Robert J. Munn

Development of IL-2-independent feline lymphoid cell lines chronically infected with feline immunodeficiency virus : importance for diagnostic reagents and vaccines

Two interleukin 2 (IL-2)-independent feline immunodeficiency virus (FIV) producer cell lines (FL-4 and FL-6) were produced by selecting cells from an IL-2-dependent culture of mixed peripheral blood lymphocytes infected with FIV. The new cell lines have been stable for over 1 year and spontaneously produce FIV with an average reverse transcriptase titer of 300,000 cpm/ml and an average sucrose gradient purified viral protein concentration of 1 mg/l. FIV produced from these cultures is highly infectious in vitro and in vivo. The FL-6 cell line was phenotyped as expressing the feline CD8 and Pan-T antigens, while the FL-4 cell line expressed the CD4, CD8, and Pan-T antigens. Both cell lines, however, express high levels of viral core and envelope proteins. Paraformaldehyde-inactivated whole virus and similarly inactivated whole-cell virus preparations induced a strong antibody response to core and envelope antigens in immunized cats. The establishment of FIV-producing feline IL-2-independent peripheral blood lymphocyte lines should be valuable for the development of FIV-diagnostic reagents and vaccines and also as a model for human acquired immunodeficiency syndrome vaccine development.

A Viral Epizootic in Cultured Populations of Juvenile Goldfish Due to a Putative Herpesvirus Etiology

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Phosphatase and Tensin Homologue Deleted on Chromosome 10 Deficiency Accelerates Tumor Induction in a Mouse Model of ErbB-2 Mammary Tumorigenesis

Loss of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and amplification or elevated expression of ErbB-2 are both involved in human breast cancer. To directly test the importance of these genetic events in mammary tumorigenesis, we have assessed whether mammary-specific disruption of PTEN could cooperate with activation of ErbB-2. Transgenic mice expressing ErbB-2 under the transcriptional control of its endogenous promoter (ErbB-2(KI)) were interbred with mice carrying conditional PTEN alleles and an MMTV/Cre transgene. Loss of one or both PTEN alleles resulted in a dramatic acceleration of mammary tumor onset and an increased occurrence of lung metastases in the ErbB-2(KI) strain. Tumor progression in PTEN-deficient/ErbB-2(KI) strains was associated with elevated ErbB-2 protein levels, which were not due to ErbB-2 amplification or to a dramatic increase in ErbB-2 transcripts. Moreover, the PTEN-deficient/ErbB-2(KI)-derived mouse mammary tumors display striking morphologic heterogeneity in comparison with the homogeneous pathology of the ErbB-2(KI) parental strain. Therefore, inactivation of PTEN would not only have a dramatic effect on ErbB-2-induced mammary tumorigenesis but would also lead to the formation of mammary tumors that, in part, display pathologic and molecular features associated with the basal-like subtype of primary human breast cancer.

Molecular analysis of metastasis in a polyomavirus middle T mouse model: the role of osteopontin

IntroductionIn order to study metastatic disease, we employed the use of two related polyomavirus middle T transgenic mouse tumor transplant models of mammary carcinoma (termed Met and Db) that display significant differences in metastatic potential.MethodsThrough suppression subtractive hybridization coupled to the microarray, we found osteopontin (OPN) to be a highly expressed gene in the tumors of the metastatic mouse model, and a lowly expressed gene in the tumors of the lowly metastatic mouse model. We further analyzed the role of OPN in this model by examining sense and antisense constructs using in vitro and in vivo methods.ResultsWith in vivo metastasis assays, the antisense Met cells showed no metastatic tumor formation to the lungs of recipient mice, while wild-type Met cells, with higher levels of OPN, showed significant amounts of metastasis. The Db cells showed a significantly reduced metastasis rate in the in vivo metastasis assay as compared with the Met cells. Db cells with enforced overexpression of OPN showed elevated levels of OPN but did not demonstrate an increase in the rate of metastasis compared with the wild-type Db cells.ConclusionsWe conclude that OPN is an essential regulator of the metastatic phenotype seen in polyomavirus middle T-induced mammary tumors. Yet OPN expression alone is not sufficient to cause metastasis. These data suggest a link between metastasis and phosphatidylinositol-3-kinase-mediated transcriptional upregulation of OPN, but additional phosphatidylinositol-3-kinase-regulated genes may be essential in precipitating the metastasis phenotype in the polyomavirus middle T model.

Feline Vaccine-associated Fibrosarcoma: An Ultrastructural Study of 20 Tumors (1996–1999)

Twenty feline vaccine-associated sarcomas were examined by transmission electron microscopy. Tumors contained pleomorphic spindle cells, histiocytoid cells, and giant cells. Most tumors contained myofibroblasts, which had morphologic features similar to those of fibroblasts. These cells were further distinguished by subplasmalemmal dense plaques and thin cytoplasmic actin myofilaments organized as elongated bundles concentrated at irregular intervals forming characteristic dense bodies. Intracellular crystalline particulate material was found in 5 of the 20 tumors. Energy dispersive X-ray spectroscopy was used to identify the crystalline material within one tumor as aluminum-based. One tumor from a feline leukemia virus-infected cat contained budding and immature retroviral particles.

In vitro macrophage tropism of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus (SIVmac)

The significance of infection of mononuclear phagocytes by immunodeficiency lentiviruses of primates is not clearly established. To explore the relationship of macrophage tropism and pathogenesis, conditions to culture and infect monocyte-derived macrophages from rhesus macaques were established and the growth properties of two molecular proviral clones of simian immunodeficiency virus (SIVmac) were studied. Rhesus macrophages supported productive infection of the nonpathogenic SIVmac-1A11 clone; extensive cytopathology characterized by formation of multinucleated giant cells and release of particle-associated reverse transcriptase activity in culture supernatant were observed. In contrast, the pathogenic SIVmac-239 did not establish a productive infection of macrophages and no cytopathology was observed. Both SIVmac-1A11 and SIVmac-239 replicated and induced cytopathic effects in cultures of rhesus peripheral blood lymphocytes and the Cemx174 lymphoid cell line. These results, together with the previously published reports on the pathogenic potential of these two clones of SIVmac, suggest that macrophage tropism measured in vitro does not correlate with in vivo virulence.

Epitheliocystis infection in cultured white sturgeon (Acipenser transmontanus): antigenic and ultrastructural similarities of the causative agent to the chlamydiae.

A mild to moderate branchial epitheliocystis infection was diagnosed in subyearling (11 months old, 250–300 g) white sturgeon (Acipenser transmontanus) from a private culture facility with a 4–8% mortality in the population. Infected branchial epithelial cells contained the coccoid to coccobacillary epitheliocystis organisms, which appeared as cytoplasmic inclusions composed of a fine, homogeneous, dense, basophilic, granular material. The infected cells were variably enlarged with spherical to oval profiles and were randomly distributed throughout the branchial epithelium. The cytoplasmic inclusions stained positive with Macchiavello stain but negative with Brown and Brenn, periodic acid-Schiff, and Gimenez stains. Expression of chlamydial antigen was demonstrated within the cytoplasmic inclusions using a standard peroxidase-antiperoxidase immunohistochemical technique. Three stages of coordinated intracellular development were recognized by electron microscopy. The reticulate bodies were oval to spherical and 0.4–0.8 × 0.5–1.4 μm but often exhibited a pleomorphic and convoluted appearance because of variable membrane invaginations and evaginations suggestive of uneven fission and budding. Separate host cells contained intermediate bodies that were spherical to oval and 0.2–0.4 × 0.3–0.6 μm although often observed in the process of apparent uneven division. The presence of a cap or plaque composed of hexagonally arrayed fibrillar surface projections was initially recognized in this stage. A homogeneous population of 0.3–0.4 μmoval elementary bodies were observed separately in individual host cells. This developmental stage had a single, dense, compact, eccentrically located cytoplasmic condensation that occurred opposite to the location of the cap of hexagonally arrayed fibrillar surface projections. Morphologic characteristics of the epitheliocystis organism in these white sturgeon were similar to those previously described in other teleosts and expands the species catalogue of epitheliocystis infection. Furthermore, the ultrastructural similarities to the chlamydiae and the immunohistochemical detection of chlamydial antigen provides further evidence that the epitheliocystis agent is related to members of the Chlamydiales. Although the infection was considered mild to moderate and could not be definitively attributed to the mortality in this population, the potential adverse impact of epitheliocystis infection on sturgeon culture should be considered especially in intensive fish culture operations.

Protective Immunity to SIV Challenge Elicited by Vaccination of Macaques with Multigenic DNA Vaccines Producing Virus-Like Particles

We utilized SIV(mne) infection of Macaca fascicularis to assess the efficacy of DNA vaccination alone, and as a priming agent in combination with subunit protein boosts. All SIV(mne) structural and regulatory genes were expressed using the human cytomegalovirus Immediate Early-1 promoter in plasmids that directed the formation of virus-like particles in vitro. Macaques (n = 4) were immunized intradermally and intramuscularly four times over 36 weeks with 3 mg plasmid DNA. A second group (n = 4) received two DNA priming inoculations followed by two intramuscular boosts consisting of 250 microg recombinant Env gp160 and 250 microg recombinant Gag-Pol particles in MF-59 adjuvant. These regimens elicited modest cellular immunity prior to challenge. Humoral immune responses to Env gp160 were elicited and sustained by both vaccine protocols, and as expected antibody titers were higher in the protein subunit-boosted animals. Neutralizing antibodies prior to challenge were measurable in two of four subunit-boosted macaques. The two vaccine regimens elicited comparable helper T cell responses at the time of challenge. Vaccinees and mock-immunized controls (n = 4) were challenged intrarectally at week 38 with uncloned SIV(mne). Following challenge all macaques became infected, but both vaccine regimens resulted in reduced peak virus loads (p = 0.07) and significantly improved maintenance of peripheral CD4(+) T cell counts postchallenge (p = 0.007, DNA alone and p = 0.01, all vaccinees). There was no significant difference between the two vaccine groups in levels of plasma viremia or maintenance of CD4(+) T cell counts postchallenge.

Comparative pathology of mammary tumorigenesis in transgenic mice

Mammary tumors arise in transgenic mice bearing growth factors, proto-oncogenes, oncogenes and tumor suppressor genes. The tumors arise from hyperplasias. The tumor natural history and histogenesis are oncogene specific. Interactions between oncogenes may impede or accelerate tumorigenesis.

Quantitation of fixative-induced morphologic and antigenic variation in mouse and human breast cancers

Quantitative Image Analysis (QIA) of digitized whole slide images for morphometric parameters and immunohistochemistry of breast cancer antigens was used to evaluate the technical reproducibility, biological variability, and intratumoral heterogeneity in three transplantable mouse mammary tumor models of human breast cancer. The relative preservation of structure and immunogenicity of the three mouse models and three human breast cancers was also compared when fixed with representatives of four distinct classes of fixatives. The three mouse mammary tumor cell models were an ER+/PR+ model (SSM2), a Her2+ model (NDL), and a triple negative model (MET1). The four breast cancer antigens were ER, PR, Her2, and Ki67. The fixatives included examples of (1) strong cross-linkers, (2) weak cross-linkers, (3) coagulants, and (4) combination fixatives. Each parameter was quantitatively analyzed using modified Aperio Technologies ImageScope algorithms. Careful pre-analytical adjustments to the algorithms were required to provide accurate results. The QIA permitted rigorous statistical analysis of results and grading by rank order. The analyses suggested excellent technical reproducibility and confirmed biological heterogeneity within each tumor. The strong cross-linker fixatives, such as formalin, consistently ranked higher than weak cross-linker, coagulant and combination fixatives in both the morphometric and immunohistochemical parameters.