bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Inhibition of LPS-Activated Human Monocyte-Induced Thrombin Generation by Unfractionated Heparin and Low Molecular Weight Heparins
Authors: Nguyên Philippe , Rémy Marie-Geneviève , Potron Gérard
Published: April 2003

Monocyte tissue factor may play a role in the physiological or pathological triggering of blood coagulation. It is well known that unfractionated heparin and low molecular weight heparins inhibit extrinsic thrombin generation. However, this notion has never been confirmed in a physiological model using tissue factor obtained from stimulated human monocytes. This is the purpose of this study. It was important to obtain a pure preparation of monocytes with no platelet contamination. This was possible by leukapheresis and elutriation. Under sterile and endotoxin-free conditions, the process does not activate tissue factor expression by monocytes. We adapted the technique of thrombin generation on an automatic analyzer and used human monocyte tissue factor to trigger thrombin generation. Our results show that unfractionated and low molecular weight heparins potently inhibit monocytic tissue factor induced thrombin generation. The comparison of low molecular weight heparins suggests that molecules with higher anti-IIa/anti-Xa ratios exert a stronger inhibitory effect. These data may be relevant to explain the therapeutic effects of unfractionated and low molecular weight heparins in cardiovascular disorders such as unstable angina.
Comparison of Different Methods to Measure Fibrinogen Concentration in Canine Plasma with Respect to Their Sensitivity towards the Fibrinogen Degradation Products X, Y and D
Authors: Mischke R. , Menzel D. , Wolling H.
Published: April 2003

In this study, fibrinogen measurements according to the Clauss method, photometric method and Jacobsson method have been investigated to find out how they are influenced by adding in vitro the purified canine fibrinogen degradation products (FDP) X, Y and D. Test results according to the Clauss method were found to be underestimated if the fragments X, Y and D were added while measurements according to the Jacobsson method turned out to underestimate the real fibrinogen concentration if the FDP Y and D were added. The Clauss method was particularly sensitive towards FDP. Results were considerably underestimated even with a quantity as little as 0.05 g FDP Y or FDP D/g fibrinogen (p < 0.05). The photometric method was only affected by FDP X leading to false high results. If FDP X was added, fibrinogen values were also overestimated with the Jacobsson method. Our results demonstrate that the photometric method is the most accurate.
How to Grow a Baby: A Reevaluation of Image-Schema and Piagetian Action Approaches to Representation
Authors: Müller, Ulrich , Overton Willis F.
Published: April 2003

This essay examines the development of representational thought from two divergent perspectives: (a) Mandler’s image-schema theory, and (b) an action-theoretical approach fundamentally derived from Piaget’s theory. Juxtaposition of the two approaches highlights conceptual problems that are inherent in image-schema theory but become resolved through an action-theoretical approach. Empirical studies in the domains of early concept development, object permanence, causality, and memory are critically reviewed with emphasis on the question of the interpretive validity of findings suggesting an early onset of mental representation. It is concluded that the empirical findings fail to support the hypotheses of early onset, and that representational development is more adequately interpreted within the context of an action-theoretical approach. Finally, the analysis examines the role of the divergent metatheoretical assumptions that contextualize image-schema theory and action theory in the understanding of the development of mental representation.
The Study of Social Interaction in Educational Settings: Accomplishments and Unresolved Issues1
Authors: Mehan Hugh
Published: April 2003

The study of face-to-face interaction in educational settings is placed in historical context. The major themes of interactional analysis – that social and cognitive structures are constructed in social interaction, human behavior is context-specific, cultural discontinuity helps explain educational inequality, and learning is a sociocultural process – are reviewed, and the contributions of these findings to theory, methodology and pedagogy are assessed. The paper concludes with a discussion of two unresolved issues: the integration of social structure and interaction in interactional analysis and the reconciliation of conflictual and consensual dimensions of learning.
Reinstating Modernity in Social Science Research – or – The Status of Bullwinkle in a Post-Postmodern Era
Authors: Kahn Jr. Peter H. , Lourenço Orlando
Published: April 2003

In recent years, postmodern critiques have enlarged in scope, and increasingly confronted traditional social scientists with challenges: epistemic, methodological, and moral. In this article, we offer a two-pronged response. First, we speak to problems within postmodern theory itself. We argue that, when taken seriously, the theory leads to contradictions in epistemology, to fragmentation in knowledge, to opportunism in interpersonal relationships, and to nihilism in moral action and commitment. Second, we demonstrate how many of the legitimate concerns of postmodernists can and are addressed in current ‘modern’ research programs. It is our hope that postmodernity – for those who believe that that is where we are – will give way to the post-postmodern era: modernity itself, reinvigorated.
Developmental Perspectives on Morningness-Eveningness and Social Interactions
Authors: Friedrich Cofer Lynette , Grice James W. , Sethre-Hofstad Lisa , Radi Christopher J. , Zimmermann Laura K. , Palmer-Seal Deborah , Santa-Maria Georgia
Published: April 2003

Developmental perspectives on sleep/wake rhythmicity have been neglected in research, yet evidence from chronobiological sources, infant sleep data, and adult light treatment studies suggest continuities in individual differences in internal synchronization to light-dark cycles. Developmental systems suggest maintenance through strong contextual constraints, perturbed only by unusual alterations in social demands and light exposure, such as shift work. Retrospective data from university students, employing the Morningness-Eveningness Questionnaire (MEQ), supported predicted continuity of sleep/wake patterns and associated states of alertness and fatigue as a function of time of day as well as relations with different patterns of social interactions with parents over daily routines in childhood and adolescence. Eveningness is related to greater difficulty meeting familial and social demands for morning performance than Morningness.
An ex vivo Study of Foam Cell Formation and Cholesterol Clearance in Macrophages Isolated from Patients with Hypertriglyceridemia and Low High-Density Lipoprotein Cholesterol Treated with High-Dose Simvastatin
Authors: Rodriguez Annabelle , Fishelevich Rita
Published: April 2003

High-density lipoproteins (HDL) are thought to be cardioprotective due to their role in decreasing cholesterol accumulation and in enhancing cholesterol removal from foam cells. Our study will examine foam cell formation and HDL-mediated cholesterol removal from macrophages isolated from patients with hypertriglyceridemia and low HDL. Peripheral blood monocytes and lipoproteins will be isolated from donors before and after 6–12 weeks on high-dose simvastatin (80 mg). The isolated lipoproteins from each donor will be incubated with their own macrophages for the study of foam cell formation and HDL-mediated cholesterol efflux. We expect to show that simvastatin increases HDL cholesterol, decreases triglyceride levels, and is associated with less foam cell formation and greater HDL-mediated cholesterol clearance.
Heterogeneous Ethnic Distribution of the 844ins68 in the Cystathionine β-Synthase Gene
Authors: Franco Rendrik F. , Elion Jacques , Lavinha João , Krishnamoorthy Ragajopal , Tavella Marli H. , Zago Marco A.
Published: April 2003

A novel mutation in the cystathionine β-synthase (CBS) gene (a 68-bp insertion in the coding region of exon 8: 844ins68) was recently described, but its prevalence in different human populations is unknown. We analyzed the prevalence of the 68-bp insertion in the CBS gene in 405 unrelated individuals (810 chromosomes) of European, African, Asian and Amerindian origins. PCR amplification of a DNA fragment containing exon 8 of the CBS gene was employed. In addition, screening for the T833C CBS mutation by BsrI digestion was carried out in all samples bearing the 844ins68 mutation, since both mutations were previously reported to be associated in cis. The insertion was found in heterozygosity in 14 out of 104 whites (13.5%), was absent among Asians, and was found solely in 1 out of 220 Amerindian chromosomes analyzed, whereas a much higher prevalence was observed among blacks (37.7% of heterozygotes and 4% of mutant homozygotes). Furthermore, the T833C CBS mutation was found to cosegregate in cis with 844ins68 in all carriers of the insertion. The finding of the double mutant among blacks and Caucasians suggests that it antedated the racial divergence between Africans and non-Africans, and provides evidence for a partly or completely neutralizing effect conferred by the 68-bp insertion, since it allows the skipping of the T833C mutation. Our study represents the first analysis of the 844ins68 insertion in the CBS gene in different human populations, and reveals an extensive ethnic and geographic variability associated with this mutation.
Apolipoprotein B 5’-Ins/Del and 3’-VNTR Polymorphisms in Chinese, Malay and Indian Singaporeans
Authors: Choong M.L. , Koay E.S.C. , Khaw M.C. , Aw T.C.
Published: April 2003

The allele frequencies for the apolipoprotein B (apo B) 5′-Ins/Del and 3′-VNTR polymorphisms varied significantly (p < 0.01) among Singaporeans of Chinese, Malay and Indian descent. We calculated the unbiased expected heterozygosities for the 5′-Ins/Del polymorphism as 0.3357, 0.1984 and 0.2418, and for the 3′-VNTR as 0.5980, 0.5260 and 0.6749, respectively, in the Chinese, Malays and Indians. Compared to heterozygosities reported for other populations, the Singaporeans differed from most Caucasians in having significantly lower values but were closely related to other non-Caucasians. Thirteen alleles, with a bimodal distribution, were observed at the 3′-VNTR polymorphic locus; the alleles occurring most frequently among the Chinese and Malays were of 35 or 53 repeats, and among the Indians, of 37 or 47 repeats. The Del allele was associated with elevated serum cholesterol (p = 0.023), LDL-cholesterol (LDL-C) (p = 0.001) in the Chinese, and apo B (p = 0.007) in the Indians. Likewise, the larger 3′-VNTR alleles (> 41 repeats) were associated with raised cholesterol (p = 0.018), LDL-C (p = 0.025), and triglyceride (p = 0.001) in the Chinese. The two polymorphisms were not in significant linkage disequilibrium (D = –0.0029, p = 0.494) in the three ethnic groups.
Epitope Specificity of Monoclonal and Polyclonal Antibodies to Human Elastin
Authors: Wei S.  M. , Katona E. , Fachet J. , Fülöp Jr. T. , Robert L. , Jacob M.P.
Published: April 2003

Polyclonal (pAb) and monoclonal (mAb) anti-human aorta elastin antibodies were reacted with a series of overlapping hexapeptides along the human tropoelastin sequence covering exons 2–7 and 23–36 from the N-terminus to the C-terminus, advancing 1 amino acid residue each time. ELISA indicated reactive epitopes. mAb A2.1 recognized sequences containing Ala-Lys, mAb G8.1, A7.1 and pAb, hydrophobic sequences. None of them reacted with the hexapeptide VGVAPG, or with desmosine or isodesmosine. pAb L85 reacted with a His-containing sequence coded in exon 26A. pAb *E(L), *E(S) and L85 reacted with the Cys-containing sequence of exon 36. A synthetic 14-residue peptide containing the three proximal tyrosines coded in exon 13 did not react with any of the antisera tested. It appears therefore that the most frequently recognized epitopes are hydrophobic sequences. One polyclonal antibody detected several isoforms of tropoelastin in the medium of cultured vascular smooth muscle cells. Monoclonal and polyclonal antibodies stained elastic fibers on tissue sections, suggesting that the epitopes recognized are available on the native fibers for reaction with the antibodies.
Experimental Study for the Development of an in vitro Test for Contact Allergens
Authors: Arimura Makoto , Yokozeki Hiroo , Katayama Ichiro , Nakamura Tsuneaki , Masuda Mitsuteru , Nishioka Kiyoshi
Published: April 2003

We have previously reported an in vitro hapten-specific sensitization method using Pam-212 cells (in vitro sensitization test) to identify the potential effectiveness of contact allergens. In the present study, we conducted comparison studies of 11 allergens and 2 irritants in order to evaluate the method as an alternative predictive test. The guinea pig maximization test (GPMT) was developed based on the test described by Magnusson and Kligman. Our assay was carried out as follows: we treated Pam-212 cells with 13 test chemical solutions, while T cells and macrophages of BALB/c mice were cultured with hapten-conjugated Pam-212 cells for 5 days. After incubation, 105 T cells were stimulated with mitomycin-C-treated spleen cells conjugated with chemicals. Three days later, the [3H]methyl thymidine incorporation was counted. The results of the GPMT were in agreement with those reported in previous studies except for benzocaine. In our GPMT experiments, benzocaine was negative, but it had been classified as a moderate sensitizer in previous studies. Our assay detected extreme, strong and moderate sensitizers as previously classified by the GPMT. They could be summarized as follows: three of five chemicals classified as moderate sensitizers, and 100% of strong or extreme sensitizers were detected by both the GPMT and the in vitro sensitization test. No irritants showed a positive reaction in our assay. These results support the view that the sensitivity of our in vitro test may be equivalent to that of the GPMT and may be useful as a rapid and objective allergen screening test.
To E or not to E?Can an IL-4-Induced B Cell Choose between IgE and IgG4?
Authors: Vercelli Donata , De Monte Lucia , Monticelli Silvia , Di Bartolo Chiara , Agresti Alessandra
Published: April 2003

Parasite immunologists had known for some time that IgE-mediated hypersensitivity reactions are rare in patients with chronic helminth infections, even though basophils and mast cells in these patients are sensitized with antiparasite IgE and exposed, often continuously, to parasite antigens. The inhibition of allergic reactivity in chronic helminth infections is mainly due to IgG4 ‘blocking antibodies’ in the serum of the infected individual. IgG4 do not fix complement and bind weakly to Fcγ receptors. Thus, antigen binding by IgG4, unlike IgE, is likely to have no or minimally harmful consequences. The discovery that, similar to IgE, expression of IgG4 is IL-4-dependent and is an intermediate step in sequential switching from IgM to IgE makes it imperative to understand how the two isotypes are coregulated and whether the two responses can be uncoupled, selectively boosting IgG4 over IgE. The ultimate goal is to apply to allergy the lesson we learnt from helminth infections.
Organs from Animals for Man
Authors: Hammer C. , Linke R. , Wagner F. , Diefenbeck M.
Published: April 2003

In the following review some of the problems of xenotransplantation shall be discussed, based on the few experimental data available so far and on reports in the literature describing investigations which may be of importance for xenotransplantation. The impact of gravity on the upright posture of man versus almost all other mammals, the dysfunction between enzymes and hormones in different species and the lack of interactions between interleukins, cytokines and vasoactive substances will be taken into consideration. The question must be asked whether different levels of carrier molecules or serum proteins play a role in the physiological network. Even though the development of transgenic animals or other imaginative manipulations may lead to the acceptance of any type of xenografted organ, it has to be established for how long the products of the xenografts are able to act in the multifactorial orchestra. We are far from understanding xenogeneic molecular mechanisms involved in toxicity, necrosis and apoptosis or even reperfusion injury and ischemia in addition to the immediate mechanisms of the hyperacute xenogeneic rejection. Here, cell adhesion, blood clotting and vasomotion collide and bring micro- and macrocirculation to a standstill. All types of xenogeneic immunological mechanisms studied so far were found to have a more serious impact than those seen in allogeneic transplantation. In addition we are now only beginning to understand that so-called immunological parameters in allogeneic mechanisms act also in a true physiological manner in the xenogeneic situation. These molecular mechanisms occur behind the curtain of hyperacute, accelerated, acute or chronic xenograft rejection of which only some folds have been lifted to allow glimpses of part of the total scene. Other obstacles are likely to arise when long-term survival is achieved. These obstacles include retroviral infections, transfer of prions and severe side effects of the massive immunosuppression which will be needed. Moral, ethical and religious concerns are under debate and the species-specific production of proteins of the foreign donor species developed for clinical use suddenly appears to be a greater problem than anticipated.
IL-8 and the Activation of Eosinophils and Neutrophils following Nasal Allergen Challenge
Authors: Jacobi Henrik H. , Poulsen Lars K. , Reimert Claus M. , Skov Per S. , Ulfgren Ann-Kristin , Jones Ilona , Elfman Lena B. , Malling Hans-Jørgen , Mygind Niels
Published: April 2003

Background: A growing body of evidence suggests that proinflammatory cytokines play a role in allergic inflammation by attracting and activating inflammatory cells. In this study, we have investigated the relationship between interleukin-8 (IL-8) in nasal lavage fluid and the local activation of eosinophils and neutrophils following nasal allergen challenge of allergic patients. Methods: Nasal challenges were performed with grass pollen extract in 14 allergic patients and 5 nonallergic controls. Nasal lavage fluid was collected repeatedly for 10 h, and the levels of eosinophil cationic protein (ECP) and myeloperoxidase (MPO) were used as markers of eosinophil and neutrophil activation, respectively. The levels of these molecules were compared with that of IL-8 in nasal lavage fluid. Results: Allergen challenge of allergic patients produced a significant late-phase increase in the levels of ECP and MPO. Furthermore, the level of MPO showed a highly significant correlation with the level of IL-8 in lavage fluid (r = 0.8, p < 0.0001), whereas there was no significant relationship between the levels of ECP and IL-8. Conclusion: Interestingly, our findings suggest that both eosinophils and neutrophils are activated following nasal allergen challenge. In addition, our results are consistent with the hypothesis that IL-8 acts as a chemoattractant/activator of neutrophils during the late phase of the allergic inflammation. In contrast, we were not able to demonstrate any significant relationship between the level of IL-8 in lavage fluid and the activation of eosinophils.
Inhibition of Pulmonary Eosinophilia Does Not Necessarily Prevent the Airway Hyperresponsiveness Induced by Sephadex Beads
Authors: Matsubara Shigeki , Fushimi Keiko , Ogawa Koji , Kikkawa Hideo , Nakata Aya , Kameda Rieko , Kikuchi Matsuo , Naito Kazuaki , Ikezawa Katsuo
Published: April 2003

Background: The Lewis rat among highly inbred strains exhibits significant airway hyperresponsiveness (AHR) following intravenous administration of Sephadex G-200 (Sephadex). The aim of this study was to investigate the association of Sephadex-induced AHR with changes in airway inflammation in Lewis rats. Methods: A suspension (0.5 mg/ml/rat) of Sephadex was intravenously administered to male Lewis rats on days 0, 2 and 5. Measurement of airway responsiveness to serotonin, bronchoalveolar lavage (BAL) and histological study were performed on day 2–11. Results: Significant AHR induced by Sephadex was recognized on day 2 (p < 0.05), and AHR reached a maximum on day 7 (p < 0.001). In the BAL study, eosinophils increased on day 2 (p < 0.01) with a peak on day 5 (p < 0.05). In the histological study, we found Sephadex beads trapped in small arteries of the lung and granulomatous arteritis on day 2 or later. Pulmonary granulomas, horseshoe-shaped multinuclear giant cells, eosinophils and goblet cell hyperplasia were observed on day 2, and the degree became intense on day 5–7. GCC-AP0341 (10 mg/kg, i.p. × 3) inhibited the recruitment of eosinophils in BAL fluid and in lung tissue, but it did not inhibit AHR. The compound also inhibited pulmonary granulomas and goblet cell hyperplasia. Conclusion: The mechanism of Sephadex-induced AHR may not be directly associated with inflammatory changes such as recruitment of eosinophils, pulmonary granulomas and hyperplasia of goblet cells in rats.
Role of CD4+ and CD8+ T cells in Regulating the Chronic Development of Liver Injury Induced by Delayed-Type Hypersensitivity to Picryl Chloride
Authors: Xu Qiang , Wu Feihua , Jiang Jieyun , Lu Jinfu , Chen Xiaochun , Zhang Baoling
Published: April 2003

In this study we first investigated the cellular immune responses in mice with chronic liver injury induced by delayed-type hypersensitivity to picryl chloride (PCl). A continuous reduction, after week 3 of liver injury, was observed in the level of PCl-induced contact sensitivity but not in sheep red blood cell-induced footpad reaction, suggesting the presence of PCl-specific suppression. When spleen cells from mice whose liver had been injured for 1 week were systemically transferred into syngeneic recipients with the liver injury, the elevation in serum lactic dehydrogenase and the decrease in alkaline phosphatase and albumin levels in recipient mice were significantly exacerbated. However, when the liver damage in the donor mouse was allowed to proceed for 3, 5 or 7 weeks, biochemical changes in recipients were reduced to near normal levels. A flow-cytometric assay demonstrated that the number of CD4+ T cells in both spleen cells and liver nonparenchymal cells decreased dramatically during the late phase of liver injury, while CD8+ counts did not. These findings suggest that CD4+ and CD8+ T lymphocytes may contribute to the positive and negative regulation, respectively, of the early and late phases in the chronic development of liver injury.
The Immunoglobulin E–Allergen Interaction: A Target for Therapy of Type IAllergic Diseases
Authors: Valenta Rudolf , Almo Steven , Ball Tanja , Dolecek Christiane , Steinberger Peter , Laffer Sylvia , Eibensteiner Petra , Flicker Sabine , Vrtala Susanne , Spitzauer Susanne , Valent Peter , Denepoux Stephan , Kraft Dietrich , Banchereau Jacques , Lebecque Serge
Published: April 2003

The interaction of immunoglobulin E and otherwise harmless antigens (allergens) leadsin sensitized individuals through effector cell activation to the immediate induction of acascade of inflammatory reactions, the hallmark of type I allergy. Recently, the molecularand structural characterization of allergens, specific IgE antibodies and their epitopes hasmade rapid progress. Here we discuss active and passive strategies for therapy of type Iallergy, which are based on interfering with the IgE–allergen interaction.
Induction of Protection against Tetanus Toxin in Mice by Tetanus Toxoid–Liposome Conjugate
Authors: Naito Seishiro , Horino Atsuko , Komiya Takako , Fukuda Tadashi , Takahashi Motohide , Ami Yasushi , Suzaki Yuriko , Oka Tetsuya , Okuma Kunio , Morokuma Masanori , Nakano Yoshio , Mori Masahito , Nishinohara Shouichi , Komuro Katsutoshi , Uchida Tetsuya
Published: April 2003

Tetanus toxoid (Ttd) was coupled to liposomes via glutaraldehyde. Intraperitoneal injection in BALB/c mice with Ttd–liposomes induced a substantial amount of anti–Ttd IgG antibody production and an extremely low level of anti–Ttd IgE antibody production. Mice immunized with Ttd–liposomes were successfully protected against a subsequent challenge with a lethal dose of tetanus toxin (Ttx). On the other hand, aluminum hydroxide–adsorbed Ttd (Ttd–alum) and plain Ttd solution induced the production of both IgG and IgE antibodies against Ttd. Moreover, secondary immunization with Ttd–liposomes in mice, in which anti–Ttd IgE antibody production was induced by Ttd–alum led to enhanced anti–Ttd IgG and a limited anti–Ttd IgE antibody production. When Ttd–liposome preparation was lyophilized, the efficacy of Ttd–liposomes was maintained for 6 months at 37°C, suggesting that this vaccine preparation would be stable without refrigeration. These results demonstrate the potential ability of Ttd–liposome conjugates to produce a tetanus vaccine which provides protection against (Ttx) while inducing the least amount of anti–Ttd IgE antibodies.
A Mouse Model of Mosquito Allergy for Study of Antigen–Specific IgE and IgG Subclass Responses, Lymphocyte Proliferation, and IL–4 and IFN–Á Production
Authors: Chen Yong Liang , Simons F. Estelle R. , Peng Zhikang
Published: April 2003

To date, there are no ideal animal models for study of natural sensitization leading to IgE– and lymphocyte–mediated hypersensitivities. We established such a model in which four BALB/c mice were each sensitized by exposure to at least 16 mosquito Aedes aegypti bites, twice a week for 4 weeks. Four non–exposed control mice were also studied. Mosquito A. aegypti head and thorax extract, saliva, and two recombinant salivary allergens (rAed a 1 and rAed a 2) were used in vitro as antigens. Intradermal tests were performed. Serum mosquito antigen–specific IgG, IgG1, IgG2a, and total IgE were measured by ELISA; specific IgE was measured by passive cutaneous anaphylaxis (PCA). IgE responses to each antigen in the saliva were analyzed using Western blotting. Spleen lymphocyte proliferation assays were performed to determine the cell–mediated hypersensitivity response. Antigen–induced IL–4 and IFN–γ production in the spleen lymphocytes were evaluated using ELISA. After 4 weeks, all 4 mosquito–sensitized mice developed a positive immediate wheal 20 min after skin tests with mosquito antigens, and a positive delayed papule 24 h later, while control mice did not. Also, the sensitized mice had a positive PCA response, which correlated significantly with total IgE levels (r = 0.84, p<0.05), confirming the presence of antigen–specific IgE, while none of control mice had a positive response. Antigen–specific IgG1, but not IgG2a, was increased in the sensitized mice (p<0.01). Western blotting showed that 5 of the 8 antigens which elicited mouse IgE responses, including 2 major antigens, also elicited human IgE responses. The mean lymphocyte proliferation response to mosquito antigens also elicited human IgE responses. The mean lymphocyte proliferation response to mosquito antigens was significantly increased in the sensitized mice (p<0.05). IL–4 production was significantly increased and IFN–γ production was decreased, further suggesting that a Th2 immune response predominates despite the development of the delayed skin reaction. This new model of natural sensitization without using an adjuvant is potentially useful for the study of other allergic disorders as well as mosquito allergy.
Food Restriction–Mediated Adrenal Influences on Antigen–Induced Bronchoconstriction and Airway Eosinophil Influx in the Guinea Pig
Authors: Underwood David C. , Matthews Jane K. , Osborn Ruth R. , Hay Douglas W.P.
Published: April 2003

The aim of this study was to measure the effects of food restriction on antigen–induced bronchoconstriction and inflammatory cell influx in guinea pigs and to determine the role of plasma cortisol and catecholamine concentrations. Ovalbumin (OA; 0.3 mg/kg, i.v.) was administered to OA–sensitized, anesthetized guinea pigs which had been allowed free access to food or had been food restricted for 18 h prior to OA challenge. In addition to higher plasma levels of epinephrine (30% increase) and cortisol (33% increase), fasted guinea pigs had significantly lower (60% decreased) maximal bronchoconstrictor responses to OA than nonfasted, sensitized litter mates. Additionally, groups of fasted or fed animals were subdivided into two additional treatment groups: (1) saline–pretreated or (2) polyethylene glycol 400 (PEG)–pretreated (1 ml/kg, p.o., 1 h prior to antigen challenge). In saline–treated, fasted animals, bronchoconstrictor responses to antigen were significantly diminished (67% decreased) and epinephrine and cortisol levels were increased (64 and 34%, respectively) compared to the corresponding fed group. In both fasted and fed groups, the PEG–treated guinea pigs had higher plasma epinephrine and cortisol levels than animals which received saline, but no significant differences were detected within the PEG–treated group. Plasma norepinephrine concentrations were lower in all fasted groups. In a separate model in conscious guinea pigs, there were no differences in aerosol OA–induced bronchoconstriction and eosinophil influx between fasted and fed groups. However, compared to the saline pretreatment group, PEG administration reduced the antigen–induced bronchoconstriction and eosinophilia in both fed and fasted guinea pigs. We speculate that the reduced responsiveness to antigen in fasted versus fed animals may result from food–restriction–induced, stress–related release of epinephrine and cortisol from the adrenal glands, thereby suppressing mast cell degranulation or reducing responsiveness to spasmogenic and chemotactic mediators. In addition, the results suggest that oral dosing with 100% PEG may enhance this phenomenon.
111In–Labelled Leukocyte Migration to the Lungs of Ovalbumin–Sensitized Guinea–Pigs after Aerosol Challenge with Ovalbumin Monitored by Gamma Scintigraphy
Authors: Lewis Christine A. , Warren Simon J. , Broadley Kenneth J.
Published: April 2003

Background: To determine whether antigen challenge is associated with increased accumulation of leukocytes in the lungs, the pulmonary accumulation of 111In–labelled neutrophils and eosinophils was monitored by gamma scintigraphy. Methods: Guinea–pigs were sensitized with ovalbumin (OA) 14–21 days before challenge with aerosolized OA (10 mg/ml) for 2 min, and protected against fatal anaphylaxis by mepyramine (30 mg/kg). Comparisons were made with OA–sensitized guinea–pigs challenged with saline. 5 or 24 h following the OA challenge, guinea–pigs were anaesthetized and the jugular vein and right carotid artery were cannulated, with the contralateral artery tied off. 2MBq 99mTc macroaggregated albumin (MAA) was injected intravenously to create a pulmonary perfusion image as a template for the lungs. 111In–labelled neutrophils or eosinophils were then injected via the carotid artery and gamma scintigraphic images obtained. Activity in the lung region of each animal was determined by superimposing the 99mTcMAA image of the lungs on the whole body image. Results: A significant increase in activitiy (p<0.05) in the lung region was observed after injection of 111In–labelled neutrophils at 5 h after the OA challenge compared with saline challenge. 24 h after OA challenge there was a significant increase in activity (p<0.05) in the lung region after injection of 111In–labelled eosinophils, but no change in activity after injection of labelled neutrophils compared with the saline–challenged animals. Conclusion: This technique models the migration of leukocyte to the lungs seen in guinea–pigs in our previous studies, namely an eosinophilia at 24 h and a neutrophilia at 5 h after OA challenge. It will therefore be useful for investigating anti–inflammatory drugs on the airways.
The Importance of Recombinant Allergens for Diagnosis and Therapy of IgE–Mediated Allergies
Authors: Kraft D. , Ferreira F. , Vrtala S. , Breiteneder H. , Ebner C. , Valenta R. , Susani M. , Breitenbach M. , Scheiner O.
Published: April 2003

In the past 10 years, a considerable number of cDNAs coding for allergens have been isolated and expressed. Intensive investigations showed that recombinant allergens and their respective natural counterparts possess comparable properties with respect to structure, function and interaction with the immune system. Recent studies documented that in vitro as well as in vivo diagnosis of IgE–mediated allergic diseases can be successfully improved by the application of recombinant allergens. In addition, new strategies for a safer specific immunotherapy (SIT) have been developed based on the knowledge of the primary structures of allergens. Naturally occurring isoforms of allergens as well as recombinant allergens with modified amino acid sequences show very low IgE binding capacity but strong T cell–stimulatory activity and represent possible candidates. In case of Bet v 1, the major birch pollen allergen, isoforms d, g and l and a Bet v 1a mutant, produced by site–directed mutagenesis resulting in 6 amino acid exchanges, fulfilled the above mentioned criteria. In a third approach, two adjacent peptides covering the entire Bet v 1a sequence were produced in an Escherichia coli expression system. These peptides contained most of the relevant T cell epitopes, but lost their IgE binding capacity and, thus, their ability to activate mast cells and basophils of sensitized patients. Our results suggest that allergen variants (isoforms, mutants, T cell epitope–containing peptides) may be used as ‘hypoallergenic agents’ in SIT.
Novel CD8 Molecule on Macrophages and Mast Cells: Expression, Function and Signaling
Authors: Hirji Nadir S. , Lin Tong-Jun , Gilchrist Mark , Nault Genevieve , Nohara Osamu , Grill Brock J. , Belosevic Mirodrag , Stenton Grant R. , Schreiber Alan D. , Befus A. Dean
Published: April 2003

Background: We previously identified, using flow cytometry and in situ RT–PCR, a novel CD8 molecule on rat alveolar macrophages (AM) and mast cells (MC). RT–PCR also demonstrated that mouse AM express CD8 mRNA. Functional studies on rat AM determined that ligation of CD8 α– and β–chains induced inducible nitric oxide synthase (iNOS) upregulation, nitric oxide (NO), TNF–α and IL–1β (CD8α only) secretion. However, CD8 did not induce AM phagocytosis of IgG–opsonized or unopsonized particles. Rat MC stimulated through CD8 secreted NO and TNF–α, but not histamine. Because of its potential role in regulating cell function, we investigated the signaling pathways involved in macrophage CD8 stimulation. Methods and Results: Inhibitor of src family kinases (PP1) significantly (p<0.05) inhibited CD8α (OX8 antibody)–induced iNOS upregulation, NO, TNF–α and IL–1β production in rat AM. In addition, Ro 31–8220 (a PKC inhibitor) inhibited OX8–induced iNOS upregulation, NO and IL–1β production, but did not inhibit TNF–α production. Using Syk antisense, we further determined that OX8 stimulation of NO is Syk kinase dependent. Conclusion: Studies on the signaling mechanisms of CD8 determined that src family kinases, PKC, and Syk kinase are involved in CD8 signaling. Additionally, CD8 may have differential signaling pathways, as an inhibitor to PKC downregulated OX8–induced IL–1β, but not TNF–α release. Our studies demonstrate that AM CD8 is similar to T lymphocyte CD8 in that src kinases are involved in CD8–mediated signaling. However, p56lck, which is expressed in T lymphocytes, has not been found in macrophages, suggesting that other src family kinases may be involved in AM and MC CD8 signaling.
Novel Autocrine and Paracrine Loops of the Stem Cell Factor/Chymase Network
Authors: de Paulis Amato , Minopoli Giuseppina , Dal Piaz Fabrizio , Pucci Pietro , Russo Tommaso , Marone Gianni
Published: April 2003

Background: The aim of this study was to investigate whether the secretory granules of human mast cells store stem cell factor (SCF). We also addressed the question whether mast cell chymase, a chymotrypsin–like protease, also present in the secretory granules of human mast cells cleaves SCF at the peptide bound between Phe 158 and Met159. Methods: The skin samples were obtained from patients with mastocytosis, undergoing skin biopsy for diagnostic purposes. Mast cells were isolated and purified from human lung parenchyma (human lung mast cells, HLMC) by countercurrent elutriation followed by discontinuous Percoll density gradient. SCF contents of human mast cells were assessed for immunoreactive SCF by ELISA. Western blot analysis of SCF and its cleavage products were performed with the MoAb anti–SCF 7H6. SCF and its proteolytic fragment were characterized by electrospray mass spectrometry (ES/MS). Results: SCF is present in the secretory granules of human skin and lung mast cells. Immunoreactive SCF (iSCF) was detected in the cell lysates of HLMC, but not in basophils. iSCF was rapidly (3 min) released after challenge with anti–IgE, and iSCF in supernatants rapidly declined after 30 min. ES/MS analysis of rhSCF1–166 treated with recombinant human chymase showed a polypeptide of 17,977.1±0.6 Da and a minor component of 697.4±0.1 Da generated by specific cleavage at Phe159. SCF1–166 and SCF1–159 similarly activated HLMC and potentiated anti–IgE–induced activation of these cells. The cleavage product SCF160–166 had no effect on mast cells. Western blot analysis of supernatants of anti–IgE activated HLMC incubated for various intervals with rhSCF1–166 showed that rhSCF1–166 was converted to a faster–migrating form with a molecular weight compatible with SCF1–159 and to several SCF species. Conclusion: SCF is stored in human mast cell secretory granules and is immunologically released by mast cells. SCF1–166 is rapidly cleaved by chymase and other proteases to several SCF species.
The CC Chemokine Receptor Antagonist Met–RANTES Inhibits Eosinophil Effector Functions
Authors: Elsner Jörn , Petering Holger , Kimmig Daniela , Wells Timothy N.C. , Proudfoot Amanda E.J. , Kapp Alexander
Published: April 2003

Eosinophils play an important role in allergic diseases such as allergic asthma, rhinoconjunctivitis and atopic dermatitis. Recruitement of eosinophils to the side of inflammation, the release of reactive oxygen species, leading to tissue damage, and the propagation of the inflammatory response are mediated by chemokines. Thus, the applicability of agents able to inhibit or antagonize chemokine–induced eosinophil activation seems to be of interest in the treatment of allergic diseases. Therefore, the effect of the CC chemokine antagonist, Met–RANTES, on its effect on human eosinophil effector functions in response to RANTES, MCP–3 and eotaxin was investigated. Met–RANTES had no intrinsic activity on [Ca2+]i transients in eosinophils and was able to dose–dependently inhibit [Ca2+]i transients in eosinophils following stimulation with RANTES, MCP–3 and eotaxin. Besides its effect on [Ca2+]i transients, Met–RANTES dose–dependently inhibited actin polymerization in eosinophils and the release of reactive oxygen species following stimulation with RANTES, MCP–3 and eotaxin. The results of this study lead to the conclusion that Met–RANTES is an effective and powerful compound to antagonize effector functions of human eosinophils following stimulation with RANTES, MCP–3 and eotaxin and is therefore a promising therapeutic approach to prevent the invasion and destructive power of eosinophils in allergic diseases.
On the Role of T Lymphocytes in Stimulation of Humoral Immunity Induced by Peptidoglycan–Monomer Linked with Zinc
Authors: Ravlić-Gulan Jagoda , Radošević-Stašić Biserka , Trobonjača Zlatko , Petković Marija , Ćuk Mira , Rukavina Daniel
Published: April 2003

Effects of peptidoglycan linked with zinc (PGM–Zn) were investigated on plaque–forming cell (PFC) generation to sheep red blood cell (SRBC) and SRBC–unrelated antibody production in primary and secondary immune response in mice depleted in vivo of CD4+ and/or CD8+ T lymphocytes. PGM–Zn in nondepleted mice stimulated the PFC generation and IgM or IgG and IgG1 production in primary and secondary reaction. Single depletion of CD4 or CD8+ T cells did not change this ability. The effects of PGM–Zn after CD8+ depletion were even greater than those in nondepleted mice. Depletion of both T cell subsets, however, completely abrogated immunostimulatory effects of PGM on PFC generation (primary and secondary response), as well as on primary SRBC–unrelated antibody production, leaving only the increase of IgG in secondary response unchanged. Immunostimulatory effects and isotype switching to IgG1 and IgG2a correlated with the changes in splenic CD4+, CD8+, CD5+ cells, pointing to the regulatory role of these cells and/or their cytokines in PGM–Zn–induced immunostimulation. Altogether the data suggest that PGM–Zn may potentiate the costimulatory signals coming from activated T cells and act on B cells without the T cell help.
Influx of Neutrophils into the Airway Lumen at 4 h after Segmental Allergen Challenge in Asthma
Authors: Nocker Rob E.T. , Out Theo A. , Weller Frank R. , Mul Erik P.J. , Jansen Henk M. , van der Zee Jaring S.
Published: April 2003

Background: Segmental allergen challenge is a powerful tool to study inflammatory reactions in asthmatic airways. There is little information on the early events at 5 min and 4 h after allergen challenge with respect to the cell influx and the chemokine interleukin–8 (IL–8). Methods: Seven mild to moderate allergic asthmatics (AA group), 5 allergic nonasthmatics (ANA group) and 5 nonallergic controls underwent segmental allergen challenge, with allergen doses based upon skin reactivity. Bronchoalveolar lavage (BAL) samples were obtained before, 5 min and 4 h postchallenge, and were analyzed for cell numbers and differential counts, eosinophil and neutrophil chemotactic activity, and levels of IL–8. Results: At 5 min postchallenge, no changes were observed compared to baseline. At 4 h postchallenge, an increase was found in the number of neutrophils and the levels of IL–8, which was dependent on the dose of allergen in the AA and ANA group. At the same allergen dose, the increases in neutrophils and levels of IL–8 were calculated to be 91 and 67 times higher, respectively, in AA than in ANA. Levels of IL–8 correlated with the number of neutrophils and with the in vitro neutrophil chemotactic activities in BAL fluid. Conclusions: Neutrophil chemotactic activity is increased in BAL fluid at 4 h after segmental allergen challenge. We suggest that apart from IgE–mediated mast cell degranulation, additional local factors in the airways determine the degree of IL–8 increase and neutrophil influx.
Cord–Blood–Derived Human Cultured Mast Cells Produce Interleukin 13 in the Presence of Stem Cell Factor
Authors: Kanbe Naotomo , Kurosawa Motohiro , Yamashita Tetsuji , Kurimoto Fumihiko , Yanagihara Yukiyoshi , Miyachi Yoshiki
Published: April 2003

Background: Mast cells have been regarded as a potential source of cytokines. Although the human mast cell line HMC–1 and human lung mast cells have been shown to produce interleukin (IL) 13, it still remains uncertain whether cord–blood–derived human cultured mast cells produce IL–13. Methods: Human cultured mast cells were raised from cord blood cells in the presence of stem cell factor (SCF) and IL–6. Levels of IL–13 mRNA were examined by a semiquantitative reverse transcriptase–polymerase chain reaction. IL–13 levels in the supernatants were measured with an enzyme–linked immunosorbent assay. Results: When the IgE–sensitized cultured mast cells were activated with anti–IgE, mRNA for IL–13 was amplified with a peak at 3 h after the stimulation. IL–13 was not detected in the supernatants of the activated mast cells in the absence of SCF, whereas the mast cells secreted significant amounts of IL–13 after the stimulation in the presence of SCF. Calcium ionophore A23187 also stimulated the mast cells to release IL–13 into the supernatant in the presence of SCF. Conclusions: These observations suggest that human mast cells can produce IL–13 under the condition with SCF. The cord–blood–derived human cultured mast cells will help in studying the functional properties of human mast cells in allergic diseases.
Topical CTLA4–Ig Suppresses Ongoing Mucosal Immune Response in Presensitized Murine Model of Allergic Rhinitis
Authors: Sato Jun , Asakura Kohji , Murakami Masaaki , Uede Toshimitsu , Kataura Akikatsu
Published: April 2003

Allergic rhinitis is thought to be mediated by CD4+ T cells producing Th2–associated cytokines. Optimal Ag–specific T–cell activation requires the engagement of T–cell receptor with antigen (Ag) in the context of MHC, and the engagement of appropriate costimulatory molecules. One of the most well–characterized costimulatory pathways is the interaction of B7/CD28–CTLA4 molecules. Recent studies have suggested that the costimulatory pathway may influence the development of Th2 immune responses. The objective of this study was the examination of the role of B7/CD28–CTLA4 costimulatory pathway in the pathogenesis of ovalbumin (OVA)–induced immune response in presensitized murine model of allergic rhinitis. Systemically presensitized BALB/c mice significantly developed Ag–induced early phase nasal symptoms, nasal hyperresponsiveness to histamine, nasal eosinophilia, serum levels of OVA– specific IgE and Th2–associated cytokines following repeated topical Ag challenges. Topical administration of CTLA4–Ig during nasal challenges inhibited Ag–induced nasal symptoms and histamine hyperresponsiveness. We also found a significant reduction in nasal lavage eosinophilia and serum levels of OVA–specific IgE. Furthermore, CTLA4–Ig treatment significantly decreased interleukin (IL)–4 content in nasal tissue, while there was no significant change in IL–5 or IFN–γ levels. These results suggest that B7/CD28–CTLA4 costimulatory pathway mediates the development of ongoing Th2 immune responses and plays a major role in regulating allergic disease, such as allergic rhinitis.
The Vascular Perspective of Systemic Sclerosis: Of Chickens, Mice and Men
Authors: Sgonc R.
Published: April 2003

Systemic sclerosis (SSc), or scleroderma, is an autoimmune connective tissue disease characterized by structural and functional vascular abnormalities, perivascular mononuclear cell infiltration, and increased deposition of extracellular matrix in skin and internal organs. The initial stages of SSc are generally not accessible for analysis in man, therefore, the availability of appropriate animal models is of great importance for the elucidation of the pathogenesis of this disease. UCD–200 chickens show the entire clinical, histopathological and serological spectrum of SSc, whereas tight skin (Tsk)1/+ and Tsk2/+ mice, other animal models of scleroderma, lack the vascular injury. A parallel comparative study of skin biopsies of UCD–200 chickens and human SSc patients revealed that endothelial cell apoptosis, induced by anti–endothelial cell antibody (AECA)– dependent cellular cytotoxicity, is a primary event in the pathogenesis of SSc. This review focuses on recently established data on endothelial cell injury in animals with spontaneous disease and humans, AECA, adhesion molecules and cytokine profiles that support a vascular pathogenesis in scleroderma.