Complete analysis of the B-cell response to a protein antigen, from in vivo germinal centre formation to 3-D modelling of affinity maturation

Somatic hypermutation of immunoglobulin variable region genes occurs within germinal centres (GCs) and is the process responsible for affinity maturation of antibodies during an immune response. Previous studies have focused almost exclusively on the immune response to haptens, which may be unrepresentative of epitopes on protein antigens. In this study, we have exploited a model system that uses transgenic B and CD4<sup>+</sup> T cells specific for hen egg lysozyme (HEL) and a chicken ovalbumin peptide, respectively, to investigate a tightly synchronized immune response to protein antigens of widely differing affinities, thus allowing us to track many facets of the development of an antibody response at the antigen-specific B cell level in an integrated system <i>in</i> <i>vivo</i>. Somatic hypermutation of immunoglobulin variable genes was analysed in clones of transgenic B cells proliferating in individual GCs in response to HEL or the cross-reactive low-affinity antigen, duck egg lysozyme (DEL). Molecular modelling of the antibody–antigen interface demonstrates that recurring mutations in the antigen-binding site, selected in GCs, enhance interactions of the antibody with DEL. The effects of these mutations on affinity maturation are demonstrated by a shift of transgenic serum antibodies towards higher affinity for DEL in DEL-cOVA immunized mice. The results show that B cells with high affinity antigen receptors can revise their specificity by somatic hypermutation and antigen selection in response to a low-affinity, cross-reactive antigen. These observations shed further light on the nature of the immune response to pathogens and autoimmunity and demonstrate the utility of this novel model for studies of the mechanisms of somatic hypermutation

Peripheral neural cell sensitivity to mTHPC-mediated photodynamic therapy in a 3D in vitro model

Background: The effect of photodynamic therapy (PDT) on neural cells is important when tumours are within or adjacent to the nervous system. The purpose of this study was to investigate PDT using the photosensitiser, meta tetrahydroxyphenyl chlorin (mTHPC), on rat neurons and satellite glia, compared with human adenocarcinoma cell (MCF-7).Methods: Fluorescence microscopy confirmed that mTHPC was incorporated into all three cell types. Sensitivity of cells exposed to mTHPC-PDT (0–10 µg ml–1) was determined in a novel 3-dimensional collagen gel culture system. Cell death was quantified using propidium iodide and cell types were distinguished using immunocytochemistry. In some cases, neuron survival was confirmed by measuring subsequent neurite growth in monolayer culture.Results: MCF-7s and satellite glia were significantly more sensitive to PDT than neurons. Importantly, 4 µg ml–1 mTHPC PDT caused no significant neuron death compared with untreated controls but was sufficient to elicit substantial cell death in the other cell types. Initially, treatment reduced neurite length; neurons then extended neurites equivalent to those of untreated controls. The protocol was validated using hypericin (0–3 µg ml–1), which caused neuron death equivalent to other cell types.Conclusion: Neurons in culture can survive mTHPC-PDT under conditions sufficient to kill tumour cells and other nervous system cells

Your space or mine? : Mapping self in time

Peer reviewedPublisher PD

The Role of Individual Variables, Organizational Variables and Moral Intensity Dimensions in Libyan Management Accountants’ Ethical Decision Making

This study investigates the association of a broad set of variables with the ethical decision making of management accountants in Libya. Adopting a cross-sectional methodology, a questionnaire including four different ethical scenarios was used to gather data from 229 participants. For each scenario, ethical decision making was examined in terms of the recognition, judgment and intention stages of Rest’s model. A significant relationship was found between ethical recognition and ethical judgment and also between ethical judgment and ethical intention, but ethical recognition did not significantly predict ethical intention—thus providing support for Rest’s model. Organizational variables, age and educational level yielded few significant results. The lack of significance for codes of ethics might reflect their relative lack of development in Libya, in which case Libyan companies should pay attention to their content and how they are supported, especially in the light of the under-development of the accounting profession in Libya. Few significant results were also found for gender, but where they were found, males showed more ethical characteristics than females. This unusual result reinforces the dangers of gender stereotyping in business. Personal moral philosophy and moral intensity dimensions were generally found to be significant predictors of the three stages of ethical decision making studied. One implication of this is to give more attention to ethics in accounting education, making the connections between accounting practice and (in Libya) Islam. Overall, this study not only adds to the available empirical evidence on factors affecting ethical decision making, notably examining three stages of Rest’s model, but also offers rare insights into the ethical views of practising management accountants and provides a benchmark for future studies of ethical decision making in Muslim majority countries and other parts of the developing world

Stabilisation of the Fc Fragment of Human IgG1 by Engineered Intradomain Disulfide Bonds

We report the stabilization of the human IgG1 Fc fragment by engineered intradomain disulfide bonds. One of these bonds, which connects the N-terminus of the CH3 domain with the F-strand, led to an increase of the melting temperature of this domain by 10°C as compared to the CH3 domain in the context of the wild-type Fc region. Another engineered disulfide bond, which connects the BC loop of the CH3 domain with the D-strand, resulted in an increase of Tm of 5°C. Combined in one molecule, both intradomain disulfide bonds led to an increase of the Tm of about 15°C. All of these mutations had no impact on the thermal stability of the CH2 domain. Importantly, the binding of neonatal Fc receptor was also not influenced by the mutations. Overall, the stabilized CH3 domains described in this report provide an excellent basic scaffold for the engineering of Fc fragments for antigen-binding or other desired additional or improved properties. Additionally, we have introduced the intradomain disulfide bonds into an IgG Fc fragment engineered in C-terminal loops of the CH3 domain for binding to Her2/neu, and observed an increase of the Tm of the CH3 domain for 7.5°C for CysP4, 15.5°C for CysP2 and 19°C for the CysP2 and CysP4 disulfide bonds combined in one molecule

The Longitudinal Relationship Between Satisfaction with Transitional Care and Social and Emotional Quality of Life Among Chronically Ill Adolescents

This study aimed to identify the relationship between satisfaction with transitional care and quality of life of chronically ill adolescents over time. This longitudinal study included adolescents with type I diabetes, juvenile idiopathic arthritis (JIA), and neuromuscular disorders (NMD). At baseline 138 respondents (response rate 31 %) filled in a questionnaire and 188 about 1 year later (response rate 43 %). Analysis of variance showed that adolescents with diabetes reported the highest physical quality of life, followed in order by those with NMD and JIA (p ≤ 0.01). Adolescents with diabetes reported the highest social quality of life, followed in order by those with JIA and NMD (both at p ≤ 0.001). Univariate analyses showed that satisfaction with transitional care at T0 was significantly related to emotional and physical quality of life at T1 (both at p ≤ 0.05). At T1, satisfaction with transitional care was significantly related to the emotional, physical, and social domains of quality of life (all at p ≤ 0.001). Multiple regression analyses revealed that satisfaction with transitional care at T1 was related to emotional (β -0.20; p ≤ 0.05) and social (β -0.35; p ≤ 0.01) quality of life domains over time. This indicates that lower gap scores, which measured differences between 'best care' and 'current care,' are associated with better social and emotional quality of life in this sample of adolescents. Satisfaction with transitional care and social and emotional quality of life are related over time

Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists, statins, and their combination

Background Vascular calcification is an organized process in which vascular smooth muscle cells (VSMCs) are implicated primarily. The purpose of the present study was to assess the effects of calcium antagonists and statins on VSMC calcification in vitro. Methods VSMC calcification was stimulated by incubation in growth medium supplemented with 10 mmol/l β-glycerophosphate, 8 mmol/l CaCl2, 10 mmol/l sodium pyruvate, 1 μmol/l insulin, 50 μg/ml ascorbic acid, and 100 nmol/l dexamethasone (calcification medium). Calcification, proliferation, and apoptosis of VSMCs were quantified. Results Calcium deposition was stimulated dose-dependently by β-glycerophosphate, CaCl2, and ascorbic acid (all P < 0.01). Addition of amlodipine (0.01–1 μmol/l) to the calcification medium did not affect VSMC calcification. However, atorvastatin (2–50 μmol/l) stimulated calcium deposition dose-dependently. Combining treatments stimulated calcification to a degree similar to that observed with atorvastatin alone. Both atorvastatin and amlodipine inhibited VSMC proliferation at the highest concentration used. Only atorvastatin (50 μmol/l) induced considerable apoptosis of VSMCs. Conclusion In vitro calcification of VSMCs is not affected by amlodipine, but is stimulated by atorvastatin at concentrations ≥10 μmol/l, which could contribute to the plaque-stabilizing effect reported for statins