bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelets Exit Venules by a Transcellular Pathway at Sites of F–Met Peptide–Induced Acute Inflammation in Guinea Pigs
Authors: Feng Dian , Nagy Janice A. , Pyne Kathryn , Dvorak Harold F. , Dvorak Ann M.
Published: April 2003

Platelets maintain the integrity of vascular endothelium, but also appear outside of blood vessels in pathological states such as acute inflammation. However, it is widely believed that platelets extravasate from blood vessels only as the result of endothelial injury and that, on contacting extravascular collagen, they undergo a morphologically defined activation sequence and release their granule contents. We here report that platelets may cross intact venular endothelium without exhibiting this release reaction or injury. Platelets became adherent to the luminal surface of venular endothelium within ∼15 min of intradermal injection of 10–5M N–formyl–methionyl–leucyl–phenylalanine in guinea pig flank skin. Individual intact platelets were noted in large endothelial cell cytoplasmic vacuoles from which they subsequently migrated abluminally. They then crossed the vascular basal lamina and entered the dermis without exhibiting evidence of a release reaction. Serial electron–microscopic sections confirmed that the cytoplasmic vacuoles within which platelets crossed endothelial cells were independent of interendothelial cell junctions which remained normally closed. Platelets extended pseudopods and gave other evidence of cell motility. These findings require a paradigm shift in our thinking about platelet movement and functions.
Serum HIV–1 RNA Levels Compared to Soluble Markers of Immune Activation to Predict Disease Progression in HIV–1–Infected Individuals
Authors: Zangerle Robert , Steinhuber Sabine , Sarcletti Mario , Dierich Manfred P. , Wachter Helmut , Fuchs Dietmar , Möst Johannes
Published: April 2003

We assessed the value of HIV–1 RNA level compared to soluble immune activation markers, namely neopterin, β2–microglobulin and soluble TNF receptor 75 (sTNFR–75), to predict the change in the number of CD4+ T cells over a 1–year period, the development of AIDS, and survival (median follow–up 54 months). The study population comprised a cohort of 47 individuals for the analysis of the change in CD4+ T cells and survival (20 died), and a subgroup of 31 individuals with a baseline CD4+ T cells above 200×106/l for the development of AIDS (11 developed AIDS). HIV–1 RNA was measured from stored sera by quantitative PCR. The CD4+ T cell count obtained at study entry strongly correlated with baseline serum HIV–1 RNA levels (r = –0.47), and to a lesser extent with neopterin (r = –0.41) and β2–microglobulin (r = –0.29). The percentage change in CD4+ T cells over a 1–year period correlated with HIV–1 RNA levels (r = –0.32, p = 0.03), however, stronger correlations were found for neopterin, β2–microglobulin and sTNFR–75 (r = –0.51, r = –0.41, r = –0.42; p<0.01). No progression to AIDS or death was observed in individuals with baseline HIV–1 RNA levels below 20,000 copies/ml (10 of 31 and 15 of 47 individuals, respectively). A Cox’s proportional hazard model to predict AIDS revealed that HIV–1 RNA, the change in CD4+ cells over a 1–year period and sTNFR–75 levels independently predict AIDS; the change in CD4+ cells, the absolute CD4+ T cell count and neopterin were jointly significant to predict death. All results were adjusted for nucleoside monotherapy. In conclusion, to improve the predictive power, quantitation of HIV–1 RNA as a 'natural history marker’ may be supplemented by measurement of sTNFR–75 for 'early’–stage disease progression and neopterin for 'late’–stage disease progression.
Inhibition of Mediator Release from Dispersed Nasal Polyp Cells by Cyclosporin A
Authors: Lebel Bernard , Crampette Louis , Vergnes Christophe , Campbell Alison M. , Bousquet Jean
Published: April 2003

The mechanisms of action of cyclosporin A require further elucidation since this drug includes anti–inflammatory properties unrelated to its previously documented effect of T cells. A study was performed using enzymatically dispersed cells from nasal polyps of 7 subjects to examine the effects of cyclosporin A on the release of histamine, leukotriene C4/D4 (LTC4/D4) and thromboxane (TxB2) following stimulation by anti–IgE. Cells were resuspended and preincubated with cyclosporin A (0.1, 1 and 10 μM) or 0.1% DMSO (the vehicle used to dissolve cyclosporin A) for 20 min prior to challenge with 10 μg/ml ε–chain–specific anti–IgE for 45 min at 37°C. Histamine, LTC4/D4 and TxB2 were measured using EIA. Cyclosporin A significantly inhibited the release of histamine, LTC4/D4, and TxB2 in a concentration–dependent manner. IC30 values, histamine (5.1 μM), LTC4/D4 (7.8 μM) and TxB2 (6.2 μM), were determined. These data demonstrate new antiallergic properties of cyclosporin A using a novel in vitro model which mimics more closely allergic inflammation.
The Genotype Determines the B Cell Response in Mercury–Treated Mice
Authors: Johansson Uno , Hansson-Georgiadis Helén , Hultman Per
Published: April 2003

Background: Mercury causes in mouse strains of the H–2s haplotype an autoimmune syndrome with antibodies to the nucleolar protein fibrillarin and systemic immune complex (IC) deposits. Other strains, like BALB/C (H–2d), develop only IC deposits, and most strains are resistant. However, mercury activates the murine immune system and causes lymphoproliferation in most strains: H–2s strains are high–responders also in this respect, while the relation between lymphoproliferation and autoimmune manifestations is unclear for other strains. We examined the B cell response to mercury in order to better understand the relation between lymphoproliferation and systemic autoimmunity, using the high–responder H–2s strains (A.SW and SJL), the intermediate responder strain BALB/C (H–2d), and the A.TL (H–2tl) and DBA/2 (H–2d) strains which are resistant to systemic autoimmunity. Methods: During 4–7 weeks of subcutaneous mercuric chloride injections, the number of B cells and the expression of cell surface activation and proliferation markers was monitored by flow cytometry. The number of cytoplasmic Ig+ splenocytes was determined by direct immunofluorescence technique on slides, and serum Ig isotype levels as well anti–ssDNA and anti–DNP antibodies were determined by ELISA. Serum ANA were monitored weekly by indirect immunofluorescence technique. Results: Mercury–treated A.SW and SJL mice (H–2s) developed an increased expression of the proliferation marker CD71 on B cells, an increased number of B cells in the spleen, and an early, strong, but transient increase in serum Ig concentrations of Th1– as well as Th2–regulated Ig isotypes. Mercury–treated H–2s mice rapidly developed a polyclonal B cell response including the IgM isotype, but also antinucleolar antibodies (ANoA) of the IgG isotype with a clumpy pattern, characteristic for antifibrillarin antibodies. The IgG ANoA response was of a long duration and high titer. The A.TL strain (H–2tl) showed only a slight, restricted B cell activation. The BALB/C strain developed a slight, transient B cell activation dominated by IgG1 and IgE, and antinuclear antibodies (ANA). The DBA/2 strain showed only a minimal B cell response without ANA. Conclusion: Mercury induces an early, transient, polyclonal B cell activation linked to the H–2A or H–2K locus in H–2s strains on the A background. This polyclonal response differs from the long–lasting, high–titered IgG autoantibody response to a nucleolar antigen with characteristics of fibrillarin in H–2s strains, which indicates that these responses arise from separate mechanisms. Another group of strains, exemplified by BALB/C (H–2d), responds to mercury with a slight, transient, Th2–dominated B cell response, a restricted antibody specificity targeting the cell nucleus, and systemic IC deposits. Another H–2d strain, DBA, is essentially resistant to mercury, illustrating the importance of non–H–2 genes for regulating the response to mercury.
Induction of Protection against Oral Infection with Cytotoxin–Producing Escherichia coli O157:H7 in Mice by Shiga–like Toxin–Liposome Conjugate
Authors: Fukuda Tadashi , Kimiya Takako , Takahashi Motohide , Arakawa Yoshichika , Ami Yasushi , Suzaki Yuriko , Naito Seishiro , Horino Atsuko , Nagata Noriyo , Satoh Sachihiro , Gondaira Fumio , Sugiyama Jun-ichi , Nakano Yoshio , Mori Masahito , Nishinohara Shouichi , Komuro Katsutoshi , Uchida Tetsuya
Published: April 2003

We have previously reported that purified Shiga–like toxins (SLT), SLT–I and SLT–II coupled with liposomes induced a substantial amount of anti–SLT–I and anti–SLT–II IgG antibody production, respectively, in mice. The levels of anti–SLT antibody in the sera of SLT–liposome–immune mice correlated well with the protection against subsequent challenge with SLT. In this study, mice were immunized intraperitoneally with the mixture of SLT–I–liposome and SLT–II–liposome and protection against oral infection with cytotoxin–producing Escherichia coli O157:H7 was evaluated. All of the mice that received immunization with the mixture of SLT–I–liposome and SLT–II–liposome were protected against subsequent intravenous challenge with 10 LD50 of either SLT–I or SLT–II. Eight weeks after primary immunization, mice were inoculated intragastrically with 109 CFU of E. coli O157:H7 strain 96–60. All SLT–liposome–immune mice tested survived without any apparent symptom while control mice died within 5 days. In addition, as shown by other antigen–liposome conjugates, SLT–liposome induced undetectable anti–SLT IgE antibody production while they induced substantial amounts of anti–SLT IgG antibodies. These results suggest that SLT–liposome conjugate may serve as a candidate vaccine that induces protection against cytotoxin–producing E. coli infection.
Denaturation of Ovalbumin Abrogates Oral Induction of Airway Hyperreactivity and IgG1, IgG2 Antibody Responses in Guinea Pigs
Authors: Peng Ho-Jen , Tsai Jaw-Ji , Chang Zo-Nan , Shen Horng-Der , Tsai Lai-Chen , Su> Song-Nan
Published: April 2003

Background: The effects of denaturation of ovalbumin (OVA) on the induction of oral sensitization in guinea pigs were examined. Methods: Guinea pig antibody and airway responses were assessed after 10 feedings of chemically or heat–denatured OVA or egg white (EW). Results: Their specific IgG, IgG1 and IgG2 antibody responses were orally sensitized by OVA or EW, but not by chemically or heat–denatured OVA or EW. When further exposed to 0.1% OVA or conalbumin aerosol, those fed OVA or EW, but not denatured OVA or EW, had increased pulmonary resistance and decreased tidal volume. On the other hand, in those fed denatured OVA, boiled EW or saline only, a second sensitization with 1% OVA aerosol generated antibody responses and airway hyperreactivity. Using a sandwich ELISA, guinea pig serum OVA was detected after feeding EW, but not chemically denatured or boiled EW. Conclusions: It is likely that guinea pig gut absorption of OVA may result in oral sensitization. Chemical or heat denaturation of proteins may minimize their intestinal uptake and thus abrogates the induction of oral sensitization in guinea pigs.
Eosinophil Development and Bone Marrow and Tissue Eosinophils in Atopic Asthma
Authors: Robinson Douglas S. , North Janet , Zeibecoglou Kyriaki , Ying Sun , Meng Qiu , Rankin Sara , Hamid Qutayba , Tavernier Jan , Kay A. Barry
Published: April 2003

Background: Eosinophils develop from bone marrow (BM) progenitors, and interleukin–5 (IL–5) and eotaxin may act in expansion and mobilisation of BM eosinophils in asthma. Methods: We have examined phenotypic changes as CD34+ cells develop to the eosinophil lineage in vitro, and have evaluated BM eosinophils from asthmatic and control subjects for expression of the eotaxin receptor, CCR3. Results: Acquisition of receptors for IL–5 and CCR3 was an early event in eosinophil development. There were increased CD34+ cells, and mature and immature CCR3+ eosinophils in BM from asthmatics. Conclusion: These data suggest that IL–5 may act early in eosinophil development, and that eotaxin has the capacity to mobilise a BM eosinophil pool in asthma.
An in vitro Model for the Allergen–IgE–FcεRI Interaction
Authors: Laffer Sylvia , Hogbom Erik , Adrianson Jonas , Grönlund Hans , Sillaber Christian , Valent Peter , Vangelista Luca , Kricek Franz , Kraft Dietrich , Valenta Rudolf
Published: April 2003

Background: The interaction of immune complexes consisting of allergens and allergen–specific IgE with the high–affinity Fcε receptor represents the key event in the induction of symptoms in type I allergic individuals. Immediate–type symptoms result from the release of biological mediators due to allergen–induced cross–linking of FcεRI receptors on mast cells and basophils, whereas FcεRI–mediated presentation of allergen–IgE complexes may contribute to late–phase symptoms through enhanced T cell activation. The interaction of allergens/allergen–specific IgE/FcεRI represents, therefore, an important target for therapeutic intervention strategies in type I allergy. Methods and Results: A molecular model of the allergen–IgE–FcεRI interaction was established. It consists of recombinant purified Bet v 1, the major birch pollen allergen, a chimeric Bet v 1 specific monoclonal IgE antibody, and the baculovirus–expressed purified human alpha chain of FcεRI. The chimeric Bet v 1–specific IgE antibody consists of the light chain and the heavy chain variable region of a mouse monoclonal Bet v 1 specific antibody, Bip 1, and the constant region of human IgE. The interaction of rBet v 1, chimeric Bip 1, and human alpha chain was investigated by overlay experiments. Nitrocellulose–immobilized recombinant alpha chains was incubated with chimeric Bip 1 and, for control purposes, with mouse–derived Bip 1. Bound chimeric Bip 1 was detected with 125I–labeled rBet v 1. The specific interaction of rBetv 1, chimeric Bip 1, and recombinant human alpha chain is demonstrated. We thus establish a molecular model of the allergen/IgE/alpha chain interaction. The usefulness of the described in vitro system is exemplified by the identification of a mouse monoclonal antihuman IgE antibody which blocked the IgE–alpha chain interaction. Conclusions: The module system consisting of rBet v 1, chimeric Bip 1, and recombinant alpha chain may be used for the identification of competitors of the allergic effector reaction by means of high throughput screening of compounds or by combinatorial chemistry.
Mimotope and Anti–Idiotypic Vaccines to Induce an Anti–IgE Response
Authors: Stadler Beda M. , Zürcher Adrian W. , Miescher Sylvia , Kricek Franz , Vogel Monique
Published: April 2003

We have defined epitopes on human IgE by screening different phage display random peptide libraries with a monoclonal anti–IgE antibody termed BSW17. The selected mimotopes and epitopes within the Cε3 and Cε4 region of IgE induced antibodies that were nonanaphylactogenic and had biological activity similar to BSW17. The chemically synthesized and KLH–coupled IgE epitopes or mimotopes were used to induce an anti–IgE response in rhesus monkeys. The immunized rhesus monkeys were subsequently protected in a PCA test when sensitized with human IgE and triggered with the corresponding allergen. Furthermore, using the same monoclonal anti–IgE antibody, we also generated an anti–idiotypic antibody that showed sequence homology with the IgE epitope in the Cε3 domain. This anti–idiotypic antibody as well as the mimotopes were then used in a mouse model to induce orally an anti–IgE immune response. For this purpose mice were fed by intragastric gavages with bacteriophages displaying the small IgE–homologous structures. Orally immunized mice produced serum anti–IgE antibodies that were inhibited by BSW17 suggesting that it may be possible to induce a systemic anti–IgE response orally.
Cloned Th Cells Confer Eosinophilic Inflammation and Bronchial Hyperresponsiveness
Authors: Kaminuma Osamu , Mori Akio , Ogawa Koji , Nakata Aya , Kikkawa Hideo , Ikezawa Katsuo , Okudaira Hirokazu
Published: April 2003

Background: The essential role of Th cells and T cell cytokines in eosinophilic inflammation has been established. Methods: To determine whether Th cells are sufficient for the development of airway eosinophilic inflammation, ovalbumin–reactive murine Th clones were established and infused into unprimed mice. Results: Eosinophilic infiltration into the lung was induced upon antigen inhalation in parallel with the rise in bronchoalveolar lavage fluid (BALF) eosinophil peroxidase activity. Neither IgG, IgA, nor IgE antibodies were present in this model. Pathological examination showed swelling and desquamation of epithelial cells, mucous plugs, and goblet cell hyperplasia, all of which well resemble human asthma. Fluorescent probe labeled Th clones migrated into the lung prior to the eosinophil accumulation. Bronchial hyperresponsiveness (BHR) was clearly induced upon antigen inhalation. Anti–IL–5 monoclonal antibody abrogated the responses. Dexamethasone and cyclosporin A suppressed cytokine production by Th cells both in vitro and in vivo, BALF eosinophilia, and BHR. The number of eosinophils recovered in the BALF correlated with the intensity of BHR. Conclusion: The results clearly indicated that monoclonal Th cells are sufficient for the development of both airway eosinophilia and BHR. Agents capable of downregulating IL–5 production seem promising for the treatment of bronchial asthma.
Allergen–Induced Sensory Neuroplasticity in Airways
Authors: Undem Bradley J. , Hunter Dawn D. , Liu Mark , Haak-Frendscho Mary , Oakragly Angela , Fischer Axel
Published: April 2003

This study investigates the influence of allergic inflammation in airway sensory innervation. We conclude that allergic inflammation in the guinea pig leads to both an increase in excitability, as manifested by an increase in the mechanical sensitivity of the airway nerve endings, and an induction of substance P production in airway sensory neurons. The data are consistent with the hypothesis that the induction of substance P occurs in fast conducting nodose sensory neurons that were previously devoid of this neuropeptide. Thus, allergen challenge is associated with a phenotypic change in the airway tachykinergic innervation. We also provide evidence that nerve growth factor is a potentially important mediator for these effects, and that it is elevated in the bronchoalveolar lavage of asthmatic subjects.
Candidate Gene and Mutational Analysis in Asthma and Atopy
Authors: Wilkinson Jane , Thomas N. Simon , Morton Newton , Holgate Stephen T.
Published: April 2003

Background: Using quantitative phenotype scores, we have genotyped four markers close to the FcεRI–β gene on chromosome 11q and 17 markers on chromosome 12. We have also determined the frequency of the I181L/V183L and E237G polymorphisms in our population. Methods: 131 randomly ascertained families and 109 families with an asthmatic proband were recruited. Written and video questionnaires, bronchial challenge, and skinprick tests were administered and IgE levels measured. Phenotype scores were derived using principal–component analysis. The asthma score incorporated the questionnaire data reduced to two variables (wheeze and video), the ratio of predicted to observed FEV1, and bronchial hyperresponsiveness calculated as the slope of the dose–response curve. Total IgE, with the highest heritability of the atopy variables, was used as the atopy score. I181L/V183L polymorphism was determined by sequencing and E237G polymorphism by the amplification refractory mutation system. The data were analyzed using the BETA programme. Results: No examples of the I181L/V183L polymorphism were identified. The E237G polymorphism was identified with a frequency of 3.5% with weak evidence for linkage (lod 1.522) to asthma. Linkage was found to markers on chromosome 12 and asthma. The largest single locus lod was achieved for D12S366 and wheeze (lod 3.307). Using multipoint analysis, a maximum lod score of 2.29 centres around D12S97 at location 173.5 cM for the asthma score. Conclusion: The linkage results for chromosome 12 justify further interest in this region. Future endeavours will be directed towards fine mapping in the hope of identifying novel candidate genes.
OX40–Mediated Cosignal Enhances the Maturation of Naive Human CD4+ T Cells into High IL–4–Producing Effectors
Authors: Delespesse G. , Ohshima Y. , Yang L.-P. , Demeure C. , Sarfati M.
Published: April 2003

Our previous studies have indicated that naive human CD4+ T cells of neonatal or adult origin may be the initial source of IL–4 which is required for their development into Th2 effectors. In addition to minute amounts of IL–4, anti–CD3/B7.1–activated naive cells also release readily detectable levels of IL–13 and IFN–γ. The production of IL–4 and IL–13 by naive T cells is differentially regulated by TGF–β and IL–12. Shortly after activation, naive T cells express surface OX40, a TNF–R family member whose ligand (OX40L) is constitutively expressed on a subset of dendritic cells. Engagement of OX40 on activated naive T cells increases their expression of IL–4 and IL–13, suppresses that of IFN–γ and promotes their development into Th2–like effectors.
IP–10 Mediated Reinforcement of Human Type 1 Cytokine Synthesis to Environmental Allergens among Non–Atopic Subjects
Authors: Gangur Venugopal , Simons F. Estelle R. , HayGlass Kent T.
Published: April 2003

Background: Non–atopic subjects do not differ from those with allergic rhinitis or asthma in their ability to respond to environmental antigens, but in the nature of their response. IP–10, a CXC (α) chemokine, is produced by a wide variety of cell types, and differs from most chemokines in its specificity for activated lymphocytes. Here, we examine its potential role in the maintenance of putatively protective, type–1–dominated cytokine responses among non–atopic individuals. Methods: The impact of rIP–10 on polyclonally driven, grass–pollen–and cat–antigen–stimulated type 1 (IFNγ) and type 2 (IL–4) cytokine responses was examined in short–term cultures of fresh PBMC from non–atopic subjects directly ex vivo. Results: rIP–10 selectively enhanced polyclonally driven and antigen–specific IFNγ, but not IL–4, responses, with median increases ranging from 100 to 400% following polyclonal activation and 20 to 30–fold in antigen–driven IFNγ responses. rIP–10 levels with maximal activity were at or below those previously reported for maximal chemotactic activity and, most importantly, were within the range of those seen in plasma obtained from non–atopic subjects during the grass pollen season. Conclusions: The data suggest a potential role for IP–10 in the maintenance of protective, type–1–dominated responses in non–atopic subjects. They raise the possibility that this chemokine could be of therapeutic value in individuals with established type–2–dominated responses, e.g. in patients with immediate hypersensitivity.
Is Enhanced Fetal Growth a Risk Factor for the Development of Atopy or Asthma?
Authors: Beasley Richard , Leadbitter Philip , Pearce Neil , Crane Julian
Published: April 2003

Background: The aetiological factors underlying the worldwide increase in the prevalence of asthma and the international patterns of the prevalence of asthma are not well understood. This has led to consideration of factors such as fetomaternal health which may programme the initial susceptibility to allergic sensitisation, or contribute to the development of asthma independently of sensitisation. Methods: A number of epidemiological studies have examined the relationship between birth anthropometric measures (as a marker of fetomaternal health) and the subsequent development of asthma and atopy in childhood or adult life. Results: Some, but not all of these studies have reported a relationship between enhanced fetal growth and an increased risk of asthma and/or atopy. Conclusion: These findings raise the hypothesis that factors responsible for fetal growth may also lead to programming of the developing respiratory or immune systems, predisposing to the subsequent development of asthma and/or atopy. This hypothesis may explain, in part, the increasing prevalence of asthma and atopy over recent decades, which has occurred concurrently with secular trends for improved fetomaternal health, as measured by anthropometric measurements at birth.
The Recognition Pattern of Sequential B Cell Epitopes of Beta–Lactoglobulin Does Not Vary with the Clinical Manifestations of Cow’s Milk Allergy
Authors: Heinzmann Andrea , Blattmann Sabine , Spuergin Peter , Forster Johannes , Deichmann Klaus A.
Published: April 2003

Background: β–Lactoglobulin (BLG) represents one of the major allergens causing cow’s milk allergy (CMA) – a disease with a wide spectrum of clinical symptoms. The aim of this study was to evaluate sequential B cell epitopes of BLG by the Pin–ELISA method. Furthermore, we wanted to investigate a possible association of the IgE recognition patterns in sera of patients with BLG sensitization and the type of clinical reactions following contact with cow’s milk. Methods: Overlapping sequential decapeptides corresponding to the amino acid sequence of BLG were used in Pin–ELISAs specific for human IgE. Tested sera were from 14 individuals with CMA, 8 of them with a history of immediate systemic reactions and 6 with delayed skin reactions following contact with cow’s milk. All of them showed specific IgE antibodies to BLG in the CAP–RAST. Control sera were from 5 healthy nonallergic individuals. Results: All sera from BLG–sensitized individuals showed IgE binding with one region of BLG corresponding to amino acids 95–113. Furthermore, individual sera showed reactions with two further regions, 12–27 and 124–135. Inhibition of IgE binding to BLG with one soluble synthetic peptide confirmed the major epitope. No differences were found in the B cell epitope recognition pattern to BLG in the two groups of patients with CMA, characterized by acute systemic or delayed skin reactions. Conclusions: Using IgE Pin–ELISAs we were able to confirm previously described sequential B cell epitopes of BLG. However, the recognition pattern of one of the major cow’s milk allergens is not predictive of the clinical type of reaction.
Antibodies to HIV-1 gp41 Recognize Synthetic Peptides of Human IFN-α and IFN-β
Authors: Bai Yun , Zhao Yingxu , Yu Tianwei , Dierich Manfred P. , Chen Ying-Hua
Published: April 2003

Based on our finding that a common epitope exists between HIV-1 gp41 and human type I interferons (IFN-α and IFN-β), and increased levels of antibodies against human IFN-α and IFN-β were observed in HIV-1-infected individuals, we tried to explain the mechanism of increased levels of antibodies. Mouse antisera recognizing HIV-1 recombinant soluble (rs) gp41 (aa 539–684) interacted with two synthetic peptides sequence-corresponding to the IFN-α/β receptor binding site on human IFN-α and IFN-β, while normal mouse serum (pooled normal sera) did not. The anti-rspg41 antisera after adsorption by IFN-β sepharose column lost the activity of interaction with both synthetic peptides. In another experiment, rsgp41 could bind to sepharose column conjugated with anti-IFN-β polyclonal antibodies (IgG). These results indicate that the common epitope on gp41 and type I interferons could induce antibodies recognizing the receptor binding site on IFN-α and IFN-β, suggesting that increased levels of antibodies against IFN-α and IFN-β in HIV-1-infected individuals could be induced by gp41.
Cleavage of Human IgE Mediated by Schistosoma mansoni
Authors: Pleass Richard J. , Kusel John R. , Woof Jenny M.
Published: April 2003

Background: IgE-mediated mechanisms are important in protection against helminth parasites. However, schistosomes are long-lived in mammalian hosts, presumably as a result of immune evasion strategies. We sought evidence for one such strategy, namely specific cleavage of host IgE. Methods: Human IgE, IgA and IgG were incubated with extracts from cercarial and schistosomular stages of Schistosoma mansoni or with schistosomular culture supernatants. The resulting products were analysed by Western blotting with Ig-specific antibodies. Numerous protease inhibitors were assessed for ability to inhibit the observed cleavage of IgE by the extracts. Partial purification of the IgE-proteolytic activity from cercarial extract was achieved by gel filtration. To test IgE function, we compared the abilities of untreated and schistosomular-treated IgE to mediate rosette formation through interaction with Fcε receptors. Results: Cercarial and schistosomular extracts were found to cleave human, mouse and rat IgE but not human IgA1, IgA2 or IgG1. Schistosomular culture supernatants displayed similar proteolytic activity towards IgE. Immunoblotting suggested that cleavage occurred close to the Cε2/Cε3 domain interface of the IgE heavy chain. PMSF and elastatinal inhibited cleavage, suggesting that the protease involved is an elastase-like serine protease, particularly since porcine pancreatic elastase also cleaved IgE to give similar-sized products. Further, the chloromethyl ketone derivatized peptide MeO-Suc-Ala-Ala-Pro-Leu- CMK, known to specifically inhibit the schistosome elastase, prevented IgE cleavage. Cleavage of human IgE rendered the antibody molecule unable to interact with U937 cells expressing FcεRII. Conclusions: An elastase-like protease in S. mansoni is able to render IgE non-functional.
Dietary Nucleotides Can Up-Regulate Antigen-Specific Th1 Immune Responses and Suppress Antigen-Specific IgE Responses in Mice
Authors: Nagafuchi Shinya , Hachimura Satoshi , Totsuka Mamoru , Takahashi Takeshi , Goto Masao , Yajima Takaji , Kuwata Tamotsu , Habu Sonoko , Kaminogawa Shuichi
Published: April 2003

Background: It has been reported that dietary nucleotides enhance T helper cell activities. In this study, we have determined the effects of dietary nucleotides on antigen-specific Th1 and Th2 responses and IgE responses. Methods: Ovalbumin (OVA)-specific T cell receptor (TCR) transgenic (OVA-TCR Tg) mice, 3 weeks old, were fed a nucleotide-free diet (NT(–) diet) or the NT(–) diet supplemented with dietary nucleotides (NT(+) diet) for 4 weeks. Cytokine production by spleen cells and macrophages obtained from these mice was measured in vitro. BALB/c mice, 3 weeks old, immunized intraperitoneally with OVA adsorbed onto alum, were fed the NT(–) diet or the NT(+) diet for 4 weeks. Serum levels of antigen-specific antibodies in the BALB/c mice were determined by ELISA. Results: The level of production of antigen-specific interferon-γ by spleen cells was significantly higher in the OVA-TCR Tg mice fed the NT(+) diet than in the control mice. The levels of secretion of bioactive IL-12 by spleen cells and peritoneal macrophages were also significantly increased in the NT(+) diet group. The serum OVA-specific IgE level was significantly decreased in BALB/c mice fed the NT(+) diet compared with those fed the NT(–) diet. Conclusion: These results show that dietary nucleotides up-regulate the antigen-specific Th1 immune response through the enhancement of IL-12 production and suppress the antigen-specific IgE response.
A Sensitive Monoclonal Antibody Sandwich ELISA for the Measurement of the Major Olive Pollen Allergen Ole e 1
Authors: van Ree Ronald , Aalbers Marja , Kea Olga , Marco de la Calle Francisco M. , Sempere Ortells José M. , Villalba Mayte , Rodriguez Rosalia , Aalberse Rob C.
Published: April 2003

Background: Olive pollen is a major cause of inhalant allergy in countries around the Mediterranean sea. The major allergen of olive pollen is Ole e 1. Measurement of the major allergen content of allergen products for diagnosis and therapy is becoming an essential element of standardization protocols. This study aimed at the development of a monoclonal antibody (mAb) sandwich ELISA for Ole e 1. Methods: Balb/c mice immunized with Ole e 1 were used for the production of mAbs. Screening of mice and hybridomas was performed in a RIA with radiolabeled purified Ole e 1. Purified mAbs were used as catching and/or (biotinylated) detecting antibodies in sandwich ELISA. Results: Four mAbs (IgG1κ) directed to nonoverlapping epitopes on Ole e 1 were obtained: 1A12, 5C1, 10A12 and 3H8. Both 1A12 and 10A12 were successfully used for affinity purification of Ole e 1 from olive pollen extract. Two sandwich ELISAs were developed, with 1A12 and 10A12 as catching, and 5C1 and 3H8 as detecting antibodies, respectively. Both catching and detecting antibodies were used in similar concentrations, ranging from 60 to 100 ng/well. For both ELISAs, the sensitivity was approximately 1 ng/ml of Ole e 1. The measuring range was from 1 to 25 ng/ml. No significant differences were observed, when the performance of both ELISAs in standardization of olive pollen extracts was compared. Conclusions: Two sensitive sandwich ELISAs for the major olive pollen allergen Ole e 1 were developed. They will prove to be useful tools in allergen standardization protocols.
Hexamethylene Diisocyanate Causes Contraction of Canine Tracheal Smooth Muscles through Activation of Muscarinic Receptors
Authors: Tomari Shinya , Shimoda Terufumi , Kawano Tetsuya , Mitsuta Kazuko , Obase Yasuishi , Matsuse Hiroto , Fukushima Chizu , Kohno Shigeru
Published: April 2003

Background: Asthma caused by occupational exposure to hexamethylene diisocyanate (HDI) is well known; however, the exact pathogenic mechanisms remain unclear. Methods: Experiments were performed using a standard canine tracheal smooth muscle (CTSM) strip preparation in an isolated bath to determine the effect of HDI on tracheal smooth muscle contraction. HDI concentration-response curves were constructed and the effects of different receptor antagonists on HDI-induced smooth muscle contraction were determined. To determine whether HDI and acetylcholine (ACh) bind to a common muscarinic receptor, ACh concentration-response curves in the absence or presence of HDI and concentration-response curves for HDI and ACh in the presence or absence of atropine were plotted. Results: HDI caused contraction of CTSM, with a threshold concentration of 10–7 M. The EC50 (HDI concentration that produced 50% of the maximal response) was 6.2 ± 0.7 × 10–7 M and the maximal contractile response (174 ± 55 g/g of tissue) occurred at a concentration of 5.0 ± 0.8 × 10–6 M. Atropine, a muscarinic blocker, significantly inhibited HDI-induced contractile responses. HDI shifted the ACh concentration-response curve to the right. The mean pA2 for atropine against ACh (8.93 ± 0.27) was not significantly different from that against HDI (8.03 ± 0.12). Conclusions: Our results indicated that HDI causes contraction of CTSM through the activation of muscarinic receptors. Direct stimulation of muscarinic receptors by HDI may play an important role in HDI-induced asthma.
Structure and Biology of Stinging Insect Venom Allergens
Authors: King Te Piao , Spangfort Michael D.
Published: April 2003

Bees, fire ants and vespids cause insect sting allergy. These insects have unique as well as common venom allergens. Vespids, including hornets, paper wasps and yellow jackets, have common allergens. Bees and vespids have one common allergen with hyaluronidase activity; they also have unique allergens with different phospholipase activities. Fire ants and vespids have one common allergen, antigen 5 of unknown biologic activity. The common venom allergens with < 70% sequence identity have barely detectable levels of antigenic cross-reactivity. Possible uses of modified allergens for immunotherapy are described.
Evolution in the Understanding of Cross-Reactivities of Respiratory Allergens: The Role of Recombinant Allergens
Authors: Pauli G.
Published: April 2003

The aim of this review is to show the impact of the use of purified and recombinant allergens to discriminate between co- and cross-sensitization to respiratory allergens. The author describes the evolution of diagnostic tests over the last decades; the tests initially allowed the detection of simultaneously positive cutaneous tests and/or simultaneous positivity of specific IgE to different allergen extracts, but they did not differentiate cross-sensitization from co-sensitization. RAST inhibition studies with crude extracts then established cross-reactivity, but did not identify the cross-reactive allergens involved. Later, immunoblot and CRIE inhibition were able to detect multiple cross-reactive allergens and to assess their physicochemical properties. But it is only since purified and recombinant allergens have been used in the different investigations that identification of cross-reactive allergens has been made possible at a molecular level. This historical approach is illustrated by examples selected from some of the main respiratory allergen sources: tree pollen, grass pollen, weed pollen, acarids, cockroaches and mammalians. For each of these allergen sources, the author gives an updated presentation of major and minor cross-reactive allergen molecules and refers to the last decade’s major publications concerning immunochemical investigations carried out in the field of cross-reactive respiratory allergens. Emphasis is placed on the clinical applications for allergic patients: improvement in the accuracy of the diagnosis of sensitization, new concepts of immunotherapy based on genetically engineered hypoallergenic variants of cross-reactive allergens used alone or in combination, evaluation of allergen load with environmental tests using monoclonal antibodies against cross-reactive allergens.
A New Bioassay Using Transient Transfection for Invasion-Related Gene Analysis
Authors: Platet Nadine , Garcia Marcel
Published: April 2003

To understand the mechanisms of tumor invasion and metastasis, model systems are required that isolate the individual steps of these complicated, multifaceted processes. We propose a new procedure to identify genes involved in cell invasion and/or motility that features the combined advantages of transient gene transfection and Matrigel invasion assays. Cancer cells were transiently cotransfected with two vectors expressing the gene of interest and luciferase, as a marker of transfected cells, and then assayed for Matrigel invasion. Luciferase cotransfection appeared to be a sensitive semiquantitative assay for transfected cells and was maximal throughout the invasion assay. The proposed transfection procedure, using calcium phosphate precipitation, did not affect cell invasiveness and allowed cellular coexpression of both genes. When applying this method, we found that transient expression of the unliganded and liganded human estrogen receptor α prevented invasiveness of MDA-MB-231 breast cancer cells. In conclusion, we propose rapid and versatile in vitro procedure for studying the effects of individual cloned genes on cellular processes, such as invasion and motility.
Maximum Effect of Urokinase Plasminogen Activator Inhibitors in the Control of Invasion and Metastasis of Rat Mammary Cancer
Authors: Evans Douglas M. , Sloan-Stakleff Kimberly D.
Published: April 2003

Experimentally induced pulmonary metastases of mammary cancer in the Fisher 344 rat can be suppressed by the inhibition of urokinase plasminogen activator (uPA). The inhibition of uPA with amiloride or B428 has been shown to be dose dependent. Increased dosage levels of inhibitors might be expected to enhance levels of suppression of metastases. The use of each of these inhibitors at equipotent concentrations that exceeded the doses administered in previous studies failed to eliminate pulmonary metastases. These results demonstrate that a maximum limit is attained for the inhibitory capacities on cells during in vitro invasion or in vivo metastasis. At increased levels, uPA inhibitors continue to suppress, but do not eradicate, experimental pulmonary metastases of MATB cell rat mammary cancer.
The A Enhancer of Polyomavirus: Protein-Protein Interactions for the Differential Early and Late Promoter Function under Nonreplicating Conditions
Authors: Shivakumar Chittari V. , Das Gokul C.
Published: April 2003

The A enhancer of the polyomavirus early promoter is a 110-bp domain located in the late region and it contains the major late RNA initiation site. The ‘core’ of this enhancer binds several cellular proteins, including proteins PEA1, PEA2 and PEA3. Another element, NF-D/YY1, is also located in this enhancer. The A enhancer is known to stimulate the early promoter, contains auxiliary elements for replication and serves as the initiator for late transcription. It may also be involved in early-to-late switch. We were interested in investigating how the A-core- and NF-D-binding proteins regulate early and late promoter activity under nonreplicating conditions and how the protein-protein interactions affect the function of the individual elements. By point mutational analysis, we show that, except for PEA1, all other proteins activate the early and late promoters differentially under nonreplicating conditions. All three core-binding proteins, and the protein bound to the NF-D site, are activators and have a combinatorial effect on early promoter activity. On late transcription, only PEA1 acts positively and inactivation of the NF-D site is without any effect. In contrast, PEA2 and PEA3 have a repressor-like activity under nonreplicating conditions, indicating that these two proteins might be involved in repressing late transcription, probably early in infection. By increasing the spacing between two consecutive elements, we further show that protein-protein interaction is important for enhancer function. Transactivation of the early promoter was affected by mutations in all four protein-binding sites. Responsiveness of these factors in regard to the late promoter was parallel to their intrinsic promoter strength. The effects of middle and large T antigens are parallel for both the early and the late promoter, suggesting that the pathway(s) for transactivation function of these oncoproteins may overlap downstream. This study with cloned viral promoter will be reflective of situations in vivo, at least partially, under nonreplicating conditions.
Nucleotide Sequence of the VP7 Gene of Human Rotavirus Isolated in Calcutta, India: Possible Emergence of a New Subtype of Serotype I
Authors: Chakladar Abhijit , Chakrabarti Sekhar
Published: April 2003

We have investigated the serotype-specific glycoprotein gene (VP7) of a short electropherotype and subgroup I rotavirus (ID 45/2) recovered from a child suffering from acute diarrhea. The nucleotide sequence of the human (ID 45/2) rotavirus genome segment 8/9, which encodes the serotype-specific glycoprotein (VP7) has been determined. Comparison of the nucleotide sequence of ID 45/2 to the sequences of other rotaviruses belonging to different serotypes (G1–14) revealed only 67–78% identity. Comparison of the deduced amino acid sequence of ID 45/2 VP7 protein to the sequences of other VP7 proteins showed that the majority of the amino acid differences are clustered at the N-terminus (residues 1–49), with a positional shift of the initiation codons as well as possession of one stretch of hydrophobic signal peptide instead of two. On the basis of other rotaviral characteristics, such as the conserved cysteine and proline residues together with the potential glycosylation site, it is proposed that this local strain (ID 45/2) might represent a new subtype related to serotype G1. Dot blot hybridization using ID 45/2 cDNA as probe indicated the prevalence of this strain in Eastern India.
Predictive Value of Serological Tests in Rubella Virus Infection during Pregnancy
Authors: Pustowoit Barbara , Liebert Uwe G.
Published: April 2003

In an attempt to define diagnostic criteria that may help to distinguish the congenital rubella syndrome (CRS) from subclinical intrauterine rubella virus (RV) infection, maternal and fetal serum samples were analyzed using (1) enzyme immunoassay employing RV synthetic peptides as antigen, (2) IgG avidity assay, and (3) immunoblot under nonreducing conditions, in addition to hemagglutination inhibition and commercial enzyme immunoassays. Infants born with CRS and their mothers were shown to reveal low or undetectable levels of E2-specific antibodies and deficient IgG recognizing the major neutralizing antibody-inducing epitope on the E1 protein (SP15). Antibody responses were normal in mothers with presumed RV reinfection as well as in asymptomatic infants born after maternal primary rubella. The results indicate that the maturation of specific humoral immune responses is obviously less efficient when intrauterine RV infection results in CRS. The detection of high avidity IgG, conformational E2-specific as well as SP15-reactive antibodies may serve as a potential predictor for a benign outcome of intrauterine RV infections.
Fetal Cells from Maternal Blood: Purpose, Biological Questions, Technical Challenges
Authors: Bohmer Ralph M.
Published: April 2003

The maternal peripheral blood circulation can serve as a source of fetal cells for the prenatal diagnosis of genetic abnormalities, eliminating the need for fetal cell sampling by invasive techniques. However, the extreme scarcity of these cells leads to a variety of biological questions and technical hurdles on the way to a clinical test. On the biological side, we need to know the numbers of fetal cells, their distinguishing properties, and the variables that affect these properties. On the technical side, we need to identify fetal cells and maximize both yield and purity of the isolation procedure. Here we review the questions and challenges as they present themselves in our specific approach to the fetal cell isolation project. We also briefly discuss the question of whether these cells could help to diagnose fetal infections.
Evaluation of Antibodies to the Epstein-Barr Virus Immediate Early Gene Product ZEBRA by a New Enzyme-Linked Immunosorbent Assay
Authors: Yamauchi Yasuyuki , Tachiband Yoichi , Maeda Akihiko , Wakiguchi Hiroshi , Usui Masahiko , Kurata Takeshi , Sairenji Takeshi
Published: April 2003

For the serodiagnosis of Epstein-Barr virus (EBV) infections, we have developed a new enzyme-linked immunosorbent assay (ELISA) for antibodies to the ZEBRA product of EBV immediate early gene BZLF1. ZEBRA protein fused with glutathione-S-transferase (GST) was expressed in Escherichia coli and purified by affinity chromatography with glutathione-Sepharose 4B. An ELISA sandwich capture system was constructed with the GST-ZEBRA immobilized on plastic microtiter plates which had been coated with a mouse monoclonal antibody to GST. ZEBRA-IgG antibodies in patients’ sera with chronic active EBV infection (CAEBV) and infectious mononucleosis (IM) had, respectively, very high and high titers. Anti-ZEBRA antibodies were also detected at low titers in sera of some healthy controls. ZEBRA-IgM antibodies were detected in sera of patients with IM and CAEBV but not in sera of healthy controls. In sera of patients with CAEBV, the titers of IgG antibodies to ZEBRA correlated with the antibody titers to early antigens obtained with an immunofluorescence assay, but not to EBV nuclear antigens. This ELISA is a useful diagnostic and prognostic test for EBV infection.
Typing of Hepatitis C Virus by a New Method Based on Restriction Fragment Length Polymorphism
Authors: Buoro Sabrina , Pizzighella Sergio , Boschetto Roberta , Pellizzari Lorenzo , Cusan Monica , Bonaguro Roberta , Mengoli Carlo , Caudai Cinzia , Padula Maria , Egisto Valensin Pier , Palù Giorgio
Published: April 2003

A new restriction fragment length polymorphism (RFLP) analysis has been developed for hepatitis C virus (HCV) typing in the viral 5′ non-coding region and contiguous core region. These genomic sequences were chosen for the relative nucleotide homology among different genotypes and for the presence of polymorphic sites. By employing two endonucleases (AccI and MboI) and, in some instances, a third one (EcoRII), we can unambiguously and reproducibly distinguish between genotypes and subtypes 1a, 1b, 1c, 2a, 2c, 2b, 3a, 3b, 4a, 5a and 6a. The method was applied for diagnosing two Italian groups of HCV-infected individuals reflecting a randomly collected population and a group of intravenous drug users. The accuracy of this method has been validated by comparison with INNOLiPA and by sequencing. Our approach represents an improvement over previous RFLP methods, since typing is accurate and simpler.