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Feline immunodeficiency virus (FIV) interacts with dendritic cells (DC) during initiation of infection, but whether DC support or transfer FIV infection remains unclear. To address this issue, we studied the susceptibility of feline myeloid DC to FIV infection and assessed potential transfer of infection from DC to CD4+ T cells. FIV was detected in membrane-bound vesicles of DC within 2 h of inoculation, although only low concentrations of FIV DNA were found in virus-exposed isolated DC. Addition of resting CD4+ T cells increased viral DNA levels; however, addition of activated CD4+ T cells resulted in a burst of viral replication manifested by FIV p27 capsid antigen generation. To determine whether transfer of FIV infection required productively infected DC (vs virus bound to DC but not internalized), virus-exposed DC were cultured for 2 days to allow for degradation of uninternalized virus and initiation of infection in the DC, then CD4+ T blasts were added. Infection of T cells remained robust, indicating that T-cell infection is likely to be mediated by de novo viral infection of DC followed by viral transfer during normal DC/T-cell interactions. We conclude that feline DC support restricted FIV infection, which nevertheless is sufficient to efficiently transfer infection to susceptible T cells and trigger the major burst of viral replication. Feline DC/FIV/T-cell interactions (similar to those believed to occur in human immunodeficiency virus and simian immunodeficiency virus infections) highlight the means by which immunodeficiency-inducing lentiviruses exploit normal DC/T-cell interactions to transfer and amplify virus infection.

The authors provide a review of current knowledge of lymphocytosis in nonneoplastic conditions. They conclude that the list of major differentials for persistent nonneoplastic lymphocyte expansion in dogs and cats is short and that most of these conditions are relatively uncommon. Persistent lymphocytosis of small, mature, or reactive lymphocytes is most commonly the result of chronic lymphocytic leukemia or lymphoma. The first step in distinguishing nonneoplastic from neoplastic lymphocytosis is immunophenotyping by flow cytometry to determine the phenotypic diversity of the circulating cells. Clonality testing using the polymerase chain reaction for antigen receptor rearrangements assay is a useful second step in cases in which the phenotype data are equivocal. Once the diagnosis of malignancy has been established, the immunophenotype also provides prognostic information in dogs.

Interleukin-12 (IL-12), a proinflammatory cytokine, shows anticancer properties. Systemically administered IL-12 causes dose-dependent toxicity. To achieve localized intratumoral gene expression, an adenoviral gene therapy vector with IL-12 controlled by a heat-inducible promoter (heat shock promoter 70B) was developed and tested in a phase I clinical trial in cats with spontaneously arising soft tissue sarcoma. A feasibility study was done in 16 cats with soft tissue sarcoma using murine IL-12 and/or enhanced green fluorescent protein adenoviral vectors under cytomegalovirus or heat shock promoter 70 control. Subsequently, we conducted a phase I clinical trial using an adenoviral feline IL-12 construct in 13 cats with soft tissue sarcoma. The soft tissue sarcomas were irradiated (48 Gy/16 fractions) followed by intratumoral injection of adenovirus. Twenty-four hours postinjection, tumors were heated (41°C, 60 min). Tumor expression of feline IL-12 and IFN-γ was determined. Cats were monitored for systemic toxicity. For the murine IL-12 construct, an association was noted between viral dose and murine IL-12 levels within tumor, whereas serum levels were minimal. Mild toxicity was noted at 1011 plaque-forming units (pfu). With the feline IL-12 construct, high levels of feline IL-12 mRNA were detected in tumor biopsies with low or absent IFN-γ mRNA following gene therapy. Hematologic and hepatic toxicities were noted at the highest viral doses and were associated with detection of IFN-γ mRNA in tumor. It is possible to localize gene expression and limit systemic toxicity of IL-12 using the hyperthermia-induced gene therapy approach. The maximum tolerated dose of the feline IL-12 adenoviral vector was 1010 pfu/tumor as dose-limiting toxicities were noted at the 4 × 1010 pfu dose. [Mol Cancer Ther 2007;6(1):380–9]

Interleukin-12 (IL-12) is a pro-inflammatory cytokine possessing anti-cancer and anti-angiogenic properties. This study quantitatively assessed the anti-angiogenic effect of IL-12 delivered using an adenoviral vector with murine IL-12 placed under control of a heat shock promoter. This approach limits systemic toxicity by restricting IL-12 delivery locally to the tumour. The kinetics of the downstream cytokines interferon-gamma (IFN-gamma) and interferon inducible protein-10 (IP-10) and other molecules affecting angiogenesis, vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) were also studied.4T1 tumours were grown in Balb/C mice and the AdhspmIL-12 construct was injected intra-tumourally. The tumours were heated after 24 h using a water bath. At various time points post-heating the tumours were collected and quantitatively assessed for cytokine production and vascularity.A significant reduction was seen in the tumour vasculature of the treated group vs. the control group mice. Systemic effects of IL-12 were limited to generalized immunostimulation. No hepatoxicity was noted.This study suggests that IL-12 can be effectively delivered using a gene-based approach with a heat shock promoter. This results in quantitatively measurable anti-angiogenesis and general immunostimulation. The complex inter-play of other pro- and anti-angiogenic factors (IFN-gamma, IP-10, VEGF and PAI-1) was also studied.

Interleukin-12 (IL-12) is a pro-inflammatory cytokine that has shown great promise as a therapeutic agent in experimental models of infectious disease and cancer. However, it is also a highly toxic molecule and for that reason has not been accepted readily into the clinic. A replication-deficient adenoviral vector was designed to deliver the feline interleukin-12 gene into tumour cells. The interleukin-12 gene has been placed under control of a heat inducible promoter, human heat shock promoter 70b, with the intent of spatially and temporally controlling the expression of IL-12, thus limiting its toxicity. In vitro, the transfection efficiency of the adenoviral vector, the effect of multiplicity of infection and the production of biologically active feline IL-12 were studied in the infected cells in response to a range of temperatures. This adenoviral vector will be a useful tool to examine the effects of intra-tumoural IL-12 delivery in a spontaneously occurring feline soft tissue sarcoma model.

Immunophenotypes in lymphoproliferative diseases (LPD) are prognostically significant, yet causative factors for these conditions, and specifically those associated with heritable risk, remain elusive. The full spectrum of LPD seen in humans occurs in dogs, but the incidence and lifetime risk of naturally occurring LPD differs among dog breeds. Taking advantage of the limited genetic heterogeneity that exists within dog breeds, we tested the hypothesis that the prevalence of LPD immunophenotypes would differ among different breeds. The sample population included 1,263 dogs representing 87 breeds. Immunophenotype was determined by the presence of clonal rearrangements of immunoglobulin heavy chain or T-cell receptor γ chain. The probability of observing the number of B-cell or T-cell tumors in a particular breed or breed group was compared with three reference populations. Significance was computed using χ2 test, and logistic regression was used to confirm binomial predictions. The data show that, among 87 breeds tested, 15 showed significant differences from the prevalence of LPD immunophenotypes seen across the dog population as a whole. More significantly, elevated risk for T-cell LPD seems to have arisen ancestrally and is retained in related breed groups, whereas increased risk for B-cell disease may stem from different risk factors, or combinations of risk factors, arising during the process of breed derivation and selection. The data show that domestic dogs provide a unique and valuable resource to define factors that mediate risk as well as genes involved in the initiation of B-cell and T-cell LPD.

Although lymphoma and leukemia usually can be diagnosed by routine cytology and histology, some cases present a diagnostic challenge for pathologists and clinicians. Often the dilemma lies in determining whether a population of lymphocytes is reactive or neoplastic. We review currently available methods for analyzing lymphocyte populations by immunophenotyping and by identifying clonally rearranged immunoglobulin and T-cell receptor genes and discuss how these tests can be used to clarify such diagnostic dilemmas. We also describe the detection of chromosomal abnormalities and methods on the horizon, such as gene expression profiling, to identify diagnostically useful oncogenes. Finally, we review the emerging concept of transitional neoplastic states, in which reactive lymphocytes transform to neoplastic lymphocytes in the presence of continued antigenic stimulation, such as that caused by infection with Helicobacter pylori. The existence of transitional neoplastic states underscores the need for an array of molecular diagnostic tools that would improve our ability to characterize lymphocyte populations in human and animal patients and enhance early detection of neoplastic lymphocytes such that eradication of the infectious or inflammatory stimulus could lead to cure. (Vet Clin Pathol. 2004;33:196-207) � 2004 American Society for Veterinary Clinical Pathology