Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase

BACKGROUND: Commercially available assays for IgE antibody provide results in international units per milliliter for many allergen extracts, but this is not easily achieved with purified or novel allergens. OBJECTIVE: To develop assays for IgE antibody suitable for purified or novel allergens by using a commercially available immunosorbent. METHODS: Streptavidin coupled to a high-capacity immunosorbent (CAP) was used to bind biotinylated purified allergens from mite (Der p 1 and Der p 2), cat (Fel d 1), and dog (Can f 1). Assays for IgE antibody to these allergens were performed on sera from children (asthma and control) as well as adults with atopic dermatitis. RESULTS: The results were validated by serial dilution of sera with high and low levels of IgE antibody and were quantitated in international units per milliliter by using a standard curve. Values for IgE antibody to Der p 1, Der p 2, and Fel d 1 correlated with values obtained with the allergen extracts (r2 = 0.80, 0.84, and 0.95, respectively; P < .001 in each case). Furthermore, the values for IgE antibody in sera from children with high exposure to mite and cat allergens demonstrated 10-fold higher levels of IgE antibody to Der p 1 and Der p 2 than to Fel d 1 (P < .001). CONCLUSION: The streptavidin immunosorbent technique provides a new method for quantifying IgE antibody to purified proteins. The results provide evidence about the high quantities of IgE antibody to purified inhalant allergens in patients with atopic dermatitis. In addition, the results demonstrate major differences in IgE antibodies specific for mite and cat allergens among children with high exposure to both allergens.     

Hyperoxia-induced DNA damage causes decreased DNA methylation in human lung epithelial-like A549 cells

The effect of hyperoxia on levels of DNA damage and global DNA methylation was examined in lung epithelial-like A549 cells. DNA damage was assessed by the single-cell gel electrophoresis (comet assay) and DNA methylation status by the cytosine extension assays. Cells exposed to ionizing radiation (0, 1, 2, 4, or 8 Gy) showed increasing rates of percentage of DNA in the tail and tail length with increasing radiation dose. When cells were exposed to room air (normoxia) for 1 day and 95% O2 (hyperoxia) for 1, 2, 3, 4, and 5 days, data indicated that hyperoxia caused time-dependent increases in levels of (a) single strand breaks, (b) double strand breaks, and (c) 8-oxoguanine. Decreased DNA methylation also was observed at day 5 of hyperoxic exposure, suggesting that hyperoxia-induced DNA damage can influence patterns of DNA methylation in a lung-derived cell line.     

The relevance of microbial allergens to the IgE antibody repertoire in atopic and nonatopic eczema

BACKGROUND: A propensity to microbial skin infections has been reported in atopic ("high IgE") and nonatopic ("low IgE") forms of eczema. However, the relationship between antimicrobial IgE antibodies and nonatopic disease is unclear. OBJECTIVE: We examined the relevance of microbial allergens to the allergen-specific IgE antibody repertoire in patients with atopic dermatitis. METHODS: Patients with IgE levels of less than 150 IU/mL were stratified according to sensitivity (n = 22) or no sensitivity (n = 27) to 11 common food allergens and aeroallergens. The prevalence and titers of antimicrobial IgE antibodies were compared with those of patients (n = 36) with increased total IgE levels (>150 IU/mL). Skin-derived serum chemokines were also analyzed. RESULTS: Patients with low IgE levels showed decreased disease severity, increased age of onset, a striking female predominance, and a distinct distribution of skin lesions. High titer IgE antibodies (sum of 8 bacterial and fungal allergens = 29.8 +/- 32.6 IU/mL) and multisensitization specific for microbial allergens was characteristic of patients with high IgE levels, with an overall 84% positivity; however, antimicrobial IgE antibodies comprised 3% or less of allergen-specific IgE antibodies. By contrast, antimicrobial IgE antibodies were detected in only 20% of patients with low IgE, and titers were negligible, irrespective of sensitization to common allergens. These patients were monosensitized, and exclusive microbial sensitivity was uncommon (10%). Patients with low IgE with no sensitivity to common allergens had lower levels of serum macrophage inflammatory protein 3alpha compared with their sensitized counterparts. CONCLUSION: Antimicrobial IgE antibodies are uncommon in patients with atopic dermatitis with low IgE levels. CLINICAL IMPLICATIONS: Hypersensitivity to microbial allergens is an unlikely trigger for eczematous eruptions in patients with low IgE levels.     

Assessing the complications and effectiveness of open carpal tunnel release in a tertiary care centre in a developing country

INTRODUCTION

Open surgical release for carpal tunnel syndrome is not devoid of complications and its quantitative assessment with the Boston questionnaire in a developing country had not been conducted, where, lack of facilities and surgical technique can influence the outcome.

PRESENTATION OF CASE

This was a prospective study in which all cases of carpal tunnel syndrome undergoing open release between June 2007 and June 2012 and who returned for follow up were included. Each patient was requested to fill out the Boston questionnaire twice both pre and post op at 3 months. All complications were recorded as well as bio-data of patients and co morbidities. Follow up was at 2 weeks and at 3 months. Those reporting complications at 3 months were further followed up until 6 months. 373 patients were included in the study. Twenty four patients developed complications. Of these, 12 experienced pain resulting from reflex sympathetic dystrophy. Three patients developed wound dehiscence, 2 cases acquired infections, 4 patients developed immediate post-operative haemorrhage and in 3 patients there was late recurrence of median nerve compression. The symptom severity score pre-operatively was 3.30 (±0.60) and it improved to 1.65 (±0.75) post-operatively indicating a significant change (p < 0.0001). The preoperative functional status score was 2.58 (±0.75) and post-op it became 1.60 (±0.80) again implying a good improvement with an effect size of 1.3.

DISCUSSION

All of the complications produced were well managed. The complication incidence was low. The open release procedure produced good improvement in hand function and in decreasing the symptom severity.

CONCLUSION

Conducting open release for carpal tunnel syndrome in a tertiary referral centre in a developing country offers a good outcome.     

Rhodopsin gene codon 106 mutation (Gly-to-Arg) in a Japanese family with autosomal dominant retinitis pigmentosa

PURPOSE: To examine rhodopsin gene mutations in Japanese patients with retinitis pigmentosa. METHODS: We performed a mutational analysis of the rhodopsin gene in 42 patients from 40 families with retinitis pigmentosa. Genomic DNA was amplified by polymerase chain reaction (PCR) and the PCR products were sequenced. Restriction enzyme analysis was performed in family members of 1 patient with a rhodopsin gene mutation (Gly106Arg) and in 100 normal individuals. RESULTS: Among the patients with retinitis pigmentosa, 3 patients in one family had a heterozygous Gly106Arg mutation of the rhodopsin gene. They had night blindness and sectorial retinal dystrophy (predominantly at the inferior fundus) in both eyes. None of the 100 individuals with normal fundi had the Gly106Arg mutation of the rhodopsin gene. CONCLUSION: The Gly106Arg mutation of the rhodopsin gene has been found in Japanese patients with sectorial retinitis pigmentosa.     

Oxidized phospholipids derived from ozone-treated lung surfactant extract reduce macrophage and epithelial cell viability

Ozone is known to be a highly toxic gas present in the urban air which exerts its effect on pulmonary tissue through its facile chemical reactions with target molecules in the airway. One of the first barriers encountered by ozone is epithelial lining fluid which contains pulmonary surfactant rich in glycerophosphocholine lipids. The reaction of ozone with calf lung surfactant extract was found to result in the production of 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (16:0a/9-al-GPCho) as an expected product of the ozonolysis of abundant unsaturated phospholipids containing unsaturated fatty acyl groups with a double bond at carbon-9. This oxidized phospholipid was identified as a biologically active product in that it reduced elicited macrophage viability by necrosis with an ED(50) of 6 microM. Further studies of the biological activity of 16:0a/9-al-GPCho revealed that concentrations from 100 to 200 nM initiated apoptosis in pulmonary epithelial-like A549 cells as assessed by TUNEL staining, nuclear size, and caspase-3 activation with loss of viability indicated by reduction of mitochondrial dehydrogenase activity. The release of IL-8, a neutrophil chemokine, from A549 cells was also stimulated by 50-100 nM 16:0a/9-al-GPCho. Exposure of calf lung surfactant to low levels of ozone (62.5, 125, and 250 ppb) for various time periods from 2 to 48 h in a feedback-regulated ozone exposure chamber resulted in a dose- and time-dependent increase in the formation of 16:0a/9-al-GPCho as measured by a specific and sensitive LC/MS/MS assay. The quantity of this biologically active chain-shortened glycerophosphocholine lipid generated even at 125 ppb ozone for 2-4 h (50-100 nM) was consistent with this product mediating the toxic effects of ozone on cells in contact with surfactant.     

Polymorphism at GSTM1, GSTM3 and GSTT1 gene loci and susceptibility to oral cancer in an Indian population

This study evaluates the influence of genetic polymorphism at GSTM1, GSTM3 and GSTT1 gene loci on oral cancer risk among Indians habituated to the use of, smokeless tobacco, bidi or cigarette. DNA extracted from white blood cells of 297 cancer patients and 450 healthy controls by the proteinase K phenol-chloroform extraction procedure were analyzed by the polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) analyses. Lifetime tobacco exposure was evaluated as a risk factor in relation to the polymorphism at the GST gene loci using logistic regression analysis. There was no significant difference in the distribution of the GSTM3 and GSTT1 genotypes between oral cancer patients and controls. In contrast, a significant 3-fold increase in risk was seen for patients with the GSTM1 null genotype (age adjusted OR = 3.2, 95% CI 2.4-4.3). The impact of the GSTM1 null genotype on oral cancer risk was also analyzed in separate groups of individuals with different tobacco habits. The odds ratio associated with the GSTM1 null genotype was 3.7 (95% CI 2.0-7.1) in tobacco chewers, 3.7 (5% CI 1.3-7.9) in bidi smokers and 5.7 (95% CI 2.0-16.3) in cigarette smokers. Furthermore, increased lifetime exposure to chewing tobacco appeared to be associated with a 2-fold increase in oral cancer risk in GSTM1 null individuals. The results suggest that the GSTM1 null genotype is a risk factor for development of oral cancer among Indian tobacco habitues.     

Genetic diversity of noroviruses and sapoviruses in children with acute sporadic gastroenteritis in New Delhi, India

BACKGROUND: Human caliciviruses (HuCVs) cause gastroenteritis throughout the world. Limited information is available on molecular epidemiology of caliciviruses from developing countries including India. OBJECTIVES: Standardization and evaluation of a two-step multiplex RT-PCR assay for HuCVs and characterization of strains. STUDY DESIGN: Two hundred and twenty-six stool samples were collected from children with acute gastroenteritis (AGE) over a one and half year to study the prevalence and diversity of HuCVs in children with AGE in New Delhi, India. A multiplex two-step RT-PCR using 3 sets of external and 4 sets of internal primers from the RdRp gene was standardized for detection of NoVs and SaVs. Molecular characterization of some HuCV strains was done by sequencing followed by phylogenetic analysis. RESULTS: Fifty-nine HuCVs strains were detected in 54 (24%) of the samples; 5 samples had mixed infections. Of these 59 HuCVs, 36 (61%) were norovirus (34 were GGII; 2 were GGI) and 23 (39%) were sapovirus (22 were GGI; 1 was GGII). Phylogenetic analysis of partial RdRp gene of 12 HuCV strains identified three genotypes (GGI/4, GGII/3 and a newly identified GIIb/Hilversum cluster) in NoVs and one genotype (GGI/1) in SaVs. CONCLUSION: This is one of the few reports from India on detection and characterization of HuCVs by multiplex RT-PCR assay. This assay can be a useful tool for epidemiological studies of HuCV infections.