Investigation of the pathophysiology of bacterial mastitis using precision-cut bovine udder slices.

Mastitis in cattle is a major health problem as well as incurring high costs for the dairy industry. To assess the suitability of precision-cut bovine udder slices (PCBUS) for bovine mastitis studies, we infected PCBUS with 2 different Staphylococcus aureus strains. Accordingly, we investigated both the tissue response to infection based on immune mediators at the mRNA and protein levels and the invasion of bacteria within the tissue. The studied proteins represent immune mediators of early inflammation [IL-1ß, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2)] and showed a time-dependent increase in concentration. Infection of PCBUS with S. aureus resulted in increased expression of proinflammatory cytokines and chemokines such as TNF-α, C-C motif chemokine ligand 20 (CCL20), IL-1ß, IL-6, and IL-10, but not C-X-C motif chemokine ligand 8 (CXCL8), lingual antimicrobial peptide (LAP), or S100 calcium binding protein A9 (S100A9) at the mRNA level. To compare the data acquired with this model, we carried out investigations on primary bovine mammary epithelial cells. Our results showed that the immune responses of both models-PCBUS and primary bovine mammary epithelial cells-were similar. In addition, investigations using PCBUS enabled us to demonstrate adherence of bacteria in the physiological cell network. These findings support the use of PCBUS in studies designed to further understand the complex pathophysiological processes of infection and inflammation in bovine mastitis and to investigate alternative therapies for mastitis.

Plasma levels of a methadone constant rate infusion and their corresponding effects on thermal and mechanical nociceptive thresholds in dogs.

BACKGROUND: The present study aimed to collect pharmacokinetic data of a methadone continuous rate infusion (CRI) and to investigate its effect on mechanical and thermal nociceptive thresholds. Seven, 47 to 54 months old beagle dogs, weighing 9.8 to 21.2 kg, were used in this experimental, randomized, blinded, placebo-controlled crossover study. Each dog was treated twice with either a methadone bolus of 0.2 mg kg- 1 followed by a 0.1 mg kg- 1 h- 1 methadone CRI (group M) or an equivalent volume of isotonic saline solution (group P) for 72 h. Mechanical and thermal thresholds, as well as vital parameters and sedation were measured during CRI and for further 24 h. Blood samples for methadone plasma concentrations were collected during this 96 h period. RESULTS: Percentage thermal excursion (%TE) increased significantly from baseline (BL) until 3 h after discontinuation of CRI in M. Within P and between treatment groups differences were not significant. Mechanical threshold (MT) increased in M until 2 h after CRI discontinuation. Bradycardia and hypothermia occurred in M during drug administration and dogs were mildly sedated for the first 47 h. Decreased food intake and regurgitation were observed in M in five and four dogs, respectively. For methadone a volume of distribution of 10.26 l kg- 1 and a terminal half-life of 2.4 h were detected and a clearance of 51.44 ml kg- 1 min- 1 was calculated. Effective methadone plasma concentrations for thermal and mechanical antinociception were above 17 ng ml- 1. CONCLUSION: A methadone CRI of 0.1 mg kg- 1 h- 1 for 3 days after a loading dose results in steady anti-nociceptive effects in an acute pain model in healthy dogs. Main side effects were related to gastrointestinal tract, hypothermia, bradycardia and sedation.

Histamine H1 receptor antagonists enhance the efficacy of antibacterials against Escherichia coli.

BACKGROUND: H1 receptor antagonists are commonly used for the treatment of allergic diseases. The aim of this study was to find out, if antihistaminic compounds like mepyramine have the ability to influence the activity of antibacterials. Therefore, the checkerboard method was chosen to detect these possible effects in vitro. Studies were performed with two different Escherichia coli (E. coli) strains as test microbes, treated with antibacterials in combination with mepyramine. RESULTS: The minimum inhibitory concentration (MIC) of E. coli ATCC® 25922™ and E. coli PIG 01 was reduced by combinations of the tested antibacterials with mepyramine. CONCLUSIONS: These results have to be confirmed in vivo, before the use of antihistamines should be considered as potential way to minimize the amount of used antibacterials for treatment of E. coli infections.

Effects of oxytocin and PGF2α on uterine contractility in cows with and without metritis-An in-vitro study.

The aim of this study was to investigate the effects of PGF2α and oxytocin in vitro on myometrial contractility in puerperal uteri. Thirteen puerperal uteri were removed and perfused after euthanasia of cows with (n=7) and without metritis (n=6). Measurement of uterine contractility was done using four piezoelectric crystals, which were implanted into the myometrium along the greater curvature of the uterine horn where fetal implantation occurred during the previous pregnancy. After 30min of equilibration, oxytocin (5 IU) or PGF2α (2.5mg Dinoprost) was administered randomly into both uterine arteries, and 30min later, the second administration of either oxytocin or PGF2α occurred. Treatment with oxytocin induced contractions in uteri with metritis and uteri without metritis (P<0.05). In uteri with metritis, greater uterine contractions occurred after stimulation with oxytocin than in uteri without metritis (P<0.05). Treatment with PGF2α did not (P>0.05) result in increased contractions in the uteri without metrtitis, however, induced an initial decrease in contractions followed by an increase (P<0.05) in contractions in uteri with metritis. Myometrial and endometrial gene expression of PGF2α (FPR) and oxytocin receptor (OTR) was greater (P<0.05) in uteri with metritis than in uteri without metritis. The results suggest that oxytocin, but not PGF2α, is an effective uterotonic drug in puerperal cows. Uteri in which metritis was diagnosed contracted more strongly after treatment with oxytocin than uteri in which metritis was not diagnosed. This effect was paralleled by greater gene expression of OTR as well as FPR in uteri with metritis compared with uteri in which metritis was not diagnosed.

Cold atmospheric pressure plasma for treatment of chronic wounds: drug or medical device?

OBJECTIVE: The use of cold atmospheric pressure plasma (CAPP) as a new therapeutic option to aid the healing of chronic wounds appears promising. Currently, uncertainty exists regarding their classification as medical device or medical drug. Because the classification of CAPP has medical, legal, and economic consequences as well as implications for the level of preclinical and clinical testing, the correct classification is not an academic exercise, but an ethical need. METHOD: A multidisciplinary team of physicians, surgeons, pharmacists, physicists and lawyers has analysed the physical and technical characteristics as well as legal conditions of the biological action of CAPP. RESULTS: It was concluded that the mode of action of the locally generated CAPP, with its main active components being different radicals, is pharmacological and not physical in nature. CONCLUSION: Depending on the intended use, CAPP should be classified as a drug, which is generated by use of a medical device directly at the point of therapeutic application.

Histamine H4 receptor knockout mice display reduced inflammation in a chronic model of atopic dermatitis.

BACKGROUND: The histamine H4 receptor (H4R) was brought into focus as a new therapeutic target for the treatment of allergic disorders such as atopic dermatitis (AD). H4R antagonists have already been tested in several animal models of AD, but these studies have yielded conflicting results. MATERIAL AND METHODS: The development of ovalbumin-induced AD-like skin lesions was analysed in H4R(-/-) mice and in H4R antagonist (JNJ28307474)-treated mice. RESULTS: H4R(-/-) mice showed a clear amelioration of the skin lesions, with a diminished influx of inflammatory cells and a reduced epidermal hyperproliferation at lesional skin sites. H4R(-/-) mice had a reduced amount of ovalbumin-specific IgE, a reduced number of splenocytes and lymph node cells with a decreased number of CD4+ T cells. The H4R modulated the cytokine secretion of CD4+ T cells and splenocytes and altered the cellular profile in the lymph nodes. The anti-inflammatory effect could only partially be mimicked by JNJ28307474 and only when the H4R antagonist was given during sensitization and challenge and not when JNJ28307474 was only given during the provocation phase of the allergic reaction. CONCLUSION: The H4R modulates inflammation in a chronic allergic dermatitis setting. However, results of this study indicate that it is necessary to block the H4R during ontogeny and development of the allergic inflammation.

MgNd2 alloy in contact with nasal mucosa: an in vivo and in vitro approach.

Biodegradable and biocompatible magnesium alloys appear to be very promising not only for temporary clinical application but also for developing deformable and degradable medical implants. This study analyzes the in vivo degradation behavior and the impact on the paranasal sinuses of the highly ductile Mg-2 wt%Nd alloy (MgNd2) in order to provide a basis for a satisfying stent system for the therapy of a chronic sinusitis. Moreover, in vitro tests were carried out on primary porcine nasal epithelial cells (PNEC). For the in vivo tests, cylindrical MgNd2 specimens were implanted into the sinus' mucosa of minipigs. During and after a total period of 180 days the long-term biodegradation and biocompatibility properties after direct contact with the physiological tissue were analyzed. Biodegradation was investigated by measuring the mass and volume losses of the MgNd2 specimens as well as by performing element analyses to obtain information about the degradation layer. The influence on the surrounding tissue of paranasal sinuses was evaluated by endoscopic and histopathological examinations of the mucosa. Here, only a locally unspecific chronic infection was found. The degradation rate showed a maximum after 45 days postsurgery and was determined to decrease subsequently. In vitro experiments using PNEC showed adequate biocompatibility of MgNd2. This study demonstrates a good in vivo biocompatibility for MgNd2 in the system of paranasal sinuses and underlines the promising properties of alloy MgNd2 for biodegradable nasal stent applications.

Prostaglandin E2 as a read out for endotoxin detection in a bovine whole blood assay.

The detection of endotoxin contamination is an essential part of drug safety testing. The rabbit pyrogen test (RPT), the limulus amoebocyte lysate (LAL) test, and the monocyte activation test (MAT) are established methods for the detection of pyrogens. However, the RPT is insufficiently standardized; the LAL test is solely capable of identifying the presence of endotoxins, whereas the use of the MAT is limited by the availability of human blood. Here, we introduce a new procedure for testing endotoxin contamination using prostaglandin E2 (PGE2 ) release from bovine whole blood. We incubated bovine whole blood overnight with lipopolysaccharide (LPS) from Escherichia coli 0111:B4, concentrations ranging from 1.56 to 12.5 pg/mL, and found significantly increased PGE2 production for even the lowest LPS concentrations. Testing the possibility of storing the blood at 4 °C before use also yielded positive results as 1.56 pg/mL still significantly increased PGE2 production, thus suggesting some flexibility of the assay regarding time. These results emphasize the potential of using bovine whole blood for highly sensitive endotoxin testing. As a perspective, currently ongoing research aims to show whether the assay is also capable of detecting nonendotoxin pyrogens.

Unique immunomodulatory effects of azelastine on dendritic cells in vitro.

Allergic contact dermatitis and atopic dermatitis are among the most common inflammatory skin diseases in western countries, and antigen-presenting cells like dendritic cells (DC) are key players in their pathophysiology. Histamine, an important mediator of allergic reactions, influences DC maturation and cytokine secretion, which led us to investigate the immunomodulatory potential of the well-known histamine H1 receptor antagonists: azelastine, olopatadine, cetirizine, and pyrilamine. Unlike other H1 antihistamines, azelastine decreased lipopolysaccharide-induced tumor necrosis factor α and interleukin-12 secretion from murine bone marrow-derived DC. This effect was independent of histamine receptors H1, H2, or H4 and may be linked to inhibition of the nuclear factor kappa B pathway. Moreover, only azelastine reduced proliferation of allogenic T cells in a mixed leukocyte reaction. We then tested topical application of the H1 antihistamines on mice sensitized against toluene-2,4-diisocyanate, a model of Th2-mediated allergic contact dermatitis. In contrast to the in vitro results, all investigated substances were efficacious in reducing allergic ear swelling. Azelastine has unique effects on dendritic cells and T cell interaction in vitro. However, this did not translate into superior in vivo efficacy for Th2-mediated allergic dermatitis, possibly due to the effects of the antihistamines on other cell types involved in skin inflammation. Future research will have to clarify whether these properties are relevant to in vivo models of allergic inflammation with a different T cell polarization.

Biodegradable polymeric coatings on cochlear implant surfaces and their influence on spiral ganglion cell survival.

To improve the electrode-nerve interface of cochlear implants (CI), the role of poly(L-lactide) (PLLA) and poly(4-hydroxybutyrate) (P(4HB)) as potential coating matrices for CI was assessed both in vitro and in vivo in terms of degradation behavior and effects on spiral ganglion neurons, the main target of the electrical stimulation with a CI. Growth rates of fibroblasts on the polymers were investigated and a direct-contact test with freshly isolated spiral ganglion cells (SGC) was performed. In addition, the effects of the polymer degradation inside the inner ear were evaluated in vivo. The polymer degradation was assessed by use of scanning electron microscopy in combination with an energy-dispersive X-ray analysis. In vitro, no influence of the polymers was detected on fibroblasts' viability and on SGC survival rate. In vivo, SGC density was decreased only 6 months after implantation in the basal and middle turns of the cochlea in comparison to normal-hearing animals but not between implanted groups (coated or uncoated). The analysis of the electrode models showed that in vivo P(4HB) is characterized by a gradual degradation completed after 6 months; whereas, the PLLA coatings burst along their longitudinal axis but showed only little degradation within the same time frame. In conclusion, both polymers seem to justify further evaluation as possible coating for CI electrodes. Of the two options, due to its excellent coating adhesion/stability and optimal degradation behavior, P(4HB) may prove to be the more promising biodegradable polymer for designing a drug delivery system from the surface of CI electrodes.

Biodegradation of metallic magnesium elicits an inflammatory response in primary nasal epithelial cells.

Resorbable magnesium-based implants hold great promise for various biomedical applications, such as osteosynthesis and coronary stenting. They also offer a new therapeutic option for the treatment of chronic rhinosinusitis, but little data is yet available regarding the use of magnesium in the nasal cavity. To model this field of application, primary porcine nasal epithelial cells were used to test the biocompatibility of degrading pure magnesium and investigate whether the degradation products may also affect cellular metabolism. Magnesium specimens did not induce apoptosis and we found no major influence on enzyme activities or protein synthesis, but cell viability was reduced and elevated interleukin 8 secretion indicated proinflammatory reactions. Necrotic damage was most likely due to osmotic stress, and our results suggest that magnesium ion build-up is also involved in the interleukin 8 release. Furthermore, the latter seems to be mediated, at least in part, by the p38 signaling pathway. These effects probably depended on the accumulation of very high concentrations of magnesium ions in the in vitro set-up, which might not be achieved in vivo, although we cannot exclude that further, as yet unknown, factors played a role in the inflammatory response during the degradation process. In conclusion, the biocompatibility of pure magnesium with cells in the immediate vicinity appears less ideal than is often supposed, and this needs to be considered in the evaluation of magnesium materials containing additional alloying elements.

The isolated perfused equine distal limb as an ex vivo model for pharmacokinetic studies.

Even though intra-articular injections play an important role in the treatment of joint-related lameness in horses, little is known about pharmacokinetic properties of substances used. Therefore, an ex vivo model for pharmacokinetic studies was developed using distal forelimbs of slaughtered horses. The extremity was perfused with gassed Tyrode solution for up to 8 h. Tissue viability was confirmed by measurements of glucose consumption, lactate production, and lactate dehydrogenase activity in the perfusate. Standard criteria for tissue viability had been determined in preliminary experiments (n = 11), which also included histological examinations of the joint capsule. As the model's first implementation, the articular efflux rate of betamethasone (BM), administered as BM disodium phosphate intra-articularly to the fetlock joint (4 mg BM/joint), was investigated. The concentration of BM in the venous perfusate of the radial vein was measured by means of high-performance liquid chromatography. The average BM efflux rate per minute was calculated to be 5.1 µg/min with values ranging from 9 µg/min to 2.9 µg/min. 7.5 h after i.a. application, 2.3 mg BM had left the joint via the radial vein. Using this inexpensive setup, the presented model allows studying a variety of pharmacological topics without the ethical limitations of animal studies.

Characterization of MgNd2 alloy for potential applications in bioresorbable implantable devices.

The aim of this study is to investigate and demonstrate the mechanical and corrosive characteristics of the neodymium-containing magnesium alloy MgNd2 (Nd2), which can be used as a resorbable implant material, especially in the field of stenting applications. To determine the mechanical characteristics of Nd2, tensile and compression tests were initially carried out in the hot extruded state. Here a unique elongation ratio (~30%) of the alloy could be observed. Subsequent T5 and T6 heat treatments were arranged to reveal their effect on the alloy's strengths and elongation values. The general degradation behaviour of Nd2 in a 0.9% NaCl solution was investigated by means of polarization curves and hydrogen evolution. In addition to this, by using various in vivo parameters, a corrosion environment was established to determine the alloy's degradation in vitro. Here, the mass loss per day in (MgF(2) and Bioglass)-coated and uncoated states and the corresponding maximum forces resulting from subsequent three-point bending tests revealed slow and steady corrosion behaviour. The cell viability and proliferation tests carried out on L-929 and MSC-P5 cells also showed good results. The mechanical and corrosive characteristics determined, as well as the in vitro test results obtained within the scope of this study, led to the development and successful in vivo testing of an MgF(2)-coated Nd2 mucosa stent which was introduced as an appropriate resorbable application.

[Comments on the Guidelines for the Prudent Use of Antibacterial Veterinary Pharmaceuticals (Guidelines for Antibiotics)]. / Anmerkungen zu den "Leitlinien für den sorgfältigen Umgang mit antimikrobiell wirksamen Tierarzneimitteln" (Antibiotika-Leitlinien).

In 2010 the German Bundestierärztekammer (Federal Chamber of Veterinarians) and the AGTAM (Working Group "Veterinary Pharmaceuticals") published the Guidelines for the prudent use of antibacterial veterinary pharmaceuticals in an updated version. Within the limits of therapeutic freedom, veterinarians are committed to take into account the latest scientific findings in veterinary medicine. These findings may, however, include conflicting interpretations if such an approach is expressed by an accredited university or anywhere else in the field of science. Hence, the state of science in veterinary medicine is not only defined by the Guidelines for Antibiotics, rather, the complete recognized scientific literature has to be considered. The Guidelines for Antibiotics are not legally-binding rules. They define the best approach and not the minimum standard for the use of antibiotics. The clinical examination provides the basis for medical treatment in each specific case. Further laboratory diagnostics represent an additional supportive instrument that is used by the veterinarian at his discretion depending on the necessity. Laboratory tests of bacterial sensitivity (identification of pathogens and antibiogram) may become necessary within the framework of diagnostics. As examples demonstrate, laboratory tests of bacterial sensitivity cannot be performed in every clinical case. It appears to be desirable to further discuss the use of antibacterial veterinary pharmaceuticals in the species-specific attachments in more concrete and specific terms, taking into consideration the standards of evidence-based medicine.

[The use of fluoroquinolones in bacterial urinary tract infections in cats]. / Einsatz von Fluorchinolonen bei bakteriellen Harnwegsinfektionen der Katze.

Older cats (>10 years) with FLUTD (Feline Lower Urinary Tract Disease) symptoms are often affected by urinary tract infections. In most of these cats organ diseases (e.g. chronic renal failure, diabetes mellitus) or iatrogenic factors (immunosuppressive drugs, indwelling catheter) are found that clearly predispose cats to this kind of infection. From a diagnostic point of view, urinalysis and urine culture are the most important tools in detecting bacteriuria. The microbiological spectrum is thereby comparable to that found in dogs, revealing Escherichia ( E. ) coli but also Staphylococcus spp. and Enterococcus spp./ Streptococcus spp. Antibiotic therapy should be based on the results of susceptibility testing. If this kind of information is not available, drug selection has to be decided on an empirical basis unless it is a complicated urinary tract infection. Preferred antibiotics should have a high renal excretion rate and thus ensure therapeutically effective drug levels in the urine. In this respect, the fluoroquinolones belong to the group of appropriate drugs to be used in cats. The relevance of therapeutical drug concentrations achievable in the urine is discussed for the example of marbofloxacin, a third-generation fluoroquinolone. New pharmacokinetic data showed that marbofloxacin concentrations of ≥2µg/ml are maintained in the urine of healthy cats for 72 and 103 hours after administration of 2 and 4mg/kg BW s.c., respectively.

X-ray computed tomography: semiautomated volumetric analysis of late-stage lung tumors as a basis for response assessments.

Background. This study presents a semiautomated approach for volumetric analysis of lung tumors and evaluates the feasibility of using volumes as an alternative to line lengths as a basis for response evaluation criteria in solid tumors (RECIST). The overall goal for the implementation was to accurately, precisely, and efficiently enable the analyses of lesions in the lung under the guidance of an operator. Methods. An anthropomorphic phantom with embedded model masses and 71 time points in 10 clinical cases with advanced lung cancer was analyzed using a semi-automated workflow. The implementation was done using the Cognition Network Technology. Results. Analysis of the phantom showed an average accuracy of 97%. The analyses of the clinical cases showed both intra- and interreader variabilities of approximately 5% on average with an upper 95% confidence interval of 14% and 19%, respectively. Compared to line lengths, the use of volumes clearly shows enhanced sensitivity with respect to determining response to therapy. Conclusions. It is feasible to perform volumetric analysis efficiently with high accuracy and low variability, even in patients with late-stage cancer who have complex lesions.