Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck.

Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR34/L98H (11 isolates), TR46/Y121F/T289A (6 isolates), TR53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing.

Preventive effect of fermented Maillard reaction products from milk proteins in cardiovascular health.

The aim of this study was to determine the dual effect of Maillard reaction and fermentation on the preventive cardiovascular effects of milk proteins. Maillard reaction products (MRP) were prepared from the reaction between milk proteins, such as whey protein concentrates (WPC) and sodium caseinate (SC), and lactose. The hydrolysates of MRP were obtained from fermentation by lactic acid bacteria (LAB; i.e., Lactobacillus gasseri H10, L. gasseri H11, Lactobacillus fermentum H4, and L. fermentum H9, where human-isolated strains were designated H1 to H15), which had excellent proteolytic and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities (>20%). The antioxidant activity of MRP was greater than that of intact proteins in assays of the reaction with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and trivalent ferric ions; moreover, the effect of MRP was synergistically improved by fermentation. The Maillard reaction dramatically increased the level of antithrombotic activity and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitory effect of milk proteins, but did not change the level of activity for micellar cholesterol solubility. Furthermore, specific biological properties were enhanced by fermentation. Lactobacillus gasseri H11 demonstrated the greatest activity for thrombin and HMGR inhibition in Maillard-reacted WPC, by 42 and 33%, respectively, whereas hydrolysates of Maillard-reacted SC fermented by L. fermentum H9 demonstrated the highest reduction rate for micellar cholesterol solubility, at 52%. In addition, the small compounds that were likely released by fermentation of MRP were identified by size-exclusion chromatography. Therefore, MRP and hydrolysates of fermented MRP could be used to reduce cardiovascular risks.

The dual effects of Maillard reaction and enzymatic hydrolysis on the antioxidant activity of milk proteins.

The objective of this study was to determine the enhanced effects on the biological characteristics and antioxidant activity of milk proteins by the combination of the Maillard reaction and enzymatic hydrolysis. Maillard reaction products were obtained from milk protein preparations, such as whey protein concentrates and sodium caseinate with lactose, by heating at 55°C for 7 d in sodium phosphate buffer (pH 7.4). The Maillard reaction products, along with untreated milk proteins as controls, were hydrolyzed for 0 to 3h with commercial proteases Alcalase, Neutrase, Protamex, and Flavorzyme (Novozymes, Bagsværd, Denmark). The antioxidant activity of hydrolyzed Maillard reaction products was determined by reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, their 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and the ability to reduce ferric ions. Further characteristics were evaluated by the o-phthaldialdehyde method and sodium dodecyl sulfate-PAGE. The degree of hydrolysis gradually increased in a time-dependent manner, with the Alcalase-treated Maillard reaction products being the most highly hydrolyzed. Radical scavenging activities and reducing ability of hydrolyzed Maillard reaction products increased with increasing hydrolysis time. The combined products of enzymatic hydrolysis and Maillard reaction showed significantly greater antioxidant activity than did hydrolysates or Maillard reaction products alone. The hydrolyzed Maillard reaction products generated by Alcalase showed significantly higher antioxidant activity when compared with the other protease products and the antioxidant activity was higher for the whey protein concentrate groups than for the sodium caseinate groups. These findings indicate that Maillard reaction products, coupled with enzymatic hydrolysis, could act as potential antioxidants in the pharmaceutical, food, and dairy industries.

Electrophysiological safety of sibutramine HCl.

Sibutramine is known to induce cardiovascular side effects such as tachycardia, vasodilation, and hypertension. The present study was aimed to examine the effects of sibutramine on action potential of guinea pig papillary muscle, recombinant hERG currents (IhERG), and inward currents (INa and ICa) of rat ventricular myocytes. Sibutramine at 30 mug/mL induced a shortening of action potential duration (APD) of guinea pig papillary muscle; on average, APD30 and APD90 were shortened by 23% and 17% at a stimulation rate of 1 Hz, respectively. Sibutramine suppressed the following currents: IhERG (IC50:2.408 +/- 0.5117 microg/mL), L-type Ca current (IC50:2.709 +/- 0.4701 microg/mL), and Na current (IC50:7.718 +/- 1.7368 microg/mL). Sibutramine blocked IhERG, ICa, and INa in a concentration-dependent manner. In conclusion, sibutramine exerted a shortening effect on APD in guinea pig papillary muscle through its more powerful blocking effects on ICa and INa rather than IhERG.

Risk Factors for Dropout From the Liver Transplant Waiting List of Hepatocellular Carcinoma Patients Under Locoregional Treatment.

BACKGROUND: In new organ allocation policy, patients with hepatocellular carcinoma (HCC) experience a 6-month delay in being granted Model for End-Stage Liver Disease exception points. However, it may not be fair for patients at risk of early progression of HCC. METHODS: All patients who were diagnosed as United Network for Organ Sharing (UNOS) stage 1 or 2 of HCC between January 2004 and December 2012 were included. Patients who received surgical resection or liver transplant (LT) as a primary treatment and who did not receive any treatment for HCC were excluded. Patients with baseline Model for End-Stage Liver Disease score ≥22 were also excluded because they have a higher chance of receiving LT. Patients who developed extrahepatic progression within 1 year were considered as high-risk for early recurrence after LT. RESULTS: A total of 586 patients were included. Mean (SD) age was 59.9 (10.3) years and 409 patients (69.8%) were men. The cumulative incidence of estimated dropout was 8.9% at 6 months; size of the maximum nodule (≥3 cm) and nonachievement of complete response were independent factors. Extrahepatic progression developed in 16 patients (2.7%) within 1 year; size of the maximum nodule (4 cm) and alpha-fetoprotein level (>100 ng/mL) were independent predictors. CONCLUSIONS: The estimated dropout rate from the waiting list within 6 months was 8.9%. Advantage points might be needed for patients with maximum nodule size ≥3 cm or those with noncomplete response. However, in patients with maximum nodule size ≥4 cm or alpha-fetoprotein level >100 ng/mL, caution is needed.

Intensity of surveillance for hepatocellular carcinoma determines survival in patients at risk in a hepatitis B-endemic area.

BACKGROUND: Data are insufficient regarding the survival benefit of surveillance for hepatocellular carcinoma (HCC). AIM: To investigate the effectiveness of HCC surveillance in a hepatitis B-endemic population. METHODS: This retrospective cohort study included 1402 consecutive patients who were newly diagnosed with HCC between 2005 and 2012 at a single tertiary hospital in Korea. The primary endpoint was overall survival. Lead-time and length-time biases were adjusted (sojourn time = 140 days) and sensitivity analyses were performed. RESULTS: The most common aetiology was hepatitis B (80.4%). Cirrhosis was present in 78.2%. HCC was diagnosed during regular surveillance (defined as mean interval of ultrasonography <8 months, n = 834), irregular surveillance (n = 104) or nonsurveillance (n = 464). Patients in the regular surveillance group were diagnosed at earlier stages ([very] early stage, 64.4%) than the irregular surveillance (40.4%) or nonsurveillance (26.9%) groups and had more chance for curative treatments (52.4%) than the irregular surveillance (39.4%) or nonsurveillance (23.3%) groups (all P < 0.001). Mortality risk was significantly lower in the regular surveillance group (adjusted hazard ratio [aHR], 0.69; 95% [CI], 0.57-0.83) but not in the irregular surveillance group (aHR, 0.94; 95% CI, 0.69-1.28) compared with the nonsurveillance group after adjusting for confounding factors and lead-time. When the subjects were restricted to cirrhotic patients or Child-Pugh class A/B patients, similar results were obtained for mortality risk reduction between groups. CONCLUSIONS: HCC surveillance was associated with longer survival owing to earlier diagnosis and curative treatment. Survival advantage was significant with regular surveillance but not with irregular surveillance.

Bio-distribution and anti-tumor efficacy of PEG/PLA nano particles loaded doxorubicin.

As a more effective in vivo drug delivery system, several methods loading anti-cancer drugs to biodegradable and biocompatible nano-particles have been explored and developed. Supposedly due to the enhanced permeability and retention (EPR) effect, systemic administration of these nano-particles have been found to result in accumulation of nano-particles into solid tumors. In this study, we prepared nano-particles using polyethylene glycol (PEG)/poly-L-lactide (PLLA) diblock copolymer and loaded doxorubicin into these nano-particles (Nano-dox). The fabricated nano-particles exhibited sustained release kinetics of the drug in vitro. To follow the in vivo biodistribution of 200-350 nm sized nano-dox particles in tumor (syngenic renal cell adenocarcinoma: RENCA) bearing mouse, the carboxylfluorescenin diacetate succinimidyl ester (CFSE) was loaded into the nano-particles. Nano-dox accumulated preferentially in tumors; however, in terms of its anti-tumor efficacy, it did not show any marked benefits, compared to freely-administered doxorubicin. This result suggests the need to re-consider and evaluate what type of anti-cancer reagents we to be used in the ongoing efforts of coupling drug delivery system with tumor EPR effects.

A nonisotopic estrogen receptor-based assay to detect estrogenic compounds.

We have used the ligand binding domain of the recombinant human estrogen receptor (hER) to develop a nonisotopic assay for detection of estrogenic compounds. The assay is based on competition of the estrogenic ligand with 17beta-estradiol for binding to the receptor, which leaves 17beta-estradiol free to bind to an anti-17beta-estradiol antibody. Unbound anti-17beta-estradiol antibody then binds to immobilized 17beta-estradiol-protein conjugate (to which hER is unable to bind for steric reasons), and is detected by an enzyme-labeled anti-rabbit IgG antibody. We used the assay to detect estrogenic compounds (mainly members of the flavonoid group of plant polyphenols) in a variety of commonly consumed plant foods.

Cellular mechanisms for trazodone-induced cardiotoxicity.

The second-generation selective 5-HT2 receptor antagonists and reuptake inhibitors (SARIs) class antidepressants are known to have fewer cardiovascular side effects than the older ones. However, several case reports showed that trazodone, one of the second-generation SARIs, induces QT prolongation, cardiac arrhythmia, and ventricular tachycardia. Although these clinical cases suggested trazodone-induced cardiotoxicity, the toxicological actions of trazodone on cardiac action potentials (APs) beyond the human ether-a-go-go related gene (hERG) remain unclear. To elucidate the cellular mechanism for the adverse cardiac effects of trazodone, we investigated its effects on cardiac APs and ion channels using whole-cell patch clamp techniques in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and transiently transfected human embryonic kidney cells (HEK293) with cardiac ion channel complementary DNA. Trazodone dose-dependently decreased the maximum upstroke velocity (Vmax) and prolonged the AP duration, inducing early after depolarizations at 3 and 10 µM that triggered ventricular arrhythmias in hiPSC-CMs. Trazodone also inhibited all of the major ion channels (IKr, IKs, INa, and ICa), with an especially high inhibitory potency on hERG. These data indicate that the prolonged AP duration and decreased Vmax due to trazodone are mainly the result of hERG and sodium ion inhibition, and its inhibitory effects on cardiac ion channels can be exhibited in hiPSC-CMs.

Nuclear magnetic resonance studies of blood plasma and urine from subjects with chronic renal failure: identification of trimethylamine-N-oxide.

We have used 1H-, 13C- and 14N-NMR spectroscopy to investigate the constituents of plasma and urine in 16 patients with chronic renal failure (CRF). Resonances not previously observed in spectra of plasma from healthy volunteers were seen in CRF plasma, including those for trimethylamine-N-oxide (TMAO) and dimethylamine (DMA). A possible analogy with the plasma of elasmobranch fishes, in which TMAO stabilizes proteins in the presence of very high urea concentrations, is noted. The intensity of the TMAO resonance for CRF subjects was correlated with the plasma concentration of urea (R = 0.55) and creatinine (R = 0.74), suggesting that the presence of TMAO is closely related to the degree of renal failure. When normal subjects ate a meal of TMAO-containing fish, TMAO appeared rapidly in the plasma and in the urine. Thus TMAO is efficiently cleared by the healthy kidney. Differences in the interaction of lactate with plasma proteins were detected by NMR, suggesting that uraemia impairs their transport roles.

Challenges facing sex preselection of stallion spermatozoa.

Since the production of the first live offspring from sex-sorted spermatozoa in 1989, there have been many developments in the fluorescence-activated cell separation (FACS) procedures to preselect X- and Y-chromosome bearing spermatozoa prior to insemination. During this time, FACS technology has been applied to a range of species and has resulted in offspring from rabbits, cattle, sheep, elk and horses. In horses, satisfactory fertility rates have been achieved after hysteroscopic insemination of 20 x 10(6) fresh or stored, sex-sorted spermatozoa. However, many of the sperm processing protocols are still based on the original protocol and components of these procedures may not necessarily be suitable for the stallion. This review examines the details of FACS protocols that have resulted in the production of live offspring and makes comparisons with the published stallion protocols in an attempt to determine how best to improve the fertility of sorted, frozen-thawed stallion spermatozoa.

An observational study on diagnoses of 3,233 Gulf Veterans (Op Granby 1990-91) who attended the Ministry of Defence's Medical Assessment Programme 1993-2004.

BACKGROUND: This is the result of an observational study on 3,233 Gulf veterans who have attended our medical assessment programme. We wanted to determine as a result of in-depth interviews, full medical examination and appropriate investigations, whether there was any unique Gulf war related medical condition. METHODS: Over a period of 10 years, 3,233 veterans have been assessed. All diagnoses have been made according to ICD-10 classifications. All psychiatric diagnoses have been confirmed by consultant psychiatrists. FINDINGS: 75% of veterans were well. Of the 25% unwell, 83% of ill health was accounted for by a psychiatric disorder. 3% of veterans had organic conditions which could be linked to Gulf deployment. The most common of these were respiratory disorders, followed by digestive disorders, injuries and skin disorders. Only 11 of these cases could be linked to the use of medical countermeasures. A further, 51 cases (41 respiratory disorders, 6 infections, 2 skin disorders and 2 eye conditions) could be linked to environmental conditions. INTERPRETATION: All veterans seen with health problems could be identified as per ICD-10 classification of disease. We did not find any medically unexplained conditions. We found no evidence of a unique 'Gulf War Syndrome'.

L-carnitine and acetyl-L-carnitine status during hemodialysis with acetate in humans: a kinetic analysis.

The effects of the acetate content of hemodialysis fluids on the relation between L-carnitine (free carnitine, cr FC) and acetyl-L-carnitine (AC) have not previously been examined in detail. The net fluxes of FC, AC, and acetate between intra- and extracellular pools during hemodialysis were calculated using a kinetic model with dialysates containing three concentrations of FC (0, 40, and 80 mumol/L) and either 40 or 3 mmol acetate/L. Radioenzymatic assays of FC and AC were optimized for use with samples taken during hemodialysis. Acetate stimulated a tissue uptake of FC (P < 0.05) that could exceed the rate of FC delivery and was related to the dialysate FC composition (P < 0.02). There were associated changes in tissue AC output. With dialysate containing 40 mmol acetate/L, AC tissue output was directly related to the dialysate FC composition (P < 0.05). The AC tissue output was less with dialysate containing 3 mmol acetate/L (P < 0.05) but the significant increase with the provision of FC in the dialysate was retained (P < 0.05). Hemodialysis may therefore represent an acute period of relative carnitine deficiency when regeneration of free coenzyme A from acetyl coenzyme A consequent to metabolism of acetate is limited.

Comparison of the effects of keto acid analogues and essential amino acids on nitrogen homeostasis in uremic patients on moderately protein-restricted diets.

Comparisons of isonitrogenous supplements (1.2 g N) of essential amino acids and five keto acid analogues with four essential amino acids were made in seven patients with stable chronic renal failure (creatinine clearance, 4.6 to 16 ml/min) on moderately protein-restricted diets (4.60 to 7.8 g N per day). Full nitrogen balance data on the four patients who have already completed studies lasting 24 weeks are presented. No benefits of keto acid over amino acid supplements were observed. Two transient episodes of hypercalcemia occurred during keto acid treatment. There was no improvement of renal function with keto acids. Also, no carry-over effects were seen after keto acid treatment. It is concluded that any beneficial effects of keto acids in patients with chronic renal failure are only likely to occur in those taking a diet of less than 30 g protein daily.

The effect of probucol and cholestyramine combination therapy in severe familial hypercholesterolaemia.

The relative effects of cholestyramine and probucol, alone and in combination, on serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and the ratio of TC to HDL-C (T/HDL-C) have been studied in a group of 10 subjects with severe familial hypercholesterolaemia. The combination of cholestyramine plus probucol was more potent than either agent alone in reducing TC. The use of probucol either alone or in combination reduced the HDL-C concentrations, but the ratio of T/HDL-C was not consistently affected. The effect of the combination therapy did not diminish after periods of at least 6 months and in some cases led to partial regression of long-standing xanthomata. The combination of probucol with cholestyramine did not give rise to additional side effects and it is considered to be a valuable addition to the regimens available for the treatment of severe familial hypercholesterolaemia.

Monoclonal anti-idiotype antibody mimicking the pesticide binding site of cutinase: potential for broad specificity organophosphate recognition.

An anti-idiotype monoclonal antibody (Mab) able to bind the organophosphate pesticides, chlorfenvinphos (CFV), ethyl paraoxon, tetrachlorfenvinphos and demeton-s-methyl, has been produced using as immunogen a Mab which binds to the active site of cutinase. The principle of using an anti-idiotype antibody as the mimic of a site on a protein able to bind a group of ligands has, therefore, been demonstrated, and may have implications for future research on broad specificity immunoanalysis of groups of compounds.

Renal transplant in a patient with major donor-recipient blood group incompatibility: reversal of acute rejection by the use of modified plasmapheresis.

A 47-year-old patient with blood group O inadvertently received a mismatched kidney from a donor of blood group A. Two days after transplantation, the clinical, biochemical manifestation of an intrarenal, intravascular coagulation was seen. This was treated by plasma exchange with the rapid reversal of all parameters, and reduction of both the IgG and IgM component of the circulating anti-A antibody. The patient, 20 months after transplantation, has normal renal function. Subsequent repeated biopsies have shown no recurrence or manifestation of the effects of the intravascular coagulopathy. This case report documents, for the first time, the reversal of the known deleterious effects of major blood group incompatibility on renal transplantation by the use of a new technique, modified plasmapheresis. Furthermore, it implies that the limitation of transplantation of kidneys on the basis of major ABO blood groups may not be justifiable in all instances.

Serum C-reactive protein. A useful and economical marker of immune activation in renal transplantation.

This study investigated whether serial daily measurements of serum C-reactive protein (sCRP) in 187 renal allograft recipients could help discriminate episodes of renal dysfunction due to rejection or cyclosporine (CsA) nephrotoxicity and help adjust immunosuppression in the early posttransplant period. Excellent primary graft function was associated with an initial peak of sCRP on day 2 after transplant (median, 29 microg/ml; range, 4 to >200 microg/ml) with a return to <20 microg/ml in all patients by day 5 (median, 7 microg/ml; range, 2-19 microg/ml). Stable graft function (mean creatinine, 155 microg/ml) was accompanied by a median sCRP of 4 microg/ml (range, 1-19 microg/ml). In 30 episodes of rejection responsive to methylprednisolone, sCRP was initially significantly raised to a median of 49 microg/ml (P<0.001) but fell rapidly in response to treatment to a median of 11 microg/ml and continued to fall. In 19 episodes of rejection unresponsive to methylprednisolone, median initial sCRP levels were significantly higher (P<0.001) at 119 microg/ml and were still at a median of 77 microg/ml at the end of the treatment. Twenty-four patients in whom renal dysfunction was associated with CsA nephrotoxicity showed no increase in sCRP concentrations; median sCRP concentrations remained at <5 microg/ml throughout the episodes. A similar pattern was seen in patients with acute tubular necrosis. Serial sCRP measurements provide economical and reproducible evidence of immune activation, help discriminate renal dysfunction due to CsA nephrotoxicity or rejection, and allow appropriate modification of immunosuppressive therapy.

Plasma exchange in acute renal allograft rejection. A controlled trial.

A controlled trial was carried out to assess the value of intensive plasma exchange in 27 renal transplant recipients with clinical and histological evidence of acute vascular rejection. In addition to standard immunosuppression, 13 patients received plasma exchange on six consecutive days at a mean exchange volume of 40.6 ml/kg of body weight each day using an intermittent cell separator. A further 14 patients received standard immunosuppression only. In addition, 10 patients exchanged prior to the controlled trial have been studied. Analysis of short-term benefit, as evidenced by a reduction in serum creatinine, and by subsequent graft survival revealed no significant difference in these parameters between the two groups in the controlled trial. This regimen of plasma exchange has not, therefore, been shown to modify acute renal allograft rejection.

Influence of 5,10-methylenetetrahydrofolate reductase gene polymorphism on plasma homocysteine concentration in patients with end-stage renal disease.

The purpose of this study is to observe the influence of the methylenetetrahydrofolate reductase (MTHFR) gene (677C-->T substitution) on plasma homocysteine levels in end-stage renal disease (ESRD) patients who received a relatively large amount of folate (2 mg/d) and are undergoing hemodialysis. A cross-sectional study of plasma homocysteine, vitamin B(12), and folate was performed in patients with ESRD. The study population for the MTHFR gene study included 312 healthy subjects and 106 patients with ESRD undergoing hemodialysis. The C677T transition in the MTHFR gene was detected by HinF 1 restriction enzyme analysis and subsequent electrophoresis in a 3% agarose gel. The genotype of the MTHFR gene in 106 patients with ESRD was homozygous C677T mutation (VV) in 17 patients (16.1%) and heterozygous (AV) in 63 patients (58.4%); 26 patients (24.5%) did not carry this mutation (AA). The mean levels of homocysteine, vitamin B(12), and folate in the patients with ESRD were 23.3 +/- 14.0 mmol/L, 620.2 +/- 98.5 pmol/L, and 138.6 +/- 55.6 nmol/L, respectively. There was no significant difference in homocysteine levels among the three genotypes: 28.2 +/- 19.4 mmol/L for VV, 22.7 +/- 14.9 mmol/L for AV, and 23.4 +/- 11.1 mmol/L for AA genotype (P > 0.05). There was no difference in genotype distribution between the patient groups of less than 25th and greater than 75th percentiles, classified according to plasma homocysteine levels (P = 0.47). In conclusion, with high-dose folate supplementation, the hyperhomocysteinemia in patients with ESRD does not seem to be caused by the 677C-->T mutation in the MTHFR gene.