Intralesional interferon alpha-2b as a novel treatment for periocular squamous cell carcinoma in horses.

OBJECTIVE: To determine the safety and efficacy of perilesional human recombinant interferon alpha-2b (IFNα2b) for treatment of periocular squamous cell carcinoma (PSCC) in horses. ANIMALS STUDIED: Eleven horses (12 eyes) with PSCC were enrolled in this prospective clinical study with owner consent. PROCEDURES: Systemically healthy horses were included in the study following confirmation of PSCC via biopsy. Every two weeks for a maximum of six treatments, horses were sedated and perilesional injection of IFNα2b (10 million IU) was performed. Tumors were measured prior to each injection and at one, three, and 12 months after treatment completion. A greater than 50% reduction in tumor size was considered positive response to treatment (i.e., partial or complete response). Development of anti-IFNα2b antibodies was assessed using serum samples obtained after treatment initiation and compared with treatment responses. Antibody concentrations were analyzed using a mixed model. Statistical significance was considered p < 0.05. RESULTS: Each horse received four to six perilesional injections of IFNα2b. Five of 12 eyes (4/11 horses) responded to treatment. Two of five eyes showed complete resolution of gross PSCC. No systemic adverse effects were seen. Local swelling occurred during treatment protocol in 6/11 horses but resolved without intervention. All horses developed serum anti-IFNα2b antibodies. There was no evidence of statistical difference in antibody concentration between responders and non-responders. CONCLUSIONS: Perilesional administration of IFNα2b was found to be well-tolerated in horses with PSCC, and induced tumor regression in 42% of treated eyes. Treatment failure appears unrelated to the development of IFNα2b antibodies.

Horses with equine recurrent uveitis have an activated CD4+ T-cell phenotype that can be modulated by mesenchymal stem cells in vitro.

Equine recurrent uveitis (ERU) is an immune-mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T-cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T-cell phenotype. Adipose-derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4+ T-cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T-cells and B-cells in horses with ERU were similar to normal horses. However, CD4+ T-cells from horses with ERU expressed higher amounts of IFNγ indicating a pro-inflammatory Th1 phenotype. When co-incubated with MSCs, activated CD4+ T-cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell-cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4+ T-cell activation phenotype through a combination of cell-cell contact and prostaglandin signaling.

Frameshift Variant in MFSD12 Explains the Mushroom Coat Color Dilution in Shetland Ponies.

Mushroom is a unique coat color phenotype in Shetland Ponies characterized by the dilution of the chestnut coat color to a sepia tone and is hypothesized to be a recessive trait. A genome wide association study (GWAS), utilizing the Affymetrix 670K array (MNEc670k) and a single locus mixed linear model analysis (EMMAX), identified a locus on ECA7 for further investigation (Pcorrected = 2.08 × 10-10). This locus contained a 3 Mb run of homozygosity in the 12 mushroom ponies tested. Analysis of high throughput Illumina sequencing data from one mushroom Shetland pony compared to 87 genomes from horses of various breeds, uncovered a frameshift variant, p.Asp201fs, in the MFSD12 gene encoding the major facilitator superfamily domain containing 12 protein. This variant was perfectly concordant with phenotype in 96 Shetland Ponies (P = 1.15 × 10-22), was identified in the closely related Miniature Horse for which the mushroom phenotype is suspected to occur (fmu = 0.02), and was absent in 252 individuals from seven additional breeds not reported to have the mushroom phenotype. MFSD12 is highly expressed in melanocytes and variants in this gene in humans, mice, and dogs impact pigmentation. Given the role of MFSD12 in melanogenesis, we propose that p.Asp201fs is causal for the dilution observed in mushroom ponies.

Two Variants in SLC24A5 Are Associated with "Tiger-Eye" Iris Pigmentation in Puerto Rican Paso Fino Horses.

A unique eye color, called tiger-eye, segregates in the Puerto Rican Paso Fino (PRPF) horse breed and is characterized by a bright yellow, amber, or orange iris. Pedigree analysis identified a simple autosomal recessive mode of inheritance for this trait. A genome-wide association study (GWAS) with 24 individuals identified a locus on ECA 1 reaching genome-wide significance (Pcorrected = 1.32 × 10-5). This ECA1 locus harbors the candidate gene, Solute Carrier Family 24 (Sodium/Potassium/Calcium Exchanger), Member 5 (SLC24A5), with known roles in pigmentation in humans, mice, and zebrafish. Humans with compound heterozygous mutations in SLC24A5 have oculocutaneous albinism (OCA) type 6 (OCA6), which is characterized by dilute skin, hair, and eye pigmentation, as well as ocular anomalies. Twenty tiger-eye horses were homozygous for a nonsynonymous mutation in exon 2 (p.Phe91Tyr) of SLC24A5 (called here Tiger-eye 1), which is predicted to be deleterious to protein function. Additionally, eight of the remaining 12 tiger-eye horses heterozygous for the p.Phe91Tyr variant were also heterozygous for a 628 bp deletion encompassing all of exon 7 of SLC24A5 (c.875-340_1081+82del), which we will call here the Tiger-eye 2 allele. None of the 122 brown-eyed horses were homozygous for either tiger-eye-associated allele or were compound heterozygotes. Further, neither variant was detected in 196 horses from four related breeds not known to have the tiger-eye phenotype. Here, we propose that two mutations in SLC24A5 affect iris pigmentation in tiger-eye PRPF horses. Further, unlike OCA6 in humans, the Tiger-eye 1 mutation in its homozygous state or as a compound heterozygote (Tiger-eye 1/Tiger-eye 2) does not appear to cause ocular anomalies or a change in coat color in the PRPF horse.

A missense mutation in damage-specific DNA binding protein 2 is a genetic risk factor for limbal squamous cell carcinoma in horses.

Squamous cell carcinoma (SCC) is the most common cancer of the equine eye, frequently originating at the limbus, with the potential to invade the cornea, cause visual impairment, and result in loss of the eye. Several breeds of horses have a high occurrence of limbal SCC implicating a genetic basis for limbal SCC predisposition. Pedigree analysis in the Haflinger breed supports a simple recessive mode of inheritance and a genome-wide association study (N = 23) identified a 1.5 Mb locus on ECA12 significantly associated with limbal SCC (Pcorrected = 0.04). Sequencing the most physiologically relevant gene from this locus, damage specific DNA binding protein 2 (DDB2), identified a missense mutation (c.1013 C > T p.Thr338Met) that was strongly associated with limbal SCC (P = 3.41 × 10-10 ). Genotyping 42 polymorphisms narrowed the ECA12 candidate interval to 483 kb but did not identify another variant that was more strongly associated. DDB2 binds to ultraviolet light damaged DNA and recruits other proteins to perform global genome nucleotide excision repair. Computational modeling predicts this mutation to be deleterious by altering conformation of the ß loop involved in photolesion recognition. This DDB2 variant was also detected in two other closely related breeds with reported cases of ocular SCC, the Belgian and the Percheron, suggesting it may also be a SCC risk factor in these breeds. Furthermore, in humans xeroderma pigmentosum complementation group E, a disease characterized by sun sensitivity and increased risk of cutaneous SCC and melanomas, is explained by mutations in DDB2. Cross-species comparison remains to be further evaluated.

Assessment of respiratory symptoms and asthma prevalence in a U.S.-Mexico border region.

The authors studied children who were 10-12 yr of age and who resided in sister cities in a U.S.-Mexico border region to determine the prevalence of asthma and respiratory symptoms. The relationship of symptoms to ambient levels of particulate matter less than 10 microm in diameter (PM10), and to several indoor environmental conditions, was assessed. The study was conducted in the border cities of Ambos Nogales (Nogales, Arizona [United States], and Nogales, Sonora [Mexico]). At the beginning of the 11-wk study, during the autumn of 1996, 631 students and their parents completed baseline questionnaires. While in school, the children completed daily symptom diaries and daily peak expiratory flow maneuvers. PM10 values and daily temperatures were also measured. The authors found that the prevalence of self-reported asthma among 5th-grade students was comparable on both sides of the border (i.e., 7.6% on the Arizona side and 6.9% on the Sonora side). Wheezing was a frequent complaint (29.5-35.6%), as was cough (16.8-29.6%). Smoking in the home was common on both sides of the border, and it was associated with a greater occurrence of self-reported asthma and respiratory complaints. Increased respiratory symptoms were also associated with increased ambient PM10 levels. The prevalence of respiratory symptoms such as wheezing and frequent cough among all children in this study, combined with the limitations inherent in self-reporting, suggest that asthma may actually be more prevalent than has been previously reported.