Open trial of recombinant Der f 2 pullulan-conjugated immunotherapy in cats.

BACKGROUND: Allermmune HDM (Zenoaq) is a recombinant Dermatophagoides farinae 2 (Der f 2) pullulan-based immunotherapy vaccine whose efficacy on house dust mite allergic dogs has been demonstrated. There is no published information on its use in cats. OBJECTIVES: The objective of the study was to evaluate the safety and short-term effects of Allermmune HDM in Dermatophagoides farinae (Df)-sensitised cats. MATERIALS AND METHODS: Eleven cats diagnosed with atopic skin syndrome received Allermmune weekly for six weeks then monthly for three months (total duration 18 weeks). On Weeks 0, 6 and 18 clinical lesions were assessed by the Feline Dermatitis Extent and Severity Index (FEDESI); owners assessed pruritus with a 10-cm Visual Analog Scale (pVAS). Concurrent medication use was recorded. The allergen-specific immunoglobulin (Ig)E were measured before study inclusion with a commercial serological assay. RESULTS: There were no evident adverse effects. FEDESI and pVAS improved significantly after six weeks (p = 0.001 and p = 0.01, respectively). The pretreatment Df-specific IgE levels were significantly higher in the cats with improved clinical scores than in the cats with no clinical score change (p = 0.009). CONCLUSIONS AND CLINICAL RELEVANCE: Allermmune HDM may be safe in cats and has the potential to alleviate signs of atopic skin syndrome. Allergen-specific IgE levels may represent an efficacy marker. Controlled studies of longer duration and larger sample size are worth pursuing.

A novel therapeutic diet can significantly reduce the medication score and pruritus of dogs with atopic dermatitis during a nine-month controlled study.

BACKGROUND: Canine atopic dermatitis (cAD) is a common chronic relapsing pruritic skin disease for which management commonly relies on life-long use of immunomodulatory drugs. A number of the medications used are associated with adverse effects and the potential for complications during long-term use. HYPOTHESIS: The goal of the study was to determine if a complete and balanced diet formulated for therapeutic benefit could contribute towards management of cAD. We hypothesised that the diet would reduce pruritus while also reducing the requirement for medication during the study period. ANIMALS, MATERIALS AND METHODS: Forty privately owned dogs, having undergone a comprehensive diagnosis for cAD, were randomly allocated to two groups, each group being fed one of two diets (test or control) for up to nine months. We assessed pruritus, Canine Atopic Dermatitis Extent and Severity Index-(4th iteration) and medication score, the latter reflecting the medication required to maintain a satisfactory quality of life for the animal. RESULTS: Both diets were well-accepted and -tolerated. There was a significant improvement in the pruritus score after three months of feeding the therapeutic diet (P = 0.0001). No such improvement was observed at any time point in the group of dogs given the control diet. There was a reduced drug requirement for dogs receiving the therapeutic diet after three months (P = 0.058), and that decrease was significant at six months (P = 0.021) and nine months (P = 0.018). No improvement was seen at any time point in the control group. CONCLUSION: The results suggest that a novel therapeutic diet can assist in the management of cAD by helping to control pruritus and reducing reliance on medication.

Phaeohyphomycosis caused by Phialophora americana in a dog.

Phaeohyphomycosis was diagnosed in a 6-year-old, male castrated Dachshund on immunosuppressive treatment. The fungus was identified by culture and PCR as Phialophora americana. This is the first reported case of infection with this pathogen in a dog. The infection was successfully managed medically, without surgical intervention.

Une phaéohyphomycose a été diagnostiquée chez un teckel mâle castré de 6 ans sous traitement immunosuppresseur. Le champignon a été identifié par culture et PCR comme Phialophora americana. Il s'agit du premier cas rapporté d'infection par cet agent pathogène chez un chien. L'infection a été prise en charge médicalement avec succès, sans intervention chirurgicale.

Se diagnosticó feohifomicosis en un macho de Teckel castrado de 6 años en tratamiento inmunosupresor. El hongo fue identificado por cultivo y PCR como Phialophora americana. Este es el primer caso reportado de infección por este patógeno en un perro. La infección se manejó con éxito médicamente, sin intervención quirúrgica.

Feohifomicose foi diagnosticada em um cão da raça Dachshund, macho castrado, de seis anos de idade, em tratamento imunossupressivo. O fungo identificado por cultura e PCR foi Phialophora americana. Este é o primeiro relato de caso de infecção por este patógeno em um cão. A infecção foi bem conduzida com tratamento medicamentoso, sem intervenção cirúrgica.

Influence of Na+ and Mg2+ ions on RNA structures studied with molecular dynamics simulations.

The structure of ribonucleic acid (RNA) polymers is strongly dependent on the presence of, in particular Mg2+ cations to stabilize structural features. Only in high-resolution X-ray crystallography structures can ions be identified reliably. Here, we perform molecular dynamics simulations of 24 RNA structures with varying ion concentrations. Twelve of the structures were helical and the others complex folded. The aim of the study is to predict ion positions but also to evaluate the impact of different types of ions (Na+ or Mg2+) and the ionic strength on structural stability and variations of RNA. As a general conclusion Mg2+ is found to conserve the experimental structure better than Na+ and, where experimental ion positions are available, they can be reproduced with reasonable accuracy. If a large surplus of ions is present the added electrostatic screening makes prediction of binding-sites less reproducible. Distinct differences in ion-binding between helical and complex folded structures are found. The strength of binding (ΔG‡ for breaking RNA atom-ion interactions) is found to differ between roughly 10 and 26 kJ/mol for the different RNA atoms. Differences in stability between helical and complex folded structures and of the influence of metal ions on either are discussed.

A comparative study of subcutaneous, intralymphatic and sublingual immunotherapy for the long-term control of dogs with nonseasonal atopic dermatitis.

BACKGROUND: Allergen-specific immunotherapy (ASIT) is the only causative treatment of canine atopic dermatitis (cAD). Different routes for administration of ASIT have been used; however, comparative studies are lacking. HYPOTHESIS/OBJECTIVES: The present study compared the efficacy and safety of subcutaneous (SCIT), intralymphatic (ILIT) and sublingual (SLIT) immunotherapy. ANIMALS: 30 atopic dogs were included and allocation to three groups (SCIT, n = 8; ILIT, n = 12; SLIT, n = 10) was determined by the owners. METHODS AND MATERIALS: ASIT was administered using routine protocols. The pruritus Visual Analog Scale (PVAS), canine atopic dermatitis extent and severity index (CADESI), concurrent medications and adverse events were recorded initially and one, three, six and 12 months later. The main outcome measure was return to a normal status, which included CADESI <12, PVAS <2.5 and medication score <10. RESULTS: Drop-outs were distributed evenly and 23 dogs finished the study (SCIT, n = 6; ILIT, n = 10; SLIT, n = 7). Adverse reactions to treatment were rare. At the start of the study, the three groups were homogeneous with respect to clinical signs and concurrent medications. After 12 months of ASIT, the CADESI and PVAS had decreased with a stable medication score in the ILIT and SCIT groups (P < 0.05), while all three scores had increased in the SLIT group. Return to normal state was achieved in one of six (17%) dogs receiving SCIT, in six of 10 (60%) dogs receiving ILIT and in one of seven (14%) dogs receiving SLIT. CONCLUSIONS AND CLINICAL IMPORTANCE: These findings suggest that SCIT and ILIT improved clinical signs of cAD, whereas ILIT had a much higher return to normal rate.

Circulating CD4(+)CD25(+)Foxp3(+) T regulatory cell levels in an experimental model of canine atopic dermatitis.

BACKGROUND: The pathogenesis of canine atopic dermatitis (cAD) is characterized immunologically by an imbalanced T-cell response. Mechanisms of immune regulation in cAD have not yet been completely elucidated. OBJECTIVES: To investigate peripheral blood T regulatory (Treg) cells and their associated cytokines (TGF-ß and IL-10) in an experimental model of cAD. ANIMALS: Eight beagle dogs that were initially naïve and subsequently sensitized to house dust mites (HDM). METHODS AND MATERIALS: T regulatory cell phenotyping was performed by flow-cytometric analysis on peripheral blood; serum cytokine levels were measured by ELISA, before sensitization and after challenge with HDM allergens. Additionally, clinical scores and allergen-specific IgE were determined. RESULTS: After challenge of sensitized dogs to HDM allergen, a significant increase of Treg cells and simultaneous decrease in the serum TGF-ß were observed. However, in most dogs, serum IL-10 values were below the detection limit. Treg cell proportions before sensitization were significantly negatively correlated with the HDM-specific IgE levels and clinical scores after induction of AD signs. CONCLUSION AND CLINICAL IMPORTANCE: The results confirm that Treg responses are involved in the pathogenesis of an experimental model cAD. Further investigations are required to clarify the precise immune modulating function of canine Treg cells and their interplay with other immune cell types.

Total and Toxocara canis larval excretory/secretory antigen- and allergen-specific IgE in atopic and non-atopic dogs.

BACKGROUND: Total IgE concentrations are higher in dogs than in humans. Persistent Toxocara canis larval infection is prevalent in dogs and is associated with substantial specific antibody reactions. A correlation, however, between total IgE and T. canis-specific antibody levels in dogs has not been evaluated. OBJECTIVES: To determine the relationship between total IgE, T. canis-specific IgG and IgE, and allergen-specific IgE levels in atopic and non-atopic dogs, and to evaluate possible confounding factors. ANIMALS: Sera of 30 atopic and 30 non-atopic client-owned dogs. METHODS: Total IgE, T. canis-specific antibody and allergen-specific IgE levels were evaluated by ELISA. RESULTS: Total IgE, T. canis-specific antibody and allergen-specific IgE levels were significantly higher in non-atopic compared to atopic dogs. A positive correlation was demonstrated between T. canis-specific IgG and T. canis-specific IgE; T. canis-specific IgG and total IgE; T. canis-specific IgE and total IgE; and allergen-specific IgE and total IgE. No differences were detected on the basis of age, gender, vaccination status; deworming or season between atopic and non-atopic dogs. Previous immunomodulatory treatment and cause of atopy did not influence antibody levels of atopic dogs. CONCLUSIONS: Toxocara canis-specific IgE appears to be a major component of total IgE in dogs. Total and T. canis-specific IgE levels are higher in non-atopic compared to atopic dogs. It is speculated that T. canis infection may have a protective effect against the development of canine atopic dermatitis and/or that elevations in total serum IgE level are often not associated with atopic dermatitis.

Macrococcus canis and M. caseolyticus in dogs: occurrence, genetic diversity and antibiotic resistance.

BACKGROUND: The discovery of a new Macrococcus canis species isolated from skin and infection sites of dogs led us to question if Macrococcus spp. are common in dogs and are resistant to antibiotics. HYPOTHESIS/OBJECTIVES: To evaluate the occurrence of Macrococcus spp. in dogs, determine antibiotic resistance profiles and genetic relationships. ANIMALS: One hundred and sixty two dogs (mainly West Highland white terriers and Newfoundland dogs) were screened for the presence of Macrococcus, including six dogs with Macrococcus infections. METHODS: Samples were taken from skin, ear canal and oral mucosa using swabs. Macrococci were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry, 16S rRNA sequencing and nuc-PCR. Minimal inhibitory concentrations of 19 antibiotics were determined using broth microdilution. Resistance mechanisms were identified by microarray and sequencing of the fluoroquinolone-determining region of gyrA and grlA. Sequence type (ST) was determined by multilocus sequence typing. RESULTS: Out of the 162 dogs, six harboured M. caseolyticus (n = 6) and 13 harboured M. canis (n = 16). Six isolates of M. canis and one of M. caseolyticus were obtained from infection sites. The 22 M. canis strains belonged to 20 different STs and the seven M. caseolyticus strains to three STs. Resistance to antibiotics was mostly associated with the detection of known genes, with mecB-mediated meticillin resistance being the most frequent. CONCLUSION AND CLINICAL IMPORTANCE: This study gives some insights into the occurrence and genetic characteristics of antibiotic-resistant Macrococcus from dogs. Presence of M. canis in infection sites and resistance to antibiotics emphasized that more attention should be paid to this novel bacteria species.

The effects of cryopreservation on the expression of canine regulatory T-cell markers.

BACKGROUND: Regulatory T (Treg) cells have been described as key regulators in various immunological processes and are of growing interest in veterinary allergy. Cryopreservation of immune cells is performed routinely in human basic science research and in clinical studies. As such, it allows batch testing of collected samples at a single time point, resulting in a significant reduction in sample variability. Data which describe the effects of cryopreservation on Treg cell frequency and functionality in the canine species are important to inform future research. HYPOTHESIS/OBJECTIVES: The purpose of this study was to establish a robust freeze/thaw procedure and flow cytometric staining protocol for canine Treg cells, and to compare the frequencies of different canine Treg cell phenotypes before and after cryopreservation. ANIMALS: Nine privately owned dogs. METHODS: Peripheral blood mononuclear cells were isolated and Treg cells stained and analysed by flow cytometry, before and after three months of cryopreservation. The recovery percentages and the corresponding correlations (fresh versus cryopreserved) for CD4+ CD25+ , CD4+ FOXP3+ and CD4+ CD25+ FOXP3+ cell populations were calculated. RESULTS: A high recovery rate of 97.2 (r = 0.94, P < 0.0001), 93.9 (r = 0.77, P < 0.01) and 101.7% (r = 0.99, P < 0.0001) for CD4+ CD25+ , CD4+ FOXP3+ and CD4+ CD25+ FOXP3+ cell populations, respectively, was observed. CONCLUSIONS: This study demonstrates an optimized protocol for freezing, thawing and quantifying canine Treg cells. These results indicate that cryopreservation does not substantially affect the expression of surface and intracellular markers of canine Treg cells; however, additional studies will be necessary to assess whether functionality of the cells is also maintained.

ballaxy: web services for structural bioinformatics.

MOTIVATION: Web-based workflow systems have gained considerable momentum in sequence-oriented bioinformatics. In structural bioinformatics, however, such systems are still relatively rare; while commercial stand-alone workflow applications are common in the pharmaceutical industry, academic researchers often still rely on command-line scripting to glue individual tools together. RESULTS: In this work, we address the problem of building a web-based system for workflows in structural bioinformatics. For the underlying molecular modelling engine, we opted for the BALL framework because of its extensive and well-tested functionality in the field of structural bioinformatics. The large number of molecular data structures and algorithms implemented in BALL allows for elegant and sophisticated development of new approaches in the field. We hence connected the versatile BALL library and its visualization and editing front end BALLView with the Galaxy workflow framework. The result, which we call ballaxy, enables the user to simply and intuitively create sophisticated pipelines for applications in structure-based computational biology, integrated into a standard tool for molecular modelling. AVAILABILITY AND IMPLEMENTATION: ballaxy consists of three parts: some minor modifications to the Galaxy system, a collection of tools and an integration into the BALL framework and the BALLView application for molecular modelling. Modifications to Galaxy will be submitted to the Galaxy project, and the BALL and BALLView integrations will be integrated in the next major BALL release. After acceptance of the modifications into the Galaxy project, we will publish all ballaxy tools via the Galaxy toolshed. In the meantime, all three components are available from http://www.ball-project.org/ballaxy. Also, docker images for ballaxy are available at https://registry.hub.docker.com/u/anhi/ballaxy/dockerfile/. ballaxy is licensed under the terms of the GPL.

Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays.

WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the 'TTGACY' W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA-protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function.

Serum antibodies and DNA indicate a high prevalence of equine papillomavirus 2 (EcPV2) among horses in Switzerland.

BACKGROUND: The DNA of equine papillomavirus type 2 (EcPV2) is consistently found in equine papillomas and squamous cell carcinomas, indicating a causal association of EcPV2 in the pathogenesis of these tumours; however, little is known about the prevalence of this virus. HYPOTHESIS/OBJECTIVES: The aim of this study was to determine the geno- and seroprevalence of EcPV2 in clinically healthy horses in Switzerland. ANIMALS: Fifty horses presented to the equine department of the university clinic, displaying no skin or mucous membrane lesions or severe signs of other diseases, were sampled. METHODS: Cytobrush samples from the penis or vulva and serum samples were collected. To determine the genoprevalence of EcPV2, DNA was extracted from cytobrush samples and tested for viral DNA with a PCR assay amplifying a 338 bp fragment of the E7/E1 region of the viral genome. Seroprevalence was tested using an enzyme-linked immunosorbent assay aimed to detect antibodies against the major capsid protein (L1) of EcPV2. RESULTS: In five of 50 horses (10%), EcPV2-specific DNA was amplified but no antibodies could be detected, whereas in 14 of 50 horses (28%), antibodies against EcPV2 but no DNA were demonstrated. Both antibodies and viral DNA were detected in four of 50 horses (8%). Neither antibodies nor viral DNA were found in 27 of 50 horses (54%). CONCLUSIONS AND CLINICAL IMPORTANCE: The seroprevalence suggests that EcPV2 is prevalent in the Swiss equine population, while the genoprevalence indicates that currently ongoing infections are less common. The discrepancy between geno- and seroprevalence probably indicates different stages of infection in the tested cohort.

A case in Europe of feline histoplasmosis apparently limited to the skin.

BACKGROUND: Histoplasma capsulatum has a worldwide distribution, but reports in Europe remain rare. We present the second report of histoplasmosis in a cat in Europe and, to the best of our knowledge, the first case of feline histoplasmosis infection apparently limited to the skin. CASE REPORT: A 6-year-old male castrated outdoor cat was presented to the dermatology service with a history of skin lesions evolving over 1 month and consisting of multiple papules and nodules on the head and neck. General examination was unremarkable. Cytological examination of the ulcerated nodules revealed a pyogranulomatous infiltrate, with numerous macrophages containing oval yeast-like cells, 2-5 µm in size, with a central, lightly basophilic core surrounded by a clear halo. A tentative diagnosis of fungal infection was made, and skin biopsy specimens were taken. Histological examination confirmed the cytology findings, and Grocott staining showed numerous organisms suggestive of Histoplasma within macrophages. Thoracic radiographs, abdominal ultrasound and routine laboratory testing were unremarkable. Fungal culture of a nodule was negative. PCR of total DNA extracted from the infected tissue and subsequent sequencing confirmed the diagnosis of H. capsulatum var. capsulatum. Surgical excision of the other nodules was performed, and the cat was treated with oral itraconazole 5 mg/kg once daily; 12 weeks after initial consultation, no lesions were visible. No recurrence was observed during an 8 month follow-up period. CONCLUSIONS AND CLINICAL IMPORTANCE: Histoplasmosis should be included in the differential diagnosis of nodular diseases of cats worldwide.

Independent COL5A1 Variants in Cats with Ehlers-Danlos Syndrome.

We investigated four cats with similar clinical skin-related signs strongly suggestive of Ehlers-Danlos syndrome (EDS). Cases no. 1 and 4 were unrelated and the remaining two cases, no. 2 and 3, were reportedly siblings. Histopathological changes were characterized by severely altered dermal collagen fibers. Transmission electron microscopy in one case demonstrated abnormalities in the collagen fibril organization and structure. The genomes of the two unrelated affected cats and one of the affected siblings were sequenced and individually compared to 54 feline control genomes. We searched for private protein changing variants in known human EDS candidate genes and identified three independent heterozygous COL5A1 variants. COL5A1 is a well-characterized candidate gene for classical EDS. It encodes the proα1 chain of type V collagen, which is needed for correct collagen fibril formation and the integrity of the skin. The identified variants in COL5A1 are c.112_118+15del or r.spl?, c.3514A>T or p.(Lys1172*), and c.3066del or p.(Gly1023Valfs*50) for cases no. 1, 2&3, and 4, respectively. They presumably all lead to nonsense-mediated mRNA decay, which results in haploinsufficiency of COL5A1 and causes the alterations of the connective tissue. The whole genome sequencing approach used in this study enables a refinement of the diagnosis for the affected cats as classical EDS. It further illustrates the potential of such experiments as a precision medicine approach in animals with inherited diseases.

Properties of Organic Liquids when Simulated with Long-Range Lennard-Jones Interactions.

In order to increase the accuracy of classical computer simulations, existing methodologies may need to be adapted. Hitherto, most force fields employ a truncated potential function to model van der Waals interactions, sometimes augmented with an analytical correction. Although such corrections are accurate for homogeneous systems with a long cutoff, they should not be used in inherently inhomogeneous systems such as biomolecular and interface systems. For such cases, a variant of the particle mesh Ewald algorithm (Lennard-Jones PME) was already proposed 20 years ago (Essmann et al. J. Chem. Phys. 1995, 103, 8577-8593), but it was implemented only recently (Wennberg et al. J. Chem. Theory Comput. 2013, 9, 3527-3537) in a major simulation code (GROMACS). The availability of this method allows surface tensions of liquids as well as bulk properties to be established, such as density and enthalpy of vaporization, without approximations due to truncation. Here, we report on simulations of ≈150 liquids (taken from a force field benchmark: Caleman et al. J. Chem. Theory Comput. 2012, 8, 61-74) using three different force fields and compare simulations with and without explicit long-range van der Waals interactions. We find that the density and enthalpy of vaporization increase for most liquids using the generalized Amber force field (GAFF, Wang et al. J. Comput. Chem. 2004, 25, 1157-1174) and the Charmm generalized force field (CGenFF, Vanommeslaeghe et al. J. Comput. Chem. 2010, 31, 671-690) but less so for OPLS/AA (Jorgensen and Tirado-Rives, Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6665-6670), which was parametrized with an analytical correction to the van der Waals potential. The surface tension increases by ≈10(-2) N/m for all force fields. These results suggest that van der Waals attractions in force fields are too strong, in particular for the GAFF and CGenFF. In addition to the simulation results, we introduce a new version of a web server, http://virtualchemistry.org, aimed at facilitating sharing and reuse of input files for molecular simulations.

Complete canine papillomavirus life cycle in pigmented lesions.

Canine papillomaviruses (CPVs) have been identified in various benign and malignant neoplastic skin disorders. The most frequent manifestations of CPV infections are classical warts and pigmented plaques. Although the etiology of canine oral papillomatosis is well established, knowledge about CPVs role in the development of pigmented plaques remains vague. Indeed, as CPV DNA may frequently be found on clinically healthy canine skin, its mere detection in lesions cannot be regarded as a sufficient indicator of causality. Whether CPVs are actually active in pigmented plaques, a requirement for any conceivable involvement, is consequently an open question. To inquire such viral activity, two distinct clinical cases of canine pigmented lesions were evaluated in greater detail. The histological findings in the two cases supported the clinical diagnosis of pigmented viral plaques. Sequencing of amplified DNA from these lesions revealed the genomes of two novel CPV types, i.e. CPV9 and CPV14, both putatively belonging to the genus Chi. Furthermore, transcription and splicing of corresponding CPV mRNA could be shown by RT-PCR in the respective lesions. Finally, viral particles were detected by electron microscopy in homogenates as well as in nuclei of keratinocytes in pigmented lesions. In conclusion, the results link clinical signs of pigmented plaques to histological changes, the presence of CPV specific DNA, viral gene transcription, and the presence of viral particles in and from the lesions. Thus, the findings outline the entire replicative cycle of CPVs in pigmented plaques, which might help understanding the relationship between these viruses and the associated disorders.