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Notes: Flow cytometric analysis of splenocytes from mice infected with lymphocytic Choriomeningitis virus revealed marked and long-standing up-regulation of LFA-1 expression on CD8+, but not on CD4+ T cells. Appearance of CD8+ T cells with a changed expression of adhesion molecules reflected polyclonal activation and expansion which was demonstrated not to depend on CD4+ T cells or their products. Cell sorting experiments defined virus-specific CTL to be included in this population (LFA-1hiMEL-14lo), but since about 80% of splenic CD8+ T cells have a changed phenotype, extensive bystander activation must take place; this is indicated also by the finding that CD8+LFA-lhi cells transiently express several markers of cellular activation, e. g. transferrin receptor, IL-2Rα and β. Analysis of cells from the cerebrospinal fluid of mice infected intracerebrally showed that virtually all T cells present belonged to the CD8LFA-lhi subset and, correspondingly, the ligand ICAM-1 was found to be up-regulated on endothelial cells in the inflamed meninges. Preincubation of LCMV-primed donor splenocytes with anti-LFA-1 markedly inhibited the transfer of virus-specific delayed-type hypersensitivity to naive recipients. Together, these findings indicate that up-regulation of LFA-1 expression is a critical factor involved in directing activated CD8+ T cells to sites of viral infection.

Notes: The yeast Pityrosporum orbiculare belongs to the normal cutaneous flora but is also considered to be one of the factors that may contribute to atopic dermatitis (AD). In the present study we investigated the possibility that P. orbiculare can act with superantigen activity in AD. P. orbiculare-reactive T-cell lines (TCLs) were obtained after stimulation of peripheral blood mononuclear cells (PBMC) with P. orbiculare extract. T-cell receptor β-chain V-segment (TCRBV) usage was investigated using monoclonal antibodies and flow cytometry. We could not find any difference in TCRBV usage between AD patients (n = 10) and healthy controls (n = 5), either in fresh PBMC or in P. orbiculare-reactive TCLs. Compared with their original PBMCs the P. orbiculare-reactive TCLs showed a decreased usage of several TCRBVs, although increased usage of certain TCRBVs could be seen in some of the individuals. Further analysis of the CDR3-length polymorphism exhibited a shift in CDR3-length distribution, indicating oligoclonal expansion of T cells specific to different antigens in the P. orbiculare extract. In conclusion we have not found any evidence for superantigen activity in P. orbiculare extract, but our data support the importance of classical major histocompatibility complex (MHC)-restricted allergens in P. orbiculare.

Notes: In a screening study concerning IgA and IgG antibodies to gliadin (IgA AGA and IgG AGA, respectively) in psoriasis, raised levels of IgA and AGA were found to be more common than in a reference group. To determine whether elevated AGA levels were associated with an increased number of intraepithelial lymphocytes. 33 patients with IgA AGA (n= 28) or IgG AGA (n= 5) values above 90% of the reference values (〉50 units/ml IgA AGA and 〈12 units/ml IgG AGA) underwent gastroduodenoscopy and duodenal biopsy in a prospective study. For comparison, six patients with low levels of both IgA AGA and IgG AGA were included. Five biopsy specimens were taken in each patient. Paraffin-embedded specimens were examined with regard to the degree of intraepithelial lymphocyte infiltration, and scored from 0 to 3. Biopsy specimens with a score of 0 had one mononuclear cell or less per four epithelial cells. The specimens were also examined with regard to the presence of intraepithelial CD3+ T lymphocytes and γ/δ+ T lymphocytes.In the six patients with low IgA/AGA and low IgG AGA, the biopsy score was 0. Fourteen of the 33 patients with raised AGA had a score of ≥1: of these. 12 had raised IgA AGA and two had slightly raised IgG AGA. Two of the patients with raised IgA AGA had partial villous atrophy, but the majority had normal villous architecture. There was a significant correlation both between the biopsy score and the number of intraepithelial CD3+ cells and between the score and the number of intraepithelial γ/δ+ positive T lymphocytes.The serum IgA AGA levels were significantly correlated with the duodenal biopsy score, the number of intraepithelial γ/δ+ T lymphocytes, and the number of CD3+ intraepithelial T lymphocytes. Most patients had no, or only mild, gastrointestinal symptoms. Of the 14 patients with biopsy scores 〈inlineGraphic alt="geqslant R: gt-or-equal, slanted" extraInfo="nonStandardEntity" href="urn:x-wiley:00070963:BJD896:ges" location="ges.gif"/〉1, seven had severe psoriasis and five moderately severe psoriasis, whereas only two bad mild psoriasis. There was no relationship between the duodenal score and haemoglobin, folate, whole blood selenium or serum zinc levels.Some of these patients improve on a gluten-free diet, but it is still too early to draw any definite conclusions concerning the type of relationship between the skin lesions, the increased number of intraepithelial lymphocytes in the duodenal mucosa and gluten hypersensitivity.

Notes: Background and Objectives Eosinophils are associated with bronchial asthma, but the role of the eosinophil is not fully understood. This study was initiated in order to study the influence of endogenous cortisol on eosinophil recruitment and activation in allergic inflammation in the lower airways in the pig.Methods Polyclonal and monoclonal antibodies against porcine eosinophil peroxidase (EPO) were raised. Detection of eosinophils in blood smears and lung biopsy specimens was achieved using the polyclonal antibody. For determination of porcine EPO in bronchoalveoiar lavage (BAL) fluid, a sandwich enzyme-linked immuno-sorbent assay (ELISA) with a detection limit of 0.15μig/L was developed. No cross-reactivity with porcine myeloperoxidase was found. Pigs that had been actively sensitized with repeated subcutaneous injections of Ascaris suum antigen were acutely challenged with antigen in the lower airways under pentobarbitone anaesthesia.Results Control animals with plasma cortisol levels of approximately 400 nM did not exhibit infiltration of eosinophils into lung parenchyma or EPO-release in the bronchial lumen within 8 h after challenge. However, in pigs treated with a cortisol-synthesis inhibitor (metyrapone), resulting in plasma cortisol levels of approximately 40 nM, there was a marked eosinophil infiltration into lung tissue at 8 h. Furthermore, EPO levels in BAL fluid were increased in some, although not all, low-cortisol animals. There was no infiltration of eosinophils into skin tissue in these animals.Conclusions It is concluded that, after allergen challenge in the lower airways of metyrapone-treated pigs, newly recruited eosinophils infiltrate lung tissue specifically. Furthermore, a cortisol-sensitive release of the eosinophil-derived cationic protein EPO, into the bronchial lumen was established. This is, to our knowledge, the first description of direct measurements of the release of an eosinophil granule protein in a large animal model of allergy.

Notes: Backgroumd Late airways obstruction and eosinophil infiltration after allergen challenge are often seen in human asthma and animal models of allergy. This inflammatory reaction, which may be a link between acute and chronic asthma, is blocked by glucocorticoid pretreatment. However, the role of eosinophils in late airways obstruction and the primary site of action of glucocorticoids, i.e. locally or systemically, have not been fully determined.Objectives This study was initiated to find out the role of eosinophils and neutrophils in allergen-induced late airways obstruction in the pig. The effect of pretreatment with budesonide (BUD) given locally or systemically on cellular responses seen within 8 h after allergen challenge was also studied.Methods Twenty-five minipigs were actively sensitized with Ascaris suum antigen and challenged under anaesthesia with antigen in the lower airways. Pigs were given BUD as an aerosol (l0μg/kg) or an intravenous infusion (5μg/kg) 1 h before allergen challenge. In one group, high doses of BUD (50μg/kg) were infused twice with a 3-h interval before allergen challenge. As a positive control, one group was given the BUD vehicle as an infusion and as a negative control, one group not treated with BUD was given the irrelevant antigen ovalbumin. Eosinophils and neutrophils in lung tissue specimens were detected and levels of eosinophil peroxidase (EPO) and myeloperoxidase (MPO) in bronchoalveolar lavage (BAL) fluid were measured using specific antibodies against porcine EPO and MPO.Results The number of eosinophils in lung tissue and BAL fluid and the level of EPO in BAL fluid were significantly increased 8 h after A. suum challenge in pigs not treated with BUD. With regard to possible recruitment and activation of neutrophils the only significant finding was an increase in the number of cells in BAL fluid. The eosinophil numbers and the level of EPO in BAL fluid were shown to be decreased by all BUD treatments in all the compartments studied compared to the positive control. However, the number of eosinophils in lung tissue and EPO levels in BAL fluid did not correlate with the magnitude of the late airways obstruction.Conclusion Although eosinophils are present in the bronchial wall and lumen and are apparently activated, a causative relationship between this granulocyte and the late bronchial obstruction could not be established in this model.

Notes: Background Atopic dermatitis (AD) is associated with increased levels of serum igE. and T-helper (Th) cells are thought to a play role in the pathogenesis. Individuals with AD often develop IgE antibodies against the yeast Pityrosporum orhiculare. a member of the normal cutaneous flora.Objective The role of P. orbiculare in atopic dermatitis was investigated by examining the T-cell reactivity for P. orbiculare.Methods Freshly isolated peripheral blood monoruclear cells (PBMC) were isolated from 10 AD patients with serum IgE antibodies against P. orbiculare, and from six healthy controls. The proliferative response after P. orbiculare stimulation, measured by [3H]thymidine incorporation, was examined in the PBMC and in T-cell clones (TCC) obtained from skin and blood of one patient. The cytokine profile of the TCC was determined by enzyme-linked immunosorhent assay (ELISA). radioimmunoassay (RIA) and reverse transcriptase-polymerase chain reaction (RT-PCR) following challenge with either P. orbiculare extract or anti-CD3 antibodies and phytohaemag-glutinin.Results The PBMC response to P. orbiculare was significantly higher in the AD patients than in the control group (P 〈 0.05). Twenty-nine out of 36 tested TCC derived from one responding patient were reactive for P. orhiculare. The clones were CD2+ and CD4+, except for one CD8+ blood clone. A majority of the TCC derived from lesional skin showed a Th2- or Th2/Th0-like cytokine profile. A co-expression of interIeukin-5 (IL-5) mRNA and II-13 mRNA was detected in five out of six P. orhicularc-reative clones analysed for their cytokinc gene expression with RT-PCR.Conclusion Our data suggest that P. orhiculare can induce a T-cell response in AD patients. The Th2-like profile of P. orbiculare-reactive TCC derived from lesional skin indicates that P. orhiculare may play a role in maintaining IgE-mediated skin inflammation in AD.

Notes: Background We have previously identified two major allergens of Pityrosporum orbiculare and characterized these as 37 kDa and 67 kDa proteins.Objective In the present study we have investigated the presence and subcellular location of the 37 kDa and 67 kDa allergen components in various members of the genus Pityrosporum as well as in Candida albicans. Candida parapsilosis and Saccharomyces cerevisiae. Methods To detect both cell surface and intracellular expression of the allergens, flow cytometry and confocal laser scanning microscopy (CLSM) were used. The cells were stained with indirect immunofluorescent (IIP) or alkaline phosphatase antialkaline phosphatase (APAAP) methods using mouse monoclonal antibodies (MoAbs)Results Ninety-five per cent of the P. orbiculare (P. ovale) cells cultured for 4 days showed cell surface-binding of the anti-37 kDa MoAb and 88% of the cells bound the anti- 67 kDa MoAb when analysed with IIF and flow cytometry. It was found that the members of the genus Pityrosporum (Malassezia). P. pachydermatis and M. sympodialis, expressed the 37 kDa and 67 kDa allergens to a similar extent as did P. orbiculare. Less than 5% of the cells of the genus Candida and S. cerevisiae showed positive staining with the MoAbs, The CLSM revealed that the 37 kDa and the 67 kDa components were located to the cell wall and could not be detected inside the acetone fixed and APAAP stained yeast cells of the genus Pityrosporum. When the yeast cells were cultured for more than 4 days the expression of both allergens decreased significantly.Conclusion All three members of the genus Pityrosporum express the 37 kDa and 67 kDa major allergens on the cell surface, whereas these proteins could virtually not be detected in the Candida genus and S. cerevisiae.

Notes: The dust mite Lepidoglyphus destructor is the dominating source of allergens giving rise to asthma and rhinitis among farmers. In a previous study of the localization of allergens in L. destructor we demonstrated that the 39 kDa allergen is associated with digestion. Here we describe the localization of the principal 15 kDa allergen and the high molecular weight allergen complex (79 and 93 kDa) in L. destructor with confocal laser scanning microscopy (CLSM). Cryostat-cut sections of mite bodies and faecal pellets were probed with mouse monoclonal antibodies (MoAbs) raised against the allergens. The 15 kDa allergen disclosed labelling of the mite body and most of the faecal pellets but left the exoskeleton unlabelled. The binding was widespread, and most intense in the mouth region. However, some staining was also observed around the gastrointestinal tract. In contrast, the 79 and 93 kDa allergen complex stained the exoskeleton and the front part of the mile. Interestingly, we detected no labelling of the faecal pellets with the MOAb against the 79/93 kDa allergen. The study indicates that the 15 kDa allergen is associated with the digestive tract whereas the function of the 79 and 93 kDa allergen complex remains to be elucidated.

Notes: Background Previous characterization studies of Pityrosporum orbiculare allergens have led to contradictory results. In immunoblotting studies a range of IgE-bitiding proteins of 10–100kDa have been identified. In another study, however, the IgE-binding structures were claimed to be associated with high-molecular-weight polysaccharides or glycoproteins, presumably mannans or mannoproleins. Objective In the present study the reasons for these discrepancies were investigated. Methods P. orbiculare preparations were compared in IgE ELISA and IgE-inhibilion ELISA, as well as in immunoblotting with sera from atopic dermatitis patients. Results It was inferred that variations in the period of in vitro culture of P. orbiculare constituted the most important factor determining the different compositions of the resulting yeast cell extracts. After 2 days of culture a wide range of allergen if proteins was present but upon more prolonged culture (〉4 days) most proteins of 10- 100kDa were lost. Accordingly, the protein concentration of the extracts gradually declined from 40% to 25% between days 4 and 15 of culture. On the other hand, the carbohydrate content remained fairly constant (approximately 30%). Using inhibition ELISA it was demonstrated that the high-molecular-weight glycoproleins or poly-saccharides presumably involved in most of the IgE-binding capacity in extracts from old cultures, were also present in comparable concentrations in all extracts tested, even after culture for only 2 or 4 days. Conclusion Preparations obtained from the exponential phase of yeast cultures (2–4 days old), should preferably be used in studies of the IgE response to P. orbicularc.

Notes: Background: To examine the influence of atopy on the different cell populations in adenoids, we investigated the presence of IgE+ cells, cells expressing the high-affinity receptor for IgE (FcεRI), and various other cell populations in adenoid tissue, in atopic and nonatopic children with otitis media with effusion (OME) or adenoid hyperplasia (AH). Methods: Cryostat sections of adenoids from 14 atopic and 16 nonatopic children suffering from long-lasting OME (n=15) or obstructive AH (n=15) were investigated with immunohistochemical markers for T-cell subsets, mast cells, eosinophils, plasma cells, CD25, CD1a, IgE, and FcεRI. Results: Sensitization to allergens was correlated to an increase of IgE+ cells in the epithelium (P〈0.01), the extrafollicular area (P〈0.0001), and the follicles (P〈0.001) of the adenoids and an increase of FcεRI+ cells in the extrafollicular area (P〈0.01). A minority of the IgE+ cells were plasma cells. No significant differences in cells stained for IgE, FcεRI, or the other markers were observed between patients with OME and AH. Conclusions: Atopy is associated with increased numbers of IgE+ and FcεRI+ cells in adenoids irrespective of whether the child has OME or AH.