Mapping the idiotopes of a monoclonal anti-myoglobin antibody with syngeneic monoclonal anti-idiotypic antibodies: detection of a common idiotope

A panel of syngeneic monoclonal anti-idiotypic antibodies was prepared by immunizing A.SW mice with keyhole limpet hemocyanin-coupled A.SW monoclonal anti-myoglobin (HAL 19, IgG1) and screening the cloned hybridomas for production of IgG2 binding to idiotype but not to certain other anti-myoglobin antibodies of the same subclass in an ELISA. With these antibodies, we identified three nonoverlapping idiotopes, based on three clusters of monoclonal anti-idiotopes that mutually inhibit within each cluster, but not between clusters (Cluster I: S2, S6, S8; Cluster II: S5, S7; Cluster III: S9). Only Cluster II antibodies block the binding of myoglobin to HAL 19 and so identify a binding site-related idiotope(s). Binding of both Cluster II monoclonals (S5 and S7) to Hal 19 is inhibited by a rabbit anti-idiotype that we previously reported detects a common cross-reactive anti-myoglobin idiotope in immune sera. However, only one of these, S7, and not S5, identifies an idiotope that is present on 20 to 30% of A.SW anti-myoglobin antibodies in immune sera and ascites. The panel of syngeneic monoclonal anti-idiotype antibodies also detects new idiotopes not detected by the rabbit anti-idiotype. The development of a panel of syngeneic monoclonal anti-idiotypic antibodies to different clusters of idiotopes on the same antibody molecule, including one that identifies a major common idiotope in immune sera, should allow the analysis of possible idiotype network regulation in vivo and in vitro in a completely syngeneic system.     

Functional and Structural Properties of a Novel Protein and Virulence Factor (Protein sHIP) in Streptococcus pyogenes

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.     

<Emphasis Type="Italic">TRAF1/C5</Emphasis>polymorphism is not associated with increased mortality in rheumatoid arthritis: two large longitudinal studies

Introduction  

Recently an association between a genetic variation in TRAF1/C5 and mortality from sepsis or cancer was found in rheumatoid arthritis (RA). The most prevalent cause of death, cardiovascular disease, may have been missed in that study, since patients were enrolled at an advanced disease stage. Therefore, we used an inception cohort of RA patients to investigate the association between TRAF1/C5 and cardiovascular mortality, and replicate the findings on all-cause mortality. As TRAF1/C5 associated mortality may not be restricted to RA, we also studied a large cohort of non-RA patients.     

Body temperature patterns and rhythmicity in free-ranging subterranean Damaraland mole-rats, Fukomys damarensis

Body temperature (T(b)) is an important physiological component that affects endotherms from the cellular to whole organism level, but measurements of T(b) in the field have been noticeably skewed towards heterothermic species and seasonal comparisons are largely lacking. Thus, we investigated patterns of T(b) patterns in a homeothermic, free-ranging small mammal, the Damaraland mole-rat (Fukomys damarensis) during both the summer and winter. Variation in T(b) was significantly greater during winter than summer, and greater among males than females. Interestingly, body mass had only a small effect on variation in T(b) and there was no consistent pattern relating ambient temperature to variation in T(b). Generally speaking, it appears that variation in T(b) patterns varies between seasons in much the same way as in heterothermic species, just to a lesser degree. Both cosinor analysis and Fast Fourier Transform analysis revealed substantial individual variation in T(b) rhythms, even within a single colony. Some individuals had no T(b) rhythms, while others appeared to exhibit multiple rhythms. These data corroborate previous laboratory work showing multiplicity of rhythms in mole-rats and suggest the variation seen in the laboratory is a true indicator of the variation seen in the wild.     

Inhibition of the interaction between protein phosphatase 1 glycogen-targeting subunit and glycogen phosphorylase increases glycogen synthesis in primary rat hepatocytes

In Type 2 diabetes, increased glycogenolysis contributes to the hyperglycaemic state, therefore the inhibition of GP (glycogen phosphorylase), a key glycogenolytic enzyme, is one of the possibilities to lower plasma glucose levels. Following this strategy, a number of GPis (GP inhibitors) have been described. However, certain critical issues are associated with their mode of action, e.g. an impairment of muscle function. The interaction between GP and the liver glycogen targeting subunit (termed G(L)) of PP1 (protein phosphatase 1) has emerged as a new potential anti-diabetic target, as the disruption of this interaction should increase glycogen synthesis, potentially providing an alternative approach to counteract the enhanced glycogenolysis without inhibiting GP activity. We identified an inhibitor of the G(L)-GP interaction (termed G(L)-GPi) and characterized its mechanism of action in comparison with direct GPis. In primary rat hepatocytes, at elevated glucose levels, the G(L)-GPi increased glycogen synthesis similarly to direct GPis. Direct GPis significantly reduced the cellular GP activity, caused a dephosphorylation of the enzyme and decreased the amounts of GP in the glycogen-enriched fraction; the G(L)-GPi did not influence any of these parameters. Both mechanisms increased glycogen accumulation at elevated glucose levels. However, at low glucose levels, only direct GPis led to increased glycogen amounts, whereas the G(L)-GPi allowed the mobilization of glycogen because it did not block the activity of GP. Due to this characteristic, G(L)-GPi in comparison with GPis could offer an advantageous risk/benefit profile circumventing the potential downsides of a complete prevention of glycogen breakdown while retaining glucose-lowering efficacy, suggesting that inhibition of the G(L)-GP interaction may provide an attractive novel approach for rebalancing the disturbed glycogen metabolism in diabetic patients.     

Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.     

The use of sparse CT datasets for auto-generating accurate FE models of the femur and pelvis

The finite element (FE) method when coupled with computed tomography (CT) is a powerful tool in orthopaedic biomechanics. However, substantial data is required for patient-specific modelling. Here we present a new method for generating a FE model with a minimum amount of patient data. Our method uses high order cubic Hermite basis functions for mesh generation and least-square fits the mesh to the dataset. We have tested our method on seven patient data sets obtained from CT assisted osteodensitometry of the proximal femur. Using only 12 CT slices we generated smooth and accurate meshes of the proximal femur with a geometric root mean square (RMS) error of less than 1 mm and peak errors less than 8 mm. To model the complex geometry of the pelvis we developed a hybrid method which supplements sparse patient data with data from the visible human data set. We tested this method on three patient data sets, generating FE meshes of the pelvis using only 10 CT slices with an overall RMS error less than 3 mm. Although we have peak errors about 12 mm in these meshes, they occur relatively far from the region of interest (the acetabulum) and will have minimal effects on the performance of the model. Considering that linear meshes usually require about 70-100 pelvic CT slices (in axial mode) to generate FE models, our method has brought a significant data reduction to the automatic mesh generation step. The method, that is fully automated except for a semi-automatic bone/tissue boundary extraction part, will bring the benefits of FE methods to the clinical environment with much reduced radiation risks and data requirement.     

Monitoring of residues of ochratoxin A in blood and kidney of chicken using HPLC-FLD

Detoxification of ochratoxin A can be achieved by chemical or enzymatic hydrolyzation, the products of such reactions are ochratoxin α and phenylalanine. Ochratoxin α like ochratoxin A, is a fluorescing molecule, therefore sensitive analysis is possible at very low concentration levels. Methods have been established that make it possible to look for residues of ochratoxin A and its main metabolite ochratoxin α in blood and tissues at very low concentration levels. Plasma is extracted by the use of small amounts of chloroform; the extract is cleaned with water and afterwards evaporated to dryness]. The residue is re-dissolved and analysed by HPLC-FLD. Using this method a limit of detection of 0.5μg/l for both ochratoxin A and ochratoxin α can be reached.     

Prospective cohort studies on risk factors for cardiovascular events in systemic lupus erythematosus: a major challenge

Cardiovascular disease (CVD) has been identified as a major contributor to morbidity and mortality in patients with systemic lupus erythematosus (SLE). The etiology of premature CVD in SLE is supposed to have many factors, including traditional coronary artery disease (CAD) risk factors, antiphospholipid antibodies, and metabolic and inflammatory factors. Despite the overwhelming interest in CVD in SLE research, prospective studies evaluating risk factors for hard endpoints (that is, cardiovascular events) are relatively scarce. The article by Gustafsson and colleagues suggests that prothrombotic factors play an important role in SLE-related CVD and that the influence of traditional CAD risk factors might be limited.     

En route to deoxygenated N-acetyllactosamine analogues employing uridyl and galactosyl transferases

All monodeoxygenated galactoses were treated with galactokinase, and for the 2-, 3-, and 4-deoxy compounds, transformation into the corresponding galactopyranosyl phosphates could be observed. In case of the 2-deoxy derivative, further reaction via UDP-2-deoxy-d-lyxo-hexose (UDP-2-deoxygalactose), which was also obtained chemically, the multiple enzymatic system could be employed to prepare 2′-deoxy-N-acetyllactosamine.