Sphingosine-1-phosphate inhibits PDGF-induced chemotaxis of human arterial smooth muscle cells: spatial and temporal modulation of PDGF chemotactic signal transduction.

Activation of the PDGF receptor on human arterial smooth muscle cells (SMC) induces migration and proliferation via separable signal transduction pathways. Sphingosine-1-phosphate (Sph-1-P) can be formed following PDGF receptor activation and therefore may be implicated in PDGF-receptor signal transduction. Here we show that Sph-1-P does not significantly affect PDGF-induced DNA synthesis, proliferation, or activation of mitogenic signal transduction pathways, such as the mitogen-activated protein (MAP) kinase cascade and PI 3-kinase, in human arterial SMC. On the other hand, Sph-1-P strongly mimics PDGF receptor-induced chemotactic signal transduction favoring actin filament disassembly. Although Sph-1-P mimics PDGF, exogenously added Sph-1-P induces more prolonged and quantitatively greater PIP2 hydrolysis compared to PDGF-BB, a markedly stronger calcium mobilization and a subsequent increase in cyclic AMP levels and activation of cAMP-dependent protein kinase. This excessive and prolonged signaling favors actin filament disassembly by Sph-1-P, and results in inhibition of actin nucleation, actin filament assembly and formation of focal adhesion sites. Sph-1-P-induced interference with the dynamics of PDGF-stimulated actin filament disassembly and assembly results in a marked inhibition of cell spreading, of extension of the leading lamellae toward PDGF, and of chemotaxis toward PDGF. The results suggest that spatial and temporal changes in phosphatidylinositol turnover, calcium mobilization and actin filament disassembly may be critical to PDGF-induced chemotaxis and suggest a possible role for endogenous Sph-1-P in the regulation of PDGF receptor chemotactic signal transduction.

Treatment of chronic hepatitis C genotype 3 with Sofosbuvir-based therpy: a real-life study.

BACKGROUND AND AIMS: Recently, Sofosbuvir was launched in India at affordable cost. We conducted a real-life study to determine the efficacy and safety of Sofosbuvir plus Ribavirin, with and without peginterferon-alfa 2a, in patients with chronic hepatitis C (CHC) genotype 3, the commonest genotype in South Asia. METHODS: This study included data of CHC patients from 11 sites in northern India between March 2015 and December 2015 (n = 1203). Patients with CHC genotype 3 (n = 931), who were treated with either Sofosbuvir 400 mg plus weight-based Ribavirin, daily ×24 weeks (n = 432) (dual therapy), or Peginterferon-α2a 180 mcg weekly, Sofosbuvir 400 mg plus weight-based Ribavirin, daily ×12 weeks (n = 499) (triple therapy) were included for analysis. Primary outcome was the proportion of patients achieving sustained viral response at 12 weeks post-therapy. RESULTS: The overall SVR rates were 91 and 92% in the dual and triple therapy arms, respectively. The SVR rates in treatment experienced were 67 and 74% versus 93 and 96% in naïve patients, on the dual and triple therapy arms, respectively. The SVR rates of cirrhotics were 73 and 75% on the dual and triple treatment arms, respectively. The SVR rates were low in the experienced cirrhotic patients: 44% (dual therapy) and 58% (triple therapy). Common adverse events were fatigue, headache, and myalgia. CONCLUSION: Both dual and triple therapy regimes resulted in SVR rates of >95% in CHC genotype 3 who were naive non-cirrhotics. However, the SVR rates were low in treatment-experienced cirrhotics.

Modulation of xenobiotic-inducible cytochrome P450 gene expression by dexamethasone in primary rat hepatocytes.

Our previous studies (Sidhu JS et al. Arch Biochem Biophys 1993: 301, 103-113; Sidhu JS et al. In Vitro Toxicol 1994: 7, 225-242) demonstrated the importance of culturing primary rat hepatocytes with an overlay of extracellular matrix (ECM), together with optimal media formulations (Williams' E or Chee's), to efficiently maintain in vivo-like responsiveness of phenobarbital (PB)-inducible cytochrome P450 genes in vitro. In the present report, we have characterized culture conditions further by examining individual and interactive effects of dexamethasone (Dex) and PB on CYP2B1, CYP2B2, and CYP3A1 expression. Dex alone was not effective in enhancing CYP2B1 or CYP2B2 expression levels. However, together with PB, addition of low concentrations (10(-9)-10(-8) M) of Dex resulted in a marked potentiation of PB-inducible P450 gene expression. In contrast, at levels > 10(-7) M, Dex profoundly inhibited PB induction of the CYP2B1 and CYP2B2 genes. The overall stimulatory response to Dex was more dramatic in cells cultured in Williams' E than in Chee's medium. Similarly, concentrations of PB > 0.5 mM resulted in substantially reduced levels of CYP2B1 and CYP2B2 induction than those attainable at lower PB concentrations. These results suggest that Dex and PB function cooperatively to regulate the CYP2B1 and CYP2B2 genes, and that composite interactions may either negatively or positively regulate expression, in a concentration-dependent manner. CYP3A1 was not regulated in a similar biphasic fashion, as this gene was fully responsive even at high dose levels of PB or Dex. With respect to other marker genes evaluated, high Dex concentrations (> 10(-7) M) were only marginally inhibitory to beta-naphthoflavone-mediated induction of CYP1A1 and CYP1A2 mRNAs, and did not perturb expression of the liver-selective serum albumin gene. Addition of Dex was critical, however, to maintain glutathione S-transferase Pi expression, a marker of hepatocyte dedifferentiation, in the repressed state. Defining optimal culture conditions for maintaining hepatocyte differentiation in vitro are requisite for establishing primary culture models enabling investigation of the molecular mechanisms of PB-mediated gene regulation.

Effects of chemical inducers on human microsomal epoxide hydrolase in primary hepatocyte cultures.

Human microsomal epoxide hydrolase (mEH; EC 3.3.2.3) is an important biotransformation enzyme and potential risk determinant for pathologies such as cancer and teratogenesis. Currently, the effects of chemical exposures on human mEH gene expression are largely unknown, but they may constitute a unique modifier of disease susceptibility. To examine this issue, we exposed cultures of primary human hepatocytes isolated from seven donors to prototypic chemical inducers [such as phenobarbital (PB), polyaromatic hydrocarbons, dexamethasone, butylated hydroxyanisole, and ciprofibrate]. Basal levels of mEH RNA and protein were detected readily in untreated cells. Chemical treatment of cultured hepatocytes resulted in variable mEH RNA and protein expression, but, in general, only modest modulatory effects were detected following these exposures. The maximum increase in mEH RNA expression observed was approximately 3.5-fold following Arochlor 1254 exposure. Immunochemical levels of mEH protein were quantified for all treatment groups in three cultures and demonstrated less overall variation and, in general, a lack of concordance with corresponding mEH RNA levels. Cytochrome P450 (CYP) 1A2 and 3A mRNA levels were measured before and following exposure to beta-naphthaflavone and PB, respectively, to permit independent evaluation of hepatocyte inducer responsiveness. Substantial increases in RNA expression levels for both the CYP1A2 and CYP3A genes demonstrated that the hepatocyte cultures were robust and highly responsive to inducer treatment. These results indicate that the mEH gene in human hepatocytes is only modestly responsive to chemical exposures.

Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.

Invasive cervical cancer in human immunodeficiency virus-infected and uninfected hospital patients.

OBJECTIVE: To compare the prevalence of invasive cervical cancer in women with, and in women without, human immunodeficiency virus (HIV) infection, so as to evaluate the inclusion of invasive cervical cancer in the AIDS surveillance case definition. METHODS: The Sentinel Hospital Surveillance System for HIV Infection collected data and serum specimens that remained after clinical testing of persons who received inpatient or outpatient care at 14 hospitals with high HIV prevalence. We analyzed data on invasive cervical cancer obtained from medical record review and HIV serostatus from white, black, and Hispanic women in the age groups 20-34, 35-44, and 45-54 years. RESULTS: In 1994 and 1995, 2684 (6.6%) of the 40,524 women sampled were HIV infected. Of the HIV-positive women, 28 had invasive cervical cancer (10.4 per 1000 women) and of the HIV-negative women, 236 had invasive cervical cancer (6.2 per 1000 women, relative risk [RR] 1.7, 95% confidence interval [CI] 1.1, 2.5). The prevalence of invasive cervical cancer was higher for HIV-positive than for HIV-negative black women aged 20-34 (RR 3.8; CI 1.7, 8.5) and Hispanic women aged 20-34 (RR 7.3; CI 1.4, 37.1) and 35-44 (RR 3.9; CI 1.1, 14.7) years. Twenty-six of the 28 cases of invasive cervical cancer in HIV-positive women were in women known to be HIV-positive during admission. CONCLUSION: The prevalence of invasive cervical cancer was higher for women who were HIV positive than for women who were HIV negative. This lends support to the inclusion of invasive cervical cancer in the revision of the surveillance case definition for AIDS in 1993.

Single-dose, comparative study of venous, capillary and salivary artemisinin concentrations in healthy, male adults.

A comparison of venous plasma, capillary plasma, and saliva pharmacokinetics of artemisinin was performed in four healthy subjects given a single 500 mg dose. Artemisinin was determined to be 88% bound in venous plasma. Saliva levels were more closely related with (unbound) capillary than with venous plasma concentrations. Matrix comparisons demonstrated artemisinin saliva/plasma and capillary/venous concentration ratios of well over one initially, which decreased with time to stabilize after a 3-hr period. Following this stabilization period, differences in capillary and saliva (relative to venous) levels were independent of both drug concentration and time. These observations, together with improved correlations of concentrations measured 3 hr following drug intake compared with correlations for the entire data set, indicate a putative arterial-venous concentration difference for the drug. The capillary and saliva matrices proved promising replacements for venous sampling in the quantitation of artemisinin concentrations. Due to their relative case of obtainment and a greater patient acceptability, capillary and saliva sampling may be of particular value in field-based and pediatric pharmacokinetic studies with artemisinin.

Papillary fibroelastoma of the left atrial appendage.

Cardiac papillary fibroelastomas are benign tumors that usually arise from the valvular endocardium. They are clinically important because of their propensity to embolize. We describe, to the best of our knowledge, the first reported case of a papillary fibroelastoma arising from the left atrial appendage, giving rise to multiple cerebral embolic events. The tumor was excised surgically, with no further embolic events.

Simple microscale high-performance liquid chromatographic method for determination of furosemide in neonatal plasma.

A simple microscale high-performance liquid chromatographic method using fluorescence detection for the quantitation of furosemide in neonatal plasma is described. Sample pre-treatment involved protein precipitation of 25 microliters of plasma with 100 microliters of acetonitrile. The mobile phase consisted of acetonitrile (460 ml) and 0.08 M orthophosphoric acid (540 ml) and was delivered at 1.1 ml/min. Calibration curves were linear from 0.1 to 25 micrograms/ml. Within-day and between-day imprecision (coefficient of variation) was 3.9-6.1, and 6.1-12.2%, respectively. Furosemide was eluted after 6.5 min and naproxen (internal standard) after 11.5 min. The assay was validated for application in neonatal plasma containing a wide range of albumin concentrations.

cAMP-associated inhibition of phenobarbital-inducible cytochrome P450 gene expression in primary rat hepatocyte cultures.

The effects of elevated intracellular cyclic adenosine monophosphate (cAMP) in regulating phenobarbital (PB)-inducible gene expression in primary rat hepatocyte cultures were investigated. Cells were exposed to various concentrations (0.1-100 microM) of cAMP analogs and/or activators of intracellular cAMP-dependent pathways. Effects of these treatments were assessed either using a 1-h pulse prior to PB (100 microM) exposure or in conjunction with PB during a 24-h exposure period. PB-inducible responses were measured in hepatocytes by hybridization to cytochrome P450 (CYP) CYP2B1, CYP2B2, and CYP3A1 mRNAs. The cAMP analogs, 8-bromo-cAMP, 8-(4-chlorophenylthio)-cAMP, dibutyryl cAMP, and (Sp)-5,6-DCl-cBiMPS ((Sp)-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-monophosphorothioate), and the activators of adenylate cyclase, forskolin and glucagon, dramatically inhibited PB-mediated induction of CYP2B1 and CYP2B2 in a concentration-dependent manner. A similar inhibition of PB-induced CYP3A1 mRNA levels was effected by the cAMP analogs and glucagon. The phosphodiesterase inhibitors isobutylmethylxanthine and RO 201724 potentiated the cAMP responses. Increasing the concentration of PB (0.05-1.00 mM) did not alleviate the cAMP-mediated repression. A requirement for protein kinase A (PKA) was demonstrated by the use of (Sp)-cAMPS, a highly specific activator of PKA, whereas the inactive diastereoisomer, (Rp)-cAMPS, was ineffective in modulating PB induction. The response to cAMP was specific since elevated intracellular cAMP levels did not perturb beta-naphtholflavone-mediated induction of CYP1A1, CYP1A2, microsomal epoxide hydrolase, or dexamethasone-mediated induction of CYP3A1 gene expression. Nor did elevated intracellular cAMP modulate the liver-selective albumin gene expression levels. The results of the present study demonstrated striking inhibition of PB-mediated CYP gene induction by cAMP and PKA activators, indicating a negative regulatory role for the cAMP signal transduction pathway on PB gene induction.

Forskolin-mediated induction of CYP3A1 mRNA expression in primary rat hepatocytes is independent of elevated intracellular cyclic AMP.

Previously, we demonstrated that elevated levels of cyclic AMP (cAMP) repressed phenobarbital (PB)-inducible cytochrome P450 (CYP)2B gene expression in primary rat hepatocyte cultures. Although CYP3A1 induction by PB was similarly repressed by most of the cAMP-enhancing strategies, forskolin additions in particular resulted in marked stimulation of CYP3A1 expression. Here we examined whether this effect was due to forskolin's ability to activate adenylate cyclase. By using a specific ELISA for assessment of intracellular cAMP levels, we determined that forskolin and a water-soluble analog (L858051; 7 beta-desacetyl-7 beta-(N-methylpiperazine)) were equipotent in stimulating adenylate cyclase activity. However, only forskolin and its inactive 1,9-dideoxy analog were active as inducers of CYP3A1. In comparative studies, both dexamethasone and PB were ineffective in stimulating production of intracellular cAMP. Furthermore, treatment of hepatocytes with glucagon, dibutyryl-cAMP, or N6O2'-dibutyryl-cyclic GMP, resulted in no detectable enhancement of CYP3A1 gene expression. These results demonstrated that CYP3A1 induction by forskolin is independent of cAMP, and instead is likely to involve a direct chemical effect of forskolin on the CYP3A1 activation pathway.

An okadaic acid-sensitive pathway involved in the phenobarbital-mediated induction of CYP2B gene expression in primary rat hepatocyte cultures.

We have previously demonstrated that specific activation of a cAMP-dependent protein kinase A (PKA) pathway resulted in complete repression of phenobarbital (PB)-inducible CYP gene expression in primary rat hepatocyte cultures. In the current investigation, we examined the role of protein phosphatase pathways as potential co-regulators of this repressive response. Primary rat hepatocytes were treated with increasing concentrations (0.1-25 nM) of okadaic acid, a potent inhibitor of serine/threonine-specific protein phosphatases PP1 and PP2A. PB induction responses were assessed by use of specific hybridization probes to CYP2B1 and CYP2B2 mRNAs. Okadaic acid completely inhibited the PB induction process in a concentration-dependent manner (IC50, approximately 1.5-2 nM). Similar repression was obtained with low concentrations of other highly specific phosphatase inhibitors, tautomycin and calyculin A. In contrast, exposure of hepatocytes to 1-nor-okadaone or okadaol, negative analogs of okadaic acid largely devoid of phosphatase inhibitory activity, was without effect on the PB induction process. At similar concentrations, okadaic acid produced only comparatively weak modulation of the beta-naphthoflavone-inducible CYP1A1 gene expression pathway. In additional experiments, hepatocytes were treated with suboptimal concentrations of PKA activators together with phosphatase inhibitors. Okadaic acid markedly potentiated the repressive effects of dibutyryl-cAMP on the PB induction process. Together, these results indicate that both PKA and protein phosphatase (PP1 and/or PP2A) pathways exert potent and complementary control of the intracellular processes modulating the signaling of PB in cultured primary rat hepatocytes.

Protein synthesis inhibitors exhibit a nonspecific effect on phenobarbital-inducible cytochome P450 gene expression in primary rat hepatocytes.

Previous investigations have indicated that de novo protein synthesis is a critical requirement for phenobarbital (PB) induction. We reexamined this issue in PB-responsive primary rat hepatocyte cultures using a broader array of protein synthesis inhibitors and experimental end points. Anisomycin, cycloheximide, emetine, puromycin, and puromycin aminonucleoside, a negative analog, were evaluated for their respective effects on protein synthesis and the PB-induction process. All of the inhibitors effectively repressed de novo protein synthesis in the cells in a concentration-dependent manner. However, anisomycin only minimally effected PB induction, ascertained though the measure of CYP2B1, CYP2B2, and CYP3A1 mRNA levels. The inactive agent, puromycin aminonucleoside, produced marked repression of the PB-induction response. Results from further experiments demonstrated that these protein synthesis inhibitors stimulated rapid and differential phosphorylation of the stress-activated protein kinase/c-Jun kinase (SAPK/JNK) pathway, indicating nonselective actions on cellular processes. Puromycin aminonucleoside was without effect on these pathways, despite its efficacy as an inhibitor of PB induction. These results demonstrate that de novo protein synthesis is not a requirement for PB induction, nor is activation of the SAPK/JNK kinase cascade responsible for down-regulating PB responsiveness in primary hepatocytes.

An alleged poisoning with methanol and formaldehyde.

It was alleged that a defendant added an unspecified amount of undyed formalin solution, containing formaldehyde and methanol, to the victim's bottle of ice and drinking water. The medical report indicated that except for a slight elevation of total creatine kinase, all other chemistry profiles were within normal ranges. The elevation of creatine kinase suggested muscle injury and inflammation; however, the significance of this elevation was not clear. Toxicological evaluations were made by conducting risk assessments. Based upon the medical report and risk assessments, the following conclusions were made: The calculated exposure doses of methanol and formaldehyde were too low to cause appreciable adverse effects; however, formaldehyde may have irritated the gastrointestinal tract causing smooth muscle and mucosal inflammation. The doses of methanol and formaldehyde were too low to cause death. The exposure scenario (a single oral exposure to formaldehyde) would not likely increase the cancer risk in the victim. The risk assessments provided resulted in a reduction in charge from attempted murder to felony.

Insulin-mediated modulation of cytochrome P450 gene induction profiles in primary rat hepatocyte cultures.

In this investigation, we examined the effects of insulin on gene induction responsiveness in primary rat hepatocytes. Cells were cultured for 72 hours either in the absence or presence of 1 microM insulin and then exposed to increasing concentrations of phenobarbital (PB; 0.01-3.5 mM). Culturing in the absence of insulin produced 1.5-2-fold increases in the induction magnitude of CYP2B1 and CYP2B2 mRNA expression resulting from PB exposures, without altering the bell-shaped dose-response curve characteristic of this agent. However, for the CYP3A1 gene, insulin removal led to a pronounced shift in both the PB-induction magnitude and dose-response relationships of the induction response, with higher levels of CYP3A1 expression resulting from exposures to lower concentrations of inducer. Insulin removal also reduced the time required to attain maximal induction of CYP2B1/2 and CYP3A1 gene expression. The insulin effects were not specific for PB induction, as insulin deprivation similarly enhanced both dexamethasone- and beta-naphthoflavone-inducible CYP3A1 and CYP1A1 expression profiles, respectively. In contrast, the level of albumin mRNA expression was reduced considerably in cells deprived of insulin. We conclude that insulin is an important regulator of inducible and liver-specific gene expression in primary rat hepatocytes.