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Background: Recent cytokine (RT-PCR, ELISA) analyses of inflammation in atopic dermatitis (AD) have suggested a role for IL-4, IL-5 and IFNgamma. Pityrosporum ovale and Candida albicans are important allergens in some patients with AD of the seborrhoic head, neck and shoulder region. In AD patients, the saprophytic yeasts induce IgE responses while they usually induce TH1 type responses. The cytokine responses induced by yeasts in AD are sparsely investigated., Objective: To characterize the P. ovale- and C. albicans-specific and non-specific humoral, lymphoproliferative and cytokine (IL-2, 4, 5 and IFNgamma) responses in AD., Methods: Fifteen AD patients and seven healthy controls (HC) were included. Ficoll-isolated PBMC were stimulated by PHA and laboratory-generated extracts of P. ovale and C. albicans. Lymphocyte proliferation was measured by 3H-thymidine incorporation and cytokine production by sandwich-ELISAs. The antigen-specific IgG and IgE antibodies were analysed by ELISA and nitrocellulose RAST., Results: Pityrosporum ovale- and C. albicans-specific IgE (both P < 0.001) and P. ovale-induced PBMC proliferation (P < 0.02) were elevated in AD. In general, the IL-4/IFNgamma ratio induced by P. ovale was higher than that induced by C. albicans (P < 0.01). The PHA-induced IL-2 (P < 0.05) and IL-4 responses (P < 0.005), and the C. albicans-induced IL-5 response (P < 0.02) and IFNgamma response (P < 0.01), were elevated in AD. A network of correlations was seen between serum total and the yeast-specific IgE, P. ovale-specific lymphoproliferation, PHA-induced IL-2, IL-4 and IL-5, and C. albicans-induced IL-5., Conclusion: The cytokine profiles found in this study support the role of TH0 or TH1 cells by the side of TH2 cells in the pathogenesis of atopic dermatitis. Pityrosporum ovale appears to be associated more with IL-4 responses and C. albicans with IFNgamma responses.

This study was designed to determine trial-to-trial and day-to-day reproducibility of isometric force and electromyogram activity (EMG) of the knee extensor muscles in water and on dry land as well as to make comparisons between the two training conditions in muscle activity and force production. A group of 20 healthy subjects (12 women and 8 men) were tested three times over 2 weeks. A measurement session consisted of recordings of maximal and submaximal isometric knee extension force with simultaneous recording of surface EMG from the vastus medialis, vastus lateralis and biceps femoris muscles. To ensure identical measurement conditions the same patient elevator chair was used in both the dry and the wet environment. Intraclass correlation coefficients (ICC) and coefficients of variation (CV) showed high trial-to-trial (ICC = 0.95-0.99, CV = 3.5%-11%) and day-to-day reproducibility (ICC=0.85-0.98, CV=11%-19%) for underwater and dry land measurements of force and EMG in each muscle during maximal contractions. The day-to-day reproducibility for submaximal contractions was similar. The interesting finding was that underwater EMG amplitude decreased significantly in each muscle during maximal (P < 0.01-P < 0.001) and submaximal contractions (P < 0.05-P < 0.001). However, the isometric force measurements showed similar values in both wet and dry conditions. The water had no disturbing effect on the electrodes as shown by slightly lowered interelectrode resistance values, the absence of artefacts and low noise levels of the EMG signals. It was concluded that underwater force and EMG measurements are highly reproducible. The significant decrease of underwater EMG could have electromechanical and/or neurophysiological explanations.

Background: The cytokine observed most often in atopic dermatitis (AD) is IL-4, but a role for IL-5 and IFN-gamma in the late and delayed phase reactions has been suggested. In AD with head, neck and shoulder distribution, hypersensitivity to saprophytic yeasts is an important pathogenetic factor. The yeast allergens include both the mannan polysaccharides and the proteins. Mannans are major cross-reacting allergens likely to be involved in the pathogenesis of AD., Objective: To characterize the humoral, lymphoproliferative and cytokine (IL-2, 4, 5 and IFN-gamma) responses of peripheral blood mononuclear cells (PBMCs) induced by Candida albicans mannan and protein antigens in AD., Methods: Fifteen AD patients and seven healthy controls were included. Ficoll-isolated PBMCs were stimulated by PHA and laboratory-generated mannan and protein extracts of C. albicans. Lymphocyte proliferation was measured and cytokine production was studied by ELISA. The antigen-specific IgG and IgE antibodies were analysed by ELISA and nitrocellulose RAST., Results: In AD mannan (P < 0.005) and protein (P < 0.002), specific IgE levels were higher than in healthy controls. Both mannan and protein-specific lymphoproliferations (both: P < 0.02) were higher in AD than in healthy controls. Mannan, but not protein, induced long lasting IL-2 and IL-4 productions from 24 h lasting up to 66-96 h and IL-5 and IFN-gamma productions with elevated levels at 66 and 96 h. The mannan-induced IL-2 (P = 0.015) and IFN-gamma (P < 0.005) were increased in AD as compared with healthy controls. Significant correlations were seen between the protein-induced proliferation responses and both serum total IgE (r = 0.59, P < 0.01) and protein-specific IgE (r = 0.65, P < 0.005). The mannan-induced IL-2 responses correlated with the specific IgE (r = 0.62, P < 0.01) and proliferation (r = 0.51, P < 0.02) and S-IgE level (r = 0.71, P < 0. 002). Mannan-induced IL-4 and IFN-gamma productions also correlated (r = 0.43, P < 0.05)., Conclusions: C. albicans mannan induced elevated IL-2 and IFN-gamma responses in AD patients. The correlations of the cytokine responses with mannan-induced IgE and proliferation responses suggest that C. albicans mannan induced TH1 type cytokine responses are involved in AD.

Purpose: The aim of this study was to investigate the effect of cyclodextrins (beta-CD, HP-beta-CD and (SBE)7m-beta-CD), and co-administration of a water-soluble polymer (HPMC) and cyclodextrins, on the oral bioavailability of glibenclamide in dogs., Methods: Effects of cyclodextrins on the aqueous solubility of glibenclamide, with and without hydroxypropylmethylcellulose (HPMC), were determined by a phase-solubility method. Solid inclusion complexes were prepared by freeze-drying. Glibenclamide was administered orally and intravenously to beagle dogs., Results: Aqueous solubility of glibenclamide increased as a function of cyclodextrin concentration, showing an AL-type diagram for beta-CD and an Ap-type diagrams for both of the beta-CD derivatives studied. HPMC enhanced the solubilising effect of cyclodextrins, but did not affect the type of phase-solubility diagram. Orally administered glibenclamide and its physical mixture with HP-beta-CD showed poor absolute bioavailability, while orally administered glibenclamide/cyclodextrin-complexes significantly enhanced the absolute bioavailability of glibenclamide. Orally administered glibenclamide/beta-CD/HPMC and glibenclamide/(SBE)7m-beta-CD/HPMC complexes showed similar absolute bioavailability compared to formulations not containing HPMC, even though 80% (in the case of (SBE)7m-beta-CD) or 40% (in the case of beta-CD) less cyclodextrin was used., Conclusions: The oral bioavailability of glibenclamide was significantly increased by cyclodextrin complexation. HPMC increased the solubilising effect of cyclodextrins and, therefore, the amount of cyclodextrin needed in the solid dosage form was significantly reduced by their co-administration. In conclusion, the pharmaceutical usefulness of cyclodextrins in oral administration may be substantially improved by co-administration of a water-soluble polymer.

Background: Environmental variation, such as an increase of mean temperature due to the greenhouse effect, as well as the genetic factors may affect the allergenicity of pollen and thus, the prevalence of allergies. The connection between these factors and the allergen content of pollen is poorly understood., Objectives: To evaluate the role of environmental and genetic factors on the allergenicity of birch pollen., Methods: Mountain birch (Betula pubescens ssp. czerepanovii (Orl.) Hämet-Ahti) pollen was studied using SDS-PAGE and IgE-immunoblotting. Pollen samples were collected from the trees of 10 half-sib families. The study trees from each family were reared in two tree line gardens where the daily mean temperatures were different during the growing season., Results: The quantitative analysis of band intensities suggested that the responses of the major birch pollen allergen, Bet v 1, were stronger in the samples collected from the garden with higher daily mean temperature. Half-sib families and individual trees differed in their Bet v 1 content., Conclusions: Our results show that both genetic and environmental factors have an effect on the amount of Bet v 1. This suggests that breeding for trees low in allergen content may be possible.

Shrew species of the subfamily Soricinae have unusually high metabolic rates when compared to Crocidurinae shrews and other similar-sized mammals. The aim of this study was to clarify whether the high basal metabolic rate of Soricinae shrews is reflected in a high capillary density in their muscles. To this end, the capillary supply of four limb muscles and diaphragm of the common shrew (Sorex araneus) was quantified from cross-sectioned muscles. The capillary densities of the limb muscles were 2575 +/- 329, 3111 +/- 299, 2812 +/- 197 and 2752 +/- 173 capillaries mm-2 fibre area in gastrocnemius lateralis, g. medialis, plantaris and soleus, respectively. Capillary density of the shrew diaphragm (6691 +/- 1057) was double that of the limb muscles. This value is among the highest ever measured in mammals. In general, the capillary supply in the hind limb of the common shrew is about 3-4 times higher than commonly found in the leg muscles of the laboratory rat or other bigger mammals, but similar to those in Crocidurinae shrews and some small rodents. Thus the high resting metabolism of the common shrew is not associated with an extraordinarily high capillary density. The apparent disparity between basal metabolic rate and muscle capillary supply in S. araneus is probably due to the small aerobic scope of shrews in the subfamily Soricinae.

This study was undertaken to analyze the differences in exposure and sensitization to five common environmental yeasts. The responses of IgG, IgA, and IgE to Candida albicans, C. utilis, Cryptococcus albidus, Rhodotorula rubra, and Saccharomyces cerevisiae and purified S. cerevisiae enolase were analyzed by immunoblotting (IgE-IB), and the cross-reactivity of their IgE-binding components by IgE-IB inhibition. Twenty atopic subjects, with asthma, allergic rhinitis, or atopic dermatitis were included. In skin prick tests (SPT), 12 of the patients showed simultaneous reactivity to at least two of the five yeasts, four reacted to one of the yeasts, and four had no responses. Antigens run in SDS-PAGE and transferred to nitrocellulose were probed with enzyme-labeled IgA-, IgG-, and IgE-specific antibodies. The IgE immunoblotting revealed most IgE-binding bands in C. albicans (11 bands) followed by C. utilis (eight bands), S. cerevisiae (five bands), R. rubra (five bands), and Cr. albidus (four bands). Six of the IgE-binding bands of C. albicans and C. utilis shared molecular weight, and only two bands shared molecular weight with other yeasts. These were the 46-kDa band, shared by all five yeasts, and a 13-kDa band shared by four yeasts. Prominent IgE binding was seen to a 46-kDa band of C. albicans (seven patients), C. utilis (five patients), and S. cerevisiae (one patient) and to corresponding weak bands of Cr. albidus and R. rubra (one patient). The possible cross-reactivity of the 46-kDa band was analyzed by IgE-IB inhibition and densitometry, revealing clear C. albicans inhibition of C. utilis (80%) and enolase (98%) (autoinhibition 100%). The strongest IgG responses were seen against S. cerevisiae and C. albicans. The responses were mainly against mannans of C. albicans and S. cerevisiae, suggesting that most of the exposure is to these yeasts. Yeasts with different types of exposure, from saprophytic growth on human mucous membranes to exposure by air and food, were shown to cross-react at the allergenic level. Atopic patients primarily sensitized by C. albicans and S. cerevisiae may develop allergic symptoms by exposure to other environmental yeasts due to cross-reacting IgE antibodies.

Background: Pityrosporum ovale is a common saprophyte on the skin capable to induce IgE antibody production in atopic dermatitis. Generally IgE response to P. ovale has been established with skin-prick test, but the stability of P. ovale skin test allergens is not known., Objective and Methods: In this study we analysed the stability of identified allergens of P. ovale in skin test solutions during storage. The stability was checked with immunoblotting and densitometry after different time periods and at various temperatures., Results: Coomassie Brilliant blue staining clearly showed, that proteins were preserved poorly at + 20 degrees C in 50% glycerol. Even extracts stored at + 4 degrees C during 6 and 12 months in 50% glycerol were degraded. Only extract stored at + 4 degrees C for 1 month in 50% glycerol was quite well preserved. According to densitometry results the 9 kDa band was the most stable main protein allergen. The 20 kDa and especially the 96 kDa bands were far more labile. The stability of low molecular allergens was in general better than the stability of high molecular weight allergens., Conclusions: The stability of P. ovale extracts stored for more than 1 month was poor even when kept at + 4 degrees C temperature. Reliability of negative skin prick results should be questioned when older extracts are used. In skin test negative cases, the sensitization should be evaluated also with the measurement of specific serum IgE antibodies to P. ovale.

A standardized, controlled procedure for preparation of an in-house reference (IHR) preparation of an allergen extract of Candida albicans is described. The procedure, based on previous studies of allergens of C. albicans, is designed to yield a maximum of allergens in optimum extraction conditions and to provide a reference preparation for further extract production. The SDS-PAGE, IgE-immunoblotting, and crossed radioimmunoelectrophoresis (CRIE) analyses showed that the procedure is reproducible with acceptable batch-to-batch variation. The variation in the content of the most important allergens, namely, proteins with molecular weights of 46, 29, and 27 kDa in the pooled final batches, is acceptable (coeff. of variation < 15%), although in the intermediate batches of different strains, the coefficient of variation may occasionally exceed 20%. A comparison with other C. albicans allergen preparations used in our previous studies is also presented. The resulting extract can be used as a reference in further extract production and also in experimental in vitro and in vivo studies.

To determine the effects of anthropogenic pollution on water-soluble proteins and specifically allergens in birch (Betula pendula and B. pubescens) pollen, we analyzed extracts of pollen from the pollution gradient around a factory complex (emitting sulfur oxides and heavy metals) by sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis (PAGE) and IgE immunoblotting. In addition, tree density-associated shading of the tree habitat, and quantity and quality of proteins and allergens in pollen of the two birch species were studied. The two studied birch species gave identical allergen profiles even though their protein profiles differed. Distance from the factory did not affect the amount of birch pollen major allergen, Bet v 1 (17 kDa), or of two other strong allergens (23 and 36 kDa). Trees growing in shaded places had significantly stronger responses to Bet v 1 and to the 23-kDa allergen than trees growing in open or half-open environments. Thus, we propose that combined heavy metal and sulfur dioxide pollution does not have an important effect on birch pollen allergens. Instead, other factors, e.g., shading and soil properties of the tree habitat, as well as the genetic background of the tree, may have a stronger influence on the quantity and relative composition of allergens.

Objective: The aim of this study was to compare the IgE response to the most commonly farmed fur animals with that to domestic animals., Methods: IgE-immunoblotting and RAST-inhibition analyses were performed using RAST-positive sera from fur workers sensitized to fur allergens and sera from patients sensitized to domestic animal allergens., Results: The urine extracts of mink, blue fox, silver fox, raccoon dog and fitchew contained more protein bands than the fur extracts did. Allergens with the same molecular weight were found in all of the fur and urine extracts. The most prominent allergenic bands had molecular weights of 62-67 kDa, 23-25 kDa and 18-19 kDa. With crossreacting sera the reciprocal RAST inhibition with all five animal extracts indicated common IgE-binding epitopes, probably common allergens (especially the 62-67 kDa bands). Urine and fur contain common allergens, since urine allergens strongly inhibited the IgE-binding to fur allergens. The IgE binding to allergenic bands of fur animal extracts was also observed in immunoblotting when dog and cat RAST-positive sera were used, but not for cow RAST-positive sera. RAST inhibition of dog-positive sera with fur animal extracts and fur-positive sera with dog extract confirmed the crossreactivity of these IgE antibodies. No such inhibition was seen with cow extract., Conclusion: The results of the RAST inhibition and immunoblotting suggest that fur animals have IgE binding epitopes or allergens in common with cat and dog--possibly albumin--but not with cow.

IgG and IgA antibodies against fungus Aspergillus umbrosus have been found in the sera of patients with farmer's lung (FL) disease and healthy exposed farmers in Finland. To determine the IgE response to antigens of A. umbrosus and Candida albicans, sera from 20 patients with FL, 20 healthy farmers and 20 nonfarming controls were tested by nitrocellulose radioallergosorbent test (RAST). The values of RAST indices were low in each group and the only statistically significant difference was found between the groups of FL patients and nonfarming controls against A. umbrosus polysaccharide antigen. Individual IgE responses to polysaccharide antigens of A. umbrosus and C. albicans correlated among FL patients, however, no cross-reacting IgE antibodies could be shown. To conclude, the IgE antibody levels of A. umbrosus polysaccharide and crude antigens were low in FL patients, healthy exposed farmers and nonfarming controls. Neither polysaccharide nor crude antigen-specific IgE antibodies of A. umbrosus have any diagnostic value in FL disease.

Background: Pityrosporum ovale is a common saprophyte on the skin capable of inducing IgE antibody production in atopic dermatitis (AD) patients. Allergens of P. ovale have been examined in several studies, but consensus on them is lacking., Objective: This study was carried out to obtain more information about the IgE antibody response against P. ovale, including mannan., Methods: Sera from 64 AD patients and 10 healthy controls were analysed with immunoblotting and the nitrocellulose radio allergosorbent test (RAST) method specifically developed to detect antimannan P. ovale IgE antibodies., Results: In immunoblotting a total of 39 different IgE stained protein bands were seen. A high molecular weight staining was also seen especially in patients who displayed elevated mannan P. ovale RAST values. The most commonly stained protein bands in immunoblotting were 9 and 96 kD bands with antibodies in 73 and 65% of AD patients who had been positive in commercial P. orbiculare RAST with total serum IgE less than 4000 kU/l. Mannan RAST appeared positive in 77% of them. Positive immunoblotting to either of these bands was seen in 90% and, if added with staining with the 20 kD band, in 100% of these AD patients. A combination of 9 kD IgE staining and mannan P. ovale RAST was positive in 92% of the patients and 96 kD and mannan P. ovale RAST in 85% of the patients., Conclusion: It is evident that P. ovale has several allergens, the 9, 96 and 20 kD regions being the most important. According to our results mannan is also an important allegen of P. ovale.

Background: Invasive candidiasis is a life-threatening complication problem in post-operative and immunocompromized patients, e.g. those treated by intensive care. Candida is frequently cultured from the mucous membranes of hospital patients and fungal cultures offer little diagnostic help. Other diagnostic methods, such as blood cultures, serology and diagnostic imaging techniques produce results too late and, if positive, low sensitivity., Objective: To study the value of Candida-specific antibodies, especially those of IgE class, in diagnosing invasive Candida infection., Methods: The immunoglobulins IgE, IgG and IgM responses to antigens of Candida albicans in the sera of 14 patients with culture, biopsy and/or autopsy proven postoperative invasive candidiasis and of 11 colonized and 19 non-colonized operated patients were studied by mannan radioallergosorbent test (RAST), mannan enzyme-linked immunosorbent assay (ELISA) and immunoblotting., Results: Detection of IgE antibodies to C. albicans polysaccharide (mannan) and protein antigens proved specific and sensitive in diagnostics of invasive candidiasis after major abdominal surgery. IgE rose early in the course of the infection and the method made a clear distinction between invasive infection and mucous colonization. Immunoblotting for protein antibodies was most sensitive while nitrocellulose-RAST for mannan antibodies was most specific. The combined use of immunoblotting and RAST increased the sensitivity and the specificity. Determinations of anti-Candida IgG and IgM antibodies had low sensitivity and specificity., Conclusion: Critically ill patients with invasive candidiasis develop IgE antibodies to Candida antigens probably because of disturbed TH1/TH2 responses. Determination of specific IgE antibodies can be used as a diagnostic aid in the early stage of invasive Candida infection.

Two chemically purified mannan preparations and one affinity purified mannan preparation of Candida albicans and Saccharomyces cerevisiae were analyzed for allergenic cross-reactivity. Simultaneous IgE binding in radioallergosorbent test (RAST) was studied with all preparations and the chemically purified mannans were also used for RAST inhibitions. The yeast mannans were purified with the Peat method, using repeated ethanol precipitations and Fehling's precipitation in alkaline conditions, and the Cetavlon method, using hexadecyltrimethylammonium bromide and ethanol precipitations. Affinity purification of the mannans was performed with concanavalin A lectin bound to a Sepharose column. Mannan from Pityrosporum ovale was purified with the Cetavlon method for analysis of simultaneous RAST reactivity. Simultaneous IgE binding to all the studied yeast mannans was generally seen in all the studied sera of patients suffering from atopic dermatitis. The strongest responses were seen against C. albicans mannan, especially the Cetavlon mannan. In the RAST inhibition studies IgE binding to S. cerevisiae mannan was completely inhibited by C. albicans mannan preparations, whereas reciprocal inhibition was not complete. These results indicate that in atopic dermatitis simultaneous IgE response to yeast polysaccharides occurs and that the major sensitizer is C. albicans and IgE antibodies against S. cerevisiae mannan are cross-reacting.

Red-toothed shrews of subfamily Soricinae are small mammals with very high mass-specific metabolic rate. Owing to their high aerobic power they are interesting objects for studies concerning the limits and constraints of skeletal muscle adaptation. In order to clarify the correlation between metabolic rate and muscle properties, we have analyzed fiber types, fiber size, and myosin heavy chain composition of the common shrew (Sorex araneus) and compared them to those of rat (Rattus norvegicus). Three distinct differences between shrew and rat muscles were noted. 1) The fibers of shrew muscles are exceptionally small in comparison to rat myofibers. 2) Electrophoretic and histochemical analysis showed that shrew muscles are composed of only fast fibers (fiber types IIB and IID), the slow type I fibers being totally absent. 3) The shrew muscles are much more homogenous than rat muscles in regard to myosin heavy chain and fiber type composition. The shrew diaphragm consists exclusively myosin heavy chain type IId (MHCIId), while masseter and soleus are composed 95% and 87% of MHCIId, respectively. Other four studied muscles contain MHCIIb and MHCIId approximately in equal proportions. The present findings show that shrew muscles are composed of small, highly aerobic, fast type II fibers, which may be sufficiently fatigue-resistant to function both as postural muscles and to power fast and high frequency movements of limbs and diaphragm.

Ultrafiltered (> 1000 Da) samples of beer, aged red wine, young white wine, sparkling wine and extracts of fresh wheat bread and dried rye bread were analysed by skin-prick test (SPT), radioallergosorbent test (RAST) inhibition, sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to find out if they contain Saccharomyces cerevisiae (S. cerevisiae, baker's yeast) allergens. Serum pool consisting of S. cerevisiae positive sera was used in the assays. The results were compared with freeze-dried reference S. cerevisiae and cereal antigens. The beer, bread, red wine and sparkling wine extracts elicited immediate reactions. However, no evident correlation with suspected symptoms was observed. White wine extract caused reactions in four out of six atopic dermatitis (AD) patients with symptoms, and in five out of seven symptom-free AD patients and in two of the 24 controls. In SDS-PAGE, protein bands were found in wheat and rye bread extracts and beer. In IgE immunoblotting, however, no staining was seen with the S. cerevisiae positive sera suggesting that they were of cereal origin. In white wine and champagne extracts a non-specific staining was seen in the region 20 kDa representing, e.g. lectin-like activity. No baker's yeast antigen could be detected in brewery and bakery products with IgE-immunoblotting even in the excessively concentrated extracts. The IgE mediated allergy to baker's yeast alone should thus not lead to denial of bakery, brewery and wine products.

The allergen extracts of wheat, rye, barley and oats flours were characterized by IgE-immunoblotting with serum samples from 40 adult patients; 35 patients with atopic dermatitis, one with rhinitis and four with urticaria. All these patients had been positive when skin-prick testing was carried out with one or more of the four flour extracts or displayed one or more positive cereal RAST results. Four non-atopic sera were used as negative controls. Acidic and neutral protein extracts of wheat, rye, barley and oats flours were processed for the immunoblotting experiments and 35 patients appeared positive in IgE immunoblotting with wheat and rye, 32 with barley and 33 with oats. The IgE immunoblots showed polyspecific binding patterns; wheat exhibited 36 IgE stained bands, rye 35, barley 33 and oats 10. Eighteen of the IgE stained bands could be classified as intermediate allergens for wheat, 23 for rye and 15 for barley. The 66 kDa protein in oats was visualized by 28 out of 33 sera (84%), however, there was evident non-specific binding to this region and thus it may also represent lectin-like binding. The most frequent staining with wheat extract was seen in the 26 kDa protein region (15/35, 43%), with rye in the 40 kDa (16/35, 46%) and with barley in the 26 and 46 kDa protein bands (14/32, 44%). Simultaneous staining with wheat, rye and barley extracts were observed with 16 bands suggesting crossreactivity between these cereals.

A nitrocellulose-based radioallergosorbent test (RAST) was developed and used for the determination of IgE antibodies to Candida albicans mannan in patients with atopic dermatitis, asthma and allergic rhinitis. The results were expressed as mannan-RAST index values (an inter- and intra-assay coefficient for variation of 8.0-10.2%). The normal range for mannan-RAST index values was determined in 102 non-atopic adults. Fifty-three of 78 (67.9%) patients with atopic dermatitis showed elevated mannan-RAST index values with a significant correlation to the severity of the dermatitis (r = 0.33, P < 0.01). Sixteen of 30 (53.3%) patients with asthma had a positive mannan-RAST index value; however, 12 of the 16 asthmatics (75%) who were positive also suffered from atopic dermatitis. Those who had allergic rhinitis but not atopic dermatitis showed a positive mannan-RAST index value in 12 of 32 (37.5%) cases. Nitrocellulose-RAST offered a sensitive method for the determination of polysaccharide-specific IgE antibodies in atopic diseases. The results show that high values are observed mainly in atopic dermatitis and less sensitization to C. albicans occurs in respiratory allergy.

Fifty patients with farmer's lung disease (FL), 20 asymptomatic exposed farmers, and 18 healthy controls were investigated for total IgG and total IgA, as well as subclass antibody levels against Aspergillus umbrosus, the most common fungus to which FL patients in Finland have been exposed. Enzyme-linked immunosorbent assay (ELISA) revealed significantly elevated levels of specific total IgG and IgA antibodies in the sera of patients during the acute phase of the disease. Both IgG1 and IgG2 subclass antibodies and both IgA1 and IgA2 subclass antibodies were significantly associated with the disease. The antibody levels decreased during the first 3 months, when mould exposure was avoided and when an oral 6-week steroid treatment was administered. During the next 12 month follow-up, the mean antibody levels continued to decrease, even though re-exposure might have occurred. The results (with sensitivity 94% and specificity 95%) imply that in addition to the measurement of A. umbrosus-specific IgG, determination of both A. umbrosus-specific IgG1 and IgA might also be useful in the serodiagnosis of acute stage of FL.

An in vitro model was established to study the stability of baker's yeast (Saccharomyces cerevisiae) allergens in conditions simulating the gastrointestinal tract. The protocol consisted of 2 hr incubation under gastric conditions (pH 1.2, +37 degrees C and gastric enzymes) and 2 hr incubation under duodenal conditions (pH 6.8, +37 degrees C and duodenal enzymes). These were studied together and separately, as well as under pure acidic conditions without gastric enzymes. The yeast extracts contained equal amounts of allergen and were analyzed by IgE-immunoblotting. The acidic conditions had partly an enhancing and slightly degrading effect on the yeast allergens, whereas the gastric enzymes destroyed several allergens, including the important intermediate allergens of 31 and 45 kD. After treatment under both gastric and duodenal conditions most of the yeast allergens were destroyed, except mannan and a 10 kD protein component. The findings suggest that the allergen exposure caused by baker's yeast takes place mainly on the mucosal surfaces orally and oesophageally and through viable baker's yeast organisms that manage to pass the stomach and duodenum and possibly lead to intestinal growth of the organism. Patients with IgE production against the 10 kD allergen and mannan are, however, moderately exposed to allergens consisting of soluble antigenic material only.

The Saccharomyces cerevisiae allergens were characterized by IgE-immunoblotting with serum samples of 83 patients; 63 represented patients with atopic dermatitis with previous positive skin prick test or RAST for S. cerevisiae, seven patients with AD but negative test results and 13 were non-atopic controls. Disrupted whole body extract of S. cerevisiae was used in the assays. From the patients tested 41 patients with atopic dermatitis appeared positive in IgE immunoblotting revealing 22 IgE stained bands. From these bands 10 represented intermediate allergens, and 12 minor allergens. The most frequent staining was obtained with the 48 kD band (39%). When the staining pattern of 45 kD and 48 kD bands and mannan was compared with Candida albicans allergens or purified baker's yeast enolase a simultaneous binding was seen with the 48 kD band of S. cerevisiae and the 46 kD band of C. albicans and enolase whereas the 45 kD band was neither associated with the 46 kD band of C. albicans nor purified enolase. High molecular weight staining was found in five samples. The staining pattern was associated with the mannose containing structures in parallel with C. albicans.

Stability of Candida albicans allergens was studied under various storage conditions. Lyophilized extract was reconstituted with human serum albumin (NSA) diluent, glycerol-free and in the presence of 10% or 50% glycerol and stored at various temperatures for different time periods. All extracts were tested at the same time with immunoblotting using C. albicans allergic patient sera and galactosidase-labelled anti-IgE. The highest number of detected allergens in the immunoblotting pattern was found in the presence of 50% glycerol at +6 degrees C. The most important allergen of C. albicans, the 46 kD protein allergen was stable up to 10 weeks at +6 degrees C in the presence of 50% glycerol but thereafter began to lose its IgE-binding capacity. After 30 weeks more than 50% of the IgE binding had disappeared. The 27 kD protein, another important allergen, was also labile but retained the allergenicity better than the 46 kD one. The 29 kD protein allergen was stable at all storage conditions, except +37 degrees C tested even after one year. More than 6 months storage at +6 degrees C or higher temperature is, however, unacceptable even in the presence of the 50% glycerol. These findings have particular importance in the diagnosis and treatment of allergic diseases.

Sera from 13 patients with positive Pityrosporum ovale skin prick test were analysed with IgE immunoblotting. Both P. ovale and Candida albicans antigens were used to reveal possible crossreactivity of these yeasts. Of the 13 sera, eight sera showed IgE binding to P. ovale and six sera to C. albicans. Simultaneous IgE-binding to both C. albicans and P. ovale was observed in five of these sera. The most common IgE-binding band pairs were, a 23 kD band of P. ovale and a 27 kD band of C. albicans, as one pair, and a 26 kD band and a 13 kD band, respectively, as another pair. In addition to these protein bands, simultaneous reactivity was observed to C. albicans mannan, a polysaccharide, and to a diffuse high molecular weight component of P. ovale, possibly also a polysaccharide. The five sera with simultaneous IgE-binding were analysed with RAST-inhibition, which revealed presence of crossreacting IgE-antibodies to P. ovale and C. albicans in two of these sera. Crossreacting epitopes were suggested to be located in the mannan polysaccharide of C. albicans and high molecular weight diffuse stain of P. ovale, based on the immunoblotting and RAST-inhibition patterns of the studied sera. Crossreacting sera were pooled and used as an IgE probe in CRIE and Tandem-CRIE. These experiments revealed a fused precipitin line indicating presence of a common structure of P. ovale and C. albicans. This suggested crossreactivity may imply an interesting phenomenon in atopics who are sensitized by C. albicans growth in the gastrointestinal tract and get exposed by P. ovale growing on the skin.(ABSTRACT TRUNCATED AT 250 WORDS)

Watersoluble antigens of Candida albicans were sequentially extracted from intact and disrupted yeast cells grown on protein-free agar, and analysed on immunoblots after SDS-PAGE. Washing of the cells in saline before proper extraction resulted in loss of 47.2% of the total carbohydrate and 1.5% of the total protein. The protein fraction contained 14 antigenic bands when analysed with hyperimmune rabbit antisera. Four of these bound IgE when probed with a RAST-positive serum pool and beta-galactosidase-labelled anti-IgE. Extraction of the disrupted cells resulted in 15% of the total carbohydrate and 94% of the total protein. The cytoplasmic protein fraction showed 69 antigenic bands, 13 of which bound IgE. The carbohydrate fraction contained mannan, which was found in the washing solutions and in the surface extract as well as in the cytoplasmic extract. Allergens found in washing solutions were also present in cytoplasmic fraction. This study suggests that the rapid release of allergens from saprophytic C. albicans cells on mucous membranes of the body may cause continuous exposure and result in sensitization.

Faecal specimens from 570 patients with gastroenteritis were studied for rotaviruses by enzyme immunoassay (EIA), electron microscopy (EM) and latex agglutination test (LX). Specimens from 127 patients were positive and 379 were negative with all 3 methods. 64 (11%) specimens gave contradictory results in the tests. EIA gave significantly more positive results than EM (168 vs 145). 30 EM-negative and EIA-positive specimens were positive in a confirmatory test. LX was positive in 161 specimens and no significant differences to EM or EIA were observed. There were 16 (2.8%) LX-positive samples that were negative both in EM and EIA. The positivity of these samples could not be confirmed and 15 of them were only slightly positive. Our results can be concluded: (1) Enzyme immunoassay may be more sensitive than electron microscopy but requires a confirmatory test and is tedious when only a few specimens per day are to be examined; (2) LX seems to be suitable for primary screening of faecal specimens for rotavirus; for definitely positive results the test is reliable.

A method was developed to facilitate the estimation of S. mutans levels in saliva. Paraffin-stimulated saliva was poured on a special slide coated with mitis-salivarius-sucrose agar. Two discs containing bacitracin (5 micrograms) were placed on the inoculated slide and the growth density of S. mutans around the bacitracin discs was scored after incubation in candle jars at 37 degrees C for 48 h. The obtained score values correlated well with the numbers of CFU of S. mutans per 1 ml of saliva estimated by a conventional method using mitis-salivarius-bacitracin agar. The experimental method was further tested by incubating the slides in an atmosphere created by CO2-generating tables that were placed into the cover tubes of the slides. These score values were similar to those after conventional incubation. The method is suggested for epidemiologic studies and in selecting persons at high risk for caries and in controlling the effectiveness of prophylactic measures of these patients.

The activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), both enzymes of collagen biosynthesis, were measured in the gastrocnemius, soleus, and tibialis anterior muscles of rats after bilateral cast-immobilization of the muscles in lengthened and shortened positions for one and three weeks. The activities of muscular proteolytic and hydrolytic enzymes cathepsin D (CD), beta-glucuronidase (beta-GU), alkaline protease (AP), and the rate of acid autolysis (AA) were also studied. The biochemical results were compared to the morphologic changes by light microscopy. Compared to the results for a control group, there was a decrease of 37% and 53% in the specific PH activity of shortened gastrocnemius and soleus, respectively, after three weeks of immobilization. The corresponding decrease in GGT of the shortened gastrocnemius was 47%. At the same time, PH and GGT in the lengthened plantarflexors were at the control level. The proteolytic activities of the shortened plantarflexors were generally higher (CD by approximately 30%, beta-GU of gastrocnemius by 81%, AP of soleus by 63%, AA of gastrocnemius by 34%, and AA of soleus by 56%) than those of the lengthened ones. Light microscopy of the shortened muscles showed numerous atrophic fibers, but no pronounced inflammatory response in the disused muscle tissue. The results suggest that increased proteolysis and wasting of muscle tissue during cast-immobilization is associated with adaptive responses in the metabolism of the muscular collagen network.

The activities of cathepsin D (CD), beta-glucuronidase (beta-GU), alkaline protease (AKP), and the rate of acid autolysis (AA) were measured in gastrocnemius, soleus, and tibialis anterior muscles of the rat after bilateral cast immobilization in the middle position for one and three weeks. The effects of daily indirect electric stimulation on the proteolytic activities of immobilized muscles were also studied. In gastrocnemius and tibialis anterior, electric stimulation partially prevented the loss of muscle weight after immobilization for one week. Activities of CD and beta-GU were highest in the soleus, but the activities did not change therein after immobilization or electric stimulation. In gastrocnemius, the activities of CD and AKP and the AA rate increased significantly after immobilization. Electric stimulation during immobilization seemed to prevent the increase of CD and AKP activities significantly after one (CD) and three (AKP) weeks immobilization. In tibialis anterior, the activities of CD and AKP and the AA rate increased significantly in the stimulated muscles after immobilization for three weeks. Both in the gastrocnemius and tibialis anterior the beta-GU activity increased significantly in the stimulated muscles. The results suggest that during the disuse atrophy process and electric stimulation of immobilized muscles, rat hind-limb muscles differ in acid and alkaline proteolytic responses.