Major allergens of date palm (Phoenix dactylifera L.) pollen

The IgE-binding components of date palm ( Phoenix dactylifera L.) pollen were determined by ELISA and Western blotting in atopic patients in order to identify its major allergens. From a pool of previously identified allergenic fractions and sera from 15 skin-test-positive, atopic subjects, four components of 12, 14.4, 57, and 65–67 kDa were found to bind IgE in 80–93% of sera. Two other components of molecular masses 28–30 and 37–40 kDa also bound 60–80 % of atopic sera. The immunologic specificity of date-pollen allergen that induced antibody response in sera of atopic patients was confirmed with ELISA. Furthermore, most of the reactivity in pooled positive atopic serum and antiserum raised in rabbits was eliminated after the sera were absorbed with the allergen. IgG immunoblot analyses showed varying degrees of cross-reactivity with common local allergens, notably Bermuda grass, but were generally of low intensity. These results indicate that date pollen has six major allergens with the 12, 14.4, 57, and 65–67 kDa bands binding 80–93%, and the 28–30 and 37–40 kDa bands 60–80% of atopic sera. We propose that these major allergens be assigned the notations Pho d I to Pho d VI in the order listed.     

Accelerating Regulated Bioanalysis for Biotherapeutics: Case Examples Using a Microfluidic Ligand Binding Assay Platform

The Gyrolab? xP is a microfluidic platform for conducting ligand binding assays (LBAs) and is recognized for its utility in discovery bioanalysis. However, few reports have focused on the technology for regulated bioanalysis. This technology has the advantage of low reagent consumption, low sample volume, and automated ligand binding methods. To improve bioanalysis testing timelines and increase the speed at which biotherapeutics are delivered to patients, we evaluated the technology for its potential to deliver high-quality data at reduced testing timelines for regulated bioanalysis. Six LBA methods were validated to support bioanalysis for GLP toxicokinetic or clinical pharmacokinetic studies. Validation, sample analysis, and method transfer are described. In total, approximately 4000 samples have been tested for regulated bioanalysis to support 6 GLP toxicology studies and approximately 1000 samples to support 2 clinical studies. Gyrolab? xP had high run pass rates (≥83%) and high incurred sample reanalysis (ISR) pass rates (>94%). The maximum total error observed across all QC levels for a given assay was <30% for all six LBAs. High instrument response precision (CV ≤5%) was observed across compact discs (CDs), and methods were validated to use a single standard curve across multiple CDs within a Gyrolab? xP run. Reduced bioanalysis timelines were achieved compared to standard manual plate-based methods, and methods were successfully transferred across testing labs, paving the way for this platform for use in late-stage clinical development.     

Dedicated teaching block for undergraduate geriatric medicine improves knowledge

Aim: This paper describes the performance of 5th year medical students in multiple choice question (MCQ) examinations before and after a geriatric medicine teaching block. Methods: A 30‐question MCQ test was administered at the start and a 45‐question one at the end of the course. Results: There was a statistically significant improvement in the MCQ scores from a mean of 62% (SD 10.4) to 75.2% (SD 7.9) (P < 0.001). Total mean scores for the University of California, Los Angeles (UCLA) Geriatrics Knowledge test improved from 65% (SD 10.4) to 73%(SD 11.7) (P < 0.001). Total mean scores for the American Geriatric Society (AGS) Geriatrics Review Syllabus MCQs improved from 59.3% (SD 17.0) to 78.1% (SD 12.1) (P < 0.001). Post‐course, students scored equally well in the new questions, the validated UCLA test and the AGS questions. Conclusion: An undergraduate geriatric medicine clinical teaching block in senior clinical years can increase students' knowledge in geriatric medicine.     

Development of a species-specific colorimetric-PCR assay for detection and species differentiation of Mycobacterium immunogenum and Mycobacterium chelonae and its comparison with quantitative real-time PCR for field metalworking fluids

Mycobacterium immunogenum and Mycobacterium chelonae are closely related species associated with occupational hypersensitivity pneumonitis (HP) and nosocomial infections. There is a need to develop specific and readily adaptable methods for detection and speciation of these agents. Here we report development of a probe-based colorimetric-PCR assay involving heat shock protein (hsp) gene amplification (228bp) and its detection in an ELISA-like reaction. A quantitative format of this assay was developed and validated on metalworking fluids (MWF). The assay showed a minimum detection limit of 10 fg genomic DNA or 1 mycobacterial cell, albeit with variations depending on type and composition of the MWF matrix. When applied to the field MWF samples, the developed assay was found to be comparable to the real-time PCR assay, and allowed direct speciation of MWF mycobacteria without sequencing and/or restriction pattern analysis. In conclusion, the developed colorimetric PCR allows detection and quantification of MWF mycobacteria without culturing and is the first probe-based assay for unambiguous differentiation between the two phylogenetically closely related species, M. immunogenum and M. chelonae. Considering that the assay offers high throughput format involving relatively simpler instrument infrastructure, it has a potential for applications in routine assessment of MWF mycobacteria in diagnostic and industrial laboratories.     

Development of a FRET-based fluorescence aptasensor for the detection of aflatoxin B1 in contaminated food grain samples

The present study aimed to develop an aptamer-based FRET detection strategy for the specific and sensitive detection of AFB1 in contaminated food grains. The study comprises generation of ssDNA aptamers against AFB1 by whole-cell SELEX and their application in a FRET-based platform utilizing graphene oxide (GO) and quantum dots (QDs). The generated aptamers were characterized to determine their specificity and sensitivity using indirect ELISA where AFB1–OVA was used as a coating antigen. Among the aptamers generated, the ATB1 aptamer showed good reactivity and selectivity against AFB1. This aptamer was further characterized to determine its secondary structure and KD value, which was found to be 5.9 kcal mol⁻¹. The characterized aptamers were conjugated onto Cd/Se quantum dots to develop a fluorimetric system for the detection of aflatoxin B1 using a graphene oxide platform. The presence of graphene oxide quenches the fluorescence ability of the quantum dots due to π–π stacking interactions between the aptamer and GO. Upon target addition, the aptamer forms a complex with aflatoxin B1 thereby restoring the fluorescence intensity. The developed assay shows a linear response from 0.002 μg μl⁻¹ to 0.2 μg μl⁻¹ with a detection limit of 0.004 μg μl⁻¹ for the AFB1 standard toxin and showed no cross-reactivity with other closely related mycotoxins. To validate the reliability of the developed method, several field samples spiked with AFB1 were included in this study and the results obtained were cross verified using a standard commercial AFB1 kit. In conclusion, the developed method may find good utility in routine food testing laboratories for risk assessment of AFB1.

The present study aimed to develop an aptamer-based FRET detection strategy for the specific and sensitive detection of AFB1 in contaminated food grains.     

A case of hypersensitivity to soluble and isophane insulins but not to insulin glargine

Insulin is an important agent for the treatment of diabetes mellitus (DM). Allergic reactions to insulin therapy, although rare, have been evident since animal insulin became available for the treatment of DM in 1922. Hypersensitivity to insulin has considerably been reduced with the introduction of human insulin produced by recombinant deoxyribonucleic acid technology. Here, we present a case of Type 2 DM who demonstrated immediate (Type 1) hypersensitivity reaction on the sites of subcutaneous injection of soluble and isophane insulin but insulin glargine was tolerated well and provided good glycemic control.KEY WORDS: Allergic reaction, diabetes mellitus, hypersensitivity, insulin     

Chylomicron particle size and number,factor VII activation and dietary monounsaturated fatty acids

This study evaluated the effects of substituting dietary saturated fatty acids (SFAs) with monounsaturated fatty acids (MUFAs) on postprandial chylomicron (triacylglycerol (TAG), apolipoprotein B-48 (apo B-48) and retinyl ester (RE)), chylomicron particle size and factor VII (FVII) response when subjects were given a standard meal. In a controlled sequential design, 51 healthy young subjects followed an SFA-rich diet (Reference diet) for 8 weeks after which half of the subjects followed a moderate MUFA diet (n=25) and half followed a high MUFA diet (n=26) for 16 weeks. Fasting lipoprotein and lipid measurements were evaluated at baseline and at 8-week intervals during the Reference and MUFA diets. In 25 of the subjects (n=12 moderate MUFA, n=13 high MUFA), postprandial responses to a standard test meal containing RE and 13C-tripalmitin were investigated at the end of the Reference and the MUFA diet periods. Although there were no differences in the postprandial lipid markers (TAG, RE, 13C-TAG) on the two diets, the postprandial apo B-48 response (incremental area under the curve (IAUC)) was reduced by 21% on the moderate MUFA diet (NS) and by 54% on the high MUFA diet (P<0.01). The postprandial peak concentrations of apo B-48 were reduced by 33% on the moderate MUFA diet (P<0.01) and 48% on the high MUFA diet (P<0.001). Fasting values for factor VII activity (FVIIc), activated factor VII (FVIIa) or factor VII antigen (FVIIag) did not differ significantly when subjects were transferred from Reference to MUFA diets. However, the postprandial increases in coagulation FVII activity (FVIIc) were 18% lower and of activated FVII (FVIIa) were 17% lower on the moderate MUFA diet (NS). Postprandial increases in FVIIc and FVIIa were 50% (P<0.05) and 29% (P<0.07) lower on the high MUFA diet and the area under the postprandial FVIIc response curve (AUC) was also lower on the high MUFA diet (P<0.05). Significantly higher ratios of RE:apo B-48 (P<0.001) and 13C-palmitic acid:apo B-48 (P<0.01) during both MUFA diets suggest that the CMs formed carry larger amounts of dietary lipids per particle, reflecting an adaptation to form larger lipid droplets in the enterocyte when increased amounts of dietary MUFAs are fed. Smaller numbers of larger chylomicrons may explain attenuated activation of factor VII during the postprandial state when the background diet is rich in MUFA.