Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a 'T7-like virus' with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10(4) and 10(6) pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.

Prostaglandin E2 (PGE2) blocks mast-cell (MC)-dependent allergic responses in humans but activates MCs in vitro. We assessed the functions of the <em>EP</em> receptors for PGE2 on cultured human MCs (hMCs). hMCs expressed the <em>EP</em>3, <em>EP</em>2, and <em>EP</em>4 receptors. PGE2 stimulated the accumulation of cyclic adenosine monophosphate (cAMP), and suppressed both Fc epsilonRI-mediated eicosanoid production and tumor necrosis factor-alpha (TNF-alpha) generation. PGE2 also caused phosphorylation of extracellular signal-regulated kinase (ERK), exocytosis, and production of prostaglandin D2 (PGD2), as well as leukotriene C4 (LTC4) when protein kinase A (PKA) was inhibited. An <em>EP</em>3 receptor-selective agonist, AE-248, mimicked PGE2-mediated ERK phosphorylation, exocytosis, and eicosanoid formation. Selective agonists of both <em>EP</em>2 and <em>EP</em>4 receptors (AE1-259-01 and AE-329, respectively) stimulated cAMP accumulation. No selective agonist, alone or in combination, was as effective as PGE2. AE-248, AE1-259-01, and AE-329 all inhibited Fc epsilonRI-mediated TNF-alpha generation, while AE1-259-01 blocked eicosanoid production. PGE2 caused the expression of inducible cAMP early repressor (ICER) by a pathway involving PKA and ERK. Thus, while PGE2 activates MCs through <em>EP</em>3 receptors, it also counteracts Fc epsilonRI-mediated eicosanoid production through <em>EP</em>2 receptors and PKA, and blocks cytokine transcription. These functions explain the potency of PGE2 as a suppressor of early- and late-phase allergic responses.

The mechanical stability of biofilms and other microbial aggregates is of great importance for both the maintenance of biofilm processes and the removal of undesired biofilms. The binding forces are weak interactions such as London dispersion forces, electrostatic interactions and hydrogen bonds. In a first attempt to rank their contribution, the viscosity of solutions of extracellular polymeric substances (EPS) from a mucoid strain of Pseudomonas aeruginosa is measured. In order to distinguish the binding forces, substances are chosen which individually address the different types of bonds. Polyacrylic acid is identified as a suitable model system for EPS when molecular interactions are studied. Electrostatic interactions and hydrogen bonds are found to be the dominating forces among macromolecules within the biofilm.

BACKGROUND

Exposure to microbial agents might inhibit the development of atopy and asthma.

OBJECTIVE

We measured the association between microbial exposure assessed at 3 months and the development of atopic sensitization and doctor-diagnosed (DD) asthma and wheeze in the first 4 years in a birth cohort study of children with atopic mothers.

METHODS

Endotoxin, fungal (1-->3)-beta-D-glucans, extracellular polysaccharides from the genera Penicillium and Aspergillus (EPS-Pen/Asp), and dust on living room floors were measured at 3 months of age. Serum IgE levels against common allergens were determined at 1 and 4 years, and questionnaire information about respiratory morbidity was collected yearly.

RESULTS

Microbial levels in mattresses were low and not associated with serum IgE levels, DD asthma, and wheeze. Floor levels of biocontaminants and dust, on the other hand, were inversely associated with DD asthma, being most pronounced for endotoxin (odds ratio [OR], 0.40; 95% CI, 0.21-0.77) and EPS-Pen/Asp (OR, 0.42; 95% CI, 0.18-0.99). Mutual adjustment for other exposures did not significantly alter the results for endotoxin and only moderately affected the results for EPS-Pen/Asp. Persistent wheeze was also consistently less common in the high-exposure group, being significant only for EPS-Pen/Asp (OR, 0.37; 95% CI, 0.15-0.96). Transient wheeze and wheeze in the past 12 months were also reduced, but effects were smaller and not significant. Relationships with serum-specific IgE levels, which could only be assessed in 41% at age 4 years, were less pronounced and statistically significant only for EPS-Pen/Asp.

CONCLUSIONS

Early exposure to common microbial contaminants, including fungal agents, might protect against asthma.

CONCLUSIONS

Microbial exposure in early life might protect against asthma and might constitute a novel target for prevention.

Caveolae are specialized domains present in the plasma membrane (PM) of most mammalian cell types. They function in signalling, membrane regulation, and endocytosis. We found that the Eps-15 homology domain-containing protein 2 (EHD2, an ATPase) associated with the static population of PM caveolae. Recruitment to the PM involved ATP binding, interaction with anionic lipids, and oligomerization into large complexes (60-75S) via interaction of the EH domains with intrinsic NPF/KPF motifs. Hydrolysis of ATP was essential for binding of EHD2 complexes to caveolae. EHD2 was found to undergo dynamic exchange at caveolae, a process that depended on a functional ATPase cycle. Depletion of EHD2 by siRNA or expression of a dominant-negative mutant dramatically increased the fraction of mobile caveolar vesicles coming from the PM. Overexpression of EHD2, in turn, caused confinement of cholera toxin B in caveolae. The confining role of EHD2 relied on its capacity to link caveolae to actin filaments. Thus, EHD2 likely plays a key role in adjusting the balance between PM functions of stationary caveolae and the role of caveolae as vesicular carriers.

With the increased use of intraoperative monitoring of the central nervous system (CNS) has come a need for better understanding of the effects of anesthetic agents on intraoperative recordings. The commonly used anesthetic agents and their effects on intraoperative electroencephalography (EEG) and evoked potentials (EP) are discussed. Halogenated inhalational anesthetics produce dose-related reduction in EEG amplitude and frequency after an initial activation. They also produce dose-related decreases in amplitude and increases in latency of sensory evoked potentials (SEP) that are most marked in cortically generated components. Subcortical, spinal, and peripheral evoked responses are less affected. Responses in the motor pathways are recordable in the epidural space; however, the relative contributions of sensory and motor tracts may be changed when both are present. Muscle responses are easily suppressed after spinal and motor cortex stimulation, probably by anesthetic effect at the anterior horn cells of the spinal cord. Intravenous analgesic agents (opioids, ketamine) are associated with less marked changes in EEG and evoked responses, with some increases in amplitude of cortically generated SEP caused by ketamine. Intravenous sedative-hypnotic drugs (droperidol, barbiturates, benzodiazepines, etomidate, propofol) produce dose-related depression of the EEG after initial activation and dose-related depression of evoked responses to a lesser extent than do the inhalation agents. Etomidate is associated with amplitude enhancement of EEG and cortically generated SEP. Muscle relaxants have minimal effect on the EEG and SEP. Their use, however, may alter muscle recordings from motor tract stimulation. These effects and their relevance to the choice of agents for specific monitoring techniques are discussed.

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors, useful for both basic research and clinical applications. Besides their characterization in colony assays, protocols exist for the cultivation of lymphoid, myeloid, and erythroid cells. With the possible exception of mast cells, however, long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here, we describe for the first time an efficient yet easy strategy to generate mass cultures of pure, immature erythroid progenitors from mouse ES cells (ES-EPs), using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived, adult, definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions, ES-EPs differentiated into mature, enucleated erythrocytes. Importantly, ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition, the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells.

The binding of labeled erythropoietin (EP) to cell surface receptors and subsequent processing of the hormone within the cell was studied in erythroid cells procured from the spleens of mice infected with the anemia strain of Friend virus. These immature erythroid cells respond to EP in culture to differentiate into reticulocytes and erythrocytes. Radiolabeled EP (both iodinated and tritiated) binds to 800-1000 cell surface receptors on these cells at 4 degrees C. Using 125I-EP, we found that 300 of these cell surface receptors have a higher affinity for EP (Kd = 0.09 nM) than the remaining receptors (Kd = 0.57 nM). The number of molecules of EP bound per cell increased about 2-fold when binding was carried out at 37 degrees C. Treatment of the cell surface with pronase or removal of surface-bound EP with a low pH wash revealed that radiolabeled EP is internalized by the cells at 37 degrees C. Pulse chase experiments showed that degradation products of radiolabeled EP are released into the medium with a corresponding loss of label from the interior of the cell. Inhibitors of lysosomal function greatly reduced this degradation of 125I-EP. Since 180 of the 300 high affinity receptors and very few of the low affinity receptors are occupied at the concentration of EP which elicits the maximum biological response in these cells, we suggest that interaction of EP with the high affinity receptors are necessary for the full biological effect of the hormone. A different murine erythroleukemia cell line which does not differentiate in response to EP was found to have only the lower affinity binding sites for the hormone.

This study was an initial phase II trial in humans of molecular magnetic resonance (MR) imaging for improved visualization of thrombi in vessel territories potentially responsible for stroke using a new fibrin-specific contrast agent (EP-2104R). Eleven patients with thrombus in the left ventricle (n = 2), left or right atrium (n = 4), thoracic aorta (n = 4) or carotid artery (n = 1) as verified by an index examination (ultrasound, computed tomograpy, or conventional MR) were enrolled. All MR imaging was performed on 1.5 T whole-body MR-system using an inversion-recovery black-blood gradient-echo sequence. The same sequence was performed before and 2-6 h after low-dose intravenous administration of 4 mumol/kg EP-2104R. Two investigators assessed image quality and signal amplification. Furthermore, contrast-to-noise ratios (CNR) between the clot and the blood pool/surrounding soft tissue before and after administration of the contrast agent were compared using Student's t-test. MR imaging and data analysis were successfully completed in 10 patients. No major adverse effects occurred. On enhanced images, thrombi demonstrated high signal amplification, typically at the clot surface, with a significantly increased contrast in comparison to the surrounding blood pool and soft tissue (CNR for clot vs. blood pool, unenhanced and enhanced: 6 +/- 8 and 29 +/- 14; CNR for clot vs. soft tissue, unenhanced and enhanced: 0 +/- 4 and 21 +/- 13; P < 0.01 for both comparisons). EP-2104R allows for molecular MR imaging of thrombi potentially responsible for stroke. High contrast between thrombus and surrounding blood and soft tissues can be achieved with enhanced imaging.

An 83-base-pair-long hepatitis B virus DNA fragment efficiently activates the transcription of the heterologous globin gene promoter. This fragment contains binding sites for at least four distinct cellular factors termed E, TGT3, EP, and NF-I. E is a positively acting factor, responsive to phorbol ester. EP is apparently identical to the factor EF-C that binds to the polyomavirus enhancer. The conservation of the binding site sequences for most of these factors in the genomes of other members of the hepadnavirus family suggests that these viruses share common enhancer elements.

METHODS

Patients (n=302) with bipolar I disorder (manic episode) were randomised to 12 weeks' double-blind treatment with quetiapine (flexibly dosed up to 800 mg/day), placebo, or haloperidol (up to 8 mg/day). The primary efficacy outcome variable was change from baseline to Day 21 in Young Mania Rating Scale (YMRS) score.

RESULTS

YMRS score improved with quetiapine at Day 21 (-12.29 versus -8.32 for placebo; P<0.01). The difference in favor of quetiapine increased by Day 84 (-17.52 versus -9.48; P<0.001). Haloperidol also showed an advantage over placebo at Days 21 and 84 (P<0.001). There was no significant difference in efficacy measures between quetiapine and haloperidol groups at any assessment except Day 21. The only common adverse event with quetiapine was somnolence (12.7%). Extrapyramidal symptoms (EPS), including akathisia, occurred at 59.6% with haloperidol, 12.7% with quetiapine, 15.8% with placebo. Most quetiapine responders (84%) received a dose of 400-800 mg/day.

CONCLUSIONS

Quetiapine was effective and well tolerated. The efficacy and tolerability profile of haloperidol (including its propensity for EPS) supported study validity.

Syntaxin 5 is a Golgi-localized SNARE protein that has been shown to be required for ER-Golgi traffic in yeast and Golgi reassembly following cell division in mammalian cells. Here, we describe the characterization of the Drosophila ortholog of syntaxin 5, dSyx5, and show that, like its mammalian and yeast counterparts, the protein is localized to the Drosophila Golgi and binds to alpha-SNAP. Null mutations in dSyx5 are larval lethal and demonstrate impaired transport of a GFP-tagged membrane protein. A hypomorphic allele of dSyx5 caused by insertion of an EP element results in impenetrant lethality, and escaping adult flies are male sterile. The male sterility results both from failure of germ cells to complete cytokinesis and from defects in spermatid elongation and maturation. Ectopic expression of dSyx5 from the EP element can rescue the cytokinesis defect, but high levels of expression are required to restore maturation and fertility. Together, these results show that dSyx5 is required for the proper function of the Golgi apparatus and that an efficiently functioning Golgi apparatus is required for the steps leading to the completion of cytokinesis and formation of mature sperm.

A common feature of human IgG1 antibodies used for cancer treatment is that their anti-tumour efficacy requires high serum trough levels and continued therapy for several months. Treatment cycles, thereby, consume several grams of IgG1 translating into significant drug needs and costs. The basis for the low in vivo efficacy, which is in contrast to high in vitro antibody-dependent cellular cytotoxicity (ADCC), is not well understood. Here, we have explored factors contributing to this discrepancy using adecatumumab (MT201), a fully human monoclonal IgG1 against epithelial cell adhesion molecule (Ep-CAM) and trastuzumab (Herceptin), a humanized IgG1 with specificity for the human epithelial growth factor receptor type 2 (HER-2) antigen. We found that physiological levels of human sera strongly inhibited ADCC of both IgG1 antibodies. Effects showed some dependence on the density of Ep-CAM and HER-2 targets, the tumour cell line tested and on effector cell and serum donors. Removal of IgG by affinity chromatography abolished the inhibitory effect of a serum pool. Inhibition of ADCC was fully restored by adding back the IgG fraction or by an equal amount of IgG from a commercial source. We further demonstrate that CD56-positive lymphocytes within human PBMC contributed >90% to ADCC and that normal serum levels of IgG effectively competed for in vitro binding of an IgG1 antibody to low-affinity Fcgamma receptor type III (CD16), as is present on natural killer (NK) cells. Competition of serum IgG for binding of therapeutic IgG1 to NK cell may be one important reason why high antibody doses are required in the clinic for treatment of cancer by an ADCC-based mechanism.

A dengue subunit vaccine candidate was developed using a mammalian cell line continuously expressing subviral extracellular particles (EPs) of the New Guinea C (NGC) strain of dengue type 2 virus. The cell line, designated D cell line, maintained envelope (E) antigen production for at least 10 passages. The EPs contained an E protein biochemically and antigenically equivalent to authentic E produced by NGC-infected Vero cells. Two immunizations of BALB/c mice with purified EPs containing 100ng or 400ng of E induced moderate levels of neutralizing antibody and anamnestic neutralizing antibody responses were produced when these animals were challenged with dengue virus. The yield of E antigen from D cells was comparable to that from NGC-infected Vero cells. When D cells were transfected with the anti-apoptotic bcl-2 gene, the E antigen release increased approximately two-fold. These results indicate that D cell EPs are a promising non-infectious vaccine antigen for dengue.

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.

In recent years, DNA vaccines have undergone a number of technological advancements that have incited renewed interest and heightened promise in the field. Two such improvements are the use of genetically engineered cytokine adjuvants and plasmid delivery via in vivo electroporation (EP), the latter of which has been shown to increase antigen delivery by nearly 1000-fold compared to naked DNA plasmid delivery alone. Both strategies, either separately or in combination, have been shown to augment cellular and humoral immune responses in not only mice, but also in large animal models. These promising results, coupled with recent clinical trials that have shown enhanced immune responses in humans, highlight the bright prospects for DNA vaccines to address many human diseases.

Only about one third of humans possess a microbiota capable of transforming the dietary isoflavone daidzein into equol. Little is known about the dietary and physiological factors determining this ecological feature. In this study, the in vitro metabolism of daidzein by faecal samples from four human individuals was investigated. One culture produced the metabolites dihydrodaidzein and O-desmethylangolensin, another produced dihydrodaidzein and equol. From the latter, a stable and transferable mixed culture transforming daidzein into equol was obtained. Molecular fingerprinting analysis (denaturing gradient gel electrophoresis) showed the presence of four bacterial species of which only the first three strains could be brought into pure culture. These strains were identified as Lactobacillus mucosae EPI2, Enterococcus faecium EPI1 and Finegoldia magna EPI3, and did not produce equol in pure culture. The fourth species was tentatively identified as Veillonella sp strain EP. It was found that hydrogen gas in particular, but also butyrate and propionate, which are all colonic fermentation products from poorly digestible carbohydrates, stimulated equol production by the mixed culture. However, when fructo-oligosaccharides were added, equol production was inhibited. Furthermore, the equol-producing capacity of the isolated culture was maintained upon its addition to a faecal culture originating from a non-equol-producing individual.

The phytopathogenic bacterium Pantoea stewartii subsp. stewartii synthesizes stewartan exo/capsular polysaccharide (EPS) in a cell density-dependent manner governed by the EsaI/EsaR quorum-sensing (QS) system. This study analyzes biofilm development and host colonization of the WT and QS regulatory mutant strains of P. stewartii. First, we show that the cell density-dependent synthesis of stewartan EPS, governed by the EsaI/EsaR QS system, is required for proper bacterial adhesion and development of spatially defined, 3D biofilms. Second, a nonvirulent mutant lacking the esaI gene adheres strongly to surfaces and develops densely packed, less structurally defined biofilms in vitro. This strain appears to be arrested in a low cell density developmental mode. Exposure of this strain to exogenous N-acyl-homoserine lactone counteracts this adhesion phenotype. Third, QS mutants lacking the EsaR repressor attach poorly to surfaces and form amorphous biofilms heavily enmeshed in excess EPS. Fourth, the WT strain disseminates efficiently within the xylem, primarily in a basipetal direction. In contrast, the two QS mutant strains remain largely localized at the site of infection. Fifth, and most significantly, epifluorescence microscopic imaging of infected leaf tissue and excised xylem vessels reveals that the bacteria colonize the xylem with unexpected specificity, particularly toward the annular rings and spiral secondary wall thickenings of protoxylem, as opposed to indiscriminate growth to fill the xylem lumen. These observations are significant to bacterial plant pathogenesis in general and may reveal targets for disease control.

The efficacies of extracting extracellular polymeric substances (EPS) from aerobic, acidogenic and methanogenic sludges using EDTA, cation exchange resin and formaldehyde under various conditions were compared. Results show that formaldehye plus NaOH was most effective in extracting EPS for all sludges; only 1.1-1.2% of DNA in the sludge samples were detected, suggesting the EPS extracted were not contaminated by intracellular substances. For each gram of volatile solids, formaldehyde-NaOH extracted 165, 179 and 102 mg of EPS from aerobic, acidogenic and methanogenic sludges, respectively. All EPS were mainly composed of carbohydrate, protein and humic substance, plus small quantities of uronic acid and DNA. Carbohydrate was predominant in the acidogenic sludge (62% in the EPS extracted by formaldehyde-NaOH), whereas protein was predominant in the methanogenic sludge (41%). Humic substance, which has often been overlooked, accounted for 30.6, 8.4 and 22.8% of the extracted EPS from aerobic, acidogenic and methanogenic sludges, respectively. However, judging from EPS quantities estimated from confocal laser scanning microscopic observations, formaldehyde-NaOH extracted only a limited portion of EPS. Optimization of extraction procedures and/or development of a more effective extraction method are warranted.

Degenerative disc disease (DDD) is still a poorly understood phenomenon because of the lack of availability of precise definition of healthy, ageing and degenerated discs. Decreased nutrition is the final common pathway for DDD and the status of the endplate (EP) plays a crucial role in controlling the extent of diffusion, which is the only source of nutrition. The vascular channels in the subchondral plate have muscarinic receptors but the possibility of enhancing diffusion pharmacologically by dilation of these vessels has not been probed. Although it is well accepted that EP damage will affect diffusion and thereby nutrition, there is no described method to quantify the extent of EP damage. Precise definitions with an objective method of differentiating healthy, ageing and degenerated discs on the basis of anatomical integrity of the disc and physiological basis of altered nutrition will be useful. This information is an urgent necessity for better understanding of DDD and also strategizing prevention and treatment. Seven hundred and thirty endplates of 365 lumbar discs from 73 individuals (26 healthy volunteers and 47 patients) with age ranging from 10-64 years were evaluated by pre-contrast and 10 min, 2, 4, 6 and 12 h post contrast MRI after IV injection of 0.3 mmol/kg of Gadodiamide. End plates were classified according to the extent of damage into six grades and an incremental score was given for each category. A total endplate score (TEPS) was derived by adding the EP score of the two endplates for each concerned disc. The base line value (SI(base)) and the signal intensity at particular time periods were used to derive the enhancement percentage for each time period (Enhancement (%) = SI(tp) - SI(base)/SI(base) x 100). The enhancement percentage for each time period, the time for peak enhancement (T-max) and the time intensity curve (TIC) over 12 h were used to study and compare the diffusion characteristics. The differences in pattern of diffusion were obvious visually at 4 h which was categorized into five patterns-Pattern A representing normal diffusion to Pattern E representing a total abnormality in diffusion. Degeneration was classified according to Pfirrmann's grading and this was correlated to the TEPS and the alterations in diffusion patterns. The relationship of TEPS on the increase in DDD was evaluated by a logistic curve and the cut point for severe DDD was found by ROC curve. The influence of the variables of age, level, Modic changes, instability, annulus fibrosis defect (DEBIT), TEPS and diffusion patterns on DDD was analyzed by multiple and stepwise regression analysis. Oral nimodipine study: Additional forty lumbar end-plates from four young healthy volunteers were studied to document the effect of oral nimodipine. Pre-drug diffusion levels were studied by pre and post contrast MRI (0.3 mmol/kg of gadodiamide) at 10 min, 2, 4, 6, 12 and 24 h. Oral nimodipine was administered (30 mg QID) for 5 days and post-contrast MRI studies were performed similarly. Enhancement was calculated at vertebral body-VB; subchondral bone-SCB; Endplate Zone-EPZ and at superior and inferior peripheral nucleus pulposus-PNP and central nucleus pulposus-CNP, using appropriate cursors by a blinded investigator. Paired sample t test and area under curve (AUC) measurements were done.The incidence of disc degeneration had a significant correlation with increasing TEPS (Trend Chi-square, P < 0.01). Only one out of 83 (1.2%) disc had either Pfirrmann Grade IV or V when the score was 4 or below when compared to 34/190 (17.9%) for scores 5-7; 41 of 72 (56.9%) for scores 8-10 and 18 of 20 (90%) for scores 11 and 12 (P < 0.001 for all groups). Pearson's correlation between TEPS and DDD was statistically significant, irrespective of the level of disc or different age groups (r value was above 0.6 and P < 0.01 for all age groups). Logistic curve fit analysis and ROC curve analysis showed that the incidence of DDD increased abruptly when the TEPS crossed six. With a progressive increase of end plate damage, five different patterns of diffusion were visualized. Pattern D and E represented totally altered diffusion pattern questioning the application of biological method of treatment in such situations. Four types of time intensity curves (TIC) were noted which helped to differentiate between healthy, aged and degenerated discs. Multiple and stepwise regression analysis indicated that pattern of disc diffusion and TEPS to be the most significant factors influencing DDD, irrespective of age. Nimodipine increased the average signal intensity for all regions-by 7.6% for VB, 8% for SCB and EPZ and 11% for CNP at all time intervals (P < 0.01 for all cases). Although the increase was high at all time intervals, the maximum increase was at 2 h for VB, SCB and EPZ; 4 h for PNP and 12 h for CNP. It was also interesting that post-nimodipine, the peak signal intensity was attained early, was higher and maintained longer compared to pre-nimodipine values. Our study has helped to establish that EP damage as a crucial event leading to structural failure thereby precipitating DDD. An EP damage score has been devised which had a good correlation to DDD and discs with a score of six and above can be considered 'at risk' for severe DDD. New data on disc diffusion patterns were obtained which may help to differentiate healthy, ageing and degenerated discs in in-vivo conditions. This is also the first study to document an increase in diffusion of human lumbar discs by oral nimodipine and poses interesting possibility of pharmacological enhancement of lumbar disc nutrition.

Electrolyte disorders can alter cardiac ionic currents kinetics and depending on the changes can promote proarrhythmic or antiarrhythmic effects. The present report reviews the mechanisms, electrophysiolgical (EP), electrocardiographic (ECG), and clinical consequences of electrolyte disorders. Potassium (K⁺) is the most abundent intracellular cation and hypokalemia is the most commont electrolyte abnormality encountered in clinical practice. The most significant ECG manifestation of hypokalemia is a prominent U wave. Several cardiac and non cardiac drugs are known to suppress the HERG K⁺ channel and hence the I(K), and especially in the presence of hypokalemia, can result in prolonged action potential duration and QT interval, QTU alternans, early afterdepolarizations, and torsade de pointes ventricular tachyarrythmia (TdP VT). Hyperkalemia affects up to 8% of hospitalized patients mainly in the setting of compromised renal function. The ECG manifestation of hyperkalemia depends on serum K⁺ level. At 5.5-7.0 mmol/L K⁺, tall peaked, narrow-based T waves are seen. At>> 10.0 mmol/L K⁺, sinus arrest, marked intraventricular conduction delay, ventricular techycardia, and ventricular fibrillation can develop. Isolated abnormalities of extracellular calcium (Ca⁺⁺) produce clinically significant EP effects only when they are extreme in either direction. Hypocalcemia, frequently seen in the setting of chronic renal insufficiency, results in prolonged ST segment and QT interval while hypercalcemia, usually seen with hyperparathyroidism, results in shortening of both intervals. Although magnesium is the second most abudent intracellular cation, the significance of magnesium disorders are controversial partly because of the frequent association of other electrolyte abnormalities. However, IV magnesium by blocking the L-type Ca(⁺⁺) current can successfully terminate TdP VT without affecting the prolonged QT interval. Finally, despite the frequency of sodium abnormalities, particularly hyponatremia, its EP effects are rarely clinically significant.

The Rhizobium meliloti Tn5 mutant Rm3131, producing galactoglucan (EPS II) instead of succinoglycan (EPS I), was complemented by a 3.6-kb EcoRI-fragment of the Rhizobium meliloti genome. Sequencing of this fragment revealed six open reading frames (ORFs). The ORF found to be affected in the mutant Rm3131 codes for a putative protein of 15.7 kDa and forms a monocistronic transcriptional unit. Further genetic analysis revealed that the gene mutated in Rm3131 is identical to the previously described R. meliloti mucR gene (H. Zhan, S.B. Levery, C. C. Lee, and J.A. Leigh, 1989, Proc. Natl. Acad. Sci. USA 86:3055-3059). By hybridization it was shown that a mucR homologous gene is present in several rhizobacteria. The deduced amino acid sequence of MucR showed nearly 80% identity to the Agrobacterium tumefaciens Ros protein, a negative regulator of vir genes and necessary for succinoglycan production. MucR contains like Ros a putative zinc finger sequence of the C2H2 type. Transcriptional fusions of genes for EPS I and EPS II synthesis, the so-called exo and exp genes, with the marker gene lacZ were used to delineate the role of mucR for exo and exp gene expression. It was found that exp genes are negatively regulated by MucR on the transcriptional level, whereas a posttranscriptional regulation by MucR is assumed for exo genes. Furthermore, mucR is negatively regulating its own transcription.

Immunogenicity of DNA vaccines varies significantly due to many factors including the inherent immunogenicity of the protein antigen encoded in the DNA vaccine, the optimal immune responses that can be achieved in different animal models and in humans with different genetic backgrounds and, to a great degree, the delivery methods used to administer the DNA vaccines. Based on published results, only the gene gun-mediated delivery approach has been able to elicit protective levels of immune responses in healthy, adult volunteers by DNA immunization alone without the use of another vaccine modality as a boost. Recent results from animal studies suggest that electroporation is also effective in eliciting high level immune responses. However, there have been no reports to identify the similarities and differences between these two leading physical delivery methods for DNA vaccines against infectious disease targets. In the current study, we compared the relative immunogenicity of a DNA vaccine expressing a hemagglutinin (HA) antigen from an H5N1 influenza virus in two animal models (rabbit and mouse) when delivered by either intramuscular needle immunization (IM), gene gun (GG) or electroporation (EP). HA-specific antibody, T cell and B cell responses were analyzed. Our results indicate that, overall, both the GG and EP methods are more immunogenic than the IM method. However, EP and IM stimulated a Th-1 type antibody response and the antibody response to GG was Th-2 dominated. These findings provide important information for the further selection and optimization of DNA vaccine delivery methods for human applications.

The large variety of biological functions governed by prostaglandin (PG) E2 is mediated by signaling through four distinct E-type prostanoid (<em>EP</em>) receptors. The availability of mouse strains with genetic ablation of each <em>EP</em> receptor subtype and the development of selective <em>EP</em> agonists and antagonists have tremendously advanced our understanding of PGE2 as a physiologically and clinically relevant mediator. Moreover, studies using disease models revealed numerous conditions in which distinct <em>EP</em> receptors might be exploited therapeutically. In this context, the <em>EP</em>4 receptor is currently emerging as most versatile and promising among PGE2 receptors. Anti-inflammatory, anti-thrombotic and vasoprotective effects have been proposed for the <em>EP</em>4 receptor, along with its recently described unfavorable tumor-promoting and pro-angiogenic roles. A possible explanation for the diverse biological functions of <em>EP</em>4 might be the multiple signaling pathways switched on upon <em>EP</em>4 activation. The present review attempts to summarize the <em>EP</em>4 receptor-triggered signaling modules and the possible therapeutic applications of <em>EP</em>4-selective agonists and antagonists.