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.

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.

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.

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.

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.

Nodal quasiparticles and classical phase dluctuations in d-wave superconductors.

We show that the nodal quasiparticles have a significant effect on the classical phase fluctuations in a quasi-two-dimensional d-wave superconductor. They give rise to singularities in the temperature behavior of some of the coupling constants in the phase-only effective action. One of the consequences is that the classical XY model is not adequate for the description of the superconducting fluctuations in d-wave superconductors at low temperatures.

Potential for the speciation of Zn using fast protein liquid chromatography (FPLC) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection.

Analytical procedures were developed for the speciation of Zn using fast protein liquid chromatography (FPLC), flame atomic absorption spectrometry (FAAS) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection. The investigation was performed on synthetic solutions (2 microg cm-3 Zn) of hydrated Zn2+ species and Zn complexes with citrate, oxalate and EDTA (ligand-to-Zn molar ratio 100:1) over a pH range from 5.4 to 7.4. It was found that Zn interacts with various buffers and the careful adjustment of the pH with diluted solutions of KOH is, therefore, required. FPLC separations were carried out on a Mono Q HR 5/5 strong anion-exchange column, applying an aqueous 1 mol dm(-3) NH4NO3 linear gradient elution over 15 min, at a flow rate of 1.0 cm3 min(-1). The separated Zn species were determined in 1.0 cm3 eluate fractions "off line" by FAAS. Speciation of Zn was also performed on a weak anion-exchange CIM DEAE fast monolithic disc by applying an aqueous 0.4 mol dm(-3) NH4NO3 linear gradient elution over 7.5 min, at a flow rate of 2.0 cm3 min(-1) and determination of the separated Zn species in 1.0 cm3 eluate fractions "off line" by FAAS. Zn-binding ligands in separated fractions were also characterized by electrospray (ES)-MS-MS analysis. The CIM DEAE disc was found to be more efficient in the separation of negatively charged Zn complexes than the Mono Q FPLC column. On the CIM DEAE disc Zn-citrate was separated from both Zn-oxalate and from Zn-EDTA. All these species were also separated from hydrated Zn2+, which was eluted with the solvent front. This method has an advantage over commonly used analytical techniques for the speciation of Zn which are only able to distinguish between labile and strong Zn complexes. Good repeatability of the measurements (RSD 2-4%), tested for six parallel determinations (2 microg cm(-3) Zn) of Zn-EDTA, Zn-citrate and Zn-oxalate was found at a pH of 6.4 on a CIM DAEA disc. The limit of detection (3s) for the separated Zn species was 10 ng cm(-3). The proposed analytical procedure was applied to the speciation of Zn in aqueous soil extracts and industrial waste water from a lead and zinc mining area.

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.

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.

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.

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.

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.

An orphan seven-transmembrane domain receptor expressed widely in the brain functions as a coreceptor for human immunodeficiency virus type 1 and simian immunodeficiency virus.

Both CD4 and an appropriate coreceptor are necessary for infection of cells by human immunodeficiency virus type 1 (HIV-1) and most strains of HIV-2. The chemokine receptors CCR5 and CXCR4 are the major HIV-1 coreceptors, although some virus strains can also utilize alternative coreceptors such as CCR3 to infect cells. In contrast, most if not all simian immunodeficiency virus (SIV) strains use CCR5 as a coreceptor, and many SIV strains can use CCR5 independently of CD4. In addition, several orphan seven-transmembrane receptors which can serve as HIV-1 and SIV coreceptors have been identified. Here we report that APJ, an orphan seven-transmembrane domain receptor with homology to the angiotensin receptor family, functions as a coreceptor for a number of HIV-1 and SIV strains. APJ was expressed widely in the human brain and in NT2N neurons. APJ transcripts were also detected by reverse transcription-PCR in the CD4-positive T-cell line C8166, but not in peripheral blood leukocytes, microglia, phytohemagglutinin (PHA)- or PHA/interleukin-2-stimulated peripheral blood mononuclear cells, monocytes, or monocyte-derived macrophages. The widespread distribution of APJ in the central nervous system coupled with its use as a coreceptor by some HIV-1 strains indicates that it may play a role in neuropathogenesis.

Inducible nitric oxide synthase and nitric oxide production by oligodendrocytes.

It has been previously demonstrated that microglia and astrocytes produce micromolar amounts of nitric oxide in vitro. In this study, we demonstrate that primary rat oligodendrocytes can be stimulated to produce iNOS mRNA as detected by Northern blot and in situ hybridization analysis and a 131-kDa iNOS protein by Western blot analysis; protein was also detected in cells by single- and double-label immunohistochemistry for iNOS and the oligodendrocyte-specific marker CNPase. NO/NOS are produced as a consequence of activation of the gene encoding the inducible nitric oxide synthase as determined by inhibition with actinomycin D and cyclohexamide. The iNOS is functional, leading to calcium/calmodulin-independent NO production in these in vitro cultures.

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.

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.