Production of Native Bispecific Antibodies in Rabbits

BACKGROUND: A natural bispecific antibody, which can be produced by exchanging Fab arms of two IgG4 molecules, was first described in allergic patients receiving therapeutic injections with two distinct allergens. However, no information has been published on the production of natural bispecific antibody in animals. Even more important, establishment of an animal model is a useful approach to investigate and characterize the naturally occurring antibody. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that a natural bispecific antibody can also be generated in New Zealand white rabbits by immunization with synthesized conjugates. These antibodies showed bispecificity to the components that were simultaneously used to immunize the animals. We observed a trend in our test animals that female rabbits exhibited stronger bispecific antibody responses than males. The bispecific antibody was monomeric and primarily belonged to immunoglobulin (Ig) G. Moreover, bispecific antibodies were demonstrated by mixing 2 purified monospecific antibodies in vivo and in vitro. CONCLUSIONS/SIGNIFICANCE: Our results extend the context of natural bispecific antibodies on the basis of bispecific IgG4, and may provide insights into the exploration of native bispecific antibodies in immunological diseases

Allergenicity assessment of genetically modified crops—what makes sense?

GM crops have great potential to improve food quality, increase harvest yields and decrease dependency on certain chemical pesticides. Before entering the market their safety needs to be scrutinized. This includes a detailed analysis of allergenic risks, as the safety of allergic consumers has high priority. However, not all tests currently being applied to assessing allergenicity have a sound scientific basis. Recent events with transgenic crops reveal the fallacy of applying such tests to GM crops

The effectiveness and cost-evaluation of manual therapy and physical therapy in patients with sub-acute and chronic non specific neck pain. Rationale and design of a Randomized Controlled Trial (RCT)

Contains fulltext : 88910.pdf (publisher's version ) (Open Access)BACKGROUND: Manual Therapy applied to patients with non specific neck pain has been investigated several times. In the Netherlands, manual therapy as applied according to the Utrecht School of Manual Therapy (MTU) has not been the subject of a randomized controlled trial. MTU differs in diagnoses and treatment from other forms of manual therapy. METHODS/DESIGN: This is a single blind randomized controlled trial in patients with sub-acute and chronic non specific neck pain. Patients with neck complaints existing for two weeks (minimum) till one year (maximum) will participate in the trial. 180 participants will be recruited in thirteen primary health care centres in the Netherlands.The experimental group will be treated with MTU during a six week period. The control group will be treated with physical therapy (standard care, mainly active exercise therapy), also for a period of six weeks.Primary outcomes are Global Perceived Effect (GPE) and functional status (Neck Disability Index (NDI-DV)). Secondary outcomes are neck pain (Numeric Rating Scale (NRS)), Eurocol, costs and quality of life (SF36). DISCUSSION: This paper presents details on the rationale of MTU, design, methods and operational aspects of the trial. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00713843

Suggested Improvements for the Allergenicity Assessment of Genetically Modified Plants Used in Foods

Genetically modified (GM) plants are increasingly used for food production and industrial applications. As the global population has surpassed 7 billion and per capita consumption rises, food production is challenged by loss of arable land, changing weather patterns, and evolving plant pests and disease. Previous gains in quantity and quality relied on natural or artificial breeding, random mutagenesis, increased pesticide and fertilizer use, and improved farming techniques, all without a formal safety evaluation. However, the direct introduction of novel genes raised questions regarding safety that are being addressed by an evaluation process that considers potential increases in the allergenicity, toxicity, and nutrient availability of foods derived from the GM plants. Opinions vary regarding the adequacy of the assessment, but there is no documented proof of an adverse effect resulting from foods produced from GM plants. This review and opinion discusses current practices and new regulatory demands related to food safety

Analysis of the Effects of Polymorphism on Pollen Profilin Structural Functionality and the Generation of Conformational, T- and B-Cell Epitopes

An extensive polymorphism analysis of pollen profilin, a fundamental regulator of the actin cytoskeleton dynamics, has been performed with a major focus in 3D-folding maintenance, changes in the 2-D structural elements, surface residues involved in ligands-profilin interactions and functionality, and the generation of conformational and lineal B- and T-cell epitopes variability. Our results revealed that while the general fold is conserved among profilins, substantial structural differences were found, particularly affecting the special distribution and length of different 2-D structural elements (i.e. cysteine residues), characteristic loops and coils, and numerous micro-heterogeneities present in fundamental residues directly involved in the interacting motifs, and to some extension these residues nearby to the ligand-interacting areas. Differential changes as result of polymorphism might contribute to generate functional variability among the plethora of profilin isoforms present in the olive pollen from different genetic background (olive cultivars), and between plant species, since biochemical interacting properties and binding affinities to natural ligands may be affected, particularly the interactions with different actin isoforms and phosphoinositides lipids species. Furthermore, conspicuous variability in lineal and conformational epitopes was found between profilins belonging to the same olive cultivar, and among different cultivars as direct implication of sequences polymorphism. The variability of the residues taking part of IgE-binding epitopes might be the final responsible of the differences in cross-reactivity among olive pollen cultivars, among pollen and plant-derived food allergens, as well as between distantly related pollen species, leading to a variable range of allergy reactions among atopic patients. Identification and analysis of commonly shared and specific epitopes in profilin isoforms is essential to gain knowledge about the interacting surface of these epitopes, and for a better understanding of immune responses, helping design and development of rational and effective immunotherapy strategies for the treatment of allergy diseases. [EN]This study was supported by the following European Regional Development Fund co-financed grants: MCINN BFU 2004-00601/BFI, BFU 2008-00629, BFU2011-22779, CICE (Junta de Andalucía) P2010-CVI15767, P2010-AGR6274 and P2011-CVI-7487, and by the coordinated project Spain/Germany MEC HA2004-0094. JCJ-L thanks Spanish CSIC and the European Marie Curie research program for his I3P-BPD-CSIC, and PIOF-GA-2011-301550 grants, respectively.Peer reviewe

Convergent Sets of Data from In Vivo and In Vitro Methods Point to an Active Role of Hsp60 in Chronic Obstructive Pulmonary Disease Pathogenesis

BACKGROUND: It is increasingly clear that some heat shock proteins (Hsps) play a role in inflammation. Here, we report results showing participation of Hsp60 in the pathogenesis of chronic obstructive pulmonary diseases (COPD), as indicated by data from both in vivo and in vitro analyses. METHODS AND RESULTS: Bronchial biopsies from patients with stable COPD, smoker controls with normal lung function, and non-smoker controls were studied. We quantified by immunohistochemistry levels of Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, and HSF-1, along with levels of inflammatory markers. Hsp10, Hsp40, and Hsp60 were increased during progression of disease. We found also a positive correlation between the number of neutrophils and Hsp60 levels. Double-immunostaining showed that Hsp60-positive neutrophils were significantly increased in COPD patients. We then investigated in vitro the effect on Hsp60 expression in bronchial epithelial cells (16HBE) caused by oxidative stress, a hallmark of COPD mucosa, which we induced with H\u2082O\u2082. This stressor determined increased levels of Hsp60 through a gene up-regulation mechanism involving NFkB-p65. Release of Hsp60 in the extracellular medium by the bronchial epithelial cells was also increased after H\u2082O\u2082 treatment in the absence of cell death. CONCLUSIONS: This is the first report clearly pointing to participation of Hsps, particularly Hsp60, in COPD pathogenesis. Hsp60 induction by NFkB-p65 and its release by epithelial cells after oxidative stress can have a role in maintaining inflammation, e.g., by stimulating neutrophils activity. The data open new scenarios that might help in designing efficacious anti-inflammatory therapies centered on Hsp60 and applicable to COP

Allergic sensitization: screening methods

Experimental in silico, in vitro, and rodent models for screening and predicting protein sensitizing potential are discussed, including whether there is evidence of new sensitizations and allergies since the introduction of genetically modified crops in 1996, the importance of linear versus conformational epitopes, and protein families that become allergens. Some common challenges for predicting protein sensitization are addressed: (a) exposure routes; (b) frequency and dose of exposure; (c) dose-response relationships; (d) role of digestion, food processing, and the food matrix; (e) role of infection; (f) role of the gut microbiota; (g) influence of the structure and physicochemical properties of the protein; and (h) the genetic background and physiology of consumers. The consensus view is that sensitization screening models are not yet validated to definitively predict the de novo sensitizing potential of a novel protein. However, they would be extremely useful in the discovery and research phases of understanding the mechanisms of food allergy development, and may prove fruitful to provide information regarding potential allergenicity risk assessment of future products on a case by case basis. These data and findings were presented at a 2012 international symposium in Prague organized by the Protein Allergenicity Technical Committee of the International Life Sciences Institute’s Health and Environmental Sciences Institute

Hyper-IgG4 disease: report and characterisation of a new disease

BACKGROUND: We highlight a chronic inflammatory disease we call 'hyper-IgG4 disease', which has many synonyms depending on the organ involved, the country of origin and the year of the report. It is characterized histologically by a lymphoplasmacytic inflammation with IgG4-positive cells and exuberant fibrosis, which leaves dense fibrosis on resolution. A typical example is idiopathic retroperitoneal fibrosis, but the initial report in 2001 was of sclerosing pancreatitis. METHODS: We report an index case with fever and severe systemic disease. We have also reviewed the histology of 11 further patients with idiopathic retroperitoneal fibrosis for evidence of IgG4-expressing plasma cells, and examined a wide range of other inflammatory conditions and fibrotic diseases as organ-specific controls. We have reviewed the published literature for disease associations with idiopathic, systemic fibrosing conditions and the synonyms: pseudotumour, myofibroblastic tumour, plasma cell granuloma, systemic fibrosis, xanthofibrogranulomatosis, and multifocal fibrosclerosis. RESULTS: Histology from all 12 patients showed, to varying degrees, fibrosis, intense inflammatory cell infiltration with lymphocytes, plasma cells, scattered neutrophils, and sometimes eosinophilic aggregates, with venulitis and obliterative arteritis. The majority of lymphocytes were T cells that expressed CD8 and CD4, with scattered B-cell-rich small lymphoid follicles. In all cases, there was a significant increase in IgG4-positive plasma cells compared with controls. In two cases, biopsies before and after steroid treatment were available, and only scattered plasma cells were seen after treatment, none of them expressing IgG4. Review of the literature shows that although pathology commonly appears confined to one organ, patients can have systemic symptoms and fever. In the active period, there is an acute phase response with a high serum concentration of IgG, and during this phase, there is a rapid clinical response to glucocorticoid steroid treatment. CONCLUSION: We believe that hyper-IgG4 disease is an important condition to recognise, as the diagnosis can be readily verified and the outcome with treatment is very good