A non-invasive method for estimating the severity of liver steatosis and the risk of fibrosis in non-obese type 2 diabetes patients with NAFLD.

Context: Prognostic considerations include assessing the risk of liver fibrosis in people with non-alcoholic fatty liver disease (NAFLD). Objectives: This study evaluates the use of hematologic and metabolic parameters regarding liver steatosis and fibrosis scores (FLI and Fib-4) in non-obese type 2 diabetes mellitus (t2DM) patients with NAFLD. Methods: Subjects underwent abdominal ultrasound examinations, and FLI and Fib-4 scores were calculated to evaluate liver steatosis and the risk of liver fibrosis non-invasively: 61 non-obese NAFLD subjects with t2DM were included in the cohort study and were divided into 2 groups depending on the t2DM treatment regimen. Results: Fib-4 and WBC count demonstrated a significant inverse correlation (OR = 0.509, p = 0.007). WBC count had an R2 of 0.237, indicating that this marker could account for up to 23.7% of a variation in Fib-4. Fib-4 and FFA had positive correlation which did not achieve statistically significant prediction (OR=7.122, p=0.062). Additionally, a significant prediction of HbA1c (OR=1.536, p=0.016) and haemoglobin (OR=1.071, p=0.020) for FLI was revealed. Conclusion: HbA1c and other haematological and metabolic parameters, such as haemoglobin and WBC, may be another non-invasive tool for determining whether non-obese NAFLD patients with t2DM are at risk of developing liver steatosis and fibrosis.

Inhibition of neurosphere proliferation by IFNgamma but not IFNbeta is coupled to neuronal differentiation.

Interferons are produced following neural damage as part of the inflammatory response and may thus affect neural stem cell function. We compared the effects of interferon-gamma and interferon-beta on the proliferation and differentiation of adult murine neural progenitors. Both interferons inhibited neurosphere proliferation due to cell cycle arrest in G1 but only interferon-gamma induced neuronal differentiation. Both interferons induced differential phosphorylation of STAT proteins and a modest and late upregulation of the cell cycle regulator p27 but not several other likely cell cycle regulators. Thus in neural progenitor cells, anti-proliferative effects of interferons are not necessarily linked to differentiation.

Natural and anthropogenic radioactivity in the environment of mountain region of Serbia.

The activity concentrations of (40)K, (238)U, (232)Th and (137)Cs have been measured using a gamma spectrometric method in different samples from the environment of two mountains in Serbia (altitude 1000-1100 m), during the period 2002-2007. The mountains Maljen and Tara (popular tourist destinations) are near Belgrade. On mountain Maljen, samples were taken at 4 different altitudes (200 m, 650 m, 1000 m and 1100 m), and on mountain Tara at altitudes of 1000 m and 1100 m. On mountain Maljen it was found that the level of (137)Cs activity increased with altitude in samples of soil, grass, hay and cow, sheep and goat milk. On the contrary, (40)K activity decreased with altitude in samples of soil, grass and hay. The highest activity concentrations of (137)Cs were found in bioindicators: sheep meat, venison, wild boar meat, moss and mushrooms. These results indicate that (137)Cs is present in mountain region of Serbia even 20 years after the nuclear accident in Chernobyl. Deposition of (137)Cs was almost two times higher on the Maljen mountain compared to Tara mountain. An average annual dose arising from (137)Cs was 7.4 microSv due to ingestion of cow milk and 6.3 microSv due to ingestion of mushrooms at the Maljen mountain.

AFCF and clinoptilolite use in reduction of (137)Cs deposition in several days' contaminated broiler chicks.

The objective of this study was to investigate the binding efficiency of AFCF and clinoptilolite, mixed to the feed and administered orally using gastric tube to chronically (137)Cs alimentary contaminated broiler chicks. Seventy-five male Hybro broiler chicks, between 35 and 47 days of age were divided into five groups (15 birds per group) reared in cages (five birds in a cage) and fed a standard diet. Every day during 13 days of the experimental period all chicks received orally 1 ml CsCl water solution with activity of 1310 Bq ml(-1)(137)Cs (gastric tube). Group 1 was the control group and received no binders. The experimental groups received the binders. Group 2 received 0.2 g of AFCF in the form of water solution (gastric tube); group 3 received 0.2% AFCF in the feed; group 4 received 2g clinoptilolite in the form of water suspension (gastric tube) and group 5 received 2% clinoptilolite in the feed. Five chicks from each group were sacrificed on days 4, 10 and 13 of the experimental period. Using gamma spectrometric methods specific activity of (137)Cs was determined in the samples of breast meat, liver and gizzard. The results obtained showed that administering binders to the chronically contaminated broiler chicks significantly (p<0.01) reduced (137)Cs transfer and deposition in breast meat, liver and gizzard. Decreasing deposition of (137)Cs in breast meat and internal organs increased with time of contamination and binders' administration. With AFCF as a cesium binder, on day 13 of measuring the (137)Cs activity in breast meat was 80-83% lower than that in the control group, 89% in liver and 83-84% in gizzard. Natural clinoptilolite demonstrated lower binding efficiency. On day 13 of measuring the (137)Cs activity in breast meat was 53-69% lower than that in the control group, 67-60% in liver and 59-71% in gizzard.

An in Vitro Model of Oligodendrocyte Destruction by Nitric Oxide and Its Relevance to Multiple Sclerosis

There is mounting evidence that nitric oxide (NO) is produced in the brains of patients with multiple sclerosis (MS) and in the experimental model of MS, experimental autoimmune encephalomyelitis, after the induction of Type II nitric oxide synthase (iNOS). Because NO can cause a variety of biological insults that compromise or even kill normal cells, we studied the effects of NO on oligodendrocytes since they are a target in MS tissue. In an in vitro model, we have been able to demonstrate that NO causes damage to oligodendrocytes preferentially, sparing microglia almost completely and affecting some but not all astrocytic functions. This article describes the types of assays used to measure morphological changes, mitochondrial dysfunction, DNA strand breaks, and cell death brought on by NO or peroxynitrite (ONOO-) as well as a comprehensive review of the various techniques and sensitivities of NO and iNOS assays that would be applicable to similar in vitro models.

The role of nitric oxide in multiple sclerosis.

During the past decade nitric oxide has emerged as an important mediator of physiological and pathophysiological processes. Elevated nitric oxide bio-synthesis has been associated with nonspecific immune-mediated cellular cytotoxicity and the pathogenesis of chronic, inflammatory autoimmune diseases including rheumatoid arthritis, insulin-dependent diabetes, inflammatory bowel disease, and multiple sclerosis. Recent evidence suggests, however, that nitric oxide is also immunoregulatory and suppresses the function of activated proinflammatory macrophages and T lymphocytes involved in these diseases. This article reviews the role of nitric oxide in the biology of central nervous system glial cells (astrocytes and microglia) as it pertains to the pathogenesis of multiple sclerosis in humans and experimental allergic encephalitis, the animal model of this disease. Although nitric oxide has been clearly implicated as a potential mediator of microglia-dependent primary demyelination, a hallmark of multiple sclerosis, studies with nitric oxide synthase inhibitors in the encephalitis model have been equivocal. These data are critically reviewed in the context of what is know from clinical research on the nitric oxide pathway in multiple sclerosis. Specific recommendations for future preclinical animal model research and clinical research on the nitric oxide pathway in patients are suggested. These studies are necessary to further define the role of nitric oxide in the pathology of multiple sclerosis and to fully explore the potential for nitric oxide synthase inhibitors as novel therapeutics for this disease.

The type IV phosphodiesterase specific inhibitor mesopram inhibits experimental autoimmune encephalomyelitis in rodents.

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease with pathological features reminiscent of those seen in multiple sclerosis and thus serves as an animal model for this disease. Inhibition of type IV phosphodiesterase (PDE IV) in animals with this disease has been shown to result in amelioration of disease symptoms. Here we describe the immunomodulatory activity of the novel potent and selective PDE IV inhibitor mesopram. In vitro, mesopram selectively inhibits the activity of type 1 helper T (Th1) cells without affecting cytokine production or proliferation of type 2 helper T (Th2) cells. Administration of mesopram to rodents inhibits EAE in various models. Clinically, EAE is completely suppressed by mesopram in Lewis rats. This is accompanied by a reduction of inflammatory lesions in spinal cord and brain. RT-PCR analysis revealed a marked reduction in the expression of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the brains of these animals. Furthermore, the ex vivo production of Th1 cytokines by activated spleen cells derived from mesopram-treated animals is significantly reduced compared to vehicle-treated controls. Amelioration of the clinical symptoms is also observed during chronic EAE in mesopram-treated SJL mice as well as in relapsing-remitting EAE in SWXJ mice using a therapeutic treatment regimen. These data demonstrate the anti-inflammatory activity of mesopram and provide a rationale for its clinical development.

Nitric oxide as a potential pathological mechanism in demyelination: its differential effects on primary glial cells in vitro.

Because we believe that macrophage-derived nitric oxide contributes to pathology of demyelinating diseases, we have determined the differential effects of nitric oxide on primary rat glial cells in vitro. Enriched cultures of microglia, astrocytes and oligodendrocytes were treated with S-nitroso,N-acetyl-DL-penicillamine, a nitric oxide-releasing chemical. There was a significantly decreased function of one of the ferrosulfur-containing mitochondrial enzymes after S-nitroso,N-acetyl-DL-penicillamine/nitric oxide treatment in oligodendrocytes and astrocytes compared to microglia, which were much less sensitive to S-nitroso,N-acetyl-DL-penicillamine/nitric oxide at all concentrations. At 0.5 mM S-nitroso,N-acetyl-DL-penicillamine/nitric oxide, astrocytes and oligodendrocytes suffered a 40% loss in succinate dehydrogenase activity, while microglia were unaffected. A control non-ferrosulfur-containing mitochondrial enzyme, isocitrate dehydrogenase, was not affected in any glial cell type. Although the per cent of mitochondrial damage in oligodendrocytes and astrocytes was the same for all concentrations of S-nitroso,N-acetyl-DL-penicillamine/nitric oxide, significant cell death occurred in oligodendrocytes at 1.0 mM; at this concentration there was no significant killing of microglia or astrocytes. Furthermore, at a 0.5 mM concentration of S-nitroso,N-acetyl-DL-penicillamine/nitric oxide, which inhibited mitochondrial respiration but did not kill oligodendrocytes, significant changes in oligodendrocyte morphology (e.g. retraction of processes) occurred. Morphological changes were not seen in microglia and astrocytes at any concentration of S-nitroso,N-acetyl-DL-penicillamine/nitric oxide. In addition, oligodendrocytes were more sensitive to S-nitroso,N-acetyl-DL-penicillamine/nitric oxide-induced single stranded DNA breaks than microglia or astrocytes. The mitochondrial damage was attributable to nitric oxide since N-acetyl-DL-penicillamine had no effect. Oxyhemoglobin, which competitively inhibits toxic effects of nitric oxide, protected these glial cells from mitochondrial damage, single stranded breaks in DNA and cell death in a time- and dose-dependent manner. Once again, oligodendrocytes were less easily rescued from nitric oxide effects by oxyhemoglobin than were astrocytes, suggesting greater vulnerability of the myelin-producing cell to nitric oxide. These findings suggest that there is differential sensitivity of glial cells to nitric oxide. Although oligodendrocytes and astrocytes are equally susceptible to nitric oxide-induced mitochondrial damage, oligodendrocytes are more sensitive to nitric oxide-induced single stranded DNA breaks, morphological changes and cell death. Compared to both oligodendrocytes and astrocytes, microglia, nitric oxide-producing cells, are resistant to nitric oxide-induced damage.

Nitric oxide induces necrotic but not apoptotic cell death in oligodendrocytes.

We have investigated the mechanism of nitric oxide-induced damage in glial cells. Genomic DNA isolated from astrocytes and microglia, treated for 18 h with varying concentrations of a nitric oxide donor, was analysed by electrophoresis. No DNA damage was evident. Oligodendrocytes, treated with 2 mM nitric oxide for 3-48 h, showed single stranded breaks at 48 h but no laddering of nucleosomic fragments of DNA. When analysed by electron microscopy, ultrastructural changes in oligodendrocytes treated with 1 mM nitric oxide for 24 h showed intact nuclei but alterations in membranes and organelles characteristic of necrosis, including disrupted mitochondria with dissolution of their christae. Astrocytes, a glial cell type that we have previously shown to be much less sensitive to nitric oxide-induced damage, did not show ultrastructural changes. DNA analysis by flow cytometry of glial cells treated with nitric oxide supported the apparent necrotic-type death in oligodendrocytes. Double staining of oligodendrocytes, using Hoechst 33342 and propidium iodide for the simultaneous assessment of both apoptotic and necrotic cells, demonstrated that, while the proportion of dead cells increased with time and increasing concentrations of nitric oxide, the death was due to necrosis and not apoptosis. In this study, we demonstrate that direct exposure to soluble nitric oxide, produced in vitro from a nitric oxide donor chemical, ultimately kills oligodendrocytes by necrosis. Microglia and astrocytes maintain DNA and organelle integrity when exposed to exogenous nitric oxide.

Neurotransmitters and cytokines in CNS pathology.

In summary, we have demonstrated an in vitro model for oligodendrocyte cell death that may be relevant to events in formation of lesions in MS. It involves cell contact to oligodendrocytes with activated, viable microglia (or inflammatory macrophages), surface TNF-alpha, surface adhesion molecules, and production of NO. Precise mechanisms of TNF-alpha and ICAM-1/LFA-1 participation and the nature of the susceptibility of the oligodendrocyte are currently being studied.

The uptake and speciation of various Al species in the Brassica rapa pekinensis.

An investigation was carried out on the uptake and speciation of Al species in Al tolerant Chinese cabbage (Brassica rapa L. ssp. pekinensis). Plants were exposed to 10 microg cm(-3) of Al in the chemical forms of Al3+, Al-citrate and Al-malate in a time span from 1 up to 24 h. In each experiment the nutrient solution and stem sap were analysed by a combination of FPLC ICP AES and ES MS MS techniques. Speciation analysis enabled determination of particular chemical forms of Al present in the nutrient solution or in stem sap. The results indicate that Al3+ added to the nutrient solution remained as Al3+ in the solution during the experiments, but in the roots transformation to Al-malate occurred. Al was transported from roots to the upper parts of the plant as Al-malate (70%) and Al3+ (30%). Al-citrate or Al-malate added to the nutrient solution were transferred to the upper parts of the plant without transformation of their chemical forms.

Changed delivery of boron to tumours using electroporation for boron neutron capture therapy with BSH.

For effective boron neutron capture therapy (BNCT) it is important that a sufficient concentration of boron (10B) is present in the tumour during irradiation. This requirement represents a specific problem. The aim of this study was to test whether electroporation can be used as a non-specific drug delivery system to increase the delivery of sodium borocaptate-10B (BSH) into MCF7 (breast carcinoma) and B16F1 (melanoma) tumour cells in vitro and in B16F1 tumours in vivo. For the in vitro determination of 10B uptake, the cells were incubated in medium containing BSH and exposed to electric pulses. Boron levels were determined by inductively coupled plasma atomic emission spectrometry. In vivo, tumours were exposed to electric pulses 3 min after intravenous BSH injection. At different times after exposure the 10B concentration was determined in tumours and in blood. A difference in the 10B accumulation in the two cell lines was observed after continuous incubation of cells with BSH. No accumulation of 10B was observed in MCF7 cells, whereas in B16F1 cells, 10B accumulated well and reached a plateau within 30 min. Electroporation of these cells resulted in an accumulation of 10B into MCF7 cells up to the level of 10B in B16F1 cells. In vivo, the application of electric pulses increased and prolonged the entrapment of 10B (BSH) in the B16F1 melanoma tumours. A sufficient concentration of 10B was present in the tumour exposed to electric pulses for up to 24 h. Boron was quickly washed out from the blood and the level was below the concentrations in the tumours exposed to electric pulses at 2 h. The results of this study show that electroporation may provide a tool to increase boron concentration in the cells that have impaired transport of BSH through the plasma membrane. Furthermore, prolonged entrapment of BSH in tumours in vivo may, in addition to electroporation, be caused by the modifying effect of electric pulses on blood flow.

Speciation of low molecular weight Al complexes in serum of CAPD patients.

Speciation of LMW--Al complexes was performed in human serum of six continuous ambulatory peritoneal dialysis (CAPD) patients in order to investigate the individual variability in the percentage and the composition of LMW--Al species. The total concentration of Al in serum ranged from 10 to 120 ng ml(-1). The samples with high total concentration of Al were analysed directly, while those of low total Al concentration were spiked with Al(3+). Spiked and non-spiked samples (100--120 ng ml(-1) of total Al) were microultrafiltered through a membrane filter (cut-off 30,000 Da) to separate Al-transferrin from LMW-Al complexes. On an anion-exchange fast protein liquid chromatography (FPLC) column, 0.2 ml of filtrate was injected. An aqueous -- 4 mol l(-1) NH(4)NO(3) linear gradient elution was applied for 10 min to separate LMW--Al complexes. Fractions of 0.2 ml collected throughout the chromatographic run were diluted 1:1 with water and Al determined 'off line' by electrothermal atomic absorption spectrometry (ETAAS). The characterisation of LMW-Al species eluted under the chromatographic peaks was performed also by electrospray tandem mass spectrometric (ES-MS-MS) analysis. It was found experimentally that the percentage of LMW--Al species in spiked and non-spiked serum ranged from 25 to 50% (in one non-spiked sample 100%). The following LMW--Al species were separated and identified during the chromatographic run: Al-phosphate and a mixture of Al-citrate and ternary Al-citrate--phosphate complexes. It was found experimentally that the distribution of these species varied among particular patients. Similar distribution of LMW--Al species was found in spiked serum of healthy volunteers.

Speciation of aluminium in forest soil extracts by size exclusion chromatography with UV and ICP-AES detection and cation exchange fast protein liquid chromatography with ETAAS detection.

Aluminium speciation was studied in forest soil extracts by size exclusion chromatography (SE) with UV and inductively coupled plasma-atomic emission spectrometric (ICP-AES) detection and cation exchange fast protein liquid chromatography (FPLC) with ETAAS detection. Size exclusion chromatography was performed on a Superdex HR75 10/30 column. Isocratic clution with 0.15 mol dm(-3) NaCl in TRIS-HCl buffer (pH = 5.5) was applied over 100 min at a flow rate of 0.35 cm(3) min(-1). The chromatographic run was followed at 278 nm and separated Al species also determined 'off line' in 0.875 cm3 fractions by ICP-AES. The analytical procedure enabled speciation of high molecular weight Al complexes. Cation exchange FPLC was performed on a Mono S HR 5/5 column. Aqueous 8 mol dm(-3) NH4NO3 linear gradient elution was applied over 10 min at a flow rate of 1 cm3 min(-1). Separated Al species were collected in 0.5 cm3 fractions and Al determined 'off line' by ETAAS. The analytical procedure enabled speciation of some positively charged monomeric Al species. Negatively charged species were eluted with the solvent front. The combination of the two analytical techniques was successfully employed in speciation of Al in forest soil extracts. Water was used as an extracting solution. It was found experimentally that 80-95% of Al in aqueous extracts of forest soils exists in monomeric Al forms. Water soluble Al (30-40%) is bound to high molecular weight complexes with humic substances. The remaining monomeric Al in the low molecular weight fraction exists as AIF2+, Al-oxalate and Al-citrate species.

In vivo analysis of kallikrein-related peptidase 6 (KLK6) function in oligodendrocyte development and the expression of myelin proteins.

Oligodendrocytes are important for not only nerve conduction but also central nervous system (CNS) development and neuronal survival in a variety of conditions. Kallikrein-related peptidase 6 (KLK6) is expressed in oligodendrocytes in the CNS and its expression is changed in several physiological and pathological conditions, especially following spinal cord injury (SCI) and experimental autoimmune encephalomyelitis. In this study, we investigated the functions of KLK6 in oligodendrocyte lineage cell development and the production of myelin proteins using KLK6-deficient (KLK6(-/-)) mice. KLK6(-/-) mice were born without apparent defects and lived as long as wild-type (WT) mice. There was no significant difference in the numbers of oligodendrocyte precursor cells and mature oligodendrocytes in the adult naive spinal cord between WT and KLK6(-/-) mice. However, there were fewer mature oligodendrocytes in the KLK6(-/-) spinal cord than in the WT spinal cord at postnatal day 7 (P7). Expression of myelin basic protein (MBP) and oligodendrocyte-specific protein/claudin-11, major myelin proteins, was also decreased in the KLK6(-/-) spinal cord compared with the WT spinal cord at P7-21. Moreover, after SCI, the amount of MBP in the damaged spinal cords of KLK6(-/-) mice was significantly less than that in the damaged spinal cords of WT mice. These results indicate that KLK6 plays a functional role in oligodendrocyte development and the expression of myelin proteins.

[Small cell neuroendocrine carcinoma of the urinary bladder].

Among neuroendocrine tumors of the urinary bladder, small cell carcinoma (SCCB) is the most common one. Less frequent is carcinoid tumour and very rare is a large-cell neuroendocrine carcinoma. Small cell neuroendocrine carcinoma is a very aggressive tumour, with major frequency in the seventh decade. In 95% of patients it presents with hematuria and muscle invasive disease. A case of a patient with the urinary bladder tumour, which had muscle invasion and extension in perivesical tissue, was presented. The patient was diagnosed with combined form of the tumour, consisting of small cell and squamous cell patterns. Some of the imunochistochemical markers used in diagnosis were chromogranin A, synaptophysin, cytokeratins, LCA and Ki-67. Consequently, neuroendocrine differentiation of small cell patterns of the tumour was proven. Neoadjuvant cisplatin- based chemotherapy followed by radical resection should be considered as the treatment of choice in surgically resectabile SCCB. Because of that it is essential to make histopathologic diagnosis of SCCB in transuretral tumour samples using, chromogranin A or synapthysin.