BACKGROUND

Burkholderia cepacia complex (Bcc) organisms produce a wide variety of potential virulence factors, including exopolysaccharides (EPS), and exhibit intrinsic resistance towards many antibiotics. In the present study we investigated the contribution of Bcc biofilm matrix components, including extracellular DNA, cepacian and poly-β-1,6-N-acetylglucosamine, to tobramycin susceptibility.

METHODS

The in vitro bactericidal activity of tobramycin in combination with recombinant human DNase (rhDNase), NaClO and dispersin B was tested against Bcc biofilms.

RESULTS

EPS degradation by NaClO pretreatment and specific PNAG degradation by dispersin B significantly increased the bactericidal effect of tobramycin towards some of the Bcc biofilms tested, including the strains of Burkholderia cenocepacia, B. cepacia and Burkholderia metallica. The presence of rhDNase during biofilm treatment and/or development had no influence on tobramycin activity.

CONCLUSIONS

These results suggest that EPS play a role in tobramycin susceptibility of Bcc biofilms and that matrix degrading combination therapy could improve treatment of Bcc biofilm infections.

Membrane and membrane skeleton proteins were examined in erythroid progenitor cells during terminal differentiation. The employed model system of erythroid differentiation was that in which proerythroblasts from mice infected with the anemia-inducing strain of Friend virus differentiate in vitro in response to erythropoietin (EP). With this system, developmentally homogeneous populations of cells can be examined morphologically and biochemically as they progress from proerythroblasts through enucleated reticulocytes. alpha and beta spectrins, the major proteins of the erythrocyte membrane skeleton, are synthesized in the erythroblasts both before and after EP exposure. At all times large portions of the newly synthesized spectrins exist in and are turned over in the cytoplasm. The remaining newly synthesized spectrin is found in a cellular fraction containing total membranes. Pulse-chase experiments show that little of the cytoplasmic spectrins become membrane associated, but that the proportion of newly synthesized spectrin which is membrane associated increases as maturation proceeds. A membrane fraction enriched in plasma membranes has significant differences in the stoichiometry of spectrin accumulation as compared to total cellular membranes. Synthesis of band 3 protein, the anion transporter, is induced only after EP addition to the erythroblasts. All of the newly synthesized band 3 is membrane associated. A two-dimensional gel survey was conducted of newly synthesized proteins in the plasma membrane enriched fraction of the erythroblasts as differentiation proceeded. A majority of the newly synthesized proteins remain in the same proportion to each other during maturation; however, a few newly synthesized proteins greatly increase following EP induction while others decrease markedly. Of the radiolabeled proteins observed in two dimensional gels, only the spectrins, band 3 and actin become major proteins of the mature erythrocyte membrane. Examination of total proteins of the plasma membrane enriched fractions of EP-treated erythroblasts using silver staining and 32P autoradiography show that many proteins and phosphoproteins are selectively eliminated from this fraction late in the course of differentiation during the reticulocyte stage. The selective removal of many proteins at the reticulocyte stage of development combined with previous selective synthesis and accumulation of some specific proteins such as alpha and beta spectrin and band 3 in the differentiating erythroblasts lead to the final mammalian erythrocyte membrane structure.

OBJECTIVE

The neonatal ventral hippocampal lesion (NVHL) rat model shows biological and behavioral abnormalities similar to schizophrenia. Disturbed sensory gating reflects a consistent neurobiological abnormality in schizophrenia. Although of critical interest, sensory gating has not been evaluated in the NVHL model.

METHODS

The N40 rat analog of the human P50 was measured to assess sensory response and gating in NVHL and sham rats. Epidural electrodes recorded evoked potentials (EPs), from which amplitudes, latencies, difference scores (S1-S2) and gating ratios (S2/S1) were assessed. Power and phase locking were computed for evoked EEG activity, to test for frequency-specific abnormalities.

RESULTS

Prolonged S1 N40 latency was detected in the NVHL group, but amplitude and power measures did not differ. NVHL rats demonstrated disturbed phase-locked sensory gating at theta and beta frequencies, as well as reduced phase-locked gamma activity across stimuli, most robustly at S1.

CONCLUSIONS

While measures of sensory gating obtained from the EP were relatively insensitive to the NVHL model, phase locking across trials was affected. NVHL rats may have increased evoked response temporal variability, similar to patients with schizophrenia. This pattern of findings likely reflects core developmental NVHL disturbances in dorsal hippocampal circuits associated with temporal and frontal areas.

The beneficial effects of fish oil on inflammation have been attributed to the content of eicosapentaenoic (EPA)/docosahexaenoic acid. EPA is also a substrate for arachidonic acid (AA) cascade enzymes, but it induces the production of alternative eicosanoids such as 3-series prostanoids and 5-series leukotrienes, which are considered to be less proinflammatory than AA metabolites. However, the molecular basis of this action is poorly understood. In this study, we compared the effects of prostaglandin (PG) E(2) and PGE(3) on endothelium permeability, and the effects of leukotriene (LT) B(4) and LTB(5) on endothelium permeability and mononuclear adhesion and migration. In our study, both prostaglandins increased trans-endothelial Evans blue-albumin (EBA) permeability in a concentration-dependent manner. It is interesting that the effect of PGE(3) was significantly more pronounced than the effect of PGE(2), and both were antagonized by EP(1) and EP(2) antagonists. LTB(4) and LTB(5) had a slight effect on EBA extravasation. However, we observed the enhancement of endothelial permeability in the presence of polymorphonuclear (PMN) cells, probably a consequence of an interplay between leukotriene and prostanoid effects. LTB(4) caused significant increases in the number of PMN cells adhering to endothelial cells, whereas LTB(5) did not induce a significant effect. This effect of LTB(4) appears BLT1 receptor-dependent and was mediated through the enhancement of lymphocyte function-associated antigen-1, membrane attack complex-1, E-selectin, and intercellular adhesion molecule-1 expression. Finally, we observed that, unlike LTB(5), which had a weak effect, LTB(4) was a highly potent chemoattractant. An understanding of the differences in the effects of LTB(4)/LTB(5) on PMN cell adhesion and migration may help to explain the beneficial impact of omega-3 fatty acids in inflammatory processes.

The Light mutation (Blt) is a dominant allele of the b-locus on mouse chromosome 4 which causes progressive dilution of coat colour. Melanocytes within the hair follicles of mutant mice develop normally but later degenerate, due to the accumulation of a toxic product, so that the hair becomes lighter with age. Previous studies on W-locus spotting mutants, from which melanocytes are absent, have shown that melanocytes in the stria vascularis of the inner ear are essential for the development and/or maintenance of the endocochlear potential (EP) which is normally around 100 mV. In this study, physiological recordings from the ears of Light mutants were correlated with strial ultrastructure. EPs recorded from all b/b controls and young homozygous and heterozygous mutants (20-22 days old) were normal (77 to 113 mV), but were reduced (19 to 59 mV) in about 30% of ears from older mutants (Blt/Blt and Blt/b). Strial function therefore appears to develop normally but later degenerates in some mutants. This suggests that strial melanocytes are affected by the Light allele and that the continued presence of melanocytes is necessary for strial function. There was no obvious association between the recorded EP value and the ultrastructural appearance of the stria. No structural abnormalities of the stria were noted in control or mutant mice aged 20 days to 4 months including those which had a reduced EP. Strial atrophy was common in old controls and mutants (1-2 years), and appeared to be an age-related process rather than an effect of the Light mutation. Similarly, pigment build-up was common in all strial cells of old mice. However, the accumulations of lipofuscin-like pigment were much larger and more abundant in aged brown non-agouti mice than those observed in old agouti mice, which suggests that this age-related process also has a genetic component.

This article reports the behavioral, biochemical, and electrophysiological effects of four therapeutic crossed-sequential double-blind trials with 60 autistic children: Trial A--vitamin <em>B</em>6 plus magnesium/magnesium; Trial <em>B</em>--vitamin <em>B</em>6 plus magnesium; Trial C--magnesium; and Trial D--vitamin <em>B</em>6. Therapeutic effects were controlled using behavior rating scales, urinary excretion of homovanillic acid (HVA), and evoked potential (<em>EP</em>) recordings. The behavioral improvement observed with the combination vitamin <em>B</em>6-magnesium was associated with significant modifications of both biochemical and electrophysiological parameters: the urinary HVA excretion decreased, and <em>EP</em> amplitude and morphology seemed to be normalized. These changes were not observed when either vitamin <em>B</em>6 or magnesium was administered alone.

The effects of beta-endorphin (beta-Ep) on plasma glucose levels in rats and on glucose metabolism in isolated rat liver cells were examined. Intravenous injection of beta-Ep (5 micrograms/100 g BW) into ether-anaesthetized rats resulted in prompt and sustained hyperglycaemia with increases in the plasma glucagon and somatostatin levels and decrease in the plasma insulin level. When liver cells isolated from fed rats were incubated in the presence of beta-Ep at concentrations of 6 X 10(-8) M to 6 X 10(-7) M, glucose release into the medium increased within 15 min in a dose-related manner. Time course experiments showed that beta-Ep increased the level of cyclic AMP within 3 min. Significant increase in gluconeogenesis in liver cells isolated from fasted rats was also observed on addition of 10(-7) M beta-Ep in the presence of 10 mM L-lactate. These results suggest that the hyperglycaemia induced by beta-Ep may be caused, at least in part, by the effects of beta-Ep on releases of pancreatic hormones and glucose production in liver cells.

OBJECTIVE

To observe the effect of Chinese herbal medicine for calming Gan (肝) and suppressing hyperactive yang (平肝潜阳, CGSHY) on arterial elasticity function and the circadian rhythm of blood pressure in patients with essential hypertension (EH).

METHODS

Adopting a parallel, randomized design, sixty-four patients with EH of stages I and II were randomly divided into two groups according to a random number table, with 32 in each group. The patients in the treatment group were treated with CGSHY and those in the control group were treated with Enalapril. All patients were given 24-h ambulatory blood pressure monitoring (ABPM) before and after a 12-week treatment. Trough/peak (T/P) ratios of systolic and diastolic blood pressure (SBP & DBP) of each group were calculated. The circadian rhythm of their blood pressure was observed at the same time. The changes in elasticity of the carotid artery in the patients, including stiffness parameter (β), pressure-strain elastic modulus (Ep), arterial compliance (AC), augmentation index (AI), and pulse wave velocity (PVWβ) were determined by the echo-tracking technique before and after a 12-week treatment. In the meantime, their levels of nitric oxide (NO) and endothelin-1 (ET-1) were measured respectively.

RESULTS

After treatment, all parameters in the 24-h ABPM and the elasticity of the carotid artery (β, Ep, AC and PVWβ) were markedly improved, the level of NO was increased, and ET-1 was decreased in both groups as compared with values before treatment (P<0.05 or P<0.01). Further, the improvements in the ratio of T/P of SBP & DBP and in the level of NO and ET-1 in the treatment group were more significant than those in the control group (P<0.05). There were no significant differences in all parameters in the ABPM monitoring and the elasticity of the carotid artery, the recovery of blood pressure circadian rhythm, and the therapeutic effect of antihypertension in EH patients between the two groups (P>0.05).

CONCLUSIONS

Chinese herbal medicine for CGSHY may lower the blood pressure smoothly and recover the circadian rhythm of blood pressure in EH patients. They may also improve the carotid elasticity of EH patients similar to that of Enalapril. The mechanism of action of Chinese herbs on EH might be related to the regulation of vascular endothelium function.

High mol wt forms of immunoreactive ACTH and beta-endorphin (beta EP) are present in cerebrospinal fluid (CSF). We have quantified these peptides directly in the CSF of 26 patients undergoing routine myelography, using a panel of monoclonal antibody-based two-site immunoradiometric assays, specific for ACTH precursors (both POMC and pro-ACTH cross-react 100%), POMC, ACTH, and beta EP. The mean +/- SD levels of POMC in CSF were 530 +/- 150 pmol/L similar to total ACTH precursor immunoreactivity (414 +/- 83 pmol/L). By comparison, the CSF levels of ACTH (3.2 +/- 0.6 pmol/L) and beta EP (6.7 +/- 2.9 pmol/L) were 100-fold lower. POMC, by virtue of its 1% cross-reactivity in the ACTH immunoradiometric assay, could have also accounted for the ACTH immunoreactivity in CSF. Sephadex G-75 chromatography of CSF confirmed the presence of a single major peak of ACTH precursors eluting at the position of POMC (31K), while ACTH immunoreactivity was not detected at the position of ACTH-(1-39) (4.5K). We also studied the effect of exogenous glucocorticoids on CSF POMC peptides by giving 2.5 mg dexamethasone (0.5 mg, orally, every 6 h for 24 h) to a similar group of age-matched patients before lumbar puncture. No significant differences in CSF peptide content were observed between the two groups. These data suggest that the unprocessed precursor molecule POMC is the predominant peptide of the POMC family in human CSF and should always be considered when interpreting data involving ACTH or other component peptide immunoreactivity in this biological fluid.

Standard electrochemical data for high-quality, boron-doped diamond thin-film electrodes are presented. Films from two different sources were compared (NRL and USU) and both were highly conductive, hydrogen-terminated, and polycrystalline. The films are acid washed and hydrogen plasma treated prior to use to remove nondiamond carbon impurity phases and to hydrogen terminate the surface. The boron-doping level of the NRL film was estimated to be in the mid 1019 B/cm3 range, and the boron-doping level of the USU films was approximately 5 x 10(20) B/cm(-3) based on boron nuclear reaction analysis. The electrochemical response was evaluated using Fe-(CN)6(3-/4-), Ru(NH3)6(3+/2+), IrCl6(2-/3-), methyl viologen, dopamine, ascorbic acid, Fe(3+/2+), and chlorpromazine. Comparisons are made between the apparent heterogeneous electron-transfer rate constants, k0(app), observed at these high-quality diamond films and the rate constants reported in the literature for freshly activated glassy carbon. Ru(NH3)6(3+/2+), IrCl6(2-/3-), methyl viologen, and chlorpromazine all involve electron transfer that is insensitive to the diamond surface microstructure and chemistry with k0(app) in the 10(-2)-10(-1) cm/s range. The rate constants are mainly influenced by the electronic properites of the films. Fe(CN)6(3-/4-) undergoes electron transfer that is extremely sensitive to the surface chemistry with k0(app) in the range of 10(-2)-10(-1) cm/s at the hydrogen-terminated surface. An oxygen surface termination severely inhibits the rate of electron transfer. Fe(3+/2+) undergoes slow electron transfer at the hydrogen-terminated surface with k0(app) near 10(-5) cm/s. The rate of electron transfer at sp2 carbon electrodes is known to be mediated by surface carbonyl functionalities; however, this inner-sphere, catalytic pathway is absent on diamond due to the hydrogen termination. Dopamine, like other catechol and catecholamines, undergoes sluggish electron transfer with k0(app) between 10(-4) and 10(-5) cm/s. Converting the surface to an oxygen termination has little effect on k0(app). The slow kinetics may be related to weak adsorption of these analytes on the diamond surface. Ascorbic acid oxidation is very sensitive to the surface termination with the most negative Ep(ox) observed at the hydrogen-terminated surface. An oxygen surface termination shifts Ep(ox) positive by some 250 mV or more. An interfacial energy diagram is proposed to explain the electron transfer whereby the midgap density of states results primarily from the boron doping level and the lattice hydrogen. The films were additionally characterized by scanning electron microscopy and micro-Raman imaging spectroscopy. The cyclic voltammetric and kinetic data presented can serve as a benchmark for research groups evaluating the electrochemical properties of semimetallic (i.e., conductive), hydrogen-terminated, polycrystalline diamond.

It is known that the same peptide can be identified in different secretory tissues and in the central nervous system (CNS). We now provide evidence that the same peptides can be found in different organs related to the control of a single function, and speculate on the possibility that this reflects a common neuroendocrine programming. Endogenous opioid peptides (EOP) inhibit the reproductive function acting via the CNS. EOP inhibit gonadotropin secretion in rodents and humans via inhibition of GnRH release and have direct inhibitory actions at the pituitary level via specific binding sites on the gonadotrophs. However, EOP can also be synthesized in the testis and in different compartments of the male genital tract. Several findings indicate that EOP of the reproductive tract have a local, paracrine role. These include: (1) the detection of significant beta-endorphin (beta-EP) production by rat Leydig cells (Lc) in cultures; (2) the hormonal regulation of Lc beta-EP production by positive (gonadotropins) and negative (steroids, glucocorticoids, GnRH) factors; (3) the presence of opioid binding sites (Kd in the nanomolar range) in tubular homogenates and Sertoli cells (Sc) in culture of adult and immature rat testes; (4) the inhibition of basal and FSH-stimulated ABP production by Sc in culture when chronically exposed to beta-EP treatment; (5) the detection of high levels of beta-EP and met-enkephalin in human semen with values 6-12 times higher than in plasma; (6) the evidence for inhibitory functions of seminal opioids on sperm motility, vas deferens muscle contraction and partner immune system. Thus the same peptides, i.e. EOP, may control the reproductive function at multiple sites, operating as a multimessenger system in which the central and peripheral level are unified by the common chemical and inhibitory nature of the message.

Brain cannulated rats were injected with the opioid peptide beta-endorphin (beta-EP) directly into the hypothalamic paraventricular nucleus (PVN) where norepinephrine (NE) is most effective in stimulating eating behavior. Beta-Endorphin (1.0 nmole) reliably increased food intake in satiated animals, and this response was blocked by local administration of the selective opiate antagonist naloxone. The eating induced by beta-EP was positively correlated in magnitude with the NE response and, like NE, was antagonized by PVN injection of the alpha-noradrenergic blocker phentolamine. Naloxone had no effect on NE-induced eating, and the dopaminergic blocker fluphenazine failed to alter either beta-EP or NE eating. When injected simultaneously, at maximally effective doses, beta-EP and NE produced an eating response which was significantly larger than either of the responses elicited separately by beta-EP or NE and was essentially equal to the sum of these two responses. The evidence obtained in this study suggests that beta-EP and NE stimulate food ingestion through their action on PVN opiate and alpha-noradrenergic receptors, respectively, and that beta-EP's action is closely related to, and in part may be dependent upon, the PVN alpha-noradrenergic system for feeding control.

Pharmacological manipulations in the Drosophila embryo have been hindered by the impermeability of the eggshell. The ultimate barrier to delivery of small molecule solutes to the embryo is the waxy layer that lies beneath the external chorion layers and encases the underlying vitelline membrane of the eggshell. Conventional protocols call for heptane or octane to permeablize the dechorionated eggshell however, these solvents are toxic and can result in low viability. Furthermore, heptane and octane require transition of the embryo between aqueous and organic phase solvents making it challenging to avoid desiccation. Here we describe an embryo permeabilization solvent (EPS) composed of d-limonene and plant-derived surfactants that is water miscible and highly effective in rendering the dechorionated eggshell permeable. EPS permeabilization enables embryo uptake of several different dyes of various molecular mass up to 995Da. We find that the embryo undergoes an age-dependent decrease in the ability to be permeabilized in the first six to eight hours after egg laying. This apparent developmental change in the vitelline membrane contributes to the heterogeneity in permeabilization seen even among closely staged embryos. However, using fluorescent properties of Rhodamine B dye and various conditions of EPS treatment we demonstrate the ability to obtain optimally permeabilized viable embryos. We also demonstrate the ability to assess teratogenic activity of several compounds applied to embryos in vitro, using both early and late developmental endpoints. Application of the method to transgenic strains carrying GFP-reporter genes results in a robust readout of pharmacological alteration of embryogenesis. The straightforward and rapid nature of the manipulations needed to prepare batches of permeabilized embryos has the potential of establishing the Drosophila embryo as an alternative model in toxicology and for small molecule screening in a high-throughput format.

Interleukin-1 (IL-1), a potent stimulant of bone resorption, has been implicated in the pathogenesis of postmenopausal osteoporosis, because monocyte IL-1 bioactivity increases after the menopause and is decreased by estrogen and progesterone (EP) replacement. As IL-1 bioactivity reflect the production of both IL-1 and the IL-1 inhibitor, IL-1 receptor antagonist (IL-1ra), EP treatment could decrease IL-1 bioactivity by regulating the secretion of either IL-1 or IL-1ra. We now report that EP treatment in vivo decreased the secretion into the medium of cultured monocytes of IL-1ra and IL-1 beta as well as the IL-1 beta/IL-1ra ratio. We also found that in normal women the production of IL-1ra was within premenopausal levels in the first 7 yr after the menopause and increased linearly thereafter. In these women, monocyte IL-1 beta, IL-1 beta/IL-1ra ratio, and IL-1 bioactivity were all increased in the first 7 yr after the menopause and within the premenopausal range thereafter. In osteoporotic women, IL-1 beta, IL-1 beta/IL-1ra ratio, and IL-1 bioactivity increased after the menopause and returned to premenopausal levels after 15 yr from the menopause. In these women monocyte IL-1ra secretion was above the premenopausal range at all times after the menopause, but did not change with the passage of time since menopause. We conclude that hormone replacement decreases the in vitro secretion of both IL-1ra and IL-1 beta. The data also suggest that in normal women a progressive increase in the secretion of IL-1ra contributes to restore a normal IL-1/IL-1ra ratio after the menopause, a phenomenon which, in turn, may play a role in limiting postmenopausal bone loss.

The authors have recently reported the presence of characteristic foamy swelling/degeneration (giant lamellar body degeneration, GLBD) of type II pneumocytes in the lungs affected by Hermansky-Pudlak syndrome (HPS)-associated interstitial pneumonia (HPSIP), and proposed the hypothesis that GLBD may be the triggering factor in the development of HPSIP (Virchows Arch 2000; 437: 304-13). The purpose of the present paper was to investigate the lung pathology of pale ear (ep) mouse, a mouse model of HPS1, and of beige (bg) mouse, a mouse model of Chediak-Higashi syndrome (CHS) with a reference to GLBD and associated pathologic changes. GLBD was found in both ep and bg mice soon after birth, and increased in severity as the mice grew older. Younger mice had only GLBD with no evidence of interstitial change. Aged bg mice showed the most prominent GLBD and patchy areas of alveolar collapse accompanied by lymphocytic infiltration and slight fibrosis. Aged ep mice with less severe GLBD than bg mice of comparable ages also had a slight tendency to develop interstitial inflammation but no fibrosis. The pneumocytes with GLBD were immunoreactive for surfactant protein B and composed of giant lamellar bodies ultrastructurally, findings which were almost identical to those of human GLBD. The results of the present study support the hypothesis that GLBD may play an important role in the development of HPSIP. Ep and bg mice, especially the latter, may be useful mouse models of HPSIP.

Exopolysaccharide (EPS) and lipopolysaccharide (LPS) from Bradyrhizobium japonicum are important for infection and nodulation of soybean (Glycine max), although their roles are not completely understood. To better understand this, we constructed mutants in B. japonicum USDA 110 impaired in galactose or galacturonic acid incorporation into the EPS without affecting the LPS. The derivative LP 3010 had a deletion of lspL-ugdH and produced EPS without galacturonic acid whereas LP 3013, with an insertion in exoB, produced EPS without galactose. In addition, the strain LP 3017, with both mutations, had EPS devoid of both galactosides. The missing galactosides were not replaced by other sugars. The defects in EPS had different consequences. LP 3010 formed biofilms and nodulated but was defective in competitiveness for nodulation; and, inside nodules, the peribacteroid membranes tended to fuse, leading to the merging of symbiosomes. Meanwhile, LP 3013 and LP 3017 were unable to form biofilms and produced empty pseudonodules but exoB suppressor mutants were obtained when LP 3013 plant inoculation was supplemented with wild-type EPS. Similar phenotypes were observed with all these mutants in G. soja. Therefore, the lack of each galactoside in the EPS has a different functional effect on the B. japonicum-soybean symbiosis.

Fungal exposure inside homes has been associated with adverse respiratory symptoms in children and adults. While fungal assessment has traditionally relied upon questionnaires, fungal growth on culture plates and spore counts, new immunoassays for extracellular polysaccharides (EPS) and beta (1-->3)-glucans have enabled quantitation of fungal agents in house dust in a more timely and cost-effective manner, possibly providing a better measure of fungal exposure. We investigated associations among measurements of EPS, beta (1-->3)-glucans and culturable fungi obtained from 23 Dutch homes. From each home, dust samples were vacuumed from the living room floor twice during the Fall, Winter and Spring seasons for a total of six collections (every 6 weeks from October 1997 to May 1998). Samples were sieved and fine dust was analyzed for EPS from Aspergillus and Penicillium spp. combined, beta (1-->3)-glucans and culturable fungi. EPS was positively associated with glucan; an increase from the 25th to the 75th percentile of glucan concentration was associated with a 1.6-fold increase in EPS concentration (95% CI = 1.3 to 2.0; p < 0.01). The most significant variables associated with EPS and glucan concentrations were the surface type that was vacuumed and the concentration of total culturable fungi (in colony forming units (CFU)/g dust), with an increase in CFU/g from the 25th to the 75th percentile associated with a 1.3 (1.1-1.6)-fold increase in glucan and a 1.7 (1.3-2.2)-fold increase in EPS concentrations. In addition, the within-home variation of EPS levels were smaller than those between homes (25,646 U/g vs. 50,635 U/g), whereas the variation of glucan levels was similar within and between homes (1,300 vs. 1,205 micrograms/g). These positive associations suggest that house dust concentrations of beta (1-->3)-glucan, and particularly those of EPS, are good markers for the overall levels of fungal concentrations in floor dust which is a surrogate for estimating airborne fungal exposure.

Although isoprostanes generally act on smooth muscle via TXA(2)-selective prostanoid receptors (TPs), some suggest other prostanoid receptors or possibly even a novel isoprostane-selective receptor might be involved. We studied contractions to several isoprostanes in porcine pulmonary vasculature using organ bath techniques. 8-iso-prostaglandin E(2) (PGE(2)) was the most potent and efficacious of the isoprostanes, with a log EC(50) of -7.0 +/- 0.2 in the pulmonary artery and -6.8 +/- 0.2 in the pulmonary vein. The responses to all the isoprostanes were essentially completely blocked by the TP receptor antagonist ICI 192605 [4(Z)-6-[(2,4,5-cis)2-(2-chlorophenyl)-4-(2-hydroxyphenyl)1,3-dioxan-5-yl]hexenoic acid], and the equilibrium dissociation constants for ICI 192605 competing with U46619 or 8-iso-PGE(2) were both approximately 2 nM, indicating that isoprostane-evoked responses involve primarily TP receptors. Only 8-iso-PGE(2) was able to evoke substantial contractions in the presence of ICI 192605 and only in the pulmonary vein. The EC(50) of these ICI 192605-insensitive responses was -6.1 +/- 0.2. Using a variety of prostanoid agonists, we found the pulmonary vein lacked excitatory PGF(2alpha)-selective prostanoid receptor (FP) or PGD(2)-selective prostanoid receptor (DP) but expressed excitatory EP(3) receptors. The ICI 192605-insensitive responses to 8-iso-PGE(2) were unaffected by the EP(1) antagonist SC-19220 [8-chloro-debenz[b,f][1,4]oxazepine-10(11H)-carboxy-(2-acetyl) hydrazine; 10(-5) M] but were antagonized by the less selective DP/EP(1)/EP(2) antagonist AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid; 10(-5) M) or by cyclopiazonic acid (10(-5) M; depletes the internal Ca(2+) store). Our data indicate that, whereas 8-iso-PGE(2) constricts pulmonary vasculature primarily through TP receptors, a substantial portion of this response is also directed through EP(3) receptors or possibly a novel isoprostane receptor.

BACKGROUND

We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers.

METHODS

HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA.

RESULTS

The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients.

CONCLUSIONS

Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use.

BACKGROUND

International Standard Randomised Controlled Trial Number (ISRCTN) 60284968.

The rhizobial bacterium Bradyrhizobium japonicum functions as a nitrogen-fixing symbiont of the soybean plant (Glycine max). Plants are capable of producing an oxidative burst, a rapid proliferation of reactive oxygen species (ROS), as a defense mechanism against pathogenic and symbiotic bacteria. Therefore, B. japonicum must be able to resist such a defense mechanism to initiate nodulation. In this study, paraquat, a known superoxide radical-inducing agent, was used to investigate this response. Genome-wide transcriptional profiles were created for both prolonged exposure (PE) and fulminant shock (FS) conditions. These profiles revealed that 190 and 86 genes were up- and downregulated for the former condition, and that 299 and 105 genes were up- and downregulated for the latter condition, respectively (>2.0-fold; P < 0.05). Many genes within putative operons for F(0)F(1)-ATP synthase, chemotaxis, transport, and ribosomal proteins were upregulated during PE. The transcriptional profile for the FS condition strangely resembled that of a bacteroid condition, including the FixK(2) transcription factor and most of its response elements. However, genes encoding canonical ROS scavenging enzymes, such as superoxide dismutase and catalase, were not detected, suggesting constitutive expression of those genes by endogenous ROS. Various physiological tests, including exopolysaccharide (EPS), cellular protein, and motility characterization, were performed to corroborate the gene expression data. The results suggest that B. japonicum responds to tolerable oxidative stress during PE through enhanced motility, increased translational activity, and EPS production, in addition to the expression of genes involved in global stress responses, such as chaperones and sigma factors.

Unicellular organisms naturally form multicellular communities, differentiate into specialized cells, and synchronize their behaviour under certain conditions. Swarming, defined as a movement of a large mass of bacteria on solid surfaces, is recognized as a preliminary step in the formation of biofilms. The main aim of this work was to study the role of a group of genes involved in exopolysaccharide biosynthesis during pellicle formation and swarming in Bacillus subtilis strain 168. To assess the role of particular proteins encoded by the group of epsI-epsO genes that form the eps operon, we constructed a series of insertional mutants. The results obtained showed that mutations in epsJ-epsN, but not in the last gene of the eps operon (epsO), have a severe effect on pellicle formation under all tested conditions. Moreover, the inactivation of 5 out of the 6 genes analysed caused total inhibition of swarming in strain 168 (that does not produce surfactin) on LB medium. Following restoration of the sfp gene (required for production of surfactin, which is essential for swarming of the wild-type bacteria), the sfp+ strains defective in eps genes (except epsO) generated significantly different patterns during swarming on synthetic B medium, as compared to the parental strain 168 sfp+.

Oenococcus oeni is the bacterial species which drives malolactic fermentation in wine. The analysis of 50 genomic sequences of O. oeni (14 already available and 36 newly sequenced ones) provided an inventory of the genes potentially involved in exopolysaccharide (EPS) biosynthesis. The loci identified are: two gene clusters named epsepseps gene clusters composition diverged from one strain to another. The soluble and capsular EPS production capacity of several strains was examined after growth in different culture media and the EPS structure was determined. Genotype to phenotype correlations showed that several EPS biosynthetic pathways were active and complementary in O. oeni. Can be distinguished: (i) a Wzy-dependent synthetic pathway, allowing the production of heteropolysaccharides made of glucose, galactose and rhamnose, mainly in a capsular form, (ii) a glucan synthase pathway (Gtf), involved in β-glucan synthesis in a free and a cell-associated form, giving a ropy phenotype to growth media and (iii) homopolysaccharide synthesis from sucrose (α-glucan or β-fructan) by glycoside-hydrolases of the GH70 and GH68 families. The eps gene distribution on the phylogenetic tree was examined. Fifty out of 50 studied genomes possessed several genes dedicated to EPS metabolism. This suggests that these polymers are important for the adaptation of O. oeni to its specific ecological niche, wine and possibly contribute to the technological performance of malolactic starters.

Improper regulation of B cell responses leads to excessive production of antibodies and contributes to the development of autoimmune disease. T helper 17 (Th17) cells also drive the development of autoimmune disease, but the role of B cells in shaping Th17 cell-mediated immune responses, as well as the reciprocal regulation of B cell responses by IL-17 family cytokines, remains unclear. The aim of this study was to characterize the regulation of IL-17A and IL-17F in a model of T cell-dependent B cell activation. Stimulation of primary human B cell and peripheral blood mononuclear cell (BT) co-cultures with α-IgM and a non-mitogenic concentration of superantigens for three days promoted a Th17 cell response as evidenced by increased expression of Th17-related gene transcripts, including Il17f, Il21, Il22, and Il23r, in CD4 T cells, as well as the secretion of IL-17A and IL-17F protein. We tested the ability of 144 pharmacologic modulators representing 91 different targets or pathways to regulate IL-17A and IL-17F production in these stimulated BT co-cultures. IL-17A production was found to be preferentially sensitive to inhibition of the PI3K/mTOR pathway, while prostaglandin EP receptor agonists, including PGE2, increased IL-17A concentrations. In contrast, the production of IL-17F was inhibited by PGE2, but selectively increased by TLR2 and TLR5 agonists. These results indicate that IL-17A regulation is distinct from IL-17F in stimulated BT co-cultures and that this co-culture approach can be used to identify pathway mechanisms and novel agents that selectively inhibit production of IL-17A or IL-17F.

Lactic acid bacteria (LAB) produce homopolysaccharides (HoPS) and heteropolysaccharides (HePS) with potential functional properties. In this work, we have performed a comparative analysis of production and purification trials of these biopolymers from bacterial culture supernatants. LAB strains belonging to four different genera, both natural as well as recombinant, were used as model systems for the production of HoPS and HePS. Two well characterized strains carrying the gft gene were used for β-glucan production, Pediococcus parvulus 2.6 (P. parvulus 2.6) isolated from cider, and the recombinant strain Lactococcus lactis NZ9000[pGTF] (L. lactis NZ9000[pGTF]). In addition, another cider isolate, Lactobacillus suebicus CUPV225 (L. suebicus CUPV225), and Leuconostoc mesenteroides RTF10 (L. mesenteroides RTF10), isolated from meat products were included in the study. Chemical analysis of the EPS revealed that L. mesenteroides produces a dextran, L. suebicus a complex heteropolysaccharide, and the β-glucan producing-strains the expected 2-substituted (1,3)-β-glucan.