Contralateral optic neuropathy and retinopathy associated with visual and afferent pupillomotor dysfunction following enucleation in six cats.

PURPOSE: To investigate contralateral optic neuropathy and retinopathy following enucleation in 6 cats. METHODS: Retrospective study. The medical records of cats with contralateral visual and afferent pupillomotor dysfunction following enucleation presented to the Animal Health Trust (AHT), Newmarket, UK, between January 1994 and January 2010 were reviewed. Information recorded included history, signalment, ophthalmic findings, electroretinography (ERG) (2/6) and MRI (3/6) findings and long-term outcome. Pearson's chi-square tests were used to compare breed proportions (P < 0.05). RESULTS: Six cats aged 1.5 to 11 (median 5.5) years presented with mydriasis and/or visual deficits noted immediately following enucleation. Enucleation involved optic nerve (ON) ligation in all of the four cases for which this information was available. Ophthalmic findings included mydriasis with absent pupillary light reflex (PLR) (4/6), incomplete PLRs (2/6), absence of dazzle reflex (4/6) and absence of menace response (4/6). Funduscopy initially revealed multifocal peripapillary retinal lesions, with subsequent progressive optic nerve head (ONH) and retinal atrophy. ERG recordings revealed normal outer retinal function at 6 and 22 weeks (2/2). On MRI, the optic chiasm (OC) ipsilateral to the enucleation could not be identified and the contralateral OC was atrophied (3/3). CONCLUSIONS: The acute afferent ON deficits following enucleation, progressive ONH atrophy, normal outer retinal function and MRI demonstrating OC pathology are consistent with chiasmal injury due to traction on the ON during enucleation. Rostral traction on the globe to facilitate ON ligation is contraindicated in cats.

A novel oral formulation of the melanocortin-1 receptor agonist PL8177 resolves inflammation in preclinical studies of inflammatory bowel disease and is gut restricted in rats, dogs, and humans.

Introduction: PL8177 is a potent and selective agonist of the melanocortin 1 receptor (MC1R). PL8177 has shown efficacy in reversing intestinal inflammation in a cannulated rat ulcerative colitis model. To facilitate oral delivery, a novel, polymer-encapsulated formulation of PL8177 was developed. This formulation was tested in 2 rat ulcerative colitis models and evaluated for distribution, in vivo, in rats, dogs, and humans. Methods: The rat models of colitis were induced by treatment with 2,4-dinitrobenzenesulfonic acid or dextran sulfate sodium. Single nuclei RNA sequencing of colon tissues was performed to characterize the mechanism of action. The distribution and concentration of PL8177 and the main metabolite within the GI tract after a single oral dose of PL8177 was investigated in rats and dogs. A phase 0 clinical study using a single microdose (70 µg) of [14C]-labeled PL8177 investigated the release of PL8177 in the colon of healthy men after oral administration. Results: Rats treated with 50 µg oral PL8177 demonstrated significantly lower macroscopic colon damage scores and improvement in colon weight, stool consistency, and fecal occult blood vs the vehicle without active drug. Histopathology analysis resulted in the maintenance of intact colon structure and barrier, reduced immune cell infiltration, and increased enterocytes with PL8177 treatment. Transcriptome data show that oral PL8177 50 µg treatment causes relative cell populations and key gene expressions levels to move closer to healthy controls. Compared with vehicle, treated colon samples show negative enrichment of immune marker genes and diverse immune-related pathways. In rats and dogs, orally administered PL8177 was detected at higher amounts in the colon vs upper GI tract. [14C]-PL8177 and the main metabolite were detected in the feces but not in the plasma and urine in humans. This suggests that the parent drug [14C]-PL8177 was released from the polymer formulation and metabolized within the GI tract, where it would be expected to exert its effect. Conclusion: Collectively, these findings support further research into the oral formulation of PL8177 as a possible therapeutic for GI inflammatory diseases in humans.

Congenital keratoconjunctivitis sicca and ichthyosiform dermatosis in Cavalier King Charles spaniel dogs. Part II: candidate gene study.

PURPOSE: To identify causative mutation(s) for congenital keratoconjunctivitis sicca and ichthyosiform dermatosis (CKCSID) in Cavalier King Charles spaniel (CKCS) dogs using a candidate gene approach. METHODS: DNA samples from 21 cases/parents were collected. Canine candidate genes (CCGs) for similar inherited human diseases were chosen. Twenty-eight candidate genes were identified by searching the Pubmed OMIM database (http://www.ncbi.nlm.nih.gov/omim). Canine orthologues of human candidate genes were identified using the Ensembl orthologue prediction facility (http://www.ensembl.org/index.html). Two microsatellites flanking each candidate gene were selected, and primers to amplify each microsatellite were designed using the Whitehead Institute primer design website (http://frodo.wi.mit.edu/primer3/). The microsatellites associated with all 28 CCGs were genotyped on a panel of 21 DNA samples from CKCS dogs (13 affected and eight carriers). Genotyping data was analyzed to identify markers homozygous in affected dogs and heterozygous in carriers (homozygosity mapping). RESULTS: None of the microsatellites associated with 25 of the CCGs displayed an association with CKCSID in the 21 DNA samples tested. Three CCGs associated microsatellites were monomorphic across all samples tested. CONCLUSIONS: Twenty-five CCGs were excluded as cause of CKCSID. Three CCGs could not be excluded from involvement in the inheritance of CKCSID.

Congenital keratoconjunctivitis sicca and ichthyosiform dermatosis in 25 Cavalier King Charles spaniel dogs. Part I: clinical signs, histopathology, and inheritance.

The clinical presentation and progression (over 9 months to 13 years) of congenital keratoconjunctivitis sicca and ichthyosiform dermatosis (CKCSID) in the Cavalier King Charles spaniel dog are described for six new cases and six previously described cases. Cases presented with a congenitally abnormal (rough/curly) coat and signs of KCS from eyelid opening. Persistent scale along the dorsal spine and flanks with a harsh frizzy and alopecic coat was evident in the first few months of life. Ventral abdominal skin was hyperpigmented and hyperkeratinized in adulthood. Footpads were hyperkeratinized from young adulthood with nail growth abnormalities and intermittent sloughing. Long-term follow-up of cases (13/25) is described. Immunomodulatory/lacrimostimulant treatment had no statistically significant effect on Schirmer tear test results, although subjectively, this treatment reduced progression of the keratitis. Histopathological analysis of samples (skin/footpads/lacrimal glands/salivary glands) for three new cases was consistent with an ichthyosiform dermatosis, with no pathology of the salivary or lacrimal glands identified histologically. Pedigree analysis suggests the syndrome is inherited by an autosomal recessive mode.

Ophthalmic and cone derived electrodiagnostic findings in outbred Miniature Long-haired Dachshunds homozygous for a RPGRIP1 mutation.

OBJECTIVE: To investigate ophthalmic and cone-derived electrodiagnostic findings in outbred Miniature Long-haired Dachshunds (MLHD) homozygous for a mutation in the RPGRIP1 gene previously associated with cone-rod dystrophy 1 (cord1). ANIMALS: A total of 36 MLHD homozygous for the RPGRIP1 mutation and 23 dogs clear of the mutation (control group). PROCEDURES: The dogs underwent ophthalmic examination and photopic electroretinogram (ERG) recordings. RESULTS: None of the control dogs presented with clinical or ophthalmic signs consistent with cord1. Amongst the dogs homozygous for the mutation one presented with bilateral symmetrical total retinal atrophy. None of the other dogs in this group showed signs consistent with cord1. Photopic ERG recordings were available in 23 control dogs and 34 dogs homozygous for the mutation. Photopic a- and b-waves following four light stimuli (3 cdS/m(2) ) at a rate of 5.1 Hz were not significantly different between groups. The amplitudes of the 30 Hz flicker (128 flashes, 3 cdS/m(2) ) response were significantly reduced in the dogs homozygous for the PRGRIP1 mutation. The difference in age between the two groups did not significantly affect the difference. CONCLUSION: Homozygosity of the RPGRIP1 mutation does not invariably result in early onset cord1. However, cone derived ERG recordings show evidence of a reduced cone or inner retinal function in homozygous but clinically normal MLHD. Modifying genes that have yet to be identified may influence an individual dog's risk of developing the blinding cord1 and also the age of onset and rate of progression.

Pharmacokinetics of the Melanocortin Type 1 Receptor Agonist PL8177 After Subcutaneous Administration.

BACKGROUND AND OBJECTIVE: PL8177 is a selective melanocortin 1 receptor agonist in development for the treatment of various immunologic and inflammatory conditions. Here we describe the pharmacokinetics of PL8177 after subcutaneous (sc) delivery in animals and humans. METHODS: Mice, rats, and dogs were administered sc PL8177 at single doses of 1.0 and 3.0 mg/kg (mice); 1.0, 5.0, and 25.0 mg/kg/day (rats); or 1.5, 8.0, and 40.0 mg/day (dogs). Blood was collected over 24 h (mice) or 28 days (rats and dogs). Safety and pharmacokinetics of single and multiple sc doses were also examined in human volunteers. Two dose levels were tested in two dosing cohorts of 1.0 and 3.0 mg/day for 7 days. Blood samples were collected through Day 1 and on Days 2 to 6 at peak and trough times based on analysis of the first two single-dose cohorts. RESULTS: In mice, 3 mg/kg PL8177 resulted in an area under the plasma concentration-time curve from 0 to infinity (AUC∞) of 1727 ng·h/mL, a maximum plasma concentration (Cmax) of 2440 ng/mL, an elimination half-life (t½) of 0.5 h, and a time to maximum concentration (tmax) of 0.25 h. Results for the 1-mg/kg dose were generally proportional. In rats, mean tmax values were independent of dose and ranged from 0.25 to 1.0 h for single and multiple dosing. Cmax values ranged from 516 to 695 ng/mL (1-mg/kg dose) and from 666 to 1180 ng/mL (25-mg/kg dose). In dogs, mean tmax values ranged from 0.4 to 1.3 h for single and multiple dosing. Values for tmax decreased with increasing dose and mean plasma Cmax increased less than dose proportionally (96-129 ng·h/mL [1.5 mg], 275-615 ng·h/mL [8.0 mg], and 633-1280 ng·h/mL [40.0 mg]). In humans, PL8177 was observed in the plasma within 15 min after a single dose and persisted for up to 48 h at higher doses. The tmax was 30-45 min (single dose) and 15-45 min (multiple doses). In multiple-dose studies, maximum steady-state plasma concentration (Cmax,ss) and AUC∞ increased with dose. Geometric mean Cmax,ss values were 20.1 ng/mL (1.0 mg) and 57.2 ng/mL (3.0 mg). AUC∞ values were 54.3 ng·h/mL (1.0 mg) and 199 ng·h/mL (3.0 mg). Unchanged PL8177 excreted in the urine was ≤ 1%, and accumulation was minimal. CONCLUSION: PL8177 administration resulted in a consistent pharmacokinetic profile. The measured exposure levels resulted in pharmacologically active PL8177 concentrations at the targeted MC1R. Rapid absorption was seen in healthy volunteers, and multiple-dose administration over 7 days resulted in pharmacokinetic characteristics similar to those observed after single-dose administration. Results support the continued development of PL8177 to treat immunologic and inflammatory conditions.

Phenotypic variation and genotype-phenotype discordance in canine cone-rod dystrophy with an RPGRIP1 mutation.

PURPOSE: Previously, a 44 bp insertion in exon 2 of retinitis pigmentosa GTPase interacting protein 1 (RPGRIP1) was identified as the cause of cone-rod dystrophy 1 (cord1), a recessive form of progressive retinal atrophy (PRA) in the Miniature Longhaired Dachshund (MLHD), a dog model for Leber congenital amaurosis. The cord1 locus was mapped using MLHDs from an inbred colony with a homogeneous early onset disease phenotype. In this paper, the MLHD pet population was studied to investigate phenotypic variation and genotype-phenotype correlation. Further, the cord1 locus was fine-mapped using PRA cases from the MLHD pet population to narrow the critical region. Other dog breeds were also screened for the RGPRIP1 insertion. METHODS: This study examined phenotypic variation in an MLHD pet population that included 59 sporadic PRA cases and 18 members of an extended family with shared environment and having six PRA cases. Ophthalmologic evaluations included behavioral abnormalities, responses to menace and light, fundoscopy, and electroretinography (ERG). The RPGRIP1 insertion was screened for in all cases and 200 apparently normal control MLHDs and in 510 dogs from 66 other breed. To fine-map the cord1 locus in the MLHD, 74 PRA cases and 86 controls aged 4 years or more were genotyped for 24 polymorphic markers within the previously mapped cord1 critical region of 14.15 Mb. RESULTS: Among sporadic PRA cases from the MLHD pet population, the age of onset varied from 4 months to 15 years old; MLHDs from the extended family also showed variable onset and rate of progression. Screening for the insertion in RPGRIP1 identified substantial genotype-phenotype discordance: 16% of controls were homozygous for the insertion (RPGRIP1(-/-)), while 20% of PRA cases were not homozygous for it. Four other breeds were identified to carry the insertion including English Springer Spaniels and Beagles with insertion homozygotes. The former breed included both controls and PRA cases, yet in the latter breed, cone ERG was undetectable in two dogs with no clinically apparent visual dysfunction. Notably, the insertion in the Beagles was a longer variant of that seen in the other breeds. Fine-mapping of the cord1 locus narrowed the critical region on CFA15 from 14.15 Mb to 1.74 Mb which still contains the RPGRIP1 gene. CONCLUSIONS: Extensive phenotypic variations of onset age and progression rate were observed in PRA cases of the MLHD pet population. The insertion in RPGRIP1 showed the strongest association with the disease, yet additional as well as alternative factors may account for the substantial genotype-phenotype discordance.

Mutation in HSF4 is associated with hereditary cataract in the Australian Shepherd.

Cataracts are a leading cause of blindness in dogs with approximately 100 breeds affected by primary hereditary forms. Despite the large number of breeds affected with hereditary cataracts (HC) little is known about the genetics of the condition, and to date only a single gene, HSF4, has been implicated in the development of the disease in dogs. We previously identified a recessively inherited 1-bp insertion in the transcription factor gene HSF4 resulting in the loss of the open reading frame in Boston terriers and Staffordshire bull terriers. While testing the insertion mutation in other breeds with HC, we identified a 1-bp deletion at the same nucleotide of HSF4 in some Australian Shepherds with cataract. Using DNA samples from almost 400 privately owned Australian Shepherds we have investigated the association between the deletion mutation in HSF4 and cataracts in this breed. We conclude that the mutation is significantly associated with cataracts and that a dog carrying the mutation is approximately 17 times more likely to develop binocular cataracts than dogs that are clear of the mutation. The data also indicate that additional mutations associated with the development of cataracts are likely to be co-segregating in the Australian Shepherd population.

Pathological and electrophysiological features of a canine cone-rod dystrophy in the miniature longhaired dachshund.

PURPOSE: To characterize the electrophysiological and histopathological features of a retinal degenerative disease in a colony of miniature longhaired dachshunds known to have a form of progressive retinal atrophy (PRA). METHODS: Serial electroretinograms were recorded from affected homozygous (n = 36) and heterozygous (n = 15) dogs. Morphologic investigations including immunohistochemistry and lectin histochemistry were performed on selected homozygous animals (n = 15). RESULTS: Clinical findings included loss of tapetal hyperreflectivity. The mode of inheritance was autosomal recessive. An early dramatic reduction of cone-specific ERG amplitude with a more modest reduction in rod b-wave amplitude was demonstrated. Progressively, rod specific responses diminished until there were no recordable responses to the ERG stimuli at 40 weeks of age. Morphologic changes confirmed early cone inner and outer segment loss. Other abnormalities included opsin mislocalization and outer nuclear layer thinning due to the subsequent loss of rod photoreceptors. CONCLUSIONS: A novel canine cone-rod dystrophy has been identified.

Mutation in HSF4 associated with early but not late-onset hereditary cataract in the Boston Terrier.

Primary hereditary cataract (HC) is one of the most common disorders in purebred dogs and is a leading cause of blindness. Boston Terriers suffer from 2 distinct forms of HC which occur at different ages and which are different in their appearance and progression. Early-onset hereditary cataract (EHC) affects dogs within the first few months of life, is always progressive and bilateral, and results in total blindness, whereas late-onset hereditary cataract (LHC) in general affects dogs over the age of 3 and is more variable in its clinical phenotype, age of onset, progression, and the degree to which vision is impaired. A mutation in HSF4 has recently been reported in a small number of Boston Terriers affected with EHC. In this study, we analyzed 22 additional Boston Terriers affected with early-onset cataract to confirm that the HSF4 mutation is causative for this form of cataract in this breed. In addition, we analyzed 40 Boston Terriers that were either clinically clear or affected with LHC for the presence or absence of the HSF4 mutation. By also sequencing HSF4 in dogs affected with LHC, we conclude that HSF4 is not associated with the development of the late-onset form of cataract and that the 2 forms of cataract in this breed are therefore genetically discrete conditions.

Cataract in the UK Leonberger population.

OBJECTIVE: To survey the Leonberger, a numerically small breed in the UK, for the presence of cataract and find statistical support for the possible presence of inherited forms of cataract. METHODS: Ocular examinations were carried out by the first author between September 1996 and September 1998 on 211 Leonbergers; results of the ocular examination of further 228 Leonbergers examined between January 1990 and September 1998 by members of the British Veterinary Association/KC/ISDS eye scheme (BVA/KC/ISDS eye scheme) panel were analyzed. Data from all dogs examined were pooled in a composite database of 365 Leonbergers and the relationships between offspring and parents for the identified forms of cataract were examined with appropriate statistical methods. RESULTS: Cataracts were diagnosed in 90 Leonbergers, the majority being nuclear (40) or posterior polar subcapsular (31). A subgroup of nuclear cataracts, diagnosed in 11 dogs, is described and named as 'posterior nuclear cataract.' For posterior polar cataract, a positive association between offspring and parents was made in the logistic regression model, supporting the suggestion of inheritance. CONCLUSIONS: The presence of several types of cataract in the UK Leonberger population is described. Statistical support for the inheritance of posterior polar subcapsular cataract is given.

Identification of mutations in HSF4 in dogs of three different breeds with hereditary cataracts.

Cataracts are a leading cause of blindness in both dogs and humans. Mutations in several genes have been associated with inherited forms of human cataract, but no mutations have been identified as the cause of any form of canine inherited cataract. We have used a candidate gene approach to investigate 20 genes, known to be associated with cataract in humans, for their potential association with the development of hereditary cataract (HC) in dogs. We have identified mutations in the HSF4 gene in Staffordshire Bull Terriers, Boston Terriers and Australian Shepherds affected by HC. Interestingly, different mutations in this single gene may be causing a recessive form of cataract in Staffordshire Bull Terriers and Boston Terriers and a dominant cataract in Australian Shepherds. Identification of the mutations that cause HC in these three breeds provides a method of controlling the disease within populations at risk using a simple diagnostic test, and also establishes cataract in these breeds as models for their human counterparts.