Marcia L. Padilla

Canine malignant hemangiosarcoma as a model of primitive angiogenic endothelium

Hemangiosarcoma (HSA) is a common untreatable cancer of dogs that resembles human angiosarcoma. Detailed studies of these diseases have been historically hindered by the paucity of suitable reagents. Here, we show that expression of CD117 (c-Kit) can distinguish primitive (malignant) from mature (benign) proliferative endothelial lesions, and we describe eight independent cell lines derived from canine HSA explants. Endothelial origin was confirmed by sustained expression of surface CD105 (endoglin), CD146 (MUC18), and CD51/CD61 (αvβ3 integrin). The cell lines showed anchorage-independent growth and were motile and invasive when cultured on a basement membrane matrix. They required endothelial growth factors for growth and survival, and they could be induced to form tubular structures resembling blood vessels when cultured under low calcium conditions. The formation of vessel-like structures was blocked by nicotine, and restored by FK506, suggesting that ‘nuclear factor of activated T cells’ activity prevents differentiation of these cells. In summary, these cell lines represent a unique and novel resource to improve our understanding of endothelial cell biology in general and canine HSA in particular.

Upregulation of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cultures by lipopolysaccharide, muramyl tripeptide, and interleukin-1

The tryptophan decyclizing enzyme indoleamine 2,3-dioxygenase (IDO) was induced in human monocyte-derived macrophages (MDM) treated with human recombinant interferon-beta (IFN-beta) or interferon-gamma (IFN-gamma). Treated cells exhibited dose-dependent increases in IDO when assayed 48 hr after treatment. Cells exposed to IFN-gamma were observed to exhibit consistently higher peak levels of IDO when compared with cells incubated in the presence of IFN-beta. When IFN-beta-treated cells were incubated in the presence of specified amounts of bacterial lipopolysaccharide (LPS) or liposome-encapsulated muramyl tripeptide (MTP), peak IDO activity increased such that enzyme activity was comparable to maximal activity observed with IFN-gamma-treated cells. LPS and MTP also upregulated IFN-gamma-mediated IDO activity when suboptimal amounts of IFN-gamma were used. When macrophages were costimulated with various concentrations of human recombinant interleukin 1 alpha (IL-1 alpha), along with either maximum-stimulating amounts of IFN-beta or suboptimal amounts of IFN-gamma, IDO activity was upregulated in a manner similar to results obtained using the microbial products as stimuli. While neither IL-1 alpha or IL-1 beta was detected in culture supernatants from macrophages treated with either LPS or MTP (alone or in combination with IFN), IL-1 alpha was detected in cell lysates of macrophages treated with these upregulators. Although neutralizing antibody to IL-1 alpha abolished the upregulatory effect of exogenous IL-1 alpha, it had no effect on upregulation by LPS or MTP. This suggests that although LPS and MTP may induce production of cell-associated IL-1 alpha, upregulation of IDO activity by these agents is independent of IL-1 alpha production and may be mediated through distinct pathways.

Factors affecting the transport of β-amino acids in rat renal brush-border membrane vesicles. The role of external chloride

The effect of a variety of ions and other solutes on the accumulation of the beta-amino acid, taurine, was examined in rat renal brush-border membrane vesicles. Initial taurine uptake (15 and 30 s) is sodium-dependent with a typical overshoot. This Na+ effect was confirmed by exchange diffusion and gramicidin inhibition of taurine uptake. External K+ or Li+ do not increase taurine accumulation more than Na+-free mannitol, except that the combination of external K+ and Na+ in the presence of nigericin enhances uptake. Of all anions tested, including more permeant (SCN- and NO3-) or less permeant (SO4(2-)), chloride supported taurine accumulation to a significantly greater degree. Preloading vesicles with choline chloride reduced taurine uptake, suggesting that external Cl- stimulates uptake. Since this choline effect could be related to volume change, due to the slow diffusion of choline into vesicles, brush-border membrane vesicles were pre-incubated with LiCl, LiNO3 and LiSO4. Internal LiCl, regardless of the final Na+ anion mixture, reduced initial rate (15 and 60 s) and peak (360 s) taurine uptake. Internal LiNO3 or LiSO4 with external NaCl resulted in similar or higher values of uptake at 15, 60 and 360 s, indicating a role for external Cl- in taurine uptake in addition to Na+ effect. Although uptake by vesicles is greatest at pH 8.0 and inhibited at acidic pH values (pH less than 7.0), an externally directed H+ gradient does not influence uptake. Similarly, amiloride, an inhibitor of the Na+/H+ antiporter, had no influence on taurine accumulation over a wide variety of concentrations or at low Na+ concentrations. Taurine uptake is blocked only by other beta-amino acids and in a competitive fashion. D-Glucose and p-aminohippurate at high concentrations (greater than 10(-3) M) reduce taurine uptake, possibly by competing for sodium ions, although gramicidin added in the presence of D-glucose inhibits taurine uptake even further. These studies more clearly define the nature of the renal beta-amino acid transport system in brush-border vesicles and indicate a role for external Cl- in this uptake system.

Enhancing antimelanoma immune responses through apoptosis

We examined the feasibility of using tumor apoptosis at accessible sites to enhance antimelanoma immune responses in a model of spontaneous canine melanoma. We show that priming peripheral blood mononuclear cells with apoptotic melanoma cells significantly enhanced autologous and allogeneic lymphokine-activated killing of tumor cells. Since various pathways required for intrinsic apoptosis are often inactivated in melanoma, we used Fas ligand (FasL) overexpression to promote extrinsic apoptosis. FasL induced apoptosis in five of six cell lines. Each of the susceptible lines, but not the resistant one, expressed Fas mRNA. In addition, direct intratumoral administration of FasL DNA to tumor-bearing dogs was safe, with no adverse events reported over 7 days of observation. A reduction of tumor burden was seen in three of five dogs treated. The reduction of tumor volume was correlated with Fas expression by the tumors, although one dog with a Fas-negative tumor survived for 82 weeks after treatment. Our data show that overexpression of FasL is suitable to promote apoptosis of Fas+ melanomas, and support the notion that priming immune responder cells with apoptotic tumor cells may enhance antitumor responses. The results also suggest that intratumoral administration of FasL offers a safe route for therapeutic gene delivery.

Developmental aspects of renal beta-amino acid transport. V: Brush border membrane transport in nursing animals--effect of age and diet.

ABSTRACT. This study examines the Na+-dependent accumulation of the β-amino acid, taurine, by brush border membrane vesicles isolated from nursing animals compared to uptake in adult animals. The diets fed to the mothers nursing these pups were altered so as to provide a low sulfur amino acid intake or a high taurine diet as well as conventional sulfur amino acid intake. Taurinuria is greater in nursing animals than in adult controls, but animals of all ages respond to exposure to the low sulfur amino acid intake by conservation of taurine and to the high taurine diet by hyperexcretion of taurine. Taurine uptake at 10 μM by brush border membrane vesicles is influenced by age in all groups and by diet in 14− and 21-day-old animals. A precession of uptake is seen both in terms of initial and peak rate of uptake with the lowest values in 7-day-old animals to the highest in adult. Greater brush border membrane vesicle uptake is found in 14− and 21-day-old rats after exposure to the low sulfur amino acid intake and reduced uptake after the HTD, whereas no dietary influence on uptake was found in 7-day-old rats. Neither the pattern of the time course of uptake nor the uptake values at equilibrium (45 min) are affected by age or diet. Kinetic analyses of concentration dependent uptake show that the maturational process involves a change in the Vmax of initial uptake. Kinetic analysis of the adaptive response reveals an increase in Vmax after low sulfur amino acid intake and a decline after high taurine diet in 14− and 21-day-old pups, but not in 7-day-old pups. Uptakes at high taurine concentrations (5 mM) which are 10-fold higher than the Km are uninfluenced by age or diet. This study indicates that the physiologic taurinuria of immature rats may relate, in part, to a lower rate of uptake at the brush border surface, but that after 1 wk and before 2 wk of age the kidney can adapt to changes in sulfur amino acid intake.

Immunostimulatory effects of human recombinant interleukin-12 on peripheral blood mononuclear cells from normal dogs

Interleukin-12 (IL-12) plays a pivotal role in regulating cellular immune responses involving autoimmunity, infectious disease, and cancer. Human recombinant (hr) IL-12 is being evaluated for therapy of human cancer. We investigated the potential of hrIL-12 to activate canine peripheral blood mononuclear cells (PBMC) using proliferation and cytotoxicity as readouts. Human rIL-12 caused increased proliferation of PBMC, and enhanced lysis of allogeneic canine tumor targets mediated by PBMC from normal dogs in vitro. In addition, antibody-dependent cellular cytotoxicity (ADCC) mediated by canine PBMC was enhanced by hrIL-12. These results indicate that hrIL-12 is recognized by canine immune cells, triggering a number of immune responses in canine PBMC, that may be important for immunotherapy of canine cancer. Information from this investigation provides impetus for evaluation of the effects of hrIL-12 on PBMC from tumor-bearing dogs and should be helpful in the development of hrIL-12 as an immune cell activator in vivo in the dog.

Potential to target dysregulated interleukin-2 receptor expression in canine lymphoid and hematopoietic malignancies as a model for human cancer.

Lymphohematopoietic malignancies are common spontaneous diseases of dogs whose clinical presentation and biologic behavior closely resemble their human counterparts. The goal of this study was to define the potential to use canine lymphoma and leukemia as suitable models to refine therapeutic approaches targeting the interleukin-2 receptor (IL-2R). The authors evaluated the patterns of IL-2R expression in 13 dogs with multicentric non-Hodgkins lymphoma (NHL) and in six dogs with leukemia (acute lymphocytic leukemia, n = 3; chronic lymphocytic leukemia in blast crisis, n = 1; acute monoblastic leukemia, n = 2). The authors first cloned and sequenced the complete coding domains of the wild-type canine IL-2R &agr;-chain gene. They next used qualitative reverse transcription polymerase chain reaction (RT-PCR) analysis to examine IL-2R &agr;, &bgr;, and &ggr; c subunit expression in the tumors. Messenger RNA (mRNA) for the interleukin-2 receptor &agr;, &bgr;, and &ggr; c subunits that comprise the high-affinity receptor was present in samples from all dogs with NHL. Expression of functional surface IL-2R also was observed flow cytometrically in NHL cells from all four dogs tested. Leukemic cells from one dog with B cell acute lymphocytic leukemia and two dogs with acute monoblastic leukemia expressed mRNA for all three subunits, whereas cells from another dog with B cell leukemia and both dogs with T cell leukemia expressed only mRNA for the &bgr; and &ggr; c subunits that comprise the intermediate-affinity receptor. These results indicate that the IL-2R is commonly expressed in canine lymphohematopoietic malignancies, and support the suitability of this large-animal model to evaluate targeted IL-2R cancer therapy using approaches of interest in the treatment of humans with hemolymphatic cancers.

Antioxidant Status in Hyperthyroid Cats before and after Radioiodine Treatment

BACKGROUND Reversible antioxidant depletion is found in hyperthyroid humans, and antioxidant depletion increases the risk of methimazole toxicosis in rats. OBJECTIVES To determine whether abnormalities in concentrations of blood antioxidants or urinary isoprostanes were present in hyperthyroid cats, and were reversible after radioiodine treatment. To determine whether or not antioxidant abnormalities were associated with idiosyncratic methimazole toxicosis. ANIMALS Hyperthyroid cats presented for radioiodine treatment (n = 44) and healthy mature adult control cats (n = 37). METHODS Prospective, controlled, observational study. Red blood cell glutathione (GSH), plasma ascorbate (AA), plasma free retinol (vitamin A), α-tocopherol (vitamin E), and urinary free 8-isoprostanes in hyperthyroid cats were compared to healthy cats and to hyperthyroid cats 2 months after treatment. RESULTS Blood antioxidants were not significantly different in hyperthyroid cats (mean GSH 1.6 ± 0.3 mM; AA 12.8 ± 4.9 μM, and vitamin E, 25 ± 14 μg/mL) compared to controls (GSH 1.4 ± 0.4 mM; AA 15.0 ± 6.6 μM, and vitamin E, 25 ± 17 μg/mL). Urinary isoprostanes were increased in hyperthyroid cats (292 ± 211 pg/mg creatinine) compared to controls (169 ± 82 pg/mg; P = .006), particularly in hyperthyroid cats with a USG < 1.035. Plasma free vitamin A was higher in hyperthyroid cats (0.54 ± 0.28 μg/mL versus 0.38 ± 0.21 in controls; P = .007). Both abnormalities normalized after radioiodine treatment. No association was found between oxidative status and prior idiosyncratic methimazole toxicosis. CONCLUSION AND CLINICAL IMPORTANCE Increased urinary isoprostane could reflect reversible renal oxidative stress induced by hyperthyroidism, and this requires additional evaluation.