Characterization of epitope specificities of reference antibodies used for the quantification of the birch pollen allergen Bet v 1.

BACKGROUND: Accurate allergen quantification is needed to document the consistency of allergen extracts used for immunotherapy. Herein, we characterize the epitope specificities of two monoclonal antibodies used in an ELISA for the quantification of the major birch pollen allergen Bet v 1, established as a reference by the BSP090 European project. METHODS: The ability of mAbs 5B4 and 6H4 to recognize Bet v 1 isoforms was addressed by immunochromatography. The capacity of each mAb to compete with patients' IgE for binding to Bet v 1 was measured by ELISA inhibition. Epitope mapping was performed by pepscan analysis, site-directed mutagenesis, and hydrogen/deuterium exchange-mass spectrometry. RESULTS: The 5B4 epitope corresponds to a peptide sequence (I56-K68) overlapping with the binding sites of patients' serum IgEs. Mutation of residues P59, E60, and K65 abolishes 5B4 binding to Bet v 1 and reduces the level of IgE recognition. In contrast, 6H4 recognizes a conformational epitope lying opposite to the 5B4 binding site, involving residues located in segments I44-K55 and R70-F79. Substitution of E45 reduces the binding capacity of 6H4, confirming that it is critical for the interaction. Both mAbs interact with >90% of Bet v 1 content present in the birch pollen extract, while displaying a weak cross-reactivity with other allergens of the PR-10 family. CONCLUSIONS: MAbs 5B4 and 6H4 recognize structurally distinct epitopes present in the vast majority of Bet v 1 isoforms. These results support the relevance as a reference method of the Bet v 1-specific quantitative ELISA adopted by the European Pharmacopoeia.

Enhancing Allergen-Presentation Platforms for Sublingual Immunotherapy.

Sublingual immunotherapy (SLIT) relies on high doses of allergens to treat patients with type I allergies. Although SLIT is commonly performed without any adjuvant or delivery system, allergen(s) could be further formulated with allergen-presentation platforms to better target oral dendritic cells eliciting regulatory immune responses. Improving the availability of allergens to the immune system should enhance SLIT efficacy, while allowing to decrease allergen dosing. Herein, we present an overview of adjuvants and vector systems that have been, or could be, considered as candidate allergen-presentation platforms for the sublingual route. Such platforms encompass adjuvants capable of stimulating allergen-specific TH1 and/or regulatory CD4+ T-cell responses, including 1,25-dihydroxy vitamin D3, glucocorticoids, Toll-like receptor ligands as well as selected bacterial probiotic strains. A limiting factor for SLIT efficacy is the number of dendritic cells capturing the allergens in the upper layers of oral tissues. Thus, adsorption or encapsulation of the allergen(s) within mucoadhesive particulate vector (or delivery) systems also has the potential to significantly enhance SLIT efficacy due to a facilitated allergen uptake by tolerogenic oral dendritic cells.

The regulatory dendritic cell marker C1q is a potent inhibitor of allergic inflammation.

The complement subunit C1q was recently identified as a marker for monocyte-derived regulatory dendritic cells supporting the differentiation of interleukin (IL)-10-secreting CD4+ T cells with a suppressive activity. Furthermore, C1q expression is upregulated in peripheral blood mononuclear cells of allergic patients in the course of successful allergen immunotherapy. Herein, we investigated a potential direct role of C1q in downregulating allergic inflammation. In mice with ovalbumin (OVA) or birch pollen (BP)-induced allergic asthma, C1q is as efficacious as dexamethasone to reduce both airway hyperresponsiveness (AHR), eosinophil, and ILC2 infiltrates in bronchoalveolar lavages, as well as allergen-specific T helper 2 cells in the lungs. Administration of C1q does not expand IL-10+/Foxp3+ regulatory T cells in the lungs, spleen, or in the blood. Depletion of plasmacytoid dendritic cells (pDCs) abrogates the capacity of C1q to reduce AHR and eosinophilic infiltrates in OVA-sensitized mice. Also C1q treatment inhibits the activation of human and mouse pDCs by CpGs, thereby demonstrating a critical role for pDCs in the anti-inflammatory activity of C1q. We conclude that regulatory dendritic cells can mediate a potent direct anti-inflammatory activity via the expression and/or secretion of molecules such as C1q, independently of their capacity to expand the pool of regulatory T cells.

Development and evaluation of a sublingual tablet based on recombinant Bet v 1 in birch pollen-allergic patients.

BACKGROUND: Sublingual immunotherapy (SLIT) applied to type I respiratory allergies is commonly performed with natural allergen extracts. Herein, we developed a sublingual tablet made of pharmaceutical-grade recombinant Bet v 1.0101 (rBet v 1) and investigated its clinical safety and efficacy in birch pollen (BP)-allergic patients. METHODS: Following expression in Escherichia coli and purification, rBet v 1 was characterized using chromatography, capillary electrophoresis, circular dichroism, mass spectrometry and crystallography. Safety and efficacy of rBet v 1 formulated as a sublingual tablet were assessed in a multicentre, double-blind, placebo-controlled study conducted in 483 patients with BP-induced rhinoconjunctivitis. RESULTS: In-depth characterization confirmed the intact product structure and high purity of GMP-grade rBet v 1. The crystal structure resolved at 1.2 Å documented the natural conformation of the molecule. Native or oxidized forms of rBet v 1 did not induce the production of any proinflammatory cytokine by blood dendritic cells or mononuclear cells. Bet v 1 tablets were well tolerated by patients, consistent with the known safety profile of SLIT. The average adjusted symptom scores were significantly decreased relative to placebo in patients receiving once daily for 5 months rBet v 1 tablets, with a mean difference of 17.0-17.7% relative to the group treated with placebo (P < 0.025), without any influence of the dose in the range (12.5-50 µg) tested. CONCLUSION: Recombinant Bet v 1 has been produced as a well-characterized pharmaceutical-grade biological drug. Sublingual administration of rBet v 1 tablets is safe and efficacious in patients with BP allergic rhinoconjunctivitis.

Characterization of oral immune cells in birch pollen-allergic patients: impact of the oral allergy syndrome and sublingual allergen immunotherapy on antigen-presenting cells.

BACKGROUND: A detailed characterization of human oral immune cells is needed to better understand local mechanisms associated with allergen capture following oral exposure. METHODS: Oral immune cells were characterized by immunohistology and immunofluorescence in biopsies obtained from three healthy individuals and 23 birch pollen-allergic patients with/without oral allergy syndrome (OAS), at baseline and after 5 months of sublingual allergen immunotherapy (AIT). RESULTS: Similar cell subsets (i.e., dendritic cells, mast cells, and T lymphocytes) were detected in oral tissues from healthy and birch pollen-allergic individuals. CD207+ Langerhans cells (LCs) and CD11c+ myeloid dendritic cells (DCs) were found in both the epithelium and the papillary layer of the Lamina propria (LP), whereas CD68+ macrophages, CD117+ mast cells, and CD4+ /CD8+ T cells were rather located in both the papillary and reticular layers of the LP. Patterns of oral immune cells were identical in patients with/without OAS, except lower numbers of CD207+ LCs found in oral tissues from patients with OAS, when compared to OAS- patients (P < 0.05). A 5-month sublingual AIT had a limited impact on oral immune cells, with only a significant increase in IgE+ cells in patients from the active group. Colocalization experiments confirmed that such IgE-expressing cells mostly encompass CD68+ macrophages located in the LP, and to a lesser extent CD207+ LCs in the epithelium. CONCLUSION: Two cell subsets contribute to antigen/allergen uptake in human oral tissues, including (i) CD207+ LCs possibly involved in the physiopathology of OAS and (ii) CD68+ macrophages likely critical in allergen capture via IgE-facilitated mechanisms during sublingual AIT.

Respiratory allergen from house dust mite is present in human milk and primes for allergic sensitization in a mouse model of asthma.

There is an urgent need to identify environmental risk and protective factors in early life for the prevention of allergy. Our study demonstrates the presence of respiratory allergen from house dust mite, Der p 1, in human breast milk. Der p 1 in milk is immunoreactive, present in similar amounts as dietary egg antigen, and can be found in breast milk from diverse regions of the world. In a mouse model of asthma, oral exposure to Der p through breast milk strongly promotes sensitization rather than protect the progeny as we reported with egg antigen. These data highlight that antigen administration to the neonate through the oral route may contribute to child allergic sensitization and have important implications for the design of studies assessing early oral antigen exposure for allergic disease prevention. The up-to-now unknown worldwide presence of respiratory allergen in maternal milk allows new interpretation and design of environmental control epidemiological studies for allergic disease prevention.

Anti-inflammatory activity of sublingual immunoglobulin (SLIG) in a murine model of allergen-driven airway inflammation.

AIM: Intravenous immunoglobulin (IVIG) displays anti-inflammatory activities in many diseases. Subcutaneous administration of anti-IgE in humans provides benefit in severe persistent allergic asthma. Given the well established efficacy of sublingual allergen immunotherapy in respiratory type I allergies, we investigated the therapeutic potential of sublingual immunoglobulin (SLIG), most particularly anti-IgE SLIG, in a murine model of allergen-driven airway inflammation. METHODS: BALB/c mice sensitized with ovalbumin (OVA) were treated sublingually with rat monoclonal IgG1 or IgG2a, either directed to mouse IgE or with no reported specificity. Airway hyperresponsiveness (AHR) was assessed by whole body plethysmography, and eosinophil infiltrates were characterized in bronchial alveolar lavages (BAL). OVA-specific antibody and T cell responses were analyzed in sera and saliva or lung and draining lymph nodes, by ELISA or CBA measurement of cytokine production, respectively. RESULTS: AHR and BAL eosinophil infiltrates were substantially decreased in mice treated sublingually with particulate OVA (positive control), as well as in animals receiving various rat IgG1, irrespective of their specificity for murine IgE. In contrast, no improvement was observed in mice treated with PBS (negative control) or various rat IgG2a. SLIG anti-inflammatory activity is not related to a downregulation of Th2, Th17 or an induction of Foxp3(+) CD4(+) regulatory T cell responses. Mass spectrometry analysis of glycan moieties, such as sialic acid, suggests that the differential efficacy of rat IgG1 and IgG2a is not related to their capacity to interact with lectins borne by oral immune cells. CONCLUSIONS: In a murine model of allergen-driven airway inflammation, SLIG exhibits an anti-inflammatory activity irrespective of the immunoglobulin specificity, and in the absence of allergen. As a noninvasive approach, SLIG deserves to be further studied as a treatment for other inflammatory diseases beyond allergic asthma.

Evaluation of therapeutic sublingual vaccines in a murine model of chronic house dust mite allergic airway inflammation.

BACKGROUND: Second generation therapeutic vaccines based upon recombinant allergens or natural extracts, potentially formulated in vector systems or adjuvants, are being developed. To this aim, preclinical studies in relevant animal models are needed to select proper allergens, formulations and administration schemes. OBJECTIVE: To develop a chronic house dust mite (HDM) allergy model to evaluate sublingual therapeutic vaccine candidates. METHODS: The BABL/c mice that were used were sensitized with Dermatophagoides pteronyssinus (Dpte) and Dermatophagoides farinae (Dfar) mite extracts by intraperitoneal injections followed by aerosol exposures. Animals subsequently underwent sublingual immunotherapy (SLIT) with either Dpte, Dfar or Dpte/Dfar extracts, twice a week for 8 weeks. SLIT efficacy was assessed by whole body plethysmography, lung histology and broncho-alveolar lavages cell counts. Specific T cell and antibody responses to major and minor HDM allergens were monitored in tissues and serum/saliva, respectively. RESULTS: Mice sensitized to Dpte and Dfar allergens exhibited strong airway hyperresponsiveness (AHR) and lung inflammatory infiltrates including eosinophils. Sensitized animals mounted Th2-biased cellular and humoral responses specific for group 1 and 2 major allergens, as well as group 5, 7 and 10 minor allergens. This phenotype was sustained for at least 2 months, allowing the evaluation of immunotherapeutic protocols with HDM extracts-based vaccines. In this model, SLIT decreased AHR and Th2 responses and induced HDM-specific IgAs in saliva. The Dpte/Dfar mix proved the most efficacious when compared to Dpte or Dfar extracts alone. CONCLUSIONS AND CLINICAL RELEVANCE: The efficacy of a sublingual vaccine based on a Dpte/Dfar allergen extract mix was demonstrated in a well standardized murine model of chronic allergic airway inflammation based on clinically relevant mite allergens. The latter will be used as a benchmark for evaluation of future vaccines, including recombinant allergens. This HDM allergic airway inflammation animal model is a useful tool to design and select candidate vaccines to be tested in humans.

Oral macrophage-like cells play a key role in tolerance induction following sublingual immunotherapy of asthmatic mice.

Sublingual allergen-specific immunotherapy (SLIT) is a safe and efficacious treatment for type 1 respiratory allergies. Herein, we investigated the key subset(s) of antigen-presenting cells (APCs) involved in antigen/allergen capture and tolerance induction during SLIT. Following sublingual administration, fluorochrome-labeled ovalbumin (OVA) is predominantly captured by oral CD11b⁺CD11c⁻ cells that migrate to cervical lymph nodes (CLNs) and present the antigen to naive CD4⁺ T cells. Conditional depletion with diphtheria toxin of CD11b⁺, but not CD11c⁺ cells, in oral tissues impairs CD4⁺ T-cell priming in CLNs. In mice with established asthma to OVA, specific targeting of the antigen to oral CD11b⁺ cells using the adenylate cyclase vector system reduces airway hyperresponsiveness (AHR), eosinophil recruitment in bronchoalveolar lavages (BALs), and specific Th2 responses in CLNs and lungs. Oral CD11b⁺CD11c⁻ cells resemble tolerogenic macrophages found in the lamina propria (LP) of the small intestine in that they express the mannose receptor CD206, as well as class-2 retinaldehyde dehydrogenase (RALDH2), and they support the differentiation of interferon-γ/interleukin-10 (IFNγ/IL-10)-producing Foxp3⁺ CD4⁺ regulatory T cells. Thus, among the various APC subsets present in oral tissues of mice, macrophage-like cells play a key role in tolerance induction following SLIT.

Mapping of the lingual immune system reveals the presence of both regulatory and effector CD4+ T cells.

BACKGROUND: Sublingual immunotherapy (SLIT) is safe and reduces both symptoms and medication requirements in patients with type I respiratory allergies. Nonetheless, immune mechanisms underlying SLIT need to be further documented. OBJECTIVE: A detailed characterization of the lingual immune system was undertaken in mice, to investigate the presence of tolerogenic and pro-inflammatory mechanisms. METHODS: Immune cells were characterized in lingual tissues from BALB/c mice using immunohistology and flow cytometry. Resident CD4(+) T cells were sorted and toll-like receptor (TLR) expression profiles as well as functional characterization were assessed by RT-PCR, T cell suppressive assays and cytokine gene expression, respectively. RESULTS: Eosinophils and mast cells were only detected in submucosal tissues. No NK, NK-T, gamma/delta, CD8(+) T cells, nor B-lymphocytes were detected. Potential antigen presenting cells include various subsets of dendritic cells (CD207(+) Langerhans cells, CD11b(+)CD11c(+) myeloid cells and 120G8(+) plasmacytoid DCs) together with F4/80(+) macrophages. Noteworthy, both CD103(-) and CD103(+) CD4(+) T cells expressing TLR2 and TLR4 receptors are present along the lamina propria, in vicinity of myeloid CD11b(+)CD11c(+/-) dendritic cells. Such resident lingual CD4(+) T lymphocytes comprise both suppressive T cells as well as cells with memory/effector functions (i.e. expressing IFN gamma, IL4, IL10 and IL17 genes following stimulation), irrespective of the presence of the mucosal addressing marker CD103. CONCLUSION: The sublingual route is pertinent to induce antigen-specific tolerance, due to (i) limited numbers of pro-inflammatory cells, rather located in submucosal tissues, (ii) co-localization of APCs and resident CD4(+) T cells with regulatory functions. Since the oral immune system can also elicit pro-inflammatory effector responses, the cytokine milieu in which allergens are presented by sublingual APCs needs to be controlled during immunotherapy (e.g. with adjuvants) in order to favour tolerance over inflammation.

Targeting the allergen to oral dendritic cells with mucoadhesive chitosan particles enhances tolerance induction.

BACKGROUND: Sublingual immunotherapy (SLIT) efficacy could be improved by formulations facilitating allergen contact with the oral mucosa and uptake by antigen-presenting cells (APCs). METHODS: Two types of chitosan microparticles, differing in size and surface charge, were tested in vitro for their capacity to improve antigen uptake and presentation by murine bone marrow-derived dendritic cells (BMDCs) or purified oral APCs. T-cell priming in cervical lymph nodes (LNs) was assessed by intravenous transfer of carboxyfluorescein diacetate succinimidyl ester-labelled ovalbumin (OVA)-specific CD4+ T cells and flow cytometry analysis. Ovalbumin-sensitized BALB/c mice were treated sublingually with soluble or chitosan-formulated OVA twice a week for 2 months. Airway hyperresponsiveness (AHR), lung inflammation and T-cell responses in cervical and mediastinal LNs were assessed by whole-body plethysmography, lung histology and Cytometric Bead Array technology, respectively. RESULTS: Only a mucoadhesive (i.e. highly positively charged) and microparticulate form of chitosan enhances OVA uptake, processing and presentation by murine BMDCs and oral APCs. Targeting OVA to dendritic cells with this formulation increases specific T-cell proliferation and IFN-gamma/IL-10 secretion in vitro, as well as T-cell priming in cervical LNs in vivo. Sublingual administration of such chitosan-formulated OVA particles enhances tolerance induction in mice with established asthma, with a dramatic reduction of both AHR, lung inflammation, eosinophil numbers in bronchoalveolar lavages, as well as antigen-specific Th2 responses in mediastinal LNs. CONCLUSIONS: Mucoadhesive chitosan microparticles represent a valid formulation for sublingual allergy vaccines.

Toll-like receptor 2 agonist Pam3CSK4 enhances the induction of antigen-specific tolerance via the sublingual route.

BACKGROUND: Sublingual immunotherapy (SLIT) has been established in humans as a safe and efficacious treatment for type I respiratory allergies. OBJECTIVE: In this study, we compared three Toll-like receptor (TLR) 2 ligands (Pam3CSK4, Porphyromonas gingivalis lipopolysaccharide and lipoteichoic acid) as potential adjuvants for sublingual allergy vaccines. METHODS: These molecules were tested in co-cultures of adjuvant-pre-treated dendritic cells (DCs) with murine naïve CD4(+) T lymphocytes. Patterns of cytokine production, phenotype, proliferation and gene expression were analysed by ELISA, cytofluorometry and quantitative PCR, respectively. TLR2 ligands were subsequently tested in a model of SLIT in BALB/c mice sensitized with ovalbumin (OVA). RESULTS: Among the three TLR2 ligands tested, the synthetic lipopeptide Pam3CSK4 is the most potent inducer of IL-12p35 and IL-10 gene expression in murine bone marrow-derived DCs, as well as in purified oral myeloid DCs. Only Pam3CSK4-treated DCs induce IFN-gamma and IL-10 secretion by naïve CD4(+) T cells. Sublingual administration of Pam3CSK4 together with the antigen in BALB/c mice sensitized to OVA decreases airway hyperresponsiveness as well as OVA-specific T-helper type 2 (Th2) responses in cervical lymph nodes dramatically. CONCLUSION: Pam3CSK4 induces Th1/regulatory T cell responses, and as such, is a valid candidate adjuvant for sublingual allergy vaccines.

Embryonic thymic epithelium naturally devoid of APCs is acutely rejected in the absence of indirect recognition.

Transplants of tissues depleted of passenger leukocytes are upon in vitro culture usually accepted in allogeneic recipients. Accordingly, fully allogeneic embryonic thymic epithelium was suggested to be poorly immunogenic. However, this tissue is capable of inducing donor-specific tolerance to peripheral tissues, when restoring T cell development in nude mice, through the production of regulatory cells. In the present work, adult immunocompetent allogeneic recipients were grafted with embryonic tissues isolated at stages before hemopoietic colonization or even before the establishment of circulation. Allogeneic thymic epithelium of day 10 embryos and heart primordium of day 8 embryonic donors were always rejected. Acute rejection of the thymic anlagen takes place in less than 12 days, with maximal CD4(+) and CD8(+) T cell infiltrates at 10 days post-transplant. In addition, a significant infiltrate of NK1.1(+) cells is observed, although without any essential role in this process. Furthermore, recipients lacking the indirect pathway of Ag presentation to CD4(+) T cells do not reveal any significant delay in rejection, even when CD8(+) T cells are also eliminated. Thus, our experimental approach reveals acute allograft rejection in the absence of all known pathways of naive T cell activation and therefore unveils a novel graft rejection mechanism that should be mediated by direct recognition of parenchymal cells. Given the importance of dendritic cells in naive T cell activation, it is likely that cross-reactive memory T cells may also drive rejection.

Increased protein synthesis after T cell activation in presence of cyclosporin A.

BACKGROUND: The immunosuppressive drug, cyclosporin A (CsA), blocks immune responses by inhibiting the calcineurin-dependent dephosphorylation of the nuclear factor of activated T cells (NFAT). We have previously reported that T cells activated in presence of CsA exhibit particular properties. In our study, we have tested the hypothesis that T cells activated in presence of CsA display a differential pattern of gene expression. METHODS: T lymphocytes were activated in vitro by Concanavalin A with or without CsA. The cells were: (1) pulsed with 35S-methionine to label the newly synthesized proteins that in turn were revealed by 2D-gel electrophoresis; (2) analyzed by flow cytometry for activation markers expression; and (3) examined by gel electrophoresis for early tyrosine phosphorylation events. RESULTS: The proteomic patterns of T lymphocytes activated by Concanavalin A, with or without CsA, were compared. In keeping with the well-known effect of the immunosuppressor, many polypeptides were not found in its presence. Remarkably, several newly synthesized polypeptides were detected only when activation was carried out in presence of CsA. In addition, immunologically relevant proteins, such as CD44 and CD69, escape CsA-inhibitory action. Furthermore, CsA did not modify the early protein tyrosine phosphorylation events resulting from T cell triggering. CONCLUSIONS: The present data show that the effect of CsA on protein synthesis is more complex than anticipated. Signaling provided by T cell activation and the blockade of the calcineurin-dependent pathway by CsA results in an altered program of gene expression.

The impact of immunosuppressive drugs on the analysis of T cell activation.

The discovery of the immunosuppressive properties of cyclosporin A (CSA) and its successful utilisation in organ transplantation was a milestone in clinics. CSA has revolutionised transplantation both in term of efficiency and quality-of-life of the patient. In addition, the analysis of the mode of action of CSA has been rewarding in the understanding the mechanisms leading to T lymphocytes activation. CSA binds to a family of cytosolic receptors, the cyclophilins, a highly conserved family of proteins. Once this complex is formed, a third protein, the calcineurin, is recruited. The calcineurin, a calcium-dependent phosphatase, loose its activity when complexed. Dephosphorylation of NFAT, a substrate of calcineurin is a mandatory step for its translocation to the nucleus where NFAT acts as a transactivator involved in the regulation of the genes encoding many cytokines. CSA preventing NFAT dephosphorylation blocks cytokine production this in turn allowing for a better engrafting. The resolution of the tertiary structure of CSA alone or complexed with cyclophilin and calcineurin has important implication in the modelling of new drugs devoid of its side effects. Indeed, the high incidence of cancer is one of the main problems linked to CSA utilisation. Recent data suggest that CSA may promote cancer inducing the transcription of the gene encoding transforming growth factor beta. Other molecules sharing with CSA its immunosuppressive activity were later described. Some of them, as FK506, have the some mode of action; others, as rapamycin, mycophenolate mofetil or leflunomide, act at different steps of T cell activation.

[Short-term effects of the isolated platform on corticosterone plasma level]. / Efectos a corto plazo del método de la plataforma aislada sobre el nivel de corticosterona plasmática en ratas.

Plasma corticosterone was measured, in rats, after a 30 min, treatment either on small platforms used to induce paradoxical sleep deprivation, on large platforms utilized as control or in home cages ("Dry Control"group). The subjects on both platforms showed similar plasma corticosterone levels, those levels being significantly higher than the ones shown by "Dry Controls". It is concluded that the use of large platforms as control of the stress induced by the small platforms in those experiments aimed at determinating the relationship between short-term post-training paradoxical sleep deprivation and memory consolidation allow to discriminate the specifical effects induced by paradoxical sleep deprivation from the putative modulatory effects of learning exerted by the stress induced by the method.

Behavioral evaluation of the stress induced by the platform method for short-term paradoxical sleep deprivation in rats.

To evaluate whether the results of short-term PSD (Paradoxical Sleep Deprivation) using the platform method can be influenced by stress, changes in body weight and in behavioral indices (food and water intake and ambulation and defecation in an open field) were measured in rats after each of four 5-hour sessions of confinement to small or large platforms. The animals of the two platform groups when compared to animals kept in home cages showed a similar decrease in body weight which was significant only after the first day of treatment, while no changes in the other measures were observed. It is concluded that 1) the effects of stress induced by short-term confinement to platforms do not seem to be a remarkable confounding factor in short-term PSD studies and 2) large platforms can be used both as an adequate stress control for small platforms and as a means of adapting the animals to the method.