mTOR kinase inhibition reduces tissue factor expression and growth of pancreatic neuroendocrine tumors.

Essentials Tissue factor (TF) isoforms are expressed in pancreatic neuroendocrine tumors (pNET). TF knockdown inhibits proliferation of human pNET cells in vitro. mTOR kinase inhibitor sapanisertib/MLN0128 suppresses TF expression in human pNET cells. Sapanisertib suppresses TF expression and activity and reduces the growth of pNET tumors in vivo. SUMMARY: Background Full-length tissue factor (flTF) and alternatively spliced TF (asTF) contribute to growth and spread of pancreatic ductal adenocarcinoma. It is unknown, however, if flTF and/or asTF contribute to the pathobiology of pancreatic neuroendocrine tumors (pNETs). Objective To assess TF expression in pNETs and the effects of mTOR complex 1/2 (mTORC1/2) inhibition on pNET growth. Methods Human pNET specimens were immunostained for TF. Human pNET cell lines QGP1 and BON were evaluated for TF expression and responsiveness to mTOR inhibition. shRNA were used to knock down TF in BON. TF cofactor activity was assessed using a two-step FXa generation assay. TF promoter activity was assessed using transient transfection of human TF promoter-driven reporter constructs into cells. Mice bearing orthotopic BON tumors were treated with the mTORC1/2 ATP site competitive inhibitor sapanisertib/MLN0128 (3 mg kg-1 , oral gavage) for 34 days. Results Immunostaining of pNET tissue revealed flTF and asTF expression. BON and QGP1 expressed both TF isoforms, with BON exhibiting higher levels. shRNA directed against TF suppressed BON proliferation in vitro. Treatment of BON with sapanisertib inhibited mTOR signaling and suppressed TF levels. BON tumors grown in mice treated with sapanisertib had significantly less TF protein and cofactor activity, and were smaller compared with tumors grown in control mice. Conclusions TF isoforms are expressed in pNETs. Sapanisertib suppresses TF mRNA and protein expression as well as TF cofactor activity in vitro and in vivo. Thus, further studies are warranted to evaluate the clinical utility of TF-suppressing mTORC1/2 inhibitor sapanisertib in pNET management.

Using 3 Tesla magnetic resonance imaging in the pre-operative evaluation of tongue carcinoma.

OBJECTIVE: This study aimed to evaluate the role of 3 Tesla magnetic resonance imaging in predicting tongue tumour thickness via direct and reconstructed measures, and their correlations with corresponding histological measures, nodal metastasis and extracapsular spread. METHODS: A prospective study was conducted of 25 patients with histologically proven squamous cell carcinoma of the tongue and pre-operative 3 Tesla magnetic resonance imaging from 2009 to 2012. RESULTS: Correlations between 3 Tesla magnetic resonance imaging and histological measures of tongue tumour thickness were assessed using the Pearson correlation coefficient: r values were 0.84 (p < 0.0001) and 0.81 (p < 0.0001) for direct and reconstructed measurements, respectively. For magnetic resonance imaging, direct measures of tumour thickness (mean ± standard deviation, 18.2 ± 7.3 mm) did not significantly differ from the reconstructed measures (mean ± standard deviation, 17.9 ± 7.2 mm; r = 0.879). Moreover, 3 Tesla magnetic resonance imaging had 83 per cent sensitivity, 82 per cent specificity, 82 per cent accuracy and a 90 per cent negative predictive value for detecting cervical lymph node metastasis. CONCLUSION: In this cohort, 3 Tesla magnetic resonance imaging measures of tumour thickness correlated highly with the corresponding histological measures. Further, 3 Tesla magnetic resonance imaging was an effective method of detecting malignant adenopathy with extracapsular spread.

Platelet glutathione and thromboxane synthesis in diabetes.

The relationship of the reduced glutathione (GSH) content in unstimulated platelets and their capacity to synthesize thromboxane A2 (TXA2), measured by radioimmunoassay of TXB2, was investigated in diabetic and matched control subjects. The GSH content in platelets from diabetic subjects (6.52 +/- 0.73 microgram/10(9) platelets, mean +/- SD) was significantly (P less than 0.001) lower than in platelets from control subjects (10.10 +/- 1.58 microgram/10(9) platelets). When platelet-rich plasma (PRP) was stimulated with 1.65 mM arachidonic acid, significantly (P less than 0.001) more TXB2 was formed in PRP from diabetic subjects (344 +/- 87 ng/2.5 X 10(8) platelets) than in PRP from control subjects (132 +/- 35 ng/2.5 X 10(8) platelets). Furthermore, the plasma level of TXB2 was increased in diabetic subjects (522 +/- 117 pg/ml) in comparison with control subjects (187 +/- 63 pg/ml). An inverse correlation (r = 0.98) was observed between the GSH content in unstimulated platelets and their capacity to synthesize TXA2 when stimulated with 1.65 mM arachidonic acid. These data suggest that platelet GSH may have an important regulatory effect on platelet TXA2 synthesis and that increased TXA2 synthesis by platelets from diabetic subjects may be the result of low intracellular GSH levels.

Bacterial tissue tropism: an in vitro model for infective endocarditis.

Since infective endocarditis may affect individuals without pre-existing valvar heart disease, and Staphylococcus aureus is the organism most commonly involved, the binding characteristics of S aureus to several components of normal vascular endothelium and subendothelium were studied. S aureus adhered specifically to endothelial monolayers (6.08(1.10)%; p less than 0.005), fibronectin (5.43(0.81)%; p less than 0.001), fibrinogen (7.13(1.43)%; p less than 0.001), and acid soluble calf skin collagen (2.38(0.90)%; p less than 0.001). S aureus also adhered specifically to Von Willebrand factor (1.62(0.28)%, p less than 0.001). Protein A containing (Cowan I) and deficient (Wood) strains of S aureus adhered similarly to all surfaces and substrates (NS). Escherichia coli adhered poorly. Immunofluorescence microscopy of preconfluent endothelial cells identified an extensive pericellular fibronectin network at regions of cell to cell contact. Light microscopy showed S aureus binding solely within these regions. Therefore, the ability of S aureus to infect valvar endothelium may be dependent on the presence of a fibronectin receptor. The existence of specific receptor for S aureus on the endothelial cell surface itself remains undetermined.

Platelet-bound IgG in systemic lupus erythematosus with and without thrombocytopenia.

A new assay for IgG bound to autologous platelets is described, in which IgG is dissociated from platelets washed at low pH, then measured by radioimmunoassay. Platelet-bound IgG was found to be elevated in thrombocytopenic patients with SLE and reduced in non-thrombocytopenic patients with this disease. There was a close inverse correlation of platelet-bound IgG and platelet count. Immune complexes did not interfere with the assay.

The contrecoup phenomenon. Reappraisal of a classic problem.

We describe briefly and comment upon the salient strengths and limitations of the major published theories that purport to explain the mechanism of contrecoup cerebrocortical contusions. Through the application of mechanical principles, we then present a modification, clarification, and expansion of selected aspects of several theories. Our final formulation emphasizes the injurious potential of nonuniform compressive stress and the relationship between brain lag and rotationally induced injury. The resulting theory remains faithful to the laws of physics while explaining the location and distribution of cerebrocortical contusions opposite the site of a moving head impact.

Morphology and cytochemistry of "microgranular" acute promyelocytic leukemia (FAB M3m).

Three cases of a newly recognized "microgranular" variant of acute promyelocytic leukemia (FAB M3) are described. This poor-prognosis variant is easily confused with myelomonocytic (M4) or monocytic (M5) leukemia, but is associated with disseminated intravascular coagulation as is "hypergranular" M3. Such patients may be inappropriately treated unless the promyelocytic nature of the leukemia is recognized.

Alternate chromogens as substitutes for benzidine for myeloperoxidase cytochemistry.

Myeloperoxidase staining methods for classification of leukemias have traditionally employed benzidine dihydrochloride as the chromogen. Recent Occupational Safety and Health Administration regulations have classified benzidine as a carcinogen, which severely restricts its use in the clinical laboratory. Twenty-two specimens from normal control subjects and leukemia patients, previously classified by FAB criteria, were stained with benzidine and two alternate chromogens, diaminobenzidine and Hanker-Yates reagent (p-phenylenediamine and pyrocatechol). In a random, blind fashion, three experienced observers scored the stains from each case according to quality of smear, degree of peroxidase positivity, tinctorial distinction between nucleus and cytoplasm, and overall acceptability of the stain. All three observers rated the substitute chromogens as adequate for routine myeloperoxidase cytochemical staining. It is concluded that either of the methods studied would have clinical utility and can substitute for benzidine as a myeloperoxidase chromogen.

A novel whole blood capillary technic for measuring the prothrombin time.

The prothrombin time (PT) is frequently performed to monitor anticoagulant therapy. Although relatively simple to perform, it requires venipuncture and laboratory resources for sample handling and analysis. A recently developed capillary whole blood device that uses fingerstick samples was evaluated. Paired capillary whole blood and reference plasma PTs were performed in 858 samples from 732 subjects. The PT for normal volunteers (n = 193) was 11.8 +/- 0.9 seconds with the use of the new instrument and 12.1 +/- 0.5 seconds with the use of the reference method. In samples from 539 patients receiving anticoagulants, the correlation coefficient between the two methods was 0.96. Venous whole blood without anticoagulant and capillary whole blood gave equivalent results, which suggests that the fingersticks do not effect the quality of the specimen. Variation in hematocrit between 23.4% (0.34) and 53.8% (0.538) did not alter the performance of the instrument. The new instrument is easy to use and may allow testing by nonlaboratory personnel and patients. It obviates the need for venipuncture, provides immediate results, and appears to be comparable in accuracy to current reference methods.

Immunophenotypic characterization of acute leukemia by immunocytology.

The potential for specific immunophenotypic characterization of the acute leukemias has been enhanced greatly by the development of monoclonal antibodies. Currently, this immunologic data is obtained most commonly by flow cytometric analysis or cellular cytotoxicity assays. The former is an expensive technic that lacks morphologic evaluation unless cell sorting is performed. The latter precludes morphologic assessment by the nature of the assay. The authors have developed an immunostaining procedure utilizing cytospin preparations and immunoperoxidase methods that are relatively inexpensive and allow simultaneous assessment of the immunologic markers and cellular morphology. Although a comparison of flow cytometry and immunocytology revealed quantitative differences for individual cell surface markers, the "qualitative" immunologic phenotype of the leukemic population was virtually identical by the two technics.

The influence of glutathione and other thiols on human platelet aggregation.

The platelet membrane contains sulfhydryl groups which are essential for normal platelet function. Reduced glutathione (GSH) and other thiols such as cysteine and 6-mercaptopurine were found to inhibit human platelet aggregation induced by adenosine diphosphate (ADP), collagen and arachidonic acid. The inhibition of ADP-induced aggregation by GSH (IC50 = 0.61 +/- 0.05 mM) was greater than that by cysteine (IC50 = 13 +/- 1 mM) or 6-mercaptopurine (IC50 = 5.4 +/- 0.2 mM). Two other thiols, dithiothreitol and beta-mercaptoethanol were found to cause platelet aggregation instead of inhibition. The interaction of GSH with the ADP receptor was noncompetitive in nature.

Stability of prostacyclin in human and rabbit whole blood and plasma.

The stability of prostacyclin (PGI2) in whole blood and plasma was studied in vitro by measuring the disappearance rate of labeled prostacyclin during a 37 degrees C incubation. Prostacyclin was assayed using a quantitative chromatographic method. The half-life of PGI2 was 6.3 +/- 0.8 minutes (mean +/- s.d., n = 6) in citrated human whole blood, significantly shorter (p less than 0.001) than the 10.7 +/- 2.3 minute half-life in citrated human plasma (n = 7). Prior freezing and thawing of plasma did not affect the rate of PGI2 hydrolysis. These values, including the prolonged half-life in plasma, were similar in the blood (5.4 +/- 1.8 min, n = 7) and plasma (9.0 +/- 1.9 min, n = 14) of diabetic patients. In plasma samples from patients with thrombotic thrombocytopenic purpura, the half-life of prostacyclin (4.9 +/- 1.0 min, n = 4) was significantly shortened (p less than 0.001) compared to that in plasma from normal volunteers. The stability of prostacyclin in rabbit blood and plasma was also quantified. The PGI2 half-life in citrated rabbit plasma (10.8 +/- 1.1 min, n = 3) was similar to that in citrated human plasma from control subjects. In contrast to the findings in human blood, the half-life of PGI2 in citrated rabbit whole blood (11.7 +/- 3.3 min, n = 4) was not different from the rabbit plasma value. Substitution of EDTA for citrate did not affect the half-life in rabbit blood or plasma.

Thromboxane synthetase inhibition in acute focal cerebral ischemia in cats.

The purpose of this investigation was to study the effects of a selective thromboxane A2 (TXA2) synthetase inhibitor (TSI) upon the evolution of cerebral infarction in the cat. Adult cats, lightly anesthetized with nitrous oxide, underwent right middle cerebral artery (MCA) occlusion for 4 hours followed by a 2-hour period of reperfusion before sacrifice. Ten cats received 3 mg/kg TSI intravenously immediately before, and 10 cats received 3 mg/kg TSI intravenously immediately after MCA occlusion. Ten cats were used as controls receiving no treatment. The bleeding time was determined at baseline and at the end of each experiment. Electroencephalographic (EEG) recordings were obtained before and after MCA clipping and MCA release, and at hourly intervals thereafter. Regional cerebral blood flow (rCBF) was measured using the xenon-133 (133Xe) clearance technique before and after MCA occlusion, after MCA reopening, and before terminating each experiment. Thirty minutes before each cat was sacrificed, Evans blue dye and sodium fluorescein were given intravenously. The animals were then perfused with colloidal carbon and the brains removed and evaluated for midline shift. Evans blue dye and sodium fluorescein extravasation, carbon staining, and infarct size. The bleeding time, arterial blood pressure, rCBF changes, brain swelling, and vital dye extravasation were not statistically different between the three treatment groups. The EEG changes, carbon staining, and infarct size differences between the three groups also failed to reach statistical significance, but there was a suggestion that these parameters were adversely affected in the cats pretreated with TSI. Ten additional cats undergoing MCA occlusion and reperfusion were used for pharmacological studies. Five of them received 3 mg/kg TSI intravenously immediately after MCA occlusion, and serial drug and thromboxane B2 (TXB2) levels (a stable metabolite of TXA2) were determined. Another five cats were not treated and serial TXB2 levels were obtained. Production of TXA2 was inhibited by 95% in cats receiving TSI. In conclusion, thromboxane synthetase inhibition failed to modify favorably the evolution of cerebral infarction. When TSI was given before MCA occlusion, cerebral infarction tended to be more extensive.

Metabolism of norethynodre, a 19-nor progestin: subcellular localization of enzyme activity.

PIP: The subcellular distribution of the enzymes involved in the metabolism of norethynodrel (17 alpha-ethynyl-17 beta-hydroxy-estr-5(10)-en-3-one) to the 3alpha and 3beta diols (17 alpha-ethynyl-3alpha (or 3beta-17 beta-dihydroxy-estr-5(10)-ene) and 17 alpha-ethinyl estradiol was studied. The purity of the male rat liver subcellular fractions was evaluated by the use of marker enzymes. Sample sections were viewed by electron microscopy. The data showed that the cytosol fraction contained the highest relative specific activity for the hydroxysteroid dehydrogenases required for the formation of the diols. The cytosol fraction also contained the highest total activity. The enzymes required for the formation of ethinyl estradiol were distributed equally among mitochondrial and microsomal fractions, however, the highest relative specific activity was associated with the heavy microsomal fraction (18,000 g).^ieng

Fibrinogen A alpha and gamma-chain dimers as potential differential indicators of atherosclerotic and thrombotic vascular disease.

Cross-linked fibrin(ogen) dimers are known to be elevated in the plasma of subjects with occlusive vascular disease, and are thought to be fibrin dimers. Immunoelectrophoretic analyses of the dimers, however, indicate that (1) they are predominantly fibrinogen rather than fibrin dimers, and (2) they contain cross-linked A alpha-chains (A alpha-dyads) instead of the gamma-chain dyads that are rapidly formed by factor XIII during blood coagulation. Furthermore, the mobilities of the A alpha-dyads differ from the cross-linked alpha-chain products that accompany the gamma-chain cross-linking by factor XIII. Instead, the mobilities coincide with the distinct A alpha-dyads that are produced by tissue transglutaminase, an intracellular enzyme not normally present in plasma. The intimal fibrinogen deposits in atherosclerotic aortas also possess fibrinopeptide A and cross-linked A alpha-chains. Thus, both the plasma fibrinogen dimers and the intimal fibrinogen deposits appear to derive from the action of released tissue transglutaminase more so than factor XIII. It is proposed that, in the absence of other indications of cytolytic processes, the levels of A alpha-dyads in plasma reflect ongoing cellular injury accompanying atherogenesis. The extent to which gamma-dyads accompany the A alpha-dyads may signal progression of the disease to advanced stages in which ulcerations and occlusive lesions trigger thrombotic complications.

Factor VIII assays. Assessment of variables.

Factor VIII assays are the most common specific coagulation factor assay performed in the United States. Interlaboratory proficiency studies have documented persistent problems with variation in results between laboratories. The Coagulation Resource Committee of the College of American Pathologists conducted a workshop to analyze variables that may affect performance of the one-stage factor assay. The results indicate that accuracy of the assay can be improved by uniform standardization of reference plasma samples and that reproducibility can be enhanced through appropriate choice of reagents and instruments. Optimizing performance of this assay should lead to more reproducible interlaboratory results.