Fate of micelles and quantum dots in cells.

Micelles and quantum dots have been used as experimental drug delivery systems and imaging tools both in vitro and in vivo. Investigations of their fate at the subcellular level require different surface-core modifications. Among the most common modifications are those with fluorescent probes, dense-core metals or radionucleids. Cellular fate of several fluorescent probes incorporated into poly(caprolactone)-b-copolymer micelles (PCL-b-PEO) was followed by confocal microscopy, and colloidal gold incorporated in poly 4-vinyl pyridine-PEO micelles were developed to explore micelle fate by electron microscopy. More recently, we have examined quantum dots (QDs) as the next-generation-labels for cells and nanoparticulate drug carriers amenable both to confocal and electron microscopic analyses. Effects of QDs at the cellular and subcellular levels and their integrity were studied. Results from different studies suggest that size, charge and surface manipulations of QDs may play a role in their subcellular distribution. Examples of pharmacological agents incorporated into block copolymer micelles, administered or attached to QD surfaces show how the final biological outcome (e.g. cell death, proliferation or differentiation) depends on physical properties of these nanoparticles.     

Protective effect of interferon-α on the DNA- and RNA-degrading pathway in anti-Fas-antibody induced apoptosis

Aim: Fas membrane-associated polypeptide antigen is a receptor molecule responsible for apoptosis-mediated signals. In animal models of acute viral hepatitis, apoptosis of hepatocytes is mediated by Fas-death receptors; therefore, the aim of this study was to evaluate the effect of interferon (IFN)-alpha on apoptotic markers and nuclease activity against different coding and non-coding single and double stranded RNAs during Fas-induced liver apoptosis. Methods: An in vivo experiment was performed with simultaneous administration of anti-Fas (CD95) antibodies and IFN-alpha, and an in vitro experiment was performed in hepatocyte cultures treated with anti-Fas antibodies and IFN-alpha. Results: Detection of apoptosis using Annexin V-FITC/propidium iodide, Bcl-2 and Bax expression in hepatocyte cultures confirmed the appearance of early apoptotic events and progression toward late apoptosis after anti-Fas antibody treatment. IFN-alpha had a tendency to retard the apoptosis process in Fas-induced apoptosis by increasing the number of viable cells and decreasing the number of cells in late apoptosis, by increasing the percentage of Bcl-2 positive cells, by decreasing the percentage of Bax positive cells, and by decreasing the nuclease activity compared to the anti-Fas antibody treated group. Total DNA and RNA concentration was much reduced in the Fas group and DNA fragmentation assay provided evidence for increased DNA degradation. Enhanced nuclease activity against DNA, rRNA, poly(A), poly(C), poly(U), poly(I:C), and poly(A:U) was manifested in the anti-Fas antibody treated group, except for the inhibitory-bound alkaline RNase. Conclusions: The results demonstrate that the RNA-degrading pathway in Fas-induced apoptosis can accelerate the liberation of the latent enzyme from the inhibitor complex. IFN-alpha prevented enormous, Fas-ligand induced degradation of all the substrates used in this experimental study, most probably due to similarities in the signal transduction pathways. Investigations of death receptor-induced apoptosis may lead to novel treatment combinations for patients with acute or chronic liver diseases.     

Effects of nerve growth factor on cortical and striatal acetylcholine and dopamine release in rats with cortical devascularizing lesions

The effects of intraventricular nerve growth factor (NGF) or saline treatments on extracellular acetylcholine (ACh), dopamine (DA) and adenosine (Ade) levels in the cortex and striatum of rats with unilateral devascularizing cortical lesions were studied in vivo with microdialysis. The devascularizing cortical lesion produced a decrease in extracellular ACh levels in both cortex and striatum as compared to those in normal rats, while the NGF treatment produced a significant increase in ACh levels in both regions. NGF could even increase cortical ACh levels in normal rats. The cortical lesion produced a decrease in extracellular DA in the cortex, while the NGF treatment appeared to reverse this effect. No significant changes in DA were observed in the striatum. The present study gives evidence that a unilateral cortical devascularizing lesion leads to changes in extracellular ACh and DA levels in cortex and striatum and that these changes could be reversed with intraventricular NGF treatment.     

Nitroglycerine effects on portal vein mechanics and oxidative stress in portal hypertension

AIM:Тo examine the effects of nitroglycerine on portal vein haemodynamics and oxidative stress in patients with portal hypertension.METHODS:Thirty healthy controls and 39 patients with clinically verified portal hypertension and increasedvascular resistance participated in the study.Liver di-ameters,portal diameters and portal flow velocities were recorded using color flow imaging/pulsed Doppler detection.Cross-section area,portal flow and index of vascular resistance were calculated.In collected blood samples,superoxide anion radical (O 2-),hydrogen per-oxide (H 2 O 2),index of lipid peroxidation (measured as TBARS) and nitric oxide (NO) as a marker of endothelial response (measured as nitrite-NO 2-) were determined.Time-dependent analysis was performed at basal state and in 10th and 15th min after nitroglycerine (sublingual 0.5 mg) administration.RESULTS:Oxidative stress parameters changed sig-nificantly during the study.H 2 O 2 decreased at the end of study,probably via O 2-mediated disassembling in Haber Weiss and Fenton reaction;O 2-increased signifi-cantly probably due to increased diameter and tension and decreased shear rate level.Consequently O 2-and H 2 O 2 degradation products,like hydroxyl radical,initi-ated lipid peroxidation.Increased blood flow was to some extent lower in patients than in controls due to double paradoxes,flow velocity decreased,shear rate decreased significantly indicating non Newtonian char-acteristics of portal blood flow.CONCLUSION:This pilot study could be a starting point for further investigation and possible implemen-tation of some antioxidants in the treatment of portal hypertension.     

Correlation between procalcitonin and intra-abdominal pressure and their role in prediction of the severity of acute pancreatitis

Background/aimsEarly assessment of disease severity and vigilant patient monitoring are key factors for adequate treatment of acute pancreatitis (AP). The aim of this study was to determine the correlation of procalcitonin (PCT) serum concentrations and intra-abdominal pressure (IAP) as prognostic markers in early stages of AP.MethodsThis prospective observational study included 51 patients, of which 29 had severe AP (SAP). Patients were evaluated with the Acute Physiology And Chronic Health Evaluation (APACHE II) score, C-reactive protein (CRP) and PCT serum concentrations and IAP at 24 h from admission. PCT was measured three times in the 1st week of disease and three times afterward, while IAP was measured daily. PCT and IAP values correlated with each other, and also compared with APACHE II score and CRP values.ResultsPCT, IAP, CRP values and APACHE II score at 24 h after hospital admission were significantly elevated in patients with SAP. There was significant correlation between PCT and IAP values measured at 24 h of admission, and between maximal PCT and IAP values. Sensitivity/specificity for predicting AP severity at 24 h after admission was 89%/69% for APACHE II score, 75%/86% for CRP, 86%/63% for PCT and 75%/77% for IAP.ConclusionsIncreased IAP was accompanied by increased PCT serum concentration in patients with AP. PCT and IAP can both be used as early markers of AP severity.     

Age-related neurodegenerative changes in the central nervous system of estrogen-deficient follitropin receptor knockout mice

Age-related neurodegenerative conditions are characterized by neuronal death and degeneration that lead to a progressive functional decline. Among the factors influencing degenerative processes during aging are altered levels of neurotrophic ovarian steroid 17beta-estradiol (E2). The follitropin receptor knockout (FORKO) female mouse displays hormonal imbalance characterized by very low levels of circulating E2 and high levels of testosterone. FORKO mice (24 days and 20 months) were used to investigate structural and functional changes in the central nervous system. We now show that the lifelong depletion of the sex hormone E2 in female FORKO mice correlates with abnormal behavior associated with defined alterations in brain morphology early in life, especially in aged animals. Immunohistochemical studies showed significant increases in the size and number of immunoreactive glial fibrillary acidic protein glial cells found in several brain regions (cortex and hippocampus) and a dramatic decline in estrogen receptors alpha and beta in the amygdala of FORKO females. These changes were associated with increased signs of anxiety in these animals. In the present study, we provide evidence that the chronic depletion of sex hormone E2 from early development leads to neural impairments in adult and aged FORKO mice that are associated with hypertrophy of glial cells, cell loss in distinct brain regions, and abnormal behavior. We suggest that the hormonal imbalance found in the female FORKO mouse provides an experimental paradigm for the study of morphological correlates of the behavioral changes that often accompany menopause in women.     

Possible impact of plasma RNase activity on immune dysfunction in juvenile diabetes mellitus

AIM: The ribonuclease (RNase) family represents important enzymes used widely in biomedical and biotechnological applications, as well as for diagnostic and therapeutic purposes. This study was undertaken to test the possibility that plasma alkaline RNase (free or inhibitory bound) determination may be useful in studying the dysregulation of nucleic acid and oligonucleotide metabolism as a possible pathogenetic mechanism in development of immune dysfunction in juvenile diabetes mellitus. PATIENTS AND METHODS: Children with type 1 diabetes (n=32, age group of 5--14 yr), together with age-matched control subjects (n=35), were enrolled in the study. None had microvascular complications. According to the metabolic regulation of the disease and the hemoglobin A1c (HbA1c) level, all patients were divided into two groups (HbA1c<7.5% and HbA1c>7.5%). According to the duration of diabetes, diabetic children were divided into two groups: duration of diabetes less than 1 yr and duration of diabetes greater than 1 yr. The control group consisted of age-matched subjects (n=35; 15 girls and 20 boys) who were clinically healthy. The activity of free and inhibitory-bound RNase and the level of acid soluble nucleotides were measured in heparinized plasma. RESULTS: The inhibitory-bound enzyme activity was higher in diabetic children, followed by sharply decreased free enzyme, especially in the group with the level of HbA1c above 7.5%. Recent-onset diabetic patients had lower free RNase activity compared with those with longer duration of the disease. The amount of pre-existing acid-soluble oligonucleotides was significantly increased in diabetic children, especially in those with poor metabolic control. CONCLUSION: Our observed preliminary results may suggest a hypothesis that a persistent increase of oligonucleotide fragments, most probably due to insufficient RNase activity, may lead to T-cell hyperactivity in type 1 diabetes through the activation of toll-like receptors (TLRs). The measurement of RNase(s) activity (free, inhibitory-bound, or specific toward different substrates), together with the well-known immunobiochemical parameters of diabetes, may help further efforts in identifying a disease-specific early biological marker of immunity dysfunction in juvenile diabetes.