Development and evaluation of mouse anti-Ara h 1 and Ara h 3 IgE monoclonal antibodies for advancing peanut allergy research.

Immediate hypersensitivity reactions to peanuts are a considerable public health concern due to the acute and severe IgE mediated reactions. To conduct research on the pathogenesis and therapeutics of peanut allergies, it is imperative to have mouse anti-crude peanut extract (CPE) IgE monoclonal antibodies (mAbs) for both in-vitro and in-vivo assays. Without these tools, it is difficult to advance research in this field. In this study, four hybridomas producing anti-CPE IgE mAbs were developed and the IgE mAbs were validated using immune-blot analysis, Sandwich ELISA, Indirect ELISA, a cell-based assay using RBL-2H3 cells, and footpad type I hypersensitivity reaction studies in mice. The results indicate that two of the four mAbs can be effectively used for both in-vitro and in-vivo peanut allergy studies, as they induce allergic reactions with sensitization alone in mice. These novel anti-Ara h1 and Ara h 3 IgE mAbs, in combination with the detailed protocols outlined in this article, offer valuable guidance for studying acute allergic reactions involving mast cells across various platforms. With some considerations, the IgE mAbs can significantly advance peanut allergy research.

Development of canine C-reactive protein assays.

C-reactive protein (CRP), which is released during tissue damage and inflammation, is a useful nonspecific inflammatory marker in both human and veterinary clinical practice. Veterinarians have often used human CRP assays to analyze samples from canine patients, but cross-reactivities between the species affect assay sensitivity and reliability, leading to inaccurate inflammation assessment. To improve the efficiency of inflammation assessment, we developed a canine CRP detection enzyme-linked immunosorbent assay (ELISA) for quantitative analysis and an immunochromatography assay (ICA) for semiquantitative point-of-care (POC) analysis. The ELISA demonstrated an assay detection limit of 0.5 ng/mL, quantitative linear assay range of 1.6-100 ng/mL, and intra- and inter-assay coefficient of variations of 0.7 to 10.0% and 6.0 to 9.0%, respectively; the recovery rates of samples spiked with purified canine CRP were 105 to 109%, and the parallelism assessments were 82.7 to 104.4%. The correlation between the CRP level results obtained with the ELISA and those of a currently available quantitative POC assay was 0.907 with the regression formula of y = 0.55x + 0.05. In addition, the ICA requires only 5 µL samples and a 10-min assay time, and clearly distinguished positive, weak positive, and negative samples (P < 0.001) at an approximately 5-10 µg/mL cut-off value. The developed canine CRP ELISA and ICA showed reliable assay results and a high correlation with a commercially available POC assay in clinical use. The ICA can be a useful canine CRP screening test for diagnostic purposes in veterinary clinics.

A double-blind placebo-controlled cross-over clinical trial of DONepezil In Posterior cortical atrophy due to underlying Alzheimer's Disease: DONIPAD study.

BACKGROUND: The study investigated whether donepezil exerts symptomatic benefit in patients with posterior cortical atrophy (PCA), an atypical variant of Alzheimer's disease. METHODS: A single-centre, double-blind, placebo-controlled, cross-over clinical trial was performed to assess the efficacy of donepezil in patients with PCA. Each patient received either donepezil (5 mg once daily in the first 6 weeks and 10 mg once daily in the second 6 weeks) or placebo for 12 weeks. After a 2-week washout period, each patient received the other treatment arm during the following 12 weeks followed by another 2-week washout period. The primary outcome was the Mini-Mental State Examination (MMSE) at 12 weeks. Secondary outcome measures were five neuropsychological tests reflecting parieto-occipital function. Intention-to-treat analysis was used. For each outcome measure, carry-over effects were first assessed. If present, then analysis was restricted to the first 12-week period. Otherwise, the standard approach to the analysis of a 2 × 2 cross-over trial was used. RESULTS: Eighteen patients (13 females) were recruited (mean age 61.6 years). There was a protocol violation in one patient, who subsequently withdrew from the study due to gastrointestinal side effects. There was statistically significant (p < 0.05) evidence of a carry-over effect on MMSE. Therefore, the analysis of treatment effect on MMSE was restricted to the first 12-week period. Treatment effect at 6 weeks was statistically significant (difference = 2.5 in favour of donepezil, 95% CI 0.1 to 5.0, p < 0.05). Treatment effect at 12 weeks was close, but not statistically significant (difference = 2.0 in favour of donepezil, 95% CI -0.1 to 4.5, p > 0.05). There were no statistically significant treatment effects on any of the five neuropsychological tests, except for digit span at 12 weeks (higher by 0.5 digits in favour of placebo, 95% CI 0.1 to 0.9). Gastrointestinal side effects occurred most frequently, affecting 13/18 subjects (72%), and were the cause of study discontinuation in one subject. Nightmares and vivid dreams occurred in 8/18 subjects (44%), and were statistically more frequent during treatment with donepezil. CONCLUSIONS: In this small study, there was no statistically significant treatment effect of donepezil on the primary outcome measure (MMSE score at 12 weeks) in PCA patients, who appear to be particularly susceptible to the development of nightmares and vivid dreams when treated. TRIAL REGISTRATION: Trial registration: Current Controlled Trials ISRCTN22636071 . Retrospectively registered 19 May 2010.

Preventing intense false positive and negative reactions attributed to the principle of ELISA to re-investigate antibody studies in autoimmune diseases.

To study the possible involvement of potential environmental pathogens in the pathogenesis of autoimmune diseases, it is essential to investigate antibody responses to a variety of environmental agents and autologous components. However, none of the conventional ELISA buffers can prevent the false positive and negative reactions attributed to its principal, which utilizes the high binding affinity of proteins to plastic surfaces. The aims of this study are to reveal all types of non-specific reactions associated with conventional buffer systems, and to re-investigate antibody responses to potential environmental pathogenic and autologous antigens in patients with autoimmune diseases using a newly developed buffer system "ChonBlock™" by ELISA. Compared to conventional buffers, the new buffer was highly effective in reducing the most intense false positive reaction caused by hydrophobic binding of immunoglobulin in sample specimens to plastic surfaces, "background (BG) noise reaction", and other non-specific reactions without interfering with antigen-antibody reactions. Applying this buffer, we found that IgG antibody responses to Escherichia coli O111:B4, E. coli lipopolysaccharide (LPS) and peptidoglycan polysaccharide (PG-PS) were significantly lower or tended to be lower in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), whereas IgA antibody responses to these antigens were equal or tended to be higher compared to normal controls. As a consequence, the IgA/IgG antibody ratios against these agents were significantly higher in patients with RA and SLE, except for Crohn's disease, which showed significantly higher IgG responses to these antigens. To assay antibodies in human sera, it is indispensable to eliminate false positive and negative reactions by using an appropriate buffer system, and to include antigen non-coated blank wells to determine BG noise reactions of invidual samples. Finally, based on our preliminary analysis in this study, we propose that low IgG antibody responses to potential pathogenic environmental factors may be the fundamental disorder in autoimmune diseases.

Involuntary admissions under the Mental Health Act 2007 (New South Wales): a comparison of patients detained by ambulance officers, medical practitioners and accredited persons in an emergency department.

OBJECTIVE: The Mental Health Act (MHA) 2007 (New South Wales [NSW]) is used to transport to, detain and treat individuals in a mental health facility. Patients are frequently taken to EDs under the MHA or detained in the ED for a mental health assessment. In NSW, medical practitioners, accredited persons, police and more recently ambulance officers, are authorised to write certificates under the MHA. There is an absence of research on the use of the MHA in NSW EDs. The primary research question for this study was: 'What is the involuntary admission rate for persons detained under the MHA in an NSW tertiary referral ED?' METHODS: After gaining ethics approval, the research team prospectively collected the MHA certificates written for patients in the ED over a 3 month period from February to May 2011. The research team reviewed the MHA certificate and medical records to characterise demographics, outcome, length of stay in ED and certificate suitability. Outcomes of interest were involuntary psychiatric admission or any inpatient admission, and were compared between the different professional groups completing the certificate. RESULTS: The involuntary admission rate for patients certified by ambulance officers under the NSW MHA was 27%, compared with a 60% involuntary admission rate when certified by medical practitioners or accredited persons. The mean average length of stay in the ED for patients certified under the MHA was 10 h. Seventy-five per cent of patients presented out of hours. CONCLUSION: Ambulance officer certification was a poor predictor of involuntary psychiatric admission in the Royal Prince Alfred Hospital Emergency Department. ED physicians should have the authority to revoke the certificate if, following assessment and treatment, the patient no longer fulfils criteria for detention under the MHA.

Symptoms of memory loss as predictors of cognitive impairment?: the use and reliability of memory ratings in a clinic population.

BACKGROUND: Symptoms of memory loss are very common with estimated prevalence between 22% and 50% in those older than 65 years of age. Those with symptoms of memory loss and impaired performance on memory tests are at high risk of progression to Alzheimer disease. The relative importance of different aspects of the clinical history in predicting cognitive impairment is uncertain. METHODS: Fifty-six patients with symptoms of memory loss were recruited from the National Hospital for Neurology and Neurosurgery. A clinician recorded type and duration of memory symptoms as perceived by the patient and their informant, and use of memory aids. All patients subsequently underwent magnetic resonance imaging (MRI) and neuropsychologic testing. FINDINGS: (i) Informant, but not patient, ratings of memory were associated with performance on tests of memory function and with hippocampal size on MRI. (ii) Decreased use of memory aids and shorter duration of memory symptoms were more common in those with memory impairment. INTERPRETATION: In a clinical setting, information gathered from the history was associated with cognitive impairment on memory testing and brain appearances on MRI. The history from a close informant is particularly important being more strongly predictive of cognitive impairment (P=0.0002) than subjective symptoms, use of memory aids or duration of symptoms.