Cell-biological effects of zinc oxide spheres and rods from the nano- to the microscale at sub-toxic levels.

Zinc oxide particles were synthesized in various sizes and shapes, i.e., spheres of 40-nm, 200-nm, and 500-nm diameter and rods of 40∙100 nm2 and 100∙400 nm2 (all PVP-stabilized and well dispersed in water and cell culture medium). Crystallographically, the particles consisted of the hexagonal wurtzite phase with a primary crystallite size of 20 to 100 nm. The particles showed a slow dissolution in water and cell culture medium (both neutral; about 10% after 5 days) but dissolved within about 1 h in two different simulated lysosomal media (pH 4.5 to 4.8). Cells relevant for respiratory exposure (NR8383 rat alveolar macrophages) were exposed to these particles in vitro. Viability, apoptosis, and cell activation (generation of reactive oxygen species, ROS, release of cytokines) were investigated in an in vitro lung cell model with respect to the migration of inflammatory cells. All particle types were rapidly taken up by the cells, leading to an increased intracellular zinc ion concentration. The nanoparticles were more cytotoxic than the microparticles and comparable with dissolved zinc acetate. All particles induced cell apoptosis, unlike dissolved zinc acetate, indicating a particle-related mechanism. Microparticles induced a stronger formation of reactive oxygen species than smaller particles probably due to higher sedimentation (cell-to-particle contact) of microparticles in contrast to nanoparticles. The effect of particle types on the cytokine release was weak and mainly resulted in a decrease as shown by a protein microarray. In the particle-induced cell migration assay (PICMA), all particles had a lower effect than dissolved zinc acetate. In conclusion, the biological effects of zinc oxide particles in the sub-toxic range are caused by zinc ions after intracellular dissolution, by cell-to-particle contacts, and by the uptake of zinc oxide particles into cells. Graphical headlights • The cytotoxicity of zinc oxide particles is mainly due to the intracellular release of zinc ions. • The size and shape of zinc oxide micro- and nanoparticles has only small effects on lung cells in the sub-toxic range. • Zinc oxide particles are rapidly taken up by cells, regardless of their size and shape. • Zinc oxide particles rapidly dissolve after cellular uptake in endolysosomes.

Metagenomic evaluation of the effects of storage conditions on the bacterial microbiota of oysters Crassostrea gasar (Adanson, 1757).

AIMS: To evaluate the influence of storage conditions on the composition of the bacterial microbiota of living oysters Crassostrea gasar. METHODS AND RESULTS: The oysters used in this study came from marine farms (Guaratuba Bay, Brazil) and were exposed to two conditions that simulated different storage situations: immersion in water (group I) and exposure to air (group II). The animals were subjected to five different temperatures (5-25°C), for 10 days. The 16S rRNA gene from oysters was amplified and sequenced to determine the taxonomic units and bacterial strains present in the samples. Group I showed higher diversity of bacteria (163 genera) rather than group II (104 genera). In all, 59 bacterial genera potentially pathogenic to humans were identified (n = 56 in group I and n = 45 in group II). CONCLUSIONS: The storage conditions having a direct influence on the oyster microbiota. Live C. gasar should be stored exposed to air at 5-25°C, because it favours a lower prevalence of bacteria potentially pathogenic to humans. SIGNIFICANCE AND IMPACT OF THE STUDY: During the oyster commercialization process, some conditions of storage, time and temperature must be followed in order to reduce the prevalence of bacteria potentially pathogenic to humans.

Kinetics of chemotaxis, cytokine, and chemokine release of NR8383 macrophages after exposure to inflammatory and inert granular insoluble particles.

Accumulation of macrophages and neutrophil granulocytes in the lung are key events in the inflammatory response to inhaled particles. The present study aims at the time course of chemotaxis in vitro in response to the challenge of various biopersistent particles and its functional relation to the transcription of inflammatory mediators. NR8383 rat alveolar macrophages were challenged with particles of coarse quartz, barium sulfate, and nanosized silica for one, four, and 16h and with coarse and nanosized titanium dioxide particles (rutile and anatase) for 16h only. The cell supernatants were used to investigate the chemotaxis of unexposed NR8383 macrophages. The transcription of inflammatory mediators in cells exposed to quartz, silica, and barium sulfate was analyzed by quantitative real-time PCR. Challenge with quartz, silica, and rutile particles induced significant chemotaxis of unexposed NR8383 macrophages. Chemotaxis caused by quartz and silica was accompanied by an elevated transcription of CCL3, CCL4, CXCL1, CXCL3, and TNFα. Quartz exposure showed an earlier onset of both effects compared to the nanosized silica. The strength of this response roughly paralleled the cytotoxic effects. Barium sulfate and anatase did not induce chemotaxis and barium sulfate as well caused no elevated transcription. In conclusion, NR8383 macrophages respond to the challenge with inflammatory particles with the release of chemotactic compounds that act on unexposed macrophages. The kinetics of the response differs between the various particles.

Response of psoriasis to interleukin-10 is associated with suppression of cutaneous type 1 inflammation, downregulation of the epidermal interleukin-8/CXCR2 pathway and normalization of keratinocyte maturation.

Psoriasis is a chronic inflammatory skin disease in which epidermal hyperplasia results from the release of cytokines by infiltrating type 1 T cells. Up- regulation of endogenous interleukin-10 controls type 1 skin responses in animal models; however, interleukin-10 production is low in psoriatic lesions. Consistent with an important role of interleukin-10 in psoriasis, we and colleagues have recently demonstrated clinical efficacy of subcutaneous administration of recombinant interleukin-10 to affected patients. Here, we studied the effects of interleukin-10 on disease-related inflammatory pathways. Patients were treated with recombinant interleukin-10 over 6 wk in an open-label phase II clinical trial. Tissue was obtained before and after therapy and examined by histology/immunohistochemistry, in situ hybridization, and quantitative real-time reverse transcription-polymerase chain reaction. Ten of 14 patients showed a marked reduction of the clinical disease activity. The clinical response was associated with a significant decrease of cutaneous T cell infiltration and the lesional expression of type 1 cytokines interferon-gamma and tumor necrosis factor-alpha. Interleukin-10 inhibited the epidermal interleukin-8 pathway by downregulating the expression of interleukin-8, its receptor CXCR2, and its inducer interleukin-17, and partially reversed the aberrant keratinocyte maturation defining psoriatic epidermal pathology. Remarkably, there was evidence that genetic factors are involved in the response to interleukin-10 as individual variations in the downregulation of tumor necrosis factor-alpha were related to the presence of polymorphisms in the tumor necrosis factor-alpha promoter. These data suggest that excessive production of type 1 cytokines in human skin disease can be counter-regulated by the administration of recombinant interleukin-10. Genotypic analysis may help to identify patients that will preferentially respond to interleukin-10 therapy.

Combined analysis of polymorphisms of the tumor necrosis factor-alpha and interleukin-10 promoter regions and polymorphic xenobiotic metabolizing enzymes in psoriasis.

Environmental and genetic factors are thought to interact in the manifestation of psoriasis, but knowledge about the involved genes and antigens is incomplete. This study has focused on the association between psoriasis and inherited variations in xenobiotic metabolism and cytokine production as two components that may influence cutaneous immune responses to foreign substances. Polymorphisms of N-acetyltransferase 2, glutathione S-transferases T1 and M1, and promoter polymorphisms of the genes encoding for tumor necrosis factor-alpha and interleukin-10 were investigated in 151 Caucasian patients with psoriasis (100 with type I and 51 with type II psoriasis) and in 123 healthy controls. Polymorphisms were detected by polymerase chain reaction-based methods, restriction enzyme analysis, and direct sequencing. There were no significant differences in the distribution of enzyme polymorphisms or point mutations at position -1082 of the interleukin-10 promoter between the psoriasis groups and the control group. The G-->A polymorphism at position -238 of the tumor necrosis factor-alpha promoter (TNF alpha-238*A allele) was more common in type I psoriasis (27%) than in the controls [9.8%; odds ratio 3.4 (95% confidence interval 1.6-7.2); p = 0.0012; pcorr = 0.018]. Surprisingly, this overrepresentation of the tumor necrosis factor alpha-238*A allele was observed in male patients [4.1 (1.5-11.0); p = 0.0046; pcorr = 0.064] but not in female patients [1.8 (0.5-6.5); p = 0.5]. The G-->A polymorphism at position -308 of the tumor necrosis factor-alpha promoter was less frequent in type I psoriasis (23%) compared with controls (35.7%), although the negative association was weak [0.54 (0.3-0.97); p = 0.041; pcorr = not significant]. The distribution of the TNF alpha-238*A and TNF alpha-238*A alleles was similar in type II patients and controls. Our results suggest that male carriers of the G-->A polymorphism at position -238 of the tumor necrosis factor-alpha promoter are at an increased risk to develop early-onset psoriasis.

PAPS-reductase of Escherichia coli. Correlating the N-terminal amino acid sequence with the DNA of gene cys H.

The DNA of the gene complementing a PAPS-reductase-deficient strain of Escherichia coli was sequenced. The N-terminal amino acid sequence of the purified PAPS-reductase confirmed that cys H is the structural gene for this enzyme. The open reading frame extends 732 bases and encodes for a peptide of Mr = 27927. The gene product is functionally active when supplemented with thioredoxin and immunologically related with the wild type enzyme.

The effect of human recombinant erythropoietin on the growth of a human neuroblastoma cell line.

Erythropoietin is a growth factor. Cancer can be described as disturbance of the fine balance of positive and negative growth control mechanisms. The effect of human recombinant erythropoietin (EPO) was studied on the cell growth and differentiation of a human neuroblastoma cell line (h-NMB). Cell growth curves, trypan blue staining and thymidine uptake were used to assess cell proliferation and death. To assess cell differentiation, neutral endopeptidase (cell membrane enzyme marker), creatine kinase (cytosolic enzyme marker), dopamine uptake (dopamine transporter marker) and cell morphology were determined. Specific EPO receptor mRNA, by RT-PCR technique, was demonstrated. The incubation of erythropoietin with the tumor cell line resulted in inhibition of cell proliferation as evidenced in a diminished cell growth. EPO was shown to have induced a differentiation process as seen from the two different enzymatic markers, membranal and cytosolic, and from the cells dopamine uptake studies. However, the morphological changes did not document a full differentiation effect. EPO specific antibodies blocked the effects of EPO on cell proliferation and creatine kinase activity. In this study, EPO did not produce any sign of proliferation in the nervous tumor cell line used.

Detection and quantification of the soluble form of the human erythropoietin receptor (sEpoR) in the growth medium of tumor cell lines and in the plasma of blood samples.

The erythropoietin receptor (EpoR) belongs to the cytokine superfamily and is a type I transmembrane protein. Like other members of this family, EpoR is also synthesized as a soluble form, and is subsequently secreted by the cell. To investigate whether soluble EpoR (sEpoR) is expressed in human tumor cell lines, we developed a sensitive quantitative ELISA, using an anti-human EpoR antibody and recombinant sEpoR as standard control. With this ELISA, sEpoR could be detected in the supernatant of human tumor cell lines. Analysis of blood samples showed that sEpoR was coprecipitated during coagulation. Therefore only plasma was suitable for analysis and first measurements of plasma samples were investigated. In conclusion, an ELISA to quantify the sEpoR was established and the expression of sEpoR in human tumor cell lines was demonstrated for the first time.

Detection of NGF-receptors TrkA and p75NTR in human tumor cell lines and effect of NGF on the growth characteristic of the UT-7/EPO cell line.

The receptors for nerve growth factor (NGF)--TrkA and p75NTR--were detected at the mRNA and the protein level in various human tumor cell lines. The NGF receptor TrkA was found on all examined tumor cell lines and is not restricted to cells belonging to the nervous system. NGF did not influence the proliferation rate of TrkA-positive cells NMB, K562, UT-7/EPO and PC-12. After NGF induction, the production level of the differentiation marker c-fos was increased in UT-7/EPO and PC- 12 cells. NGF-treatment of the UT-7/EPO cells and deprivation of erythropoietin (EPO) led to the new adherent cell line UT-7/NGF. Although UT-7/NGF showed a similar growth curve as UT-7/EPO, there were differences in the pattern of adhesion molecules and of the cytoskeleton. The effect of NGF on the cytoskeleton could not be induced in other human cell lines like NMB or KTCTL-30. TrkA inhibition with K252a--a blocker of Trk-induced receptor kinase--suggests, that the NGF signal may be transduced by the TrkA receptor in UT-7/NGF cells. This indicates that NGF is a decisive mediator of cellular adhesion.

Inhibition of the human erythrocytic glutathione-S-transferase T1 (GST T1) by thimerosal.

We have investigated the interaction of thimerosal, a widely used antiseptic and preservative, with the human erythrocytic GST T1 (glutathione-S-transferase T1). This detoxifying enzyme is expressed in the erythrocytes of solely the human species and it displays a genetic polymorphism. Due to this polymorphism about 25% of the individuals of the caucasian population lack this activity ("non-conjugators"), while 75% show it ("conjugators") (Hallier, E., et al., 1993). Using our newly developed HPLC-fluorescence detection assay (Müller, M., et al., 2001) we have profiled the kinetics of enzyme inhibition in erythrocyte lysates of two individuals previously identified as "normal conjugator" (medium enzyme activity) and "super-conjugator" (very high activity). For the normal conjugator we have determined a 2.77 mM thimerosal concentration to inhibit 50% of the GST T1 activity. In the case of the super-conjugator a 2.3 mM thimerosal concentration causes a 50% inhibition of the enzyme activity. For both phenotypes a 14.8 mM thimerosal concentration results in residual enzyme activities equal to those typically detected in non-conjugator lysates. Thus, sufficiently high doses of thimerosal may be able to change the phenotypic status of an individual--at least in vitro--by inhibition of the GST T1 enzyme.

Application of short-lived radionuclides in neutron activation analysis of biological and environmental samples.

The application of short-lived nuclides, especially in connection with the 6LiD-converter, in biological and environmental samples is demonstrated on I and Br determination in human urine, on I in pet food, and on the analysis of all the halogens in volcanic gases in a single activation. Trace element determination in lichens indicates polluted and unpolluted areas. The use of the .74-s 38mCl enables the rapid screening of great number of samples.

[The clinical value of radioimmunological oestradiol assays in the diagnosis of menstrual disorders. Evidence suggesting the presence of an ovarian "inhibin" (author's transl)]. / Der klinische Wert der radioimmunologischen Ostradiolbestimmung für die Diagnostik von Zyklusstörungen

The radioimmunoassay of oestradiol 17 beta (E2) from the blood without chromatography was evaluated in regard to the diagnosis of menstrual cycle disturbances. Two significantly different classes were distinguished, viz. 1. Women with higher E2 values, consisting of three groups: normal controls, cases of oligomenorrhoea, and WHO II (normogonadotropic, clomiphen-positive amenorrhoea). 2. Women with lower E2 values, consisting of two groups: WHO I (hypogonadotropic, clomiphen-negative amenorrhoea) and WHO III (hypergonadotropic amenorrhoea). Within these classes no significant differences were found between the groups. Only values below 43.1 pg/ml can be assigned with 95% certainty to the low-value class and only values above 108.8 pg/ml can be assigned with 95% certainty to the high-value class. The fact that 40% of all E2 values in hypergonadotropic, (i.e. ovarian) amenorrhoea fell within the range of the double standard deviation of the normal group and, likewise, the fact that physiological doses of oestrogen cannot reduce postmenopausal FSH to the level found in women of reproductive age suggest that E2 is not the only FSH-reducing factor, which leads us to postulate the existence of an ovarian "inhibin".

[Potential virostatics. Part 3: NMR Studies in quinoxalines and s-triazolo (4,3-a) quinoxalines (author's transl)]. / Potentielle Virostatica. Teil 3: NMR-Untersuchungen an Chinoxalinen und s-Triazolo[4.3-a]chinoxalinen.

Examples of different tautomeric forms were detected in a series of modified quinoxalines by means of nmr spectrometry. Apart from tautomeric equilibria, certain specially favoured conformers may play a part in biological systems, as illustrated by an example.

[Antimicrobial activity of selected quinoxalines (author's transl)]. / Antimikrobielle Wirksamkeit ausgewählter Chinoxaline.

After a survey of the literature on biological effects of quinoxalines experimental results of 2,3- and 2,3,6-substituted quinoxalines against Bacillus subtilis, Staphylococcus aureus, Candida albicans, Candida tropicalis, Trichophyton mentagrophytes and Microsporon gypseum were demonstrated. The most active compound was 2-chlor-3-methyl-6-nitroquinoxaline. Its MIC values against Trichophyton mentagrophytes and Candida albicans were determined with 54 and 223 micromol/l.

Guidelines for maintenance of adult patients with brain death and potential for multiple organ donations: the Task Force of the Brazilian Association of Intensive Medicine the Brazilian Association of Organs Transplantation, and the Transplantation Center of Santa Catarina.

INTRODUCTION: The organ shortage for transplantation, the principal factor that increases waiting lists, has become a serious public health problem. In this scenario, the intensivist occupies a prominent position as one of the professionals that first has a chance to identify brain death and to be responsible for the maintenance of the potential deceased donor. OBJECTIVE: This report attempts to establish guidelines for care and maintenance of adult deceased donor organs guiding and standardizing care provided to patients with brain death. METHOD: These guidelines were composed by intensivists, transplant coordinators, professionals from various transplant teams, and used transplant center. The formulated questions were forwarded to all members and recommendations were constructed after an extensive literature review selecting articles with the highest degree of evidence. RESULTS: Guidelines were developed in the form of questions reflecting frequent experiences in clinical intensive care practices. The main questions were: Is there an optimal interval for keeping organs of deceased donors viable? What actions are considered essential for maintaining deceased donors in this period? What are the limits of body temperature? How should the patient be warmed? Which laboratory tests should be performed? What is the collection interval? What are the limits in the laboratory and the capture scenario? What are the limits of blood pressure? When and how should one use catecholamines? CONCLUSIONS: This pioneer project involved a multidisciplinary team working in organ transplantation seeking to provide treatment guidance to increase the number of viable organs from deceased adult donors.

Ether oxygenate additives in gasoline reduce toxicity of exhausts.

UNLABELLED: Fuel additives can improve combustion and knock resistance of gasoline engines. Common additives in commercial fuels are "short-chain, oxygen containing hydrocarbons" such as methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE). Since these additives change the combustion characteristics, this may as well influence toxic effects of the resulting emissions. Therefore we compared toxicity and BTEX emissions of gasoline engine exhaust regarding addition of MTBE or ETBE. Non-reformulated gasoline served as basic fuel. This fuel was supplemented with 10%, 20%, 25% and 30% ETBE or 15% MTBE. The fuels were combusted in a gasoline engine at idling, part load and rated power. Condensates and particulate matter (PM) were collected and PM samples extracted with dichloromethane. Cytotoxic effects were investigated in murine fibroblasts (L929) using the neutral red uptake assay and mutagenicity using the bacterial reverse mutation assay. BTEX emissions were analyzed by gas chromatography. RESULTS: PM-extracts showed mutagenicity with and without metabolic activation. Mutagenicity was reduced by the addition of MTBE and ETBE, 10% ETBE being most effective. The condensates produced no significant mutagenic response. The cytotoxicity of the condensates from ETBE- and MTBE-reformulated fuels was reduced as well. The BTEX content in the exhaust was lowered by the addition of MTBE and ETBE. This effect was significantly related to the ETBE content at rated power and part load. CONCLUSIONS: Addition of MTBE and ETBE to fuels can improve combustion and leads to decreased toxicity and BTEX content of the exhaust. Reduction of mutagenicity in the PM-extracts is most probably caused by a lower content of polycyclic aromatic hydrocarbons.