Biomedical subjects

Randall M Goldblum

Publications and source records attributed to Randall M Goldblum.

14 recordsLinked to original sources

PubMed2006

Estradiol activates mast cells via a non-genomic estrogen receptor-alpha and calcium influx.

BACKGROUND: Allergic airway diseases are more common in females than in males during early adulthood. A relationship between female hormones and asthma prevalence and severity has been suggested, but the cellular and molecular mechanisms are not understood. OBJECTIVE: To elucidate the mechanism(s) by which estrogens enhance the synthesis and release of mediators of acute hypersensitivity. METHODS: Two mast cell/basophil cell lines (RBL-2H3 and HMC-1) and primary cultures of bone marrow derived mast cells, all of which naturally express estrogen receptor-alpha, were examined. Cells were incubated with physiological concentrations of 17-beta-estradiol with and without IgE and allergens. Intracellular Ca(2+) concentrations and the release of beta-hexosaminidase and leukotriene C(4) were quantified. RESULTS: Estradiol alone induced partial release of the preformed, granular protein beta-hexosaminidase from RBL-2H3, BMMC and HMC-1, but not from BMMC derived from estrogen receptor-alpha knock-out mice. The newly synthesized LTC(4) was also released from RBL-2H3. Estradiol also enhanced IgE-induced degranulation and potentiated LTC(4) production. Intracellular Ca(2+) concentration increased prior to and in parallel with mediator release. Estrogen receptor antagonists or Ca(2+) chelation inhibited these estrogenic effects. CONCLUSION: Binding of physiological concentrations of estradiol to a membrane estrogen receptor-alpha initiates a rapid onset and progressive influx of extracellular Ca(2+), which supports the synthesis and release of allergic mediators. Estradiol also enhances IgE-dependent mast cell activation, resulting in a shift of the allergen dose response.

Allergens↗

PubMed2006

Major mountain cedar allergen, Jun a 1, contains conformational as well as linear IgE epitopes.

We have previously identified four linear IgE epitopes on Jun a 1, the dominant allergen in mountain cedar pollen, and mapped these to the surfaces of a molecular model and to the crystal structure of this glycoprotein. The aim of the present study was to determine if Jun a 1 also displays conformational IgE epitopes. Jun a 1 was denatured by heating at 75 degrees C for 1h, exposure to 6M guanidine or by reductive alkylation in the presence and absence of guanidine. The effects of these manipulations on the binding to IgE from patients with mountain cedar hypersensitivity was evaluated by an ImmunoCAP inhibition assay, using Jun a 1-specific caps. Treatment-associated changes in the 3D-structure were assessed by dynamic light scattering and CD spectroscopy. IgE binding to native Jun a 1 was inhibited 92+/-9% by soluble native protein and 92+/-9% by reduced and alkylated Jun a 1. However, the capacity of Jun a 1 to inhibit the binding of IgE antibodies was significantly diminished upon denaturation by heat, guanidine alone, or reduction and alkylation in guanidine, compared to native Jun a 1. Reductive alkylation treatment under denaturing conditions also increased the Stoke's radius, suggesting that the protein was partially unfolded. Analysis of the circular dichroism (CD) spectra suggested that heating and treatment with guanidine caused a loss of alpha-helical structure. Guanidine also caused an increase in random coil structure. Thus, at least a portion of the anti-Jun a 1 IgE antibodies produced by allergic humans recognize conformational epitopes and it is likely that some of these epitopes reside in alpha-helical structures of Jun a 1.

Allergens↗

PubMed2006

Identification of italian cypress (Cupressus sempervirens) pollen allergen Cup s 3 using homology and cross-reactivity.

BACKGROUND: The prevalence of seasonal allergic diseases of the upper airways is increasing in industrialized countries. The Cupressaceae are important causes of pollinosis, particularly in Europe. OBJECTIVE: To determine whether the pollen from Cupressus sempervirens (Italian cypress) contains a pathogenesis-related group 5 (PR-5) protein, similar to that found in other allergenic Cupressaceae pollens. METHODS: Messenger RNA was purified from Italian cypress pollen, and complementary DNA (cDNA) was synthesized. cDNAs for PR-5 proteins were amplified by polymerase chain reaction and extended by rapid amplification of cDNA ends methods. Recombinant Cup s 3 was expressed in Escherichia coli as a fusion protein. Inhibition enzyme-linked immunosorbent assays were used to test the allergenicity of Cup s 3. RESULTS: Three cDNAs were cloned. These clones had approximately 95% identity to Jun a 3 and Cup a 3. Recombinant Cup s 3.0102 maltose-binding protein inhibited the IgE from most patients from binding to an extract of Italian cypress. The extent of inhibition suggested that antibodies to Cup s 3 were a prominent component of the IgE response to Italian cypress pollen. CONCLUSION: Cup s 3, an allergen of Italian cypress pollen, was identified based on cross-reactivity and homology with other pollen PR-5 proteins, despite an apparently low level of protein expression. Variations in the content of Cup s 3 in the pollen from different regions or trees should be considered in the choice of extracts for diagnosis and specific immunotherapy for Italian cypress pollen hypersensitivity.

Adult↗

PubMed2006

Effect of sodium sulfite on mast cell degranulation and oxidant stress.

BACKGROUND: Sulfur dioxide is 1 of 6 environmental pollutants monitored by the Environmental Protection Agency. Its ability to induce bronchoconstriction is well documented. It is highly soluble, initially forming sulfite ions in solution. Sulfur oxides are important constituents of other pollutants, such as diesel exhaust and fine particulates. OBJECTIVE: To investigate the cellular responses of sulfite on cultured mast cells (rat basophilic leukemia [RBL-2H3] cells) and human peripheral blood basophils. METHODS: Sulfite-induced mast cell degranulation and intracellular production of reactive oxygen species were evaluated in the presence and absence of antioxidants and inhibitors of redox metabolism. Degranulation was determined using beta-hexosaminidase, serotonin, and histamine release assays. Induction of intracellular reactive oxygen species generation was determined using the redox-sensitive dye 2',7'-dichlorofluorescein diacetate. RESULTS: Sodium sulfite induced degranulation and the generation of intracellular reactive oxygen species in RBL-2H3 cells. These responses were inhibited by the free radical scavenger tetramethylthiourea and the flavoenzyme inhibitor diphenyliodinium but not by depletion of extracellular calcium. Peripheral blood basophils also showed histamine release after exposure to sodium sulfite CONCLUSIONS: Sulfite, the aqueous ion of sulfur dioxide, induces cellular activation, leading to degranulation in mast cells through a non-IgE-dependent pathway. The response also differs from IgE-mediated degranulation in that it is insensitive to the influx of extracellular calcium. The putative pathway seems to rely on activation of the reduced form of nicotinamide adenine dinucleotide phosphate oxidase complex, leading to intracellular oxidative stress.

Animals↗

PubMed2005

Interleukin-18 primes human basophilic KU812 cells for higher leukotriene synthesis.

The human basophilic cell line KU812 that is an established tool for studying the function of human basophils, is differentiated into mature basophils by interleukin (IL-3) or other agents. However, whether leukotrienes (LTs)-synthesis is affected by cytokines in KU812 cells remains unknown. KU812 cells were incubated with IL-3, IL-4, IL-6, IL-13 or IL-18 for up to 14 days. The A23187 stimulated- and IgE cross-linked-synthesis of LTC(4) and LTB(4) were measured using an enzyme immunoassay (EIA). The expression of messenger RNA (mRNA) for LT-synthesizing enzymes was examined by reverse transcriptase polymerase chain reaction (RT-PCR), and the expression of 5-lipoxygenase (5-LO) was examined by immunostaining. Incubation with IL-3 (10 ng/ml) and IL-18 (10 ng/ml) induced the expression of 5-LO. A23187stimulated LT-synthesis and IgE cross-linked LT-synthesis were enhanced after incubation with IL-3 or IL-18. These results indicated that IL-3 and IL-18 primed human basophils for higher LT-synthesis. Thus, both IL-3 and IL-18 might be important factors for regulating LT-synthesis during the differentiation of human basophils.

5-Lipoxygenase-Activating Proteins↗

PubMed2005

Pyelonephritic Escherichia coli expressing P fimbriae decrease immune response of the mouse kidney.

P fimbriae are proteinaceous appendages on the surface of Escherichia coli bacteria that mediate adherence to uroepithelial cells. E. coli that express P fimbriae account for the majority of ascending urinary tract infections in women with normal urinary tracts. The hypothesis that P fimbriae on uropathic E. coli attach to renal epithelia and may regulate the immune response to establish infection was investigated. The polymeric Ig receptor (pIgR), produced by renal epithelia, transports IgA into the urinary space. Kidney pIgR and urine IgA levels were analyzed in a mouse model of ascending pyelonephritis, using E. coli with (P+) and without (P-) P fimbriae, to determine whether P(+) E. coli regulate epithelial pIgR expression and IgA transport into the urine. (P+) E. coli establish infection and persist to a greater amount than P(-) E. coli. P(+)-infected mice downregulate pIgR mRNA and protein levels compared with P(-)-infected or PBS controls at > or =48 h. The decrease in pIgR was associated with decreased urinary IgA levels in the P(+)-infected group at 48 h. pIgR mRNA and protein also decline in P(+) E. coli-infected LPS-hyporesponsive mice. These studies identify a novel virulence mechanism of E. coli that express P fimbriae. It is proposed that P fimbriae decrease pIgR expression in the kidney and consequently decrease IgA transport into the urinary space. This may explain, in part, how E. coli that bear P fimbriae exploit the immune system of human hosts to establish ascending pyelonephritis.

Animals↗

PubMed2005

Structural basis for epitope sharing between group 1 allergens of cedar pollen.

The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved beta-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development.

Adolescent↗

PubMed2005

ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation.

Pollen exposure induces allergic airway inflammation in sensitized subjects. The role of antigenic pollen proteins in the induction of allergic airway inflammation is well characterized, but the contribution of other constituents in pollen grains to this process is unknown. Here we show that pollen grains and their extracts contain intrinsic NADPH oxidases. The pollen NADPH oxidases rapidly increased the levels of ROS in lung epithelium as well as the amount of oxidized glutathione (GSSG) and 4-hydroxynonenal (4-HNE) in airway-lining fluid. These oxidases, as well as products of oxidative stress (such as GSSG and 4-HNE) generated by these enzymes, induced neutrophil recruitment to the airways independent of the adaptive immune response. Removal of pollen NADPH oxidase activity from the challenge material reduced antigen-induced allergic airway inflammation, the number of mucin-containing cells in airway epithelium, and antigen-specific IgE levels in sensitized mice. Furthermore, challenge with Amb a 1, the major antigen in ragweed pollen extract that does not possess NADPH oxidase activity, induced low-grade allergic airway inflammation. Addition of GSSG or 4-HNE to Amb a 1 challenge material boosted allergic airway inflammation. We propose that oxidative stress generated by pollen NADPH oxidases (signal 1) augments allergic airway inflammation induced by pollen antigen (signal 2).

Aldehydes↗

PubMed2005

Clearing the air: a model for investigating indoor air quality in Texas schools.

This pilot project focused on the assessment of indoor air quality at a local high school in Galveston, Texas, using methods based on guidelines for the U.S. Environmental Protection Agency's Indoor Air Quality Tools for Schools program. Tools for Schools, developed for evaluating and ensuring acceptable air quality for schools, takes a low-cost, minimal-involvement, primarily educational approach. The authors also compared the findings from this approach with the results of an air-sampling program. The overall goal was to determine if use of Tools for Schools was sufficient to identify conditions with the potential to cause adverse health effects. The primary objectives were to 1) establish an indoor air quality committee for the school to implement Tools for Schools assessments and management strategies, 2) collect air quality data in high-risk areas identified within the school by the indoor air quality committee, 3) collect outdoor air quality data at or in close proximity to the school, and 4) develop methods and instruments for assessing environmental risks associated with daily school attendance. Data were gathered on levels of formaldehyde and other volatile organic compounds (VOCs), ozone, particulate matter (PM10), mold, relative humidity, and temperature. Data values for each sampled pollutant were compared with federal standards, recommended values established by the American Conference of Governmental Industrial Hygienists for non-industrial populations, and effects screening levels developed by the Texas Commission on Environmental Quality. Levels of all VOCs except formaldehyde were found to be well within guidelines, as were ozone and particulate-matter levels. Mold, however, was widespread, including both common species and species associated with allergy and asthma, such as Aspergillus and Alternaria. In general, Tools for Schools provides an excellent foundation for a school indoor air quality program, although the authors did find it necessary to streamline data collection and did find that mold with the potential for adverse health effects was present, albeit not visible in some areas.

Air Pollutants↗

PubMed2004

Crystal structure of Jun a 1, the major cedar pollen allergen from Juniperus ashei, reveals a parallel beta-helical core.

Pollen from cedar and cypress trees is a major cause of seasonal hypersensitivity in humans in several regions of the Northern Hemisphere. We report the first crystal structure of a cedar allergen, Jun a 1, from the pollen of the mountain cedar Juniperus ashei (Cupressaceae). The core of the structure consists primarily of a parallel beta-helix, which is nearly identical to that found in the pectin/pectate lyases from several plant pathogenic microorganisms. Four IgE epitopes mapped to the surface of the protein are accessible to the solvent. The conserved vWiDH sequence is covered by the first 30 residues of the N terminus. The potential reactive arginine, analogous to the pectin/pectate lyase reaction site, is accessible to the solvent, but the substrate binding groove is blocked by a histidine-aspartate salt bridge, a glutamine, and an alpha-helix, all of which are unique to Jun a 1. These observations suggest that steric hindrance in Jun a 1 precludes enzyme activity. The overall results suggest that it is the structure of Jun a 1 that makes it a potent allergen.

Allergens↗

PubMed2003

Detecting potential IgE-reactive sites on food proteins using a sequence and structure database, SDAP-food.

The high incidence of food allergies, including oral allergy syndrome, represent major considerations when introducing new crops and foods. A new structural database of allergenic proteins, SDAP-Food, http://fermi.utmb.edu/SDAP/, has been developed to aid in predicting the IgE-binding potential of novel food proteins and cross-reactivities among known allergens. The site is designed to facilitate the first steps of a decision tree approach to determine the allergenicity of a given protein, based on the sequence and structural similarity to known allergens and their IgE binding sites. Immunological tests can then be used to confirm the predictions. A hierarchical procedure for identifying potential allergens, using a physical property-based sequence similarity index, has been designed to identify regions that resemble known IgE binding sites. As an example, SDAP tools were used to find food allergen sequences similar to an IgE binding site of the Jun a 3 allergen from mountain cedar pollen. The SDAP sequence similarity search matched the Jun a 3 epitope to regions in several food allergens, including cherry (Pru av 2), apple (Mal d 2) and pepper (Cap a 1), which are, like Jun a 3, members of the plant pathogenesis-related (PR-5) protein family. Homology modeling, using our EXDIS/DIAMOD/FANTOM program suite, indicated a similar surface location and structure for the potential epitope region on all of these allergens. The quantitative approach presented here can be used as part of a screening process for potential allergenicity of recombinant food products.

Allergens↗

PubMed2003

Crystallization and preliminary X-ray diffraction analysis of Jun a 1, the major allergen isolated from pollen of the mountain cedar Juniperus ashei.

Jun a 1, the major allergen of pollen from the mountain cedar Juniperus ashei, has been crystallized using the hanging-drop vapor-diffusion method at 277 K. The crystals are monoclinic, space group P2(1), with unit-cell parameters a = 53.38, b = 113.48, c = 72.44 A, beta = 96.36 degrees and four molecules in the unit cell. A complete 2.5 A data set has been collected at 100 K with X-rays from a Cu Kalpha rotating-anode generator.

Allergens↗

PubMed2003

Major linear IgE epitopes of mountain cedar pollen allergen Jun a 1 map to the pectate lyase catalytic site.

Resolution of the 3D structures and IgE epitopes of allergens may identify common or conserved features of allergens. Jun a 1, the predominant allergen in mountain cedar pollen, was chosen as a model for identifying common structural and functional features among a group of plant allergens. In this study, synthetic, overlapping peptides of Jun a 1 and sera from patients allergic to mountain cedar pollen were used to identify linear epitopes. A 3D model of Jun a 1 was produced using the Bacillus subtiles pectate lyase (PL) as a template and validated with biophysical measurements. This allowed mappings of four IgE binding sites on Jun a 1. Two of the epitopes mapped to turns or loops on the surface of the model structure. The other two epitopes mapped to the beta-sheet region, homologous to the catalytic site of PL. This region of Jun a 1 is highly conserved in the group 1 allergens from other cedar trees as well as microbial PLs. The finding that two out of three major IgE epitopes map to highly conserved catalytic regions of group 1 cedar allergens may help to explain the high degree of cross-reactivity between cedar pollen allergens and might represent a pattern of reactivity common to other allergens with catalytic activity.

Allergens↗

PubMed2002

Isolation and characterization of cDNAs that encode homologs of a pathogenesis-related protein allergen from Cryptomeria japonica.

Many plant pathogenesis-related (PR) proteins are allergenic. We isolated three cDNAs, Cry j 3.1, Cry j 3.2, and Cry j 3.3, that encoded homologs of Jun a 3, a PR protein allergen in Juniperus ashei, from a cDNA library derived from the pollen of Cryptomeria japonica. The predicted amino acid sequences encoded by the three cDNAs were more than 85% identical to each other and about 57% identical to the sequence of Jun a 3. The Cry j 3 genes seemed to form a small multigene family in the genome of C. japonica. Expression of Cry j 3 was strong in roots and in female and male strobili; expression was weaker in cotyledons, leaves, stems, and pollen grains.

Allergens↗