Photodynamic fungicidal efficacy of hypericin and dimethyl methylene blue against azole-resistant Candida albicans strains.

Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to treat infections based on the use of photosensitisers (PSs) and visible light. To investigate the fungicidal effect of PDT against azole-resistant Candida albicans strains using two PSs with a different mechanism of action, hypericin (HYP) and 1,9-dimethyl methylene blue (DMMB), comparing their efficacy and the reactive oxygen species (ROS) species involved in their cytotoxicity. Azole-resistant and the azole-susceptible C. albicans strains were used. Solutions of 0.5 and 4 McFarland inoculum of each Candida strain were treated with different concentrations of each PS, and exposed to two light-emitting diode light fluences (18 and 37 J cm⁻²). Mechanistic insight was gained using several ROS quenchers. The minimal fungicidal concentration of HYP for ≥3 log10 CFU reduction (0.5 McFarland) was 0.62 µmol l⁻¹ for most strains, whereas for DMMB it ranged between 1.25 and 2.5 µmol l⁻¹. Increasing the fluence to 37 J cm⁻² allowed to reduce the DMMB concentration. Higher concentrations of both PSs were required to reach a 6 log10 reduction (4 McFarland). H2O2 was the main phototoxic species involved in the fungicidal effect of HYP-aPDT whereas ¹O2 was more important for DMMB-based treatments. aPDT with either HYP or DMMB is effective in killing of C. albicans strains independent of their azole resistance pattern. HYP was more efficient at low fungal concentration and DMMB at higher concentrations.

Improved systemic antitumor therapy with oncolytic adenoviruses by replacing the fiber shaft HSG-binding domain with RGD.

Retargeting oncolytic adenoviruses from their systemic preeminent liver tropism to disseminated tumor foci would highly improve the efficacy of these agents at eradicating tumors. We have replaced the KKTK fiber shaft heparan sulfate glycosaminoglycan-binding domain with an RGDK motif in order to achieve simultaneously liver detargeting and tumor targeting. When inserted into a wild-type backbone, this mutation palliated liver transaminase elevation and hematological alterations in mice. Importantly, when tested in a backbone that redirects E1A transcription towards pRB pathway deregulation, RGD at this novel shaft location also improved significantly systemic antitumor therapy compared with the broadly used RGD location at the HI-loop of the fiber knob domain.

Effect of oxysterol-induced apoptosis of vascular smooth muscle cells on experimental hypercholesterolemia.

Smooth muscle cells (SMCs) undergo changes related to proliferation and apoptosis in the physiological remodeling of vessels and in diseases such as atherosclerosis and restenosis. Recent studies also have demonstrated the vascular cell proliferation and programmed cell death contribute to changes in vascular architecture in normal development and in disease. The present study was designed to investigate the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, using an in vivo/in vitro cell model in which SMCs were isolated and culture from chicken exposed to an atherogenic cholesterol-rich diet (SMC-Ch) and/or an antiatherogenic fish oil-rich diet (SMC-Ch-FO). Cells were exposed in vitro to 25-hydroxycholesterol to study levels of apoptosis and apoptotic proteins Bcl-2, Bcl-X(L) and Bax and the expression of bcl-2 and bcl-x(L), genes. The quantitative real-time reverse transcriptase-polymerase chain reaction and the Immunoblotting western blot analysis showed that 25-hydroxycholesterol produces apoptosis in SMCs, mediated by a high increase in Bax protein and Bax gene expression. These changes were more marked in SMC-Ch than in SMC-Ch-FO, indicating that dietary cholesterol produces changes in SMCs that make them more susceptible to 25-hydroxycholesterol-mediated apoptosis. Our results suggest that the replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of cholesterol-induced changes in the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, making SMCs more resistant to apoptosis.

Urinary excretion of low molecular weight proteins in patients with pure monoclonal light chain proteinuria.

BACKGROUND: Because urinary low molecular weight protein (LMWP) measurement shows changes in renal integrity at an early stage, beta2-microglobulin (B2m), retinol-binding protein (RBP) and alpha1-microglobulin (A1m) were evaluated in 24-hour urine collection of 65 patients with pure monoclonal light chain (MLC) proteinuria and in 47 patients with different kidney diseases (DKDs) for comparison. METHODS AND RESULTS: Albumin, kappa, lambda, A1m and B2m were measured by immunonephelometry. RBP was determined by ELISA. The mean values of LMWP quantitation were significant for origin of the disease (MLC and DKD) (p<0.05) and renal failure (RF) (p<0.001) (MANOVA). Tukey HSD test only showed significant differences for LMWP between MLC patients with RF and DKD patients without RF. The mean value of A1m was different between patients with and without RF in each group (p<0.05 for MLC, and p<0.01 for DKD). In the group without RF, the frequency of A1m excretion above 12 mg/L differed between MLC patients and DKD patients (p<0.01). CONCLUSION: A tubular dysfunction occurred in a great number of patients excreting pure MLC even in those with well-preserved renal function, as it did in patients with DKDs. In patients with MLC without RF, A1m might be measured for the early recognition of tubular involvement.

Induction of experimental phenylketonuria-like conditions in chick embryo. Effect on amino acid concentration in brain, liver and plasma.

Experimental hyperphenylalaninemia has been induced in chick embryos between 11-20 days of incubation by daily injection of ?-methylphenylalanine and phenylalanine. Brain and liver weight decreased after 8 days of treatment. An increase of nearly 14-fold in the brain phenylalanine/tyrosine ratio was observed after 9 days of treatment. Similar results were obtained in liver, although the increase found in this case was smaller than in brain. Chronic hyperphenylalaninemia induced a clear rise in the levels of plasma and liver valine, leucine and isoleucine, while in brain these levels did not change significantly. Plasma and brain glycine content was also enhanced by this treatment. Brain tyrosine concentration was clearly decreased in these conditions, in contrast to the enhancement reported after this and other treatments in various animal species. Thus, the higher value of the brain phenylalanine/tyrosine ratio obtained by ?-methylphenylalanine plus phenylalanine administration was due to both an increase in the phenylalanine and a decrease in the tyrosine levels, conditions that have been also found in human phenylketonurics. Therefore, the treatment here reported was an excellent method for imitating the conditions of phenylketonuria during the period of rapid myelination in the chick, one of the most dramatic in nervous system development.

The reversal of the effects on HMG-CoA reductase activity and microsome lipid composition in chick livers caused by short-period cholesterol feeding.

Chicks of different ages were fed on a cholesterol diet for 48 hours and then switched to a standard diet. HMG-CoA reductase activity and microsomal cholesterol levels, altered by the cholesterol diet, returned to control values in younger chicks after 48 hours on the standard diet. This study clearly shows that early age is an important factor to be taken into account when considering the reversibility of cholesterol-induced membrane alterations.

Development of the diurnal rhythm of chick 3-hydroxy-3-methylglutaryl-CoA reductase.

Chick liver and intestine 3-hydroxy-3-methylglutaryl-CoA reductase did not show diurnal rhythm at hatching. Differences in activity between light and dark periods appeared during the first week and remained more or less constant between 10-14 days after hatching. Hepatic and intestinal reductase activities were maximal during the light period and minimal during the dark period. Amplitude of the rhythm was practically similar in both tissues (about 3-fold) although specific activities and differences between peak and nadir values were always higher in liver. Chick brain reductase did not show significant diurnal variations in the age range assayed.

Alterations in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA concentration in cultured chick aortic smooth muscle cells.

We observed and compared alterations in 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase at the transcriptional level in unsynchronized, three-passage cultures of smooth-muscle cells from the aorta of chicks fed on a control diet (C-SMC) and those of chicks fed on a similar diet plus cholesterol (Ch-SMC). Alterations in reductase mRNA concentrations in senescent cultures were much lower. We used a modification of the competitive (c) reverse transcription polymerase chain reaction method, using a Thermus thermophilus DNA polymerase (Tth pol) to quantify the very scarce species of HMG-CoA reductase mRNA in samples of cytoplasmic SMC mRNA. We cloned and sequenced a 199 bp cDNA fragment of chicken HMG-CoA reductase, which encoded a region of 66 amino acids belonging to the catalytic domain of the enzyme. HMG-CoA reductase mRNA concentrations from young C-SMC cultures rose 3.89-fold 4 h after the change of medium and returned to base levels between 8 to 12 h afterward. Concentrations in Ch-SMC cultures increased less (2.36-fold) 8 h after the change to fresh medium. Increases in reductase mRNA in senescent cultures of Ch-SMC and C-SMC measured under similar conditions were only 1.28- and 1.39-fold, respectively.

Different thermal behavior of neonatal hepatic and cerebral 3-hydroxy-3-methylglutaryl-CoA reductase.

The Arrhenius plots of hepatic and cerebral 3-hydroxy-3-methylglutaryl-CoA reductase activity were studied in neonatal chicks fed with a standard diet. Supplementation of the diet with 2% cholesterol from hatching has no effect on the thermal characteristics of the brain enzyme. The Arrhenius plot of brain reductase was practically similar to that found in control chicks. However, hepatic reductase was inhibited by cholesterol feeding. Dietary cholesterol increased the cholesterol/lipidic phosphorus molar ratio in liver microsomes, whereas no significant differences were observed in brain microsomes. These results are in agreement with the hypothesis that activity of hepatic reductase is regulated by the fluidity of microsomal membrane and show that cholesterol feeding does not alter the fluidity of microsomal membranes in neonatal chick brain having, thus, no effect on the thermal behavior of cerebral reductase.

Comparative activity of ceftizoxime against aminoglycoside-resistant aerobic gram-negative bacilli.

The in vitro activity of ceftizoxime was compared with that of other beta-lactam antibiotics against 331 aminoglycoside (AG)-resistant clinical isolates. Two hundred and six AG-resistant, beta-lactamase producing, R-plasmid harbouring Enterobacteriaceae strains had MICs ranging from 0.0125 to 0.063 mg/l. AG-resistant Escherichia coli (36 strains) and Klebsiella pneumoniae (19) had MIC 90 values of 8 mg/l. Proteus rettgeri and P. vulgaris as well as Morganella morganii, resistant to several AGs, had MICs ranging from 0.5 to 4 mg/ml. Against all six isolates of AG-resistant Salmonella enteritidis the MIC90 was 0.5 mg/l. Twenty-seven strains of Serratia marcescens, most of which were resistant to beta-lactam and AG antibiotics, had MICs ranging from 0.5 to 8 mg/l. The AG-resistant strains of Enterobacteriaceae producing several AG-modifying enzymes (AAC(3); AAC(2'); AAC(6'); APH(3')) showed MICs ranging from 0.6 to 4 mg/l. Against 10 AG-resistant strains of Pseudomonas aeruginosa producing AAC(3), AAC(6') and APH(3') enzymes, the MICs ranged from 16 to 64 mg/l. In conclusion, ceftizoxime was equally or more active than cefotaxime, cefoperazone, ceftazidime and moxalactam against AG-resistant E. coli, Klebsiella, Morganella, Proteus, Serratia, Salmonella and R-plasmid harbouring Enterobacteriaceae. Ceftizoxime was less active than cefotaxime, moxalactam and ceftazidime against P. aeruginosa.

[Chronic autoimmune type II hepatitis with various extrahepatic clinical manifestations]. / Hepatitis crónica autoimmune tipo II con diversas manifestaciones clínicas extrahepáticas.

We present the case of a patient with autoimmune chronic hepatitis and anti-LKM antibodies, who developed associated autoimmune diseases, cyclic nodose erythema, bilateral peripheric paralysis, idiopathic thrombocytopenic purpura and diabetes mellitus. We describe the first signs of the disease and how three different forms can be differentiated depending on the type of autoantibodies present in the patients' serum. Finally, we list several forms of presentation of the disease, the potential clinical associations with other autoimmune processes and the potential immunological basis for the development of the hepatic lesion.

Cutaneous sporotrichosis treated with photodynamic therapy: an in vitro and in vivo study.

BACKGROUND: Sporotrichosis is a fungal infection caused by Sporothrix schenckii complex, usually restricted to the skin, subcutaneous cellular tissue, and adjacent lymphatic vessels. Antimicrobial photodynamic therapy (aPDT) could be a good alternative to manage localized, superficial infections. CASE REPORT: A 65-year-old African woman was diagnosed with a fixed cutaneous sporotrichosis on her left arm, treated with itraconazol and oral terbinafine with partial improvement. Topical 16% methyl aminolevulinate (MAL, Metvix(®))-PDT was used without success. METHODS: An in vitro photoinactivation test with the isolated microorganism revealed phenothiazinium salts to be more effective than MAL. CONCLUSIONS: PDT with intralesional 1% methylene blue (MB) in combination with intermittent low doses of itraconazole obtained complete microbiological and clinical response.

Nutritional control, gene regulation, and transformation of vascular smooth muscle cells in atherosclerosis.

Contractile-state smooth muscle cells (SMC), the only cell type in the arterial media, undergoes migration to the intima, proliferation, and abundant extracellular matrix production during the early stages of atherosclerosis. This involves the ingestion of low-density lipoprotein (LDL) and modified or oxidised LDL by macrophages together with SMC by several pathways including a scavenger pathway leading to accumulation of cholesterol esters and formation of foam cells. High-plasma cholesterol levels constitute a major causative risk for atherosclerosis. The membrane-bound transcription factor called sterol regulatory element binding protein (SREBP) activates gene-encoding enzymes of cholesterol and fatty acid biosynthesis. The SREBP expression, in response to diet, shows that are involved in both lipogenesis and cholesterol homeostasis, moreover SREBPs are regulated directly by cholesterol. Animal models were used in trials of atherosclerosis, and cholesterol feeding has been described elsewhere as producing atherosclerotic lesions. We have examined the morphological, molecular and proliferative change in arterial SMC mimicking such a cholesterol diet, this transformed SMC is a good model to study the alterations of the differentiated state of SMC, and the transformation into foam cell, caused by cholesterol-rich diet. Despite the complexity of the interactions in atherosclerosis, there are many opportunities to affect the homeostatic balance of the artery wall at SMC levels. We have considered here some of the possible targets for intervention with promising strategies for the nutritional control of the genes, and, in a general way, the possibilities for modulating the expression of genes influencing atherosclerosis.

Homeostatic restoration of microsomal lipids and enzyme changes in HMG-CoA reductase and acyl-CoA: cholesterol acyltransferase in chick liver.

We have studied the correlation between changes in the lipid composition in chick liver microsomes and the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acyl-CoA: cholesterol acyltransferase (ACAT) by in vivo and in vitro experiments with 21-day-old chicks. A 5% cholesterol diet for 3 hr produced an increase in the microsomal and plasmatic cholesterol content, a decrease in HMG-CoA reductase activity and a concomitant increase in ACAT activity. The effect produced by the short-term treatment virtually disappeared 27 hr after ending the cholesterol diet. In vitro experiments were carried out by using vesicles constituted by phosphatidylcholine/cholesterol and phosphatidylcholine.

Characterization of mevalonate metabolism in the sea bass (Dicentrarchus labrax L) liver.

The activities of mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase, were examined in sea bass (Dicentrarchus labrax L) liver. The activities of the three enzymes were studiedin vitro in relation to the influence of protein content, time of incubation, pH, temperature, mevalonate, ATP and Mg(++) concentration. Protein content in the assay medium affected the three enzymes differently. Mevalonate kinase, mevalonate 5-phosphate kinase, and mevalonate 5-pyrophosphate decarboxylase activities were linear up to 0.2, 0.4 and 0.8 mg protein, respectively. With respect to the time course studies, the enzymes also behaved differently. Mevalonate kinase activity increased over forty minutes, reaching a plateau thereafter, while mevalonate 5-phosphate kinase and decarboxylase increased over the entire assay period. All the three enzymes showed a maximum in activity at pH 7.5. The effect of reaction temperature showed that phosphorylation increased to maximum around 35°C for mevalonate kinase and 30°C for mevalonate 5-phosphate kinase while decarboxylation rates remained constant well until 30°C temperature decreasing afterwards. The enzymes behaved differently as a function of mevalonate concentration. Mevalonate 5-phosphate formed was maximal when the initial mevalonate concentration was 272 µM, whereas mevalonate 5-pyrophosphate and CO2 were formed maximally at mevalonate concentrations of 136 µM and 68µM, respectively. Optimal ATP concentration in the medium was 3 mM for decarboxylase and 6 mM for kinases, and Mg(++) requirements varied from 4 mM for decarboxylase to 6 mM for kinases.

Differentiation between Trichophyton mentagrophytes and T. rubrum by sorbitol assimilation.

Trichophyton rubrum was easily differentiated from T. mentagrophytes by its ability to assimilate sorbitol with an API 20C AUX strip. One hundred percent of 36 T. rubrum strains and none of 147 T. mentagrophytes strains assimilated sorbitol.

The role of age on the cholesterol-metabolizing enzymes and lipid levels in chick plasma and liver microsomes after cholesterol enriched diet cessation.

Chicks of two age groups (11 and 21 days-old) were fed a cholesterol enriched diet administered either from hatching or for only 6 hours (9:00-15:00). Afterwards, the cholesterol was removed from the diet and we analysed hepatic Acyl-coenzyme A:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activities, plasma cholesterol levels and hepatic microsomal cholesterol content up to a day and a half. The treatments raised plasma and microsomal cholesterol levels in both age groups, increases in the older group exceeding those of the younger chicks. After removal of the cholesterol diet, both groups recovered by the middle of the next day, reaching values similar to those of control-chick groups. The ACAT activity showed a relationship with the microsomal cholesterol levels although did not return to similar levels than those of control microsomes. This correlation was not observed in HMG-CoA reductase. In general, differences in returning to the control values depended on the developmental stage of the chick.

Correlation between changes in membrane lipid composition induced by dietary lipid and membrane-bound enzyme activity in chick liver.

The activity of acyl-CoA: cholesterol acyltransferase in the liver-microsomal fraction was considerably reduced in chicks fed on diet containing unsaturated fat, whereas the activity of HMG-CoA reductase and NADPH cytochrome c reductase was not affected. The fatty acid composition of the microsomes was modified appreciably by this dietary condition and there was no change in the phospholipid or cholesterol levels. The addition of cholesterol to the fat supplemented diet resulted in a considerable increase in the microsomal cholesterol content. A decrease in HMG-CoA reductase and an increase ACAT activity was observed compared with the corresponding values from both the groups fed on a standard diet and a fat supplemented diet with no cholesterol. These results suggest that acyl-CoA: cholesterol acyltransferase is modulated by alteration in the fatty acid composition of the microsomal membrane, while the cholesterol content of the microsomes shows a close relationship with the HMG-CoA reductase activity.

The reversal of the inhibition on lipids synthesis by L-659,699 in arterial smooth muscle cells cultures.

The beta-lactone isolated from Fusarium sp. termed L-659,699 is a potent specific inhibitor of the enzyme 3-hydroxi-3-methylglutaril coenzyme A (HMG-CoA) synthase. In cultures of smooth muscle cells (SMC) isolated from aortic-arch of control (C-SMC) and 5% of cholesterol diet (Ch-SMC) treated chicks, the incorporation of (14C)-acetate to lipids (cholesterol, triacylglycerides and cholesterol ester) were greater in Ch-SMC cultures than in C-SMC and the presence of 0.05 microM L-659,699 for 2 h in the incubation medium decrease the synthesis of cholesterol however the triacylglycerides synthesis increase. The effect of inhibitor is stronger in young cultures (3-4 steps) than in the older ones (11-12 steps). In young C-SMC and Ch-SMC cultures the inhibition of cholesterol and triacylglycerides synthesis by L-659,699 was reversal.

3-hydroxy-3-methylglutaryl-CoA reductase from neonatal chick.

The optimal conditions for identification of mevalonic acid as the product of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase are described, as well as the effect of different buffer constituents on the enzyme activity. Under the chosen assay conditions, reductase activity from neonatal chick liver increased with the incubation time up to 60 min and was proportional to the amounts of protein added in a range of 0.1-0.5 mg. The specific activity was maximal in brain and liver and lower in intestine of 6-day-old chicks. Thermostability of hepatic reductase was studied. When microsomal preparations were maintained at 4 degrees C, reductase activity remained unchanged for 6 hr and decreased afterwards. Addition of 50 mM KF to the homogenization medium had no effect on the reductase activity. Similarly, preincubation of microsomal preparations with 105,000 g supernatants in the presence or absence of KF did not significantly increase the reductase activity. These results suggest that HMG-CoA reductase was isolated from neonatal chick in the fully activated form.