Clinical Development of Sphingosine as Anti-Bacterial Drug: Inhalation of Sphingosine in Mini Pigs has no Adverse Side Effects.

BACKGROUND/AIMS: Pulmonary infections with Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus) are of utmost clinical relevance in patients with cystic fibrosis, chronic obstructive pulmonary disease, after trauma and burn, upon ventilation or in immuno-compromised patients. Many P. aeruginosa and S. aureus strains are resistant to many known antibiotics and it is very difficult or often impossible to eradicate the pathogens in patient´s lungs. We have recently shown that the sphingoid base sphingosine very efficiently kills many pathogens, including for instance P. aeruginosa, S. aureus or Acinetobacter baumannii, in vitro. In vivo experiments of our group on cystic fibrosis mice indicated that inhalation of sphingosine prevents or eliminates existing acute or chronic pneumonia with P. aeruginosa or S. aureus in these mice. We also demonstrated that sphingosine is safe to use for inhalation up to high doses, at least in mice. To facilitate development of sphingosine to an anti-bactericidal drug that can be used in humans for inhalation, safety data on non-rodents, larger animals are absolutely required. METHODS: Here, we inhaled mini pigs with increasing doses of sphingosine for 10 days and analyzed the uptake of sphingosine into epithelial cells of bronchi as well as into the trachea and lung and the systemic circulation. Moreover, we measured the generation of ceramide and sphingosine 1-phosphate that potentially mediate inflammation, the influx of leukocytes, epithelial cell death and disruption of the epithelial cell barrier. RESULTS: We demonstrate that inhalation of sphingosine results in increased levels of sphingosine in the luminal membrane of bronchi and the trachea, but not in systemic accumulation. Inhaled sphingosine had no side effects up to very high doses. CONCLUSION: In summary, we demonstrate that inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no systemic or local side effects.

What Can Laboratory Animal Facility Managers Do to Improve the Welfare of Laboratory Animals and Laboratory Animal Facility Staff? A German Perspective.

Laboratory animal facility managers must ensure that animal experiments can be carried out under optimal scientific conditions, that all legal requirements are met, and that animal welfare is maximized. Animal experimentation is stressful not only for the animals involved but also for the people who maintain these animals or carry out the experiments. Many of those involved find themselves in a constant conflict between scientific necessity, care, and harm. Under the term Culture of Care, procedures have been developed to reduce the burden of animal experimentation on the animals and the staff involved. The focus here is on what laboratory animal facility managers can do to improve the welfare of laboratory animals and the people working with them. Exemplary measures are the improvement of the housing conditions of laboratory animals, the introduction of uniform handling measures, clear and transparent structures via a quality management system, implementation of a no-blame culture of error (e.g., via Critical Incident Reporting System in Laboratory Animal Science [CIRS-LAS]), and open and respectful communication with all parties involved in animal experimentation, including the public and representatives of the authorities (public webpage, open house policy). The 6 Rs must be considered at all times: replacement, reduction, refinement, respect, responsibility, and reproducibility. We are writing this article from the perspective of laboratory animal facility managers in Germany.

Treatment of Staphylococcus aureus infection with sphingosine in ex vivo perfused and ventilated lungs.

BACKGROUND: Ex vivo lung perfusion (EVLP) has expanded the donor pool for lung transplantation. Pulmonary Staphylococcus aureus infection, especially that caused by multidrug-resistant strains, is a severe threat to posttransplantation outcomes. Sphingosine is a lipid compound that exhibits broad-spectrum antibacterial activity. Therefore, we aimed to evaluate the effects of S aureus infection on EVLP and whether sphingosine administration during EVLP prevents infection with S aureus. METHODS: Eighteen pigs were randomly assigned to 3 groups: uninfected, infected with S aureus with NaCl treatment, or infected with sphingosine treatment. Bacterial numbers were determined before and after treatment. Sphingosine concentrations in the lung tissues were determined using biochemical assays. Lung histology, lung physiological parameters, perfusate content, lung weight, and cell death were measured to analyze the effects of infection and sphingosine administration on EVLP. RESULTS: Sphingosine administration significantly reduced the bacterial load. The concentration of sphingosine in the bronchial epithelium was elevated after sphingosine administration. S aureus infection increased pulmonary artery pressure and pulmonary vascular resistance. Lung edema, histology scores, lactate and lactate dehydrogenase levels in the perfusate, ΔPO2 in the perfusate, static lung compliance, and lung peak airway pressure did not differ among the groups. CONCLUSIONS: Infection of S aureus did not affect the lung function during EVLP but induced higher pulmonary artery pressure and pulmonary vascular resistance. Administration of sphingosine effectively eliminated S aureus without side effects in isolated, perfused, and ventilated pig lungs.

Wolfram-silicone implants as effective radiation shielding for ocular brachytherapy: dosimetric features and in vivo animal study on biocompatibility.

OBJECTIVE: To evaluate wolfram as a photon and beta absorber in the management of uveal melanoma with radiotherapy, examining its potential ocular adverse effects and physiologic tolerance using an in vivo rabbit ocular model. METHODS: A method of manufacturing implants from mixtures of wolfram and silicone was developed. Their shielding effect on the radiation of sources used in ocular brachytherapy was investigated by dosimetric measurement in an eye phantom as well as numerical simulations. Different wolfram implantation techniques, such as extraocular fixation of a wolfram-silicone implant (n = 1), vitrectomy with silicone oil and intravitreal injection of a wolfram-silicone oil suspension (n = 2), and concurrent attachment of a wolfram implant onto the sclera (n = 2), were tested to investigate the long-term effects of wolfram. A vitrectomy with silicone oil without wolfram implantation was carried out in 2 rabbits (n = 2), constituting the control group. The eyes were enucleated after 3 months for histologic analysis. RESULTS: Wolfram-silicone mixtures have been dosimetrically proven to be very effective radiation absorbers for use in ocular brachytherapy. Severe complications, such as endophthalmitis, secondary glaucoma, cornea decompensation, and vessel occlusion, were not documented in the tested rabbit eyes after the application of wolfram. Histologic examination of the bulbi after enucleation showed epiretinal gliosis without further pathologic findings in all eyes after vitrectomy. CONCLUSIONS: The results of this study show that wolfram and wolfram-silicone implants constitute a promising candidate as potential radiation shielding substrates.

Effects of castration and sterilization on baseline and response levels of cortisol-A case study in male guinea pigs.

An uncontrolled reproduction of animals in human hands should be avoided. To meet this goal, animals are widely castrated, i.e., the gonads are completely removed. Since the gonads are the most important source of sex hormones, this is a serious intervention in the entire endocrine system of an organism. Sterilization is a much less invasive procedure. Thus, it could have advantages over castration. Therefore, the overall aim of this study was to analyze the effect of castration vs. sterilization on the release of glucocorticoids, i.e., an important indicator for welfare. Taking domestic guinea pigs as a model system, we studied baseline and response cortisol values (cortisol is the main glucocorticoid in guinea pigs) in castrated, sterilized, sham-operated and intact males and baseline values in their cohoused females. Whereas baseline values of males did not differ between the groups, castrated males showed significantly higher cortisol response levels than intact, sham-operated and sterilized males. Females housed with castrated, sterilized, sham-operated or intact males did not differ in their cortisol concentrations, neither shortly after being placed with the respective male or after being co-housed for several weeks. Overall, the results support the hypothesis that castrated males exhibited a higher cortisol responsiveness during acute challenge which could point to a generalized impaired welfare of castrated males in comparison to intact, sham-operated and sterilized males. Our results provide first evidence for a potential negative impact of castration on the animals' welfare, while at the same time pointing toward sterilization representing a less invasive, promising alternative. Therefore, the results may stimulate future research on this topic to further detect potential welfare-related side effects of castration.

In-vivo comparison of the Ni-free steel X13CrMnMoN18-14-3 and titanium alloy implants in rabbit femora - A promising steel for orthopedic surgery.

A variety of metallic biomaterials is used for fracture fixation. Allergic reactions towards nickel-containing steels urge the need for alternatives. The present study investigated the suitability of the nickel-free stainless steel P2000 in comparison to titanium alloy implants for bone surgical applications in a rabbit femora defect model. Thirty-six rabbits received two different cylindrical implants press-fit inserted into the distal femoral metaphysis. At day 0, 28, and 56, implant ingrowth was monitored by radiography; implant stability was assessed by pull-out torque measurements while bone-to-implant contact (BIC) was determined histomorphometrically. Radiography revealed comparable implant ingrowth after 1 and 2 months for both implant materials. The pull-out force of P2000 tended to be higher than that for titanium at day 28 (p = .076) but the values were comparable at day 56 (p = .905). At day 56, implant fixation was significantly increased compared to the day of surgery for both, P2000 (p = .030) and for titanium alloy (p = .026). Microscopic examination revealed that both implant types appeared to be well integrated and firmly anchored in the bone. BIC ratio of titanium alloy tended to be higher at day 28 (p = .079) but they did not differ significantly at day 56 (p = .711). In the present rabbit femora defect model, the nickel-free stainless steel P2000 provides primary stability and osseointegration comparable to that of titanium alloy implants.

Single nucleotide polymorphisms of the serotonin transporter gene in migraine--an association study.

We report a case-control study with 251 unrelated migraine patients and 192 unrelated healthy controls to evaluate an association between the polymorphisms of the 5-HT transporter (5-HTT) gene rs2066713 and rs1979572 and different migraine phenotypes. We found a genetic association for the A allele of rs1979572 for migraine with aura (MA) especially in women as well as a significant lower prevalence for MA for carrier of the A allele of rs2066713 in women. These findings support previous results suggesting that the 5-HTT gene is involved in the polygenic etiology of MA. These data further suggest that women are more likely to be clinically affected by mutations in the 5-HTT gene than men.

Establishment of a swine model for validation of perfusion measurement by dynamic contrast-enhanced magnetic resonance imaging.

The aim of the study was to develop a suitable animal model for validating dynamic contrast-enhanced magnetic resonance imaging perfusion measurements. A total of 8 pigs were investigated by DCE-MRI. Perfusion was determined on the hind leg musculature. An ultrasound flow probe placed around the femoral artery provided flow measurements independent of MRI and served as the standard of reference. Images were acquired on a 1.5 T MRI scanner using a 3D T1-weighted gradient-echo sequence. An arterial catheter for local injection was implanted in the femoral artery. Continuous injection of adenosine for vasodilation resulted in steady blood flow levels up to four times the baseline level. In this way, three different stable perfusion levels were induced and measured. A central venous catheter was used for injection of two different types of contrast media. A low-molecular weight contrast medium and a blood pool contrast medium were used. A total of 6 perfusion measurements were performed with a time interval of about 20-25 min without significant differences in the arterial input functions. In conclusion the accuracy of DCE-MRI-based perfusion measurement can be validated by comparison of the integrated perfusion signal of the hind leg musculature with the blood flow values measured with the ultrasound flow probe around the femoral artery.

Anti-allodynic effect of the flavonoid myricetin in a rat model of neuropathic pain: Involvement of p38 and protein kinase C mediated modulation of Ca²+ channels.

Flavonoids are increasingly ingested by the population as chemotherapeutic and anti-inflammatory agents. Myricetin is a naturally occurring flavonoid known for its anti-neoplastic and anti-inflammatory effects. Recently, behavioral studies indicate a potential analgesic effect in animal models of pain. Pilot studies suggest a flavonoid-induced modulation of intracellular protein kinases and interactions with voltage activated calcium channels. The aim of this study was to investigate the analgesic effect of myricetin in a neuropathic pain model (spinal nerve ligation, SNL) in rats. To identify potential mechanisms of action, in vitro whole cell patch-clamp recordings of isolated rat dorsal root ganglia (DRG) neurons were performed to analyze the modulation of voltage activated calcium channel currents (I(Ca(V))) and the influence of intracellular kinase phosphorylation such as p38 mitogen-activated protein kinase (p38) or protein kinase C (PKC). In vivo, a single injection of myricetin (0.1-10 mg/kg i.p.) reduced SNL-induced mechanical allodynia and thermal hyperalgesia lasting for several hours. In vitro, I(Ca(V)) (depolarization from -80 to 0 mV) were reduced (10-56%) by low (0.1-5 µM) concentrations of myricetin. This decrease was abolished by blockade of PKC (20 µM chelerythrine for 30 min), but not of p38 (10 µM SB203580 for 30 min). In contrast, higher (10-100 µM) concentrations of myricetin induced an increase of I(Ca(V)) (20-40%), which was blocked by inhibition of p38, but not of PKC. We conclude that myricetin transiently reduces established neuropathic pain behavior. This analgesic effect may be related to its PKC-induced decrease of I(Ca(V)) in DRG neurons.