Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex.

Systemic administration of ISIS 2302, a 20-mer antisense phosphorothioate oligonucleotide targeting human intercellular adhesion molecule-1 mRNA, causes prolongation of plasma clotting times in both monkey and human studies. The anticoagulant effects of ISIS 2302 were investigated with both in vitro coagulation assays in human plasma and purified enzyme systems. At high oligonucleotide plasma concentrations (>100 microgram/mL), prolongation of the prothrombin and thrombin times was observed. In a thrombin time assay using purified components, high concentrations of ISIS 2302 inhibited thrombin clotting activity both by stimulating inhibition by heparin cofactor II and directly competing with fibrinogen for binding to anion binding exosite I. In contrast, low concentrations of ISIS 2302 (<100 microgram/mL) showed a selective, linear prolongation of the activated partial thromboplastin time (PTT). The rate limiting effect of 50 microgram/mL ISIS 2302, which prolonged the PTT to 1.5 times control, was identified by sequential modification of the clotting assay. Delaying addition of oligonucleotide until after contact activation failed to correct prolongation of the PTT. The calcium-dependent steps of the intrinsic pathway were individually assessed by adding sufficient activated coagulation factor to correct the PTT in plasma deficient in that specific factor. Addition of factor XIa, IXa, VIIIa, or Va failed to correct the PTT in the presence of ISIS 2302. In contrast, 0.2 nmol/L factor Xa corrected prolongation of the PTT in factor X-deficient plasma with or without oligonucleotide present. ISIS 2302 (50 microgram/mL) did not prolong a modified Russel viper venom time, suggesting no significant inhibition of prothrombinase. Thus, 50 microgram/mL ISIS 2302 prolonged the PTT by selectively inhibiting intrinsic tenase activity. ISIS 2302 showed partial inhibition of intrinsic tenase activity (to approximately 35% of control) at clinically relevant oligonucleotide concentrations in a chromogenic assay. This activity was oligonucleotide sequence-independent but required the phosphorothioate backbone, suggesting that inhibition of intrinsic tenase is a general property of this class of oligonucleotides. These results are relevant to both the therapeutic use of phosphorothioate oligonucleotides and the potential design of inhibitors of the intrinsic tenase complex, a novel target for anticoagulation.

Cytokine production in vitro by macrophages of goats with caprine arthritis-encephalitis.

The response of caprine macrophages to exposure to caprine arthritis-encephalitis virus (CAEV) and lipopolysaccharide (LPS) was investigated in female Nubian and Nubian crossbreed of goats. Macrophages were matured in vitro from monocytes isolated from blood of control and CAEV-infected goats and the concentrations of tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6) in culture supernatant after exposure of cells to LPS and virus were assayed. Though barely detectable in unstimulated cells, TNF-alpha and IL-6 levels showed peak values of 420 +/- 28 to 530 +/- 32 and 70 +/- 27 to 93 +/- 29, respectively, in supernatants of control goat cells [corrected], remaining high through 24 hrs. post treatment with LPS stimulation. Exposure of these control goat cells [corrected] to virus induced lower secretions (p<0.05) of the cytokines. The peak values occurred between 6 and 12 hrs. post treatment with LPS or virus. Cells prepared from virus-infected goats and treated with the mitogen or virus showed significantly (p<0.05) lower response than those from control goats. The present results suggest a dysregulation, possibly downregulation of the production of both cytokines in macrophages of goats chronically diseased by lentivirus infection.

Effect of bovine herpesvirus-1 on expression of interleukin-2 receptors and effect of interleukin-12 on lymphocyte proliferation.

Expression of the interleukin-2 receptor alpha chain (IL-2R alpha) by peripheral blood mononuclear cells (PBMC) from Holstein calves, both experimentally-infected with bovine herpesvirus-l (BHV-l) and controls, was measured by flow cytometry. Expression of IL-2R alpha was 35 percent and 23 percent higher in infected calves than controls, on days 2 and 3 postinfection (PI), respectively. Concurrent with this increase in IL-2R alpha expression, a significant decrease (P < 0.001) was observed in the PHA-induced proliferative responses of PBMC from infected compared with control calves. In vitro treatment with recombinant human (rhu) IL-12 enhanced PHA-induced proliferative responses of PBMC from both infected and control calves. This rhuIL-12 enhancement of mitogen-induced proliferative responses was significant (P < 0.001) in infected calves on day 2 PI and was sufficient to abrogate the decrease observed due to BHV-1 infection. Since the expression of the beta and gamma chains of IL-2R was not measured it is difficult to speculate as to the status of high affinity receptor expression during BHV-1 infection. However results of the present study suggest that the decrease in proliferative responses observed during infection may not be due to a decrease in IL-2R alpha expression but may possibly be due to a selective down-regulation of signal transduction through IL-2R and/or by modulation of the expression of other cytokines involved in lymphocyte activation and proliferation.

Effect of stress on interleukin-2 receptor expression by bovine mononuclear leukocytes.

Holstein calves given three consecutive i.m. injections of dexamethasone (DEX) (0.04 mg kg-1) showed lymphopenia and neutrophilia with increased numbers of mature neutrophils on post-injection Days 1 and 2, but these values returned to normal levels by post-injection Day 3. Interleukin-2 receptor (IL-2R) expression by peripheral blood mononuclear cells (PBMC) was evaluated by flow cytometry using a monoclonal antibody specific for bovine IL-2R alpha. Treatment with DEX significantly decreased expression of IL-2R alpha in Concanavalin A (Con A)-activated PBMC on Day 1 (P < 0.02) and on Day 2 (P < 0.1). On Day 3, expression of IL-2R alpha by PBMC was similar in control and DEX-treated calves. This decrease in IL-2R alpha expression correlated with decreased proliferative responses of PBMC to the T-cell mitogens, phytohemagglutinin (PHA) and Con A. Following in vitro treatment with recombinant human (rhu) interleukin-12 (IL-12) Con A-induced proliferative responses of PBMC tended to be higher in both groups. However, the rhu IL-12 induced increase of Con A activated proliferative responses were significantly greater in DEX-treated calves than in control calves. IL-2R alpha expression by PBMC was found to be less in calves transported 800 km in a truck as compared to that in PBMC from controls. These data suggest that stress-induced immunosuppression in calves may involve decreased IL-2R alpha expression and decreased IL-12 production. Serum chemistry results indicated a trend toward higher creatine kinase (CK) levels in DEX-treated calves. This may be due to the lysis of corticosteroid sensitive lymphocytes.

[Sonographic patterns and pathological correlation of neuroblastoma].

This study prospectively correlated pathological findings with sonographic results in two cases of neuroblastoma, and retrospectively analyzed the sonographic pattern of neuroblastomas encountered in our hospital in past years. Prospectively, among the five pathologically proved neuroblastomas, only two received operation. The removed specimens were scanned by high resolution ultrasound, and selected areas were guided by a needle for pathological correlation. Pathologically, the hyperechoic region represented tumor necrosis, hemorrhage and calcifications, as well as hypercellularity and irregular arrangement of the neurofilaments. The hypoechoic areas showed less cellularity with loose tumor cells. No pseudorosette formation was noted. In the retrospective study, 26 neural crest tumors were collected, but only 14 cases received sonographic examination. The most common sonographic findings were heterogenous mixed pattern (65%) and heterogenous hyperechoic pattern (21%). Eleven cases showed calcifications. Two cases presented with echogenic lobule. Most of the tumors were larger than 8 cm, ill-defined and grew across the midline (73%). Sonography is advantageous for diagnosis of abdominal mass in children and might be helpful in differentiating neuroblastomas from other tumors.

[High resolution real-time sonographic evaluation of superficial lesions].

One hundred and thirty-one cases of suspected superficial soft tissue lesions, excluding parotid, thyroid, parathyroid, breast and scrotum, were collected in 7-year-period in Veterans General Hospital-Taichung. High resolution (10MHz transducer) real-time ultrasound was applied as imaging modality. Among them, 46 cases were proved pathologically or clinically. We retrospectively evaluated the sonographic features and divided them into five major patterns. I. Cystic lesion with smooth margin, II. Cystic lesion with blurred margin, III. Solid lesion with smooth margin, IV. Solid lesion with blurred margin, V. Diffuse infiltrative lesion. In addition, gas or calcification in the lesion and the echogenicity were included in the evaluation. Some of the lesions considered to be of vascular origin were evaluated with doppler. Ultrasonography can accurately recognize cystic, solid, or infiltrative lesions. In spite of the fact that most of the findings are non-specific, several lesions do have characteristic sonographic patterns that can easily be diagnosed, especially in conjunction with the anatomical location, ie. Backer's cyst, branchial cyst, lymphangioma, and abscess. Lesions with cystic or hyperechoic solid patterns can be considered as benign, if the margin is smooth. Lesions with hypoechoic solid pattern and blurred margin, however except for abscess, can be considered as malignant. We conclude that high resolution real-time sonography is a good imaging modality for evaluating superficial soft tissue lesions.