Enhanced wound healing using a 3D printed VEGF-mimicking peptide incorporated hydrogel patch in a pig model.

There is a need for effective wound healing through rapid wound closure, reduction of scar formation, and acceleration of angiogenesis. Hydrogel is widely used in tissue engineering, but it is not an ideal solution because of its low vascularization capability and poor mechanical properties. In this study, gelatin methacrylate (GelMA) was tested as a viable option with tunable physical properties. GelMA hydrogel incorporating a vascular endothelial growth factor (VEGF) mimicking peptide was successfully printed using a three-dimensional (3D) bio-printer owing to the shear-thinning properties of hydrogel inks. The 3D structure of the hydrogel patch had high porosity and water absorption properties. Furthermore, the bioactive characterization was confirmed by cell culture with mouse fibroblasts cell lines (NIH 3T3) and human umbilical vein endothelial cells. VEGF peptide, which is slowly released from hydrogel patches, can promote cell viability, proliferation, and tubular structure formation. In addition, a pig skin wound model was used to evaluate the wound-healing efficacy of GelMA-VEGF hydrogel patches; the results suggest that the GelMA-VEGF hydrogel patch can be used for wound dressing.

Analysis of contrast time-enhancement curves to optimise CT pulmonary angiography.

AIM: To assess enhancement profiles of the pulmonary artery (PA) and determine optimal scan timing in PA computed tomography (CT) angiography. MATERIALS AND METHODS: One hundred consecutive patients referred for contrast-enhanced chest CT were prospectively studied. Fifteen low-radiation monitoring images were acquired at 2-second intervals, 5 seconds after the start of an injection of 370 mg iodine/I contrast medium for 1 ml/kg of body weight injected over 20 seconds. Contrast time-enhancement data were measured over the PA. The time and magnitude of peak as well as times to five different enhancement thresholds (50, 100, 150, 200, 250 HU) were calculated. A set of candidate fixed and circulation-adjusted scan delays were analysed and compared in terms of the quality of contrast enhancement over the 4-second diagnostic scan duration. RESULTS: The mean degree of peak PA enhancements was 431.4±65.2HU (range, 263.8-575.3HU). The mean time to peak enhancement was 22.4±3.1 seconds (range, 11-27 seconds). From potential fixed delays ranging 11-27 seconds, 19 seconds showed the highest enhancement quality. For the circulation-adjusted delays, the combination of 150 HU bolus-track threshold with diagnostic delay of 10 seconds had the highest enhancement quality. CONCLUSION: Peak enhancement of PA occurred, on average, right after completion of contrast injection for 20 seconds. The fixed scan delay of 19 seconds or circulation-adjusted delay with the bolus-threshold of 150 HU and diagnostic delay of 10 seconds appear optimal.

Identification of human UGT isoforms responsible for glucuronidation of efavirenz and its three hydroxy metabolites.

Uridine 5'-diphosphate-glucuronosyltransferases (UGTs) involved in the glucuronide formation of efavirenz (EFV) and its three hydroxy metabolites, 8-hydroxyefavirenz (8-OH EFV), 7-hydroxyefavirenz (7-OH EFV), and 8,14-dihydroxyefavirenz (8,14-diOH EFV), were assessed. Among 12 recombinant UGT isoforms tested, only UGT2B7 showed catalytic activity in the formation of EFV-N-glucuronide (EFV-G) as previously reported. On the other hand, almost all UGT isoforms were involved in the glucuronidation of the three hydroxy metabolites, although their relative contribution is unclear. The catalytic activities in the formation of EFV-G by 17 different human liver microsomes exhibit a more than 40-fold inter-individual variability, whereas those of glucuronidation of the three hydroxy metabolites showed almost identical activity. The formation of EFV-G showed a significant correlation (r = 0.920; p < 0.0001) with UGT2B7-catalysed azidothymidine glucuronidation in 17 different human liver microsomes. Furthermore, fluconazole, a known UGT2B7 inhibitor, potently inhibited the formation of EFV-G up to 80%. This suggests that EFV might be a specific UGT2B7 substrate in vitro. This is the first study identifying specific UGT isozymes that glucuronidate EFV and its three hydroxy metabolites. Continued identification and characterisation of these pathways may help reduce adverse effects such as CNS toxicity in EFV therapy.

Pharmacokinetics of sildenafil and its metabolite, N-desmethylsildenafil, in rats with liver cirrhosis and diabetes mellitus, alone and in combination.

Pharmacokinetics of sildenafil and its metabolite, N-desmethylsildenafil, in humans and rats with liver cirrhosis (LC) and diabetes mellitus (DM), alone and in combination (LCD) did not seem to be reported. Sildenafil was administered intravenously (10 mg/kg) and orally (20 mg/kg) to control, LC, DM, and LCD rats. Expression of intestinal CYP isozymes in those rats was also measured. In LC, DM, and LCD rats, the areas under the curve (AUCs) of intravenous sildenafil were significantly greater (by 195%, 54.2%, and 127%, respectively) than controls. In LC and LCD rats, AUCs of oral sildenafil were significantly greater (3010% and 2030%, respectively) than controls. In LC, DM, and LCD rats, significantly greater AUCs of intravenous sildenafil were due to the slower hepatic extraction of sildenafil (because of decrease in the protein expression of hepatic CYP2C11 and 3A subfamily in LC and LCD rats, and CYP2C11 in DM rats). In LC and LCD rats, greater magnitude of increase in AUCs of oral sildenafil than those after the intravenous administration could be mainly due to the decrease in the intestinal extraction of sildenafil (because of decrease in the protein expression of intestinal CYP2C11 in LC and LCD rats).

Sonographic differentiation of asymptomatic diffuse thyroid disease from normal thyroid: a prospective study.

BACKGROUND AND PURPOSE: There is no useful guide or study related to the differentiation of asymptomatic diffuse thyroid disease from normal thyroid by using thyroid US. This study was prospectively designed to evaluate the efficacy of the use of real-time thyroid sonography as performed by an experienced radiologist for the identification of asymptomatic DTD. MATERIALS AND METHODS: From January 2008 to December 2008, 2267 patients underwent thyroid sonography in our hospital by 1 radiologist. Each patient's thyroid was prospectively classified as being in 1 of 4 of the following diagnostic categories on the basis of the sonographic features as determined with the use of real-time sonography: suggestive for DTD, suspicious for DTD, indeterminate, and no evidence of DTD. We calculated the diagnostic efficacy of the sonographic classifications compared with the pathology results. RESULTS: Sonographic classifications for DTD in 340 patients who underwent thyroid surgery because of thyroid malignancy or other causes included the following: suggestive for DTD (n = 32), suspicious for DTD (n = 39), indeterminate (n = 18), and no evidence of DTD (n = 251). On the pathology, HT (n = 33), chronic lymphocytic thyroiditis (n = 27), diffuse hyperplasia (n = 2), and NTP (n = 278) were identified. There were true-positive cases (n = 50), true-negative cases (n = 244), false-positive cases (n = 21), and false-negative cases (n = 7). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for a diagnosis of asymptomatic DTD were 87.7%, 92.1%, 70.4%, 97.2%and 91.3%, respectively. CONCLUSIONS: The present sonographic classification based on real-time sonography of the thyroid is a useful tool for differentiating asymptomatic DTD from normal thyroid.

Interaction between udenafil and tamsulosin in rats: non-competitive inhibition of tamsulosin metabolism by udenafil via hepatic CYP3A1/2.

BACKGROUND AND PURPOSE: Orthostatic hypotension has been observed when PDE 5 (cGMP-specific phosphodiesterase type 5) inhibitors are co-administered with alpha-adrenoceptor antagonists. Here we assessed the pharmacokinetic and haemodynamic interactions between udenafil and tamsulosin in rats, as both drugs are metabolized via rat hepatic cytochrome P450 3A1/2. EXPERIMENTAL APPROACH: Interactions between the two drugs were evaluated in rats after simultaneous 1 or 15 min i.v. infusion or after p.o. administration of udenafil (30 mg x kg(-1)) and/or tamsulosin (1 mg x kg(-1)). In vitro metabolism of tamsulosin with udenafil was measured to obtain the inhibition constant (K(i)) and [I]/K(i) ratio of udenafil. KEY RESULTS: The total area under the plasma concentration-time curve from time zero to time infinity (AUC)s (or AUC(0-4 h)) of tamsulosin were significantly greater after 15 min of i.v. infusion or after oral administration with udenafil, compared with tamsulosin alone. The hepatic first-pass metabolism of tamsulosin was inhibited by udenafil, and the inhibition in vitro was in a non-competitive mode. The arterial systolic blood pressure was significantly lower at 5, 10 and 60 min after oral co-administration of the drugs. CONCLUSIONS AND IMPLICATIONS: The significantly greater AUC of tamsulosin after i.v. and p.o. administration of both drugs may be attributable to non-competitive inhibition of cytochrome P450 3A1/2-mediated hepatic tamsulosin metabolism by udenafil. The inhibition was also observed in human liver S9 fractions, suggesting that a reassessment of the oral dosage of tamsulosin is necessary when udenafil and tamsulosin are co-administered to patients with benign prostatic hyperplasia.

Pharmacokinetics of oltipraz in diabetic rats with liver cirrhosis.

BACKGROUND AND PURPOSE: The incidence of diabetes mellitus is increased in patients with liver cirrhosis. Oltipraz is currently in trials to treat patients with liver fibrosis and cirrhosis induced by chronic hepatitis types B and C and is primarily metabolized via hepatic cytochrome P450 isozymes CYP1A1/2, 2B1/2, 2C11, 2D1 and 3A1/2 in rats. We have studied the influence of diabetes mellitus on pharmacokinetics of oltipraz and on expression of hepatic, CYP1A, 2B1/2, 2C11, 2D and 3A in rats with experimental liver cirrhosis. EXPERIMENTAL APPROACH: Oltipraz was given intravenously (10 mg x kg(-1)) or orally (30 mg x kg(-1)) to rats with liver cirrhosis induced by N-dimethylnitrosamine (LC rats) or with diabetes, induced by streptozotocin (DM rats) or to rats with both liver cirrhosis and diabetes (LCD rats) and to control rats, and pharmacokinetic variables measured. Protein expression of hepatic CYP1A, 2B1/2, 2C11, 2D and 3A was measured using Western blot analysis. KEY RESULTS: After i.v. or p.o. administration of oltipraz to LC and DM rats, the AUC was significantly greater and smaller, respectively, than that in control rats. In LCD rats, the AUC was that of LC and DM rats (partially restored towards control rats). Compared with control rats, the protein expression of hepatic CYP1A increased, that of CYP2C11 and 3A decreased, but that of CYP2B1/2 and 2D was not altered in LCD rats. CONCLUSIONS AND IMPLICATIONS: In rats with diabetes and liver cirrhosis, the AUC of oltipraz was partially restored towards that of control rats.

Contribution of cytochrome P450 3A4 and 3A5 to the metabolism of atorvastatin.

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor that is mainly metabolized by cytochrome P450 (CYP) 3A4. A recent study showed that the lipid-lowering effect of statins is affected by the CYP3A5 polymorphism. Therefore, it was investigated whether CYP3A5 contributes to the metabolism of atorvastatin. Two metabolites of atorvastatin, para- and ortho-hydroxyatorvastatin, were produced by human liver microsomes and human recombinant CYP3A enzymes, and the enzyme kinetic pattern exhibited substrate inhibition. The intrinsic clearance (CL(int)) rates of para- and ortho-hydroxyatorvastatin by CYP3A4 were 2.4- and 5.0-fold of the respective CL(int) rates of CYP3A5, indicating that CYP3A4 is the major P450 isoform responsible for atorvastatin metabolism. These results suggest that atorvastatin is preferentially metabolized by CYP3A4 rather than by CYP3A5, and thus the genetic CYP3A5 polymorphism might not be an important factor in the inter-individual variation of atorvastatin disposition and pharmacodynamics in human.

Pharmacokinetic and pharmacodynamic consequences of inhibition of terazosin metabolism via CYP3A1 and/or 3A2 by DA-8159, an erectogenic, in rats.

BACKGROUND AND PURPOSE: Recently, orthostatic hypotension was observed in patients with benign prostatic hyperplasia who are taking vardenafil (a PDE 5 inhibitor) and terazosin (a long acting alpha blocker). Therefore, this study was performed with DA-8159 (a long acting PDE 5 inhibitor) and terazosin in rats to find whether or not pharmacokinetic and pharmacodynamic interactions between the two drugs were observed. EXPERIMENTAL APPROACH: Pharmacokinetic and pharmacodynamic (changes in blood pressure) interactions between DA-8159 and terazosin were evaluated after simultaneous i.v. and p.o. administration of DA-8159 (30 mg kg(-1)) and terazosin (5 mg kg(-1)) to male Sprague-Dawley rats. KEY RESULTS: After simultaneous i.v. and p.o. administration of terazosin and DA-8159, the total area under the plasma concentration-time curve from time zero to time infinity (AUC) of terazosin became significantly greater (57.4 and 75.4% increase for i.v. and p.o. administration, respectively) than those of without DA-8159. The blood pressure dropping effect was considerable after simultaneous p.o. administration of DA-8159 and terazosin compared with each drug alone. CONCLUSIONS AND IMPLICATIONS: The significantly greater AUC of terazosin after both simultaneous i.v. and p.o. administration of both drugs could be due to the hepatic (both i.v. and p.o.) and intestinal (p.o.) inhibition of the metabolism of terazosin via CYP3A1 and/or 3A2 by DA-8159, since both DA-8159 and terazosin are metabolized via CYP3A1 and/or 3A2 in rats. The blood pressure lowering effect after simultaneous p.o. administration of both drugs could be due to significant increase in plasma concentrations of terazosin.

Bioequivalence of pravastatin tablet formulations assessed in Korean males.

AIM: Determination of the bioequivalence of 2 pravastatin tablet formulations manufactured in Korea. PATIENTS AND METHODS: Twenty-three healthy male Korean volunteers received each of the 2 pravastatin formulations at a dose of 20 mg in a 2 x 2 crossover study. There was a 1-week washout period between doses. Plasma concentrations of pravastatin were monitored using high-performance liquid chromatography over a period of 8 hours after administration. AUC(0-8h) (the area under the plasma concentration-time curve from time zero to the last measured time in plasma, 8 h) was calculated using the linear-log trapezoidal method. Cmax (maximum plasma drug concentration) and tmax (time to reach Cmax) were compiled from the plasma concentration-time data. Analysis of variance was carried out using logarithmically transformed AUC(0-8h) and Cmax and untransformed tmax. RESULTS: The point estimates and 90% confidence intervals for AUC(0-8h) (parametric) and Cmax (parametric) were 1.067 (0.968 to approximately 1.176) and 1.074 (0.999 to approximately 1.155), respectively, satisfying the bioequivalence criteria of the European Committee for Proprietary Medicinal Products and the US Food and Drug Administration guidelines. The corresponding value of tmax was 0.000 (-0.250 to approximately 0.250). CONCLUSION: These results indicate that the 2 medications of pravastatin are bioequivalent and, thus, may be prescribed interchangeably.

Math6, a bHLH gene expressed in the developing nervous system, regulates neuronal versus glial differentiation.

BACKGROUND: Whereas multiple basic helix-loop-helix (bHLH) genes are expressed in the developing nervous system, they account for the differentiation of only subsets of neurones, suggesting that there may be as-yet unidentified bHLH genes. RESULTS: We have isolated a novel bHLH gene, designated Math6, a distant mammalian homologue of the Drosophila proneural gene atonal. Structural analysis of the Math6 gene demonstrated that the coding region is divided into three exons, whereas that of other atonal homologues is present in a single exon, indicating that the genomic structure of Math6 is unique among the atonal homologues. Math6 is initially expressed by neural precursor cells in the ventricular zone, but later by subsets of differentiating and mature neurones such as hippocampal neurones and cerebellar Purkinje cells. Mis-expression of Math6 with retrovirus in the developing retina induced neurogenesis, while inhibiting gliogenesis, without affecting cell proliferation and death. CONCLUSIONS: These results show that cells which would normally differentiate into glia adopted the neuronal fate by mis-expression of Math6, indicating that Math6 promotes neuronal vs. glial fate determination in the nervous system.

The usefulness of serum transferrin receptor and ferritin for assessing anemia in rheumatoid arthritis: comparison with bone marrow iron study.

OBJECTIVES: This study was aimed at investigating the usefulness of serum transferrin receptor (sTfR) and ferritin in anemic patients with rheumatoid arthritis (RA) compared with bone marrow storage iron and other tests for anemia. METHODS: Fifty-five anemic RA patients underwent anemia study. Bone marrow iron stain was performed in 18 patients. sTfR and serum ferritin levels were compared with bone marrow iron stores. RESULTS: (1) Mean sTfR concentration was 2.63+/-1.91 mg/L, (2) sTfR correlated with most indicators of anemia, (3) sTfR showed no correlation with CRP and ESR, whereas ferritin did, and (4) sTfR was higher in the "iron depleted" subgroup than in the "iron nondepleted" subgroup in bone marrow study. CONCLUSION: The measurement of sTfR and ferritin is useful in finding the cause of anemia in RA and is a possible substitute for invasive bone marrow iron study.

hGFRalpha-4: a new member of the GDNF receptor family and a candidate for NBIA.

Hallervorden-Spatz syndrome (neurodegeneration with brain iron accumulation type 1; OMIM entry 234200) is a rare inherited neurodegenerative disease. In this article, evidence for a newly identified gene as a candidate for Hallervorden-Spatz syndrome is given. Previously Hallervorden-Spatz syndrome was mapped to a 4-cm region in 20p12.3-13. During positional cloning efforts a new member of the glial-derived neurotrophic factor receptor family was discovered in this region. Like other members of this receptor family, this new gene is predicted to be secreted and glycosyl-phosphatidylinositol linked, and it maintains conserved cysteine residues. However, cDNA and genomic studies in both humans and mice indicate that this gene lacks the sequence corresponding to exons 2 and 3 in other family members. In situ hybridization reveals that it is expressed primarily in the brain and bladder in the embryonic mouse. Mutation analysis of patients with Hallervorden-Spatz syndrome revealed two potentially significant amino acid changes in two patients but failed to identify mutations in the remaining 10 subjects. The implication of these findings for the relationship between this gene and Hallervorden-Spatz syndrome is discussed.

A case of hypothermia combined with systemic lupus erythematosus.

Systemic lupus erythematosus(SLE) is a prototypic autoimmune disease affecting various organ systems. Hypothermia is a rare manifestation of SLE. We experienced a case of SLE combined with hypothermia. A 36-year-old woman, who had been diagnosed as SLE 3 days before admission, admitted complaining of mental confusion. After admission, her body temperature, initially 36.1 degrees C, became 32.6 degrees C. Her core body temperature was less than 35.0 degrees C. Despite of warming with heating lamp and blankets, her core temperature did not reach 35.0 degrees C during 18 hours. Ten days later, her temperature exceeded 36.0 degrees C.

The basic helix-loop-helix gene hesr2 promotes gliogenesis in mouse retina.

Members of a subclass of hairy/Enhancer of split [E(spl)] homologs, called hesr genes, are structurally related to another subclass of hairy/E(spl) homologs, Hes genes, which play an important role in neural development. To characterize the roles of hesr genes in neural development, we used the retina as a model system. In situ hybridization analysis indicated that all hesr genes are expressed in the developing retina, but only hesr2 expression is associated spatially with gliogenesis. Each member was then misexpressed with retrovirus in the retinal explant cultures prepared from mouse embryos or neonates, which well mimic in vivo retinal development. Interestingly, hesr2 but not hesr1 or hesr3 promoted gliogenesis while inhibiting rod genesis without affecting cell proliferation or death, suggesting that the cells that normally differentiate into rods adopted the glial fate by misexpression of hesr2. The gliogenic activity of hesr2 was more profound when it was misexpressed postnatally than prenatally. In addition, double mutation of the neuronal determination genes Mash1 and Math3, which increases Müller glia at the expense of bipolar cells, upregulated hesr2 expression. These results indicate that, among structurally related hesr genes, only hesr2 promotes glial versus neuronal cell fate specification in the retina and that antagonistic regulation between hesr2 and Mash1-Math3 may determine the ratios of neurons and glia.

Determination of hypoxic region by hypoxia marker in developing mouse embryos in vivo: a possible signal for vessel development.

Hypoxia is a well-known signal for angiogenesis, but the recent proposal that hypoxia exists in developing embryonic tissues and that it induces vascular development remains to be proven. In the present study, we demonstrate the presence of hypoxia in normal developing embryos by means of a hypoxia marker, pimonidazole, and its associated antibody. Our data clearly show that hypoxia marker immunoreactivity was highly detected in developing neural tubes, heart, and intersomitic mesenchyme at an early stage of organogenesis, suggesting that hypoxia may exist in the early stages of embryo development. We also found that hypoxia inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) were spatiotemporally co-localized with possible hypoxic regions in embryos. Investigation of platelet endothelial cell adhesion molecule (PECAM) expression provides evidence that endothelial cells proliferate and form the vessels in the hypoxic region in developing organs. Furthermore, we found that hypoxia induced both HIF-1alpha and VEGF in F9 embryonic stem and differentiated cells. Thus, we suggest that hypoxia may exist widely in developing embryonic tissues and that it may act as a signal for embryonic blood vessel formation in vivo.

Determination of a chemopreventive agent, Oltipraz, in rat plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the determination of a chemopreventive agent, Oltipraz, in rat plasma and urine. The sample preparation was simple; 2 volumes of acetonitrile were added to deproteinize the biological sample. A 50-microl aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase, acetonitrile : 0.5 mM ammonium acetate (55: 45, v/v for rat plasma and 45 : 55, v/v for rat urine), was run at a flow-rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector set at 305 nm. The retention times for Oltipraz in rat plasma and urine were approximately 5.8 and 8.6 min, respectively. The detection limits of Oltipraz in rat plasma and urine were 20 and 50 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 4.65%) in concentration ranges from 0.02 (0.05) to 10 microg/ml for rat plasma and urine. No interference from endogenous substances was found.

Human hepatitis B virus X protein is a possible mediator of hypoxia-induced angiogenesis in hepatocarcinogenesis.

The hepatitis B virus (HBV)-encoded transcriptional activator HBV-X protein (HBx) was known to be involved in hepatocarcinogenesis. Hepatocarcinogenesis generally included an active angiogenesis that was mainly considered to be due to a local hypoxia in liver tissues. However, the exact mechanisms of HBx-induced hepatocarcinogenesis were poorly understood. In this study, we examined the role of HBx in the increased angiogenesis and the possible regulating mechanisms of HBx by hypoxia. We demonstrated that HBx stimulated the transcription of vascular endothelial growth factor (VEGF), a potent angiogenic factor, in HBx-stable transfectants. HBx-induced angiogenesis was confirmed by in vivo tumor angiogenesis assay, resulting in that the HBx transfectants increased the formation of new blood vessels compared to the control transfectants. Then, we demonstrated that the expression of HBx was enhanced after incubating HBV-infected hepatoma cells under hypoxia. Moreover, the activity of HBV enhancer 1 (Enh1) was increased when hepatoma cells transfected with the reporter plasmid containing HBV Enh1 were exposed to hypoxic conditions. These results strongly suggest that HBx may play a critical role in the hypoxia-induced angiogenesis through transcriptional activation of VEGF during hepatocarcinogenesis.