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Biomedical subjects

S Elsayed

Publications and source records attributed to S Elsayed.

At least 19 recordsLinked to original sources

Novel B and T cell epitopes of chicken ovomucoid (Gal d 1) induce T cell secretion of IL-6, IL-13, and IFN-gamma.

BACKGROUND: Chicken ovomucoid (OM, Gal d 1) has an important role in the pathogenesis of IgE-mediated allergic reactions to hen's egg white. OBJECTIVES: The purpose of this study was to clarify the mechanisms of T cell recognition of ovomucoid using intact OM and chemically modified, characterized and homogeneous solid phase synthetic peptides covering the whole molecule. METHODS: Eighteen overlapping peptides were prepared by solid phase F-moc polyamide peptide synthesis (SPPS), characterized and high-pressure liquid chromatography (HPLC) purified. The peptides, together with intact, denatured and oxidized OM, were used to stimulate patient-derived cell cultures for mapping T cell epitopes. Proliferation responses, T cell phenotype and cytokine secretion using peripheral blood mononuclear cells (PBMC) from eight individuals and T cell lines (TCL) derived from six hen's egg-allergic patients, were examined. In addition, intact, denatured, oxidized and deglycosylated OM, as well as the peptides solely or with their keyhole limpet haemocyanin (KLH) complexes, were tested. For locating IgE and IgG B cell epitopes, seven egg-allergic patient sera and three OM-polyclonal sera were used. Healthy non-allergic individuals were included as controls. RESULTS: Seven peptides were recognized by specific IgE, while OM-specific TCL recognized 10 peptides. Six of the OM peptides were commonly recognized both by patient S-IgE and blood-derived TCL. Among those, one novel epitope, peptide OM 61-74, had the ability to bind IgE. Another peptide, OM 101-114, was recognized by IgE and IgG sera, but not by any of the TCLs. In contrast, the peptides OM 41-56, OM 71-84, OM 131-144 and OM 171-186 were exclusively T cell epitopes with no affinity to specific antibodies. Abundant TCL secretion of IFN-gamma, IL-6, IL-4, IL-13, IL-10 and TNF-alpha in response to OM stimulation indicates the contribution of Th2 as well as Th1/Th0 CD4+ cell subsets. For allergic patients moderate amounts of IFN-gamma, IL-13, and high amounts of IL-6, were secreted in response to TCL stimulation by OM peptides. High amounts of IL-6 were secreted in response to all molecular forms of OM (intact-, modified-OM and the peptides 71-84 and 51-64) when TCLs from two non-allergic donors were used. CONCLUSIONS: One novel B cell epitope (OM 61-74) and 10 T cell epitopes have been identified. The most reactive epitopes of the OM molecule comprise the motifs 1-14 to 71-84, the overlapping peptide-pairs OM 121-134 and OM 131-144 and peptides OM 161-174 and 171-186. Peptides OM 1-14 and 171-186 are the only ones capable of inducing IL-4 secretion. Only one peptide (OM 11-24) induces IL-10 secretion. Those peptides recognized as both T and B cell epitopes or only T cell epitopes, have the potential to induce T cell secretion of moderate to high amounts of IL-13, IFN-gamma and particularly IL-6.

Adult↗

Comparison of PCR to histology for the diagnosis of invasive candidiasis in a murine model.

The ability of a PCR method to diagnose invasive candidiasis in comparison to histology was determined using a murine model. One hundred and sixty mice were divided into 16 groups of 10 mice each. One group served as a negative control while 15 groups were infected with Candida albicans. Of the infected groups, 14 (A1-A7 and B1-B7) received fluconazole treatment. Surviving animals of group A1 were killed on day 3, and animals of successive groups were sacrificed every other day thereafter. Fluconazole treatment was stopped in the corresponding B group. All surviving mice were killed on day 43. One hundred and thirty-eight mice were evaluable. Yeast were seen in the kidney specimens of 77 mice; 66/77 of these, plus an additional 7 histologically-negative mice, were positive by PCR. The sensitivity and specificity of the PCR test compared with histology were 85.71% and 88.52%, respectively. In combination with blood cultures, PCR could be a useful diagnostic tool for invasive candidiasis in clinical settings.

Animals↗

Evaluation of the Candigen enzyme-linked immunosorbent assay for quantitative detection of Candida species antigen.

OBJECTIVE: To assess the clinical utility of an enzyme-linked immunosorbent assay method for the quantitative detection of Candida species antigen (Candigen; Biomerica Inc, Newport Beach, Calif) in patients with suspected disseminated candidiasis. METHODS: Specimens of blood or cerebrospinal fluid from 75 patients with suspected disseminated candidiasis were analyzed by the Candigen test. Results were compared with those obtained by culture and polymerase chain reaction (PCR) analysis. RESULTS: Thirty-seven patients had specimens positive for Candida species by either culture or PCR. Of these specimens, 4 were positive by both culture and PCR, 21 were culture positive but PCR negative, and 12 were PCR positive but culture negative. Five specimens were positive by the Candigen test, all of which were PCR positive but culture negative. The sensitivity, specificity, and positive and negative predictive values of the Candigen test compared to culture plus PCR were 13.5%, 100%, 100%, and 54.3%, respectively. Turnaround time for the Candigen test was approximately 3 hours. CONCLUSION: The Candigen test showed excellent specificity and turnaround time, but its poor sensitivity coupled with its inability to provide species information or susceptibility data make its clinical utility questionable.

Adolescent↗

Expression and analysis of recombinant salmon parvalbumin, the major allergen in Atlantic salmon (Salmo salar).

The parvalbumin from white muscle of Atlantic salmon was previously found to be a major allergen, and designated Sal s1. Two distinct cDNAs, 14.1 and 24.1, which comprise the entire parvalbumin-encoding regions, were cloned, revealing transcripts from two different parvalbumin genes. In the present study, the protein-coding regions of these cDNAs were subcloned into an Escherichia coli expression vector (pET-19b). Both proteins were expressed and the generated target proteins were localized in both soluble and insoluble fractions of the expression host. The recombinant products in the soluble fraction were purified using the His tag-purification system and analysed on Western blots with anti-salmon parvalbumin polyclonal rabbit sera and sera from patients allergic to fish. Both recombinant products (His10-14.1 and His10-24.1) reacted positively with salmon parvalbumin-specific immunoglobulin G (IgG) from rabbits, and with specific immunoglobulin E (IgE) from the sera of six fish-allergic patients. The allergenicity of His10-14.1 was confirmed using enzyme-linked immunosorbent assay (ELISA). The 14.1 cDNA of salmon parvalbumin was shown to be the dominant type represented in a muscle cDNA library.

Allergens↗

[Immunoglobulin E, viral antibodies and obstructive lung disease in adults. The relation between antibody level in serum, lung function and non-specific bronchial reactivity].

We have measured immunoglobulin E levels and respiratory virus antibodies and examined their possible role as risk factors for obstructive lung disease in adults. We observed that increased total serum IgE levels were associated with reduced lung function in subjects with obstructive lung disease, but not in asymptomatic subjects. Subjects sensitised to indoor allergens (house dust mites, cats and mould) had reduced lung function and increased, non-specific, bronchial responsiveness compared with individuals who were not sensitised to indoor allergens. Similar relationships were not observed for subjects sensitised to outdoor allergens (birch and timothy). The presence of respiratory virus antibodies was vaguely associated with reduced lung function, but was not related to increased, non-specific, bronchial responsiveness. In adults in this community sensitisation to indoor allergens is a strong predictor of reduced lung function and increased, non-specific, bronchial responsiveness, which are again closely associated with obstructive lung disease.

Adolescent↗

Cloning and characterisation of a group II allergen from the dust mite Tyrophagus putrescentiae.

The complete cDNA encoding a major allergen from the dust mite Tyrophagus putrescentiae, Tyr p 2, has been sequenced and expressed. A degenerate primer was designed to the N-terminal amino acid sequence of the 16-kDa protein. The complete cDNA sequence was achieved by using reverse transcriptase PCR, PCR+1, standard cloning and sequencing techniques. The cDNA of Tyr p 2 is 552 nucleotides in length from the start codon including 126 nucleotides after the stop codon up to the beginning of the poly(A) tail. The leader sequence consists of 15 amino acids. Regarding the predicted amino acid sequence, there are no potential N-glycosylation sites (N-X-S/T). The sequence showed similarity to group II allergens from other mite species, and some regions are completely conserved. To show that the cloned cDNA sequence was coding for an allergen, Tyr p 2 was expressed in Escherichia coli and shown to react with a T. putrescentiae-positive serum pool.

Allergens↗

Effects of beta2 adrenoceptor agonists on T-cell subpopulations.

The aim of the present communication is to determine the effects of beta2 adrenoceptor agonists on growth and cytokine secretion using allergen-specific T cells. Four beta2 adrenoceptor agonists were administered at therapeutically relevant doses (salbutamol 1-2 microM; salmeterol 0.03-0.06 microM; terbutaline 0.56-1.12 microM, and fenoterol 0.7-1.4 microM to: a) Cultures of human peripheral mononuclear cells (PBMC) b) Positively selected CD4+ and CD8+ subsets, c) Allergen-specific T-cell lines (TCL). Drug effects on growth kinetics and the secretion of IL-4, IL-5, INF-gamma and IgE following T-cell stimulation were investigated. Comparing the growth inhibitory effect of the 4 beta2 agonists at 2 different concentrations, using 12 PBMC, 10 CD4+ and CD8+ and 10 TCL cultures, the following patterns were observed: PBMC-, CD4+- and CD8+-cultures: salmeterol, followed by salbutamol and fenoterol, was a more potent inhibitor than terbutaline. In long-term TCL-cultures, salmeterol was the most potent drug, followed by fenoterol. No significant differences were observed between salbutamol and terbutaline. TCL secretion of IL-4 and IL-5 (TH2 cytokines) was also significantly inhibited. In one patient, INF-gamma secretion (TH1/THO cytokine) could be enhanced by drug administration. High IgE secretion, from 1% remaining B cells in one of the patients, following PHA+IL-2 stimulation, could be reduced by the drugs. The results showed that the beta2 agonists could influence T-cell growth and function. The changes regarding cell function were individual and related to T-cell phenotypes secreting TH1/THO or TH2 cytokines. These results suggest that administration of beta2 adrenoceptor agonists could be beneficial, not only for bronchodilation, but also for suppressing the underlying inflammatory process dominated by TH2-like cytokine secretion.

Adrenergic beta-2 Receptor Agonists↗

Allergen content in dust from homes and schools in northern Norway in relation to sensitization and allergy symptoms in schoolchildren.

BACKGROUND: Previous studies have shown a high prevalence of atopic diseases among school children in the community of Sør-Varanger. Moreover, animal dander followed by pollen and house dust mite, were the most common allergens in skin prick tests. OBJECTIVE: To assess the allergen content in homes (living-rooms and mattresses) and classrooms of children living in an arctic area at 70 degrees. The presence of allergens in homes and schools and their relationship to atopy was of particular interest. METHODS: Dust samples from 38 homes and seven schools in northern Norway were collected by vacuum cleaning. The presence of allergens of dog, birch, timothy, Cladosporium herbarum, codfish and hen egg-white was investigated by radio-allergosorbent test (RAST) inhibition and the presence of major allergens of cat Felis domesticus (Fel d I) and house dust mites (HDM) Dermatophagoides pteronyssinus (Der p I) and Dermatophagoides farinae (Der f I) by enzyme-linked immunosorbent assay (ELISA). RESULTS: Mattresses contained significantly more dust per unit area than living-rooms and classrooms. No statistically significant differences in allergen content for dog, birch, timothy. Cladosporium, codfish and hen egg-white were seen between HDM-sensitized and non-atopic children. Most dust samples contained dog allergens with the highest allergenic activity found in living-rooms of those keeping dogs. An increased level of Fel d I was detected in only one of 38 samples from living-rooms (this family kept a cat) and in 25 of 38 samples from mattresses with ranges from 24 to 84 ng/m2. The highest concentrations were found in mattresses of children keeping cats. Increased levels (> or = 25 ng/m2) of Der p I were found only in homes and virtually only in mattresses of HDM-sensitized children. An increased level of Der f I was found in only one case, i.e. in the mattress of an HDM-sensitized child where additionally Der p I and HDMs were demonstrated microscopically. When relating Der p I to HDM-sensitization an odds ratio of more than 16 (95% CI: 1.6-394.3) was found. All extracts from living-rooms included codfish allergens. Low RAST inhibition values were detected for hen egg-white. Cladosporium, birch and timothy pollen in most samples. Furthermore, the study demonstrated that dust from schools was relatively free of allergens. CONCLUSION: Previous findings indicating that the main allergen exposure problem in this geographical area is that of pet allergens were confirmed.

Allergens↗

Cloning of two distinct cDNAs encoding parvalbumin, the major allergen of Atlantic salmon (Salmo salar).

Allergy to fish is common in Northern Europe. Variable reactions to different fish species are usually experienced among fish allergic patients. The allergens of cod fish and particularly the major allergen parvalbumin beta (Gadus callarias) have been extensively studied in Norway. In the present communication, the white muscle parvalbumin was similarly found to be a major allergen in Atlantic salmon (Salmo salar, Sal sl). A purified salmon parvalbumin was obtained by anion exchange chromatography, gel filtration chromatography (GFC) and high-performance liquid chromatography (HPLC) of the muscle extracts. The antigenicity and allergenicity of salmon parvalbumin were confirmed using various immunologic and electrophoretic techniques. The protein is representative for several isoallergens judged by the amino acid (AA) sequence variance at certain sites in the AA sequence of CNBr cleavage peptides. Using sera from patients with cod and salmon allergy Sal sl was demonstrated to be the major allergen of Atlantic salmon, as judged by RAST- and ELISA-inhibitions and crossed radioimmunoelectrophoresis (CRIE) techniques. The protein was also demonstrated to be antigenic by the use of polyclonal cod and salmon antibodies in IgG ELISA and immunoelectrophoretic methods. Cloning of parvalbumin cDNA from Atlantic salmon was performed based on an alignment of parvalbumin AA sequences from other species. A probe was generated by PCR and used for screening a salmon muscle cDNA-library. Subcloning and sequencing of two hybridizing clones revealed transcripts from two different parvalbumin genes. The translated sequences of both clones belong to the beta-lineage of parvalbumins and include the entire coding region.

Allergens↗

Serum eosinophil cationic protein and interleukin-5 in children with bronchial asthma and acute bronchiolitis.

The aim of our study was to evaluate the clinical applicability of serum eosinophil cationic protein (ECP), interleukin-5 (IL-5) and total eosinophil counts in childhood asthma and bronchiolitis. These parameters were measured in 44 children aged 12-84 months with moderate and mild asthma during symptomatic and asymptomatic phases of disease. Fifteen of the patients were included at the time of admission to hospital due to an acute asthmatic attack, and ten of these were also examined one month after discharge. None of the patients were treated with glucocorticoids or cromoglycate at any time during the study. Serum ECP was significantly increased in the children with acute asthma compared to children with stable moderate asthma, stable mild asthma, as well as to controls. There was no difference between the groups with stable asthma or between stable asthma and controls, and there was large overlap between all groups of asthmatics and controls. Detectable levels of circulating IL-5 were demonstrated in eight of 15 children with acute asthma, with significantly higher levels in atopic children, whereas all samples from children with stable asthma and controls were negative. The results suggest that even though serum ECP and IL-5 increases during acute asthmatic attacks, these parameters cannot alone be used to discriminate between different groups of young children with stable asthma, nor between asthmatics and healthy controls. In addition, the same parameters of eosinophil inflammation were examined in serum samples from 25 children aged 1-17 months undergoing their first episode of acute bronchiolitis. Children with acute respiratory syncytial virus (RSV) bronchiolitis had significantly higher levels of serum ECP than those with RSV negative disease, whereas the total eosinophil counts were significantly decreased in all patients with acute bronchiolitis. Serum IL-5 was only detected in two children with acute bronchiolitis. The results suggest that the inflammation in RSV bronchiolitis differs from that induced by other viruses.

Acute Disease↗

Serum house dust mite antibodies: predictor of increased bronchial responsiveness in adults of a community.

The purpose of this study was to determine whether the presence of serum specific immunoglobulin E (IgE) antibodies was associated with increased bronchial responsiveness in adults. We studied cross-sectionally a random community sample of 18-73 year old adults, of whom 83% (n = 489) performed bronchial responsiveness testing as well as serum measurements of five specific IgE antibodies. In the crude data, 39% of those with house dust mite antibodies (n = 18) had a bronchial responsiveness < or = 32 g.L-1 methacholine compared with 19% in subjects without any of the five specific IgE antibodies (n = 453). The corresponding percentages for subjects with timothy antibodies (n = 16) was 25%, birch antibodies (n = 13) 23%, cat antibodies (n = 10) 40% and mould antibodies (n = 2) 50%. When assessing the multivariate relationship between the presence of one specific IgE antibody and degree of bronchial responsiveness we used a semi-proportional hazards model with the response as a 20% fall in forced expiratory volume in one second (FEV1) from pretest value. Covariates included in the model were: gender, age, pretest FEV1, smoking habits, pack-years, season and other specific IgE antibodies than that examined. The presence of house dust mite antibodies was a significant predictor (p < 0.01) of increased bronchial responsiveness in never- and ex-smokers. Indoor allergic sensitization (house dust mite, cat and mould) was a significant predictor of increased bronchial responsiveness, while outdoor allergic sensitization (timothy and birch) was not. Excluding subjects with obstructive lung disease (n = 39) or including the covariate log total serum IgE as a potential confounder yielded the same result. Thus, in this community, indoor allergic sensitization rather than allergic sensitization per se was related to increased bronchial responsiveness after adjusting for other relevant covariates.

Adolescent↗

Specific T cell lines for ovalbumin, ovomucoid, lysozyme and two OA synthetic epitopes, generated from egg allergic patients' PBMC.

BACKGROUND: Proteins of hen egg whites are common ingredients in food and difficult to eliminate. Allergens of egg white induce allergic symptoms among relatively high numbers of patients suffering from food allergy. B cell epitopes to hen egg white major allergens have been reported. Considering that IgE antibody formation is mostly T cell dependent, the study of T cell epitopes is essential for both T cell dependent and independent IgE response. OBJECTIVES: Little information on T cell epitopes recognizing food allergens has been reported. T cell responses to hen egg white allergens and two synthetic OA peptides located at amino acid residues No. 105-122 and 323-339 were investigated. METHODS: Peripheral blood mononuclear cells from hen egg allergic patients were investigated. Various allergens of hen egg white were used for stimulation. Primary proliferation responses were detected followed by the generation of long-term cultures which were examined for their specificity, phenotype, cytokine profile and IgE production. The allergen specific T cell lines were mapped using a panel of 13 synthetic peptides of ovalbumin. RESULTS: Human T cells recognizing ovomucoid, lysozyme and ovalbumin epitope 105-122 are reported for the first time. The cell lines were enriched CD4+/CD8+ T cells (CD2+ > 95%). Ovomucoid and ovalbumin induced IgE synthesis by a small fraction of B cells (1%) present in the ovalbumin and ovomucoid specific T cell lines. CONCLUSIONS: Human T cells recognized several egg white allergens and epitopes within the ovalbumin molecule. Specific IgE was produced in cultures stimulated with ovalbumin and ovomucoid. OA peptides 105-122 and 323-339 have no affinity to the specific IgE of the two patients; an observation which could be of particular interest regarding the mechanisms of peptide-based immunotherapy.

Cell Line↗

Total serum IgE and FEV1 by respiratory symptoms and obstructive lung disease in adults of a Norwegian community.

BACKGROUND: The importance of total serum IgE level on lung function impairment has not been established in a general population. OBJECTIVE: The aim of this cross-sectional community study was to examine the relationship between total serum IgE and level of lung function in adults, and whether this relationship differed by sex, age, smoking habits or by respiratory symptoms and disease status. METHODS: A stratified random sample of 18-73 year old adults from the general population were invited to spirometry and serum analyses of total and specific IgE. Of 1512 subjects invited, 82% met and performed complete examinations. RESULTS: Increasing level of total serum IgE was related to reduced lung function (P < 0.01) given as sex, age, and height standardized residuals of one second forced expiratory volume (SFEV1). Subjects with total serum IgE in the highest vs the lowest tertile had a mean SFEV1 of -0.28, corresponding to age and height adjusted FEV1 differences of 120 and 150 mL in women and men, respectively. The relationship between IgE and lung function impairment did not differ significantly by sex, age or smoking habits. In subjects with obstructive lung disease increasing level of total serum IgE was more negatively associated with lung function level than in subjects with respiratory symptoms alone. No relationship was observed in asymptomatic subjects. This was confirmed in a multiple linear regression analysis adjusting for sex, age, smoking habits and lifetime smoking consumption showing that SFEV1 was predicted by an interaction between total serum IgE level and symptom and disease status (P < 0.01). This interaction remained after excluding subjects (n = 105) having specific IgE antibodies. CONCLUSION: Increasing total serum IgE level was associated with progressively lower lung function in a general adult population after taking other predictors of impaired spirometric lung function into account, though dependent on the subjects' respiratory symptom and disease status. Variation in prevalences of respiratory symptoms and obstructive lung disease in previous examined populations may thus explain conflicting observations of the association between total IgE and airflow impairment.

Adolescent↗

The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4+, CD8+, and CD19+ cell populations derived from allergic and healthy donors.

The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4+ and CD8+ T cells and CD19+ B cells, positively selected by immunomagnetic separation, was investigated. The cells were obtained from allergic patients with moderate serum IgE levels and mild to moderate atopic dermatitis, and healthy controls. The different cell subfractions were stimulated with mitogens or specific allergens, as well as cell supernatants from the lymphoblastoid B- (RPMI 8866) and T-hybridoma (166 A2) cell lines. Proliferative responses of T- and B-cell subsets stimulated with mitogens together with recombinant interleukin-2 (rIL-2) or accessory cells (AC) could be inhibited by DSCG. In allergic individuals, significant allergen-specific stimulation could be observed in the CD8-depleted peripheral blood mononuclear cell (PBMC) fractions. Isolated CD4+ T cells, without AC or IL-2, could also be stimulated with specific allergen, but the responses were rather low. DSCG inhibited, concentration dependently, all allergen-induced responses. Interestingly, only atopic derived CD4+ and CD8+ T cells were stimulated by soluble low-affinity IgE receptor (Fc epsilon RII/sCD23) and IgE binding factor (IgEBF), including IgE enhancing factor, present in culture supernatants from RPMI 8866 and 166 A2, respectively. These responses were also inhibited by DSCG. This was in contrast to the amplifying effect of DSCG on spontaneously proliferating RPMI 8866 and 166 A2 cells, cultured in fresh cRPMI 1640 medium without sCD23 and IgE enhancing factor. Our results show that DSCG delivers an inhibitory signal or signals to PBMC subpopulations expressing Fc epsilon RII/sCD23, either upregulated by phytohemagglutinin in normal and atopic cells, or by allergens or sCD23 in atopic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗

The effect of H1 receptor antagonists on peripheral blood mononuclear cells, adenoid cells and primary cell lines.

This study describes the in vitro effect of three H1 receptor antagonists (dexchlorpheniramine, terfenadine and loratadine) on human peripheral blood mononuclear cells (PBMC, n = 30) from allergic patients and healthy individuals. The three H1 receptor antagonists significantly inhibited antigen/mitogen-induced PBMC proliferation in a concentration-dependent manner. Allergen-specific T-cell responses in allergic individuals were similarly inhibited. The effect of the three drugs was also tested in cultures of mononuclear cells derived from adenoid tissue. The growth kinetics were investigated using spontaneously proliferating cell lines to examine whether the inhibition was caused by general toxicity. Three cell lines, HCT 8 (an ileocaecal adenocarcinoma) RPMI 8866 (B-cell line) and 166 A2 (T hybridoma) were tested. Loratadine (< 0.03 microM) and dexchlorpheniramine (< 0.62 microM) altered the kinetics of HCT 8 and RPMI 8866, respectively. When testing RPMI 8866 and 166 A2, the growth-inhibitory effect of terfenadine and loratadine could be neutralized by addition of cell culture filtrate from RPMI 8866 or 166 A2. These culture filtrates are rich in soluble low-affinity IgE receptor (sCD23) and IgE-binding factor (IgEBF), respectively. Our findings show that the antihistamines investigated display some non-convential in vitro anti-allergic properties possibly not related to their interaction with the H1 receptor. In addition, our results suggest: a) The H1 receptor antagonists used differ in their pattern of cell inhibition; b) The inhibitory effect is completely reversible at low drug concentrations.

Adenoids↗