Effect of clinical signs, endocrinopathies, timing of surgery, hyperlipidemia, and hyperbilirubinemia on outcome in dogs with gallbladder mucocele.

Gallbladder mucocele (GBM) is a common extra-hepatic biliary syndrome in dogs with death rates ranging from 7 to 45%. Therefore, the aim of this study was to identify the association of survival with variables that could be utilized to improve clinical decisions. A total of 1194 dogs with a gross and histopathological diagnosis of GBM were included from 41 veterinary referral hospitals in this retrospective study. Dogs with GBM that demonstrated abnormal clinical signs had significantly greater odds of death than subclinical dogs in a univariable analysis (OR, 4.2; 95% CI, 2.14-8.23; P<0.001). The multivariable model indicated that categorical variables including owner recognition of jaundice (OR, 2.12; 95% CI, 1.19-3.77; P=0.011), concurrent hyperadrenocorticism (OR 1.94; 95% CI, 1.08-3.47; P=0.026), and Pomeranian breed (OR, 2.46; 95% CI 1.10-5.50; P=0.029) were associated with increased odds of death, and vomiting was associated with decreased odds of death (OR, 0.48; 95% CI, 0.30-0.72; P=0.001). Continuous variables in the multivariable model, total serum/plasma bilirubin concentration (OR, 1.03; 95% CI, 1.01-1.04; P<0.001) and age (OR, 1.17; 95% CI, 1.08-1.26; P<0.001), were associated with increased odds of death. The clinical utility of total serum/plasma bilirubin concentration as a biomarker to predict death was poor with a sensitivity of 0.61 (95% CI, 0.54-0.69) and a specificity of 0.63 (95% CI, 0.59-0.66). This study identified several prognostic variables in dogs with GBM including total serum/plasma bilirubin concentration, age, clinical signs, concurrent hyperadrenocorticism, and the Pomeranian breed. The presence of hypothyroidism or diabetes mellitus did not impact outcome in this study.

The release of microparticles by apoptotic cells and their effects on macrophages.

Microparticles are small membrane vesicles released from the cell membrane by exogenous budding. To elucidate the interactions of microparticles with macrophages, the effect of microparticles released from Jurkat T cells on RAW 264.7 cells was determined. Microparticles were isolated by differential centrifugation, using FACS analysis with annexin V and cell surface markers for identification. Various inducers of apoptosis increased the release of microparticles from Jurkat cells up to 5-fold. The released microparticles were then cultured with RAW 264.7 cells. As shown by confocal microscopy and FACS analysis, RAW 264.7 macrophages cleared microparticles by phagocytosis. In addition, microparticles induced apoptosis in RAW 264.7 cells in a dose-dependent manner with up to a 5-fold increase of annexin V positive cells and 9-fold increase in caspase 3 activity. Cell proliferation as determined by the MTT test was also reduced. Furthermore, microparticles stimulated the release of microparticles from macrophages. These effects were specific for macrophages, since no apoptosis was observed in NIH 3T3 and L929 cells. These findings indicate that microparticles can induce macrophages to undergo apoptosis, in turn resulting in a further increase of microparticles. The release of microparticles from apoptotic cells may therefore represent a novel amplification loop of cell death.

Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro.

DNA is a nuclear macromolecule that circulates in the blood where its levels can reflect the activity of inflammatory and malignant diseases. While dead and dying cells have usually been considered the source of blood DNA, the mechanisms for its release during apoptosis and necrosis are not well defined. To elucidate DNA release, an in vitro model system was used, assessing DNA in the media of living, apoptotic or necrotic Jurkat and U937 cells. Apoptosis was induced by etoposide, camptothecin or staurosporine, while necrosis was induced by heating at 56 degrees C. DNA release was measured by fluorometry with the dye PicoGreen while the extent of death was measured by fluorescence-activated cell sorter analysis with propidium iodide and annexin. Apoptotic Jurkat cells released significantly more DNA in the media than untreated cells while necrotic cells did not show significant DNA release. U937 cells showed similar findings. Pretreatment of Jurkat cells with z-VAD-fmk, a caspase inhibitor, reduced both apoptosis and DNA release. By gel electrophoresis, extracellular DNA from apoptotic cells showed laddering with low molecular weight fragments. These studies suggest that extracellular release of DNA is a consequence of apoptosis and may account for some of the DNA in the blood.

The role of the macrophage scavenger receptor in immune stimulation by bacterial DNA and synthetic oligonucleotides.

To assess the role of the macrophage scavenger receptor type A (SRA) in immune activation by CpG DNA, cytokine induction and DNA uptake were tested in vitro and in vivo using SRA knockout (SRA-/-) and wild type (WT) mice. As a source of CpG DNA, Escherichia coli DNA (EC DNA) and a 20-mer phosphorothioate oligodeoxynucleotide with two CpG motifs (CpG ODN) were used. In vitro, both EC DNA and the CpG ODN induced dose-dependent increases of interleukin (IL)-12 production by spleen cells and bone-marrow-derived macrophages (BMMPhi) from both SRA-/- and WT mice. The levels of cytokines produced by SRA-/- spleen cells and BMMPhi were similar to those of WT spleen cells and BMMPhi. When injected intravenously with CpG ODN and EC DNA, both SRA-/- and WT mice showed elevated serum levels of IL-12. To investigate further the role of the SRA, flow cytometry and confocal microscopy were performed to examine the uptake of fluorescently labelled oligonucleotides. SRA-/- and WT BMMPhi showed similarity in the extent of uptake and distribution of oligonucleotides as assessed by these two techniques. Together, these findings indicate that, while the SRA may bind DNA, this receptor is not essential for the uptake of CpG DNA or its immunostimulatory activity.

The role of surface ig binding in the activation of human B cells by phosphorothioate oligodeoxynucleotides.

Phosphorothioate oligodeoxynucleotides (sODNs) can induce T-cell-independent polyclonal activation of human B cells by a mechanism that depends on both sequence and back-bone structure. Because matrix-bound as well as soluble sODNs are mitogenic, this stimulation may result from the engagement of surface receptor(s). In order to investigate whether surface immunoglobin (Ig) could be a receptor for sODNs, the interaction of sODNs-fluorescein isothiocyanate (FITC) with Ig-coated beads was examined. sODNs specifically bound to human IgM and IgG. Moreover, binding of sODN to human B cells induced temperature-dependent capping of bound receptors and colocalization of FITC-sODN and IgM into aggregated caps on the surface of human B cells. A role of surface Ig was furthermore shown by observations that antibody-mediated capping of B-cell surface IgM or IgD inhibited subsequent binding of sODNs and that the capacity of sODN to stimulate human B cells was blocked by excess IgM or IgG, by nonstimulatory antibodies to sIgM, as well as by a variety of negatively charged molecules. Together, these results indicate that sODNs engage surface Ig by charge-charge interactions that lead to activation of human B cells.

Inhibition of murine macrophage IL-12 production by natural and synthetic DNA.

DNA is a complex macromolecule whose immunological properties vary with sequence and structure. To determine whether DNA can inhibit immune responses, the effects of mammalian DNA and synthetic phosphodiester (Po) and phosphorothioate (Ps) oligonucleotides (ODNs) on IL-12 production were tested using murine macrophages. With bacterial DNA as a stimulant, calf thymus DNA and human placenta DNA blocked IL-12 production by splenic and bone marrow macrophages. A (dG)(30) Po ODN and all single-base Ps 30 mer ODNs were also effective inhibitors. The Ps ODNs also blocked IL-12 production induced by lipopolysaccharide (LPS) and a stimulatory Ps ODN. With the J774 cell line, single-base Ps ODNs inhibited IL-12 production induced by bacterial DNA, LPS, and a stimulatory Ps ODN. Together, these results indicate that DNA has inhibitory properties, suggesting that mammalian DNA could limit immune activation during inflammation and counteract the effects of bacterial DNA.

The role of cell surface receptors in the activation of human B cells by phosphorothioate oligonucleotides.

Phosphorothioate oligodeoxynucleotides (sODN) containing the CpG motif or TCG repeats induce T cell-independent polyclonal activation of human B cells. To elucidate the mechanism of this response, the role of cell surface receptors was investigated. Sepharose beads coated with stimulatory but not nonstimulatory sODNs induced B cell proliferation comparably with soluble sODNs. The B cell stimulatory activity of Sepharose-bound sODN did not result from free sODN released from the beads since media incubated with coated beads were inactive. Using FITC-labeled sODNs as probes, binding to human B cells could be detected by flow cytometry. Binding was rapid, saturable, initially temperature independent, but with a rapid off-rate. Competition studies indicated that both stimulatory sODNs and minimally stimulatory sODNs bound to the same receptor. By contrast, phosphodiester oligonucleotides with the same nucleotide sequence as sODNs and bacterial DNA inhibited the binding of sODNs to B cells minimally. Charge appeared to contribute to the binding of sODNs to B cells since binding of sODNs was competitively inhibited by negatively charged molecules, including fucoidan, poly I, and polyvinyl sulfate. These data indicate that human B cells bind sODNs by a receptor-mediated mechanism that is necessary but not sufficient for polyclonal activation.

Influence of backbone chemistry on immune activation by synthetic oligonucleotides.

Depending on base sequence, DNA displays immunological activities relevant to the design of novel therapeutic agents. To determine the influence of backbone structure on these activities, we tested a series of synthetic phosphodiester and phosphorothioate oligonucleotides in in vitro cultures of murine spleen cells. These compounds were 30 bases long and consisted of either a single base or an immunostimulatory sequence (AACGTT) flanked on 5' and 3' ends by 12 nucleotides of each base. Cell activation was assessed by both thymidine incorporation and expression of cell surface CD69; production of interleukin-6 and interleukin-12 was used as a measure of cytokine stimulation. In these assays, phosphorothioate oligonucleotides induced much higher levels of proliferation, CD69 expression, and cytokine production than the comparable phosphodiester compounds and had activity at lower concentrations. The sequence for optimal stimulation by phosphorothioates varied among responses, however. For example, whereas compounds containing an immunostimulatory sequence all induced similar levels of proliferation and CD69 expression, cytokine production was greatest with compounds with dA and dT flanks. Furthermore, while single base dG oligonucleotides stimulated proliferation as both phosphodiesters and phosphorothioates, they failed to stimulate cytokine production. Together, these findings indicate that base sequence as well as backbone chemistry influence immune activation by synthetic oligonucleotides, with the effects varying among responses. While suggesting differences in the structure-function relationships of nucleic acids in their immune activities, these findings also raise the possibility of the design of agents with specific patterns of immune modulation.

IgG binding of monoclonal anti-nuclear antibodies from MRL-lpr/lpr mice.

To assess the specificity of anti-nuclear antibodies with cross-reactive rheumatoid factor (RF) activity, monoclonal anti-DNA and anti-Sm antibodies from MRL-lpr/lpr mice were tested for binding to a variety of IgG antigens. These antibodies had all been previously identified as binding heterologous IgG. By ELISA, antibodies in this panel all bound BALB/c myeloma proteins representing the different IgG subclasses, indicating broad reactivity with murine IgG as well as heterologous IgG. The determinant recognized by these antibodies was further investigated using the Fab, F(ab')2 and Fc fragments of both human as well as rabbit origin. All antibodies bound well to fragments as well as intact IgG antigens. These antibodies were further analysed by Western blotting, demonstrating that most bound to both heavy and light chains of human origin. Together, these observations suggest that some anti-nuclear antibodies bind a conserved antigenic determinant present widely on immunoglobulin chains. This determinant may represent a common sequence important in immunoglobulin domain structure.

Establishment and characterization of a cell line (SS78) from a human renal cell carcinoma.

A new cell line, SS78, was established from a primary renal cell carcinoma of a Caucasian male. The tissue was dispersed with collagenase, and viable cells were separated by flotation on a Ficoll-Hypaque gradient. In culture, the SS78 cells retained a distinct epithelial morphology, and no fibroblast like cells were seen. The cultured cells were aneuploid with a modal chromosome number of 80 and had several marker chromosomes. Inoculation of the cultured cells into athymic nude mice caused tumors at the sites of inoculation.

Clastogenic effect of 4-nitroquinoline-1-oxide on swine urothelial cells in culture.

Swine bladder epithelial cells in culture were treated with 4-nitroquinoline-1-oxide (4NQO) for 4 hr with three different concentrations to examine dose-dependent effects. To study time course effects, they were treated with one concentration for 4 hr and then incubated in 4NQO-free medium for three different periods before harvesting. Three different effects were recorded: (i) a suppression of mitotic activity that was dose-dependent and which continued for more than 30 hr posttreatment; (ii) no marked changes in the proportions of different ploidy classes; and (iii) chromatid gaps, breaks, and exchanges that were dose-dependent. This clastogenic effect decreased with increasing time after treatment.

Polyploidy in mammalian urothelial cells.

The mitotic indices and the extent of polyploidy in urothelial cells of baboons, dogs and swine were studied. All three species had very low mitotic activity in vivo but short-term culturing of these cells in vitro stimulated mitosis thus enabling chromosome counts. Tetraploid cells were found in the urothelium of all three species, and higher ploidies also in dog and swine. There were substantial differences in the proportions of diploidy and higher ploidies among the three species and among individuals within each species. Dog urothelial cells were predominantly tetraploid (70%) while more swine cells were diploid (68%). Baboon urothelial cells had only two ploidy classes and 92% were diploid.

Canine urinary bladder epithelial cells: preparation for cell culture by enzyme dispersion.

In a qualitative and quantitative study of enzymic dispersion of cells from the mucosal layer stripped from canine urinary bladder, trypsin was found to be equal or superior to the other enzymes tested for dispersal of urothelial cells specifically. Collagenase or collagenase plus trypsin served to disperse the whole tissue. A procedure for recovering the urothelial cells as a single-cell suspension and establishing them in culture is presented. The morphology, culture behaviour, and chromosome complement of these cells is described.