Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent.

BACKGROUND: Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown. RESULTS: In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment. In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups. In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis. CONCLUSION: Treatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10. The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated.

[Equine anaplasmosis and equine piroplasmosis in Germany, Austria and Switzerland - previously anecdotal, now relevant?] / Equine Anaplasmose und equine Piroplasmose in Deutschland, Österreich und der Schweiz ­ früher anekdotisch, heute relevant?

INTRODUCTION: Equine granulocytic anaplasmosis (EGA) and equine piroplasmosis (EP) are triggered by tick-borne pathogens - the intracellular bacterium Anaplasma phagocytophilum and the intracellular protozoa Babesia caballi and Theileria equi. These pathogens attack cells in the blood stream and cause similar clinical symptoms and changes in laboratory values. Although the treatment principles are naturally different, similarities in prophylaxis exists due to the transmission route. Tick transmitted pathogens can play a greater role in equine medicine in the future due to various factors, such as the tendency of relevant tick species to spread, but also the increasing import and travel activities of and with pets (both in the context of sporting events and as a leisure activity). While EGA is endemic in Central Europe, EP is a sporadic disease in Switzerland, Austria and Germany. However, EP must be viewed as underdiagnosed, as horses persistently infected with T. equi are also repeatedly detected in Central Europe. These diseases should be considered in horses with a fever and corresponding laboratory changes. Available diagnostic tests are direct pathogen detection by blood smear or PCR, and, indirect antibody detection, which is considered to be highly sensitive and (as a competitive ELISA) also very specific. Acute infections can be detected with PCR, serology is more suitable for chronic infections. A pathogen-free condition after treatment can be demonstrated with decreasing antibody titers in combination with repeated PCR tests. In addition, clinically healthy horses infected with T. equi should be identified by antibody detection and appropriate preventative transmission measures must be initiated. The prophylaxis of tick bites in horses is difficult due to the high exposure, and long-term tick bite prevention can hardly be guaranteed. Monitoring of tick activity and strict measures to prevent the spread of the pathogen within the tick population are therefore of great importance.

INTRODUCTION: L'anaplasmose granulocytaire équine (EGA) et la piroplasmose équine (EP) sont causées par des agents pathogènes transmis par les tiques ­ la bactérie intracellulaire Anaplasma phagocytophilum et les protozoaires intracellulaires Babesia caballi et Theileria equi. Ces agents pathogènes attaquent les cellules sanguines et provoquent des symptômes cliniques similaires et des modifications des valeurs de laboratoire. Bien que les principes de traitement soient naturellement différents, des similitudes dans la prophylaxie existent en raison de la voie de transmission. Les agents pathogènes transmis par les tiques pourraient jouer un rôle plus important en médecine équine à l>avenir en raison de divers facteurs, tels que la tendance des espèces de tiques concernées à se propager, mais aussi l>augmentation des activités d>importation et de voyage d>animaux de compagnie avec eux (à la fois dans le contexte d>événements sportifs et comme activité de loisir). Alors que l>EGA est endémique en Europe centrale, l>EP est une maladie sporadique en Suisse, en Autriche et en Allemagne. Cependant, l'EP doit être considérée comme sous-diagnostiquée, car des chevaux infectés de manière persistante par T. equi ont également été détectés à plusieurs reprises en Europe centrale. Ces maladies doivent être envisagées chez les chevaux présentant de la fièvre et des modifications biologiques correspondantes. Les tests de diagnostic disponibles sont la détection directe des agents pathogènes par frottis sanguin ou par PCR et la détection indirecte des anticorps, qui est considérée comme très sensible et, en tant qu'ELISA compétitif, également très spécifique. Les infections aiguës peuvent être détectées par PCR, la sérologie est plus adaptée aux infections chroniques. Une condition sans agent pathogène après le traitement peut être démontrée avec des titres d'anticorps décroissants en combinaison avec des tests PCR répétés. De plus, les chevaux cliniquement sains infectés par T. equi doivent être identifiés par détection d'anticorps et des mesures appropriées pour prévenir la transmission doivent être initiées. La prophylaxie des morsures de tiques chez les chevaux est difficile en raison de la forte exposition et la prévention des morsures de tiques à long terme peut difficilement être garantie. La surveillance de l'activité des tiques et des mesures strictes pour empêcher la propagation de l'agent pathogène au sein de la population de tiques sont donc d'une grande importance.

Identification of hypoglycin A binding adsorbents as potential preventive measures in co-grazers of atypical myopathy affected horses.

BACKGROUND: Intestinal absorption of hypoglycin A (HGA) and its metabolism are considered major prerequisites for atypical myopathy (AM). The increasing incidence and the high mortality rate of AM urgently necessitate new therapeutic and/or preventative approaches. OBJECTIVES: To identify a substance for oral administration capable of binding HGA in the intestinal lumen and effectively reducing the intestinal absorption of the toxin. STUDY DESIGN: Experimental in vitro study. METHODS: Substances commonly used in equine practice (activated charcoal composition, di-tri-octahedral smectite, mineral oil and activated charcoal) were tested for their binding capacity for HGA using an in vitro incubation method. The substance most effective in binding HGA was subsequently tested for its potential to reduce intestinal HGA absorption. Jejunal tissues of 6 horses were incubated in Ussing chambers to determine mucosal uptake, tissue accumulation, and serosal release of HGA in the presence and absence of the target substance. Potential intestinal metabolism in methylenecyclopropyl acetic acid (MCPA)-conjugates was investigated by analysing their concentrations in samples from the Ussing chambers. RESULTS: Activated charcoal composition and activated charcoal were identified as potent HGA binding substances with dose and pH dependent binding capacity. There was no evidence of intestinal HGA metabolism. MAIN LIMITATIONS: Binding capacity of adsorbents was tested in vitro using aqueous solutions, and in vivo factors such as transit time and composition of intestinal content, may affect adsorption capacity after oral administration. CONCLUSIONS: For the first time, this study identifies substances capable of reducing HGA intestinal absorption. This might have major implications as a preventive measure in cograzers of AM affected horses but also in horses at an early stage of intoxication.

In vitro anticancer activity of Betulinic acid and derivatives thereof on equine melanoma cell lines from grey horses and in vivo safety assessment of the compound NVX-207 in two horses.

Betulinic acid, a pentacyclic triterpene, and its derivatives are promising compounds for cancer treatment in humans. Melanoma is not only a problem for humans but also for grey horses as they have a high potential of developing melanoma lesions coupled to the mutation causing their phenotype. Current chemotherapeutic treatment carries the risk of adverse health effects for the horse owner or the treating veterinarian by exposure to antineoplastic compounds. Most treatments have low prospects for systemic tumor regression. Thus, a new therapy is needed. In this in vitro study, Betulinic acid and its two derivatives B10 and NVX-207, both with an improved water solubility compared to Betulinic acid, were tested on two equine melanoma cell lines (MelDuWi and MellJess/HoMelZh) and human melanoma (A375) cell line. We could demonstrate that all three compounds especially NVX-207 show high cytotoxicity on both equine melanoma cell lines. The treatment with these compounds lead to externalization of phosphatidylserines on the cell membrane (AnnexinV-staining), DNA-fragmentation (cell cycle analysis) and activation of initiator and effector caspases (Caspase assays). Our results indicate that the apoptosis is induced in the equine melanoma cells by all three compounds. Furthermore, we succeed in encapsulating the most active compound NVX-207 in 2-Hydroxyprolyl-ß-cyclodextrine without a loss of its activity. This formulation can be used as a promising antitumor agent for treating grey horse melanoma. In a first tolerability evaluation in vivo the formulation was administered every one week for 19 consecutive weeks and well tolerated in two adult melanoma affected horses.

Influences of age and sex on leukocytes of healthy horses and their ex vivo cytokine release.

Leukocytes and their functional capacities are used extensively as biomarkers in immunological research. Commonly employed indicators concerning leukocytes are as follows: number, composition in blood, response to discrete stimuli, cytokine release, and morphometric characteristics. In order to employ leukocytes as biomarkers for disease and therapeutic monitoring, physiological variations and influencing factors on the parameters measured have to be considered. The aim of this report was to describe the ranges of selected leukocyte parameters in a sample of healthy horses and to analyse whether age, sex, breed, and sampling time point (time of day) influence peripheral blood leukocyte composition, cell morphology and release of cytokines ex vivo. Flow cytometric comparative characterisation of cell size and complexity in 24 healthy horses revealed significant variance. Similarly, basal release of selected cytokines by blood mononuclear cells also showed high variability [TNFα (65-16,624pg/ml), IFNγ (4-80U/ml), IL-4 (0-5069pg/ml), IL-10 (49-1862pg/ml), and IL-17 (4-1244U/ml)]. Each animal's age influenced leukocyte composition, cell morphology and cytokine release (TNFα, IL-4, IL-10) ex vivo. Geldings showed smaller monocytes and higher spontaneous production of IL-10 when compared to the mares included. The stimulation to spontaneous release ratios of TNFα, IL-4 and IL-17 differed in Warmblood and Thoroughbred types. Sampling time influenced leukocyte composition and cell morphology. In summary, many animal factors - age being the dominant one - should be considered for studies involving the analysis of equine leukocytes. In addition, high inter-individual variances argue for individual baseline measurements.

[Examination of horses with acute colic - clinical pathology and diagnostic imaging]. / Untersuchung des akut kolikkranken Pferdes -labordiagnostische und bildgebende Verfahren.

The article summarizes the relevant clinical pathological assessment of horses with acute colic. A minimal laboratory evaluation should include the patient's haematocrit (or packed cell volume), total protein, and lactate concentration in the blood. Haematocrit and total protein provide an indication of the severity of dehydration (haematocrit < 0.45 l/l is evidence of no to mild dehydration whereas > 0.5 l/l points to a severe dehydration). The degree of dehydration is often associated with the severity of the colic. Additionally, the blood lactate concentration rises with increasing intestinal compromise with a concentration of > 4 mmol/l indicating a guarded prognosis. However, it is crucial to assess laboratory values only in the context of the clinical findings. If an abdominocentesis is performed, the leukocyte count and the protein and lactate concentrations offer valuable information regarding the type of colic, the severity of the lesion, further therapy, and prognosis of the colic. Reddish discolouration of peritoneal fluid may be a sign of a strangulating obstruction. Transcutaneous abdominal ultrasonography may provide a crucial insight into the colic cause and severity in a relatively short time, even for inexperienced examiners. In regards to small intestinal lesions, dilated small intestinal loops can often be imaged ultrasonographically before they can be palpated transrectally. The occurrence of free peritoneal fluid and dilated small intestine as well as the evaluation of the intestinal wall and the extent of the gastric wall, allow a better management of the acute colic patient. In ponies and foals, radiography as a further diagnostic imaging modality of the abdomen is of great value. It can help to visualise sand impactions, meconium impactions, or gastrointestinal atresia in the neonate.

Evaluation of the reactivity of commercially available monoclonal antibodies with equine cytokines.

Research on equine cytokines is often performed by analyses of mRNA. For many equine cytokines an analysis on the actual protein level is limited by the availability of antibodies against the targeted cytokines. Generation of new antibodies is ongoing but time consuming. Thus, testing the reactivity of commercially available antibodies for cross-reactivity with equine cytokines is of particular interest. Fifteen monoclonal antibodies against IL-1ß, IL-6, IL-8, IL-12, IL-18 and Granulocyte Macrophage Colony stimulating factor (GM-CSF) of different species were evaluated for reactivity with their corresponding equine cytokines. Dot Blot (DB) and Western Blot (WB) analyses were performed using recombinant equine cytokines as positive controls. Immunohistochemistry (IHC) was carried out on equine tissue and flow cytometry on equine PBMC as positive controls. As expected, three equine IL-1ß antibodies detected equine IL-1ß in DB, WB and IHC. For these, reactivity in IHC has not been described before. One of them was also found to be suitable for intracellular staining of equine PBMC and flow cytometric analysis. Two antibodies raised against ovine GM-CSF cross-reacted with equine GM-CSF in DB, WB and IHC. For these anti-GM-CSF mAbs this is the first experimental description of cross-reactivity with equine GM-CSF (one mAb was predicted to be cross-reactive in WB in the respective data sheet). The other clone additionally proved to be appropriate in flow cytometric analysis. Two mAbs targeting porcine IL-18 cross-reacted in IHC, but did not show specificity in the other applications. No reactivity was shown for the remaining five antibodies in DB, although cross-reactivity of two of the antibodies was described previously. The results obtained in this study can provide beneficial information for choosing of antibodies for immunological tests on equine cytokines.