesis. Recently described fully human anti-MIF antibodies were tested in vitro and in vivo for their ability to influence growth rate and invasion of the human PC3 prostate cancer cell line. In vitro, the selected candidate antibodies BaxG03, BaxB01, and BaxM159 reduced cell growth and viability by inhibiting MIF-induced phosphorylation of the central kinases p44/42 mitogen-activated protein kinase [extracellular signal–regulated kinase-1 and -2 (ERK1/2)] and protein kinase B (AKT). Incubation of cells in the presence of the antibodies also promoted activation of caspase-3/7. The antibodies furthermore inhibited MIF-promoted invasion and chemotaxis as transmigration through Matrigel along a MIF gradient was impaired. In vivo, pharmacokinetic parameters (half-life, volume of distribution, and bioavailability) of the antibodies were determined and a proof-of-concept was obtained in a PC3-xenograft mouse model. Treatment with human anti-MIF antibodies blunted xenograft tumor growth in a dose-dependent manner. We therefore conclude that the anti-MIF antibodies described neutralize some of the key tumor-promoting activities of MIF and thus limit tumor growth in vivo. Mol Cancer Ther; 12(7); 1223–34. ©2013 AACR.

Human factor VIIa (FVIIa) is commonly used as bypassing therapy to treat bleeding episodes in hemophilia patients with neutralizing antibodies to factors VIII (FVIII) or IX (FIX). There is a need for a suitable animal model to assess the immunogenicity of new FVIIa products during preclinical development. The aim of this study was the design of a novel transgenic mouse model with immune tolerance to human FVIIa.The model was generated by transgenic expression of human F7 cDNA. FVIIa-specific immune responses after treatment with human FVIIa were assessed by analyzing circulating antibodies, antibody producing plasma cells and CD4(+) T cells.In contrast to wild-type mice, human FVII transgenic mice did not develop antibodies when treated with human FVIIa. The immune tolerance was specific and could be broken by application of human FVIIa together with a strong stimulus of the innate immune system. Break of tolerance was associated with increased numbers of pro-inflammatory FVIIa-specific CD4(+) T cells.The new mouse model is suitable to study the influence of the innate immune system on maintenance and break of immune tolerance against FVIIa and could be used to assess the immunogenicity of new FVIIa products during pre-clinical development.

Adrenal hypoplasia congenita (AHC) is an X-linked disorder characterized by primary adrenal insufficiency. Hypogonadotropic hypogonadism (HHG) is frequently associated with this disorder but is thought not to be caused by the low adrenal androgen levels due to adrenal hypoplasia. It is uncertain whether there are two distinct yet physically linked genes responsible for AHC and HHG or a single gene responsible for both diseases. AHC can occur as a part of a contiguous deletion syndrome together with Duchenne muscular dystrophy (DMD) and/or glycerol kinase deficiency (GKD). From the analysis of deletions, the following gene order has been deduced: Xpter-AHC-GKD-DMD-cen. An AHC critical region of 200-500 kilobases has been defined by physical mapping and partially overlaps with a 160-kilobase dosage-sensitive sex (DSS) reversal critical region. The DAX-1 (DSS-AHC critical region on the X, gene 1) gene was isolated and found to encode a new member of the nuclear hormone receptor family. Here we report that DAX-1 is deleted in 14 patients and point mutations were found in the coding region in DNA from 12 unrelated individuals. All AHC patients over 14 years old and with only point mutations in DAX-1 were also diagnosed with HHG, confirming that the DAX-1 gene is responsible for both X-linked AHC and HHG. But in four sporadic cases and a single familial case, no point mutations were found, suggesting genetic heterogeneity or differential expression of DAX-1.

BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor for the serine protease factor IX, which actives factor X. This method type, termed modification-dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. In contrast to all other methods described so far, it allows measurement of the biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle can be extended to protein modifications other than PEGylation and to a variety of functional activity assays.

OBJECTIVE We aimed at evaluating BMI changes during GH therapy pause, to assess the transfer pattern between children and adult medical services in our clinic and to confirm the previous growth hormone deficiency (GHD) diagnosis. DESIGN Retrospective cohort study. METHODS We identified 75 transition patients (age at first visit

Deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a VWF-cleaving protease, is the key factor in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), a life-threatening thrombotic microangiopathy. It is well established that ADAMTS13 deficiency results in elevated plasma levels of ultra-large VWF multimers (ULVWF), which are prone to induce platelet aggregation; however, the actual trigger of TTP development remains uncertain. Here we describe a new animal model in which some TTP-like symptoms can be triggered in ADAMTS13 knockout mice by challenge with 2000 units/kg body weight of recombinant human VWF containing ULVWF multimers. Animals rapidly showed clinical symptoms and developed severe thrombocytopenia. Schistocytosis, a decrease in hematocrit, and elevated serum lactate dehydrogenase levels were observed. The heart was identified as the most sensitive target organ with rapid onset of extensive platelet aggregation in the ventricles and myocardial necrosis. Prophylactic administration of 200 units/kg recombinant human ADAMTS13 protected ADAMTS13 knockout mice from developing TTP. Therapeutic administration of 320 units/kg rhADAMTS13 reduced the incidence and severity of TTP findings in a treatment interval-dependent manner. We therefore consider this newly established mouse model of thrombotic microangiopathy highly predictive for investigating the efficacy of new treatments for TTP.

Summary Objective Poor growth early in life is associated with numerous adverse conditions including decreased bone mass. The aim is to investigate bone and body composition in young adults born small for gestational age (SGA). Design Observational study. Participants A total of 76 young adults born SGA (34f) at a mean age of 19·68 ± 0·5 years were enrolled. Method Bone mineral density (BMD), bone geometry and body composition were analysed using peripheral quantitative computed tomography. Results Adults born SGA had significantly lower z-score for height (−0.86 ± 0·87), weight (−0·61 ± 0·78) and BMI (−0·38 ± 1·04) as well as fat cross-sectional area (CSA) (−0·62 ± 0·80) compared with a healthy reference population (P

Recombinant VWF (rVWF) is a candidate for therapy of von Willebrand disease and the largest known multimeric glycoprotein. In this study rVWF was covalently linked with a 20 kDa branched polyethylene glycol (PEG) to obtain PEGylated rVWF (PEGrVWF). This conjugation results in a further increase of heterogeneity besides glycoheterogeneity and a challenge in analyzing of such a bioconjugate, particular when investigated on the intact molecule level. Four different techniques including SDS-PAGE, MALDI-TOF-MS, capillary-gel-electrophoresis-on-a-chip (CGE-on-a-chip) and nano electrospray gas-phase electrophoretic mobility molecular analysis (nES-GEMMA) were applied to determine the molecular weight (MW) and the PEGylation degree of the monomeric rVWF. The degree and distribution of PEGylation of rVWF obtained by CGE-on-a-chip were in good agreement with results obtained by MALDI-TOF-MS with a special high mass detector. An average PEGylation degree of 3.1 PEG chains coupled to the monomeric glycoforms was found. MW determination by MALDI-TOF-MS (317.4 ± 1.0 kDa; 3 PEG chains attached) showed in comparison to CGE-on-a-chip (413.4 ± 2.1 kDa) the highest precision. Furthermore the orthogonal method, nES-GEMMA provided first information on the globular size (11.3 ± 0.1 nm) and based on that the MW (251 ± 7.2 kDa for the average PEGylation) of the PEGrVWF.

Recently there have been major developments in understanding the basic mechanisms of actions of natural and adaptive antibodies, especially with regard to their anti-inflammatory and immunoregulatory properties, and the art of their clinical application in a variety of inflammatory and autoimmune disorders. This was the major theme of the International Forum of Immunoglobulin Research, held in Miami Florida on October 3–6, 2013. There have been several recent developments in the understanding of the role of natural autoantibodies in immune homeostasis, tolerance, and antimicrobial activity. Natural IgM antibodies to apoptotic-cell membrane determinants represent a distinct subset of antibodies that potentiate the phagocytic clearance of apoptotic cells and apoptotic bodies. Furthermore, these autoantibodies display potent antiinflammatory activities both in vitro and in vivo, which are mediated by MAPK phosphatase 1 signaling pathway (Caroline Gronwell, New York, USA). A role of natural IgM to be protective in human diseases comes from patients with systemic lupus erythematosus in which high levels of natural IgM to phosphocholine correlate with less active lupus disease, less organ damage, and lower risk of cardiovascular events. Srini Kaveri (Paris, France) and associates have identified and characterized the natural autoantibodies of the IgG isotype directed against the human Fc receptors. They show that the F(ab′)2 of IVIG recognizes the FcγRIII (CD16) and FcγRII (CD32). Furthermore, the immunopurified anti-FcγIII and anti-FcγII antibodies present in IVIG recognized soluble and membrane-bound FcR. These observations provide further evidence of the existence of natural autoantibodies towards a wide range of self-motifs, including those present in apoptotic bodies in immunoglobulin preparations that may participate in regulation of the immune response. Peter Lobo (Virginia, USA), using in vitro and murine knock out models, demonstrated an anti-inflammatory role of natural IgM antileucocyte antibodies (ALA). This anti-inflammatory effect of IgM is mediated, in part, by inhibiting TLR-4-induced translocation of NF-κB into the nucleus and inhibiting differentiation of naive Th0 cells into Th1 and Th17 cells. He also showed that intra-peritoneal administration of IgM prevents NODmice from developing autoimmune insulitis, which also involves Th1 and Th17 cells. Therefore, anti-inflammatory and immunoregulatory properties of natural antibodies may justify the use of polyclonal IgM preparations and/or development of monoclonal IgM for the treatment of inflammatory and autoimmune disease. Although a role of both ‘natural’ and ‘immune’ IgM antibodies in immune homeostasis, removal of self-antigen containing apoptotic cells and apoptotic bodies, and in protective immunity against pathogens is well-recognized, the mechanisms of IgM-mediated protection against microbes and immune-regulation; however, are not completely understood, Recent recognition and characterization of long since elusive Fc receptor for IgM (FcμR), in both humans and mice have shed light on some of these mechanisms. Hiromi Kubagawa (Alabama, USA) presented data on the cellular distribution of FcμR and potential functions. FcμR knock out mice are unable to secrete IgM; however, express surface IgM and other Ig isotypes on B cells, and secrete other immunoglobulins. Studies of these mice have reinforced the importance of both ‘natural’ and ‘immune’ IgM in microbial defense and in the regulation of autoimmunity. The sugar moieties attached to the IgG constant fragment (Fc) are essential for IgG functionality such as binding to cellular Fc receptors and complement activation. Presence or absence of distinct residues such as fucose and sialic acid can S. Gupta (*) University of California at Irvine, Irvine, CA, USA e-mail: sgupta@uci.edu

Vor dem Hintergrund kategorialer und nichtkategorialer Ansatze der psychosozialen Beeintrachtigung durch eine chronische Erkrankung wurden Kinder mit Diabetes mellitus Typ 1 (DM), mit adrenogenitalem Syndrom (AGS) und mit der Diagnose Mangelgeburt (SGA) hinsichtlich ihrer psychosozialen Adaptation, ihrer Stressbewaltigungsfahigkeiten sowie der Lebensqualitat und Bewaltigung ihrer Eltern verglichen. An einer Befragung mit psychometrischen Fragebogen nahmen 38 Kinder mit DM, 37 Kinder mit AGS und 39 Kinder mit SGA und ihre Eltern teil. Zum Einsatz kamen die Instrumente SDQ-D (Strength and Difficulties Questionnaire, deutsche Version), SSKJ 3–8 (Fragebogen zu Stress und Stressbewaltigung im Kindes- und Jugendalter, Altersgruppe 3–8 Jahre), SVF-KJ (Stressverarbeitungsfragebogen fur Kinder und Jugendliche), ULQIE (Ulmer Lebensqualitatsinventar fur Eltern eines chronisch kranken Kindes) und der Fragebogen zur elterlichen Bewaltigung. Signifikante Gruppenunterschiede zeigten sich hinsichtlich der Stressbelastung und -bewaltigung der Kinder sowie der elterlichen Lebensqualitat und Bewaltigungsstrategien. Unterschiedliche Erkrankungen bringen unterschiedliche Faktoren der Beeintrachtigung und Bewaltigungsanforderungen mit sich, konnen aber zu einem ahnlichen Muster der psychosozialen Anpassung fuhren. Strategien der Bewaltigung sind trainierbar.

The human IgG repertoire contains endogenous antibodies against beta amyloid (Aβ) that may be relevant to the pathogenesis and treatment of Alzheimer's disease. There have been widely disparate estimates of the levels of these antibodies in human plasma. We identify factors that have contributed to these disparities and describe improved methods for measuring anti-Aβ antibodies in blood. These methods include isolating immunoglobulin by thiophilic chromatography and using chaotropic salts to dislodge weakly bound antibodies without significantly reducing the binding of specific anti-Aβ antibodies. Using these methods, we show that human blood contains polyvalent IgG antibodies that bind to Aβ with relatively low avidity and specificity, as well as IgG antibodies that bind to linear and conformational epitopes on amyloid monomers and aggregates with moderate to high avidity.

The tail cut bleeding model (CUT) is routinely used in factor VIII-deficient mice to assess pharmacodynamic effects of therapeutic strategies for haemophilia A. Results from this model are highly variable, many modifications to the model are reported and at times the animals’ wellbeing may be compromised by recording survival as an endpoint. We therefore investigated if the ferric chloride carotid occlusion model (COM) used for thrombosis research can be applied to enhance data quality and animal welfare in haemophilia A research. Relative dose effects and relative dose variations were calculated for the CUT and COM. The requisite sample sizes were estimated and the importance of survival rates to assess rebleeds during recovery was evaluated by correlating initial blood loss to mortality. Relative dose effects increased with higher doses in both models. The COM was more sensitive at lower doses than the CUT, had up to 82% less variation across doses and clearly showed superior accuracy. Only 5% of the sample size required for the CUT would be needed to establish non-inferiority between a specific therapeutic dose in haemophilia A mice and healthy wild-type animals. A strong statistically significant correlation was found between initial blood loss and mortality within 24 h. Our findings clearly suggest that the COM is a valid tool for assessing haemophilia A treatment in vivo. The highly reproducible data means that significantly fewer animals are required and a more humane endpoint can be used by directly assessing clot stability instead of survival rate.

In 21-hydroxylase (CYP21A2) deficiency (21OHD), the level of in vitro enzymatic function allows for classification of mutation groups (null, A, B, C) and prediction of disease severity. However, genital virilization in affected females correlates only weakly with CYP21A2 mutation groups, suggesting the influence of genetic modifiers.The objective of the study was to investigate the influence of the polymorphic CAG and GGn repeats of the androgen receptor (AR) gene on the degree of genital virilization in 21OHD females.Design of the study was the determination of CYP21A2 genotype, degree of genital virilization (Prader stage), and X-weighted biallelic mean of AR CAG and GGn repeat length in 205 females with 21OHD.Correlation of AR CAG and GGn repeat lengths with Prader stages using nested stepwise logistic regression analysis was measured.CYP21A2 mutation groups null and A showed significantly higher levels of genital virilization than groups B and C (P0.01). However, Prader stages varied considerably within mutation groups: null, Prader I-V (median IV); A, Prader I-V (median IV); B, Prader I-V (median III); C, 0-III (median I). Mean GGn repeat length of patients was not significantly associated with Prader stages, classified as low (0-I), intermediate (II-III), or severe (IV-V) (odds ratio per repeat: 0.98, 95% confidence interval 0.71-1.35). In contrast, patients with Prader 0-I showed a trend toward longer CAG repeats without reaching statistical significance (P = 0.07, odds ratio per repeat: 0.82, 95% confidence interval 0.65-1.02).Neither CAG nor GGn repeat lengths are statistically significant modifiers of genital virilization in females with 21OHD.

Introduction We have previously shown that a subpopulation of naturally occurring human IgGs has therapeutic potential for the amyloid-associated disorders. These molecules cross-react with conformational epitopes on amyloidogenic assemblies, including amyloid beta (Aβ) protein fibrils that are a pathological hallmark of Alzheimer’s disease. Materials and Methods Using our europium-linked immunosorbant assay, we established that ∼95% of 260 screened donor plasma samples had amyloid fibril-reactive IgGs and Aβ conformer-reactive IgGs with minimal binding to Aβ monomers. Anti-amyloidogenic reactivity was diverse and attributed to Aβ targeting multiple fibril-related binding sites and/or variations in multidentate binding. Results and Discussion There was no correlation between anti-fibril and anti-oligomer reactivity and donor age (19 to 60 years old) or gender. These findings demonstrate the inherent but diverse anti-amyloidogenic activity of natural IgGs contained in normal plasma. Conclusion Our studies provide support for investigating the clinical significance and physiological function of this novel class of antibodies.

Dalla Pozza R, Netz H, Schwarz HP, Bechtold S. Subclinical atherosclerosis in diabetic children: results of a longitudinal study. Context: Cardiovascular disease caused by atherosclerosis is a major cause of morbidity and mortality in adult diabetic patients. In children, we detected signs of subclinical atherosclerosis in a large patient cohort. This study reports the results of a longitudinal observation in this patient group. Patients and methods: Of the 37/150 diabetic children in whom an increased intima-media thickness (IMT) of the carotid artery had been found, 27 (mean age 14.6 ± 2.6 yrs) could be reevaluated 2 yrs after the initial study. Of the 27, 5 patients were on medication with angiotensin-converting enzyme (ACE) inhibitors, and all patients underwent detailed counselling of their lifestyle, sports activity, and nutritional habits. Results: Mean IMT increased significantly (0.49 ± 0.02 mm vs. 0.51 ± 0.026 mm, p < 0.05) However, there was no significant change compared to normal values (mean IMT z-score 2.4 ± 0.3 vs. 2.6 ± 0.5). Of the 27, 13 patients (48%) showed a progression of the IMT whereas in 14/27 patients the IMT values remained stable. In these subgroups, patients with IMT progression showed a higher hemoglobin A1c (HbA1c) (7.5 ± 0.8 vs. 7.1 ± 0.7, p < 0.05) and a slightly higher systolic blood pressure (120 ± 14.4 vs. 113.9 ± 12.1, p = 0.08). Conclusions: In a well-selected group of diabetic children, mean IMT progression during a 2-yr period did not exceed the physiological increase. Children with a higher HbA1c and a higher systolic blood pressure showed a progression of the IMT. Control of atherogenic risk factors in diabetic children may help to avoid subclinical atherosclerosis progression.

Von Willebrand factor (VWF), an adhesive glycoprotein with an approximate molecular weight (MW) of the monomer of 260 kDa, circulates in human blood plasma as a series of multimers ranging in size up to 20.000 kDa; thus the determination of the accurate MW of the monomer is of great importance and due to its high MW quite challenging. In this study accurate MW determination of intact recombinant VWF monomer (rVWF) was performed with GEMMA (gas-phase electrophoretic mobility macromolecular analysis) and MALDI TOF MS (matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry). Three rVWF preparations with differing buffer systems and glycoprotein concentrations were analyzed. First investigations directed towards heterogeneity determination by means of capillary gel electrophoresis (CGE)-on-the-chip with a laser-induced fluorescence detector revealed two compounds (MW of 277 kDa (migration time 44.3 s) and 341 kDa (migration time 49.5 s)) present in each sample to varying extents, namely mature and pro-rVWF. MALDI MS analysis in the linear positive ion mode allowed the detection of mature rVWF with an exact MW of 256.1 kDa (±0.8%) and pro-rVWF with a MW of 349.8 kDa (±0.8%). Two samples containing pro-rVWF in very minor concentration resulted in GEMMA detection of the mature rVWF with a MW of 227.4 kDa (±2.5%), derived from the measured globular size of 10.9 nm. For one sample containing both rVWF species in almost equal concentrations no differentiation of the two species was possible with GEMMA. Due to its lower resolution only a peak representing a mixture of both species at 11.8 nm could be observed, yielding a MW of 298.8 kDa (±1.6%). Copyright © 2010 John Wiley & Sons, Ltd.

Polyspecific human IgG preparations are indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. In addition, intraveneous IgG (IVIG) is used to treat patients with autoimmune and systemic inflammatory diseases. Lectin chromatography on Sambucus nigra agglutinin stood at the cradle of the hypothesis that the anti-inflammatory properties depend on sialylation of the N-glycans in the Fc region of IgG. A detailed analysis of fractions obtained by lectin chromatography revealed that binding of IVIG is essentially mediated by Fab glycosylation. Moreover, experiments with a monoclonal antibody from a human cell line and IVIG Fc fragments indicated that at least two sialic acids in the Fc region of an antibody are required for lectin binding. Such glycoforms contain either two monosialylated glycans or a disialylated glycan and constitute 1% or less of the total human IgG. Arguably this small proportion holds the entire anti-inflammatory potency. A new mass spectrometric quantification method of IgG subclass ratio revealed that the IVIG Fc preparation essentially consists of IgG1. This observation may be relevant when studying the effect of human Fc in murine models of inflammation because mouse IgG subclasses differ substantially in their interaction with receptors.

To evaluate insulin sensitivity in short children born small for gestational age (SGA) treated with growth hormone (GH), and to study the relationship between growth response and insulin levels.In 29 children (16 female, 13 male) who were short and SGA, an oral glucose tolerance test was performed before (mean age, 8.8 years; range, 4.5-14.3 years) and after 1 year of GH treatment (33 microg/kg/day). Insulin sensitivity was calculated with the homeostasis model assessment (HOMA) and the insulin sensitivity index (ISI) of Matsuda.The mean height increased from -3.1 to -2.4 SD. Insulin resistance (ISI5) was seen in 17.2% of children before and in 48.3% (mainly pubertal) children after GH treatment. Insulin sensitivity decreased significantly: ISI fell from 12.2 to 6.1 (P= .02) and HOMA increased from 1.2 to 2.2 (P= .001). Glucose and HbA1c levels did not change significantly. ISI after 1 year did not correlate with height gain, but it did correlate with age (r= -0.469; P= .01) and body mass index (r= -0.52; P= .004).Insulin sensitivity is impaired in some children who are SGA already at baseline and decreases with GH treatment in most of them. Children close to puberty and children who are less underweight have the highest risk to become insulin resistant.

The objective of this study was to identify genes that are differentially expressed in peripheral T cells of patients with MS exacerbation receiving treatment with IVIG. Using microarray analysis, we identified 360 genes that were at least two-fold up- or down-regulated. The expression of four representative genes (PTGER4, CXCL5, IL11 and CASP2) was confirmed by quantitative PCR. Four of the differentially expressed genes encode chemokines (CXCL3, CXCL5, CCL13 and XCL2) that are involved in directing leukocyte migration. We suggest that the modulation of chemokine expression in peripheral T cells contributes to the beneficial activity of IVIG in patients with MS exacerbation.

Background: Steroid 11-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Herein, we describe two novel CYP11B1 mutations (g659_660dupTG, p.M92X; g.4817G>A, p.R453Q) found in a patient diagnosed with classic 11OHD, after presenting with borderline elevated 17-hydroxyprogesterone concentrations in CAH newborn screening. Methods: A novel CYP11B1 variant (p.R453Q) identified in a patient with classic 11OHD was characterized employing a COS7 cell assay and a computational three-dimensional CYP11B1 model. Results: The in vitro expression analysis revealed an almost complete loss of function of p.R453Q. This finding was consistent with the clinical presentation of classic 11OHD as the patient was compound heterozygous for p.R453Q and a nonsense mutation (p.M92X) on the other allele. Inserting the p.R453Q mutation into our CYP11B1 model provided two potential explanations for the almost complete loss of enzyme activity. Firstly, the heme coordination is most likely disturbed. A second possibility could be an altered interaction with the redox partner adrenodoxin. Conclusion: Results indicate that both novel mutations are disease-causing mutations. Proving the pathogenic effect of a missense sequence variation is of particular importance for clinical genetic counseling as this provides essential information on the prediction of recurrence risk and disease severity.

Objective: Type 1 diabetes mellitus (T1DM) is associated with other autoimmune diseases such as celiac disease (CD) and Hashimoto thyroiditis. The aim of this study was to evaluate the screening frequency for CD and thyroid antibodies in a multicentre survey. Methods: The Diabetes Patienten Verlaufsdokumentationssystem (DPV) initiative is based on standardized, prospective, multicentre documentation in children and adolescents with diabetes. Data from 31 104 patients

Human butyrylcholinesterase (hBChE) is a highly glycosylated protein present in human plasma. The enzyme hydrolyses choline esters, for example benzoylcholine, butyrylthiocholine and acetylthiocholine as well as noncholine esters like heroin and aspirin. hBChE is primarily involved in neuronal transmission and is a potential bioscavenger of toxic organophosphates to protect acetylcholinesterase. A prerequisite for the therapeutic use of hBChE is a detailed characterization of this glycoprotein purified from human plasma. In this study, MS/MS could confirm most of the protein backbone, including the N- and the C-terminus. Site-specific analysis of all nine potential N-glycosylation sites revealed mainly mono- and disialylated N-glycans to be present on this glycoprotein. Sialic acids (Neu5Ac) are mainly alpha2,6-linked, however a fraction of the N-glycans contained Neu5Ac also in alpha2,3 linkage. On monosialylated N-glycans, sialic acid is exclusively located on the 3-arm and in alpha2,6 linkage, as verified by 2D-HPLC and exoglycosidase digests of 2-aminopyridine (PA)-labelled N-glycans. This first comprehensive glycoproteomic analysis of the important human plasma glycoprotein BChE did not give any indication of O-glycosylation or any other kind of PTMs as previously postulated.