An hGM‐CSF DNA‐Cationic Lipid Complexed Autologous Tumor Cell Vaccine did not Improve Remission Duration in Dogs with Lymphoma

Introduction:  Lymphoma is one of the most common cancers in dogs and while clinical remission can be induced using chemotherapy, very few dogs are cured. Since cytokine‐adjuvanted autologous whole‐tumor‐cell vaccines (ATCV) can induce potent antitumor immune responses against otherwise non‐immunogenic cancers we initiated a study of such an approach in dogs with lymphoma.
Methods:  Following achievement of a complete remission using a 19‐week CHOP‐based chemotherapy protocol, 51 dogs with B‐cell lymphoma were randomized to receive 8 treatments (4 weekly, then 4 additional at q2wk intervals) of vaccine or lipid‐equivalent placebo. Dogs were followed monthly for assessment of remission duration and survival. Surrogate indices of immune response (delayed‐type hypersensitivity, interferon‐γ quantitative RT‐PCR, lymphocyte proliferation, and flow cytometry for lymphoma‐specific antibodies) were also investigated before and after vaccination.
Results:  No significant difference in median remission duration was observed between dogs receiving vaccine (277 days) or placebo (258 days); the Kaplan‐Meier curves were virtually super‐imposable. No significant differences in surrogate indices of immune response were noted pre‐ and post‐vaccination.
Conclusions:  In this context, an hGM‐CSF DNA‐cationic lipid complexed ATCV vaccine did not enhance remission duration in dogs with lymphoma, likely due to lack of vaccine‐induced tumor‐specific immunity.     

Changes in Regulatory T Cells in Dogs with Cancer and Associations with Tumor Type

Background: Regulatory T cells (Treg) have been shown to suppress antitumor immunity and often are increased in humans and rodents with cancer. However, Tregs have not been well studied in dogs with cancer and it is not known if certain tumor types are associated with increased Tregs.
Hypothesis: We hypothesized that Treg percentages would be increased in dogs with cancer and that Treg percentages would be higher in dogs with certain types of cancer.
Animals: The percentages and numbers of Tregs and nonregulatory T cells and B cells were assessed in 34 dogs with cancer and 9 age-matched control dogs. Dogs evaluated included 14 dogs with sarcoma, 7 dogs with carcinoma, 7 dogs with lymphoma, and 6 dogs with mast cell tumor.
Methods: Numbers and percentages of Tregs, CD4⁺, and CD8⁺ T cells and B cells were determined using flow cytometry and compared between control dogs and dogs with cancer.
Results: The percentage of Tregs was significantly increased overall in dogs with cancer compared with control dogs. When tumor types were compared, Treg percentages were significantly increased in dogs with carcinoma. The Treg/CD8 T cell ratio was significantly higher in dogs with cancer compared with control dogs and was also significantly increased in 2 dogs with T-cell lymphoma.
Conclusions: Treg percentages in blood were increased in dogs with cancer, particularly in dogs with carcinoma. The Treg/CD8 ratio also identified tumor-specific abnormalities in dogs with cancer. These findings indicate that tumor-specific factors may affect Tregs in dogs.     

Decreased Ratio of CD8+ T Cells to Regulatory T Cells Associated with Decreased Survival in Dogs with Osteosarcoma

Background: Increased numbers of regulatory T cells (Treg) and decreased ratios of CD8+ T cells to Treg have been shown to correlate with decreased survival times (ST) in humans with certain malignancies. A possible connection between Treg and ST in dogs with cancer has not been investigated previously. Hypothesis: The purpose of this study was to compare numbers of Treg and T lymphocyte subsets in dogs with osteosarcoma (OSA) to those of healthy dogs and to determine whether pretreatment values were associated with disease‐free interval or with ST. We hypothesized that Treg numbers would be increased in dogs with cancer and that dogs with a high percentage of Treg would have a poorer prognosis. Animals: Twelve client‐owned dogs with appendicular OSA were entered into a prospective clinical trial. Twenty‐two healthy dogs were used as controls. Methods: The percentages and numbers of Treg and CD4+ and CD8+ T cells in blood, lymph nodes, and tumors were determined with flow cytometry and compared between dogs with OSA and control dogs. Results: Dogs with OSA had significantly fewer circulating CD8+ T cells and significantly more Treg compared with healthy dogs. The CD8/Treg ratio also was significantly lower in dogs with OSA compared with control dogs. In dogs with OSA, a decreased CD8/Treg ratio was associated with significantly shorter STs. Conclusions: These data support a role for Treg in the immune control of canine OSA and suggest that determination of the CD8/Treg ratio may be useful for assessing outcomes.     

The Utility of Flow Cytometry for the Diagnosis of Cranial Mediastinal Malignancy in Canine Patients

Introduction:  The most common neoplasms located in the anterior mediastinum in the canine are thymoma and lymphoma. Distinguishing between the two is a diagnostic challenge. Treatment and prognosis for these diseases differs significantly. Thymomas contain a population of normally developing T cells. The majority of these T cells exhibit an immature phenotype, characterized by co‐expression of CD4 and CD8. This phenotype is rarely seen on neoplastic lymphocytes. The purpose of this study was to determine if analysis by flow cytometry could discriminate thymoma from lymphoma based on these cell surface markers.
Methods:  Fine needle aspirates were obtained from ten canine patients with mediastinal masses. Cells were analyzed by flow cytometry using a panel of T and B cell markers.
Results:  Six cases with 10% or greater CD4 + CD8+ cells were diagnosed with thymoma and were confirmed by histopathology. Four cases had fewer than 5% CD4 + CD8+ cells, having lymphocytes expressing CD4 only (3 cases) or CD21, a B cell marker(1 case). These were confirmed as lymphoma by cytology and/or a clonality assay. The sensitivity and specificity of this assay when used in the diagnostic work‐up for suspected thymoma was 100%.
Conclusion:  Flow cytometry may provide important, complementary information in the diagnostic work‐up of the canine patient with a mediastinal mass.     

Efficacy and Toxicity of Intracavitary Administration of Pegylated Liposomal Encapsulated Doxorubicin (DOXIL) in Dogs with Hemangiosarcoma

Introduction:  Canine hemangiosarcoma (HSA) is a fatal malignancy and most dogs die within 6–8 months of diagnosis. The spleen is a common primary site, representing 50% of all cases. These dogs typically present with clinical signs due to tumor rupture and intra‐abdominal dissemination; the abdomen is also the main site of disease progression when these patients fail. Direct delivery of chemotherapy into the abdominal cavity may therefore be a rational approach in this malignancy.
Methods:  14 dogs with stage 2 or 3 splenic HSA were recruited. Doxil at a dose of 1 mg/kg was diluted in saline and administered via ultrasound‐guidance into the abdominal cavity. The dogs were scheduled to receive 4 treatments every 3 weeks. Samples of plasma and abdominal fluid were collected for pharmacokinetic analysis. All dogs were monitored for recurrence and complete necropsies were requested at death.
Results:  8 dogs with stage 3 and 6 dogs with stage 2 HSA were enrolled. All 14 dogs have died, 12/14 due to tumor and 2 from other causes. There was no difference in median survival days between stages (stage 2: 244, stage 3: 125, p = .22). All 12 dogs that died due to tumor‐related causes failed with intra‐abdominal recurrence. Necropsies showed that the dogs in this study had relatively fewer extra‐abdominal metastasis compared to dogs treated with systemic chemotherapy. Pk analysis showed detectable plasma doxorubicin 1 and 2 weeks after treatment.
Conclusion:  Direct abdominal administration of Doxil did not prevent intra‐abdominal recurrence; however, it appeared to provide effective systemic coverage.     

Efficacy of C/B‐OPP rescue for the treatment of relapsed canine lymphoma (1998–2004)

Introduction:  MOPP chemotherapy is useful for relapsed canine lymphoma. This study evaluates the efficacy of this protocol after substitution of CCNU (lomustine) or BiCNU (carmustine) for mechlorethamine (C/B‐OPP).
Methods:  Patient signalment, response to chemotherapy, toxicity and survival data were abstracted from medical records of dogs from receiving C/B‐OPP between 1998 and 2004.
Results:  Fifty‐eight dogs received C/B‐OPP rescue chemotherapy during the study period. The median remission duration after initial chemotherapy, consisting of CHOP‐based therapy in 91% of dogs, was 133 days (range, 10 to 932 days). Thirty‐eight of fifty‐eight dogs (66%) responded to C/B‐OPP rescue after relapse (22 CR, 16 PR), for a median of 48 days (range, 2 to 359 days). Overall, C/B‐OPP extended survival by a median of 90 days (range, 2 to 426 days). Twenty‐four dogs (41%) experienced one or more episodes of Grade II or higher gastrointestinal toxicity. Forty‐one dogs (71%) experienced one or more episodes of Grade II or higher hematologic toxicity. Twelve dogs (20%) developed regenerative anemia with diarrhea consistent with gastrointestinal hemorrhage. Treatment delays due to hematologic toxicity occurred in 37 dogs (63%). There were 16 nonfatal treatment‐related episodes of sepsis requiring hospitalization. 5 dogs died due to sepsis and/or chemotherapy‐related complications.
Conclusions:  C/B‐OPP chemotherapy has activity against relapsed canine lymphoma which is similar to that of traditional MOPP rescue therapy. Moderate to severe hematologic toxicity was observed. Further work is warranted to optimize drug doses and scheduling.     

Effect of age on immune parameters and the immune response of dogs to vaccines: a cross-sectional study

The evaluation of anti-aging intervention strategies in dogs would benefit from reliable quantitative biomarkers of aging. In the present study, the expression of various immune parameters was measured in young and old dogs to identify potential biomarkers of aging. The second goal of the study was to determine the effect of age on the immune response to vaccines. The immune function, including the antibody response to vaccines, was determined in 32 young adult (3.15+/-0.8 years of age) and 33 old dogs (12.1+/-1.3 years of age) of various breeds. Old dogs had a significantly lower lymphocyte proliferative response and a lower percentage of CD4+ T cells and CD45R+/CD4+ T cells, and a higher percentage of CD8+ T cells and a higher concentration of serum and salivary IgA. The most significant differences (P<0.001) occurred in the lymphocyte proliferative responses to ConA and PHA, the CD4:CD8 ratio, and the percentage of CD45R+/CD4+ T cells suggesting that these parameters are potential biomarkers of aging. There was no difference in the percentage of total T and B lymphocytes and the concentration of serum IgM and IgG. Both groups of dogs had protective titers against distemper virus, parvovirus and rabies virus before annual revaccination. The pre-vaccination titer against rabies virus was higher in the old dogs than in the young dogs, and there were no differences in post-vaccination titers against any of the viruses. This suggests that annual vaccination protocols provide adequate protection for old dogs.     

Association of p53 Gene Mutation with the Prognosis of Canine Lymphoid Tumors

Introduction:  Many dogs with lymphoid tumors develop resistance to chemotherapy. As a mechanism of drug resistance in canine lymphoma, ATP‐dependent drug efflux by P‐glycoprotein was reported, however, inhibition of apoptosis mediated by P53 inactivation has not been investigated. In this study, we investigated the relationship between p53 gene mutation and clinical drug resistance in canine lymphoid tumors.
Methods:  Tumor specimens were obtained from 44 dogs with lymphoid tumors. Mutations of p53 gene at exon 4–8 of these tumor tissues were examined by PCR‐SSCP (single strand conformational polymorphism) analysis, followed by nucleotide sequencing of the abnormal bands. The cases were treated with UW‐Madison protocol, and its response was evaluated by the tumor size or the number of peripheral leukemic cells.
Results:  Of the 44 dogs, 15 dogs (34%) had p53 mutation, whereas 29 dogs (66%) were devoid of p53 mutation, before or during the chemotherapeutic protocol. Rate of good response (CR and PR) to chemotherapy was significantly lower in the dogs with p53 mutation (20%) than those without p53 mutation (55%)(p = 0.022). Median overall survival duration after examination of p53 mutation was significantly shorter in dogs with p53 mutation (101days) than those without p53 mutation (223days)(p = 0.008).
Conclusions:  Lymphoid tumors with p53 mutations were shown to have worse prognosis than those without p53 mutation.     

Xenogeneic Human Tyrosinase DNA Vaccination Induces Specific Immune Response in Dogs with Advanced Malignant Melanoma

Introduction:  Xenogeneic melanosomal differentiation antigens, delivered in the form of a plasmid DNA vaccine, can overcome host immune ignorance/tolerance in preclinical animals to melanoma by: 1) generating humoral and cytotoxic T cell responses and 2) inducing protection from tumor challenge. Initial trials of human tyrosinase (huTyr) DNA vaccination of dogs with advanced malignant melanoma (Bergman et al , 2003) demonstrated safety and prolongation of survival with this therapeutic modality. We investigated antigen‐specific immunity in dogs receiving huTyr DNA vaccination.
Methods:  Three cohorts of three dogs each with advanced (WHO stage II‐IV) canine malignant melanoma (CMM) received four biweekly IM injections (dose levels 100, 500, or 1,500 ug, respectively/vaccination) of huTyr plasmid DNA via the Biojector2000 jet delivery device. Sera samples were taken before and after vaccination to detect specific antibody formation to huTyr by Enzyme‐Linked ImmunoSorbent Assays (ELISAs) and flow‐cytometry.
Results:  Three dogs have measurable, 2 to 4 fold huTyr‐specific antibody titers. Preliminary studies by flow‐cytometry have confirmed antibody response to huTyr by positive binding to endogenous human tyrosinase in SK‐Mel188 cells using post‐vaccinate serum.
Conclusions:  Xenogeneic (huTyr) DNA vaccination generates antigen‐specific humoral responses, which may partially explain the previously reported clinical efficacy and anti‐tumor responses. Ongoing studies include: 1) a comprehensive analysis by flow‐cytometry to detect huTyr‐specific antibodies and 2) a quantitative measure of potential cytotoxic T‐cell responses in these dogs by the DNA vaccination.     

A Candidate Second‐Generation Photosensitizer for Photodynamic Therapy in Veterinary Medicine

Introduction:  Photodynamic therapy (PDT) involves the light activation of a drug within a tumor causing selective tumor cell death. Unfortunately, some photosensitizing drugs have been associated with adverse reactions in veterinary patients. Zinc phthalocyanine tetrasulfonate (ZnPcS4) is a promising second‐generation photosensitizer for use in veterinary medicine, however, it cannot be applied clinically until safety and efficacy data are available.
Methods:  Increasing intraperitoneal doses of ZnPcS4 were given to Swiss Webster mice to assess acute toxicity. Based on mouse toxicity data, a phase I clinical trial of ZnPcS4‐based PDT in tumor‐bearing dogs was designed, using an accelerated titration scheme starting at 0.5% of the minimum toxic dose in mice. 24‐hours after ZnPcS4 administration tumors were irradiated with 675 nm light and dogs were evaluated by routine hematology and serum biochemistry at regular intervals after PDT.
Results:  Doses >125 mg/kg were associated with acute toxicity and mortality in Swiss Webster mice, suggesting the minimum toxic dose is 120–125 mg/kg. One dog, a Golden retriever with a massive malignant fibrous histiocytoma, has been entered into the phase I clinical trial. No deleterious effects were noted after ZnPcS4 administration. Within 48 hours of PDT, the tumor was dark and necrotic, with no grossly visible changes to the surrounding normal tissues. Histological examination of the PDT‐treated tumor confirmed widespread necrosis and thrombosis consistent with PDT‐mediated damage. The owner reported no adverse effects after treatment.
Conclusions:  Although preliminary data are encouraging, additional evaluation of ZnPcS4‐based PDT is required to determine its role in veterinary medicine.     

Results of the Combined Treatment with Piroxicam and Carboplatin in Canine Oral Non‐Tonsillar Squamous Cell Carcinoma

Introduction:  Over‐expression of COX‐2 has been observed in several human and animal malignancies and is implicated in carcinogenesis through the conversion of arachidonic acid to PGE‐2. Use of platinum‐containing cytostatic agents and/or (non‐)specific COX‐2 inhibitors, has been reported as a treatment option for canine oral non‐tonsillar squamous cell carcinomas (ONT‐SCC). However, no study describes the effect of a combination of carboplatin and piroxicam on this tumor type.
Methods:  7 dogs with a T3 (WHO‐TNM) ONT‐SCC were treated with piroxicam and carboplatin. Five had bone involvement and no detectable metastasis. Two dogs without bone involvement had metastasis in the regional lymph nodes. Piroxicam was given orally 0.3 mg/kg s.i.d. Each dog was scheduled to receive between 6 and 12 carboplatin infusions (300 mg/m² i.v.) at 3 week intervals. Ondansetron and metoclopramide were used as anti‐emetic agents. The dogs are planned to receive piroxicam on a lifelong basis.
Results:  Complete response (CR) without adjuvant surgery was achieved in 4 of the 7 dogs. Two dogs needed adjuvant surgery to achieve CR. One dog had progressive disease and was euthanised 231 days after start of therapy. All the others were still alive and in CR at date of analysis. Median follow‐up was 335 days (107–689 days).
Conclusions:  Our study suggests that a combination of piroxicam and carboplatin is a useful treatment option for canine ONT‐SCC. All dogs tolerated therapy well and the 57% response rate for reaching a complete and durable remission without adjuvant surgery is promising.     

Induction of remission results in spontaneous enhancement of anti-tumor cytotoxic T-lymphocyte activity in dogs with B cell lymphoma

Characterization of the tumor microenvironment, particularly the immune cells that infiltrate tumors, provides important predictive and prognostic information in humans with lymphoma and other types of cancer. Tumor associated T lymphocytes have not been previously described in dogs with lymphoma. Therefore, we investigated the phenotype and function of T cells in the lymph nodes of dogs with B cell Non-Hodgkin's lymphoma (NHL), as well as the function of T cells in circulation of these dogs. We found that CD4+ and CD8+ T lymphocytes were few in number and minimally responsive to mitogenic stimuli compared to T cells in lymph nodes of normal dogs. Additionally, regulatory T cells (Treg) were significantly increased in tumor tissues compared to lymph nodes of healthy dogs. To better understand cell mediated antitumor immune responses we developed a non-radioactive assay to measure cytotoxic T lymphocyte (CTL) mediated killing of autologous tumor cells. Using this assay, we found that spontaneous CTL activity in the blood of dogs with lymphoma improved significantly following induction of tumor remission using doxorubicin. Coincident with the improvement in CTL activity, circulating Treg numbers were significantly decreased compared to pretreatment levels. We conclude from these studies that CTL activity in dogs with lymphoma can be significantly improved following induction of tumor remission using chemotherapy, as assessed using a new non-radioactive CTL assay.     

Coarse Fractionated Radiation Therapy for Pituitary Tumours in Cats: A Retrospective Study of 10 Cases

Introduction:  Pituitary tumours are uncommon in cats. Signs may be due to either an expansile mass or paraneoplastic effects (acromegaly and/or unstable diabetes mellitus). There are a few small case series providing evidence of efficacy for radiotherapy of pituitary tumours in cats. This retrospective study describes the outcome of ten cats with pituitary tumours treated with course‐fractionated radiation.
Methods:  The medical records of cats with MRI‐confirmed pituitary tumours that underwent radiotherapy were reviewed. A standard coarse‐fractioned radiation protocol was used; 37 Gy in 5 once‐weekly fractions using two parallel‐opposed 4MeV X‐ray beams. Survival times were calculated from date of first radiation dose.
Results:  Ten cats with pituitary tumours underwent radiotherapy. 5 cats had CNS signs and 5 had evidence of growth hormone excess (1 cat also showed CNS signs). 2 cats with pre‐existing moderate to severe CNS signs died of unknown causes before completing the radiation course. Of the remaining 4 with CNS signs, 3 had complete resolution of signs and the fourth showed partial improvement. Of the 5 cats with unstable diabetes, 2 no longer required insulin and 3 became stable at a lowered dose. The median survival time was 77.6 weeks. 6 cats died: 2 without completing the radiation course, 2 from unrelated causes (CRF, VAFS) and 2 from relapse and/or progression of CNS signs. 4 cats remain alive (range 34–191 weeks).
Conclusions:  Radiation therapy is confirmed as an effective treatment for pituitary tumours in cats giving extended survival and control of both direct mass effect and paraneoplastic signs.     

Induction of Chemoresistance by Transfection of the Full‐Length Canine mdr1 Gene in Canine Cell Lines

Introduction:  In the chemotherapy for treatment of lymphoid tumors in dogs, myelosuppression is a frequently encountered dose‐limiting factor. One possible approach to overcome myelosuppression is induction of chemoresistance in hematopoietic stem cells through expression of the mdr1 gene. A full‐length canine mdr1 cDNA clone was isolated in our laboratory. The present study was carried out to assess whether it confers multidrug resistance in canine cell lines.
Materials and methods:  The full‐length canine mdr1 cDNA was inserted into an expression plasmid vector. A canine mammary tumor cell line (CIPP) and osteosarcoma cell line (OOS) were transfected with the canine mdr1 expression vector. Expression of P‐gp was examined by immunoblotting. Function of ATP‐dependent drug efflux was measured by flow cytometric analysis using Rhodamine 123. Sensitivity to chemotherapeutic drugs was shown by estimation of 50% inhibitory concentrations (IC₅₀) of vincristine or doxorubicin.
Results:  Immunoblotting of the transfected cells revealed a strong band of P‐gp detected by a monoclonal antibody directed to P‐gp. Rhodamine 123 efflux test showed an apparent drug efflux activity in the transfected cells. From the IC₅₀ of the chemotherapeutic agents, the transfected cells showed a remarkable increase (20 to 60‐fold) in the resistance to vincristine or doxorubicin.
Conclusion:  Transfection of canine mdr1 gene induced P‐gp expression and strong drug resistance in canine cell lines.     

Lymphocyte subsets in peripheral blood of dogs--a flow cytometric study

Slight differences in the results of papers describing lymphocyte subsets distribution in the peripheral blood of healthy dogs may be explained by differences in monoclonal antibody clones and sources, breed and age of animals examined, methods of sample treatment, or methods of result analysis. In this paper, we described the effect of sample processing and of sample storage as well as the effect of age, breed, and gender of dogs on lymphocyte subset distribution. No significant differences were found between samples processed following a whole-blood lysis method and samples processed after density gradient separation. Furthermore, no significant differences were found between samples processed within 2h after collection and those stored at 4 degrees C for 12-16 h before processing. Age-related changes were evident in lymphocyte subset distribution in the peripheral blood of 38 Beagles divided according to their age into the six groups: (1) 5-6 days; (2) 2 months; (3) 6 months; (4) 1-2 years; (5) 3-5 years; and (6) >5 years. The percentage of B-lymphocytes (CD21-like positive cells) in the peripheral blood of newborn pups was 39.5+/-5.7 and decreased with advancing age. The percentage of CD8+ lymphocytes was 7.7+/-3.4 after birth and increased with advancing age. No age-related changes were observed in the percentages of CD4+ lymphocytes. The CD4+:CD8+ ratio decreased with advancing age. No significant age-related change was observed for lymphocytes bearing the gammadelta-TCR. Some breed differences were evident. Adult (1-5-year-old) Beagles, German Shepherds, Dalmatians, and Dachshunds were examined. The percentages of lymphocytes were higher in Beagles and Dachshunds than in Dalmatians and German Shepherds. The highest and the lowest absolute lymphocyte counts were found in Beagles and German Shepherds, respectively. As a consequence, German Shepherds showed the lowest absolute counts of the individual lymphocyte subpopulations and the widest neutrophil:lymphocyte ratio. Dalmatians showed the lowest percentage of CD3+ cells, the highest percentage of CD21+ cells, and the lowest CD4+:CD8+ ratio. German Shepherds showed the lowest percentage of CD21+ cells and the highest CD4+:CD8+ ratio. Females in Beagles and Dachshuns had nonsignificantly higher percentages of total lymphocytes, CD3+, CD4+, and nonsignificantly lower percentages of CD21+ lymphocytes. We concluded that there are age-, breed-, and perhaps also gender-related differences in lymphocyte subset distribution in the peripheral blood of dogs. Therefore, there is need to use appropriate control group in the experimental protocols. Among-breed differences could explain, at least partly, breed predisposition for some diseases.     

Quantitation of canine regulatory T cell populations, serum interleukin-10 and allergen-specific IgE concentrations in healthy control dogs and canine atopic dermatitis patients receiving allergen-specific immunotherapy

Canine atopic dermatitis (AD) shares many clinical and immunological similarities with human AD. Regulatory T cells (Treg) are a distinct lineage of T lymphocytes with various immunosuppressive properties including the down-regulation of allergic inflammation associated with IgE production. Antigen-induced Treg typically regulate immune homeostasis via productions of cytokines such as interleukin-10. Given the immunological similarities with human AD, it is likely that Tregs and the cytokines they produce play an important role in diseases of dogs as well. A cross-reactive FoxP3 antibody was used to identify a subset of CD4(+) T cells in the blood of both healthy dogs and dogs with atopic dermatitis undergoing immunotherapy over a year period. There was no significant difference in the Treg percentage over time in the healthy dogs. The immunotherapy group showed a significant increase in Treg percentage at 6, 9, and 12 months when compared to the healthy dogs. For the immunotherapy group, the mean Treg percentage at the beginning of the study was 4.94+/-0.71 and 10.86+/-2.73 at the completion. A commercially available ELISA kit was also used to quantitate the concentration of IL-10 in the serum of the same subsets of dogs. There was no significant difference in the IL-10 concentrations over time in the healthy dogs. The immunotherapy group showed a significant increase in serum IL-10 concentrations at 6, 9, and 12 months when compared to the control group. The mean serum IL-10 concentration at the initiation of immunotherapy was 20.40+/-3.52ngL(-1) and 37.26+/-15.26ngL(-1) at the completion of the study. The immunotherapy group also showed a significant decrease in serum IgE levels over the 1-year treatment period for specific allergens identified during ASIT. We conclude from these studies that similar to humans undergoing immunotherapy, increasing Treg populations likely play a significant role in the success of this particular type of therapy for atopic dermatitis and other allergic conditions.     

Antitumor Activity of Mycobacterial Cell Wall‐DNA Complex (MCC) Against Canine Urinary Bladder Transitional Cell Carcinoma Cells

Introduction:  Mycobacterial cell wall‐DNA complex (MCC) is a bifunctional anticancer agent that induces cancer cell apoptosis and stimulates cytokine synthesis by immune cells. Intravesical MCC is currently being evaluated in humans with high‐grade urinary bladder cancer. Evaluation of MCC in dogs with transitional cell carcinoma (TCC) will allow mechanistic studies in a natural animal model of TCC, and a potentially beneficial therapy for dogs with this cancer. In this study, we have determined the anticancer activity of MCC against canine TCC cells in vitro .
Methods:  Canine TCC cells (K9TCC cell line) were incubated with MCC (0.05–100 μg/ml, 0.5–72 hours). Cellular proliferation was measured by MTT reduction. Cell cycle was analyzed by flow cytometry with propidium iodide. Apoptosis was identified by flow cytometry using anti‐active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies. Apoptosis‐inducing activity of 100 μg/ml MCC in combination with piroxicam (0.1–1.0 uM) was evaluated.
Results:  MCC inhibited K9TCC cell proliferation in a concentration‐dependent manner (maximal activity – 45% at 100 μg/ml MCC) in association with the presence of activated caspase‐3 and cleaved PARP. Inhibition of proliferation and apoptosis‐inducing activities of MCC were independent of cell cycle phase. A thirty‐minute exposure of MCC was sufficient for optimal activity. Piroxicam (0.5 uM) enhanced apoptosis‐inducing activity of MCC.
Conclusions:  MCC induces apoptosis in K9TCC cells. This activity is potentiated by piroxicam. Following positive results in vitro , in vivo studies have been initiated. One dog, treated to date, has had a minor reduction in tumor volume following the first course of treatment with no treatment‐related toxicity.     

Proliferative responses to canine thyroglobulin of peripheral blood mononuclear cells from hypothyroid dogs

The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 microg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4+ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4+ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4+ T cells may play an important role in the development of canine hypothyroidism.     

Long‐Term Totally Implantable Venous Access Ports in Dogs and Cats Receiving Chemotherapy

Introduction:  We evaluated the totally implantable subcutaneous vascular access port (VAP) in 16 cancer patients undergoing intermittent chemotherapy for more than 30 months.
Methods:  Ports were surgically placed (The CompanionPort, Norfolk Vet Products, Skokie, Illinois 60076) in the jugular vein of 12 dogs and 4 cats between 1/2002 and 7/2004. Body weight determined polyurethane catheter size (4, 5, 7 fr.). The polysulfone port, surrounded by titanium, was anchored to subcutaneous tissue in the dorsolateral neck and confirmed with C‐arm fluoroscopy. All blood samples were obtained via VAP. Nine anticancer agents, other medications, crystalloids/colloids, and whole blood were administered. Ports were flushed every 4–5 weeks with heparinized saline solution (100 IU/ml). Removed catheters were submitted for bacteriology.
Results:  Seven of 16 animals are still alive. VAP were used for 1.5 to more than 30 months with 4–60 injections/port. Catheter tips were visualized from the left atrium distally into the caudal vena cava. Adverse events included post‐operative subcutaneous bruising and/or hematoma (4/16), difficult aspiration (4/16), catheter malposition (1/16), positional flushing (1/16), and occlusion requiring replacement (1/16). No thrombus formation or extravasation was evident. Bacterial colonization without signs of septicemia was observed in 3/4 catheters.
Conclusions:  VAP are an effective way of achieving long‐term venous access in the dog and cat. Complications are typically minor and infrequent.     

Altered surface antigen expression on peripheral blood mononuclear cells in cats infected with feline immunodeficiency virus.

Expression of CD4, CD8, IL-2 receptor alpha chain (IL-2R alpha), and MHC class II (MHC-II) on peripheral blood mononuclear cells were examined in cats infected with feline immunodeficiency virus (FIV). CD4/CD8 T cell ratio in FIV-infected cats was slightly decreased, as compared with that in specific-pathogen-free (SPF) cats. However, there was no statistical differences between them. The number of circulating IL-2R alpha+ cells in FIV-infected cats was higher than that in healthy cats, whereas induction of IL-2R alpha expression by concanavalin A (Con A) stimulation was depressed in FIV-infected cats. By using two-color cytofluorometry, Con A-induced enhancement of IL-2R alpha expression was found to be reduced in both CD4+ and CD8+ populations in PBMC from FIV-infected cats. The circulating MHC-II+ cells were also increased in FIV-infected cats. Furthermore, the induction of IL-2R alpha expression on PBMC after Con A-stimulation significantly depressed by FIV inoculation in vitro. These results suggest that FIV activates PBMC in vivo via direct and/or indirect mechanisms, leading to the unresponsive state of T cells to further stimuli in vitro.