bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Approach to Search for Identity by Descent in Small Samples of Patients and Controls from the Same Mendelian Breeding Unit: A Pilot Study on Myopia
Authors: Heath Simon , Robledo Renato , Beggs William , Feola Gaetano , Parodo Carlo , Rinaldi Antoniettina , Contu Licinio , Dana Debra , Stambolian Dwight , Siniscalco Marcello
Published: April 2003

Autosomal dominant high myopia, a genetic disorder already mapped to region 18p11.31, is common in Carloforte (Sardinia, Italy), an isolated village of 8,000 inhabitants descending from a founder group of 300 in the early 1700s. Fifteen myopic propositi and 36 normal controls were selected for not having ancestors in common at least up to the grandparental generation, although still descendants of the original founders. All subjects were genotyped for 14 markers located on autosome 18 at a resolution of about 10 cM. Allelic distributions were found to be similar at all tested loci in propositi and controls, except for the candidate marker D18S63 known to segregate in close linkage association with high myopia. In particular, the frequency of allele 85 among the propositi was almost double that of the controls (Fisher’s exact test, p = 0.037). The association is more striking when the frequency of the genotype 85/85 in the two groups is compared (Fisher’s exact test, p = 0.005). This conclusion was further evaluated through a bootstrap analysis by computing the overall probability of the observed data under the null hypothesis (i.e. no difference between the two groups in frequency distributions for the chromosome 18 markers). Again, marker D18S63 was found to have a sample probability lower than 0.004, which is significant at the 0.05 level after correcting for simultaneous testing of multiple loci. The study demonstrates the efficiency of our novel strategy to detect identity by descent (IBD) in small numbers of patients and controls when they are both part of well-defined Mendelian breeding units (MBUs). The iterative application of our strategy in separate MBUs is expected to become the method of choice to evaluate the ever-growing number of reported associations between candidate genes and multifactorial traits and diseases.
The Null Distribution of the Heterogeneity Lod Score Does Depend on the Assumed Genetic Model for the Trait
Authors: Huang Jian , Vieland Veronica J.
Published: April 2003

It is well known that the asymptotic null distribution of the homogeneity lod score (LOD) does not depend on the genetic model specified in the analysis. When appropriately rescaled, the LOD is asymptotically distributed as 0.5 χ2₀ + 0.5 χ21, regardless of the assumed trait model. However, because locus heterogeneity is a common phenomenon, the heterogeneity lod score (HLOD), rather than the LOD itself, is often used in gene mapping studies. We show here that, in contrast with the LOD, the asymptotic null distribution of the HLOD does depend upon the genetic model assumed in the analysis. In affected sib pair (ASP) data, this distribution can be worked out explicitly as (0.5 – c)χ2₀ + 0.5χ21 + cχ22, where c depends on the assumed trait model. E.g., for a simple dominant model (HLOD/D), c is a function of the disease allele frequency p: for p = 0.01, c = 0.0006; while for p = 0.1, c = 0.059. For a simple recessive model (HLOD/R), c = 0.098 independently of p. This latter (recessive) distribution turns out to be the same as the asymptotic distribution of the MLS statistic under the possible triangle constraint, which is asymptotically equivalent to the HLOD/R. The null distribution of the HLOD/D is close to that of the LOD, because the weight c on the χ22 component is small. These results mean that the cutoff value for a test of size α will tend to be smaller for the HLOD/D than the HLOD/R. For example, the α = 0.0001 cutoff (on the lod scale) for the HLOD/D with p = 0.05 is 3.01, while for the LOD it is 3.00, and for the HLOD/R it is 3.27. For general pedigrees, explicit analytical expression of the null HLOD distribution does not appear possible, but it will still depend on the assumed genetic model.
The Involvement of CD80 and CD86 Costimulatory Molecules in the Induction of Eosinophilia in Mice Infected with Nipponstrongylus brasiliensis
Authors: Huang Yong , Watanabe Naohiro , Ohtomo Hiroshi
Published: April 2003

The costimulatory signal provided by the interaction between CD28 and its ligands, CD80 and CD86, is critical for T cell activation. The requirement of CD80 and CD86 in T cell activation for eosinophilia and IgE production was examined in mice infected with the nematode parasite Nippostrongylus brasiliensis. Combined treatment with anti-CD80 and anti-CD86 suppressed eosinophilia in the blood and the small intestine and suppressed IgE production. However, administration of either anti-CD80 or anti-CD86 alone had little effect on eosinophilia and on the elevation of IgE levels. These results suggest that CD80 and CD86 costimulation is required and either CD80 or CD86 can provide a sufficient costimulatory signal for induction of eosinophilia in mice infected with N. brasiliensis.
Identification of cDNA for Rat Homologues of Human Major Basic Protein and Eosinophil Cationic Protein
Authors: Nittoh Takeaki , Hirakata Mikito , Mue Suetsugu , Ohuchi Kazuo
Published: April 2003

We have determined the complete nucleotide sequence for cDNA of rat homologues of human eosinophil major basic protein (MBP) and eosinophil cationic protein (ECP) using the rapid amplification of cDNA ends (RACE) procedure. Nucleotide sequence of cDNA of rat MBP revealed that mRNA of rat MBP encodes a protein containing 227 amino acids which has three functional domains; namely, the signal peptide, the acidic peptide that contains numerous acidic amino acids and the mature MBP, as in human, guinea pig and mouse MBP. In addition, cDNA of a rat homologue of human ECP was also cloned. The deduced amino acid sequence revealed that this gene encodes a putative protein with a molecular weight of 15.5 kD which has ribonuclease activity. The homology of amino acid sequence between the rat homologue and the murine eosinophil-associated ribonucleases (EARs) was high (65%). Therefore, we named this rat homologue ‘rat EAR-1’.
Involvement of β1 and β2 Integrin Stimulation in RNA Synthesis in an Eosinophilic Cell Line (EoL-1)
Authors: Tsuda Akira , Saito Norihiro , Kayaba Hiroyuki , Kamada Yumiko , Oyamada Hajime , Chihara Junichi
Published: April 2003

In allergic inflammatory disease, especially in bronchial asthma, eosinophils play important roles as essential inflammatory cells. In the accumulation of eosinophils in airway inflammation, eosinophils receive very diverse stimuli. In this study, we investigated the influences of the signal between β2 integrin/intercellular adhesion molecule-1 (ICAM-1) or β1 integrin/fibronectin (FN) and RNA synthesis on proteins in eosinophilic cell line-1 (EoL-1), using recombinant soluble ICAM-1 (r-sICAM-1) as a global response of eosinophils. 3H-thymidine incorporation and 3H-uridine incorporation were used as indices for DNA synthesis and RNA synthesis, respectively. In a comparison of the RNA/DNA ratio with various durations of stimulation of 1 µg/ml of r-sICAM-1 and 100 µg/ml of FN, a time-dependent increase was observed, but the increase induced by FN rose slower than that induced by r-sICAM-1. From this result, a β2 integrin/ICAM-1 signal induced an increase in the RNA/DNA ratio in EoL-1, implying that the signal promotes RNA synthesis, which suggests that various types of protein synthesis, such as the synthesis of various cytokines, are induced by the β2 integrin/ICAM-1 signal. Similar results were obtained with a β1 integrin signal using FN, but there was a difference in the time course between β2 integrin/ICAM-1 and β1 integrin/FN signals. This experimental method may be useful for understanding these manifestations as a global response of eosinophils.
IL-4 Induces Eotaxin in Human Dermal Fibroblasts
Authors: Mochizuki Mitsuru , Schröder Jens-M. , Christophers Enno , Yamamoto Shoso
Published: April 2003

A common feature of Th2-mediated skin diseases is selective eosinophil (Eo) infiltration in the affected dermis. However, the mechanisms of selective Eo recruitment are not yet fully understood. Our recent findings indicated that dermal fibroblasts actively participate in the accumulation of Eo in Th2-mediated skin inflammation. Following stimulation with IL-4, dermal fibroblasts produce eotaxin, which is the only biologically active Eo attractant detected in the supernatants. Natural eotaxin is comprised of a mixture of N-terminal-truncated and O-glycosylated variants. The expression of eotaxin mRNA is upregulated within 1 h after stimulation. TNF-α markedly enhances eotaxin mRNA expression and release of the protein product from IL-4-stimulated fibroblasts. As mast cells and Th2 cells produce both cytokines, it is probable that Eo recruitment into sites of allergen-induced, Th2-mediated skin reactions is, at least in part, due to IL-4-stimulated induction of eotaxin in dermal fibroblasts.
Whole-Blood Flow-Cytometric Analysis of Induction of Adhesion Molecules Expression on Eosinophils Stimulated by Phorbol-12-Myristate-13-Acetate/Ionomycin
Authors: Saito Norihiro , Kayaba Hiroyuki , Honda Kohei , Yamada Yoshiyuki , Cui Chang-Hao , Kamada Yumiko , Kuwasaki Tomoe , Oyamada Hajime , Chihara Junichi
Published: April 2003

Background: Activation of eosinophils is closely associated with the pathology of allergic inflammatory disease, especially bronchial asthma. We recently investigated the activation of eosinophils by applying whole blood to a flow cytometer. We measured here β1 and β2 integrin on eosinophils stimulated by phorbol-12-myristate-13-acetate (PMA)/ionomycin to evaluate eosinophil activation in vitro using whole blood. Methods: Heparinized whole blood was diluted with the same volume of RPMI 1640, then cells were incubated in the presence or absence of PMA and ionomycin for 45 min at 37°C. After hemolyzation with lysing solution, flow-cytometric findings for CR3, LFA1-α, LFA1-β and VLA-4 expression on eosinophils were examined. Results: Mean fluorescent intensity (MFI) of CR3 and LFA1-β stimulated by PMA and ionomycin was significantly higher than that of the unstimulated control. MFI of LFA1-α showed no significant difference from the unstimulated control. On the other hand, MFI of VLA-4 tended to decrease. Conclusions: Our method to distinguish eosinophils from various cell groups in whole blood is simple and time-saving, similar to conditions in vivo and may allow intensive investigation of eosinophils in clinical laboratories as well as in research laboratories. We are currently investigating the influence of different kinds of stimulations, regulation factors or agents on eosinophils using this method.
Effect of Eotaxin on the Generationof Reactive Oxygen Species from Eosinophil Cell Line, YY-1
Authors: Honda Kohei , Yamada Yoshiyuki , Cui Chang-Hao , Saito Norihoro , Kayaba Hiroyuki , Kobayashi Yoshimi , Oyamada Hajime , Kamada Yumiko , Kuwasaki Tomoe , Tsurufuji Susumu , Chihara Junichi
Published: April 2003

Background: The CC chemokine eotaxin is a selective chemoattractant for eosinophils. Eosinophils have been considered to be the major effector cells in allergic inflammation, since not only eosinophil-specific granule proteins but also reactive oxygen species (ROS) from eosinophils may cause the damage to the cells or tissue of the mucosal epithelium. In this study, we examined the effect of eotaxin on ROS from an eosinophil cell line, YY-1. Methods: ROS in luminol-dependent reaction were examined. Calcium ionophore A23187 were added to the mixture of YY-1 cells with luminol, and then ROS were determined. Results: Eotaxin primed the production of ROS from YY-1 cells. ROS from untreated YY-1 cells evoked with calcium ionophore A23187 in luminol-dependent chemiluminescence gave a maximal value of 1,928 ± 223 intensity counts (IC; mean ± SE, n = 4) and an integral value of 17.04 ± 1.51 IC (×10–4), while eosinophils that were treated with eotaxin gave a maximal value of 2,264 ± 86 IC (10 nM), 2,691 ± 124 IC (100 nM) and an integral value of 21.22 ± 0.67 IC (×10–4; 10 nM), 26.20 ± 1.41 IC (×10–4; 100 nM). Conclusion: Eotaxin might play important roles in the pathogenesis of allergic inflammation through eosinophil activation by priming of eosinophil oxidative metabolism as well as involvement in selective eosinophil chemotaxis.
Intracellular Signaling Pathways in Human Eosinophil Activation: Role of a β2 Integrin, αMβ2
Authors: Kato Masahiko , Kimura Hirokazu , Tachibana Atsushi , Motegi Youichi , Tokuyama Kenichi , Kita Hirohito , Morikawa Akihiro
Published: April 2003

Cellular adhesion is crucial roles for eosinophil effector functions such as degranulation. Here, we focused on the role of a β2 integrin, αMβ2, in intracellular signaling pathways of human eosinophil activation. We found that the ligation of αMβ2 triggers the activation of an intracellular signaling cascade, including protein tyrosine phosphorylation and phosphoinositide turnover, and subsequent cellular degranulation in human eosinophils. These signaling events may play important roles in adhesion-dependent cellular functions of eosinophils.
Tumor Necrosis Factor-α Mediates Antiapoptotic Signals Partially via p38 MAP Kinase Activation in Human Eosinophils
Authors: Tsukahara Kayo , Nakao Atsuhito , Hiraguri Masaki , Miike Satoshi , Mamura Mizuko , Saito Yasushi , Iwamoto Itsuo
Published: April 2003

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine with many biological effects on a variety of cells. In particular, TNF-α has been shown to act as a death or survival factor which mediates apoptosis or antiapoptotic signals in various types of cells. In eosinophils, TNF-α has been reported to activate eosinophil functions. However, it is not clearly defined whether TNF-α delivers antiapoptotic signals in eosinophils. In order to determine whether TNF-α prevents eosinophil apoptosis, we examined the effect of TNF-α on eosinophil apoptosis by the survival assay and cell cycle analysis. We also determined whether intracellular MAP kinases (ERKs, Jun kinase/JNK, and p38 MAP kinase) are involved in the TNF-α-induced signaling for the prevention of eosinophil apoptosis. We showed that TNF-α mediated antiapoptotic signals in human eosinophils in part via activation of p38 MAP kinase, but not via activation of ERKs and JNK. Our data suggest that TNF-α/p38 MAP kinase pathways are involved in the regulation of eosinophil survival and, thus, would be important for the development of allergic eosinophil-rich inflammation.
Atopic Asthma Is Dominant in Elderly Onset Asthmatics: Possibility for an Alteration of Mast Cell Function by Aging through Fc Receptor Expression
Authors: Atsuta Ryo , Akiyama Kazuo , Shirasawa Takuji , Okumura Ko , Fukuchi Yoshinosuke , Ra Chisei
Published: April 2003

The morbidity and mortality of elderly onset asthma have recently been on the increase. To evaluate the allergic status of the elderly onset asthmatics, we performed skin tests for allergens and estimated serum total IgE levels of the patients. The proportion of the skin-test-positive patients among the elderly onset asthmatics was significantly larger than that in the younger onset group, whereas no significant difference was observed in the frequency of patients with serum total IgE levels higher than the normal range (287.2 IU/ml). On the other hand, in the experiments with aged mice, isolated peritoneal mast cells (PMC) showed a considerable decrease in the FcγRIIB/III expression and higher degranulation triggered by 2.4G2, as compared to the PMC derived from young mice. Since FcγRIIB has been shown to act as a negative regulator, we speculate that in the aged mice Fc receptor-mediated mast cell function may be upregulated by a release from the negative regulation as a result of decreased FcγRIIB expression. Our results obtained from a mouse model system may help to understand pathophysiological mechanisms underlying elderly onset asthma.
Heterogeneity in Expression of Cytokine mRNA in Freshly Isolated Peripheral Blood Eosinophils of Patients with Cutaneous-Disease-Associated Eosinophilia
Authors: Hatano Yutaka , Katagiri Kazumoto , Takayasu Susumu
Published: April 2003

Background: It is thought that there are subpopulations of eosinophils concerning cytokine production: some of them predominantly produce Th1 cytokines, while others produce Th2. Methods: By semiquantitative RT-PCR, the in vivo levels of the cytokine mRNA were measured in freshly isolated eosinophils and peripheral blood mononuclear cells (PBMCs) of 19 patients with eosinophilia, including 6 with atopic dermatitis, 4 with drug eruption, 1 with chronic dermatitis, 2 with erythroderma, 3 with bullous pemphigoid, 1 each with urticaria, adult T cell lymphoma, and hypereosinophilic syndrome, and 7 healthy individuals. Eosinophils and PBMCs were isolated by centrifugation over Ficoll-Paque and magnetic cell separation using anti-CD16 antibody. Results: The IL-4/IFN-γ ratio in eosinophils as well as PBMCs was increased and correlated well with IgE levels in atopic dermatitis. In other cytokines, such as IL-8, TNF-α and TGF-β1, the levels of mRNA expression were variable among each sample. Conclusion: Not only helper T lymphocytes but also eosinophils may take part in the pathogenesis of cutaneous diseases by virtue of heterogeneity of Th1 and Th2 cytokine production.
Overexpression of CD11b on Eosinophils in Atopic Dermatitis: Downregulation by Cyclosporin A and Upregulation by Interleukin 5
Authors: Yamada Hidekazu , Kurashimo Shinji , Chihara Junichi , Matsukura Masaharu , Yudate Tatsuo , Tezuka Tadashi
Published: April 2003

We examined the level of expression of CD11b on eosinophils in pripheral blood samples from patients with atopic dermatitis (AD) and non-AD volunteers. Eosinophils were defined using a new method employing CD14/CD45 and a backgate technique. Overexpression of CD11b was noted in eosinophils of AD patients. Treatment of AD with cyclosporin A resulted in clinical improvement as well as reduction in the expression of CD11b. Stimulation of eosinophils from patients with inactive AD by interleukin 5 upregulated the expression of CD11b on these cells. Our results suggest that the expression of CD11b surface molecule on eosinophils may play an important role in the activity of AD.
Analysis of the Prolongation of Rat Eosinophil Survival Induced by Recombinant Rat Interleukin-5
Authors: Ishihara Kenji , Satoh Ikuko , Ohuchi Kazuo
Published: April 2003

Rat eosinophil survival was prolonged by recombinant rat IL-5 prepared by the baculovirus expression system. The IL-5-induced prolongation of eosinophil survival was dose-dependently inhibited by the protein synthesis inhibitor cycloheximide, the DNA-dependent RNA synthesis inhibitor actinomycin D, and the tyrosine kinase inhibitor herbimycin A. The MEK-1 inhibitor PD98059 inhibited IL-5-induced phosphorylation of both p44 and p42 MAP kinases, but the IL-5-induced prolongation of eosinophil survival was not inhibited. In contrast, the JAK2 inhibitor AG490 inhibited the IL-5-induced prolongation of eosinophil survival. Treatment of eosinophils with IL-5 resulted in phosphorylation of STAT5 but not STAT1, and the IL-5-induced phosphorylation of STAT5 was inhibited by AG490. These findings suggest that recombinant rat IL-5 activates JAK2 tyrosine kinase, which phosphorylates STAT5, and induces protein synthesis required for the prolongation of rat eosinophil survival.
T Cell Receptor Mimic Peptidesand Their Potential Application in T-Cell-Mediated Diseas e
Authors: Enk Alexander H. , Knop Jürgen
Published: April 2003

Background: A T cell receptor (TCR) peptide was designed that mimics the intramembranous amino acid sequence of the TCR chain. Prior studies had shown that this mimic peptide would inhibit TCR signaling. This study was designed to investigate the use of this mimic peptide for the treatment of T-cell-mediated skin diseases. Methods: Synthesized mimic peptides were first tested for their T-cell-inhibitory effect in proliferation assays. Afterwards, mimic peptides were applied to murine ear skin prior to application of a contact allergen and tested for their inhibitory effect in the model of murine allergic contact sensitivity. The effect of epicutaneous treatment with the peptide was also tested on patients with T-cell-mediated skin disease. Results: Mimic peptide potently inhibited proliferation of CD4+ and CD8+ T cells when added to allogeneic proliferation assays using dendritic cells as antigen-presenting cells (APCs). Suppression of the proliferative capacity could be overcome by addition of an anti-CD3 monoclonal antibody. When applied to murine ear skin prior to application of a contact allergen, mimic peptide efficiently blocked ear swelling responses in mice. In humans, application of mimic peptide for the treatment of various diseases resulted in amelioration or even cure in patients suffering from atopic dermatitis, psoriasis or lichen planus. Conclusions: TCR mimic peptide efficiently abrogates T-cell-mediated immune responses in mice and man in vitro and in vivo. The potential immunosuppressive effect of the drug is discussed.
Identification and Characterization of Cross-Reactive Natural Rubber Latex and Ficus benjamina Allergens
Authors: Chen Zhiping , Düser Maria , Flagge Angelika , Maryska Silke , Sander Ingrid , Raulf-Heimsoth Monika , Baur Xaver
Published: April 2003

Background: An association between allergy to Ficus benjamina and natural rubber latex (NRL) has been suspected based on clinical and immunological observations. The responsible cross-reactive allergens have not been identified yet. This study was undertaken to investigate the cross-reactivity between hevein (Hev b 6.02, 4.7 kD), a major allergen of NRL, and F. benjamina and identify its counterpart in F. benjamina. Methods: 89 serum samples from subjects allergic to NRL were used in the study. Skin prick tests were performed with highly purified hevein and sap extract of F. benjamina. Specific IgE antibodies to NRL, F. benjamina and Hev b 6.02 were determined by the Pharmacia CAP method. Cross-reactivity among these allergens was investigated by means of CAP and immunoblot inhibition experiments. Two-dimensional gel electrophoresis separation and protein microsequencing were performed to identify the cross-reactive allergens in F. benjamina. Results: 67 out of 89 (75%) sera showed elevated IgE to hevein. Specific IgE to Ficus were found in 22 (24.7%) sera, and with 1 exception, all these sera also had IgE to Hev b 6.02. Results of CAP inhibition assays using 11 sera showing IgE to both Hev b 6.02 and Ficus demonstrated that the IgE to Ficus could be completely inhibited by Hev b 6.02 in 6 of 11 sera. Immunoblots and immunoblot inhibition assays revealed that a protein of about 45 kD in F. benjamina is strongly recognized by serum IgE. In addition, the IgE-binding reactivity to this 45-kD protein could be completely inhibited by preincubation of the sera with Hev b 6.02. N-terminal protein sequencing of 14 amino acids indicated that this 45-kD protein has a hevein-like domain at the N-terminal region and may belong to the endochitinase family. Conclusion: Latex-allergicpatients are at higher risk of becoming sensitized to Ficus. Hev b 6.02 in latex is a major cross-reactive allergen and its counterpart in F. benjamina is an acidic protein with a molecular weight of about 45 kD and a hevein-like N-terminal domain.
Different HBs Antibody versus Lymphoproliferative Responses after Application of a Monovalent (Hepatitis B) or Combined (Hepatitis A + Hepatitis B) Vaccine
Authors: Wiedermann U. , Kundi M. , Vollmann U. , Kollaritsch H. , Ebner Ch. , Wiedermann G.
Published: April 2003

Background: The aim of the study was to evaluate a possible adjuvanticity of simultaneous hepatitis A (HAV) vaccination for the development of HBs-specific antibodies and lymphoproliferative responses in prophylactic immunization with hepatitis B (HBV). Methods: Thirty-nine volunteers were vaccinated (schedule: 0/1/6 months) either with a bivalent HAV/HBV (18 individuals) or with HBV (recombinant HBs-antigen) vaccine alone (21 individuals). Anti-HBs antibody titers and lymphoproliferative responses as consequence of stimulation of peripheral blood mononuclear cells (PBMC) with HBs were evaluated and compared between the two groups before second vaccination, before and 1 month after booster. Results: Geometric mean titers were higher at all time points in the group treated with the combined vaccine. On the other hand, after the booster injection, HBs-induced stimulation indices in PBMC were higher in the group vaccinated with HBs alone. Neither the difference in antibody titers nor in proliferative responses reached the level of statistical significance. Interestingly, the inverse relation between cellular proliferation and antibodies was significant, indicating that cellular reactivity is not in all cases a useful marker to evaluate the intensity of the induced immunity. Conclusions: The magnitude of the T-lymphocyte response may eventually not be decisive for the subsequent antibody response. Both vaccination strategies led to a cellular and humoral immune response and resulted in protective levels of HBs-specific antibodies.
ENU Mouse Mutagenesis: Generation of Mouse Mutants with Aberrant Plasma IgE Levels
Authors: Alessandrini Francesca , Jakob Thilo , Wolf Alexander , Wolf Eckhard , Balling Rudolf , de Angelis Martin Hrabé , Ring Johannes , Behrendt Heidrun
Published: April 2003

Background: The ENU Mouse Mutagenesis Project aims at a large-scale, systematic production of mouse mutants using the alkylating agent ethyl-nitrosourea (ENU). Offspring of mutagenized mice are subjected to a multiparameter screen to detect alterations in various phenotypes with the ultimate goal of identifying novel genes relevant for the expression of the phenotype. Using this approach, we have analyzed plasma IgE concentrations to identify mouse mutants with aberrant plasma IgE levels. Methods and Results: ENU-mutagenized male C3HeB/FeJ were mated to wild-type females to produce F1 offspring. F1 animals were analyzed for alterations in their plasma IgE concentrations that showed a dominant mode of inheritance, or bred further to screen for recessive phenotypes. Plasma IgE concentrations were determined by ELISA and a normal range for plasma IgE was established using C3HeB/FeJ wild-type animals. So far we have tested 6568 F1 animals. Repeated testing confirmed a stable aberrant IgE phenotype in 124 animals. To confirm the genetic basis of the observed phenotype, these mice were subjected to confirmation crossing. Currently we have established 9 independent mutant mouse lines (3 with high plasma IgE and 6 with plasma IgE below detection limit) that have been genetically confirmed and additional 24 variant mouse lines are currently undergoing confirmation testing. Conclusion: ENU mouse mutagenesis allowed us to generate and identify mouse mutants with aberrant plasma IgE levels, which may be used to characterize novel genes involved in IgE regulation and may serve as animal models for IgE-mediated diseases.
Unexpected Functions of FcεRI on Antigen-Presenting Cells
Authors: Kraft Stefan , Katoh Norito , Novak Natalija , Koch Susanne , Bieber Thomas
Published: April 2003

In contrast to mast cells and basophils, the high affinity IgE receptor (FcΕRI) on antigen-presenting cells (APC) shows structural and functional differences. It consists only of a minimal structure of one α- and two γ-chains and enables APC to efficiently take up and present antigen in IgE-mediated delayed-type hypersensitivity reactions that are thought to play a pivotal role in atopic diseases. However, recent studies of FcΕRI signal transduction and function on APC suggest additional mechanisms by which FcΕRI engagement on APC could affect inflammatory reactions. FcΕRI ligation is able to induce major signaling events like protein tyrosine kinase activation including p72syk leading to PLC-γ1 phosphorylation and consecutive calcium influx. Late signaling events like the activation of transcription factors such as NF-ĸB provide a link to the release of proinflammatory cytokines and chemokines, Th-polarizing factors such as IL-12 and the induction of antiapoptotic factors. FcΕRI-mediated IL-10 production in monocytes could also influence their differentiation. Since there are hints that in vivo a functional FcΕRI signaling pathway only exists in individuals from an atopic background, we suggest that these unexpected mechanisms may have an effect on inflammatory reactions in atopic diseases.
Tapping Allergen Repertoires by Advanced Cloning Technologies
Authors: Crameri Reto , Kodzius Rimantas , Konthur Zoltan , Lehrach Hans , Blaser Kurt , Walter Gerald
Published: April 2003

Background: Complex allergenic sources such as moulds, foods and mites contain complex panels of IgE-binding molecules which need to be cloned, produced and characterized in order to mimic the entire allergenicity of whole extracts reconstituted by mixing single standardized recombinant allergens. Methods: Phage surface display of cDNA libraries selectively enriched for allergen-expressing clones using IgE from allergic patients allows rapid isolation of large panels of allergens. For the characterization of all different clones present in enriched cDNA libraries in a fast and cost-effective way, high-throughput screening technology is required. Results: The combination of selective enrichment of cDNA libraries based on biopanning against serum IgE from sensitized patients and automated robot technology for picking and high-density gridding of clones onto filter membranes, followed by hybridization, enables fast identification of all the different clones present in an enriched library. The consequent application of selective enrichment and robotic-based screening allows, within weeks, cloning and characterization of the whole allergenic repertoire of any organisms. Conclusions: Robotic-based high-throughput screening of clones selected for IgE-binding capacity from phage surface-displayed cDNA libraries of Aspergillus fumigatus, Cladosporium herbarum, Coprinus comatus, Malassezia furfur, peanut and human lung tissue allowed rapid characterization of 81, 28, 37, 27, 8 and 151 different sequences, respectively. All these cDNAs bear a high probability to encode allergens derived from the respective allergenic source.
Reduction in Allergenicity of Grass Pollen by Genetic Engineering
Authors: Bhalla Prem L. , Swoboda Ines , Singh Mohan B.
Published: April 2003

Background: Hay fever and allergic asthma triggered by grass pollen allergens affect ∼20% of the population in cool temperate climates. Ryegrass is the dominant source of allergens due to its prodigious airborne pollen production. Lol p 5 or group 5 is among the most important and widespread grass pollen allergen because it reacts with IgE antibodies of more than 90% of grass pollen-allergic patients, contains most of the grass pollen-specific IgE epitopes and elicits strong biological responses. Significant efforts have been made in developing diagnostic and therapeutic reagents for designing new and more effective immunotherapeutic strategies for treatment of allergic diseases. An alternative approach to this problem could be to reduce the amount of allergen content in the source plant. Methods: High velocity microprojectile bombardment was used to genetically engineer ryegrass. Antisense construct targeted to one of major allergen, Lol p 5, was introduced. The expression of antisense RNA was regulated by a pollen-specific promoter. Pollen was analysed for IgE reactivity. Results: Analysis of proteins with allergen-specific monoclonal and polyclonal antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE binding capacity of pollen extract as compared to control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development. Conclusions: Our studies showed that it is possible to selectively ‘switch off’ allergen production in pollen of ryegrass demonstrating feasibility of genetic engineering of plants for reduced allergenicity.
Engineering, Characterization and in vitro Efficacy of the Major Peanut Allergens for Use in Immunotherapy
Authors: Bannon Gary A. , Cockrell Gael , Connaughton Cathie , West C. Michael , Helm Ricki , Stanley J. Steven , King Nina , Rabjohn Pat , Sampson Hugh A. , Burks A. Wesley
Published: April 2003

Background: Numerous strategies have been proposed for the treatment of peanut allergies, but despite the steady advancement in our understanding of atopic immune responses and the increasing number of deaths each year from peanut anaphylaxis, there is still no safe, effective, specific therapy for the peanut-sensitive individual. Immunotherapy would be safer and more effective if the allergens could be altered to reduce their ability to initiate an allergic reaction without altering their ability to desensitize the allergic patient. Methods: The cDNA clones for three major peanut allergens, Ara h 1, Ara h 2, and Ara h 3, have been cloned and characterized. The IgE-binding epitopes of each of these allergens have been determined and amino acids critical to each epitope identified. Site-directed mutagenesis of the allergen cDNA clones, followed by recombinant production of the modified allergen, provided the reagents necessary to test our hypothesis that hypoallergenic proteins are effective immunotherapeutic reagents for treating peanut-sensitive patients. Modified peanut allergens were subjected to immunoblot analysis using peanut-positive patient sera IgE, T cell proliferation assays, and tested in a murine model of peanut anaphylaxis. Results: In general, the modified allergens were poor competitors for binding of peanut-specific IgE when compared to their wild-type counterpart. The modified allergens demonstrated a greatly reduced IgE-binding capacity when individual patient serum IgE was compared to the binding capacity of the wild-type allergens. In addition, while there was considerable variability between patients, the modified allergens retained the ability to stimulate T cell proliferation. Conclusions: These modified allergen genes and proteins should provide a safe immunotherapeutic agent for the treatment of peanut allergy.
A Highly Allergenic Fragment of the Major Timothy Grass Pollen Allergen, Phl p 5, Defined by a Human Monoclonal IgE Antibody
Authors: Flicker S. , Vrtala S. , Steinberger P. , Vangelista L. , Kraft D. , Valenta R.
Published: April 2003

We report the characterization of a domain of the major timothy grass pollen allergen, Phl p 5A, which contains the binding site for a human monoclonal IgE antibody. The human monoclonal IgE antibody fragment (Fab) was obtained from an IgE combinatorial library constructed from lymphocytes of a grass pollen-allergic patient. An 11.2-kD N-terminal fragment representing approximately one third of the complete Phl p 5A allergen could be identified to contain the binding site for the IgE Fab. The rPhl p 5A fragment revealed an extremely high allergenic activity in skin test experiments which in some cases equaled that of the complete Phl p 5A allergen. The rPhl p 5A domain thus represents an allergen fragment containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation.
Can Mycobacterium tuberculosis Infection Prevent Asthma and Other Allergic Disorders?
Authors: Suzuki Naohito , Kudo Koichiro , Sano Yasuyuki , Ito Koji
Published: April 2003

It is generally considered that tuberculosis (TB) is a disease which upregulates Th1 cell function. There is a hypothesis that infection of Mycobacterium tuberculosis may prevent allergic disorders such as bronchial asthma. However, our clinical experience of patients with TB somewhat conflicts this hypothesis. Hence, we investigated Th1/Th2 balance in the peripheral blood of patients with active TB by measuring serum levels of IgE antibody and by intracellular cytokine assay. We found that serum levels of IgE in the patients with active TB were significantly higher than in those with lung cancer or with COPD. In the TB patients, titers of IgE tended to correlate with disease severity. Intracellular cytokine assay demonstrated that IFN-γ-positive cells were significantly decreased in the patients with active TB compared to normal controls. The ratio of IFN-γ-positive (Th1-like)/IL-4-positive (Th2-like) cells was remarkably reduced in the TB patients (p < 0.0001). This ratio showed a significant negative correlation with erythrocyte sedimentation rate and with C-reactive protein. Therapy against TB for 2–3 months did not result in significant changes of the Th1/Th2 ratio. These findings suggest that infection of M. tuberculosis does not systematically upregulate Th1 cells in some patients, and is unlikely to prevent allergic disorders like asthma.
Reduced Interferon-γ Production and Mutations of the Interleukin-12 Receptor β2 Chain Gene in Atopic Subjects
Authors: Kondo Naomi , Matsui Eiko , Kaneko Hideo , Fukao Toshiyuki , Teramoto Takahide , Inoue Ryosuke , Watanabe Mizuho , Kasahara Kimiko , Morimoto Naoko
Published: April 2003

Background: The atopic patient has a predisposition to selective synthesis of IgE antibodies to common environmental antigens. IgE production is upregulated by interleukin-4 (IL-4) and downregulated by interferon-γ (IFN-γ). IL-12 is a cytokine that induces IFN-γ production. The signal of IL-12 is transduced through the IL-12 receptor (IL-12R) and Stat4. Methods: We examined IFN-γ production in peripheral blood mononuclear cells (PBMCs) following stimulation with IL-12 or phytohemagglutinin (PHA) in healthy controls and atopic patients. Moreover, sequences of the IL-12R β2 chain gene were analyzed. Results: The serum IgE levels were negatively correlated (p < 0.001) with IFN-γ production. In 24 out of 75 atopic patients, IFN-γ production in PBMCs following stimulation with IL-12 was under the detection limit, but PHA stimulation elicited detectable IFN-γ production. Sequence analysis of the cDNA of IL-12R β2 revealed three kinds of distinct genetic mutations (2496 del 91, 1577 A to G and 2799 A to G) in 10 unrelated subjects of the 24 whose IFN-γ production following IL-12 stimulation was under the detection limit. PBMCs cultured with IL-12 and PHA in these 10 subjects showed decreased tyrosine phosphorylation of Stat4. Conclusions: The results of our study indicate that atopic diseases are caused, in part, by impairment of the IL-12 signal cascade, which downregulates IgE production, and that the mutation of the IL-12R β2 chain gene is one of the causative genes for atopy.
Studies of Different Aspects of the Role of Protein Kinase C in Mast Cells
Authors: Nechushtan Hovav , Razin Ehud
Published: April 2003

Mast cells induce the inflammatory process when their FcΕRI receptors aggregate in response to an antigen binding to immunoglobulin E. Direct interactions between FcΕRI receptor cytoplasmic domains and various intracellular proteins initiate diverse signal transduction pathways resulting in the immediate release of proinflammatory agents. A delayed response also occurs that includes the release of various cytokines. It is clear that the activation of kinases, such as protein kinase C (PKC), is a requirement for both the early and delayed responses of this inflammatory process. In this review we present the results of various studies investigating the role of PKC isozymes in mast cells.
Nonspecific B and T Cell-Stimulatory Activity Mediated by Mast CellsIs Associated with Exosomes
Authors: Skokos Dimitris , Le Panse Sophie , Villa Irène , Rousselle Jean-Claude , Peronet Roger , Namane Abdelkader , David Bernard , Mécheri Salaheddine
Published: April 2003

Bone marrow-derived mouse mast cells (BMMC) and mast cell lines P815 and MC9 have recently been shown to induce antigen-independent B and T lymphocyte activation. It has been demonstrated that a physical contact between mast cells and B and T lymphocytes is not necessary since mast cell supernatants contain full activity. Electron microscopy studies revealed the presence in mast cell supernatants of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induce B and T cell blast formation, proliferation as well as IL-2 and IFN-γ production. In contrast to P815 and MC9 mast cell lines, a pretreatment with IL-4 is required for BMMC to produce active exosomes. Structurally, these exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1 and ICAM-1. Here we provide for the first time the evidence that mast cells use exosomes as sophisticated messengers to communicate with cells of the immune system.
Genetics of Allergic Disease: Evidence for Organ-Specific Susceptibility Genes
Authors: Nishimura Akio , Campbell-Meltzer Robert Scott , Chute Kathy , Orrell Jeff , Ono Santa Jeremy
Published: April 2003

Background: While previous studies have probed the genetics of asthma and serum IgE levels, there have been no studies on the genetics of allergic conjunctivitis. This paper describes the initial phase of a genetic study of allergic conjunctivitis. Methods: Approximately 117 families with probands with allergic conjunctivitis were recruited generating 245 affected sib pairs. Each family member completed a detailed questionnaire on atopic symptoms, and results from skin testing were obtained. Genomic DNA was extracted and genotyped using thirty-five polymorphic markers spanning human chromosomes 5, 6, 11, 12, 16 and 17. Heritability was assessed using the POINTER program, and nonparametric linkage analysis using the BETA program. Results: Evidence for genetic linkage of allergic conjunctivitis was obtained for chromosomes 5, 16 and 17. Weak linkage was detected for chromosome 6 when studies were restricted to specific allergens. No evidence for linkage was detected for chromosomes 11 and 12. Conclusions: Consistent with heritability analysis discussed in this paper, genetic linkage for allergic conjunctivitis is shown to differ from that reported for atopic asthma. This indicates that there are likely to be organ-specific disease susceptibility genes, which together with general atopy genes target the allergic response to specific mucosal tissues.
Inhibition of Allergic Inflammation by C-Terminal Peptides of the Prohormone Submandibular Rat 1 (SMR-1)
Authors: Dery R.E. , Mathison R. , Davison J. , Befus A.D.
Published: April 2003

Background: The C-terminal of the prohormone submandibular rat 1 protein (SMR-1) contains several small peptides that reduce the severity of allergic inflammation and septic shock, and are part of the cervical sympathetic trunk-submandibular gland (SMG) axis of neuroendocrine immunology. These peptides include the heptapeptide, submandibular gland peptide-T and the tripeptide FEG. The D-isomeric form of this tripeptide, feG, which is active when administered orally, reduces LPS-provoked leukocyte rolling on mesenteric venules and influx of inflammatory cells into the peritoneum and intestinal muscle. Methods: To investigate the mechanism of action of these peptides, the influx of inflammatory molecules into the airways, and several properties of human neutrophils were examined. Results: Oral feG (1 mg/kg) inhibited the influx of inflammatory cells into the airways lumen of allergen challenged, sensitized Brown Norway rats. This inhibition occurred whether feG was given 30 min prior to 6 h post allergen challenge. Moreover, feG in picomolar to nanomolar concentrations inhibited PAF elicited chemotaxis by 30–40%, but the peptides did not affect superoxide production or phagocytosis by neutrophils. feG reduced PAF-stimulated expression of CD11b. Conclusions: feG may exert its anti-inflammatory effects by modulating the expression and functions of β2 integrins. The CST-SMG axis may be a major neuroendocrine pathway that modulates allergic asthma and other inflammatory responses.
Aminooxypentane-RANTES Induces CCR3 Activation and Internalization of CCR3 from the Surface of Human Eosinophils
Authors: Elsner Jörn , Dulkys Yasmin , Kimmig Daniela , Wells Timothy N.C. , Proudfoot Amanda E.I. , Kapp Alexander
Published: April 2003

Eosinophils are predominant effector cells in allergic diseases attracted by several CC chemokines into the inflammatory tissue. According to their important role in attracting leukocytes, several kinds of chemokine receptor antagonists have been developed. Therefore, the aim of this study was to investigate the effect of aminooxypentane (AOP)-RANTES on the activation of the CC chemokine receptor 3, CCR3, exemplary on human eosinophils, because they represent the dominant CCR3+ cell type. AOP-RANTES dose-dependently induced an increase of intracellular calcium concentration ([Ca2+]i) and a release of reactive oxygen species, which could be inhibited by pertussis toxin, in human eosinophils from normal nonatopic donors. AOP-RANTES was as effective as RANTES but less effective than eotaxin and eotaxin-2 in the activation of the respiratory burst. Flow-cytometric analyses revealed that eosinophils constitutively expressed the CC chemokine receptors CCR1 and CCR3, whereas CCR5 was not expressed. AOP-RANTES, RANTES, eotaxin and eotaxin-2, but not Met-RANTES, induced a downregulation of CCR3 at 37°C. Reexpression of CCR3 on eosinophils was observed within 120 min. Whereas no differences of CCR3 downregulation and recycling after stimulation with AOP-RANTES, RANTES, eotaxin and eotaxin-2 were found there exists a distinct profile of activity with respect to the activation of the respiratory burst in human eosinophils.
A Naturally-Processed Universal T Cell Epitope Is Located Within a Conserved Region of Der p 2: Implications for Allergen Sensitization and Immunotherapy
Authors: Wu Bo , Vander Elst Luc , Jacquemin Marc , Saint-Remy Jean-Marie R.
Published: April 2003

Background: Major allergens could represent a ‘port of entry’ to allergen polysensitization. The immunogenic properties of Der p 2 T cell epitopes were evaluated to test this hypothesis. Material and Methods: T cells were obtained from regional lymph nodes of mice belonging to four different haplotypes and used in proliferation assays using Der p 2 or synthetic peptides. Results: T cells from recombinant (r) Der p 2-immunized Balb/c mice (H-2d) proliferated to an immunodominant region encompassing residues 21–35, which also stimulates T cells of mice expressing three alternative haplotypes (H-2b, H-2k and H-2s), indicating the presence of a universal T cell epitope. Epitope mapping identified Ile28 as a critical H-2-binding residue for all four different haplotypes. p21–35 presents a sequence homology with FIS, another universal T cell epitope. FIS priming boosts immune responses to distinct weak immunogens. Balb/c mice primed with p21–35 or FIS produced stronger immune responses to transferrin upon administration of the latter than unprimed mice. Besides, this procedure profoundly affected the overall B cell response to rDer p 2. Conclusion: An immunodominant T cell epitope of Der p 2 behaves as a universal epitope and enhances responses to related and unrelated coadministered antigens. These findings have implications for the understanding of allergen polysensitization and for immunotherapy.
Dolphin-Assisted Seawater Therapy for Severe Atopic Dermatitis: An Immunological and Psychological Study
Authors: Iikura Y. , Sakamoto Y. , Imai T. , Akai L. , Matsuoka T. , Sugihara K. , Utumi M. , Tomikawa M.
Published: April 2003

We performed seawater therapy on atopic dermatitis patients for 6 days with dolphins. The presence of the dolphins seemed to alleviate the pain patients experienced while bathing in seawater. The skin condition improved dramatically, and immunologically, serum IL-8 levels decreased but MIP-1α and eosinophil cationic protein did not change.