The Journal of laboratory and clinical medicine最新文献

Elevations in plasma angiotensin II (AngII) are associated with evidence of vascular hyperpermeability expressed as efflux of plasma macromolecules into the perivascular and interstitial space. This exudative response is followed by a series of fibrogenic events that lead to a perivascular fibrosis of involved vessels. Mediators of hyperpermeability and fibrogenesis are unknown. In dogs receiving intravenous AngII, hemodynamic factors (i.e., arterial hypertension or coronary venoconstriction) were discounted as being responsible for the rise in cardiac lymph-to-plasma protein ratio. Accordingly, we investigated the relationship between AngII-induced coronary hyperpermeability and the release of prostaglandin E2 (PGE2) and activation of the basement membrane degrading matrix metalloproteinase, gelatinase/type IV collagenase. In dogs, cardiac lymph was monitored over the course of a 90-minute intravenous infusion of either AngII (0.2 to 0.3 micrograms/kg/min; n = 8) or saline solution (n = 6). Lymph was examined at 30-minute intervals for the following: total protein (Lowry's method), albumin (sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)), plasma fibronectin (SDS-PAGE and enzyme-linked immunosorbent assay); PGE2 (radioimmunoassay) and gelatinase/type IV collagenase (zymography). In comparison with baseline we found a consistent rise in lymph flow (p = 0.02), total protein (p = 0.02), albumin, fibronectin, PGE2 (p = 0.03), and gelatinase/type IV collagenase (p = 0.019), which began after 30 minutes of AngII infusion. Similar trends were not observed in dogs receiving saline solution alone. We therefore conclude that AngII-induced coronary vascular hyperpermeability is associated with an early release of PGE2 and gelatinase.

Here the cognate interactions between human eosinophils and lymphocytes are studied. These interactions were measured in a double-colored FACS analysis by applying fluorescent red eosinophils (stained with hydroethidine, 40 mumol/L) and fluorescent green lymphocytes (stained with sulfidofluorescein diacetate, 100 mumol/L) in a ratio of 1:3. When normal eosinophils were mixed with a total lymphocyte preparation in stirred suspensions (37 degrees C), no physical interaction was present between both cell types. However, the addition of phytohemagglutinin and PMA resulted in a clear aggregation response between both cell types (up to 30% of the eosinophils interacted with lymphocytes after 15 minutes). CD8(+)- and CD4(+)-positive T cells contributed equally to the heterotypic aggregation response. It is interesting that when lymphocytes were pretreated with phytohemagglutinin or eosinophils with phorbol myristate acetate and subsequently washed, the cells still interacted with unstimulated counterparts. The heterotypic interaction between lymphocytes and eosinophils is blocked by monoclonal antibodies directed against the beta-chain of the beta 2-integrins (CLB LFA1/1; CD18) and the alpha-chain of very late antigen-4 (VLA-4) (HP2/1; CD49d), indicating that both the beta 2-integrins and VLA-4 contributed to this heterotypic interaction. When eosinophils bound to PHA-treated lymphocytes, the cells exhibited an activated phenotype that was characterized by an enhanced expression of CD66b and CD11b. The cells interacted with each other provided that an intact cellular metabolism was present--that is, no interaction was seen after treatment with the glycolysis inhibitor sodium mono-iodoacetate.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the effects of insulin, insulin-like growth factor-I (IGF-I), and phorbol 12-myristate 13-acetate (PMA) on neutral cholesteryl esterase activity in cultured rat vascular smooth muscle cells. Insulin and IGF-I at concentrations between 10(-9) mol/L and 10(-6) mol/L significantly decreased neutral cholesteryl esterase activity in growth-arrested vascular smooth muscle cells in a dose-dependent manner but with no influences on the intracellular concentration of 3',5'-adenosine monophosphate (cyclic AMP). Treatment of cells with KT5720 (10(-7) mol/L to 10(-5) mol/L), a specific inhibitor of cyclic AMP-dependent protein kinase, significantly decreased neutral cholesteryl esterase activity in a dose-dependent manner. Incubation of cells for 6 to 12 hours with PMA (10(-9) mol/L to 10(-6) mol/L), an activator of protein kinase C, significantly increased neutral cholesteryl esterase activity in a dose-dependent manner. However, down-regulation of protein kinase C activity by long-term incubation (18 to 48 hours) with PMA resulted in a significant decrease in neutral cholesteryl esterase activity. Treatment of cells with UCN-01 (10(-7) mol/L to 10(-5) mol/L), a specific protein kinase C inhibitor, decreased the enzyme activity in a dose-dependent manner and completely blocked the activation of the enzyme by PMA. When insulin or IGF-I at a concentration of 10(-6) mol/L was present in the medium containing CL 277,082--an inhibitor of acyl coenzyme A:cholesterol acyltransferase--cellular cholesteryl ester content of the cells significantly increased. In contrast, after the treatment with PMA at a concentration of 10(-6) mol/L in the presence of CL 277,082, the net cholesteryl ester content of the cells significantly declined. These data suggest that both insulin and IGF-I may increase cholesteryl ester accumulation in arterial smooth muscle cells by decreasing arterial cholesteryl ester hydrolysis. The data also suggest that neutral cholesteryl esterase is activated not only by cyclic AMP-dependent protein kinase but also by protein kinase C. Thus growth factors may exert their antilipolytic or lipolytic actions specifically by modulating neutral cholesteryl esterase activity in vascular smooth muscle cells. Neutral cholesteryl esterase of vascular smooth muscle cells may be regulated by recholesteryl esterase of vascular smooth muscle cells may be regulated by reversible phosphorylation, with the phosphorylated form being the active form.

We present evidence for the presence of specific, high-affinity binding sites for tritiated phorbol 12,13-dibutyrate on osteosarcoma-derived (HT-3) cells. Activation of protein kinase C by a phorbol ester resulted in an inhibition of alkaline phosphatase activity and the accumulation of prostaglandin E2. Indomethacin blocked prostaglandin E2 production and enhanced alkaline phosphatase activity. These data suggest that prostaglandin E2 is enhanced by activation of protein kinase C, and in turn, alkaline phosphatase activity is reduced.

We have previously shown that cyclic adenosine monophosphate (cAMP) increases thrombomodulin (TM) mRNA and protein in vascular smooth muscle cells (VSMCs). The mechanism of that enhancement is now further defined. A time course evaluation of this effect by Northern blot analysis showed that exposure to the cAMP analog dibutyryl-cAMP and theophylline (CT) amplified TM mRNA sixfold by 3 hours. This effect was sustained through 9 hours and began to decline by 24 hours of CT exposure. In vitro exposure of VSMCs either to CT and actinomycin D or to actinomycin D alone showed equivalent half-lives for TM mRNA. This indicates that the increase in TM mRNA with CT supplementation was not the result of enhanced mRNA stability. Nuclear run-off analysis of VSMCs grown in the presence of control or CT-supplemented medium showed that the increase in TM mRNA in VSMCs with CT exposure was transcriptional. CT exposure was associated with an eightfold increase in measured TM transcription at 90 minutes. As previously reported, cAMP induced a decrease in tropomyosin and in alpha-actin mRNA species, a change that paralleled the enhancement of TM. Thus cAMP enhances transcription of this antithrombotic species while simultaneously causing diminished expression of these myogenic mRNA species. Addition of cycloheximide prevented the cAMP-mediated increase in TM mRNA and curtailed the down-regulation of myogenic mRNA species, alpha-actin, and tropomyosin. This suggests that the cAMP-mediated down-regulation of some smooth muscle-specific mRNA, including tropomyosin mRNA and alpha-actin mRNA, like the enhancement of TM transcription, is dependent on de novo protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Human polymorphonuclear leukocytes (PMNLs) have recently been shown to release cytokines in response to a variety of inflammatory stimuli. Because adenosine (AR) modulates numerous functions of human PMNLs, the effect of the metabolic stable AR derivative 2-chloro-adenosine was tested on the production of tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide-stimulated PMNLs. In addition, the highly selective A1-receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and the A2 receptor agonist 5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA) were compared for their effects on the lipopolysaccharide-stimulated TNF-alpha production. All AR agonists inhibited the lipopolysaccharide-stimulated production of TNF-alpha in a concentration-dependent fashion. The rank order of the half-maximal inhibitory concentration (IC50) of the different agonists was as follows: IC50 (2-chloro-adenosine) approximately 10(-6) mol/L > IC50 (CCPA) approximately 5 x 10(-7) mol/L >> IC50 (CPCA) = 5 x 10(-10) mol/L. Because the A2 receptor agonist was 1000 times more effective than the A1 agonist, adenosine analogs inhibited the lipopolysaccharide-stimulated production of TNF-alpha of human PMNLs most likely via an A2 receptor site. Of note, CPCA inhibited the TNF-alpha production even when PMNLs had been stimulated by lipopolysaccharide for 2 hours previously. The potential pathophysiologic implications for patients with sepsis are discussed.

It is generally accepted that the anemia of uremia is caused by decreased production of erythropoietin. Nevertheless, the erythropoietin titers are not lower than but equal to or higher than in normal non-anemic individuals. To examine this discrepancy, erythrokinetic studies were made of 22 hematologically stable dialysis patients without clinical or laboratory evidence of extrarenal inflammation, infection, or neoplastic disorders. The red cell life span was normal in 14, and because of stable hematocrits, their daily rate of red cell production had to equal their daily rate of red cell destruction, which could be determined by dividing the red cell mass by red cell life span. These rates were about one half the rates of normal stable individuals, despite the same or higher erythropoietin titers. This suggests that the anemia of uremia is caused in part by a decreased bone marrow response to endogenous erythropoietin.