Stability of an epidural analgesic admixture of levobupivacaine, fentanyl and epinephrine.

WHAT IS KNOWN AND OBJECTIVE: Admixtures of levobupivacaine, fentanyl and epinephrine are increasingly used in epidural pain management. Neither the compatibility nor the stability of levobupivacaine with fentanyl and epinephrine is known and therefore we examined the chemical, physical and microbiological stability of levobupivacaine-fentanyl-epinephrine and levobupivacaine-fentanyl admixtures prepared in the hospital pharmacy. METHODS: Fentanyl and epinephrine were added into commercial levobupivacaine infusion bags. The components were analysed by HPLC and assays were performed up to 60 days of storage of the bags both protected and exposed to light at room temperature and stored in the refrigerator. In addition, sterility, bacterial endotoxins, organoleptic properties, pH and mass of the admixture were determined. RESULTS AND DISCUSSION: Levobupivacaine, fentanyl and epinephrine concentrations remained within the ± 10% specification limit during 60 days storage in the refrigerator in tightly closed secondary packing material and protected from light and for at least 40 days at room temperature. The degradation of epinephrine exceeded 10% within 60 hours when exposed to light. The solutions were microbiologically and physically stable. WHAT IS NEW AND CONCLUSION: Epidural analgesic admixtures of levobupivacaine and fentanyl with or without epinephrine have to be stored in a tightly closed secondary package protected from light. The extended stability, up to 60 days, in a refrigerator enables the centralized preparation in the hospital pharmacy.

Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds.

The aim of this study was to examine the occurrence of bacterial, mycoplasmal and viral pathogens in the lower respiratory tract of calves in all-in all-out calf-rearing units. According to clinical status, non-medicated calves with and without respiratory disease signs were selected of the 40 herds investigated to analyse the micro-organisms present in healthy and diseased calves. Tracheobronchial lavage (TBL) and paired serum samples were analysed for bacteria, mycoplasmas, respiratory syncytial virus (RSV), parainfluenza virus 3 (PIV3), bovine corona virus (BCV) and bovine adenovirus (BAV). Pasteurella multocida was the most common bacterial pathogen. It was isolated from 34% of the TBL samples in 28 herds and was associated with clinical respiratory disease (p < 0.05) when other pathogenic bacteria or mycoplasma were present in the sample. Mannheimia spp. and Histophilus somni were rarely found. Mycoplasma bovis was not detected at all. Ureaplasma diversum was associated with clinical respiratory disease (p < 0.05). TBL samples from healthy or suspect calves were more often negative in bacterial culture than samples from diseased calves (p < 0.05). No viral infections were detected in six herds, while 16-21 herds had RSV, BCV, BAV or PIV3. In the herds that had calves seroconverted to BCV, respiratory shedding of BCV was more frequently observed than faecal shedding. This study showed that the microbial combinations behind BRD were diverse between herds. M. bovis, an emerging pathogen in many countries, was not detected.

Tissue localization of bovine dander allergen Bos d 2.

BACKGROUND: Domestic mammals are important sources of indoor allergens. However, the origin at the tissue level and the biologic function of mammalian allergens are largely unknown. OBJECTIVE: The aim of this study was to localize the source of the major bovine dander allergen, Bos d 2, in bovine tissues. METHODS: Samples from several organs were tested for the presence of mRNA encoding Bos d 2 and Bos d 2 protein by using the reverse transcriptase polymerase chain reaction and immunohistochemical staining, respectively. RESULTS: Skin proved to be the only tissue where mRNA encoding Bos d 2 was detected. This observation was confirmed by immunohistochemistry with a monoclonal anti-Bos d 2 antibody as the primary antibody. In the skin sections, Bos d 2 was found in the secretory cells of apocrine sweat glands and the basement membranes of the epithelium and hair follicles. Bos d 2 belongs to the family of lipocalins comprising a number of pheromone carrier proteins that are present, for example, in the secretions of the apocrine sweat glands. CONCLUSION: Together with earlier data, our findings suggest that Bos d 2 is produced in sweat glands and transported to the skin surface as a carrier of the pheromone ligand. Because dander allergens of a number of mammalian species are lipocalins, the common biologic function of being pheromone carriers seems to be a common feature of an important group of aeroallergens.

Molecular analysis of allergenic proteins in bovine dander.

An analytic procedure was established to characterize bovine dander proteins with allergenic properties. The proteins from dander extract were separated by size-exclusion gel filtration, and the fractions were studied with SDS-PAGE followed by immunoblotting. An 11-kDa allergen was found in the same gel filtration fractions as 20- and 22-kDa allergens, and this suggests that the 11-kDa allergen is a dimer in its native form. Our method also detected two separate 22-kDa allergens. The primary structure of the major bovine dander allergen (BDA20) was also studied. A protein sequencer was used to determine the amino acid sequences of enzymatically cleaved peptides. The homology searches revealed that BDA20 is not a previously known bovine protein.

Complementary DNA cloning of the predominant allergen of bovine dander: a new member in the lipocalin family.

BACKGROUND: A number of allergenic proteins in animal danders have been characterized at the molecular level, but little is known of their biologic functions. We have found that the prevalence of IgE antibodies among patients with cattle-associated asthma is highest against a dander protein referred to as BDA20. OBJECTIVE: The study was performed to characterize the molecular structure of BDA20,* the predominant allergen in bovine dander. METHODS: Clones encoding allergens were identified and isolated from a complementary DNA library by immunoblotting and DNA hybridization and sequenced. Recombinant proteins were produced in Escherichia coli. Immunoreactivity of the recombinant proteins and amino acid sequences of peptides obtained from native BDA20 after Lys-C cleavage were used to identify clones coding for BDA20. RESULTS: In this article we report the cDNA and amino acid sequences of BDA20. Homology comparisons showed that BDA20 belongs to the family of lipocalins. CONCLUSIONS: The results link a dander allergen to a group of functionally important proteins. Lipocalins are present in various body fluids and secretions of several animal species in which they function as carriers of small hydrophobic molecules, such as retinoids and pheromones. If allergenicity proves to be a property shared by lipocalins, our results will have considerable implications for allergen research.

Immune reactivity of cow-asthmatic dairy farmers to the major allergen of cow (BDA20) and to other cow-derived proteins. The use of purified BDA20 increases the performance of diagnostic tests in respiratory cow allergy.

BACKGROUND: Cow dust is one of the most important inducers of occupational allergic diseases in Finland. For example, in 1991 it accounted for almost 40% of the new occupational asthma cases. OBJECTIVE: This study compares the performance of the purified major cow allergen (BDA20) and crude bovine epithelial extract (BEA) in diagnostic tests and examines the role of milk allergy-associated bovine proteins (bovine serum albumin, alpha-lactalbumin, beta-lactoglobulin, casein) in respiratory cow allergy. METHODS: The humoral responses of cow-asthmatic and healthy farmers to the various components of BEA were analysed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The levels of specific IgE and IgG antibodies were quantificated with enzyme-linked immunosorbent assays (ELISAs). The cellular responses were analysed with antigen-specific lymphocyte proliferation tests. RESULTS: The specific anti-BDA20 IgE measurement was found to be best in distinguishing between the asthmatic farmers and their healthy colleagues. It proved possible to determine a cut-off value that gave the analysis a specificity and sensitivity of 100%; the distinction between the two groups was highly significant (P < 0.0001). In the lymphocyte proliferation analysis, cow asthma was more closely associated with reactivity to BDA20 than to BEA. In the measurement of anti-BDA20 and anti-BEA IgG antibody levels, considerable overlap between the groups was observed, suggesting that these antibodies are not directly involved in cow allergy. When proteins associated with milk allergy were used as test reagents, no statistically significant differences could be observed between the groups, except for anti-casein IgE antibodies the level of which, however, overlapped considerably between the farmer groups. CONCLUSION: These findings suggest that purified BDA20 is better than BEA for diagnosing cow asthma and that proteins associated with milk allergy are of only marginal significance in this disease.

cDNA cloning and protein analysis of a bovine dermal allergen with homology to psoriasin.

Immunoscreening of a cDNA library from bovine skin led to isolation of clones coding for an allergen named BDA11. Sequence analysis of the clones revealed that they can encode a protein of 11.6 kDa with a predicted pI of 5.19. Allergenicity of BDA11 was verified by the IgE reactivity in cattle-allergic patients' sera with the recombinant protein produced in Escherichia coli. A biochemically purified native allergen of 11 kDa from bovine dander was identified as BDA11 by peptide sequencing. Homology comparisons showed that BDA11 had a 63.4% amino acid identity with human psoriasin. Psoriasin is a calcium-binding protein expressed in keratinocytes, and it is strongly up-regulated in psoriatic skin. BDA11 also had segments homologous with calcium-binding proteins from three other species.

Homology of a bovine allergen and the oligomycin sensitivity-conferring protein of the mitochondrial adenosine triphosphate synthase complex.

We have characterized bovine allergens by constructing and analyzing a complementary DNA library from bovine skin. Clones producing proteins that reacted with IgE antibodies from persons with allergy were purified and sequenced. One set of the allergen-coding clones showed an almost complete homology with the bovine oligomycin sensitivity-conferring protein of the mitochondrial adenosine triphosphate synthase complex. The IgE antibodies adsorbed with the recombinant allergen reacted with an 11 kd protein in the cow dander extract. Binding of the IgE from patients allergic to the recombinant allergen expressed in Escherichia coli confirmed the allergen-coding ability of the complementary DNA sequence. The prevalence of the IgE-positive sera among patients with cow allergy and control subjects suggests that the recombinant allergen represents one of the minor allergens in cow dander. This is the first time a mammalian allergen has been identified as a protein with a known function.