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Glucocorticoids are important drugs in the treatment of many inflammatory, autoimmune and allergic diseases in humans and animals. We investigated the effects of hydrocortisone and dexamethasone on TNF-α, IL-1Ra and INF-γ release in stimulated whole blood cell culture from healthy horses. Whole blood cell cultures proved to be useful for the characterization of the anti-inflammatory properties of new drugs. Diluted equine whole blood was exposed to lipopolysaccharide (LPS) and PCPwL (a cocktail consisting of phythemagglutinin E, concanavalin A, pokeweed mitogen and lipopolysaccharide) in the presence or absence of hydrocortisone and dexamethasone (10-12 - 10-5 M). TNF-α and IL-1Ra (LPS) as well as IFN-γ (PCPwL) levels were measured in the supernatants using specific enzyme-linked immunosorbent assay (ELISA). The LPS-induced TNF-α and IL-1Ra as well as the PCPwL-induced IFN-γ levels were more potently suppressed by dexamethasone than by hydrocortisone in a concentration-dependent manner. Dexamethasone inhibited TNF-α, IL-1Ra and IFN-γ with the half maximal inhibition concentration (IC50) values of 0.09 μM, 0.453 μM and 0.001 μM, respectively, whereas hydrocortisone inhibited these cytokines with lower IC50 values of 1.45 μM, 2.96 μM and 0.09 μM, respectively. Our results suggest that the equine whole blood test system is useful and reliable to evaluate drug effects and immunological alterations and offers several advantages including simple and cheap performance in physiological and pathological conditions.

Trichophyton (T.) verrucosum is a highly pathogenic dermatophyte causing zoonotic bovine ringworm that is transmissible to humans. The virulence factors subtilisin (Sub)3 and Sub6 are discussed to contribute to disease manifestation but no protein expression study is available for T. verrucosum. We used customized antibodies (against Trichophyton-species, Sub3 and Sub6) to examine skin biopsies of infected cattle via immunofluorescence stainings. Both virulence factors Sub3 and 6 were solely expressed by conidia and not only found in epidermal but also in dermal and hair structures. The anti-T-antibody reliably detected the fungus and proved more sensitive compared to histological stains. Lay Summary We examined the zoonotic dermatophyte Trichophyton (T.) verrucosum in bovine skin and studied two important virulence factors called subtilisin (Sub)3 and Sub6 that T. verrucosum produces and secretes using immunolabeling.

Gut integrity impairment leading to increased intestinal permeability (IP) is hypothesized to be a trigger of critically illness. Approximately 15–20% of human ischemic stroke (IS) victims require intensive care, including patients with impaired level of consciousness or a high risk for developing life-threatening cerebral edema. Local and systemic inflammatory reactions are a major component of the IS pathophysiology and can significantly aggravate brain tissue damage. Intracerebral inflammatory processes following IS have been well studied. Until now, less is known about systemic inflammatory responses and IS consequences apart from a frequently observed post- IS immunosuppression. Here, we provide a hypothesis of a crosstalk between systemic acute phase response (APR), IP and potential secondary brain damage during acute and subacute IS stages supported by preliminary experimental data. Alterations of the acute phase proteins (APPs) C-reactive protein and lipopolysaccharide-binding protein and serum level changes of antibodies directed against Escherichia coli-cell extract antigen (IgA-, IgM-, and IgG-anti-E. coli) were investigated at 1, 2, and 7 days following IS in ten male sheep. We found an increase of both APPs as well as a decrease of all anti- E. coli antibodies within 48 h following IS. This may indicate an early systemic APR and increased IP, and underlines the importance of the increasingly recognized gut-brain axis and of intestinal antigen release for systemic immune responses in acute and subacute stroke stages.

Bacteremia is a hallmark of invasive Streptococcus suis infections of pigs, often leading to septicemia, meningitis, or arthritis. An important defense mechanism of neutrophils is the generation of reactive oxygen species (ROS). In this study, we report high levels of ROS production by blood granulocytes after intravenous infection of a pig with high levels of S. suis-specific antibodies and comparatively low levels of bacteremia. This prompted us to investigate the working hypothesis that the immunoglobulin-mediated oxidative burst contributes to the killing of S. suis in porcine blood. Several S. suis strains representing serotypes 2, 7, and 9 proved to be highly susceptible to the oxidative burst intermediate hydrogen peroxide, already at concentrations of 0.001%. The induction of ROS in granulocytes in ex vivo-infected reconstituted blood showed an association with pathogen-specific antibody levels. Importantly, inhibition of ROS production by the NADPH oxidase inhibitor apocynin led to significantly increased bacterial survival in the presence of high specific antibody levels. The oxidative burst rate of granulocytes partially depended on complement activation, as shown by specific inhibition. Furthermore, treatment of IgG-depleted serum with a specific IgM protease or heat to inactivate complement resulted in >3-fold decreased oxidative burst activity and increased bacterial survival in reconstituted porcine blood in accordance with an IgM-complement-oxidative burst axis. In conclusion, this study highlights an important control mechanism of S. suis bacteremia in the natural host: the induction of ROS in blood granulocytes via specific immunoglobulins such as IgM.

Recently, C-reactive protein (CRP) was shown to affect intracellular calcium signaling and blood pressure in vitro and in vivo, respectively. The aim of the present study was to further investigate if a direct effect on G-protein coupled receptor (GPCR) signaling by CRP can be observed by using CRP in combination with different GPCR agonists on spontaneously beating cultured neonatal rat cardiomyocytes. All used agonists (isoprenaline, clenbuterol, phenylephrine, angiotensin II and endothelin 1) affected the beat rate of cardiomyocytes significantly and after washing them out and re-stimulation the cells developed a pronounced desensitization of the corresponding receptors. CRP did not affect the basal beating-rate nor the initial increase/decrease in beat-rate triggered by different agonists. However, CRP co-incubated cells did not exhibit desensitization of the respective GPCRs after the stimulation with the different agonists. This lack of desensitization was independent of the GPCR type, but it was dependent on the CRP concentration. Therefore, CRP interferes with the desensitization of GPCRs and has to be considered as a novel regulator of adrenergic, angiotensin-1 and endothelin receptors.

Trichophyton benhamiae is an emerging zoonotic dermatophyte. We present a case of a small animal stock infected with two Trichophyton species. T. benhamiae was isolated from 15 out of 26 (58%) guinea pigs including two morphologically different phenotypes. Eight guinea pigs were infected with T. benhamiae and T. mentagrophytes simultaneously. The animals showed alopecia and crusts or no clinical signs at all. T. benhamiae was not detected in rats, rabbits and mice kept in the same stock. Keywords: Trichophyton benhamiae, Trichophyton mentagrophytes, Zoonotic dermatophytes, Guinea pig, Mass spectrometry

BACKGROUND Dermatophytoses rank among the most frequent communicable diseases in humans, and the zoonotic transmission is increasing. The zoophilic dermatophyte Trichophyton (T.) benhamiae is nowadays one of the main causes of tinea faciei et corporis in children. However, scientific data on molecular pathomechanisms and specific virulence factors enabling this ubiquitous occurrence are scarce. OBJECTIVES To study tissue invasion and the expression of important virulence factors of T. benhamiae, isolates that were recovered from two groups of hosts (humans vs. guinea pigs (GP)) using an ex vivo skin model. METHODS After confirmation of species identity by ITS sequencing, CFU suspensions of dermatophyte isolates (n = 20) were applied to the skin infection model and cultured. Employing specific immunofluorescence staining techniques, the expression of subtilisin 3 and 6 and metallocarboxypeptidase A was analysed. The general mode of invasion was explored. Results were compared with biopsies of naturally infected GP. RESULTS All isolates were successfully recovered and proliferated well after application to the infection model. Progressive invasion of hyphae through all skin structures and destruction of explants were observed with early events being comparable to natural infection. An increasing expression of the examined virulence factors towards the end of culture was noticed but no difference between the two groups of isolates. CONCLUSIONS For the first time, important in vivo markers of dermatophytosis were visualised immunohistochemically in an ex vivo skin infection model and in skin biopsies of GP naturally infected with T. benhamiae. More research on the underlying pathomechanisms of dermatophyte infection is urgently needed.

Dermatophyte infections are a growing health concern worldwide with increasing patient numbers, especially in children. However, detailed knowledge about infection mechanisms and virulence factors are scarce. This study aimed to establish an infection model based on guinea pig skin explants mimicking the in vivo situation as closely as possible to survey the pathogenesis of dermatophytoses. A fundamental prerequisite was the detailed description of native guinea pig skin and its morphological changes during tissue culture because comprehensive data on guinea pig skin characteristics were not available. Skin explants were harvested from healthy, adult guinea pigs and transferred to cell culture inserts. One group was inoculated with defined suspensions of colony-forming units of zoonotic Trichophyton benhamiae isolates; others served as controls to assess the tissue viability during the 10-day culture. Samples were taken on days 3, 5, 7 and 10 and processed for histological and immunohistochemical analysis. Standard tissue culture conditions provoked acantholysis and regional orthokeratotic alterations. The reduced desquamation caused hyperkeratosis paralleled by hypogranulosis or regional hyperplasia. During T. benhamiae infection, keratinocyte proliferation came to a complete halt on day 5 whereas the number of terminal deoxynucleotidyl transferase dUTP nick end labeling assay-positive cells increased moderately up to day 7. Hyphae grew massively into the skin explants causing strong keratinolysis and tricholysis. By the end of the culture, complete disintegration of the basement membrane and dermal tissue was observed. A realistic and reliable skin infection model was established to study dermatophytoses in general and cutaneous T. benhamiae infections in particular.

Streptococcus suis (S. suis) causes meningitis, arthritis and endocarditis in piglets. The aim of this study was to characterize the IgM degrading enzyme of S. suis (IdeSsuis) and to investigate the role of IgM cleavage in evasion of the classical complement pathway and pathogenesis. Targeted mutagenesis of a cysteine in the putative active center of IdeSsuis abrogated IgM cleavage completely. In contrast to wt rIdeSsuis, point mutated rIdeSsuis_C195S did not reduce complement-mediated hemolysis indicating that complement inhibition by rIdeSsuis depends on the IgM proteolytic activity. A S. suis mutant expressing IdeSsuis_C195S did not reduce IgM labeling, whereas the wt and complemented mutant showed less IgM F(ab’)2 and IgM Fc antigen on the surface. IgM cleavage increased survival of S. suis in porcine blood ex vivo and mediated complement evasion as demonstrated by blood survival and C3 deposition assays including the comparative addition of rIdeSsuis and rIdeSsuis_C195S. However, experimental infection of piglets disclosed no significant differences in virulence between S. suis wt and isogenic mutants without IgM cleavage activity. This work revealed for the first time in vivo labeling of S. suis with IgM in the cerebrospinal fluid of piglets with meningitis. In conclusion, this study classifies IdeSsuis as a cysteine protease and emphasizes the role of IgM cleavage for bacterial survival in porcine blood and complement evasion though IgM cleavage is not crucial for the pathogenesis of serotype 2 meningitis.

Biotyper analysis of Nannizziopsis guarroi, a fatal fungal pathogen in lizards, was described recently. Hypocrealean fungal infections in captive reptiles appear with an increasing frequency during the last decade. Therefore, the aim of this study was to proof Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) as diagnostic tool for the identification of reptile pathogenic hypocrealean fungi. Ten fungal isolates obtained from nine reptiles with fungal glossitis, disseminated visceral mycosis, pneumomycosis, and fungal keratitis were analyzed. Phylogeny consisted of fragments of the large subunit of nuclear encoded ribosomal DNA (D1/D2, LSU) and the internal transcribed spacer region 1 of nuclear encoded ribosomal DNA (ITS1) as well as the protein coding gene translation elongation factor 1 alpha (TEF). Results revealed unanimously two Metarhizium granulomatis genotypes in a total of three isolates, various M. viride genotypes (n = 3), two different Purpureocillium lilacinum isolates as well as one isolate of each P. lavendulum and Beauveria bassiana. Purpureocillium lilacinum and B. bassiana are likewise frequently employed as a mycoinsecticide and mycoacaricide in agriculture on a worldwide scale and have occasionally been reported in man, causing fungal keratitis, sclerokeratitis, nosocomial infections in immunosuppressed patients, as well as cavitary pulmonary disease and cutaneous hyalohyphomycosis in immunocompetent patients. According to the results establishment of Biotyper analysis for faster differentiation of reptile-associated fungal pathogens is entirely justified.

Streptobacillus (S.) moniliformis is a rat-associated zoonotic pathogen that occasionally causes disease in other species. We investigated the working hypothesis that intranasal infection might lead to different immune responses in C57BL/6 and BALB/c mice associated with distinct pathologies. This study confirmed with 75% mortality the known high susceptibility of C57BL/6 mice to Streptobacillus moniliformis infection in comparison to BALB/c mice which did not develop signs of disease. Main pathologies in C57BL/6 mice were purulent to necrotizing lymphadenitis and pneumonia. Significant seroconversion was recorded in surviving mice of both strains. Differentiation of IgG-subclasses revealed mean ratios of IgG2b to IgG1 below 0.5 in sera of all mice prior to infection and of BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 2.5 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice. Evaluation of different sentinel systems revealed that cultural and serological investigations of these animals might not be sufficient to detect infection. In summary, an intranasal S. moniliformis infection model in C57BL/6 mice leading to purulent to necrotizing inflammations in the lung, the lymph nodes and other organs associated with a Th1 immune response is described.

The present work was directed to investigate the relationship between Downer cow syndrome (DCS) and chronic botulism in dairy cattle. For this purpose, a total of 52 fresh calving downer cows and 206 apparently healthy cows at 14 dairy farms were investigated for Clostridium botulinum ABE and CD antibody levels, C. botulinum and botulinum neurotoxin in rumen fluids as well as in faeces. Results indicated that the downer cows had higher IgG titers for C. botulinum ABE and CD than the healthy cows. All tested rumen fluids were negative for BoNT and C. botulinum. BoNT/D, however, and C. botulinum type D spores were detected in faecal samples of healthy and downer cows in the selected farms. In conclusion, the presence of a significantly higher C. botulinum ABE and CD antibody levels in DCS cows than in the healthy animals suggests that chronic C. botulinum toxico-infection could be a predisposing factor for DCS.

Biogas plants have been considered as a source for possible amplification and distribution of pathogenic bacteria capable of causing severe infections in humans and animals. Manure and biogas wastes could be sources for spore-forming bacteria such as Clostridium botulinum. In the present study, 24 liquid manure and 84 biogas waste samples from dairies where the majority of the cows suffered from chronic botulism were investigated for the presence of botulinum neurotoxins (BoNT) and C. botulinum spores. The prevalence of BoNT/A, B, C, D, and E in biogas wastes was 16.6, 8.3, 10.7, 7.1, and 10.8 %, respectively, while in manure, the prevalence was 0.0, 0.0, 0.0, 8.3, and 4.1 %, respectively. After enrichment of samples in reinforced cultural medium, they were tested for C. botulinum BoNT/A, B, C, D, and E using ELISA (indirect C. botulinum detection). The prevalence of C. botulinum type A, B, C, D, and E samples in biogas wastes was 20.2, 15.5, 19, 10.7, and 34.8 %, respectively, while the prevalence in liquid manure was 0.0, 0.0, 0.0, 8.3, and 12.5 %, respectively. In conclusion, the occurrence of BoNT and C. botulinum spores in biogas waste of diseased animals indicates an increased and underestimated hygienic risk. Application of digestates from biogas fermentations as fertilizers could lead to an accumulation of long lifespan spores in the environment and could be a possible health hazard.

To compare the efficacy of an herbal toothpaste with two other chemically active toothpastes regarding plaque and gingivitis control.Seventy-six (27 females and 49 males, mean age 47.8 years, range 40-58 years) of 84 initial participants with slight and moderate chronic periodontitis used standardised manual toothbrushes and their usual technique for daily manual mechanical plaque control for 24 weeks of supportive periodontal therapy. The volunteers were randomly assigned to one of 3 groups: group 1 used the herbal toothpaste, group 2 a triclosan/copolymer toothpaste, and group 3 an amine/stannous fluoride toothpaste. OHI, API, SBI, BOP, PD and AL were recorded at baseline and after 6, 12 and 24 weeks (PD and AL only at baseline). The Kruskal-Wallis, Mann-Whitney U-, Friedman, and Wilcoxon tests were used for statistical analysis.Moderate changes occurred in API and OHI in all groups. The herbal toothpaste resulted in significantly lower API and OHI in comparison to the fluoride toothpaste during the study period (p = 0.001 and 0.049, minimum and maximum of cases, respectively). SBI was significantly improved in all groups starting after 12 weeks (p = 0.001 and 0.033). BOP remained largely unchanged in all groups and was always significant lower in the herbal toothpaste group (p = 0.001 and 0.036).During the study period of 24 weeks, the herbal toothpaste was as good as the control toothpastes. No side effects were seen. In terms of improving periodontal conditions, the tested herbal toothpaste could be a suitable alternative to conventional toothpastes with artificial chemical ingredients.

OBJECTIVE To measure immunologic responses of snakes after experimentally induced infection with ferlaviruses. ANIMALS 42 adult corn snakes (Pantherophis guttatus) of both sexes. PROCEDURES Snakes were inoculated intratracheally with genogroup A (n = 12), B (12), or C (12) ferlavirus (infected groups) or cell-culture supernatant (6; control group) on day 0. Three snakes from each infected group were euthanized on days 4, 16, 28, and 49, and 3 snakes from the control group were euthanized on day 49. Blood samples were collected from live snakes on days −6 (baseline), 4, 16, 28, and 49. Hematologic tests were performed and humoral responses assessed via hemagglutination-inhibition assays and ELISAs. Following euthanasia, gross pathological and histologic evaluations and virus detection were performed. RESULTS Severity of clinical signs of and immunologic responses to ferlavirus infection differed among snake groups. Hematologic values, particularly WBC and monocyte counts, increased between days 4 and 16 after infection. A humoral response was identified between days 16 and 28. Serum IgM concentrations increased from baseline earlier than IgY concentrations, but the IgY relative increase was higher at the end of the study. The hemagglutination-inhibition assay revealed that the strongest reactions in all infected groups were against the strain with which they had been infected. Snakes infected with genogroup A ferlavirus had the strongest immune response, whereas those infected with genogroup B had the weakest responses. CONCLUSIONS AND CLINICAL RELEVANCE Results of this experimental study suggested that the ferlavirus strain with the highest virulence induced the weakest immune response in snakes.

The zoophilic dermatophyte Trichophyton verrucosum is the most important causative agent of bovine dermatophytosis. Additionally, it causes profound and poorly healing skin infections in humans indicating the high zoonotic potential. The objective of this study was to establish differentiation of T. verrucosum from other dermatophytes by mass spectrometry and to identify distinct features of the mass spectra. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was successful for identification of this pathogen only after extension of the database of the manufacturer with spectra from T. verrucosum strains, which were identified as such by sequencing of the internal transcribed spacer (ITS) region. MALDI-TOF MS analysis was conducted with 46 field isolates from cattle, two live vaccine strains, and 10 isolates from humans identified as T. verrucosum by sequence analysis of the ITS region. The results suggest a very good agreement of both methods. Comparison with the mass spectra of 68 strains of other keratinophilic fungi revealed that most T. verrucosum wild-type isolates showed a characteristic peak at 7950-7954 m/z, which was missing in the spectra of other keratinophilic fungi and the live vaccine strains. The spectra of T. verrucosum were most similar to the spectra of T. benhamiae, an emerging zoophilic dermatophyte. In summary, MALDI-TOF MS is a powerful and reliable tool to identify T. verrucosum.

Fungal infections in captive as well as in free-living reptiles caused by emerging obligate pathogenic fungi appear with increasing frequency and give occasion to establish new and fast methods for routine diagnostics. The so-called yellow fungus disease is one of the most important and common fungal dermatomycoses in central bearded dragons (Pogona vitticeps) and green iguanas (Iguana iguana) and is caused by Nannizziopsis guarroi. The aim of this study was to prove reliability in identification of N. guarroi with Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in comparison to molecular biological analysis of ribosomal DNA genes. In seven lizards from three different species, including central bearded dragons, green iguanas, and a European green lizard (Lacerta viridis), dermatomycoses caused by N. guarroi were diagnosed by isolation of the fungal pathogen as well as histopathological confirmation of the granulomatous inflammatory reaction in deep skin biopsies. With this survey, we proved that MALDI-TOF MS is a diagnostic tool for accurate identification of N. guarroi. Besides small subunit 18S rDNA (SSU) and internal transcribed spacer (ITS)1-5.8S rDNA, a large fragment of the large subunit of the 28S rDNA (LSU), including the domain (D)1 and D2 have been sequenced, for phylogenetical analysis. Large fragment of the LSU from N. guarroi has been sequenced for the first time. Yellow fungus disease in a European lizard species is described for the first time to our knowledge as well, which could be of importance for free-ranging populations of European lizards.

The aim of the present study is to investigate the impact of glyphosate on the microbiota and on the botulinum neurotoxin (BoNT) expression during in vitro ruminal fermentation. This study was conducted using two DAISY(II)-incubators with four ventilated incubation vessels filled with rumen fluid of a 4-year-old non-lactating Holstein-Friesian cow. Two hundred milliliter rumen fluid and 800 ml buffer solution were used with six filter bags containing 500 mg concentrated feed or crude fiber-enriched diet. Final concentrations of 0, 1, 10, and 100 µg/ml of glyphosate in the diluted rumen fluids were added and incubated under CO2-aerated conditions for 48 h. The protozoal population was analyzed microscopically and the ruminal flora was characterized using the fluorescence in situ hybridization technique. Clostridium botulinum and BoNT were quantified using most probable number and ELISA, respectively. Results showed that glyphosate had an inhibitory effect on select groups of the ruminal microbiota, but increased the population of pathogenic species. The BoNT was produced during incubation when inoculum was treated with high doses of glyphosate. In conclusion, glyphosate causes dysbiosis which favors the production of BoNT in the rumen. The global regulations restrictions for the use of glyphosate should be re-evaluated.

Glyphosate (N-phosphonomethyl glycine) is registered as a herbicide for many food and non-food crops, as well as non-crop areas where total vegetation control is desired. Glyphosate influences the soil mycobiota; however, the possible effect of glyphosate residues in animal feed (soybean, corn, etc.) on animal mycobiota is almost unknown. Accordingly, the present study was initiated to investigate the mycological characteristics of dairy cows in relationship to glyphosate concentrations in urine. A total of 258 dairy cows on 14 dairy farms in Germany were examined. Glyphosate was detected in urine using ELISA. The fungal profile was analyzed in rumen fluid samples using conventional microbiological culture techniques and differentiated by MALDI-TOF mass spectrometry. LPS-binding protein (LBP) and antibodies (IgG1, IgG2, IgA, and IgM) against fungi were determined in blood using ELISA. Different populations of Lichtheimia corymbifera, Lichtheimia ramosa, Mucor, and Rhizopus were detected. L. corymbifera and L. ramosa were significantly more abundant in animals containing high glyphosate (>40 ng/ml) concentrations in urine. There were no significant changes in IgG1 and IgG2 antibodies toward isolated fungi that were related to glyphosate concentration in urine; however, IgA antibodies against L. corymbifera and L. ramosa were significantly lower in the higher glyphosate groups. Moreover, a negative correlation between IgM antibodies against L. corymbifera, L. ramosa, and Rhizopus relative to glyphosate concentration in urine was observed. LBP also was significantly decreased in animals with higher concentrations of glyphosate in their urine. In conclusion, glyphosate appears to modulate the fungal community. The reduction of IgM antibodies and LBP indicates an influence on the innate immune system of animals.

In piglet production, Escherichia coli (E. coli) and the presence of lipopolysaccharides (LPS) were considered as predominant causative agents in the aetiology of important diseases of sows and piglets. The aim of the study was to assess the effects of feeding different roughage sources to sows in gestation on the microbiota and endotoxin concentration in colostrum and on the specific immune response of their piglets. In two trial runs, a total of 144 sows were assigned to one of the six dietary treatments: the Control group receiving a concentrate diet and five groups with a restrictive supply of the control diet in combination with straw (S), hay (H), clover grass silage (GS), maize silage (MS) or Jerusalem artichoke (JA). During lactation no roughage was offered. Colostrum samples were analysed for bacteriological and immunological parameters. Blood samples were taken from two piglets per corresponding litter to examine the concentration of C-reactive protein (CRP) and of specific antibodies to LPS of E. coli (strain J5). Roughage feeding had an effect neither on the total bacterial count nor on the content of E. coli in colostrum of sows. The concentration of LPS in colostrum was reduced in Group MS. The concentration of CRP in the colostrum samples was low and independent of the feeding regime (max. 9.3 µg/ml). However, the administration of roughage components rich in crude fibre (Groups H and S) decreased the level of CRP in colostrum significantly. The analysis of the specific immunoglobulin to LPS of E. coli (strain J5) showed a higher concentration of IgG-anti-LPS in blood serum of piglets from sows of Group JA. The results suggest that the inclusion of selected roughage components may have the potential to affect the immunocompetence of sows and their corresponding piglets.

In the present study the efficacy Botulism vaccine (formalinised aluminum hydroxide gel adsorbed toxoid of Clostridium botulinum types C and D) was evaluated in four Danish dairy cows under field conditions. Other four dairy herds were unvaccinated. Blood serum of all animals was analyzed for specific C. botulinum types A, B, C, D and E antibodies using a developed ELISA. Feces of all animals were analyzed for botulinum neurotoxins (BoNTs) and C. botulinum spores. C. botulinum types C and D antibodies were significantly (p < 0.05) increased in vaccinated animals. Vaccination with botulism vaccine significantly reduced (p < 0.001) BoNTs and C. botulinum spores in cattle feces. Our findings represent that C. botulinum vaccination increases specific blood serum antibodies and reduces free BoNTs and C. botulinum spores in feces.

In the present study, efficacy of the toltrazuril treatment for prevention of coccidiosis and necrotic enteritis was tested. Ninety-six 14-day-old commercial broiler chickens were caged and divided into eight groups (n=12), designated groups 1 to 8. Chickens of groups 1 to 6 were inoculated orally at 18 days of age with 25,000 oocysts of Eimeria tenella and 75,000 oocysts of Eimeria brunetti. At 22 days of age, chickens of groups 1 to 6 were infected with 10(9) colony-forming unit Clostridium perfringens. Chickens of group 1 were treated with 75 parts/10(6) toltrazuril in drinking water for 8 h on two consecutive days up to 12 h before Eimeria infection, while chickens of groups 2 to 5 were treated with the same dose of toltrazuril at 12 h, 36 h, 60 h and 84 h after Eimeria infection, respectively. The non-treated group 6 served as a positive control. Chickens in group 7 were treated with toltrazuril at 17 and 18 days of age, and those of group 8 remained uninfected and non-treated as a negative control. The feed conversion ratio was higher in the positive control compared with other groups. The mortality rates were 16.8% and 41.7% in the late toltrazuril-treated (at 84 h) and infected non-treated chickens, respectively. Lesions scores of necrotic enteritis or coccidiosis in infected, non-treated chickens were significantly more severe compared with negative controls (P

Human C-reactive protein (CRP) and serum amyloid component P (SAP) are well-characterised ligands for dying and dead cells, while facets of their physiological function still need to be unravelled. We partially characterised CRP and SAP from different species with similar acute-phase systems. Human, rabbit and porcine CRP bound phosphocholine (PC) and phosphoethanolamine (PEt). Human and porcine SAP bound PEt while rabbits seem to have very low levels of SAP or rabbit SAP does not bind PEt. Porcine serum additionally contained other ligands for PC and PEt. Some of them were immunoglobulins. Therefore, rabbits, pigs and humans cover the ability to bind PC and PEt with different extents.

Acute phase proteins (APPs) are highly conserved plasma proteins that are increasingly secreted by the liver in response to a variety of injuries, independently of their location and cause. APPs favor the systemic regulation of defense, coagulation, proteolysis, and tissue repair. Various APPs have been applied as general diagnostic parameters for a long time. Through proteomic techniques, more and more APPs have been discovered to be differentially altered. Since they are not consistently explainable by a stereotypic hepatic expression of sets of APPs, most of these results have unfortunately been neglected or attributed to the nonspecificity of the acute phase reaction. Moreover, it appears that various extrahepatic tissues are also able to express APPs. These extrahepatic APPs show focally specific roles in tissue homeostasis and repair and are released primarily into interstitial and distal fluids. Since these focal proteins might leak into the circulatory system, mixtures of hepatic and extrahepatic APP species can be expected in blood. Hence, a selective alteration of parts of APPs might be expected. There are several hints on multiple molecular forms and fragments of tissue-derived APPs. These differences offer the chance for multiple selective determinations. Thus, specific proteoforms might indeed serve as tissue-specific disease indicators.

Zygomycetes of the order Mucorales can cause life-threatening infections in humans. These mucormycoses are emerging and associated with a rapid tissue destruction and high mortality. The resistance of Mucorales to antimycotic substances varies between and within clinically important genera such as Mucor , Rhizopus , and Lichtheimia . Thus, an accurate diagnosis before onset of antimycotic therapy is recommended. Matrix-assisted laser desorption ionization (MALDI)–time of flight (TOF) mass spectrometry (MS) is a potentially powerful tool to rapidly identify infectious agents on the species level. We investigated the potential of MALDI-TOF MS to differentiate Lichtheimia species, one of the most important agents of mucormycoses. Using the Bruker Daltonics FlexAnalysis (version 3.0) software package, a spectral database library with m/z ratios of 2,000 to 20,000 Da was created for 19 type and reference strains of clinically relevant Zygomycetes of the order Mucorales (12 species in 7 genera). The database was tested for accuracy by use of 34 clinical and environmental isolates of Lichtheimia comprising a total of five species. Our data demonstrate that MALDI-TOF MS can be used to clearly discriminate Lichtheimia species from other pathogenic species of the Mucorales. Furthermore, the method is suitable to discriminate species within the genus. The reliability and robustness of the MALDI-TOF-based identification are evidenced by high score values (above 2.3) for the designation to a certain species and by moderate score values (below 2.0) for the discrimination between clinically relevant ( Lichtheimia corymbifera , L. ramosa , and L. ornata ) and irrelevant ( L. hyalospora and L. sphaerocystis ) species. In total, all 34 strains were unequivocally identified by MALDI-TOF MS with score values of >1.8 down to the generic level, 32 out of 34 of the Lichtheimia isolates (except CNM-CM 5399 and FSU 10566) were identified accurately with score values of >2 (probable species identification), and 25 of 34 isolates were identified to the species level with score values of >2.3 (highly probable species identification). The MALDI-TOF MS-based method reported here was found to be reproducible and accurate, with low consumable costs and minimal preparation time.

Zusammenfassung Gegenstand und Ziel: Ziel dieser Studie war die Beurteilung des perioperativen Verlaufs der Konzentration des kaninen C-reaktiven Proteins (CRP) im Blutserum bei komplikationsloser Wundheilung und bei postoperativen Wundheilungsstörungen. Material und Methoden: Entsprechend dem postoperativen Verlauf der Wundheilung wurden 99 Hunde in folgende Gruppen eingeteilt: komplikationslose Wundheilung (Kontrollgruppe, n = 57), Wundinfektion (n = 11), Nahtdehiszenz (n = 5), Serom (n = 6), Hämatom (n = 7), passagerer Reizzustand (n = 12) und sonstige Komplikationen (n = 1). Ergebnisse: Patienten mit komplikations-loser Wundheilung zeigten klinisch im postoperativen Verlauf eine stetige Abschwächung der initialen lokalen Entzündungsreaktion. Auch die CRP-Konzentration war durch einen initialen Gipfel und einen sich anschließenden stetigen Abfall charakterisiert. Bei Patienten, deren Wundheilung durch eine fortbestehende oder wiederaufflammende Entzündung charakterisiert war, ergaben sich postoperativ fast immer unverändert hohe CRP-Konzentrationen oder ein deutlicher Wiederanstieg sowie höhere CRP-Konzentrationen als bei den Kontrolltieren. Dies konnte nicht nur bei Patienten mit klinisch sichtbarer Wundinfektion oder Nahtdehiszenz nachgeweisen werden, sondern auch bei Patienten ohne eindeutige klinische Anzei-chen einer Nahtdehiszenz oder Wundinfektion (passagerer Reizzustand). Demgegenüber zeigten Patienten mit Serom oder Hämatom im weiteren postoperativen Verlauf weder klinisch eine ausgeprägte Entzündungsreaktion noch erhöhte CRP-Konzentrationen. Schlussfolgerung: Die serielle Bestimmung der CRPKonzentration stellt einen objektiven Parameter zur Beurteilung der Wundheilung dar. Klinische Relevanz: Unverändert hohe oder deutlich wiederansteigende CRP-Konzentrationen sind, unter Beachtung bestimmter Voraussetzungen, eine Indikation, weitere diagnostische Maßnahmen einzuleiten und therapeutische Gegenmaßnahmen zu ergreifen.

Zusammenfassung Gegenstand: Untersuchung des Einflusses konservativ nicht therapierbarer Torsio uteri intra partum mit Sectio caesarea auf den Blutplasmagehalt des C-reaktiven Proteins (CRP) im Vergleich mit Dystokien anderer Genese und Sectio caesarea sowie Normalgeburten beim Rind. Material und Methode: In die Studie gingen 55 Rinder ein, eingeteilt in drei Gruppen. Gruppe 1 bestand aus 31 Rindern mit operativ beendeter Torsio uteri intra partum. In Gruppe 2 befanden sich 13 Tiere mit durch Kaiserschnitt beendeter Dystokie anderer Genese und in Gruppe 3 11 Kühe mit Normalgeburten und ungestörtem Puerperium. Im Abstand von 24 Stunden, beginnend unmittelbar präoperativ, bis 10 Tage post operationem wurden Blutproben aus der V. jugularis externa entnommen und das CRP im Plasma mittels ELISA quantitativ bestimmt. Ergebnisse: Schwere intrazelluläre Ödeme und Nekrosen am Uterus als Folge einer Torsio uteri intra partum führten zu einem Anstieg der CRPKonzentration, eine Ovariohysterektomie bedingte einen Abfall des CRP-Spiegels. Der alleinige chirurgische Eingriff einer Sectio caesarea verursachte keine wesentliche akute Veränderung der CRP-Konzentration. Aufgrund der großen individuellen Variation der CRP-Konzentrationen war eine Definition eindeutiger Grenzwerte bei den einzelnen Tiergruppen nicht möglich. Eine prognostische Aussage zur Fertilität nach Torsio uteri und Kaiserschnitt lässt sich anhand des CRP-Werts nicht ableiten. Der positive Einfluss der Milchleistung auf die CRP-Konzentration im Blutplasma konnte bestätigt werden. Schlussfolgerung: Nekrosen und schwere intrazelluläre Ödeme am Uterus beeinflussen die CRP-Blutplasmakonzentration deutlich. Klinische Relevanz: Hohe CRP-Werte (> 90 μg/ml) sprechen bei Rindern mit Torsio uteri intra partum für eine schwere Uterusschädigung, die mit einer drastischen Verschlechterung der Prognose quo ad vitam einhergeht. Eine Ovariohysterektomie kann in diesen Fällen eine gute Therapieoption sein.

Background Horses are much predisposed and susceptible to excessive and acute inflammatory responses that cause the recruitment and stimulation of polymorphnuclear granulocytes (PMN) together with peripheral blood mononuclear cells (PBMC) and the release of cytokines. The aim of the study is to develop easy, quick, cheap and reproducible methods for measuring tumor necrosis factor alpha (TNF-α) and interleukin-1 receptor antagonist (IL-1Ra) in the equine whole blood cultures ex-vivo time- and concentration-dependently. Results Horse whole blood diluted to 10, 20 and 50 % was stimulated with lipopolysaccharide (LPS), PCPwL (a combination of phytohemagglutinin E, concanavalin A and pokeweed mitogen) or equine recombinant TNF-α (erTNF-α). TNF-α and IL-1Ra were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). Both cytokines could be detected optimal in stimulated 20 % whole blood cultures. TNF-α and IL-1Ra releases were time-dependent but the kinetic was different between them. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24–48 h, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8–12 h and started to decrease thereafter, whereas IL-1Ra peaked later between 12–24 h and rather continued to accumulate over 48 h. The equine recombinant TNF-α could induce also the IL-1Ra release. Conclusions Our results demonstrate that similar to PCPwL, LPS stimulated TNF-α and IL-1Ra production time-dependently in whole blood cultures, suggesting the suitability of whole blood cultures to assess the release of a variety of cytokines in health and diseases of horse.

The clinical and anti-inflammatory effects of a single treatment of 0·4 mg meloxicam/kg bodyweight on pigs that had been challenged with Escherichia coli endotoxin were investigated. Significantly lower total clinical scores were recorded in pigs treated with meloxicam than in pigs treated with a placebo. Significantly higher mean serum concentrations of thromboxane B 2 were also recorded in pigs treated with a placebo for up to 24 hours after the challenge. The serum concentrations of acute phase proteins and specific antibody titres to E coli lipopolysaccharide were unaffected by the meloxicam. The meloxicam treatment was well tolerated.

Zusammenfassung: Ziel: Zur Beurteilung von intestinaler Ischämie wurden spezifische Parameter im Blut und Bauchpunktat (BP) bei Pferden mit verschiedenen Kolikformen untersucht. Material und Methoden: Bei acht gesunden und 26 Kolikpferden (Dislocatio coli [geringgradige Ischämie], n = 14; Dünndarmstrangulation [hochgradige Ischämie], n = 12) erfolgte im Blut und/oder BP die Bestimmung folgender Parameter: Leukozytenzahl, Hämatokrit (HK), pH-Wert, Basenüberschuss, Harnstoff, Kreatinin,Totalprotein (TP), Albumin, Laktat, LDH, AP, CK und C-reaktives Protein (CRP). Für TP, Albumin, Laktat, LDH, AP, CK, CRP wurde das Verhältnis der Werte in BP und Blut (BP/B-Ratio) berechnet und mit den Light-Kriterien zum Nachweis eines Exsudatsverglichen. Eine Differenzierung und Beurteilung der Leukozyten im BP nach spezifischen Apoptose- und Ne- krosemerkmalen erfolgte an gefärbten Ausstrichen. Die Statistik beinhaltete u. a. die Prüfung auf Zuverlässigkeit der Light-Kriterien und die Berechnung der Korrelationen der Parameter zum Ischämiegrad. Ergebnisse: Mit zunehmendem Schweregrad der Kolik ergab sich eine signifikante Erhöhung und positive Korrelation von Laktat, LDH und CK im Blut sowie von TP, Albumin, Laktat, LDH, AP, CK und CRP im BP. Auch die BP/B-Ratio dieser Parameter korrelierte signifikant mit dem Ischämiegrad. Nur bei Pferden mit Dünndarmstrangulation entsprach das BP bezüglich der Light-Kriterien einem Exsudat. Die Wahrscheinlichkeit, dass ein BP ein Exsudat darstellte, war bei einer LDH-Aktivität von > 200 U/l am größten. Die CRP-Konzentration im BP stieg bei Pferden mit hochgradiger Ischämie signifikantan. Die Leukozytenbeurteilung ergab bei allen Kolikpferden einen erhöhten prozentualen Anteil apoptotischer Zellen, v. a. polymorphkerniger Zellen, und eine signifikante Zunahme nekrotischer Leukozyten im BP bei Pferden mit Dünndarmstrangulation. Schlussfolgerung und klinische Relevanz: TP, Albumin, Laktat, LDH, AP, CK im BP und die BP/B-Ratio dieser Parameter sind für die Beurteilung ischämischer Prozesse hilfreich. CRP im BP und die Light-Kriterien können zur Charakterisierung hochgradiger intestinaler Schädigungen genutzt werden. Eine gesteigerte Apoptose und Nekrose der Leukozyten im BP stellen Indikatoren für intraabdominale Entzündungen dar.

Title of the paper: Application of Echinacea-extract in pregnant and suckled sows In an experiment with thirteen sows (echinacea: n = 6; control: n = 7), the influence of Echinacea purpurea treatment of pregnant and suckled sows on immune system, health status, growth performance, and carcass quality of the offspring (echinacea: n = 69; control: n = 79) was investigated. Echinacea was given as liquid feed supplement during the whole pregnant and suckling period of sows in six intervalls (5 days of treatment, followed by 2 weeks of break, respectively). During the treatment time the daily dosage per sow in echinacea-group was 0.125 ml preparation of echinacea extract (incl. solution)/kg body weight. To measure the concentrations of immunoglobulin G (IgG), immunoglobulin A (IgA) and C-reactive protein (CRP) in serum, blood samples were taken by punction of V. cava cranialis (sows and piglets) at different times. The treatment of sows resulted in immunostimulatory effects both in sows and piglets with highest changes in peripartal period. In one day old piglets, the concentrations of IgG, IgA and CRP were significantly increased in echinacea-group (P = 0.0004, P < 0.0001, P = 0.05, respectively). Up to day 70 of age, the rate of therapeutical treatments of piglets from echinacea-treated sows was decreased in tendency (P = 0.08). The growth performance and carcass quality of offspring was not influenced by echinacea treatment of sows.

Aims: To examine the immune consequences of chronic alcoholism in man, in relation to the known association between alcoholism and raised incidence and severity of infections. Methods: In 36 alcoholics without liver disease, at the point of commencing withdrawal from alcohol, the following measures of immune competence were measured: the immunophenotypes of cells, acute phase proteins, the endotoxin-neutralizing capacity (ENC) of the serum, titers of anti-lipopolysaccharide (LPS) antibodies, and ex vivo cytokine inducibility in T cells and monocytes (TNFα, IL1β, IL1RA, IL4, IL6, IL8, IL10 and IL12). The results were compared to those from healthy volunteers (day controls). Measures were repeated after 8-13 days of abstinence. Results: LPS-binding protein (LBP) and soluble CD14 (sCD14) were significantly increased in patients' sera at the outset of withdrawal, whereas reduced titers of anti-LPS IgG (P = 0.012) and a reduced ENC (P = 0.001) were measured. Only ENC rapidly returned to normal values after withdrawal therapy. Cytokine induction with phorbol ester showed no significant alterations in patients' T cells. Patients' monocytes, however, responded to LPS stimulation with enhanced IL1β-, but reduced TNFα- and IL12-production (P = 0.004, P = 0.0042 and P = 0.001, respectively). While IL1- and TNFα-responses normalized after the withdrawal period, impairment of the IL12 response persisted throughout the observation period of 2 weeks. Conclusions: Alcoholism results in a prolonged LPS-mediated hypoinflammatory conditioning of the innate but not the adaptive immune system, which is not reversed immediately after withdrawal. This alcohol- induced status of the immune system predisposes to infections and sepsis by blunting initial response to the pathogens.

From 41 dairy farms in Schleswig Holstein, Germany, 196 fecal specimens of diseased cows, 77 fecal specimens of farmers and family members from 26 of these farms, 35 animal feed specimens and 7 house dust specimens were investigated for Clostridium botulinum and its antigens, respectively. Four of the humans under study (one child, 8 month, and three adults) showed symptoms of infant/visceral botulism. Specimens were cultivated in reinforced clostridial medium (RCM). C. botulinum antigens were detected by ELISA. The aim of the study was to obtain information on the relationship of detected C. botulinum toxin-types in cows, in the feces of attending humans, and in the immediate environment. The results revealed that C. botulinum toxin-types were different for cows and humans. Toxin-type A was dominant in cow feces while type E was found in humans. Type E was also present in some animal feed specimens. Conversely, toxin-type A was prevalent in the house dust of farms. It may be assumed that the feeds were the source of human colonization with C. botulinum.

AIM To test the effects of humic acids on innate microbial communities of the colon. METHODS We followed the effects of oral supplementation with humic acids (Activomin®) on concentrations and composition of colonic microbiome in 14 healthy volunteers for 45 d. 3 × 800 mg Activomin® were taken orally for 10 d followed by 3 × 400 mg for 35 d. Colonic microbiota were investigated using multicolor fluorescence in situ hybridization (FISH) of Carnoy fixated and paraffin embedded stool cylinders. Two stool samples were collected a week prior to therapy and one stool sample on days 10, 31 and 45. Forty-one FISH probes representing different bacterial groups were used. RESULTS The sum concentration of colonic microbiota increased from 20% at day 10 to 30% by day 31 and remained stable until day 45 (32%) of humic acid supplementation (P < 0.001). The increase in the concentrations in each person was due to growth of preexisting groups. The individual microbial profile of the patients remained unchanged. Similarly, the bacterial diversity remained stable. Concentrations of 24 of the 35 substantial groups increased from 20% to 96%. Two bacterial groups detected with Bac303 (Bacteroides) and Myc657 (mycolic acid-containing Actinomycetes) FISH probes decreased (P > 0.05). The others remained unaffected. Bacterial groups with initially marginal concentrations (< 0.1 × 109/mL) demonstrated no response to humic acids. The concentrations of pioneer groups of Bifidobacteriaceae, Enterobacteriaceae and Clostridium difficile increased but the observed differences were statistically not significant. CONCLUSION Humic acids have a profound effect on healthy colonic microbiome and may be potentially interesting substances for the development of drugs that control the innate colonic microbiome.

Einleitung: Chlamydia psittaci (Cp)-Infektionen konnen bei Mensch und Tier respiratorische Erkrankungen unterschiedlichen Schweregrades verursachen. Unter Verwendung eines etablierten Kalber-Modells [1, 2] waren die Ziele der vorliegenden Studie 1.) die pathophysiologische Charakterisierung der pulmonalen Reaktionsmechanismen sowie 2.) der klinische Vergleich mit naturlich infizierten Kalbern. Methoden: Je 21 Kalbern wurden 108 Einschluss-bildende Einheiten Cp oder Zellsuspension intrabronchial inokuliert. 1 – 2 Tage nach Inokulation (dpi) wurden drei gesunde, naive Sentinel-Kalber mit den Cp-infizierten Tieren vergesellschaftet. Ausgangswerte wurden ab einer Woche vor Inokulation gewonnen. Analysiert wurden Ergebnisse des klinischen Scorings, der nicht-invasiven Lungenfunktionsdiagnostik (Spirometrie, Kapnovolumetrie, Impulsoszillo-resistometrie) und der Blutproben (Zellkomposition, Konzentration des Lipopolysaccharid-bindenden Proteins (LBP), Gehalt chlamydialer DNA, Antikorper Bildung). Wahrend der Sektionen (n = 3 an 2, 3, 4, 7, 10, 14 & 35 dpi) wurde broncho-alveolare Lavageflussigkeit (BALF) gewonnen und zytologisch untersucht. Ergebnisse: Nach experimenteller Inokulation erreichte die fieberhafte, von respiratorischen Symptomen gepragte Allgemeininfektion ihren Hohepunkt 2 – 3 dpi und klang im Laufe einer Woche ab, ohne das Ausgangsniveau wieder zu erreichen. Dieser Verlauf spiegelte sich auch in der LBP-Konzentration wider. Trotz kurzen, flachen Atmungsmusters war 2 – 3dpi das Atemminutenvolumen erhoht. Anderungen der Atmungsmechanik resultierten aus Obstruktionen und Restriktionen, die bis 11dpi nachweisbar waren. Der Gehalt an Lymphozyten (L) im Blut sank nach Cp Inokulation signifikant (bis 4 dpi), wahrend stab- und segmentkernige neutrophile Granulozyten (N) anstiegen (2 dpi Leukozytose). Beide Zellfraktionen wurden zum Entzundungsherd Lunge rekrutiert (BALF: ↑N: 2 – 7 dpi, ↑L : 7 – 10 dpi). Nach Erregerkontakt war chlamdiale DNA bei 67% der Cp-exponierten Kalber im Blut nachweisbar, wobei der Gehalt in der experimentell infizierten Gruppe signifikant hoher war als bei den Sentinels, deren Infektion auch deutlich milder verlief. In beiden Gruppen fanden sich geringe DNA-Mengen bis zum Studienende (35 dpi) im Blut. Eine humorale Immunantwort war nur bei 67% der experimentell inokulierten Tiere nachweisbar, begann fruhestens 10 dpi und sank teilweise schon zum Studienende hin wieder ab. Diskussion: Die Freiheit von Symptomen nach Cp-Infektion erlaubt keinen Ruckschluss bezuglich der Elimination des Erregers oder dem Aufbau einer humoralen Immunantwort. Literatur: [1] Ostermann C et al. Vet J 2013; 196:351 – 359. [2] Reinhold P et al. PLoS One. 2012;7:e30125.7.

It is not clear whether increased asthma severity associated with long-term use of β2-adrenoceptor (β2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between β-AR agonist-mediated effects on β2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o− cells grown in the presence and absence of β-AR agonists and/or antagonists, the β2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in β2-AR density. Both Salbu and ISO reduced cAMP generation in a concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the β2-AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered β2-AR expression and function in airway epithelial cells. β2-AR desensitization and downregulation induced by long-term treatment with β2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy.