Upregulated MYC expression and p53 mutations may contribute to the oncogenesis of canine Meibomian gland carcinomas.

Sebaceous carcinomas of the human ocular adnexa commonly exhibit pagetoid spread, mutations in tumor-suppressor genes, and protooncogene copy number gain. Sebaceous carcinomas are rarely reported in other species, and while the Meibomian gland (MG) represents the most common ocular adnexal structure of the canine eyelid to develop neoplasia, most are clinically and histologically benign. The objective of this study was to compare molecular features of canine MG carcinomas and adenomas. Two retrospectively identified MG carcinomas were subject to immunohistochemistry and qPCR. When compared with normal glands, MYC was upregulated in benign and malignant MG neoplasms. Aberrant p53 expression was restricted to the nuclei of intraepithelial neoplastic cells in MG carcinomas. Adipophilin expression was diminished in MG neoplasms compared with the normal MG. Our findings, if confirmed in a larger cohort of cases, could suggest that MG oncogenesis in a dog may exhibit similar molecular features as their human counterparts.

Feline Epitheliotropic Mastocytic Conjunctivitis in 15 Cats.

Mast cell infiltration occurs in malignant, inflammatory (eg, allergic, infectious), and idiopathic disease processes in humans and animals. Here, we describe the clinical and histological features of a unique proliferative conjunctivitis occurring in 15 cats. Ocular specimens were examined histologically, and polymerase chain reaction (PCR) for feline herpesvirus 1 (FHV-1) was performed on ocular tissues obtained from 10 cats. Cats had a median age of 8 years (range: 7 months-17.5 years). The known median duration of ocular lesions prior to biopsy was 4 months (range: 1 week-3 years). Ocular disease was unilateral in 12 cats, and 9 cats had coexisting corneal disease. Clinically and histologically, proliferative or nodular conjunctival lesions were noted in 13 cats. The nictitating membrane was affected in 10 cats. Histologically, lesions were characterized by mixed inflammatory infiltrates with an abundance of Giemsa-positive and toluidine blue-positive intraepithelial and subepithelial mast cells, marked edema, and papillary epithelial hyperplasia. Feline herpesvirus 1 was demonstrated by PCR in 1 of 10 cats tested. Follow-up information was available for 14 cats: 8 had no recurrence during a median follow-up period of 17.5 months (range: 4.5-30 months), 2 underwent orbital exenteration, 3 had recurrence that was medically managed, and 1 cat had diffuse conjunctivitis at the time of biopsy and recurrence was deemed irrelevant. Various ocular medications were administered before and after surgical biopsy. This condition was designated as feline epitheliotropic mastocytic conjunctivitis, with intraepithelial mast cells being an essential feature and papillary epithelial proliferation being characteristic but not diagnostic alone. The condition appears to be uncommon and benign. Although the cause is unknown, an allergic component is possible.

Ultrastructural abnormalities of the trabecular meshwork extracellular matrix in Cyp1b1-deficient mice.

Cytochrome P450 1B1 (CYP1B1) is highly expressed in human and murine ocular tissues during development. Mutations in this gene are implicated in the development of primary congenital glaucoma (PCG) in humans. Mice deficient in Cyp1b1 (Cyp1b1(-/-) ) present developmental abnormalities similar to human primary congenital glaucoma. The present work describes the ultrastructural morphology of the iridocorneal angle of 21 eyes from 1-week-old to 8-month-old Cyp1b1(-/-) mice. Morphometric and semiquantitative analysis of the data revealed that 3-week-old Cyp1b1(-/-) mice present a significantly (P < .005) decreased amount of trabecular meshwork (TM) collagen and higher TM endothelial cell and collagen lesion scores (P < .005) than age-matched controls. Collagen loss and lesion scores were progressively increased in older animals, with 8-month-old animals presenting severe atrophy of the TM. Our findings advance the understanding of the effects of CYP1B1 mutations in TM development and primary congenital glaucoma, as well as suggest a link between TM morphologic alterations and increased intraocular pressure.

Malignant uveal schwannoma with peripheral nerve extension in a 12-week-old color-dilute Labrador Retriever.

The formalin-fixed, amber-colored right globe from a 12-week-old female silver Labrador Retriever dog was submitted to the Comparative Ocular Pathology Laboratory of Wisconsin for light microscopic evaluation. The clinical history described a collapsed anterior chamber and multifocal nodular lesions in the peripheral iris. Histologically, immunohistochemically, and ultrastructurally, the uveal mass was consistent with a malignant schwannoma; there was extension along peripheral nerves within the sclera. The signalment and behavior of the neoplasm distinguish it from the uveal schwannoma of blue-eyed dogs and bear some resemblance to the ocular lesions in human neurofibromatosis. The dilute color mutation may contribute to the cause. Six weeks later, the dog did not develop any additional masses.

Squamous Papillomas of the Conjunctiva in Dogs: A Condition Not Associated With Papillomavirus Infection.

Papillomas of the conjunctival surface in people can be of viral or nonviral origin and are found in high association with human papillomavirus. Canine conjunctival papillomas are seldom described, and published accounts have mostly been associated with canine oral papillomavirus infection. Here, we describe conjunctival squamous papillomas that do not express papillomavirus proteins and compare them with papillomavirus-associated conjunctival papillomas. Conjunctival squamous papillomas presented a distinct histopathologic profile and lacked the cytopathic effects seen in viral papillomas. They appeared as exophytic, papilliferous, pedunculated lesions with delicate fronds and angular terminal margins. Squamous papillomas presented with a delicate fibrovascular core and were associated both clinically and grossly with a feeder vessel. Pigmentation was variable within the epithelium and stroma of these lesions, and inflammatory infiltrates were characteristically minimal. Conjunctival squamous papillomas resembled squamous papillomas of the skin; however, they lacked significant hyperkeratosis. Compared with conjunctival viral papillomas, these masses occurred in older dogs and were smaller and solitary. Furthermore, polymerase chain reaction and immunohistochemistry failed to demonstrate papillomavirus genetic material and antigens in conjunctival squamous papillomas. Both viral and nonviral conjunctival papillomas were considered benign.

Clinical, morphologic, and immunohistochemical features of canine orbital hibernomas.

Hibernomas are uncommon benign tumors of brown fat that occur in humans and various animal species. They have not been observed in the orbit of dogs, humans, or other animals. Here we report clinical, light and electron microscopic, and immunohistochemical features of a series of 7 hibernomas arising in the orbital region of dogs. These neoplasms occurred in adult dogs with no breed predilection. The mean age of the affected dogs was 10.4 years (range, 8-13 years). All neoplasms presented as soft lobular masses composed of predominantly round or polygonal neoplastic cells with granular eosinophilic and vacuolated cytoplasm resembling adipocytes. The cytoplasm contained large numbers of pleomorphic mitochondria with dense matrices and indistinct cristae. Immunohistochemical evaluation confirmed positive labeling of neoplastic cells from all cases with uncoupling protein 1 (UCP-1) consistent with brown fat differentiation. Interestingly, rare neoplastic cells also expressed myogenin and myoD, possibly suggesting a common progenitor cell for neoplastic brown adipose and skeletal muscle cells.

Bilateral buphthalmia in a 4-month-old Texas longhorn steer.

Congenital ocular disease occurs uncommonly in cattle, with multiple abnormalities reported only sporadically in the literature. This report describes a case of anterior segment dysgenesis resulting in glaucoma in a 4-month-old Texas Longhorn steer. On clinical exam, bilateral buphthalmia was present and intraocular pressures exceeded 47 mm Hg in both eyes. On histopathologic examination, the iridocorneal angle and filtration apparatus were distorted due to collapse of the ciliary cleft and anterior displacement of the anterior portion of the ciliary body. No evidence of inflammation or other causes of glaucoma were recognized.

Systemic distribution of viral antigen in alpacas persistently infected with bovine pestivirus.

Recently, confirmed occurrences of persistent bovine viral diarrhea virus (BVDV) infection in North American alpacas have raised concerns about the role of persistently infected (PI) alpacas in transmission of virus among herds, yet only limited pathological descriptions of persistent infections in alpacas have been reported. The objective of this study was to characterize BVDV antigen distribution in 10 PI alpacas of varying age and to compare viral antigen distribution and localization in tissues of PI alpacas with 5 PI calves of varying age. Ocular dysplasia was evident in 1 PI alpaca, constituting the first reported congenital ocular lesion in PI alpacas. Viral antigen was widely distributed in alpaca tissues and was prominent in neurons, endothelial cells, and vascular tunica media myocytes but had limited distribution in lymphoid tissues and moderate distribution in epithelium of several organ systems of alpacas. Macrophages in the alpaca gastrointestinal system submucosa and lymph node medullary sinuses often had prominent labeling. In addition, only 1 alpaca had antigen labeling in the bone marrow in contrast to PI cattle. Labeled cells in calf tissues were more widely distributed, occurring prominently in lymphoid and epithelial tissues. Common features of the 2 host species were widespread antigen labeling and absence of lymphoid depletion.

Blind sterile 2 (bs2), a hypomorphic mutation in Agps, results in cataracts and male sterility in mice.

Blind sterile 2 (bs2) is a spontaneous autosomal recessive mouse mutation exhibiting cataracts and male sterility. Detailed clinical and histological evaluation revealed that bs2 mice have cataracts resulting from severely disrupted lens fiber cells. Analysis of bs2 testes revealed the absence of mature sperm and the presence of large multinucleate cells within the lumens of seminiferous tubules. Linkage analysis mapped the bs2 locus to mouse chromosome 2, approximately 45cM distal from the centromere. Fine mapping established a 3.1Mb bs2 critical region containing 19 candidate genes. Sequence analysis of alkylglycerone-phosphate synthase (Agps), a gene within the bs2 critical region, revealed a G to A substitution at the +5 position of intron 14. This mutation results in two abundantly expressed aberrantly spliced Agps transcripts: Agps(∆exon14) lacking exon 14 or Agps(exon∆13-14) lacking both exons 13 and 14 as well as full-length Agps transcript. Agps is a peroxisomal enzyme which catalyzes the formation of the ether bond during the synthesis of ether lipids. Both aberrantly spliced Agps(∆exon14) and Agps(exon∆13-14) transcripts led to a frame shift, premature stop and putative proteins lacking the enzymatic FAD domain. We present evidence that bs2 mice have significantly decreased levels of ether lipids. Human mutations in Agps result in rhizomelic chondrodysplasia punctata type 3 (RCDP3), a disease for which bs2 is the only genetic model. Thus, bs2 is a hypomorphic mutation in Agps, and represents a useful model for investigation of the tissue specificity of ether lipid requirements which will be particularly valuable for elucidating the mechanism of disease phenotypes resulting from ether lipid depletion.

Diagnostic features of feline restrictive orbital myofibroblastic sarcoma.

A progressive debilitating disease of the orbit and adjacent connective tissues of cats has historically been called feline orbital pseudotumor. The authors reviewed clinical, histopathologic, and diagnostic imaging features of this disease in 12 cases from the Comparative Ocular Pathology Laboratory of Wisconsin. The cats' ages ranged from 7 to 16 years (mean, 10.8 years). All cats had a history of severely restricted mobility of the globe and eyelids with secondary corneal disease. Eleven cats (92%) had concurrent involvement of the contralateral eye and/or the oral cavity. Diffuse scleral or episcleral thickening was seen with computed tomography in all clinically affected eyes. Histologically, an insidious infiltration of neoplastic spindle cells in the orbit, eyelids, and periorbital skin and soft tissues, with collagen deposition and a few perivascular lymphocytes, led to entrapment and restricted mobility of the eyelids and orbital tissues. The tumor failed to form a discrete mass, and it spread along fascial planes to the contralateral orbit and eyelids and/or the lips and oral cavity. In all tested cases (n = 10), neoplastic cells were immunohistochemically positive for vimentin, S100 protein, and smooth muscle actin. The authors adopted the term feline restrictive orbital myofibroblastic sarcoma to reflect the restricted mobility of the eyelids and globe and the imaging and histologic features of an invasive yet low-grade myofibroblastic sarcoma.

Characterizing a rat Brca2 knockout model.

Evidence exists that BRCA2 carriers may have an elevated risk of breast, ovarian, colon, prostate, and pancreatic cancer. In general, carriers are defined as individuals with protein truncating mutations within the BRCA2 gene. Many Brca2 knockout lines have been produced and characterized in the mouse. We previously produced a rat Brca2 knockout strain in which there is a nonsense mutation in exon 11 between BRC repeats 2 and 3, and a truncated protein is produced. Interestingly, while such a mutation in homozygous mice would lead to limited survival of approximately 3 months, the Brca2-/- rats are 100% viable and the vast majority live to over 1 year of age. Brca2-/- rats show a phenotype of growth inhibition and sterility in both sexes. Aspermatogenesis in the Brca2-/- rats is due to a failure of homologous chromosome synapsis. Long-term phenotypes include underdeveloped mammary glands, cataract formation and lifespan shortening due to the development of tumors and cancers in multiple organs. The establishment of the rat Brca2 knockout model provides a means to study the role of Brca2 in increasing cancer susceptibility and inducing a novel ocular phenotype not previously associated with this gene.

Histopathological features of ocular leishmaniosis in the dog.

Canine leishmaniosis (CL) can present with multiple clinical signs and ocular disease is reported to occur in almost 25% of affected dogs. The purpose of the present study was to characterize the nature of inflammation within the eyes of dogs with leishmaniosis and to determine whether parasites were present in these lesions. Eyes from 60 dogs with confirmed leishmaniosis that died or were humanely destroyed over a 4 year period were included in the study. Sections of formalin-fixed globes were stained with haematoxylin and eosin (HE) and subjected to immunohistochemistry using a Leishmania-specific antibody. Clinically evident ocular signs were present in 15 of 60 dogs (13 bilaterally and 2 unilaterally). Thirty-five of 60 dogs received some form of anti-protozoal treatment. In 36 of 120 eyes (30%) a granulomatous inflammatory infiltrate was found and in 32 of 120 eyes (26.6%) the parasite was identified immunohistochemically within the globe. Ocular tissues affected, in order of frequency, were conjunctiva and limbus, ciliary body, iris, cornea, sclera and iridocorneal angle, choroid and the optic nerve sheath. Different microscopical patterns were defined in each of these structures. Leishmania organisms and associated inflammation can be found in different ocular tissues, accounting for some of the ocular clinical signs described for this disease.

Metastasis-associated microRNA expression in canine uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is the most common primary intraocular tumour in dogs. There is no effective means of predicting whether a tumour will metastasize. microRNA (miRNA) metastasis signatures have been identified for several human cancers, including UM. AIMS: In this study we investigated whether metastasizing and non-metastasizing canine UMs can be distinguished by miRNA expression levels. MATERIALS AND METHODS: miRNA microarray profiling was used to compare miRNA expression in 8 metastasizing and 12 non-metastasizing formalin-fixed, paraffin-embedded (FFPE) primary UM biopsies. RESULTS: Fourteen miRNAs exhibited statistically significant differences in expression between the metastasizing and non-metastasizing tumours. Class prediction analysis pinpointed 9 miRNAs which categorized tumours as metastasizing or non-metastasizing with an accuracy of 89%. Of the discriminating miRNAs, 8 were up-regulated in metastasizing UM, and included 3 miRNAs implicated as potential "metastasis activators" in human cutaneous melanoma. The expression of 4 of the miRNAs was subsequently measured using the quantitative reverse transcription polymerase chain reaction (RT-qPCR), and their up-regulation in metastasizing tumours validated. CONCLUSION: miRNA expression profiles may potentially be used to identify UMs that will metastasize, and miRNAs that are up-regulated in metastasizing tumours may be targets for therapeutic intervention.

Lentoid bodies in the avian retina.

In-vitro studies suggest that, in avian retina, lentoid bodies arise from Müller cells or developing neuroretina. This report describes lentoid bodies in adult avian retinas in association with retinal trauma or degeneration. Retinal lentoids were identified in four birds (three owls and one parrot) in the course of routine diagnostic histopathology. Sections were stained with periodic acid-Schiff for the purposes of descriptive histology, and immunolabelled for a Müller cell marker (glial fibrillary acidic protein; GFAP) and a lens-specific marker (crystallin alpha-A). Intraretinal lentoids of varying size were identified, the constituent cells resembling bladder cells similar to those seen in cataracts. The process of lentoid formation followed a consistent pattern, characterized by progressive Müller cell hypertrophy in damaged areas, culminating in lentoid formation. GFAP immunoreactivity was strongest in Müller cells in the early stages of hypertrophy and receded as Müller cell hypertrophy advanced and lentoids developed. In contrast to GFAP immunoreactivity, crystalline alpha-A labelling increased in distribution and intensity as Müller hypertrophy became more prominent and lentoids were formed. This represents the first report of intraretinal lentoids in birds in vivo. The immunohistochemical data suggest that they arise from Müller cells. Association of lentoids with retinal damage supports the assertion that they arise following disruption of normal cell-cell communication.

Adjuvant therapy for osteosarcoma in dogs: results of randomized clinical trials using combined liposome-encapsulated muramyl tripeptide and cisplatin.

Two randomized, double-blind clinical trials in dogs with spontaneous appendicular osteosarcoma treated with combination chemoimmunotherapy are reported. In both trials, dogs without overt metastasis underwent complete amputation of the affected limb. In trial 1, 40 dogs were treated with cisplatin chemotherapy [(CDDP), 70 mg/m2 i.v. every 28 days x 4]. Following CDDP, dogs without evidence of overt metastasis (n = 25) were randomized to receive liposome-encapsulated muramyl tripeptide phosphatidylethanolamine ](L-MTP-PE), 2 mg/m2 i.v.) or placebo liposomes (lipid equivalent) twice weekly for 8 weeks. Of 14 dogs in the placebo group, 13 (93%) died of metastasis; the median survival time was 9.8 months. Of 11 dogs in the L-MTP-PE group, 8 (73%) developed metastasis; the median survival time was 14.4 months, which was significantly longer than that of the placebo group (P < 0.01). In trial 2, 64 dogs received CDDP (70 mg/m2 i.v. every 21 days x 4) and were randomized to concurrently receive L-MTP-PE (2 mg/m2 i.v.) twice or once weekly, or placebo liposomes once weekly for 8 weeks. Median survival times were 10.3, 10.5, and 7.6 months, respectively. There were no significant differences among the three treatment groups in trial 2. Survival times for dogs receiving L-MTP-PE in trial 1 were significantly longer than those for dogs in trial 2 that received four doses of CDDP concurrently with twice weekly L-MTP-PE (P < 0. 04). The results of the first trial confirm our previous observation that L-MTP-PE has antimetastatic activity in dogs with osteosarcoma when given following amputation. The results of the second trial demonstrate that there is no survival advantage of administering L-MTP-PE concurrently with CDDP.

Liposome-encapsulated muramyl tripeptide phosphatidylethanolamine adjuvant immunotherapy for splenic hemangiosarcoma in the dog: a randomized multi-institutional clinical trial.

Canine splenic hemangiosarcoma (HSA) is a spontaneous tumor with high metastatic potential. Despite surgical excision, most dogs die within 2 months of diagnosis as a result of widespread visceral metastasis. This study was designed to determine the efficacy of liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) when used in combination with splenectomy and systemic chemotherapy for the treatment of HSA in the dog. Thirty-two dogs with HSA and without gross evidence of metastases were treated with splenectomy, stratified by clinical stage, and randomized to receive doxorubicin/cyclophosphamide chemotherapy and either L-MTP-PE immunotherapy or lipid equivalent (placebo liposomes). Dogs were subsequently followed to determine disease-free survival and overall survival times. The effects of L-MTP-PE on serum tumor necrosis factor-alpha and interleukin 6 activity were assessed on a small subset of dogs. Dogs receiving L-MTP-PE had significantly prolonged disease-free survival (P = 0.037) and overall survival (P = 0.029) compared with dogs receiving placebo. Dogs with clinical stage I disease had significantly prolonged disease-free survival (P = 0. 026) and overall survival (P = 0.017) compared with dogs with clinical stage II disease. Dogs receiving L-MTP-PE had significantly greater serum tumor necrosis factor-alpha (P < 0.001) and interleukin 6 (P = 0.007) activities compared with placebo-treated dogs. L-MTP-PE has significant antimetastatic activity in highly malignant, spontaneously occurring, splenic HSA in the dog. Canine HSA may have potential as a large animal model for additional investigation of antimetastatic chemoimmunotherapy.

Adjuvant therapy for melanoma in dogs: results of randomized clinical trials using surgery, liposome-encapsulated muramyl tripeptide, and granulocyte macrophage colony-stimulating factor.

Spontaneous canine oral melanoma (COM) is a highly metastatic cancer, resistant to chemotherapy, and can serve as a model for cancer immunotherapy. Liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (L-MTP-PE) can activate the tumoricidal activity of the monocyte-macrophage system following i.v. injection. The objective of these studies was to evaluate the therapeutic effectiveness of L-MTP-PE administered alone and combined with recombinant canine granulocyte macrophage colony-stimulating factor (rcGM-CSF) in dogs undergoing surgery for oral melanoma. Ninety-eight dogs with histologically confirmed, clinically staged, oral melanoma were entered into two randomized, double-blind, surgical adjuvant trials. In trial 1, 50 dogs were stratified based on clinical stage and randomized to once a week L-MTP-PE or lipid equivalent (control). When all of the clinical stages were combined, no difference in disease-free survival or in survival time (ST) were detected. However, within stage I, dogs receiving L-MTP-PE had a significant increase in ST compared with control, with 80% of the dogs treated with L-MTP-PE still alive at >2 years. Within each stage II and stage III, there was no difference detected between the treatment groups. In trial 2, 48 dogs were stratified on the basis of clinical stage and extent of surgery (simple resection or radical excision), treated with L-MTP-PE two times a week, and randomized to rcGM-CSF or saline (placebo) given s.c. daily for 9 weeks. Within each stage and when all of the stages were combined, there was no difference between the treatment groups. In both studies, stage I COM is associated with a better prognosis. No effect on survival was observed with regard to tumor location in the oral cavity, sex, type/extent of surgery, or age. In a subset of dogs tested, pulmonary alveolar macrophage cytotoxicity was enhanced with combined rcGM-CSF and L-MTP-PE but not in dogs treated with L-MTP-PE alone. The present study indicates that after surgery, L-MTP-PE administered alone or combined with rcGM-CSF showed no significant antitumor activity in treating advanced stage COM. In early stage COM, L-MTP-PE was shown to result in a prolongation of ST. Furthermore, this study provides additional rationale for the use of the dog model for human malignant melanoma.

Telomerase activity with concurrent loss of cell cycle regulation in feline post-traumatic ocular sarcomas.

Paraffin wax-embedded ocular globes of cats with post-traumatic ocular sarcomas were examined for the presence of TERT, the active subunit of telomerase. The latter is a ribonucleoprotein complex essential for immortalization and expressed by most malignant tumours, germ line cells, lens epithelial cells, and some stem cells. Due to the frequent loss of cell cycle control with the increased expression of telomerase activity, post-traumatic ocular sarcomas were also examined for loss of p16 expression and alterations in p53, the findings being related to mitotic score, tumour grade, and proliferating cell nuclear antigen. These sarcomas expressed telomerase at a high frequency (62.5%); in addition, the majority showed alterations in cell cycle control, as evaluated by lack of p16 immunolabelling (66.7%). Alterations in p53 were the sole mechanism by which cell cycle control was dysregulated in only two tumours expressing TERT (13%). These findings suggest that p16, and not p53, represents the primary mechanism by which post-traumatic ocular sarcomas that express telomerase activity escape cell cycle control.

Development of human granulocyte-macrophage colony-stimulating factor-transfected tumor cell vaccines for the treatment of spontaneous canine cancer.

Cytokine gene-engineered tumor vaccines are currently an area of intense investigation in both basic research and clinical medicine. Our efforts to utilize tumor vaccines in an immunotherapeutic manner involve canines with spontaneous tumors. We hypothesized that canine tumor cells, transfected with human granulocyte-macrophage colony-stimulating factor (hGM-CSF) cDNA in a plasmid vector, would prove nontoxic following intradermal administration, generate biologically relevant levels of protein, effect local histological changes at the sites of vaccination, and create a systemic antitumor response. Sixteen tumor-bearing dogs were admitted to a study of ex vivo gene therapy. Tumor tissue was surgically removed, enzymatically and mechanically dissociated, irradiated, transfected, and intradermally injected back into the patients. The dogs were vaccinated with primary autologous tumor cells transfected with hGM-CSF or a reporter control gene. hGM-CSF protein was detected (0.07 to 14.15 ng/vaccination site) at 24 hr postinjection and dramatic histological changes were observed, characterized by neutrophil and macrophage infiltration at the sites of injection of hGM-CSF-transfected tumor cells. This was in stark contrast to the lesser neutrophilic and eosinophilic infiltrates found at control vaccination sites. Objective evidence of an antitumor response was observed in three animals. These data, in a large animal translational model of spontaneous tumors, demonstrate in vivo biological activity of hGM-CSF-transfected autologous tumor cell vaccines.

Preclinical development of human granulocyte-macrophage colony-stimulating factor-transfected melanoma cell vaccine using established canine cell lines and normal dogs.

Tumor vaccines and gene therapy have received significant attention as means of increasing cellular and humoral immune responses to cancer. We conducted a pilot study of seven research dogs to determine whether intradermal injection of canine tumor cells transfected via the Accell particle-mediated gene transfer device with the cDNA for human granulocyte-macrophage colony-stimulating factor (hGM-CSF) would generate biologically relevant levels of protein and result in demonstrable histological changes at sites of vaccination. Tumor cell vaccines of 10(7) irradiated canine melanoma cells were nontoxic, safe, and well tolerated. No significant alterations in blood chemistry values or hematological profiles were detected. A histological review of control vaccine sites revealed inflammatory responses predominated by eosinophils, whereas vaccine sites with hGM-CSF-transfected tumor cells had an influx of neutrophils and macrophages. Enzyme-linked immunosorbent assays of skin biopsies from vaccine sites had local hGM-CSF production (8.68-16.82 ng/site of injection) at 24 hours after injection and detectable levels (0.014-0.081 ng/site) for < or =2 weeks following vaccination. Flow cytometric analysis of hGM-CSF-transfected cells demonstrated < or =25% transfection efficiency, and hGM-CSF levels obtained during time-course assays demonstrated biologically relevant levels for both irradiated and nonirradiated samples. These data demonstrate the in vivo biological activity of irradiated hGM-CSF-transfected canine tumor cells and help provide evidence for a valid translational research model of spontaneous tumors.