RESUMEN
Recent discoveries suggest that the critical events leading to the anti-proliferative activity of the IMiD immunomodulatory agents lenalidomide and pomalidomide in multiple myeloma (MM) cells are initiated by Cereblon-dependent ubiquitination and proteasomal degradation of substrate proteins Ikaros (IKZF1) and Aiolos (IKZF3). By performing kinetic analyses, we found that the downregulation or proteasomal degradation of Ikaros and Aiolos led to specific and sequential downregulation of c-Myc followed by IRF4 and subsequent growth inhibition and apoptosis. Notably, to ensure growth inhibition and cell death, sustained downregulation of Ikaros and Aiolos, c-Myc or IRF4 expression was required. In addition, we found that the half-maximal rate, rather than the final extent of Ikaros and Aiolos degradation, correlated to the relative efficacy of growth inhibition by lenalidomide or pomalidomide. Finally, we observed that all four transcription factors were elevated in primary MM samples compared with normal plasma cells. Taken together, our results suggest a functional link between Ikaros and Aiolos, and the pathological dysregulation of c-Myc and IRF4, and provide a new mechanistic understanding of the relative efficacy of lenalidomide and pomalidomide based on the kinetics of substrate degradation and downregulation of their downstream targets.
Asunto(s)
Antineoplásicos/farmacología , Factor de Transcripción Ikaros/metabolismo , Factores Reguladores del Interferón/metabolismo , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Regulación hacia Abajo , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Humanos , Immunoblotting , Inmunohistoquímica , Lenalidomida , Mieloma Múltiple/patología , ARN Interferente Pequeño , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Talidomida/análogos & derivados , Talidomida/farmacologíaRESUMEN
Platelet satellitosis of polymorphonuclear cells is a phenomenon induced or enhanced by the anticoagulant EDTA. In contrast with previously reported studies, the subject in the present case did not demonstrate platelet satellitism but was profoundly pseudothrombocytopenic owing to platelet phagocytosis. Virtually all polymorphonuclear leukocytes and monocytes contained numerous ingested platelets in contrast with previous cases in which phagocytosis was observed only rarely and involved ingestion of single cells. The phenomenon was documented by immunocytochemical staining and transmission electron microscopy. Autoantibodies were detected in EDTA-anticoagulated blood. However, neither platelet antibody nor phagocytosis was present when heparin, acid-citrate dextrose, or citrate was used as an alternative anticoagulant. The antibody was not temperature dependent. Mixing studies showed the transfer of the phagocytosis phenomenon to healthy donors. Although platelet function assays are typically normal in EDTA-dependent platelet satellitism, this subject showed no secondary aggregation wave in response to adenosine diphosphate and depressed adenosine triphosphate release with collagen, adenosine diphosphate, and arachidonic acid.
Asunto(s)
Anticoagulantes/farmacología , Artefactos , Plaquetas/inmunología , Ácido Edético/farmacología , Fagocitosis , Trombocitopenia/etiología , Adulto , Autoanticuerpos/sangre , Plaquetas/ultraestructura , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Directa , Humanos , Inmunohistoquímica , Masculino , Microscopía Electrónica , Monocitos/inmunología , Neutrófilos/inmunología , Neutrófilos/ultraestructura , Agregación Plaquetaria , TemperaturaRESUMEN
Avasimibe, a novel inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), is currently being developed as an antiatherosclerotic agent. The preclinical safety and toxicokinetics of the compound were assessed in beagle dogs in an escalating-dose study and in repeated-dose studies of 2-, 13-, and 52-week duration. Oral (capsule) doses up to 1000 mg/kg b.i.d. were assessed in the escalating dose study and once-a-day doses up to 300 mg/kg, 1000 mg/kg, and 1000 mg/kg were assessed in the 2-, 13-, and 52-week studies, respectively. Avasimibe was found to be a substrate and inducer of hepatic CYP 3A, producing pronounced decreases in plasma drug concentrations subsequent to Day 1. Plasma drug concentrations plateaued markedly at doses above 100 mg/kg. Significant toxicologic findings were restricted to the higher doses (> or =300 mg/kg) and included emesis, fecal consistency changes, salivation, body weight loss, microscopic and clinical pathologic evidence of hepatic toxicity, and red blood cell (RBC) morphology changes. Mortality occurred at 1000 mg/kg due to hepatic toxicity. Toxicity was more closely associated with the exaggerated pharmacodynamic effects of the compound (e.g., marked serum cholesterol decreases) seen at the high doses of avasimibe used in these studies rather than with measures of systemic exposure (Cmax or AUC). Adrenal effects were noted only in the 52-week study and consisted of minimal to mild cortical cytoplasmic vacuolization and fibrosis at doses > or =300 mg/kg, with no change in adrenal weight. In conclusion, avasimibe is an ACAT inhibitor that has minimal adrenal effects in dogs, with dose-limiting toxicity defined by readily monitored and reversible changes in hepatic function.
Asunto(s)
Acetatos/toxicidad , Glándulas Suprarrenales/efectos de los fármacos , Inhibidores Enzimáticos/toxicidad , Hipolipemiantes/toxicidad , Esterol O-Aciltransferasa/antagonistas & inhibidores , Ácidos Sulfónicos/toxicidad , Acetamidas , Acetatos/administración & dosificación , Acetatos/farmacocinética , Administración Oral , Glándulas Suprarrenales/patología , Alanina Transaminasa/sangre , Animales , Área Bajo la Curva , Arteriosclerosis/prevención & control , Colesterol/sangre , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacocinética , Eritrocitos/efectos de los fármacos , Eritrocitos/patología , Femenino , Hipolipemiantes/administración & dosificación , Hipolipemiantes/farmacocinética , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Longevidad/efectos de los fármacos , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Sulfonamidas , Ácidos Sulfónicos/administración & dosificación , Ácidos Sulfónicos/farmacocinética , Pruebas de ToxicidadRESUMEN
Troglitazone, a thiazolidinedione, is a novel agent for the oral treatment of non-insulin-dependent (Type II) diabetes mellitus; it works by increasing cell sensitivity to available insulin. Previous studies have shown that rodents treated with high doses of troglitazone develop increased heart weight and increased interscapular brown fat. This study investigated cellular proliferation in heart and brown fat of troglitazone-treated mice as well as possible interactions with an angiotensin-converting enzyme inhibitor (quinipril). B6C3F1 female mice were treated daily with either vehicle control, 125 mg/kg quinipril, 1,200 mg/kg troglitazone, or troglitazone/quinipril combination per os for up to 14 days. Four days before necropsy, mice were dosed with bromodeoxyuridine (BrdU) using osmotic pumps. Cell proliferation in heart, brown fat, and retroperitoneal white fat was investigated by means of light microscopic anti-BrdU immunolabeling techniques. Immunoelectron microscopy was used to determine the cell phenotypes and cellular distribution of BrdU label in heart and brown fat. Treatment with troglitazone for 2 wk resulted in increased heart and brown fat weights but in decreased white fat weight. Combination treatment with troglitazone and quinipril also resulted in decreased white fat weight compared with controls. Histologically, brown fat adipocytes in troglitazone- and troglitazone/quinipril-treated mice had coalescent lipid vacuoles and increased eosinophilia of the cytoplasm. White fat adipocytes in troglitazone- and troglitazone/quinipril-treated mice had decreased cell size and increased cytoplasmic eosinophilia. BrdU labeling revealed increased cell proliferation in troglitazone-treated hearts after 1 wk but did not reveal increased cell proliferation in quinipril- or troglitazone/quinipril-treated animals. Brown fat BrdU labeling after 1 wk was increased in troglitazone- and troglitazone/quinipril-treated mice. Ultrastructural anti-BrdU immunogold labeling demonstrated that troglitazone-treated heart and brown fat had greater populations of BrdU-labeled cells that were identified as endothelial cells. These results demonstrated that troglitazone-induced increased cardiac weight in mice can be prevented by quinipril and that increased cardiac weight coincides with early increased endothelial cell proliferation.
Asunto(s)
Tejido Adiposo Pardo/efectos de los fármacos , División Celular/efectos de los fármacos , Cromanos/farmacología , Corazón/efectos de los fármacos , Hipoglucemiantes/farmacología , Tetrahidroisoquinolinas , Tiazoles/farmacología , Tiazolidinedionas , Adipocitos/química , Adipocitos/patología , Adipocitos/ultraestructura , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/crecimiento & desarrollo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Peso Corporal/efectos de los fármacos , Bromodesoxiuridina/análisis , Bromodesoxiuridina/metabolismo , Femenino , Inmunohistoquímica , Isoquinolinas/farmacología , Ratones , Ratones Endogámicos , Microscopía Inmunoelectrónica , Miocardio/citología , Miocardio/ultraestructura , Tamaño de los Órganos/efectos de los fármacos , Quinapril , TroglitazonaRESUMEN
An epizootic of subclinical lymphoplasmacytic gastritis occurred in cynomolgus monkeys maintained at our research facility. Gastric pathology data and histologic sections of 63 adolescent monkeys (2.5-3.5 years old) sacrificed during the epizootic were reviewed. Localized to multifocal reddening of the gastric mucosa was noted grossly in 7 of 44 (16%) monkeys harboring Helicobacter pylori, but not in any of 19 monkeys in which these bacteria were not seen. Gastritis, characterized by accentuation of lymphoplasmacytic infiltrates in antral and to a lesser degree cardiac mucosa, occurred in 42 of 63 (67%) monkeys evaluated and in 42 of 44 (93%) monkeys in which H. pylori was observed microscopically. Two monkeys with H. pylori infection had infiltrate scores that overlapped with the upper limit of scores of H. pylori-negative animals. Coincident with accentuated infiltrates were gastric gland epithelial hyperplasia, reduction in mucin content of surface and gland epithelia, and comparatively minor infiltrates of neutrophils in superficial lamina propria and gastric glands. Antral mucosa thickness often exceeded 1.5 to 2 times normal. Antral mucosal erosions occurred in 7 of 44 (16%) monkeys with H. pylori. Argyrophilic bacteria morphologically consistent with H. pylori were present in antral and less commonly cardiac mucosal glands. Intensity of bacterial colonization correlated with lymphoplasmacytic infiltrates (r = 0.754) and hyperplasia (r = 0.700), although responses were quite variable. These bacteria were not detected in fundic mucosa except in instances where parietal cells were substantially depleted in glands coincident with localized increases in lamina propria inflammatory cell infiltrates. Helicobacter heilmannii-like organisms (HHLOs) were present in fundic glands of all 63 monkeys; colonization was often pronounced. Scores for fundic mucosal inflammation did not correlate with presence or intensity of colonization with HHLOs (r = 0.005). Rather, fundic inflammation scores positively correlated with the antral inflammation scores (r = 0.548). Bacteria morphologically, biochemically, and genetically consistent with H. pylori were cultured from gastric mucosal specimens confirming bacterial identification. These findings demonstrate that adolescent cynomolgus monkeys are susceptible to natural infection with H. pylori and develop many morphologic hallmarks of H. pylori-related gastritis in humans.
Asunto(s)
Mucosa Gástrica/patología , Gastritis/veterinaria , Infecciones por Helicobacter/veterinaria , Helicobacter pylori , Helicobacter/aislamiento & purificación , Linfocitos/patología , Enfermedades de los Primates/patología , Animales , Brotes de Enfermedades/veterinaria , Mucosa Gástrica/microbiología , Gastritis/microbiología , Gastritis/patología , Infecciones por Helicobacter/patología , Infecciones por Helicobacter/fisiopatología , Helicobacter pylori/aislamiento & purificación , Hiperplasia , Inflamación , Macaca fascicularis , Neutrófilos/patología , Enfermedades de los Primates/microbiología , Enfermedades de los Primates/fisiopatologíaRESUMEN
Intravenously administered nitro-imidazole radiosensitizer and alkylating anticancer compound CI-1010, designated as (R)-alpha-[[(2-bromoethyl)amino]methyl]-2-nitro-1H-imidazole-1-ethanol monohydrobromide, causes multiorgan toxicity in rodents, including retinal degeneration. This study determined the potential of CI-1010 to induce similar effects in nonhuman primates. One male and 1 female cynomolgus monkey were given single daily doses of CI-1010 intravenously for 5 consecutive days each week for 3 wk. Doses were escalated from 5 mg per kilogram of body weight in week 1 to 40 and 60 mg/kg in week 3. Postdosing emesis occurred in both monkeys at 5 mg/kg, and clinical signs at 40 and 60 mg/kg included more pronounced emesis, reduced food consumption, pallor, weakness, and body weight loss. At study termination, both monkeys had markedly reduced peripheral blood lymphocytes and moderately lowered erythrocyte, hemoglobin, and hematocrit levels, which correlate with a decreased total nucleated bone marrow cell count. At necropsy, the monkeys had pancytic bone marrow hypocellularity, multiorgan lymphoid depletion, pancreatic acinar cell apoptosis, testicular seminiferous tubular degeneration, and bilateral multifocal retinal degeneration involving the photoreceptor and outer nuclear layers. Ultrastructurally, selected inner and outer retinal rod segments were swollen and fragmented, a state associated with cytoplasmic condensation and pyknosis of the outer nuclear cell layer. Thus, CI-1010 induced toxicity of hematopoietic/lymphoid organs, retina, testes, and pancreas in monkeys, findings similar to those of previous studies in rodents.
Asunto(s)
Nitroimidazoles/toxicidad , Profármacos/toxicidad , Fármacos Sensibilizantes a Radiaciones/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Enfermedades de la Médula Ósea/inducido químicamente , Femenino , Enfermedades Hematológicas/inducido químicamente , Macaca fascicularis , Masculino , Enfermedades Pancreáticas/inducido químicamente , Enfermedades de la Retina/inducido químicamente , Enfermedades Testiculares/inducido químicamenteRESUMEN
We examined whether troglitazone and pioglitazone, antidiabetic thiazolidinediones, would directly induce endothelial cell proliferation or influence cytokine-driven proliferation in vitro. Monolayers of Balb/c mouse aortic endothelial cells were treated with troglitazone or pioglitazone in the absence of fetal bovine serum. Endothelial cells also were exposed to varying concentrations of basic fibroblast growth factor (bFGF) or insulin with or without either thiazolidinedione. After 48 h, 3-[4,5-dimethylthiozol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays were performed to quantitate endothelial cell proliferation by using the various treatment regimens. The data demonstrate that the antidiabetic thiazolidinediones troglitazone and pioglitazone negligibly affect direct endothelial cell proliferation in vitro. Furthermore, troglitazone and pioglitazone significantly inhibit bFGF-induced endothelial cell mitogenesis, whereas only high concentrations of troglitazone affect insulin-mediated proliferation.
Asunto(s)
Cromanos/farmacología , Endotelio Vascular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Hipoglucemiantes/farmacología , Insulina/farmacología , Tiazoles/farmacología , Tiazolidinedionas , Animales , División Celular/efectos de los fármacos , Endotelio Vascular/citología , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Pioglitazona , TroglitazonaRESUMEN
The anti-cancer compound CI-1010, designated as (R)-alpha-([(2-bromoethyl)amino]methyl)-2-nitro-1H-imidazole-1-ethanol monohydrobromide, has a proposed dual mechanism of action due to alkylating and radiosensitizing activities. To assess potential toxicity, adult Wistar rats were treated with a single intravenous injection (0, 50, 100, 150, 225, or 350 mg/kg) and necropsied at 4 or 29 days following treatment. In a repeated dose experiment, rats were injected daily (0, 10, 40, or 80 mg/kg; 5 doses/wk) for 3 wk and necropsied at the end of week 3 or 7. CI-1010 induced retinal degeneration by 4 days after a single injection of > or = 225 mg/kg or by 3 wk of repeated injections of > or = 40 mg/kg. The locally extensive to diffuse retinal degeneration involved the photoreceptor and outer nuclear layer. The photoreceptor layer was vacuolated and compressed corresponding to ultrastructural evidence of inner segment swelling and outer segment fragmentation. The outer nuclear layer was thinned due to loss of nuclei and contained numerous pyknotic or karyorrhectic nuclei. These nuclear changes were morphologically consistent with apoptosis and many outer nuclear layer nuclei labeled with in situ TdT-mediated dUTP-digoxigenin nick end labeling (Apoptag). The retinal degeneration was nonreversible, evidenced by increased lesion severity and incidence after CI-1010 was withdrawn for either 25 or 28 days.
Asunto(s)
Ojo/efectos de los fármacos , Nitroimidazoles/toxicidad , Profármacos/toxicidad , Fármacos Sensibilizantes a Radiaciones/toxicidad , Degeneración Retiniana/inducido químicamente , Administración Tópica , Animales , Ojo/patología , Ojo/ultraestructura , Femenino , Masculino , Ratas , Ratas Wistar , Degeneración Retiniana/patologíaRESUMEN
Orally administered dextran sodium sulfate (DSS) produces an acute colitis in rodents. The pathogenesis is unknown but may relate to DSS-mediated toxicity of colonic crypt epithelium and/or DSS-induced inflammation. The purpose of this study was to determine when colonic mucosal inflammation, as indicated by histopathology and intercellular adhesion molecule-1 (ICAM-1) expression, occurs relative to crypt epithelial damage. Groups of eight adult male Wistar rats were administered 5.0% DSS solution in the drinking water for 2-6 days. Clinical signs at 3 days consisted of loose stool, progressing to marked rectal hemorrhage by days 5 and 6 that correlated with marked intraluminal colonic hemorrhage at necropsy. Histological lesions of predominantly the distal colon consisted of multifocal areas of mucosal erosion, reduction in goblet cells, dilated crypts, crypt collapse, increased lamina propria neutrophils, and submucosal edema on days 2 and 3, progressing to locally extensive ulceration and marked mixed inflammatory infiltrates by days 4-6. Enhanced expression of ICAM-1, demonstrated by both immunohistochemical and northern blot analysis, was evident in colonic mucosa as early as day 2, with consistent increases through days 3-6. Results demonstrate that enhanced colonic mucosal endothelial cell ICAM-1 expression is an early event in the inflammatory cascade of DSS-induced colitis.
Asunto(s)
Colitis/inducido químicamente , Colitis/metabolismo , Colon/química , Sulfato de Dextran/toxicidad , Molécula 1 de Adhesión Intercelular/análisis , Enfermedad Aguda , Animales , Northern Blotting , Colitis/fisiopatología , Colon/metabolismo , Colon/patología , Modelos Animales de Enfermedad , Células Epiteliales/química , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica/fisiología , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Mucosa Intestinal/química , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Ratas WistarRESUMEN
Gastric effects of subchronic treatment with the cholecystokinin-B (CCK-B)/gastrin receptor antagonist CI-988 were investigated in cynomolgus monkeys. In preliminary range-finding studies, CI-988 was given orally to 1 monkey per sex for 14 days at doses of 50, 100, 200, and 500 mg/kg/day. Subchronic studies of CI-988 were subsequently conducted using 5 monkeys per sex at doses of 0, 5, 25, and 75 mg/kg for 4 or 13 wk. High-dose monkeys were dosed initially at 100 mg/kg, but the dose was not well tolerated and was decreased to 75 mg/kg after 8 days of treatment. One male monkey at 75 mg/kg was euthanatized in extremis on day 23. In the range-finding study, minimal to moderate, multifocal to diffuse degeneration of gastric glands, primarily in the fundic region, was observed at 100 mg/kg and above, with frank gastric mucosal atrophy occurring at 200 and 500 mg/kg. Minimal to mild gastric gland degeneration was also observed in the subchronic study after 4 wk at 25 and 75 mg/kg, but histopathologic gastric changes were remarkably absent after 13 wk. Mucosal height in the stomach fundus was decreased 19.8% in 75-mg/kg males at week 4, and although gastric mucosa appeared histologically normal after 13 wk, mucosal height remained 28.6% less than that of controls. In females at 75 mg/kg, fundic mucosal height was decreased 7% and 5% at weeks 4 and 13, respectively, but decreases were not statistically significant. Mean serum gastrin concentrations were increased 10-fold in males only after 4 wk at 75 mg/kg, but were comparable to controls during week 13. CI-988-induced gastric gland degeneration is consistent with antagonism of gastrin's trophic activity toward gastric mucosa. Notwithstanding decrements in gastric mucosal height, disappearance of mild histopathologic findings despite continued treatment with the ligand suggests some degree of adaptation to subchronic CCK-B/gastrin inhibition, although the mechanism of accommodation has yet to be delineated.
Asunto(s)
Mucosa Gástrica/efectos de los fármacos , Antagonistas de Hormonas/toxicidad , Indoles/toxicidad , Meglumina/análogos & derivados , Receptores de Colecistoquinina/antagonistas & inhibidores , Administración Oral , Animales , Atrofia , Tamaño de la Célula/efectos de los fármacos , Femenino , Mucosa Gástrica/patología , Gastrinas/sangre , Macaca fascicularis , Masculino , Meglumina/toxicidad , Receptor de Colecistoquinina BRESUMEN
In this study, we determined in vivo morphologic effects of continuous intravenous infusion of recombinant human epidermal growth factor (EGF) in adult Wistar rats. The EGF used consisted of the amino acid residues 1-48 of the human 53-amino-acid EGF molecule, purified from transfected Escherichia coli. Doses of 25, 100, or 250 micrograms/kg body weight were administered using Harvard digital syringe infusion pumps for 4 weeks. At necropsy, the submandibular salivary glands, Harderian glands, liver, kidneys (females only), and ovaries were enlarged and urinary bladders were thickened in 100- and 250-micrograms/kg rats. Numerous tissues of the 100- and 250-micrograms/kg rats contained hyperplastic epithelial cells, and selected organs also had mesenchymal cell proliferation. Epithelial proliferation was most pronounced in the trachea, nasal cavity, nasolacrimal duct, tongue, stomach, small intestine, large intestine, urinary tract, salivary gland ducts, and Harderian gland. Periportal hepatocytes were hypertrophic, correlating with increased liver weight. In addition, mesenchymal cell proliferation was evident in the gastric mucosa lamina propria and in heart valves in 100- and 250-micrograms/kg rats. Increased ovarian weight correlated with increased number and size of corpora lutea and an increased incidence of luteal cysts. Continuous systemic exposure of adult Wistar rats to high doses of EGF resulted in generalized epithelial hyperplasia and tissue-selective mesenchymal proliferation.
Asunto(s)
Sistema Digestivo/patología , Factor de Crecimiento Epidérmico/administración & dosificación , Sistema Respiratorio/patología , Sistema Urogenital/patología , Secuencia de Aminoácidos , Animales , Peso Corporal/efectos de los fármacos , Recuento de Células/efectos de los fármacos , Femenino , Gastrinas/sangre , Gastrinas/efectos de los fármacos , Humanos , Hiperplasia/inducido químicamente , Infusiones Intravenosas , Masculino , Datos de Secuencia Molecular , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Wistar , Proteínas Recombinantes/administración & dosificaciónRESUMEN
Bacterial endotoxin (lipopolysaccharide, LPS) has potent proinflammatory properties toward many cell types, including vascular endothelial cells. Bovine endothelial cells are often used for investigations involving the vascular endothelium in vitro, and other bovine products such as fetal bovine serum are also widely utilized in research laboratories. Evidence is presented that soluble CD14 (sCD14) is present in bovine serum and that LPS-mediated activation and cytotoxicity to bovine endothelial cells in vitro are dependent on sCD14. LPS-mediated activation of endothelial cells was quantitated by measuring tissue factor expression using an activated factor X-related chromogenic assay. Concentrations of 0.1-5.0% fetal bovine serum in the culture medium promoted LPS-induced tissue factor expression on bovine endothelial cells, and anti-CD14 monoclonal antibody (mAb) (20 micrograms/ml) inhibited tissue factor expression, whereas control antibodies did not. LPS-mediated damage to endothelial cells was assayed using the MTT tetrazolium assay. We found that either serum or recombinant human soluble CD14 (rsCD14, 20-2000 ng/ml) was required for LPS-related endothelial cell damage and that anti-CD14 mAb inhibited cytotoxicity. In addition, bovine LPS-binding protein (LBP, 20 ng/ml) purified from bovine serum had no effect on LPS-mediated cytotoxicity, but bovine LBP greatly enhanced the cytotoxic effect of LPS plus rsCD14. Western blot analysis performed on fractionated bovine serum samples with anti-CD14 mAb revealed immunoreactivity with a 50-55-kd protein, a size consistent with sCD14. Evidence of endothelial cell-associated CD14 was not detected using an immunofluorescence technique on cell preparations, nor by Northern blot analysis. These results indicate the existence of sCD14 in bovine serum and that soluble bovine serum factors including sCD14 and LBP facilitate presentation of LPS to receptive cells.
Asunto(s)
Proteínas de Fase Aguda , Proteínas Portadoras/toxicidad , Endotelio Vascular/efectos de los fármacos , Receptores de Lipopolisacáridos/toxicidad , Lipopolisacáridos/toxicidad , Glicoproteínas de Membrana , Animales , Anticuerpos/farmacología , Western Blotting , Proteínas Portadoras/sangre , Bovinos , Células Cultivadas , Sinergismo Farmacológico , Técnica del Anticuerpo Fluorescente , Humanos , Receptores de Lipopolisacáridos/análisis , Receptores de Lipopolisacáridos/sangre , Solubilidad , Estimulación QuímicaRESUMEN
Itraconazole, a third-generation azole, was evaluated for treatment of resistant nasal mycotic infections in horses. Two horses with Aspergillus spp nasal granulomas and 1 horse with Conidiobolus coronatus nasal infection were treated with itraconazole (3 mg/kg PO bid). One of the horses with nasal aspergillosis was also treated by surgical resection of the nasal septum. The treatment time for the horses ranged from 3 to 4.5 months. No adverse effects were noted in any of the horses during the treatment period. Peak and trough serum itraconazole concentrations were < 0.5 micrograms/mL in all 3 horses. Itraconazole (3 mg/kg PO bid) appears to be effective in the treatment of nasal Aspergillus spp infections in horses because the fungal infection was eliminated in both horses. One horse still had excessive nasal sounds during exercise and was retired from training, whereas the other horse returned to normal. The nasal C. coronatus infection appeared resistant to itraconazole treatment in the affected horse because the granulomas were still present after 4.5 months of treatment.
Asunto(s)
Enfermedades de los Caballos/tratamiento farmacológico , Enfermedades de los Caballos/microbiología , Itraconazol/uso terapéutico , Micosis/veterinaria , Rinitis/veterinaria , Animales , Aspergilosis/veterinaria , Entomophthora/aislamiento & purificación , Femenino , Caballos , Masculino , Micosis/tratamiento farmacológico , Micosis/microbiología , Rinitis/tratamiento farmacológico , Rinitis/microbiologíaRESUMEN
Increased procoagulant activity of vascular endothelial cells may be an important component in the pathogenesis of intravascular coagulation associated with gram-negative bacterial diseases. Two bovine endothelial cell (BEC) lines isolated from pulmonary arteries (ENS-2 and ENT-18) were used in this study to investigate procoagulant signal transduction pathways of endotoxin (lipopolysaccharide, LPS)--stimulated BECs. The endothelial cell line ENS-2 was sensitive to LPS as demonstrated by tissue factor (TF) expression, but in contrast, the ENT-18 endothelial cell line was unusually resistant to the effects of LPS. No remarkable quantitative difference in binding of radiolabeled LPS was detected between the two endothelial cell lines. A protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA) failed to induce TF expression in either cell line at concentrations ranging from 0.05 to 1.00 microM when used as a sole stimulus for the endothelial cells. However, when PMA was used in combination with LPS, PMA enhanced the stimulatory effect of LPS on the endothelial cells. In parallel experiments, PKC inhibitors (H-7 and GF 109203X) interfered with the stimulatory effect of LPS on the cells by decreasing tissue factor expression. We also found that an activator of adenylate cyclase, forskolin, similarly inhibited LPS-induced tissue factor activity. In contrast, protein tyrosine kinase inhibitors (genistein, lavendustin A) had no inhibitory effect on LPS-induced endothelial cell tissue factor expression. Our results collectively suggest that activation of PKC is an important step in stimulation of endothelial cells by LPS, and that LPS and phorbol esters may synergize to produce an enhanced stimulatory effect. Our results also suggest participation of cAMP in controlling LPS-mediated stimulation of endothelial cells, but fail to demonstrate a role for protein tyrosine kinase activity.
Asunto(s)
Endotelio Vascular/fisiología , Lipopolisacáridos/farmacología , Transducción de Señal , Adenilil Ciclasas/metabolismo , Animales , Bovinos , Células Cultivadas , Colforsina/farmacología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Activación Enzimática , Lipopolisacáridos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacología , Tromboplastina/metabolismoRESUMEN
Effective antitumor photodynamic therapy (PDT) may be related to damage of vasculature within the tumor. The purpose of this study was to determine if tumor cells secrete factors that stimulate proliferation of human umbilical vein endothelial cells (HUVEC) and result in enhanced sensitivity of HUVEC to aluminum-sulfonated phthalocyanine (AlSPc)-PDT. Three human tumor cell lines--pharyngeal squamous carcinoma, colonic carcinoma, and mammary carcinoma--were used in this study. Co-culture of HUVEC and either squamous carcinoma or colonic carcinoma, but not mammary carcinoma, significantly increased HUVEC proliferation and AlSPc-PDT mediated cell damage. In addition, supernatant from squamous carcinoma and colonic carcinoma cultures also stimulated HUVEC proliferation and sensitivity to AlSPc-PDT. Both supernatant and cell lysate from squamous carcinoma cells contained angiogenic factors consistent with basic and acidic fibroblast growth factors, as evidenced by Western blot analysis and BALB/c 3T3 fibroblast cell proliferation assays. Collectively, these results suggest that selected tumor cell lines produce angiogenic factors that induce HUVEC proliferation and subsequently enhance sensitivity to AlSPc-PDT.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Carcinoma/metabolismo , Endotelio Vascular/efectos de los fármacos , Rayos Láser , Fotoquimioterapia , Células 3T3/efectos de los fármacos , Aluminio/farmacología , Animales , Western Blotting , Carcinoma/patología , Carcinoma de Células Escamosas/patología , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Endotelio Vascular/patología , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Factor 1 de Crecimiento de Fibroblastos/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Indoles/farmacología , Ratones , Ratones Endogámicos BALB C , Neovascularización Patológica , Compuestos Organometálicos/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Células Tumorales Cultivadas , Venas Umbilicales/citologíaRESUMEN
Pasteurella haemolytica in cattle produces fibrino-hemorrhagic pleuropneumonia characterized by extensive pulmonary microvascular thrombosis and parenchymal necrosis. The purpose of this in vitro study was to determine if P. haemolytica lipopolysaccharide (LPS) promotes vascular thrombosis by inducing a procoagulant state in vascular endothelial cells. After treatment of confluent monolayers of bovine pulmonary artery endothelial cells with various concentrations of either P. haemolytica LPS or Escherichia coli LPS, the procoagulant activity of the endothelial cells was determined using a chromogenic assay dependent on cellular tissue factor expression. The LPS treatment induced significant increases in cellular tissue factor expression in a LPS concentration- and time-dependent manner. Highest levels of tissue factor were present at 22 hours after treatment, although high LPS concentrations induced moderate tissue factor levels at 5 hours after treatment. Interleukin-1 also induced tissue factor expression in endothelial cells and enhanced the LPS-induced effects. This interleukin-1 effect could be diminished by concurrent use of an interleukin-1 receptor antagonist. These results demonstrate that LPS and cytokine promotion of a procoagulant state in endothelial cells occurs in vitro. Similar mechanisms may play a role in P. haemolytica-mediated pulmonary vascular thrombosis.
Asunto(s)
Endotelio Vascular/metabolismo , Interleucina-1/farmacología , Lipopolisacáridos/farmacología , Mannheimia haemolytica , Tromboplastina/metabolismo , Animales , Bovinos , Sinergismo Farmacológico , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Tromboplastina/efectos de los fármacos , Factores de TiempoRESUMEN
We tested the hypothesis that the two common oxidant air pollutants, ozone and nitrogen dioxide, modulate the development of respiratory tract tumors in Syrian golden hamsters. The animals received subcutaneous injections of the carcinogen diethylnitrosamine (20 mg/kg) twice a week while being exposed continuously to an atmosphere of 0.8 parts per million (ppm)* of ozone or 15 ppm of nitrogen dioxide. Animals were killed 16 weeks or 24 to 32 weeks after the beginning of the treatment. Ozone delayed the appearance of tracheal tumors and reduced the incidence of tumors in the lung periphery. A suspected neuroendocrine differentiation of those lung tumors could not be established by immunocytochemistry due to overfixation of tissues. On the other hand, ozone seemed to mitigate development of hepatotoxic lesions mediated by diethylnitrosamine. In animals treated with diethylnitrosamine and exposed to nitrogen dioxide, fewer tracheal tumors and no lung tumors were found. Only a few lung tumors were produced in animals treated with diethylnitrosamine and kept in an atmosphere of 65% oxygen. The previously observed neuroendocrine nature of tumors induced by simultaneous exposure to diethylnitrosamine and hyperoxia could not be established because the long fixation of tissues precluded immunocytochemical stains. Animals treated with diethylnitrosamine and kept in filtered air while being housed in wire-mesh cages developed fewer lung tumors than animals given the same treatment and kept on conventional bedding in shoebox cages. Although all inhalants tested are known to produce substantial cell proliferation in the respiratory tract, it was not possible to document whether this would enhance lung tumor development. The role of the two common air pollutants, ozone and nitrogen dioxide, as possible additional risks in the pathogenesis of lung cancer in animals continues to remain uncertain.
Asunto(s)
Neoplasias Pulmonares/patología , Dióxido de Nitrógeno/farmacología , Ozono/farmacología , Animales , Cricetinae , Dietilnitrosamina , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/inducido químicamente , Mesocricetus , Neoplasias de la Tráquea/inducido químicamente , Neoplasias de la Tráquea/patologíaRESUMEN
The purpose of this study was to determine if recombinant angiogenic cytokines modulate the sensitivity of endothelial cells to the toxic effects of chloroaluminum sulphonated phthalocyanine (AlSPc) photodynamic therapy (PDT). Bovine pulmonary artery endothelial cells in 24-well tissue culture plates were pretreated for 24 hr with AlSPc and either acidic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), tumor necrosis factor-alpha (TNF), interleukin-1-alpha (IL-1), or transforming growth factor-beta (TGF) followed by argon-pumped dye laser. Endothelial cell damage was monitored with 51chromium release. FGF, TGF, and, to a lesser extent, IL-1, enhanced the PDT-mediated damage to endothelial cells, whereas PDGF and TNF did not significantly modulate toxicity. The enhanced endothelial cell damage was seemingly not related to rate of cell proliferation or amount of photoactive drug uptake by the EC. These results suggest that presence of tumor secreted cytokines may enhance PDT-mediated toxicity of tumor associated endothelial cells.
Asunto(s)
Aluminio/farmacología , Citocinas/farmacología , Endotelio Vascular/patología , Indoles/farmacología , Terapia por Láser , Compuestos Organometálicos/farmacología , Fotoquimioterapia , Fármacos Sensibilizantes a Radiaciones/farmacología , Aluminio/administración & dosificación , Aluminio/farmacocinética , Animales , Argón , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Citocinas/administración & dosificación , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Factor 1 de Crecimiento de Fibroblastos/administración & dosificación , Factor 1 de Crecimiento de Fibroblastos/farmacología , Indoles/administración & dosificación , Indoles/farmacocinética , Interleucina-1/administración & dosificación , Interleucina-1/farmacología , Compuestos Organometálicos/administración & dosificación , Compuestos Organometálicos/farmacocinética , Factor de Crecimiento Derivado de Plaquetas/administración & dosificación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Fármacos Sensibilizantes a Radiaciones/farmacocinética , Factor de Crecimiento Transformador beta/administración & dosificación , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/administración & dosificación , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Alveolar macrophages (AMs) are important in the host response to aerogenous pulmonary bacterial infections, such as Pasteurella haemolytica-induced pneumonia in cattle. Previous work has shown that AMs enhance P. haemolytica-mediated pulmonary endothelial cell (EC) damage in vitro. The purpose of this study was to determine the mechanism of AM-enhanced EC damage using an in vitro AM-EC coculture system consisting of AMs cultured on culture plate insert membranes and ECs in the underlying chamber. The addition of lipopolysaccharide (LPS) to the culture plate insert chamber resulted in EC damage indicated by 51Cr release, which was enhanced in the presence of AMs. To determine the role of AM-secreted cytokines, recombinant human interleukin 1 alpha (IL-1) or tumor necrosis factor alpha (TNF) was added to ECs simultaneously with varying concentrations of LPS. Although TNF and IL-1 alone had only marginal toxic effects on ECs, the simultaneous treatment of TNF or IL-1 with LPS greatly increased the LPS cytotoxic effect on ECs. In addition, IL-1 receptor antagonist eliminated the IL-1 enhancement of LPS-mediated EC toxicity. These results suggest that macrophage-secreted cytokines synergistically enhance LPS-mediated pulmonary EC damage.