Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope “tags”

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system''s ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.     

Calcium signalling and pancreatic cell death: apoptosis or necrosis?

Secretagogues, such as cholecystokinin and acetylcholine, utilise a variety of second messengers (inositol trisphosphate, cADPR and nicotinic acid adenine dinucleotide phosphate) to induce specific oscillatory patterns of calcium (Ca(2+)) signals in pancreatic acinar cells. These are tightly controlled in a spatiotemporal manner, and are coupled to mitochondrial metabolism necessary to fuel secretion. When Ca(2+) homeostasis is disrupted by known precipitants of acute pancreatitis, for example, hyperstimulation or non-oxidative ethanol metabolites, Ca(2+) stores (endoplasmic reticulum and acidic pool) become depleted and sustained cytosolic [Ca(2+)] elevations replace transient signals, leading to severe consequences. Sustained mitochondrial depolarisation, possibly via opening of the mitochondrial permeability transition pore (MPTP), elicits cellular ATP depletion that paralyses energy-dependent Ca(2+) pumps causing cytosolic Ca(2+) overload, while digestive enzymes are activated prematurely within the cell; Ca(2+)-dependent cellular necrosis ensues. However, when stress to the acinar cell is milder, for example, by application of the oxidant menadione, release of Ca(2+) from stores leads to oscillatory global waves, associated with partial mitochondrial depolarisation and transient MPTP opening; apoptotic cell death is promoted via the intrinsic pathway, when associated with generation of reactive oxygen species. Apoptosis, induced by menadione or bile acids, is potentiated by inhibition of an endogenous detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), suggesting its importance as a defence mechanism that may influence cell fate.     

Are patients with non-ST elevation myocardial infarction undertreated?

Background

We have previously documented significant differences in orthogonal P wave morphology between patients with and without paroxysmal atrial fibrillation (PAF). However, there exists little data concerning normal P wave morphology. This study was aimed at exploring orthogonal P wave morphology and its variations in healthy subjects.

Methods

120 healthy volunteers were included, evenly distributed in decades from 20–80 years of age; 60 men (age 50+/-17) and 60 women (50+/-16). Six-minute long 12-lead ECG registrations were acquired and transformed into orthogonal leads. Using a previously described P wave triggered P wave signal averaging method we were able to compare similarities and differences in P wave morphologies.

Results

Orthogonal P wave morphology in healthy individuals was predominately positive in Leads X and Y. In Lead Z, one third had negative morphology and two-thirds a biphasic one with a transition from negative to positive. The latter P wave morphology type was significantly more common after the age of 50 (P < 0.01). P wave duration (PWD) increased with age being slightly longer in subjects older than 50 (121+/-13 ms vs. 128+/-12 ms, P < 0.005). Minimal intraindividual variation of P wave morphology was observed.

Conclusion

Changes of signal averaged orthogonal P wave morphology (biphasic signal in Lead Z), earlier reported in PAF patients, are common in healthy subjects and appear predominantly after the age of 50. Subtle age-related prolongation of PWD is unlikely to be sufficient as a sole explanation of this finding that is thought to represent interatrial conduction disturbances. To serve as future reference, P wave morphology parameters of the healthy subjects are provided.     

Skewed distribution of proinflammatory CD4+CD28null T cells in rheumatoid arthritis

Expanded populations of CD4+ T cells lacking the co-stimulatory molecule CD28 (CD4+CD28null T cells) have been reported in several inflammatory disorders. In rheumatoid arthritis, increased frequencies of CD4+CD28null T cells in peripheral blood have previously been associated with extra-articular manifestations and human cytomegalovirus (HCMV) infection, but their presence in and contribution to joint manifestations is not clear. In the present article we investigated the distribution of CD4+CD28null T cells in the synovial membrane, synovial fluid and peripheral blood of RA patients, and analysed the association with erosive disease and anti-citrullinated protein antibodies. CD4+CD28null T cells were infrequent in the synovial membrane and synovial fluid, despite significant frequencies in the circulation. Strikingly, the dominant TCR-Vbeta subsets of CD4+CD28null T cells in peripheral blood were often absent in synovial fluid. CD4+CD28null T cells in blood and synovial fluid showed specificity for HCMV antigens, and their presence was clearly associated with HCMV seropositivity but not with anti-citrullinated protein antibodies in the serum or synovial fluid, nor with erosive disease. Together these data imply a primary role for CD4+CD28null T cells in manifestations elsewhere than in the joints of patients with HCMV-seropositive rheumatoid arthritis.     

Age and Gender-Based Comparison of Nickel Content of Scalp Hair of Edible Oil- and Hydrogenated Oil-Consuming Populations

Nickel concentrations (μ g/g, dry weight) in the scalp hair of vegetable oil– and hydrogenated oil–consuming categories of male and female donors, ages between 1–66 years, were estimated by the atomic absorption method to assess the contribution of nickel as a contaminant in the hydrogenated oil. Comparative estimates of hair Ni content revealed enhanced metal levels for donors consuming hydrogenated oil both for male (29.33 μg/g, dry weight) and female (27.09 μg/g, dry weight) population segments, whereas for oil-consuming donors the corresponding levels were 11.51 μg/g and 13.49 μg/g, respectively. The Ni content of hair of elderly donors consuming hydrogenated oil was found significantly higher than that of younger male/female donors. Hair Ni levels as high as 63.59 μg/g and 68.40 μg/g were estimated for hydrogenated oil–consuming males and females, respectively. The Ni concentrations exhibited strong positive correlation with age for the hydrogenated oil–consuming male (r = 0.713) and female (r = 0.707) categories, whereas negative correlations were found for both oil-consuming categories. The regression relationships linking hair nickel content with the donor age of either sex indicated a negative dependence for oil-consuming donors, whereas strong direct dependence was observed for hydrogenated oil-consuming donors. The overall results evidenced an index of elevated nickel levels in the hair of population segment consuming hydrogenated oil, believed to arise from excessive residual nickel in the hydrogenated oil, present at concentrations surpassing the limit recommended by the World Health Organization for the safe ingestion of nickel in food commodities.     

Multivariate analysis of the selected metals in the hair of cerebral palsy patients versus controls

Seventeen metals were measured in scalp hair samples from cerebral palsy patients (CPPs) and controls. Samples were collected from 95 CPPs and 93 controls. The nitric acid-perchloric acid wet digestion procedure was used for quantification of the selected metals by flame atomic absorption spectrophotometry. The concentrations of Ag, Ca, Cd, Co, Cr, Li, and Mg were significantly higher and those of Cu, Fe, K, Mn, Na, Ni, Pb, and Sb were lower in the hair of CPPs compared with controls. A strong positive correlation was found between Ca and Mg in the hair of controls but not in that of CPPs. Antimony was found significantly negative in terms of its correlation with Co and Cu in CPPs group but not in the controls. Principal component analysis (PCA) of the data extracted seven factors for CPPs and six factors for controls. Cluster analysis (CA) was also used to support the PCA results. The study evidenced some specific source of Mg and Sb in the hair of CPPs.     

The growth and some physiological responses of rice to Cd toxicity as affected by nitrogen form

A hydroponic experiment was conducted to examine the effect of Cd stress on anti-oxidative enzyme activities at heading stage, yield components, root exudation and Cd and N uptake of rice plants grew in different N source i.e. (NH₄)₂SO₄, NH₄NO₃ and Ca(NO₃)₂. The results show that the effect of Cd stress on all measured parameters were N source dependent. Cd stress (1 μM) caused a remarkable reduction in grain yield and shoot biomass, an increase in root exudation, glutathione content, Cd concentration and catalase (CAT) and peroxidase (POD) activities of rice plants. In the plants under the control (without Cd addition) N source had no distinctive effect on the above measured parameters, but the differences among the three N forms in these parameters became significant when plants were exposed to Cd stress. Cd stress significantly increased POD and CAT activities, and gultathione content, with Ca(NO₃)₂-fed plants having the greatest POD and CAT activities and lowest glutathione content, and (NH₄)₂SO₄-fed plants being just opposite. Moreover, organic acid exudation varied also with N form for the Cd-stressed plants. In comparison with other two N forms, (NH₄)₂SO₄,-treated plants had higher grain yield, N concentration and lower Cd concentration in plants. The current results indicated that (NH₄)₂SO₄ is a better fertilizer for use in Cd contaminated soil.     

Nitric oxide/cGMP protects endothelial cells from hypoxia-mediated leakiness

Leakiness of the endothelial bed is attributed to the over-perfusion of the pulmonary bed, which leads to high altitude pulmonary edema (HAPE). Inhalation of nitric oxide has been successfully employed to treat HAPE patients. We hypothesize that nitric oxide intervenes in the permeability of the pulmonary macrovascular endothelial bed to rectify the leaky bed under hypoxia. Our present work explores the underlying mechanism of 'hypoxia-mediated' endothelial malfunction by using human umbilical cord-derived immortalized endothelial cells, ECV-304, and bovine pulmonary artery primary endothelial cells. The leakiness of the endothelial monolayer was increased by two-fold under hypoxia in comparison to cells under normoxia, while optical tweezers-based tethering assays reported a higher membrane tension of endothelial cells under hypoxia. Phalloidin staining demonstrated depolymerization of F-actin stress fibers and highly polarized F-actin patterns in endothelial cells under hypoxia. Nitric oxide, 8-Br-cGMP and sildenafil citrate (phosphodiesterase type 5 inhibitor) led to recovery from hypoxia-induced leakiness of the endothelial monolayers. Results of the present study also suggest that 'hypoxia-induced' cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability under hypoxia. We conclude that nitric oxide-based recovery of hypoxia-induced leakiness of endothelial cells is a cyclic guanosine monophosphate (cGMP)-dependent phenomenon.