Intra- and inter-rater consistency of dual assessment by radiologist and neurologist for evaluating DWI-ASPECTS in ischemic stroke.

OBJECTIVES: To estimate the intra -and inter-rater consistency of radiologist and neurologist working in pairs attributing DWI-ASPECTS (Diffusion Alberta Stroke Program Early CT Score) in patients with acute middle cerebral artery ischemic stroke referred for mechanical thrombectomy, intravenous thrombolysis or bridging therapy. METHODS: Five neurologists and 5 radiologists working in pairs and in hour period scored independently and in two reading sessions anonymized DWI-ASPECTS of 80 patients presenting with acute anterior ischaemic stroke in our center. We measured agreement between pairs using intraclass correlation coefficients (ICCs). A Fleiss kappa was used for dichotomized (0-6;7-10) and trichotomized (0-3;4-6;7-10) ASPECTS. The interrater distribution of the score in the trichotomized (0-3;4-6;7-10) ASPECTS was calculated. We determined the interrater (Cohen kappa) and intrarater (Fleiss kappa) agreement on the ASPECTS regions. RESULTS: The average DWI-ASPECTS was 6.35 (SD±2.44) for the first reading, and 6.47 (SD±2.44) for the second one. The ICC was 0.853 (95%CI, 0.798-0.896) for the interrater, and 0.862 (95%CI, 0.834-0.885) for the intrarater evaluation. Kappa coefficients were high for dichotomized (k=0.75) and trichotomized (k=0.64) ASPECTS. Evaluators agreement on the ASPECTS category (0-3), (4-6) and (7-10) was 88, 76 and 93% respectively. The anatomic region infarcted was well identified (k=0.70-0.77), except for the internal capsula (k=0.57). Interrater agreement was fair for M5 (k=0.37), moderate for internal capsula (0.52) and substantial for the other regions (0.60-0.79). CONCLUSIONS: Reliability of DWI-ASPECTS is good when determined by radiologist and neurologist working in pairs, which corresponds to our current clinical practice. However, discrepancies are possible for cut-off determination, which may impact the indication of thrombectomy, and for the determination of the exact infarcted region. Agreement to propose category (4-6) is lower than for (0-3) and (8-10) ASPECTS categories.

Adenosine: a sensitive marker of myocardial ischaemia in man.

OBJECTIVE: In the human heart, the significance of adenosine as an indicator of myocardial ischaemia is controversial. Since adenosine fulfils key functions in the regulation of cardiac metabolism, its sensitivity as a marker of tissue ischaemia was investigated in this study in relation to other metabolites such as hypoxanthine and lactate. METHODS: Cardiac metabolite production was studied in 18 patients with left coronary obstruction (> 90%) undergoing percutaneous transluminal coronary angioplasty (PTCA). Three balloon inflation procedures per patient were performed for 30, 60, and 90 s (+/- 5 s each) and coronary sinus adenosine, hypoxanthine, uric acid, and lactate were determined. RESULTS: Before PTCA, coronary sinus concentrations of adenosine and hypoxanthine were 176(SEM 34) and 723(73) nM, respectively, and the lactate concentration was 0.47(0.07) mM. Lactate was extracted by cardiac tissue during normoxia, and adenosine and hypoxanthine were in the physiological range of healthy volunteers. During reperfusion the concentrations of all myocardial metabolites were temporarily increased. In particular, adenosine was enhanced in close proportion to the duration of coronary occlusion. Moreover, coronary sinus adenosine, but not lactate, was significantly lowered during reperfusion when nifedipine (0.2 mg) was given by intracoronary injection before PTCA. CONCLUSIONS: With longer periods of coronary occlusion (> 30 s) the relative rank order of sensitivity indicating myocardial ischaemia was adenosine > lactate > hypoxanthine > uric acid. Coronary sinus concentrations of adenosine are quantitatively sufficient to be responsible for some of the changes in coronary blood flow occurring during reactive hyperaemia.

Thrombin augments vascular cell-dependent migration of human mast cells: role of MGF.

Recent data suggest that auricular thrombosis is associated with accumulation of mast cells (MC) in the upper endocardium (where usually no MC reside) and local expression of MGF (mast cell growth factor) (25). In this study, the role of vascular cells, thrombin-activation and MGF, in MC-migration was analyzed. For this purpose, cultured human auricular endocardial cells (HAUEC), umbilical vein endothelial cells (HUVEC) and uterine- (HUTMEC) and skin-derived (HSMEC) microvascular endothelial cells were exposed to thrombin or control medium, and the migration of primary tissue MC (lung, n = 6) and HMC-1 cells (human MC-line) against vascular cells (supernatants) measured. Supernatants (24 h) of unstimulated vascular cells (monolayers of endocardium or endothelium) as well as recombinant (rh) MGF induced a significant migratory response in HMC-1 (control: 3025 +/- 344 cells [100 +/- 11.4%] vs. MGF, 100 ng/ml: 8806 +/- 1019 [291 +/- 34%] vs. HAUEC: 9703 +/- 1506 [320.8 +/- 49.8%] vs. HUTMEC: 8950 +/- 1857 [295.9 +/- 61.4%] vs. HSMEC: 9965 +/- 2018 [329.4 +/- 66.7%] vs. HUVEC: 9487 +/- 1402 [313.6 +/- 46.4%], p < 0.05) as well as in primary lung MC. Thrombin-activation (5 U/ml, 12 h) of vascular cells led to an augmentation of the directed migration of MC as well as to a hirudin-sensitive increase in MGF synthesis and release. Moreover, a blocking anti-MGF antibody was found to inhibit MC-migration induced by unstimulated or thrombin-activated vascular cells. Together, these data show that endocardial and other vascular cells can induce migration of human MC. This MC-chemotactic signal of the vasculature is associated with expression and release of MGF, augmentable by thrombin, and may play a role in the pathophysiology of (auricular) thrombosis.

The incidence of neoplasms in Syrian hamsters with particular emphasis on intestinal neoplasia.

The incidence of neoplasms is presented for 600 Syrian hamsters (Mesocricetus auratus) used as controls in the evaluation of carcinogenicity of various experimental viral vaccines administered as a single subcutaneous injection at birth. Approximately 30% of the animals had neoplasms with no appreciable sex difference in the overall tumor incidence. The most frequent tumor types were those of the adrenal cortex (13.5%), the lymphoreticular system (3%), and the endometrium (3%). Small intestinal adenocarcinomas occurred in 0.8% of the animals. In this laboratory, the incidence of intestinal tumors is greater and more variable than that in the mouse or the rat. Furthermore, several unusual morphologic and biologic features of this tumor type were identified. These findings raise questions about the suitability of the Syrian hamster in carcinogenic bioassays.

Nodular hyperplasia of the liver in the beagle dog.

Gross and light microscopic features of focal nodular hyperplasia of the liver were investigated in beagle dogs used for the study of the long-term effects of low-dose, whole-body, 60Co gamma radiation. The nodules were morphologically similar in the irradiated and unirradiated dogs. Microscopically, and normal lobular architecture, although retained, often was distorted. Hepatocytes in the nodules often were vacuolated and the distribution of vacuolated cells varied from focal to diffuse. There were more hepatocytes per unit area within nodules than in the adjacent parenchyma. Nodule hepatocytes were more variable in size. A lower proportion were binucleate and a higher proportion were in mitosis. The cells in the nodules contained less hemosiderin. The incidence of hyperplastic nodules was directly related to age but not to sex, was higher in irradiated dogs than in controls. Nodular hyperplasia occurred in association with primary hepatocellular neoplasms in only two dogs.

Stimulation of tissue factor expression in human microvascular and macrovascular endothelial cells by cultured vascular smooth muscle cells in vitro.

The effect of conditioned media obtained from different smooth muscle cells (SMC) on tissue factor (TF) expression in endothelial cells (EC) in vitro was investigated. We could show that conditioned media from cultured human aortic SMC, human umbilical artery SMC or human umbilical vein SMC all resembling the synthetic phenotype of SMC induced TF activity in human umbilical vein EC and human skin microvascular EC in a dose- and time-dependent fashion. This induction was also seen at the level of specific TF mRNA as evidenced by Northern blotting. The TF inducing activity was heat-labile and acid-stable and had an approximate molecular mass of 38 kD. This activity was found to be distinct from known inducers of TF expression in EC such as interleukin-1, tumor necrosis factor-alpha, bacterial lipopolysaccharide or vascular endothelial growth factor. Such as factor, if released by SMC in vivo, could contribute to the activation of EC under conditions such as when EC are in close contact with SMC of the synthetic (nondifferentiated) phenotype seen in processes like vessel development or neo-intima formation.

Human fibroblasts downregulate plasminogen activator inhibitor type-1 in cultured human macrovascular and microvascular endothelial cells.

We have previously reported that plasminogen activator inhibitor type-1 (PAI-1) expression in endothelial cells (ECs) can be modulated differently by smooth muscle cells depending on their origin. Human pulmonary artery smooth muscle cells (HPASMCs) strongly downregulated PAI-1 expression in ECs. Fibroblasts (FBs) are another cell type that could come in close contact with ECs. Therefore, it was the aim of this study to investigate whether FBs could also influence the fibrinolytic potential of ECs. As in the case of HPASMCs, PAI-1 antigen produced by human umbilical vein ECs (HUVECs) cocultured with human skin FBs (HSFBs) was significantly lower as compared with the sum of PAI-1 secreted by the respective cell types cultured separately. Not only HUVECs but also human skin microvascular ECs (HSMECs) responded in a dose-dependent way to serum-free conditioned media (CM) from HSFBs from one individual donor. Similar results were obtained when CM from HSFBs from four other individual donors were used. PAI-1 mRNA decreased in HUVECs incubated for 6 hours with HSFB-CM to 24% to 55% of control, depending on the preparation of HSFBs used. A significant PAI-1 downregulatory effect was only observed when CM from low-passage HSFBs (up to passage no. 5) was used, whereas no reduction in EC PAI-1 production was observed with CM obtained from HSFBs in passage no. 8. This PAI-1 downregulatory activity present in HSFB-CM was heat-labile and had a molecular mass of approximately 5 kD. When CM from HPASMCs was analyzed in the same way, an almost identical elution profile was found. In conclusion, our data showed that FBs can decrease the expression of PAI-1 in ECs. Such an effect could be operative during wound-healing and at other capillary sites where FBs could render ECs profibrinolytic, thereby facilitating processes requiring an increase in proteolytic activity such as EC migration and proliferation.

Hepatocyte growth factor stimulates expression of plasminogen activator inhibitor type 1 and tissue factor in HepG2 cells.

HGF is a powerful mitogen for both rat and human hepatocytes, epithelial cells and endothelial cells in vitro, and is angiogenic in vivo. It has considerable homology with plasminogen and has been shown to upregulate urokinase-type plasminogen activator (u-PA) in endothelial cells as well as u-PA and its receptor in kidney epithelial cells. In this study, we report that human recombinant HGF stimulates expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue factor (TF) in the human hepatoma cell line HepG2. PAI-1 antigen as determined by a specific enzyme-linked immunosorbent assay increased up to threefold in conditioned media of HepG2. This increase was dose dependent with maximum stimulation achieved with a concentration of 50 ng/mL of hepatocyte growth factor (HGF). PAI-1 antigen also increased up to fourfold in the extracellular matrix in HGF treated HepG2. The production of the PAI-1 binding protein vitronectin (Vn) was not affected by HGF. In contrast, TF activity in HepG2 treated with HGF increased up to twofold. As determined by Northern blotting, PAI-1 and TF-specific mRNA were increased significantly in the presence of HGF, whereas Vn mRNA was not affected. The increase in PAI-1 and TF mRNA was also seen when HepG2 were incubated with HGF in the presence of cycloheximide, thereby indicating that de novo protein synthesis is not required to mediate the effect. u-PA could be detected neither in unstimulated or HGF-stimulated HepG2 cells on the antigen level nor on the mRNA level. In conclusion, our data give evidence that HGF, in addition to its proliferative effect for different cell types, is also involved in the local regulation of fibrinolysis and coagulation. One could speculate that HGF might modulate processes requiring matrix degradation by increasing the expression of the protease u-PA in one cell type and by upregulating the expression of the serine protease inhibitor PAI-1 in a different cell type. Because u-PA has been shown to activate latent HGF to the active form, it could furthermore be speculated that by upregulating PAI-1, which in turn could inhibit u-PA, HGF might regulate its own activation.

Estrogen does not induce the calcium-dependent nitric oxide synthase in cultured human uterine endothelial and myometrial smooth muscle cells.

In many tissues, estrogen-induced vasodilatation is mediated, at least in part, by the release of nitric oxide (NO). We determined whether human myometrial endothelial and smooth muscle cells express estrogen receptors (ERs) and whether endothelial NO synthase (eNOS) expression in these cells was affected by 17beta-estradiol (10[-13]-10[-6]M). ER was strongly expressed in myometrial smooth muscle cells but was absent from endothelial cells. Expression of eNOS mRNA was strong in endothelial cells, but weak in muscle cells. 17beta-estradiol administration for 24 or 72 h failed to increase eNOS in both cell types. Thus, an increase of human uterine blood flow by estrogens appears not to be mediated by stimulation of myometrial eNOS expression.

Serum albumin is a specific inhibitor of apoptosis in human endothelial cells.

Excess blood vessels are removed by apoptosis of endothelial cells, however, the signals responsible for this have not been defined. Apoptosis of cultured human umbilical vein endothelial cells is induced by deprivation of serum or adhesion. In this paper, apoptosis in human umbilical vein and microvascular endothelium was induced by deprivation of serum and or adhesion. Apoptosis was confirmed on the basis of morphology, ultrastructure and internucleosomal cleavage of DNA. Loss of endothelial adhesion was found to be an early event in cultured endothelial cell apoptosis and was exploited to quantitate apoptosis. The effect of: bovine serum albumin; human serum albumin; recombinant human albumin; dithiothreitol reduced human and bovine albumin; CNBr treated human and bovine albumin as well as ovalbumin upon endothelial apoptosis was determined. Native bovine and human albumin as well as recombinant human material inhibited apoptosis at physiological concentrations with identical dose response curves in both umbilical vein and microvascular cells. Dithiothreitol treatment destroyed all protective activity while bovine but not human albumin was partially inactivated by CNBr treatment. The unrelated protein ovalbumin was not protective. Albumin did not inhibit apoptosis if cells were also deprived of adhesion. The data suggest that albumin is a specific inhibitor of human endothelial apoptosis but does not protect cells also deprived of adhesion. Reduced supply of albumin to endothelium in poorly perfused blood vessels may provide a mechanism for the removal of excess blood vessels in remodelling tissues. Also, the failure of albumin to protect endothelial cells deprived of adhesion from apoptosis may reflect the need to remove potentially micro-embolic cells detached due to trauma.

Preclinical safety evaluation of the benzodiazepine quazepam.

7-Chloro-5-(2-fluorophenyl)-1,3-dihydro-1-(2,2,2-trifluoroethyl)-2H-1,4- benzodiazepine-2-thione (quazepam, Sch 16134, Dormalin) was evaluated for evidence of systemic toxicity, carcinogenicity and reproductive toxicity in several laboratory animal species including the hamster. Mutagenic potential was also assessed in one in vivo and three in vitro assays. In some studies, diazepam was used as a comparative control. Oral LD50 values were greater than 5000 mg/kg in the mouse and rat while i.p. LD50 values were approximately 900 and 2900 mg/kg in the mouse and rat, respectively. Studies in hamsters for 4 weeks at doses up to 500 mg/kg/d and for 51 weeks at doses up to 120 mg/kg/d demonstrated that the liver was the principal target organ in this species with the effects upon the liver related to dose and duration of dosing. Studies in the squirrel monkey for 13 and 52 weeks at doses up to 50 mg/kg/d demonstrated a transient ataxia, hypoactivity and somnolence during the initial two weeks of dosing. No unusual necropsy or microscopic observations were noted in the 13-week study. Male reproductive organs of quazepam-dosed monkeys were reduced in weight after 52 weeks. Moderate to marked impairment of spermatogenesis and higher liver weights with moderate to marked fatty change in both sexes were observed in groups given diazepam. Abrupt withdrawal of quazepam or diazepam after 52 weeks of dosing was associated at all dose levels with excitability, hyperactivity and convulsions. Two quazepam- and all diazepam-dosed monkeys died.(ABSTRACT TRUNCATED AT 250 WORDS)