Paul R. Hess

Safety and efficacy of a xenogeneic DNA vaccine encoding for human tyrosinase as adjunctive treatment for oral malignant melanoma in dogs following surgical excision of the primary tumor

OBJECTIVEnTo evaluate the safety and efficacy of a vaccine containing plasmid DNA with an insert encoding human tyrosinase (ie, huTyr vaccine) as adjunctive treatment for oral malignant melanoma (MM) in dogs.nnnANIMALSn111 dogs (58 prospectively enrolled in a multicenter clinical trial and 53 historical controls) with stage II or III oral MM (modified World Health Organization staging scale, I to IV) in which locoregional disease control was achieved.nnnPROCEDURESn58 dogs received an initial series of 4 injections of huTyr vaccine (102 μg of DNA/injection) administered transdermally by use of a needle-free IM vaccination device. Dogs were monitored for adverse reactions. Surviving dogs received booster injections at 6-month intervals thereafter. Survival time for vaccinates was compared with that of historical control dogs via Kaplan-Meier survival analysis for the outcome of death.nnnRESULTSnKaplan-Meier analysis of survival time until death attributable to MM was determined to be significantly improved for dogs that received the huTyr vaccine, compared with that of historical controls. However, median survival time could not be determined for vaccinates because < 50% died of MM before the end of the observation period. No systemic reactions requiring veterinary intervention were associated with vaccination. Local reactions were primarily limited to acute wheal or hematoma formation, mild signs of pain at the injection site, and postvaccination bruising.nnnCONCLUSIONS AND CLINICAL RELEVANCEnResults support the safety and efficacy of the huTyr DNA vaccine in dogs as adjunctive treatment for oral MM.nnnIMPACT FOR HUMAN MEDICINEnResponse to DNA vaccination in dogs with oral MM may be useful in development of plasmid DNA vaccination protocols for human patients with similar disease.

Allelic diversity at the DLA-88 locus in Golden Retriever and Boxer breeds is limited.

In the dog, previous analyses of major histocompatibility complex class I genes suggest a single polymorphic locus, dog leukocyte antigen (DLA)-88. While 51 alleles have been reported, estimates of prevalence have not been made. We hypothesized that, within a breed, DLA-88 diversity would be restricted, and one or more dominant alleles could be identified. Accordingly, we determined allele usage in 47 Golden Retrievers and 39 Boxers. In each population, 10 alleles were found; 4 were shared. Seven novel alleles were identified. DLA-88*05101 and *50801 predominated in Golden Retrievers, while most Boxers carried *03401. In these breeds, DLA-88 polymorphisms are limited and largely non-overlapping. The finding of highly prevalent alleles fulfills an important prerequisite for studying canine CD8+ T-cell responses.

Chronic lymphocytic leukaemia in the cat: 18 cases (2000-2010).

There is little information regarding the presentation, biologic behaviour, treatment and prognosis in cats with chronic lymphocytic leukaemia (CLL), and further investigation is needed to characterize this disease in cats. The goal of this study was to describe the clinical presentation, response to treatment and prognosis of feline CLL. A multi-institutional retrospective study of 18 cats diagnosed with CLL between 2000 and 2010 was performed. CLL was defined as the presence of a mature lymphocytosis (>9000 lymphocytes µL(-1) ) and confirmation of an immunophenotypically monomorphic or clonal lymphoid population. Each patient was required to also have at least one of the two following criteria: (1) concurrent cytopenia of at least one cell line and/or (2) >15% mature lymphocytes in the bone marrow. Data on signalment, history, clinical signs, clinicopathologic features and response to treatment were reviewed. Median age of the cats at initial presentation was 12.5 years (range: 5-20 years). The most common presenting complaint was chronic weight loss, which was present in 8/18 (44%) cats. Sixteen of 18 (89%) cats were treated with chlorambucil and prednisolone; four of these cats also received vincristine. Two (11%) cats were treated with multi-agent injectable chemotherapy (L-CHOP, l-asparaginase, cyclophosphamide, doxorubicin, vincristine, prednisolone). Eighty-eight percent of cats evaluable for response achieved a complete (nine cats) or partial (six cats) remission. Median overall remission was 15.7 months (range: 1.3-22.8 months). The median overall survival in the 17 cats with follow-up data was 14.4 months (range: 0.9-25.3 months). Results of this study suggest that CLL affects older-aged cats and responds favourably to treatment with oral chlorambucil and prednisolone.

Evaluation of an in vitro telomeric repeat amplification protocol assay to detect telomerase activity in canine urine

OBJECTIVEnTo evaluate the usefulness of a PCR-based telomeric repeat amplification protocol (TRAP) assay for detecting telomerase activity in cells from a canine transitional cell carcinoma (TCC) cell line and, ultimately, in the urine of dogs with TCC.nnnANIMALSn11 dogs with histologic or cytologic evidence of TCC, 10 dogs with benign lower urinary tract disease, and 9 healthy dogs.nnnPROCEDURESnTelomerase activity was initially evaluated in cells from canine TCC (K9TCC) and telomerase-negative (WI-38) cell lines. Following assay optimization, telomerase stability was evaluated at various storage durations and temperatures. Urine samples were then obtained prospectively from study dogs.nnnRESULTSnTelomerase activity was detected in the K9TCC cell line. The TRAP assay detected telomerase activity in as few as 10 K9TCC cells alone and as low as 2% of a total cell population in K9TCC and WI-38 mixing experiments. A loss of telomerase activity was detected with increasing urine storage durations at various temperatures. Telomerase activity was clearly detected in samples collected from 10 of 11 dogs with TCC, 2 of 10 dogs with benign lower urinary tract disease, and none of the 9 healthy dogs.nnnCONCLUSIONS AND CLINICAL RELEVANCEnThe TRAP-based assay detected telomerase activity in the canine TCC cell line and revealed that the telomerase ribonucleoprotein complex was inherently unstable at various storage durations and conditions. Telomerase activity was also detectable in urine samples obtained from dogs with TCC, which suggested the TRAP assay may be useful in diagnosing TCC in dogs.

Management of Portal Hypertension and Its Consequences

Increased pressure in the protal venous system results from impedance to blood flow at any point along its course from the splanchnic circulation through the liver to the right heart. Typical manifestations of sustained increases in portal venous pressure commonly may include accumulation of abdominal fluid and development of acquired portosystemic shunts. Pathophysiology of altered portal vascular dynamics, diagnostic approach for animals suspected of having an intra-abdominal source of portal hypertension and treatment options are discussed.

Canine acute leukaemia: 50 cases (1989-2014).

Acute leukaemia (AL) is a bone marrow malignancy of hematopoietic progenitors that historically is poorly responsive to treatment. With the widespread adoption of dose-intense chemotherapy, more human patients attain long-term survivals, but whether comparable progress has been made in canine AL is unknown. To investigate this question, medical records from three academic veterinary hospitals were reviewed. Fifty dogs met the criteria for AL, having excess circulating or marrow blasts, a major cytopenia(s), and no substantial lymphadenopathy. Thirty-six dogs received cytotoxic chemotherapy; 23 achieved a complete or partial response for a median of 56 days (range, 9-218). With failure or relapse, 14 dogs were rescued. Median survival with treatment was poor at 55 days (range, 1-300). Untreated (nu2009=u20096) and palliatively-treated (nu2009=u20098) dogs lived a median of 7.5u2009days. Most dogs developed chemoresistance within weeks of initiating treatment, and consequently, survival times for AL remain disappointingly short.

A cell-based MHC stabilization assay for the detection of peptide binding to the canine classical class I molecule, DLA-88.

Identifying immunodominant CTL epitopes is essential for studying CD8+ T-cell responses in populations, but remains difficult, as peptides within antigens typically are too numerous for all to be synthesized and screened. Instead, to facilitate discovery, in silico scanning of proteins for sequences that match the motif, or binding preferences, of the restricting MHC class I allele - the largest determinant of immunodominance - can be used to predict likely candidates. The high false positive rate with this analysis ideally requires binding confirmation, which is obtained routinely by an assay using cell lines such as RMA-S that have defective transporter associated with antigen processing (TAP) machinery, and consequently, few surface class I molecules. The stabilization and resultant increased life-span of peptide-MHC complexes on the cell surface by the addition of true binders validates their identity. To determine whether a similar assay could be developed for dogs, we transfected a prevalent class I allele, DLA-88*50801, into RMA-S. In the BARC3 clone, the recombinant heavy chain was associated with murine β2-microglobulin, and importantly, could differentiate motif-matched and -mismatched peptides by surface MHC stabilization. This work demonstrates the potential to use RMA-S cells transfected with canine alleles as a tool for CTL epitope discovery in this species.

Polymorphisms and tissue expression of the feline leukocyte antigen class I loci FLAI-E, FLAI-H, and FLAI-K

Cytotoxic CD8+ T-cell immunosurveillance for intracellular pathogens, such as viruses, is controlled by classical major histocompatibility complex (MHC) class Ia molecules, and ideally, these antiviral T-cell populations are defined by the specific peptide and restricting MHC allele. Surprisingly, despite the utility of the cat in modeling human viral immunity, little is known about the feline leukocyte antigen class I complex (FLAI). Only a few coding sequences with uncertain locus origin and expression patterns have been reported. Of 19 class I genes, three loci—FLAI-E, FLAI-H, and FLAI-K—are predicted to encode classical molecules, and our objective was to evaluate their status by analyzing polymorphisms and tissue expression. Using locus-specific, PCR-based genotyping, we amplified 33 FLAI-E, FLAI-H, and FLAI-K alleles from 12 cats of various breeds, identifying, for the first time, alleles across three distinct loci in a feline species. Alleles shared the expected polymorphic and invariant sites in the α1/α2 domains, and full-length cDNA clones possessed all characteristic class Ia exons. Alleles could be assigned to a specific locus with reasonable confidence, although there was evidence of potentially confounding interlocus recombination between FLAI-E and FLAI-K. Only FLAI-E, FLAI-H, and FLAI-K origin alleles were amplified from cDNAs of multiple tissue types. We also defined hypervariable regions across these genes, which permitted the assignment of names to both novel and established alleles. As predicted, FLAI-E, FLAI-H, and FLAI-K fulfill the major criteria of class Ia genes. These data represent a necessary prerequisite for studying epitope-specific antiviral CD8+ T-cell responses in cats.

Characterization and allelic variation of the transporters associated with antigen processing (TAP) genes in the domestic dog (Canis lupus familiaris)

The function of the transporters associated with antigen processing (TAP) complex is to shuttle antigenic peptides from the cytosol to the endoplasmic reticulum to load MHC class I molecules for CD8(+) T-cell immunosurveillance. Here we report the promoter and coding regions of the canine TAP1 and TAP2 genes, which encode the homologous subunits forming the TAP heterodimer. By sampling genetically divergent breeds, polymorphisms in both genes were identified, although there were few amino acid differences between alleles. Splice variants were also found. When aligned to TAP genes of other species, functional regions appeared conserved, and upon phylogenetic analysis, canine sequences segregated appropriately with their orthologs. Transfer of the canine TAP2 gene into a murine TAP2-defective cell line rescued surface MHC class I expression, confirming exporter function. This data should prove useful in investigating the association of specific TAP defects or alleles with immunity to intracellular pathogens and cancer in dogs.

Oral melphalan for the treatment of relapsed canine lymphoma

Oral melphalan has been included in multi-agent rescue protocols for canine lymphoma but its activity as a single-agent for this purpose has not been established. Inexpensive cost, ease of administration and tolerability make oral melphalan an attractive candidate for single-agent rescue therapy of canine lymphoma. Retrospective evaluation of 19 cases of relapsed canine lymphoma treated with oral melphalan was performed. Melphalan was primarily administered (nu2009=u200916) via a high dose protocol (HDM) with a median dosage of 19.4u2009mgu2009m-2 . Fifteen dogs (78.9%) were treated concurrently with corticosteroids. Response evaluation was possible for all dogs with a calculated overall clinical benefit (partial response [PR]u2009+u2009stable disease [SD]) of 31.6% (PR 3/19; SD 3/19). Times to progression following melphalan (TTP-M) were 14, 24 and 34u2009days for responders and 20, 28 and 103u2009days for dogs experiencing SD. Twelve of 17 dogs evaluable for toxicity experienced an adverse event (AE) with only 3 dogs experiencing a grade III or higher AE. Haematologic toxicity was common (11/17) while gastrointestinal toxicity was rare (1/17). Although treatment resulted in limited clinical benefit and non-durable responses, oral melphalan was well-tolerated and may be a reasonable rescue option in cases where minimal effective agents remain.