Tumor necrosis factor alpha and interleukin 1 alpha enhance lipopolysaccharide-mediated bovine endothelial cell injury.

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.

Soluble CD14 and lipopolysaccharide-binding protein from bovine serum enable bacterial lipopolysaccharide-mediated cytotoxicity and activation of bovine vascular endothelial cells in vitro.

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.

Rate of CRL4(CRBN) substrate Ikaros and Aiolos degradation underlies differential activity of lenalidomide and pomalidomide in multiple myeloma cells by regulation of c-Myc and IRF4.

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.

EDTA-dependent platelet phagocytosis. A cytochemical, ultrastructural, and functional characterization.

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.

Preclinical safety evaluation of avasimibe in beagle dogs: an ACAT inhibitor with minimal adrenal effects.

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.

Interaction of bovine neutrophils in Pasteurella haemolytica mediated damage to pulmonary endothelial cells.

The purpose of these studies was to determine mechanisms of pulmonary tissue damage mediated by Pasteurella haemolytica and interaction with bovine neutrophils. Bovine pulmonary artery endothelial cell monolayers were treated with various combinations of P. haemolytica factors including bacterial culture supernatant (CS) and purified LPS, with and without bovine neutrophils. Damage to endothelial cells was monitored by 51Cr release, cell detachment rate, and morphological changes. At 5 h post-treatment (PT) bacterial factors produced very little toxic change in cells, however, by 22 h PT both crude leukotoxin and LPS caused high levels of cytotoxicity and detachment. Neutrophils did not augment toxicity mediated by LPS, but actually protected endothelial cells from low levels of LPS. When the LPS component of CS was neutralized with polymyxin B, leukotoxin mediated neutrophil killing resulted in extensive endothelial cell damage. These results suggest that LPS may directly injure endothelial cells and this toxic effect may be reduced by neutrophils. However, neutrophil killing by leukotoxin may also contribute to endothelial cell damage in the absence of LPS.

Signal transduction pathways of bacterial lipopolysaccharide-stimulated bovine vascular endothelial cells.

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.

Detection of Ehrlichia risticii using an avidin-biotin-immunoperoxidase staining system.

An indirect immunoperoxidase procedure using a specific anti-Ehrlichia risticii monoclonal antibody and an avidin-biotin-peroxidase staining method was used to detect E. risticii antigen in infected P388D1 murine monocytes. Several different methods of cytological fixation were used, including acetone (15 min), 95% ethanol (15 min), Bouin's fixative (5 hr), and 10% buffered neutral formalin (24 hr). The E. risticii organisms were labeled effectively and identified in cells fixed with acetone and ethanol. However, infected P388D1 cells fixed in 10% formalin or Bouin's fixative required enzymatic digestion with 1.0% trypsin for 15 min at 37 C before positive results were evident. This indirect immunoperoxidase avidin-biotin staining procedure proved to be a sensitive assay for the detection of intracellular E. risticii and may be an effective diagnostic procedure for formalin-fixed paraffin-embedded tissue.

Failure of ozone and nitrogen dioxide to enhance lung tumor development in hamsters.

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.

Treatment of mycotic rhinitis with itraconazole in three horses.

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.

Pulmonary lesions induced by Pasteurella haemolytica in neutrophil sufficient and neutrophil deficient calves.

The role of neutrophils in the development of peracute lung lesions of bovine pneumonic pasteurellosis was investigated. Eight calves were divided into two groups of four calves each. Group I was treated with intravenous phosphate-buffered saline and served as the neutrophil sufficient calves. Group II was treated with intravenous hydroxyurea which produced a state of neutropenia. When peripheral blood neutrophil numbers dropped below 300 cells/microL in group II, all calves were challenged with an intrabronchial bolus of Pasteurella haemolytica in the log phase of growth. An acute inflammatory process occurred in both groups of calves indicated by a rise in body temperature. While pulmonary lesions occurred in both groups by six hours postinoculation, they varied in pathological characteristics. Pulmonary lesions in the neutrophil sufficient calves consisted of fibrinopurulent alveolitis-bronchiolitis with associated alveolar septal necrosis, interlobular edema, and intravascular thrombi. The neutrophil deficient calves had extensive intra-alveolar edema, interlobular edema, intraalveolar hemorrhage, atelectasis, and focal areas of alveolar septal necrosis. These results show that P. haemolytica can induce severe pulmonary tissue damage through both neutrophil dependent and neutrophil independent mechanisms.

Influence of interferon in natural resistance of mice to Sendai virus pneumonia.

The influence of interferon (IFN) on the natural resistance to Sendai virus was investigated in resistant (C57BL/6NCr) and susceptible (DBA/2NCr) mouse strains. After inoculation with strain 52 Sendai virus, the pulmonary titers of Sendai virus were similar in both mice strains, indicating that the quantity of virus in the lungs may not be a major factor in determination of resistance. The pulmonary IFN titers were higher in DBA/2 mice, indicating that increased susceptibility of DBA/2 mice is not a result of inadequate production of IFN. Treatment of both strains with exogenous IFN before and during viral infection induced an increased survival time in C57BL/6 mice, but eventual mortality was the same in both mouse strains. Administration of anti-IFN antibodies to both mouse strains during Sendai infection decreased mortality equally in both strains, compared with mortality in control mice. Seemingly, the difference in resistance to Sendai virus in DBA/2 and C57BL/6 mice was probably not a result of the beneficial effects of IFN. On the contrary, administration of anti-IFN antibodies decreased the susceptibility of both mouse strains.

Effect of Pasteurella haemolytica saline capsular extract on bovine pulmonary endothelial cells.

The purpose of this in vitro study was to determine whether Pasteurella haemolytica capsular extract (CE) damages bovine pulmonary endothelial cells (EC) directly or through neutrophil-mediated mechanisms. Chromium 51-labeled EC were treated with the following variables: CE (1, 10, and 100 ng of protein/ml), CE and bovine neutrophils (10(6) cells/well), and CE and polymyxin B (500 U/ml). Although only minimal damage to EC occurred by 5 hours after treatment, by 22 hours after treatment, the 10-ng and 100-ng CE dose produced severe damage to EC, as indicated by 51Cr release, cellular detachment, and loss of monolayer confluency. The component in the CE that was toxic to the EC was lipopolysaccharide, evidenced by effective neutralization of the toxic effect with polymyxin B. Neutrophils inhibited the CE-mediated EC toxicity and were activated, as indicated by shape change and adhesion to EC monolayers. We concluded that the lipopolysaccharide component of CE causes direct damage to EC, which can be attenuated by neutrophils and polymyxin B.

Early renal ultrasonographic findings in dogs with experimentally induced ethylene glycol nephrosis.

Renal ultrasonographic changes were evaluated in 5 dogs administered 10 ml of commercial antifreeze (95% ethylene glycol)/kg of body weight, PO, and in 2 dogs given placebos. Studies were made prior to and after ingestion on an hourly basis over a period of 8 to 10 hours. All dogs were anesthetized immediately after toxin or placebo ingestion for the duration of the study. Renal cortical echogenicity was evaluated in comparison with that of the adjacent liver and spleen. Echogenicity of the renal medulla and definition of the corticomedullary junction were assessed. Within 4 hours after ethylene glycol administration, renal cortical echogenicity of all intoxicated dogs increased from normal to surpass that of liver and approach or equal that of the spleen. Medullary echogenicity in all intoxicated dogs progressively increased over the course of the study, with changes recognized within 5 hours after ethylene glycol administration. An ultrasonographic pattern consisting of nearly equal, marked increase in cortical and medullary echogenicity and relatively hypoechoic corticomedullary junction and central medullary regions was recognized concurrent with the development of anuria in 3 of the 5 intoxicated dogs. Mild, transient increases in cortical and medullary echogenicity were observed in anesthetized control dogs. However, no statistical difference (P less than 0.05) was detected between baseline, peak, and terminal echogenicity values in these dogs. Blood and urine samples were collected hourly from intoxicated dogs to coincide with ultrasonographic studies. Most clinicopathologic values derived from these samples were not statistically different (P less than 0.05) from those reported in a study that used a similar intoxication protocol in nonanesthetized dogs.

Cardiovascular changes after bone marrow transplantation in dogs with mucopolysaccharidosis I.

Five dogs with mucopolysaccharidosis I, 3 of which had been treated with bone marrow transplantation (BMT), were evaluated for 20 months with electrocardiography, thoracic radiography, and M-mode and 2-dimensional echocardiography. Treated and untreated (control) dogs had widened P waves on ECG. Thoracic radiographs remained normal for all dogs throughout the study. Thickening of the mitral valve was observed on echocardiograms of dogs in both groups, but the untreated dogs appeared to have thicker valves. Concentrations of glycosaminoglycans in the mitral valves and myocardium were higher in control dogs than in treated dogs. Markedly large aortic root diameters were observed on echocardiograms in both untreated dogs, but aortic root diameters remained normal in treated dogs. Echocardiography, but not electrocardiography, was useful in monitoring heart enlargement in each dog. Dogs treated with BMT generally had less severe cardiac changes and slower disease progression than control dogs.

Ultrasonographic findings in dogs and cats with oxalate nephrosis attributed to ethylene glycol intoxication: 15 cases (1984-1988).

Renal ultrasonographic findings in 12 dogs and 3 cats determined to have oxalate nephrosis presumed to be secondary to ethylene glycol intoxication were examined. Ultrasonographic changes varied from mild to marked increases in renal cortical echogenicity. A pattern of greater than normal cortical and medullary echogenicity with persistence of areas of lesser echo intensity at the corticomedullary junction and central medullary regions was observed. This pattern, termed the halo sign, was recognized in 7 dogs and 1 cat concurrent with the development of clinical anuria. Ultrasonographic patterns in these clinical cases were similar to those observed in a previous study of dogs with experimentally induced ethylene glycol nephrosis. Ultrasonographic findings were not considered pathognomonic of ethylene glycol nephrosis. Due to the high death rate reported in the cases surveyed, detection of ultrasonographic changes was considered to warrant a guarded to poor prognosis. Because of the association of the halo sign with anuria, its detection was considered to warrant a grave prognosis.

Chronic eosinophilic pancreatitis and ulcerative colitis in a horse.

A generalized debilitating disease in a horse was believed to be related to hypersensitivity to migrating strongyle larvae. The clinical signs included weight loss, diarrhea, and ulcers on all 4 coronary bands. The mare's condition deteriorated rapidly, so the mare was euthanatized and necropsied. The major histopathologic findings were chronic multifocal eosinophilic pancreatitis, hepatic portal fibrosis, biliary hyperplasia, and chronic ulcerative eosinophilic colitis. This case was similar to previously reported cases of chronic eosinophilic gastroenteritis in horses. Although the etiologic agent was not evident, the distribution and character of the lesions were consistent with a hypersensitivity response to migrating parasitic larvae, most probably Strongylus equinus.

Blastomycosis in cats: five cases (1979-1986).

Medical records of 5 cats with blastomycosis examined at the University of Tennessee Veterinary Teaching Hospital from 1979 to 1986 were reviewed. Clinical signs of blastomycosis varies depending on the organ(s) affected, but respiratory tract disease was most common, followed by CNS signs and ocular problems. A definitive diagnosis was made by identification of characteristic fungal organisms in biopsy or necropsy specimens. Two cats treated with amphotericin B did not respond to treatment and died or were euthanatized. The lungs, brain, eyes, and lymph nodes commonly were affected, but one cat had only cutaneous and regional lymph node involvement. The respiratory tract appeared to be a common primary site of infection, with dissemination to other organ systems. The typical host response was a pyogranulomatous cellular infiltrate with numerous fungal organisms evident.