Isolation, establishment, and characterization of ex vivo equine melanoma cell cultures

Gray horses spontaneously develop metastatic melanomas that resemble human disease, and this is often accompanied with metastasis to other organs. Unlike in other species, the establishment of primary equine melanoma cultures that could be used to develop new therapeutic approaches has remained a major challenge. The purpose of the study was to develop a protocol for routine isolation and cultivation of primary equine melanocytes. Melanoma tissues were excised from 13 horses under local anesthesia, mainly from the perianal area. The melanoma cells were isolated from the melanoma tissue by serial enzymatic digestion using dispase and collagenase. Out of the 13 excised melanomas, cell cultures from eight melanomas were established, which corresponded to a success rate 62%. These cells showed different degrees of melanin pigmentation. Characterization of these cells using confocal microscopy, FACs analysis and western blotting showed that they expressed melanoma-associated antigens; Melan-A, MAGE-1, and MAGE-3, and PCNA expression was higher in fast-proliferating isolates. The protocol we developed and established proved successful for routine isolation and cultivation of primary equine melanoma cells. This method provided a large number of primary equine melanoma cells that could be used to study new therapeutic approaches for treatment of equine melanoma

Ambulatory sleep scoring using accelerometers—distinguishing between nonwear and sleep/wake states

Background. Differentiating nonwear time from sleep and wake times is essential forthe estimation of sleep duration based on actigraphy data. To efficiently analyze large-scale data sets, an automatic method of identifying these three different states is re-quired. Therefore, we developed a classification algorithm to determine nonwear, sleepand wake periods from accelerometer data. Our work aimed to (I) develop a new patternrecognition algorithm for identifying nonwear periods from actigraphy data based onthe influence of respiration rate on the power spectrum of the acceleration signal andimplement it in an automatic classification algorithm for nonwear/sleep/wake states;(II) address motion artifacts that occur during nonwear periods and are known to causemisclassification of these periods; (III) adjust the algorithm depending on the sensorposition (wrist, chest); and (IV) validate the algorithm on both healthy individuals andpatients with sleep disorders. Methods. The study involved 98 participants who wore wrist and chest accelerationsensors for one day of measurements. They spent one night in the sleep laboratoryand continued to wear the sensors outside of the laboratory for the remainder of theday. The results of the classification algorithm were compared to those of the referencesource: polysomnography for wake/sleep and manual annotations for nonwear/wearclassification. Results. The median kappa values for the two locations were 0.83 (wrist) and 0.84(chest). The level of agreement did not vary significantly by sleep health (good sleepersvs. subjects with sleep disorders) (p=0.348,p=0.118) or by sex (p=0.442,p=0.456).The intraclass correlation coefficients of nonwear total time between the referenceand the algorithm were 0.92 and 0.97 with the outliers and 0.95 and 0.98 after theoutliers were removed for the wrist and chest, respectively. There was no evidence of anassociation between the mean difference (and 95% limits of agreement) and the meanof the two methods for either sensor position (wrist p=0.110, chest p=0.164), and themean differences (algorithm minus reference) were 5.11 [95% LoA−15.4–25.7] and1.32 [95% LoA−9.59–12.24] min/day, respectively, after the outliers were removed. Discussion. We studied the influence of the respiration wave on the power spectrum ofthe acceleration signal for the differentiation of nonwear periods from sleep and wakeperiods. The algorithm combined both spectral analysis of the acceleration signal and rescoring. Based on the Bland-Altman analysis, the chest-worn accelerometer showed better results than the wrist-worn accelerometer

Two different hematocrit detection methods: Different methods, different results?

BACKGROUND: Less is known about the influence of hematocrit detection methodology on transfusion triggers. Therefore, the aim of the present study was to compare two different hematocrit-assessing methods. In a total of 50 critically ill patients hematocrit was analyzed using (1) blood gas analyzer (ABLflex 800) and (2) the central laboratory method (ADVIA(R) 2120) and compared. FINDINGS: Bland-Altman analysis for repeated measurements showed a good correlation with a bias of +1.39% and 2 SD of +/- 3.12%. The 24%-hematocrit-group showed a correlation of r2 = 0.87. With a kappa of 0.56, 22.7% of the cases would have been transfused differently. In the-28%-hematocrit group with a similar correlation (r2 = 0.8) and a kappa of 0.58, 21% of the cases would have been transfused differently. CONCLUSIONS: Despite a good agreement between the two methods used to determine hematocrit in clinical routine, the calculated difference of 1.4% might substantially influence transfusion triggers depending on the employed method

Isolation, establishment, and characterization of ex vivo equine melanoma cell cultures

Gray horses spontaneously develop metastatic melanomas that resemble human disease, and this is often accompanied with metastasis to other organs. Unlike in other species, the establishment of primary equine melanoma cultures that could be used to develop new therapeutic approaches has remained a major challenge. The purpose of the study was to develop a protocol for routine isolation and cultivation of primary equine melanocytes. Melanoma tissues were excised from 13 horses under local anesthesia, mainly from the perianal area. The melanoma cells were isolated from the melanoma tissue by serial enzymatic digestion using dispase and collagenase. Out of the 13 excised melanomas, cell cultures from eight melanomas were established, which corresponded to a success rate 62%. These cells showed different degrees of melanin pigmentation. Characterization of these cells using confocal microscopy, FACs analysis and western blotting showed that they expressed melanoma-associated antigens; Melan-A, MAGE-1, and MAGE-3, and PCNA expression was higher in fast-proliferating isolates. The protocol we developed and established proved successful for routine isolation and cultivation of primary equine melanoma cells. This method provided a large number of primary equine melanoma cells that could be used to study new therapeutic approaches for treatment of equine melanomas

Fallbericht eines Osteolipoms im ventralen Nasengang beim Pferd

A five year old Swiss Warmblood gelding was presented with unilateral, chronic, mucopurulent nasal discharge. Clinical, radiographic and computertomographic examinations showed a mass in the ventral meatus of the nasal cavity. The mass had connection to the root of the fourth premolar tooth. During surgery, the fourth premolar tooth and the mass were completely removed. A rectangular bone flap of the maxillary bone was performed to gain access to the apex of the tooth. The fourth premolar tooth had to be repelled initially before the neoplasia could be isolated and removed through the surgical approach. The large opening between the oral and the nasal cavities was closed with a silicon-dental packing. The histologic examination of the neoplastic tissue revealed an ostelipoma. Eight weeks later the silicon-plug was removed orally. Four months after the surgery the horse was presented again with an unilateral, purulent nasal discharge. In the mean time a fistula developed between the oral cavity and the maxillary sinus. Clinically as well as radiographically the patient exhibited all the signs of a chronic sinusitis of the maxillary and frontal sinus. In a second surgery the fistula was closed with a methylmethacrylat-plug. After another 2 months the horse was presented without any complications. The methylmetacrylat-packing was removed 3 months later

Transendoskopische Therapie eines intermittierenden Epiglottic Entrapment am stehenden Pferd

Epiglottic entrapment can be associated with poor performance in- and exspiratory stertor and occasionally with coughing (Honnas and Wheat 1988; Robertson, 1991; Lumsden et al., 1994; Lumsden at al. 1995). But it can also be an incidental finding (Ferraro, 1990). A 14 year old Trakehner stallion was presented to the Veterinary Hospital of the University of Zurich with a history of an abnormal inspiratory noise increasing with exercise. The general physical examination showed no abnormalities. By endoscopy during exercise on a high-speed treadmill the diagnosis of intermittent epiglottic entrapment could be made. Therapy consisted of two transendoscopic surgeries two days apart. In the first session subepiglotteal tissue was resected via a transoral approach, in a second session the aryepiglottic folds were partly removed. Both manipulations were performed electrosurgically with a wire snare in the standing, sedated horse and local anaesthesia of the mucosal membranes. No medication was given after surgery. The horse was fed fresh gras and was walked daily for 15 minutes for one week. Six days after the second surgery no abnormalities on general physical examination and during an endoscopy at rest were present. By exercise endoscopy on the treadmill the Epiglottic entrapment could not be induced any more neither at the same or at higher exercise intensities than before surgery. The main advantages of the surgical technique used in this case are the easy to perform procedure, minimal trauma by the transoral approach and a short reconvalescence period

Experimental cross-species infection of donkeys with equine hepacivirus and analysis of host immune signatures

$\bf Background$ The Equine Hepacivirus (EqHV) is an equine-specific and liver-tropic virus belonging to the diverse genus of Hepaciviruses. It was recently found in a large donkey $\textit {(Equus asinus)}$ cohort with a similar seroprevalence (30%), but lower rate of RNA-positive animals (0.3%) compared to horses. These rare infection events indicate either a lack of adaptation to the new host or a predominantly acute course of infection. $\bf Methods$ In order to analyze the susceptibility and the course of EqHV infection in donkeys, we inoculated two adult female donkeys and one control horse intravenously with purified EqHV from a naturally infected horse. Liver biopsies were taken before and after inoculation to study changes in the transcriptome. $\bf Results$ Infection kinetics were similar between the equids. All animals were EqHV PCR-positive from day three. EqHV RNA-levels declined when the animals seroconverted and both donkeys cleared the virus from the blood by week 12. Infection did not have an impact on the clinical findings and no significant histopathological differences were seen. Blood biochemistry revealed a mild increase in GLDH at the time of seroconversion in horses, which was less pronounced in donkeys. Transcriptomic analysis revealed a distinct set of differentially expressed genes, including viral host factors and immune genes. $\bf Conclusion$ To summarize, our findings indicate that donkeys are a natural host of EqHV, due to the almost identical infection kinetics. The different immune responses do however suggest different mechanisms in reacting to hepaciviral infections