Novel approach to gene expression profiling in Sézary syndrome.

BACKGROUND: Microarray hybridization studies in Sézary syndrome (SS) have compared T lymphocytes from patients with cutaneous T-cell lymphoma with those of normal controls; a major limitation of this design is that significant inherent genetic variability of lymphocyte populations between individuals may produce differences in gene expression unrelated to disease state. OBJECTIVE: The objective of this study was to minimize the heterogeneity of information derived from whole-genome expression analysis and to identify specific genetic differences between highly purified malignant and nonmalignant (control) T cells from the same patient with SS. METHODS: Peripheral blood mononuclear cells were obtained from a patient with SS, stained with anti-T-cell receptor Vb (TCR-Vb) antibodies, and sorted by multiparameter flow cytometry. Malignant cells expressed the dominant TCR-Vb; control T cells lacked the dominant TCR-Vb but were otherwise phenotypically identical (CD3+CD4+CD45RO+). These cell populations were compared using the Illumina Inc. Sentrix Human-6 expression BeadChip system. RESULTS: Transcriptome analysis using the J5 test, which was selected for data analysis based on an efficiency analysis of competing statistical methods, showed differential expression of 44 genes between the malignant and nonmalignant cell subsets. Promyelocytic leukaemia zinc finger protein (ZBTB16) was the most profoundly upregulated gene in the malignant cell population, while interferon regulatory factor 3 (IRF3) and interferon-induced protein 35 (IFI35), which are important elements of the cellular response to viral infection, were significantly downregulated. CONCLUSIONS: The results of this study suggest the feasibility of this novel comparative approach to genomic profiling in SS. Using this method, we identified several differentially expressed genes and pathways not previously described in SS. While these findings require validation in larger studies, they may be important in SS pathogenesis.

[Neuromonitoring and neuroprotection in cardiac anaesthesia. Nationwide survey conducted by the Cardiac Anaesthesia Working Group of the German Society of Anaesthesiology and Intensive Care Medicine]. / Neuromonitoring und Neuroprotektion in der Kardioanästhesie: Bundesweite Umfrage des Arbeitskreises Kardioanästhesie der Deutschen Gesellschaft für Anästhesiologie und Intensivmedizin e.V.

OBJECTIVE: The primary objective of this nationwide survey carried out in department of cardiac anesthesia in Germany was to identify current practice with regard to neuromonitoring und neuroprotection. METHODOLOGY: The data are based on a questionnaire sent out to all departments of cardiac anesthesia in Germany between October 2007 und January 2008. The anonymized questionnaire contained 26 questions about the practice of preoperative evaluation of cerebral vessels, intra-operative use of neuromonitoring, the nature und application of cerebral protective measures, perfusion management during cardiopulmonary bypass, postoperative evaluation of neurological status, and training in the field of cerebral monitoring. RESULTS: Of the 80 mailed questionnaires 55% were returned and 90% of department evaluated cerebral vessels preoperatively with duplex ultrasound. The methods used for intra-operative neuromonitoring are electroencephalography (EEG, 60%) for type A dissections (38.1%), for elective surgery on the thoracic and thoraco-abdominal aorta (34.1% and 31.6%, respectively) and in carotid surgery (43.2%) near infrared spectroscopy (40%), evoked potentials (30%) and transcranial Doppler sonography (17.5%), with some centers using combined methods. In most departments the central nervous system is not subjected to monitoring during bypass surgery, heart valve surgery, or minimally invasive surgery. Cerebral protective measures used comprise patient cooling on cardio-pulmonary bypass (CPB 100%), extracorporeal cooling of the head (65%) and the administration of corticosteroids (58%), barbiturates (50%) and antiepileptic drugs (10%). Neuroprotective anesthesia consists of administering inhalation anesthetics (32.5%; sevoflurane 76.5%) and intravenous anesthesia (20%; propofol and barbiturates each accounting for 46.2%). Of the departments 72.5% cool patients as a standard procedure for surgery involving cardiovascular arrest and 37.5% during all surgery using CPB. In 84.6% of department CPB flow equals calculated cardiac output (CO) under normothermia, while the desired mean arterial pressure (MAP) varies between 60 and 70 mmHg (43.9%) and between 50 and 60 mmHg (41.5%), respectively. At body temperatures less than 18 degrees C CPB flow is reduced below the calculated CO (70%) while 27% of departments use normothermic flow rates. The preferred MAP under hypothermia is between 50 and 60 mmHg (59%). The results of intra-operative neuromonitoring are documented on the anesthesia record (77%). In 42.5% of the departments postoperative neurological function is estimated by the anesthesiologist. Continuing education sessions pertaining to neuromonitoring are organized on a regular basis in 32.5% of the departments and in 37.5% individual physicians are responsible for their own neuromonitoring education. CONCLUSION: The present survey data indicate that neuromonitoring and neuroprotective therapy during CPB is not standardized in cardiac anesthesiology departments in Germany. The systemic use of available methods to implement multimodal neuromonitoring would be desirable.

[Anesthesia in patients with glucose-6-phosphate dehydrogenase deficiency: case report and perioperative anesthesiologic management]. / Anästhesie bei Patienten mit Glukose-6-Phosphat-Dehydrogenase-Mangel: Fallbericht und perioperatives anästhesiologisches Management.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a frequent congenital human enzyme defect, is the most frequent cause of hemolytic anemia triggered by drugs or infectious diseases. Drugs which induce acute hemolysis in patients with G6PD deficiency are often used in anesthesia and perioperative pain therapy. Considering the fact that patients from geographic regions with a high prevalence of the disease are often treated in European hospitals, special attention should be paid to this problem. We report a case of a 30-year-old female patient with favism and review the disease and anesthesia-related implications.

Hearts and mouths: perceptions of oral hygiene by at-risk heart surgery patients.

OBJECTIVE: To assess and use the attitudes of patients who are placed at risk after valvular heart surgery due to the connection between poor oral hygiene, valvular heart disease/surgery and the risk of developing infective endocarditis. DESIGN: A qualitative (focus group) design based study carried out on subjects three months post heart surgery. METHOD: There were five focus groups of five participants each convened by an experienced moderator. RESULTS: These portrayed an apparent pressing desire by most patients to talk about their experiences. However, patients did not accept the link between their oral health and their general health. Oral hygiene practices were not necessarily oral health related. CONCLUSIONS: The importance of the study in understanding the reasons for a patient's behaviour is evident when there is a clear need to modify the behaviour patterns of the patients effectively. Clinical trials can now be developed based on these results.

[Anaesthesiological management of mediastinal tumors]. / Anästhesiologisches Management bei mediastinaler Raumforderung. Aktuelle Konzepte der perioperativen Versorgung.

The perioperative management of patients with mediastinal masses is a special clinical challenge in our field. Even though regional anaesthesia is normally the first choice, in some cases it is not feasible due to the method of operation. In these cases general anaesthesia is the second option but can lead to respiratory and haemodynamic decompensation due to tumor-associated compression syndrome (mediastinal mass syndrome). The appropriate treatment begins with the preoperative risk classification on the basis of clinical and radiological findings. In addition to anamnesis, chest radiograph, and CT, dynamical methods (e.g. pneumotachography and echocardiography) should be applied to verify possible intraoperative compression syndromes. The induction of general anaesthesia is to be realized in awake-fiberoptic intubation with introduction of the tube via nasal route while maintaining the spontaneous breathing of the patient. The anaesthesia continues with short effective agents applied inhalative or iv. If possible from the point of operation, agents of muscle relaxation are not to be applied. If the anaesthesia risk is classified as uncertain or unsafe, depending on the location of tumor compression (tracheobronchial tree, pulmonary artery, superior vena cava), alternative techniques of securing the respiratory tract (different tubes, rigid bronchoscope) and cardiopulmonary bypass with extracorporal oxygen supply are prepared. For patients with severe clinical symptoms and extensive mediastinal mass, the preoperative cannulation of femoral vessels is also recommended. In addition to fulfilling technical and personnel requirements, an interdisciplinary cooperation of participating fields is the most important prerequisite for the optimal treatment of patients.

[Hypoglycemia despite hyperglycemia. Is a cerebral glucose deficiency possible even with raised blood sugar levels?]. / Hypoglykämie trotz Hyperglykämie. Ist eine zerebrale Minderversorgung mit Glukose auch bei erhöhtem Blutzuckerspiegel möglich?

Hypoglycemia represents the most frequent endocrinologic emergency situation in prehospital patient care. As the patients are usually unconscious on arrival of emergency medical personnel, often the only way to establish a diagnosis is by determination of the blood glucose concentration. However, even normoglycemic or hyperglycemic levels cannot definitively exclude the diagnosis of a previous hypoglycemia as the cause of the acute cerebral deficiency. Therefore, and especially in the case of insulin-dependent diabetes mellitus, a differential diagnosis should be considered. We report a case of emergency treatment of a hypoglycemic episode in a female patient with prolonged neuroglycopenia together with cerebrovascular dementia and Alzheimer's disease.

Cassini magnetometer observations during Saturn orbit insertion.

Cassini's successful orbit insertion has provided the first examination of Saturn's magnetosphere in 23 years, revealing a dynamic plasma and magnetic environment on short and long time scales. There has been no noticeable change in the internal magnetic field, either in its strength or its near-alignment with the rotation axis. However, the external magnetic field is different compared with past spacecraft observations. The current sheet within the magnetosphere is thinner and more extended, and we observed small diamagnetic cavities and ion cyclotron waves of types that were not reported before.

Direct transfection and activation of human cutaneous dendritic cells.

Gene therapy techniques can be important tools for the induction and control of immune responses. Antigen delivery is a critical challenge in vaccine design, and DNA-based immunization offers an attractive method to deliver encoded transgenic protein antigens. In the present study, we used a gene gun to transfect human skin organ cultures with a particular goal of expressing transgenic antigens in resident cutaneous dendritic cells. Our studies demonstrate that when delivered to human skin, gold particles are observed primarily in the epidermis, even when high helium delivery pressures are used. We demonstrate that Langerhans cells resident in the basal epidermis can be transfected, and that biolistic gene delivery is sufficient to stimulate the activation and migration of skin dendritic cells. RT-PCR analysis of dendritic cells, which have migrated from transfected skin, demonstrates the presence of transgenic mRNA, indicating direct transfection of cutaneous dendritic cells. Importantly, transfected epidermal Langerhans cells can efficiently present a peptide derived from the transgenic melanoma antigen MART-1 to a MART-1-specific CTL. Taken together, our results demonstrate direct transfection, activation, and antigen-specific stimulatory function of in situ transduced human Langerhans cells.

Crystal-cell interaction and apoptosis in oxalate-associated injury of renal epithelial cells.

Two renal epithelial cell lines, LLC-PK1 and Madin-Darby canine kidney (MDCK), were grown in monolayers and exposed to oxalate (Ox) and/or calcium oxalate (CaOx) crystals to investigate cellular responses to these challenges. In addition, LLC-PK1 cells were exposed to high concentrations of Ox for various time periods to investigate the role of apoptosis in Ox-associated cell injury. Both cell types showed signs of damage when exposed to Ox. However, LLC-PK1 cells appeared more sensitive than MDCK cells. There was a significant increase in release of lactate dehydrogenase into the medium and decrease in trypan blue exclusion by cells in the monolayer. Most noticeable was the detachment of cells from the substrate. Exposure of cells to CaOx crystals resulted in their attachment to cell surfaces followed by internalization. Using flow cytometry for quantification of apoptotic cells, transmission electron microscopy for morphology, and electrophoresis for DNA laddering detection, we observed significant apoptotic changes including condensation and margination of nuclear chromatin, DNA fragmentation, and migration of phosphatidylserine of the plasma membrane from inside to the cell surface. However, these cells also showed some necrotic changes such as loss of plasma membrane integrity and release of lactate dehydrogenase, indicating that the apoptotic process was interrupted.

The genotoxicity and cytotoxicity of dermally-administered cadmium: effects of dermal cadmium administration.

Cadmium, unlike zinc, selenium and copper, has no known biological importance, and therefore, it is classified as a carcinogen in humans, as well as in animals. The effect(s) of levels of dermally-administered cadmium on cadmium genotoxicity and cytotoxicity was investigated in Harlan Sprague-Dawley rats for 14, 21, 28, 35 and 42 days at concentrations of 14 and 28 mg/kg/day. Exposure of rats to cadmium via dermal application caused lesions on the skin (hyperkeratosis, acanthosis and scabbing, alopecia and erythema) and tumors in the scrotum. Anatomical changes, such as distention of the stomach, atrophy of kidney and liver and loss of body weight were also observed in these rats. The toxic effects of cadmium on cell ultrastructure were nuclear membrane damage, chromatin condensation, regression of mitochondrial cristae and ultimately cell death. Analyses of the brain, kidney and liver cells of rats exposed to cadmium, clearly showed DNA damage. Of the three organs examined, DNA from kidney cells sustained the most damage followed by DNA in liver cells. There is a positive correlation between Cd dose(s) and duration of exposure and the extent of DNA damage.

DQAsomes: a novel potential drug and gene delivery system made from Dequalinium.

PURPOSE: Dequalinium, a drug known for over 30 years, is a dicationic amphiphile compound resembling bolaform electrolytes. The purpose of our work was to determine the state of aggregation of dequalinium in aqueous medium and to investigate both, its ability to bind DNA and its potential to serve as a novel non-viral transfection vector. METHODS: The form of aggregation was determined employing electron microscopic techniques. The DNA binding capacity of dequalinium was assayed using SYBR Green I stain. For in vitro cell transfection experiments plasmid DNA encoding for firefly luciferase was used. RESULTS: Dequalinium forms in aqueous medium liposome-like aggregates, which we term DQAsomes. These dequalinium vesicles bind DNA and they are able to transfect cells in vitro with an efficiency comparable to Lipofectin. CONCLUSIONS: Based on the intrinsic properties of dequalinium such as the in vivo selectivity for carcinoma cells and selective accumulation in mitochondria we propose DQAsomes as a novel and unique drug and gene delivery system.

[Molecular effects of a microbicidal substance on relevant microorganisms: electron microscopic and biochemical studies on povidone-iodine]. / Molekulare Effekte eines mikrobiziden Wirkstoffes auf relevante Mikroorganismen: Elektronenmikroskopische und biochemische Untersuchungen zu Polyvidon-Iod.

The microbicidal activity of the broad spectrum antimicrobial agent povidone-iodine is due to the strong oxidizing effects of free iodine on functional groups of amino acids, nucleotides and double bonds of unsaturated fatty acids. While the chemical mechanism of action of PVP-iodine is well understood, the actual sequence of events on the cellular and molecular level that causes rapid cell death has not been fully understood. The aim of this study was to elucidate effects of povidone-iodine on cell ultrastructure by electron microscopy and to monitor changes in enzyme activity and nucleotide efflux. Staphylococcus aureus, E. coli and C. albicans, medically relevant gram-positive, gram-negative and yeast micro-organisms, served as models. In the presence of povidone-iodine, rapid partitioning of the cytoplasm and pronounced coagulation of nuclear material was noted. Especially C. albicans exhibited a rapid, dose-dependent "loosening" of the cell wall; cells remained intact without lysis, rupture or wall breakage. Changes in beta-galactosidase and nucleotide concentrations were measured in E. coli. A rapid and dose-dependent loss of cellular beta-galactosidase activity was found, with no increase in the supernatant; loss of cellular nucleotides corresponded with an increase in the supernatant. Electron microscopy and biochemical observations support the conclusion that povidone-iodine interacts with cell walls of micro-organisms causing pore formation or generating solid-liquid interfaces at the lipid membrane level which lead to loss of cytosol material, in addition to enzyme denaturation. The chemical mechanism of action explains the fact that povidone-iodine does never generate resistance in micro-organisms.

Physicochemical properties and in vitro toxicity of cationic liposome cDNA complexes.

The purpose of this study was to elucidate the interaction of cationic liposomes and plasmid cDNA by examining their ultrastructure, zeta potential, stability in aqueous media and protection from DNaseI digestion; their potential for hemolysis and platelet aggregation was evaluated as it may serve as an in vitro toxicity screen. Liposomes consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and dioleylphosphatidylethanolamine (DOPE) were complexed with plasmid constructs of ovine prostaglandin G/H synthase (pCMV4-PGH) or human alpha 1-antitrypsin (pCMV4-AAT) at lipid:plasmid (L/P) ratios of 3:1-8:1 (w/w). The electron micrographs showed bead-like attachment of liposomes to cDNA and coating of plasmid strands. The zeta potential showed isoelectric points at L/P ratios of 3.5-4 (DOTMA/DOPE) and 5.5-6.5, corresponding to a pKa of 6.45 (DC-Chol/DOPE). Liposome cDNA complexes were stable in water, saline and 5% dextrose for 48 h, but precipitated instantaneously in PBS. An increase in the L/P ratio corresponded with increased protection from DNaseI digestion. DOTMA/DOPE liposomes alone were highly hemolytic and DC-Chol/DOPE liposomes moderately hemolytic; hemolysis was abolished by cDNA complexation, with the exception of very high (> or = 7:1) L/P ratios. Both liposomes alone and cDNA complexes caused transient serum turbidity, while none caused platelet aggregation. It was concluded that current cationic lipid cDNA formulations are metastable and appear to have very little if any toxicity with respect to hemolytic potential and untoward interaction with other blood components.

Intraerythrocytic inclusions associated with iridoviral infection in a fer de lance (Bothrops moojeni) snake.

Intraerythrocytic inclusions associated with infection by an iridovirus were observed in a fer de lance (Bothrops moojeni) snake that was being evaluated for the presence of renal carcinoma. The erythrocytes contained two types of inclusions, one viral and one crystalline, usually concomitantly. The snake was markedly anemic and exhibited a marked regenerative response. Ultrastructural analysis identified the virus to be an iridovirus consistent with snake erythrocyte virus and the crystalline structures to be of a different nature than hemoglobin.

Molecular effects of povidone-iodine on relevant microorganisms: an electron-microscopic and biochemical study.

The aim of this study was to elucidate the effects of povidone-iodine (PVP-I) on cell ultrastructure by electron microscopy and to monitor changes in enzyme activity and nucleotide efflux. Staphylococcus aureus, Escherichia coli and Candida albicans, medically relevant gram-positive, gram-negative and yeast microorganisms, served as models. In the presence of PVP-I, rapid partitioning of the cytoplasm and pronounced coagulation of nuclear material was noted. E. coli and S. aureus showed no major structural wall damage. C. albicans exhibited a rapid, dose-dependent 'loosening' of the cell wall; cells remained intact without lysis, rupture or wall breakage. Changes in beta-galactosidase and nucleotide concentrations were measured in E. coli. A rapid and dose-dependent loss of cellular beta-galactosidase activity was found, with no increase in the supernatant; loss of cellular nucleotides corresponded with an increase in the supernatant. Electron-microscopic and biochemical observations support the conclusion that PVP-I interacts with cell walls of microorganisms causing pore formation or generating solid-liquid interfaces at the lipid membrane level which lead to loss of cytosol material, in addition to enzyme denaturation.

Localization of 14-3-3 proteins in the nuclei of arabidopsis and maize.

It has been demonstrated that 14-3-3 proteins are present in the nuclei of Arabidopsis thaliana and Zea mays cells using laser scanning confocal microscopy and immunocytochemistry with monoclonal antibodies against plant 14-3-3 proteins. Confirmation of nuclear localization provides insight into the range of functions normally attributed to 14-3-3 proteins, especially since the association of 14-3-3s with transcription factors is (thus far) a phenomenon unique to plants, and since 14-3-3 proteins do not possess a recognizable nuclear targeting sequence.

The influence of molecular conformation upon the self-assembly of cyclohexane diamide diacids.

BACKGROUND: Information regarding the self-association of small peptide motifs can be used in the design of peptide microstructures. Previous work in our laboratories illustrated the self-association of certain diamide diacids into microcapsules. In this report a series of cyclohexane diamide diacids are investigated. The cyclohexylene (R-C6H10-R) system (with its axial and equatorial requirements) provided an opportunity to study the influence of molecular conformation upon the self-aggregation process. RESULTS: Condensation of the respective cis- and trans-1,2-, 1,3-, and 1,4- cyclohexane dicarboxylic acid platforms with two equivalents of a L-Phe ester followed by deprotection gave the desired diamide diacids. Basic solutions of cis-1,2-, trans-1,3-, and cis-1,4-diamide diacids generated solid microspheres when acidified to pH 2.4. Molecular modeling revealed that 1,3-diaxial interactions favor a helical turn within these diamides. CONCLUSIONS: Access to 'complementary' molecular geometries is needed to self-associate into microscopic architectures.

Self-assembly of the brain MAP-2 microtubule-binding region into polymeric structures resembling Alzheimer filaments.

The neuronal microtubule-associated protein known as MAP-2 has not been considered to be a subunit of paired helical filaments (PHFs) in neurofibrillary tangles seen in Alzheimer's Disease. We now describe the assembly of paired helical filament-like structures from MAP-2's 203-residue microtubule-binding region (MTBR). SDS gel electrophoresis and equilibrium ultracentrifugation suggest that a dimeric form, cross-linked by an interchain disulfide, is involved in polymerization. MAP-2 MTBR polymers bind thioflavin-S, a dye used to histochemically localize Alzheimer neurofibrillary tangles. Our finding that PHF-like structures assemble from a MAP-2 fragment raises new questions about MAP-2's role in the etiology of Alzheimer's Disease.

SP75 is encoded by the DP87 gene and belongs to a family of modular Dictyostelium discoideum outer layer spore coat proteins.

Highly purified spore coats of Dictyostelium discoideum each contained about 5 x 10(6) protein molecules as determined by amino acid composition analysis. By two-dimensional gel electrophoresis the coats were found to contain nine major-abundance and numerous minor protein species, most of which were highly enriched relative to the adjacent interspore matrix. Protein was nearly quantitatively eluted by denaturants and 2-mercaptoethanol, showing that it was not irreversibly cross-linked. Because a reducing agent is required together with denaturants to elute most proteins if their free thiol groups have been prealkylated, it was concluded that the D. discoideum spore coat proteins are disulfide cross-linked into the matrix. One major coat protein, SP75, was partially sequenced and found to be encoded by the previously identified DP87 gene; this finding was supported by additional physical, genetic, biochemical and microscopic evidence. The five major proteins for which genes have been cloned were associated with the outer layer of the coat. In coats missing one or more of four of these proteins as a result of gene disruption, there were physical changes but, with one exception, the other major coat proteins appeared to be incorporated normally. Sequence analysis showed that these five outer layer coat proteins are homologous and consist of alternating sequence motifs related to epithelial mucin repeats, basic proline repeats found in salivary acidic proline-rich proteins, the NH2-terminal subdomain of epidermal growth factor modules and other cysteine repeats. Based on these and other observations, outer layer coat proteins are predicted to organize indeterminately to form a cell surface microenvironment supportive of cellulose morphogenesis during spore coat formation.

Hypoglycemia-induced AP-1 transcription factor and basic fibroblast growth factor gene expression in multidrug resistant human breast carcinoma MCF-7/ADR cells.

We investigated the effect of hypoglycemic treatment on the activation of the AP-1 transcription factors and the regulation of basic fibroblast growth factor (bFGF) gene expression in multidrug resistant human breast carcinoma MCF-7/ADR cells. Northern blot and gel mobility shift assays showed that hypoglycemic treatment induced c-jun and c-fos gene expression, AP-1 binding activity, as well as bFGF gene expression. Moreover, transfected cells expressing high levels of abnormal c-Jun protein exhibited a reduction in the bFGF protein levels compared to parental cells. A potent protein kinase C (PKC) inhibitor, H-7 (60 micrograms/ml) suppressed the stress-induced bFGF gene expression. Our study also demonstrated that H-7 did not facilitate the decay of bFGF mRNA. Thus, the suppression of bFGF gene expression by treatment with H-7 was due to the effect of the drug on the synthesis of bFGF mRNA rather than the stability of bFGF mRNA. Our data suggest that hypoglycemia-induced bFGF gene expression is mediated through the activation of PKC and the AP-1 transcription factors.