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Find video protocols related to scientific articles indexed in Pubmed.
Copper(i)-assisted red-shifted phosphorescence in Au(i)Cu(i) heteropolynuclear complexes.
Dalton Trans
PUBLISHED: 09-25-2014
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Reactions between [Au(C6Cl2F3)(tht)] and P,N-donor bridging ligands of the type PPh2py and (PPh2)2phen lead to the homonuclear gold complexes [Au(C6Cl2F3)(PPh2py)] () and [Au2(C6Cl2F3)2{(PPh2)2phen}] (). Subsequent addition of [Cu(CH3CN)4](BF4) leads to the formation of the corresponding gold-copper heterometallic complexes [Au2Cu(C6Cl2F3)2(PPh2py)2](BF4) () and [Au2Cu(C6Cl2F3)2{(PPh2)2phen)}(CH3CN)](BF4) (). The four complexes have been structurally characterized and are luminescent. The gold precursors show emissions arising from metal-perturbed intraligand transitions. The heterometallic complexes show a red shift of the emissions that is proposed to arise from an admixture of IL (intraligand) and MLCT (metal-to-ligand-charge-transfer) transitions. DFT and TD-DFT calculations agree well with these results.
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The yrpAB operon of Yersinia ruckeri encoding two putative U32 peptidases is involved in virulence and induced under microaerobic conditions.
Virulence
PUBLISHED: 05-27-2014
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In an attempt to dissect the virulence mechanisms of Yersinia ruckeri two adjacent genes, yrpA and yrpB, encoding putative peptidases belonging to the U32 family, were analyzed. Similar genes, with the same genetic organization were identified in genomic analysis of human-pathogenic yersiniae. RT-PCR studies indicated that these genes form an operon in Y. ruckeri. Transcriptional studies using an yrpB::lacZY fusion showed high levels of expression of these genes in the presence of peptone in the culture medium, as well as under oxygen-limited conditions. These two factors had a synergic effect on gene induction when both were present simultaneously during bacterial incubation, which indicates the important role that environmental conditions in the fish gut can play in the regulation of specific genes. LD 50 experiments using an yrpA insertional mutant strain demonstrated the participation of this gene in the virulence of Y. ruckeri.
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Regulatory T-cell vaccination independent of auto-antigen.
Exp. Mol. Med.
PUBLISHED: 03-15-2014
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To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine's capacity to protect against travelers' diarrhea or salmonellosis. By adapting the vaccine's anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25(+) Treg cells are stimulated, but for arthritis CD39(+) Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-? and interleukin-10.
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Experimental and theoretical comparison of the metallophilicity between d(10)-d(10) Au(I)-Hg(II) and d(8)-d(10) Au(III)-Hg(II) interactions.
Inorg Chem
PUBLISHED: 01-14-2014
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The heteronuclear Au(I)/Hg(II) complexes [Hg{AuR(?-2-C6H4PPh2)}2] [R = C6F5 (1), C6Cl2F3 (2)] were prepared by reacting [Hg(2-C6H4PPh2)2] with [AuR(tht)] (1:2) and further transformed into the Au(III)/Hg(II) species [Hg{Au(C6F5)Cl2(?-2-C6H4PPh2)}2] [R = C6F5 (3), C6Cl2F3 (4)] by the addition of 2 equiv of PhI·Cl2. The crystal structures of 1-3 display Au···Hg(II) interactions, which in the case of 3 is the first Au(III)···Hg contact described to date. Theoretical calculations on model systems of the C6F5 derivatives evidence that the attraction between Au(I) or Au(III) and Hg(II) arise from dispersion-type interactions and that both contacts are of the same strength.
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Oral Escherichia coli Colonization Factor Antigen I Fimbriae Ameliorate Arthritis via IL-35, Not IL-27.
J. Immunol.
PUBLISHED: 12-11-2013
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A Salmonella therapeutic expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) fimbriae protects against collagen-induced arthritis (CIA) by eliciting two regulatory T cell (Treg) subsets: TGF-?-producing Foxp3(-)CD39(+)CD4(+) T cells and IL-10-producing Foxp3(+)CD39(+)CD4(+) T cells. However, it is unclear whether CFA/I fimbriae alone are protective and whether other regulatory cytokines are involved, especially in the context for the EBI3-sharing cytokines, Treg-derived IL-35 and APC-derived IL-27, both capable of suppressing Th17 cells and regulating autoimmune diseases. Subsequent evaluation revealed that a single oral dose of purified, soluble CFA/I fimbriae protected against CIA as effectively as did Salmonella-CFA/I and found that Foxp3(+)CD39(+)CD4(+) T cells were the source of secreted IL-35, whereas IL-27 production by CD11c(+) cells was inhibited. Inquiring into their relevance, CFA/I fimbriae-treated IL-27R-deficient (WSX-1(-/-)) mice were equally protected against CIA as were wild-type mice, suggesting a limited role for IL-27. In contrast, CFA/I fimbriae-mediated protection was abated in EBI3(-/-) mice, accompanied by the loss of TGF-?- and IL-10-producing Tregs. Adoptive transfer of C57BL/6 CD39(+)CD4(+) T cells to EBI3(-/-) mice with concurrent CFA/I plus IL-35 treatment effectively stimulated Tregs suppressing proinflammatory collagen II-specific Th cells. In contrast, recipients cotransferred with C57BL/6 and EBI3(-/-) CD39(+)CD4(+) T cells and treated with CFA/I plus IL-35 were not protected, implicating the importance of endogenous IL-35 for conferring CFA/I-mediated protection. Thus, CFA/I fimbriae stimulate IL-35 required for the coinduction of TGF-? and IL-10.
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Incidence of lower eyelid asymmetry: an anthropometric analysis of 204 patients.
Aesthet Surg J
PUBLISHED: 07-03-2013
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To avoid complications and improve patient satisfaction with lower lid blepharoplasty, a precise assessment of any preoperative eyelid asymmetry is essential.
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Interleukin-17 protects against the Francisella tularensis live vaccine strain but not against a virulent F. tularensis type A strain.
Infect. Immun.
PUBLISHED: 06-17-2013
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Francisella tularensis is a highly infectious intracellular bacterium that causes the zoonotic infection tularemia. While much literature exists on the host response to F. tularensis infection, the vast majority of work has been conducted using attenuated strains of Francisella that do not cause disease in humans. However, emerging data indicate that the protective immune response against attenuated F. tularensis versus F. tularensis type A differs. Several groups have recently reported that interleukin-17 (IL-17) confers protection against the live vaccine strain (LVS) of Francisella. While we too have found that IL-17R?(-/-) mice are more susceptible to F. tularensis LVS infection, our studies, using a virulent type A strain of F. tularensis (SchuS4), indicate that IL-17R?(-/-) mice display organ burdens and pulmonary gamma interferon (IFN-?) responses similar to those of wild-type mice following infection. In addition, oral LVS vaccination conferred equivalent protection against pulmonary challenge with SchuS4 in both IL-17R?(-/-) and wild-type mice. While IFN-? was found to be critically important for survival in a convalescent model of SchuS4 infection, IL-17 neutralization from either wild-type or IFN-?(-/-) mice had no effect on morbidity or mortality in this model. IL-17 protein levels were also higher in the lungs of mice infected with the LVS rather than F. tularensis type A, while IL-23p19 mRNA expression was found to be caspase-1 dependent in macrophages infected with LVS but not SchuS4. Collectively, these results demonstrate that IL-17 is dispensable for host immunity to type A F. tularensis infection, and that induced and protective immunity differs between attenuated and virulent strains of F. tularensis.
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Progress in Brucella vaccine development.
Front Biol (Beijing)
PUBLISHED: 06-05-2013
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Brucella spp. are zoonotic, facultative intracellular pathogens, which cause animal and human disease. Animal disease results in abortion of fetuses; in humans, it manifests flu-like symptoms with an undulant fever, with osteoarthritis as a common complication of infection. Antibiotic regimens for human brucellosis patients may last several months and are not always completely effective. While there are no vaccines for humans, several licensed live Brucella vaccines are available for use in livestock. The performance of these animal vaccines is dependent upon the host species, dose, and route of immunization. Newly engineered live vaccines, lacking well-defined virulence factors, retain low residual virulence, are highly protective, and may someday replace currently used animal vaccines. These also have possible human applications. Moreover, due to their enhanced safety and efficacy in animal models, subunit vaccines for brucellosis show great promise for their application in livestock and humans. This review summarizes the progress of brucellosis vaccine development and presents an overview of candidate vaccines.
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Attenuating gene expression (AGE) for vaccine development.
Virulence
PUBLISHED: 05-07-2013
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Live attenuated vaccines are adept in stimulating protective immunity. Methods for generating such vaccines have largely adopted strategies used with Salmonella enterica. Yet, when similar strategies were tested in other gram-negative bacteria, the virulence factors or genes responsible to incapacitate Salmonella often failed in providing the desired outcome. Consequently, conventional live vaccines rely on prior knowledge of the pathogens virulence factors to successfully attenuate them. This can be problematic since such bacterial pathogens normally harbor thousands of genes. To circumvent this problem, we found that overexpression of bacterial appendages, e.g., fimbriae, capsule, and flagella, could successfully attenuate wild-type (wt) Salmonella enterica serovar Typhimurium. Further analysis revealed these attenuated Salmonella strains conferred protection against wt S. Typhimurium challenge as effectively as genetically defined Salmonella vaccines. We refer to this strategy as attenuating gene expression (AGE), a simple efficient approach in attenuating bacterial pathogens, greatly facilitating the construction of live vaccines.
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Immunomodulatory activity of polysaccharides isolated from Clerodendrum splendens: beneficial effects in experimental autoimmune encephalomyelitis.
BMC Complement Altern Med
PUBLISHED: 02-22-2013
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Extracts of leaves from Clerodendrum have been used for centuries to treat a variety of medicinal problems in tropical Africa. However, little is known about the high-molecular weight active components conferring therapeutic properties to these extracts.
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Experimental and theoretical evidence of the existence of gold(I)···mercury(II) interactions in solution through fluorescence-quenching measurements.
Chemistry
PUBLISHED: 02-21-2013
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Heteronuclear complexes {[Hg(R)2][Au(R)(PMe3)]2}n (R=R=C6Cl2F3 (3); R=R=C6F5 (4); R=C6Cl2F3, R=C6F5 (5); R=C6F5, R=C6Cl2F3 (6)) were prepared by the treatment of the corresponding organomercury compounds, [Hg(C6X5)2], with two equivalents of [Au(C6X5)(PMe3)]. Their crystal structures, as determined by using X-ray diffraction methods, display Au···Hg interactions. Although only compound 4 and 5 show luminescence in the solid state, all of these compounds quench the fluorescence of naphthalene in solution. Solution studies of these derivatives suggest a cooperative effect of the gold(I) center in switching on the quenching capabilities of the [Hg(C6X5)2] synthon with naphthalene. Theoretical studies confirmed the quenching ability of the organomercury species in the presence of gold.
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A polymer/oil based nanovaccine as a single-dose immunization approach.
PLoS ONE
PUBLISHED: 01-01-2013
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The recognized necessity for new antigen delivery carriers with the capacity to boost, modulate and prolong neutralizing immune responses prompted our approach, in which we describe a multifunctional nanocarrier consisting of an oily nanocontainer protected by a polymeric shell made of chitosan (CS), named CS nanocapsules (CSNC). The CS shell can associate the antigen on its surface, whereas the oily core might provide additional immunostimulating properties. In this first characterization of the system, we intended to study the influence of different antigen organizations on the nanocarriers surface (using the recombinant hepatitis B surface antigen -rHBsAg- as a model antigen) on their long-term immunopotentiating effect, without any additional immunostimulant. Thus, two prototypes of antigen-loaded CSNC (CSNC+ and CSNC-), exhibiting similar particle size (200 nm) and high antigen association efficiency (>80%), were developed with different surface composition (polymer/antigen ratios) and surface charge (positive/negative, respectively). The biological evaluation of these nanovaccines evidenced the superiority of the CSNC+ as compared to CSNC- and alum-rHBsAg in terms of neutralizing antibody responses, following intramuscular vaccination. Moreover, a single dose of CSNC+ led to similar IgG levels to the positive control. The IgG1/IgG2a ratio suggested a mixed Th1/Th2 response elicited by CSNC+, in contrast to the typical Th2-biased response of alum. Finally, CSNC+ could be freeze-dried without altering its physicochemical properties and adjuvant effect in vivo. In conclusion, the evaluation of CSNC+ confirms its interesting features for enhancing, prolonging and modulating the type of immune response against subunit antigens, such as rHBsAg.
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Sublingual immunization with adenovirus F protein-based vaccines stimulates protective immunity against botulinum neurotoxin A intoxication.
Int. Immunol.
PUBLISHED: 12-29-2011
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Sublingual (s.l.) vaccination is an efficient way to induce elevated levels of systemic and mucosal immune responses. To mediate mucosal uptake, ovalbumin (OVA) was genetically fused to adenovirus 2 fiber protein (OVA-Ad2F) to assess whether s.l. immunization was as effective as an alternative route of vaccination. Ad2F-delivered vaccines were efficiently taken up by dendritic cells and migrated mostly to submaxillary gland lymph nodes, which could readily stimulate OVA-specific CD4(+) T cells. OVA-Ad2F + cholera toxin (CT)-immunized mice elicited significantly higher OVA-specific serum IgG, IgA and mucosal IgA antibodies among the tested immunization groups. These were supported by elevated OVA-specific IgG and IgA antibody-forming cells. A mixed T(h)-cell response was induced as evident by the enhanced IL-4, IL-10, IFN-? and TNF-?-specific cytokine-forming cells. To assess whether this approach can stimulate neutralizing antibodies, immunizations were performed with the protein encumbering the ?-trefoil domain of C-terminus heavy chain (Hc?tre) from botulinum neurotoxin A (BoNT/A) as well as when fused to Ad2F. Hc?tre-Ad2F + CT-dosed mice showed the greatest serum IgG, IgA and mucosal IgA titers among the immunization groups. Hc?tre-Ad2F alone also induced elevated antibody production in contrast to Hc?tre alone. Plasma from Hc?tre + CT- and Hc?tre-Ad2F + CT-immunized groups neutralized BoNT/A and protected mice from BoNT/A intoxication. Most importantly, Hc?tre-Ad2F + CT-immunized mice were protected from BoNT/A intoxication relative to Hc?tre + CT-immunized mice, which only showed ?60% protection. This study shows that s.l. immunization with Ad2F-based vaccines is effective in conferring protective immunity.
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Segregated regulatory CD39+CD4+ T cell function: TGF-?-producing Foxp3- and IL-10-producing Foxp3+ cells are interdependent for protection against collagen-induced arthritis.
J. Immunol.
PUBLISHED: 10-03-2011
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Oral immunization with a Salmonella vaccine vector expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) can protect against collagen-induced arthritis (CIA) by dampening IL-17 and IFN-? via enhanced IL-4, IL-10, and TGF-?. To identify the responsible regulatory CD4(+) T cells making the host refractory to CIA, Salmonella-CFA/I induced CD39(+)CD4(+) T cells with enhanced apyrase activity relative to Salmonella vector-immunized mice. Adoptive transfer of vaccine-induced CD39(+)CD4(+) T cells into CIA mice conferred complete protection, whereas CD39(-)CD4(+) T cells did not. Subsequent analysis of vaccinated Foxp3-GFP mice revealed the CD39(+) T cells were composed of Foxp3-GFP(-) and Foxp3-GFP(+) subpopulations. Although each adoptively transferred Salmonella-CFA/I-induced Foxp3(-) and Foxp3(+)CD39(+)CD4(+) T cells could protect against CIA, each subset was not as efficacious as total CD39(+)CD4(+) T cells, suggesting their interdependence for optimal protection. Cytokine analysis revealed Foxp3(-) CD39(+)CD4(+) T cells produced TGF-?, and Foxp3(+)CD39(+)CD4(+) T cells produced IL-10, showing a segregation of function. Moreover, donor Foxp3-GFP(-) CD4(+) T cells converted to Foxp3-GFP(+) CD39(+)CD4(+) T cells in the recipients, showing plasticity of these regulatory T cells. TGF-? was found to be essential for protection because in vivo TGF-? neutralization reversed activation of CREB and reduced the development of CD39(+)CD4(+) T cells. Thus, CD39 apyrase-expressing CD4(+) T cells stimulated by Salmonella-CFA/I are composed of TGF-?-producing Foxp3(-) CD39(+)CD4(+) T cells and support the stimulation of IL-10-producing Foxp3(+) CD39(+)CD4(+) T cells.
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Tolerogen-induced interferon-producing killer dendritic cells (IKDCs) protect against EAE.
J. Autoimmun.
PUBLISHED: 07-26-2011
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Natural killer (NK) cells and dendritic cells (DCs) have been shown to link the innate and adaptive immune systems. Likewise, a new innate cell subset, interferon-producing killer DCs (IKDCs), shares phenotypic and functional characteristics with both DCs and NK cells. Here, we show IKDCs play an essential role in the resolution of experimental autoimmune encephalomyelitis (EAE) upon treatment with the tolerizing agent, myelin oligodendrocyte glycoprotein (MOG), genetically fused to reovirus protein ?1 (termed MOG-p?1). Activated IKDCs were recruited subsequent MOG-p?1 treatment of EAE, and disease resolution was abated upon NK1.1 cell depletion. These IKDCs were able to kill activated CD4(+) T cells and mature dendritic DCs, thus, contributing to EAE remission. In addition, IKDCs were responsible for MOG-p?1-mediated MOG-specific regulatory T cell recruitment to the CNS. The IKDCs induced by MOG-p?1 expressed elevated levels of HVEM for interactions with cognate ligand-positive cells: LIGHT(+) NK and T(eff) cells and BTLA(+) B cells. Further characterization revealed these activated IKDCs being MHC class II(high), and upon their adoptive transfer (CD11c(+)NK1.1(+)MHC class II(high)), IKDCs, but not CD11c(+)NK1.1(+)MHC class II(intermediate/low) (unactivated) cells, conferred protection against EAE. These activated IKDCs showed enhanced CD107a, PD-L1, and granzyme B expression and could present OVA, unlike unactivated IKDCs. Thus, these results demonstrate the interventional potency induced HVEM(+) IKDCs to resolve autoimmune disease.
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Protective live oral brucellosis vaccines stimulate Th1 and th17 cell responses.
Infect. Immun.
PUBLISHED: 07-18-2011
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Zoonotic transmission of brucellosis often results from exposure to Brucella-infected livestock, feral animals, or wildlife or frequently via consumption of unpasteurized milk products or raw meat. Since natural infection of humans often occurs by the oral route, mucosal vaccination may offer a means to confer protection for both mucosal and systemic tissues. Significant efforts have focused on developing a live brucellosis vaccine, and deletion of the znuA gene involved in zinc transport has been found to attenuate Brucella abortus. A similar mutation has been adapted for Brucella melitensis and tested to determine whether oral administration of ?znuA B. melitensis can confer protection against nasal B. melitensis challenge. A single oral vaccination with ?znuA B. melitensis rapidly cleared from mice within 2 weeks and effectively protected mice upon nasal challenge with wild-type B. melitensis 16M. In 83% of the vaccinated mice, no detectable brucellae were found in their spleens, unlike with phosphate-buffered saline (PBS)-dosed mice, and vaccination also enhanced the clearance of brucellae from the lungs. Moreover, vaccinated gamma interferon-deficient (IFN-?(-/-)) mice also showed protection in both spleens and lungs, albeit protection that was not as effective as in immunocompetent mice. Although IFN-?, interleukin 17 (IL-17), and IL-22 were stimulated by these live vaccines, only RB51-mediated protection was codependent upon IL-17 in BALB/c mice. These data suggest that oral immunization with the live, attenuated ?znuA B. melitensis vaccine provides an attractive strategy to protect against inhalational infection with virulent B. melitensis.
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DNA vaccination of bison to brucellar antigens elicits elevated antibody and IFN-? responses.
J. Wildl. Dis.
PUBLISHED: 07-02-2011
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Brucella abortus remains a threat to the health and well-being of livestock in states bordering the Greater Yellowstone Area. During the past several years, cohabitation of infected wildlife with cattle has jeopardized the brucellosis-free status of Idaho, USA; Wyoming, USA; and Montana, USA. Current livestock B. abortus vaccines have not proven to be efficacious in bison (Bison bison) or elk (Cervus elaphus nelsoni). One problem with the lack of vaccine efficacy may stem from the failure to understand wildlife immune responses to vaccines. In an attempt to understand their immune responses, bison were vaccinated with eukaryotic DNA expression vectors encoding the Brucella periplasmic protein, bp26, and the chaperone protein, trigger factor (TF). These DNA vaccines have previously been shown to be protective against Brucella infection in mice. Bison were immunized intramuscularly at weeks 0, 2, and 4 with bp26 and TF DNA vaccines plus CpG adjuvant or empty vector (control) plus CpG. Blood samples were collected before vaccination and at 8, 10, and 12 wk after primary vaccination. The results showed that bison immunized with bp26 and TF DNA vaccines developed enhanced antibody, proliferative T cell, and interferon-gamma (IFN-?) responses upon in vitro restimulation with purified recombinant bp26 or TF antigens, unlike bison immunized with empty vector. Flow cytometric analysis revealed that the percentages of CD4(+) and CD8(+) T lymphocytes from the DNA-vaccinated groups were significantly greater than they were for those bison given empty vector. These data suggest that DNA vaccination of bison may elicit strong cellular immune responses and serve as an alternative for vaccination of bison for brucellosis.
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Influence of the electronic characteristics of N-donor ligands in the excited state of heteronuclear gold(I)-copper(I) systems.
Inorg Chem
PUBLISHED: 06-30-2011
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By reaction of the heterometallic gold-silver complexes [{AuAg(C(6)F(5))(2)(N?C-Me)}(2)](n) or [{AuAg(C(6)Cl(5))(2)(N?C-Me)}(2)](n) and CuCl in the presence of pyrimidine and different nitrile ligands (acetonitrile, benzonitrile, and cinnamonitrile), the heteronuclear complexes {[Au(C(6)X(5))(2)][Cu(L)(?(2)-C(4)H(4)N(2))]}(n) (X = F and L = N?C-Me (1), L = N?C-Ph (2) or N?C-CH?CH-Ph (3); X = Cl and L = N?C-Me (4), N?C-Ph (5), N?C-CH?CH-Ph (6)) have been prepared. The crystal structures of complexes {[Au(C(6)X(5))(2)][Cu(L)(?(2)-C(4)H(4)N(2))]}(n) (X = F; L = N?C-CH?CH-Ph (3), X = Cl; L = N?C-Ph (5)) have been determined by X-ray diffraction studies. The crystal structures of both complexes consists of polymeric chains formed by the repetition of [Au(C(6)X(5))(2)][Cu(L)(?(2)-C(4)H(4)N(2))] units through copper-pyrimidine bonds. Complexes 1, 2, 4, and 5 are brightly luminescent in the solid state at room temperature and at 77 K with lifetimes in the microseconds range. These compounds are also luminescent in solution, displaying different photophysical behaviors depending on the donor characteristics of the solvents used. The distortion in the excited state allows an associative attack by donor solvents quenching one of the emitting excited states. DFT optimizations of the ground (S(0)) and lowest triplet excited state (T(1)) display the structure distortion of the complexes upon electronic excitation. The molecular orbitals involved in the electronic transitions responsible for the phosphorescence in the case of the complexes 1, 2, 4, and 5 are related to metal (gold-copper) to ligand (pyrimidine) charge transfer transitions, while in the case of the nonluminescent complexes 3 and 6, the nonradiative electronic transition arises from metal (gold-copper) to ligand (cinnamonitrile) charge transfer transitions.
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Apple polyphenols require T cells to ameliorate dextran sulfate sodium-induced colitis and dampen proinflammatory cytokine expression.
J. Leukoc. Biol.
PUBLISHED: 06-21-2011
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Human IBD, including UC and Crohns disease, is characterized by a chronic, relapsing, and remitting condition that exhibits various features of immunological inflammation and affects at least one/1000 people in Western countries. Polyphenol extracts from a variety of plants have been shown to have immunomodulatory and anti-inflammatory effects. In this study, treatment with APP was investigated to ameliorate chemically induced colitis. Oral but not peritoneal administration of APP during colitis induction significantly protected C57BL/6 mice against disease, as evidenced by the lack of weight loss, colonic inflammation, and shortening of the colon. APP administration dampened the mRNA expression of IL-1?, TNF-?, IL-6, IL-17, IL-22, CXCL9, CXCL10, CXCL11, and IFN-? in the colons of mice with colitis. APP-mediated protection requires T cells, as protection was abated in Rag-1(-/-) or TCR?(-/-) mice but not in IL-10(-/-), IRF-1(-/-), ?MT, or TCR?(-/-) mice. Administration of APP during colitis to TCR?(-/-) mice actually enhanced proinflammatory cytokine expression, further demonstrating a requirement for TCR?? cells in APP-mediated protection. APP treatment also inhibited CXCR3 expression by TCR?? cells, but not B or NK cells, in the colons of mice with colitis; however, depletion of CD4(+) or CD8(+) T cells alone did not abolish APP-mediated protection. Collectively, these results show that oral administration of APP protects against experimental colitis and diminishes proinflammatory cytokine expression via T cells.
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Application of suppressive subtractive hybridization to the identification of genetic differences between two Lactococcus garvieae strains showing distinct differences in virulence for rainbow trout and mouse.
Microbiology (Reading, Engl.)
PUBLISHED: 05-05-2011
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Lactococcus garvieae is the causative microbial agent of lactococcosis, an important and damaging fish disease in aquaculture. This bacterium has also been isolated from vegetables, milk, cheese, meat and sausages, from cow and buffalo as a mastitis agent, and even from humans, as an opportunistic infectious agent. In this work pathogenicity experiments were performed in rainbow trout and mouse models with strains isolated from human (L. garvieae HF) and rainbow trout (L. garvieae UNIUDO74; henceforth referred to as 074). The mean LD(50) value in rainbow trout obtained for strain 074 was 2.1 × 10(2) ± 84 per fish. High doses of the bacteria caused specific signs of disease as well as histological alterations in mice. In contrast, strain HF did not prove to be pathogenic either for rainbow trout or for mice. Based on these virulence differences, two suppressive subtractive hybridizations were carried out to identify unique genetic sequences present in L. garvieae HF (SSHI) and L. garvieae 074 (SSHII). Differential dot-blot screening of the subtracted libraries allowed the identification of 26 and 13 putative ORFs specific for L. garvieae HF and L. garvieae 074, respectively. Additionally, a PCR-based screening of 12 of the 26 HF-specific putative ORFs and the 13 074-specific ones was conducted to identify their presence/absence in 25 L. garvieae strains isolated from different origins and geographical areas. This study demonstrates the existence of genetic heterogeneity within L. garvieae isolates and provides a more complete picture of the genetic background of this bacterium.
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Amalgamating at the molecular level. A study of the strong closed-shell Au(I)···Hg(II) interaction.
Chem. Commun. (Camb.)
PUBLISHED: 04-04-2011
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Complex {[Hg(C(6)F(5))(2)][Au(C(6)F(5))(PMe(3))](2)}(n)2 displays unsupported Au(I)···Hg(II) and Au(I)···Au(I) interactions. Its crystal structure displays a polymeric -(Au-Hg-Au-Au-Hg-Au)(n)- disposition. Ab initio calculations show very strong Au(I)···Hg(II) and Au(I)···Au(I) closed-shell interactions of -73.3 kJ mol(-1) and -57.0 kJ mol(-1), respectively, which have a dispersive (van der Waals) nature and are strengthened by large relativistic effects (>20%).
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Serum antibodies protect against intraperitoneal challenge with enterotoxigenic Escherichia coli.
J. Biomed. Biotechnol.
PUBLISHED: 03-15-2011
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To assess whether anticolonization factor antigen I (CFA/I) fimbriae antibodies (Abs) from enterotoxigenic Escherichia coli (ETEC) can protect against various routes of challenge, BALB/c mice were immunized with a live attenuated Salmonella vaccine vector expressing CFA/I fimbriae. Vaccinated mice elicited elevated systemic IgG and mucosal IgA Abs, unlike mice immunized with the empty Salmonella vector. Mice were challenged with wild-type ETEC by the oral, intranasal (i.n.), and intraperitoneal (i.p.) routes. Naïve mice did not succumb to oral challenge, but did to i.n. challenge, as did immunized mice; however, vaccinated mice were protected against i.p. ETEC challenge. Two intramuscular (i.m.) immunizations with CFA/I fimbriae without adjuvant conferred 100% protection against i.p. ETEC challenge, while a single 30??g dose conferred 88% protection. Bactericidal assays showed that ETEC is highly sensitive to anti-CFA/I sera. These results suggest that parenteral immunization with purified CFA/I fimbriae can induce protective Abs and may represent an alternative method to elicit protective Abs for passive immunity to ETEC.
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Murine and bovine ?? T cells enhance innate immunity against Brucella abortus infections.
PLoS ONE
PUBLISHED: 02-12-2011
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?? T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human ?? T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of ?? T cells in vivo during experimental brucellosis has not been studied. Here we report TCR?(-/-) mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCR?? cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. ?? T cells were the major source of IL-17 following infection and also produced IFN-?. Depletion of ?? T cells from C57BL/6, IL-17R?(-/-), and GMCSF(-/-) mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when ?? T cells were depleted from IFN-?(-/-) mice, enhanced susceptibility was observed. Neutralization of ?? T cells in the absence of TNF-? did not further impair immunity. In the absence of TNF-? or ?? T cells, B. abortus-infected mice showed enhanced IFN-?, suggesting that they augmented production to compensate for the loss of ?? T cells and/or TNF-?. While the protective role of ?? T cells was TNF-?-dependent, ?? T cells were not the major source of TNF-? and activation of ?? T cells following B. abortus infection was TNF-?-independent. Additionally, bovine TCR?? cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-?. Collectively, these results demonstrate ?? T cells are important for early protection to B. abortus infections.
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Comparative analysis and mutation effects of fpp2-fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum.
Microbiology (Reading, Engl.)
PUBLISHED: 02-03-2011
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Flavobacterium psychrophilum is a very significant fish pathogen that secretes two biochemically characterized extracellular proteolytic enzymes, Fpp1 and Fpp2. The genes encoding these enzymes are organized as an fpp2-fpp1 tandem in the genome of strain F. psychrophilum THC02/90. Analysis of the corresponding encoded proteins showed that they belong to two different protease families. For gene function analysis, new genetic tools were developed in F. psychrophilum by constructing stable isogenic fpp1 and fpp2 mutants via single-crossover homologous recombination. RT-PCR analysis of wild-type and mutant strains suggested that both genes are transcribed as a single mRNA from the promoter located upstream of the fpp2 gene. Phenotypic characterization of the fpp2 mutant showed lack of caseinolytic activity and higher colony spreading compared with the wild-type strain. Both characteristics were recovered in the complemented strain. One objective of this work was to assess the contribution to virulence of these proteolytic enzymes. LD(50) experiments using the wild-type strain and mutants showed no significant differences in virulence in a rainbow trout challenge model, suggesting instead a possible nutritional role. The gene disruption procedure developed in this work, together with the knowledge of the complete genome sequence of F. psychrophilum, open new perspectives for the study of gene function in this bacterium.
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Mucosal targeting of a BoNT/A subunit vaccine adjuvanted with a mast cell activator enhances induction of BoNT/A neutralizing antibodies in rabbits.
PLoS ONE
PUBLISHED: 01-27-2011
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We previously reported that the immunogenicity of Hc?tre, a botulinum neurotoxin A (BoNT/A) immunogen, was enhanced by fusion to an epithelial cell binding domain, Ad2F, when nasally delivered to mice with cholera toxin (CT). This study was performed to determine if Ad2F would enhance the nasal immunogenicity of Hc?tre in rabbits, an animal model with a nasal cavity anatomy similar to humans. Since CT is not safe for human use, we also tested the adjuvant activity of compound 48/80 (C48/80), a mast cell activating compound previously determined to safely exhibit nasal adjuvant activity in mice.
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Design, development and application of a bioelectrochemical detection system for meat tenderness prediction.
Biosens Bioelectron
PUBLISHED: 01-25-2011
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The analytical method described, based on antibody-antigen bio-recognition and the measuring system for amperometric detection, was designed for accurate, easy to use and cost effective quantification of calpastatin, a meat tenderness biomarker. The novel assay for calpastatin quantification was integrated in a portable electrochemical device known as the Tendercheck system and was used to analyze meat samples collected from animals of different breeds and ages. The data obtained were correlated (R² = 0.62) with Warner Bratzler Shear Force (WBSF) measurements, a routinely used method for meat tenderness determination.
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A novel cdsAB operon is involved in the uptake of L-cysteine and participates in the pathogenesis of Yersinia ruckeri.
J. Bacteriol.
PUBLISHED: 12-17-2010
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Application of in vivo expression technology (IVET) to Yersinia ruckeri, an important fish pathogen, allowed the identification of two adjacent genes that represent a novel bacterial system involved in the uptake and degradation of l-cysteine. Analysis of the translational products of both genes showed permease domains (open reading frame 1 [ORF1]) and amino acid position identities (ORF2) with the l-cysteine desulfidase from Methanocaldococcus jannaschii, a new type of enzyme involved in the breakdown of l-cysteine. The operon was named cdsAB (cysteine desulfidase) and is found widely in anaerobic and facultative bacteria. cdsAB promoter analysis using lacZY gene fusion showed highest induction in the presence of l-cysteine. Two cdsA and cdsB mutant strains were generated. The limited toxic effect and the low utilization of l-cysteine observed in the cdsA mutant, together with radiolabeled experiments, strongly suggested that CdsA is an l-cysteine permease. Fifty percent lethal dose (LD(50)) and competence index experiments showed that both the cdsA and cdsB loci were involved in the pathogenesis of the bacteria. In conclusion, this study has shown for the first time in bacteria the existence of an l-cysteine uptake system that together with an additional l-cysteine desulfidase-encoding gene constitutes a novel operon involved in bacterial virulence.
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The yctCBA operon of Yersinia ruckeri, involved in in vivo citrate uptake, is not required for virulence.
Appl. Environ. Microbiol.
PUBLISHED: 12-03-2010
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A three-gene operon, named yctCBA (Yersinia citrate transporter), induced by citrate and repressed by glucose was identified from a previously selected in vivo-induced (ivi) clone in the fish pathogen Yersinia ruckeri. Interestingly, despite being an ivi clone, the drastic growth reduction of the yctC mutant in the presence of citrate, and the relatively high content of this compound in rainbow trout serum, the operon was not required for virulence.
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[Development and validation of a questionnaire about the main variables affecting the individual investors behavior in the Stock Exchange].
Psicothema
PUBLISHED: 11-04-2010
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Development and validation of a questionnaire about the main variables affecting the individual investors behavior in the Stock Exchange. There is a considerable lack of information about the methodology usually used in most of the studies about individual investors behavior. The studies reviewed do not show the method used in the selection of the items or the psychometric properties of the questionnaires. Because of the importance of investment in the Stock Exchange nowadays, it seems relevant to obtain a reliable instrument to understand individual investors behavior in the Stock Exchange. Therefore, the goal of the present work is to validate a questionnaire about the main variables involved in individual investors behavior in the Stock Exchange. Based on previous studies, we elaborated a questionnaire using the Delphi methodology with a group of experts. The internal consistency (Cronbach alpha=.934) and validity evidence of the questionnaire show that it may be an effective instrument and can be applied with some assurance.
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Active immunization using a single dose immunotherapeutic abates established EAE via IL-10 and regulatory T cells.
Eur. J. Immunol.
PUBLISHED: 09-27-2010
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Stimulation of Ag-specific inducible Treg can enhance resolution of autoimmune disease. Conventional methods to induce Treg often require induction of autoimmune disease or subjection to infection. Reovirus adhesin, protein ?1 (p?1), can successfully facilitate tolerance when fused to a tolerogen. We tested whether myelin oligodendrocyte glycoprotein (MOG) fused to p?1 (MOG-p?1) can stimulate Ag-specific Treg. We show that C57BL/6 mice treated nasally with MOG-p?1 fail to induce MOG-specific Abs and delayed-type hypersensitivity (DTH) responses and resist EAE. Such resistance was attributed to stimulation of Foxp3(+) Treg, as well as Th2 cells. MOG-p?1s protective capacity was abrogated in IL-10(-/-) mice, but restored when adoptively transferred with MOG-p?1-induced Treg. As a therapeutic, MOG-p?1 diminished EAE within 24?h of nasal application, unlike recombinant MOG (rMOG), p?1, or p?1+rMOG, implicating the importance of Ag specificity by p?1-based therapeutics. MOG-p?1-treated mice showed elevated IL-4, IL-10, and IL-28 production by CD4(+) T cells, unlike rMOG treated or control mice that produced elevated IFN-? or IL-17, respectively. These data show the feasibility of using p?1 as a tolerogen platform for Ag-specific tolerance induction and highlight its potential use as an immunotherapeutic for autoimmunity.
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Risk factors associated with moderate-to-severe renal dysfunction among heart transplant patients: results from the CAPRI study.
Clin Transplant
PUBLISHED: 07-06-2010
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The longer survival of patients with heart transplantation (HT) favors calcineurin inhibitor-related chronic kidney disease (CKD). It behoves to identify risk factors. At 14 Spanish centers, data on 1062 adult patients with HT (age 59.2 ± 12.3 yr, 82.5% men) were collected at routine follow-up examinations. Glomerular filtration rate, GFR, was estimated using the four-variable MDRD equation, and moderate-or-severe renal dysfunction (MSRD) was defined as K/DOQI stage 3 CKD or worse. Time since transplant ranged from one month to 22 yr (mean 6.7 yr). At assessment, 26.6% of patients were diabetic and 63.9% hypertensive; 53.9% were taking cyclosporine and 33.1% tacrolimus; and 61.4% had MSRD. Among patients on cyclosporine or tacrolimus at assessment, multivariate logistic regression identified male sex (OR 0.44), pre- and post-HT creatinine (2.73 and 3.13 per mg/dL), age at transplant (1.06 per yr), time since transplant (1.05 per yr), and tacrolimus (0.65) as independent positive or negative predictors of MSRD. It is concluded that female sex, pre- and one-month post-HT serum creatinine, age at transplant, time since transplant, and immunosuppression with cyclosporine rather than tacrolimus may all be risk factors for development of CKD ? stage 3 by patients with HT.
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IL-35 stimulation of CD39+ regulatory T cells confers protection against collagen II-induced arthritis via the production of IL-10.
J. Immunol.
PUBLISHED: 05-10-2010
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IL-35 is produced by regulatory T cells, and this novel cytokine can downregulate Th17 cell development and inhibit autoimmune inflammation. In this work, an rIL-35, as a single-chain fusion between murine IL-12p35 and EBV-induced gene 3, was expressed in yeast. This rIL-35 inhibited OVA-specific cellular and Ab responses in OVA-challenged recipients of DO11.10 CD4+ T cells. Likewise, IL-35 inhibited clinical manifestation of collagen-induced arthritis or could cease further disease exacerbation upon initiation of IL-35 treatment. Exogenous IL-35 treatments suppressed Th1 and Th17 cells and promoted CD39 expression by CD4+ T cells. Sorted CD25-CD39+CD4+ T cells from IL-35-treated mice produced IL-10 and, upon adoptive transfer, were sufficiently potent to inhibit subsequent development of inflammation in mice with collagen-induced arthritis, whereas sorted CD25+CD39+CD4+ T cells showed reduced potency. IL-35 treatments of IL-10-/- mice failed to induce protective CD39+CD4+ T cells, demonstrating the effector role of IL-10 by IL-35 immunosuppression.
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Antinociceptive effect of three common analgesic drugs on peripheral neuropathy induced by paclitaxel in rats.
Pharmacol. Biochem. Behav.
PUBLISHED: 02-15-2010
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Nowadays, there are no validated drugs to control the neuropathic pain induced by paclitaxel, one of the most effective antineoplastic drugs. The aim was to study the involvement of opioid and NMDA receptor on established paclitaxel-induced pain, testing three common analgesics drugs morphine, ketamine and methadone. Animals received four intraperitoneal (i.p.) injections on alternate days of paclitaxel (1mg/kg). Three weeks later, animals showed a mechanical and heat allodynia/hyperalgesia. Morphine (1, 2.5, 5 and 10mg/kg) abolished the reduction in the mechanical and thermal withdrawal thresholds in a dose dependent manner. This effect was blocked by naloxone. Only highest dose of ketamine (50mg/kg) was able to increase the mechanical and thermal threshold and returned to basal values. Subanalgesic doses of morphine (1mg/kg) and ketamine (12.5mg/kg) produced an additive effect on heat hyperalgesia reaching an antinociceptive effect. This combination did not induce any change on tactile allodynia. Methadone (2.5 and 5mg/kg) produced an analgesic effect that was completely antagonized by naloxone in both tests. Our results confirm that: the activation of opioids receptor produced analgesia; the blockade of NMDA receptors produce antinociception but at high doses with motor impairments and low doses of ketamine enhancing the effect of opioids.
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A parenteral DNA vaccine protects against pneumonic plague.
Vaccine
PUBLISHED: 02-10-2010
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The chemokine, lymphotactin (LTN), was tested as a molecular adjuvant using bicistronic DNA vaccines encoding the protective Yersinia capsular (F1) antigen and virulence antigen (V-Ag) as a F1-V fusion protein. The LTN-encoding F1-V or V-Ag vaccines were given by the intranasal (i.n.) or intramuscular (i.m.) routes, and although serum IgG and mucosal IgA antibodies (Abs) were induced, F1-Ag boosts were required for robust anti-F1-Ag Abs. Optimal efficacy against pneumonic plague was obtained in mice i.m.-, not i.n.-immunized with these DNA vaccines. These vaccines stimulated elevated Ag-specific Ab-forming cells and mixed Th cell responses, with Th17 cells markedly enhanced by i.m. immunization. These results show that LTN can be used as a molecular adjuvant to enhance protective immunity against plague.
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IL-28 supplants requirement for T(reg) cells in protein sigma1-mediated protection against murine experimental autoimmune encephalomyelitis (EAE).
PLoS ONE
PUBLISHED: 01-14-2010
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Conventional methods to induce tolerance in humans have met with limited success. Hence, efforts to redirect tolerogen uptake using reovirus adhesin, protein sigma 1 (psigma1), may circumvent these shortcomings based upon the recent finding that when reovirus psigma1 is engineered to deliver chicken ovalbumin (OVA) mucosally, tolerance is obtained, even with a single dose. To test whether single-dose tolerance can be induced to treat EAE, proteolipid protein (PLP(130-151)) was genetically fused to OVA to psigma1 (PLP:OVA-psigma1) and shown to significantly ameliorate EAE, suppressing proinflammatory cytokines by IL-10(+) forkhead box P3 (FoxP3)(+) CD25(+)CD4(+) T(reg) and IL-4(+)CD25(-)CD4(+) Th2 cells. IL-10R or IL-4 neutralization reversed protection to EAE conferred by PLP:OVA-psigma1, and adoptive transfer of Ag-specific T(reg) or Th2 cells restored protection against EAE in recipients. Upon assessment of each relative participant, functional inactivation of CD25 impaired PLP:OVA-psigma1s protective capacity, triggering TGF-beta-mediated inflammation; however, concomitant inactivation of TGF-beta and CD25 reestablished PLP:OVA-psigma1-mediated protection by IL-28-producing FoxP3(+)CD25(-)CD4(+) T cells. Thus, psigma1-based therapy can resolve EAE independently of or dependently upon CD25 and assigns IL-28 as an alternative therapy for autoimmunity.
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Adenovirus F protein as a delivery vehicle for botulinum B.
BMC Immunol.
PUBLISHED: 01-06-2010
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Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination.
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DeltaznuADeltapurE Brucella abortus 2308 mutant as a live vaccine candidate.
Vaccine
PUBLISHED: 08-24-2009
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To create a new, safe brucellosis live vaccine, a double mutant strain was constructed from Brucella abortus 2308. Using the DeltaznuA B. abortus 2308 mutant, a second mutation was introduced by deleting purE gene. The DeltaznuA DeltapurE B. abortus 2308 strain was less capable of surviving in macrophages. When evaluated in vivo, it was cleared within 8 weeks (wks) from mice, causing significantly less inflammation than spleens obtained from wild-type B. abortus 2308-infected mice. Furthermore, two doses of DeltaznuA DeltapurE B. abortus 2308 conferred 0.79 log protection, similar to S19 as did a single dose of DeltaznuA B. abortus 2308. Thus, this study shows the DeltaznuA DeltapurE B. abortus 2308 strain to be a potential livestock vaccine candidate.
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Antibody selection for immobilizing living bacteria.
Anal. Chem.
PUBLISHED: 08-18-2009
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We report a comparative study of the efficacy of immobilizing living bacteria by means of seven antibodies against bacterial surface antigens associated with Salmonella enterica Serovar Typhimurium. The targeted bacterial antigens were CFA/I fimbriae, flagella, lipopolysaccharides (LPS), and capsular F1 antigen. The best immobilization of S. Typhimurium was achieved with the antibody against CFA/I fimbriae. The immobilization of bacteria using antiflagellin showed significant enhancement if the flagella rotary motion was paralyzed. Of the four antibodies targeting LPS structures, only one, the antibody against the O-antigen polysaccharides, showed a relatively efficient bacterial immobilization. No bacterial immobilization was achieved using the antibody against F1 antigen, presumably because F1 protein can detach from the bacterial surface easily. The results suggest that an antibody for bacterial immunoimmobilization should target a surface antigen which extends out from the bacterial surface and is tightly attached to the bacterial cell wall. The microarrays of living S. Typhimurium cells immobilized in this manner remained viable and effective for at least 2 weeks in growth medium before a thick biofilm covered the whole surface.
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Spreading versus biomass production by colonies of the fish pathogen Flavobacterium psychrophilum: role of the nutrient concentration.
Int. Microbiol.
PUBLISHED: 03-24-2009
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Colonies of the fish pathogen Flavobacterium psychrophilum have gliding motility in media with low agar concentrations. Although gliding motility, particularly in Flavobacterium johnsoniae, has been well-studied, little is known about its regulation by environmental factors. The work described here shows that the ability of F. psychrophilum to spread over surfaces depends on nutrient availability. In fact, as the nutrient contents of the medium decreased, spreading was favored and the diameter of the colonies increased. Macroscopy examination revealed modifications in colony morphology as nutrient depletion increased: from a dense and defined colony to the formation of microcolonies inside a general colony structure. Additionally, colony expansion dynamics and population density across the colony radius varied inversely with bacterial biomass production. Motility was an immediate response when bacteria were transferred from a rich to a more diluted medium. Our results suggest that, when nutrients are limiting, F. psychrophilum activates a specific growth mode that enables it to colonize surfaces by means of gliding motility. The use of diluted media allowed the differentiation, among previously isolated F. psychrophilum non-gliding mutants, of those completely unable to glide and those with only partially impaired gliding ability.
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An IL-12 DNA vaccine co-expressing Yersinia pestis antigens protects against pneumonic plague.
Vaccine
PUBLISHED: 03-12-2009
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Pneumonic plague remains problematic in endemic areas, and because it can be readily transmitted and has high mortality, the development of efficacious vaccines is warranted. To test whether stimulation of cell-mediated immunity with IL-12 will improve protective immunity against plague, we constructed two IL-12 DNA vaccines using a bicistronic plasmid encoding the protective plague epitopes, capsular (F1) antigen and virulence antigen (V-Ag) as F1-V fusion protein and V-Ag only, respectively. When applied intramuscularly, antibody responses to F1- and V-Ag were detectable beginning at week 6 after 3 weekly doses, and F1-Ag protein boosts were required to induce elevated Ab responses. These Ab responses were supported by mixed Th cell responses, and the IL-12/V-Ag DNA vaccine showed greater cell-mediated immune bias than IL-12/F1-V DNA vaccine. Following pneumonic challenge, both IL-12 DNA vaccines showed similar efficacy despite differences in Th cells simulated. These results show that IL-12 can be used as a molecular adjuvant to enhance protective immunity against pneumonic plague.
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Bacteria survive multiple puncturings of their cell walls.
Langmuir
PUBLISHED: 03-06-2009
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A bacterial cell wall is a highly dynamic multilayer structure interfacing the cytoplasm to the outside environment. It supports a multitude of chemical and biological processes necessary for life. It is therefore postulated that damage to the structure of bacterial cell wall would threaten cell integrity and result in cell death. We tested this hypothesis by repeatedly puncturing the cell wall of a live Gram negative bacterium Salmonella typhimurium at different locations using a sharp atomic force microscope nanotip and conducting multiple viability tests. Our study demonstrated that a S. typhimurium survives repeated puncturings of its cell wall and retains its integrity, viability, and ability to divide. The results are explained on the basis of the concept of the self-repairing of lipid bilayers and the peptidoglycan layer.
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Antinociceptive effect of the cannabinoid agonist, WIN 55,212-2, in the orofacial and temporomandibular formalin tests.
Eur J Pain
PUBLISHED: 02-03-2009
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Orofacial pain disorders are frequent in the general population and their pharmacological treatment is not always adequately resolved. Cannabinoids have demonstrated their analgesic effect in several pain conditions, both in animal models and in clinical situations. The aim of the present study was to evaluate the cannabinoid-mediated antinociception in two inflammatory models of orofacial pain (orofacial and temporomandibular joint (TMJ) formalin test) and to compare it with a spinal inflammatory model (paw formalin test). WIN 55,212-2 (0.5, 1mg/kg), a synthetic cannabinoid agonist, was intraperitoneally (i.p.) administered prior to formalin and significantly reduced the nociceptive behavioural responses in these inflammatory tests. To elucidate which subtype of receptor could be involved in such effect, two selective cannabinoid antagonists were administered prior to WIN. SR141716A (1mg/kg i.p.), the CB1 receptor-selective antagonist, was able to prevent the cannabinoid-induced analgesia in all three models, whereas SR144528 (1mg/kg i.p.), the CB2 receptor-selective antagonist, only prevented it in the paw formalin test. A comparison with the antinociceptive effects of morphine (2.5, 5, 10mg/kg, i.p.), indomethacin (2.5, 5mg/kg, i.p.) and ketamine (25, 50mg/kg, i.p.) was also performed. Morphine displayed a dose-dependent reduction of acute and inflammatory pain in all three models, whereas indomethacin and ketamine only attenuated inflammatory pain at the highest tested doses. These results indicate that the cannabinoid-induced antinociception in the orofacial region is mediated by activation of CB1 cannabinoid receptor. Moreover WIN was as effective as morphine and more effective than indomethacin and ketamine, in oral inflammatory pain.
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18?-glycyrrhetinic acid delivered orally induces isolated lymphoid follicle maturation at the intestinal mucosa and attenuates rotavirus shedding.
PLoS ONE
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Glycyrrhizin, an abundant bioactive component of the medicinal licorice root is rapidly metabolized by gut commensal bacteria into 18?-glycyrrhetinic acid (GRA). Either or both of these compounds have been shown to have antiviral, anti-hepatotoxic, anti-ulcerative, anti-tumor, anti-allergenic and anti-inflammatory activity in vitro or in vivo. In this study, the ability of GRA to modulate immune responses at the small intestinal mucosa when delivered orally was investigated. Analysis of cytokine transcription in duodenal and ileal tissue in response to GRA treatment revealed a pattern of chemokine and chemokine receptor gene expression predictive of B cell recruitment to the gut. Consistent with this finding, GRA induced increases in CD19(+) B cells in the lamina propria and B220(+) B cell aggregates framed by CD11c(+) dendritic cells in structures resembling isolated lymphoid follicles (ILF). Using a mouse model of rotavirus infection, GRA reduced the duration of viral antigen shedding, and endpoint serum antibody titers were higher in GRA-treated animals. Together the data suggest GRA delivered orally augments lymphocyte recruitment to the intestinal mucosa and induces maturation of B cell-rich ILF independently of ectopic antigenic stimulus. These results provide further support a role for dietary ligands in modulation of dynamic intestinal lymphoid tissue.
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Flagella overexpression attenuates Salmonella pathogenesis.
PLoS ONE
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Flagella are cell surface appendages involved in a number of bacterial behaviors, such as motility, biofilm formation, and chemotaxis. Despite these important functions, flagella can pose a liability to a bacterium when serving as potent immunogens resulting in the stimulation of the innate and adaptive immune systems. Previous work showing appendage overexpression, referred to as attenuating gene expression (AGE), was found to enfeeble wild-type Salmonella. Thus, this approach was adapted to discern whether flagella overexpression could induce similar attenuation. To test its feasibility, flagellar filament subunit FliC and flagellar regulon master regulator FlhDC were overexpressed in Salmonella enterica serovar Typhimurium wild-type strain H71. The results show that the expression of either FliC or FlhDC alone, and co-expression of the two, significantly attenuates Salmonella. The flagellated bacilli were unable to replicate within macrophages and thus were not lethal to mice. In-depth investigation suggests that flagellum-mediated AGE was due to the disruptive effects of flagella on the bacterial membrane, resulting in heightened susceptibilities to hydrogen peroxide and bile. Furthermore, flagellum-attenuated Salmonella elicited elevated immune responses to Salmonella presumably via FliCs adjuvant effect and conferred robust protection against wild-type Salmonella challenge.
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Research participation improves students exam performance.
Span J Psychol
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Although there have been several attempts to explore for beneficial effects of research participation in social sciences, most of them have mainly explored satisfaction and students learning perceptions (e.g., Bowman & Waite, 2003). Very few works have studied learning by measuring exam performance. Moreover, participation has been usually conceptualized as a mixture of active and passive participation, including in the same measure different practices such as filling up questionnaires, running experiments or reading and answering questions about a journal article or a scientific conference. The present work tries to determine if there is an advantage due to research participation comparing exam performance, satisfaction and perceived learning of the matter Research Methods in Psychology, in three different groups (non-participating, passive and active participating). As we can see in the results, the mere participation benefits exam performance. Results are discussed in terms of the use of research participation as a new powerful active method in education.
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IFN-?-deficient mice develop IL-1-dependent cutaneous and musculoskeletal inflammation during experimental brucellosis.
J. Leukoc. Biol.
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Human brucellosis exhibits diverse pathological manifestations that can affect almost any organ. In particular, osteoarticular complications are the most common focal manifestation of brucellosis and occur in 40-80% of patients. In immunocompetent mice, Brucella replication is generally restricted to the spleen, liver, and to a lesser extent, LNs, thereby limiting their use for study of focal inflammation often found in brucellosis. Here, we report that nasal, oral, or peritoneal infection of IFN-?(-/-) mice with WT Brucella melitensis or Brucella abortus results in joint and periarticular tissue inflammation. Histological analysis of the affected joints revealed inflammatory infiltrates and debris within the joint space colocalizing with Brucella antigen. Osteoarthritis, necrosis, periarticular soft tissue inflammation, and substantial brucellae burdens were observed. Oral rifampicin was effective in clearing infection and halting further progression of focal inflammation from infected IFN-?(-/-) mice, although some symptoms and swelling remained. Elevated IL-1 ?, but not TNF-?, IL-6, or IL-17, was detected in joint homogenates from infected IFN-?(-/-) mice. Whereas more susceptible to systemic infection, IL-1R(-/-) mice depleted of IFN-? were more resistant to focal inflammation than WT mice similarly depleted of IFN-?. Collectively, these results show IFN-?(-/-) mice represent a potential model for study of focal inflammation attributed to Brucella infection and will allow evaluation of intervention strategies targeting IL-1, IL-1R, or other inflammatory mediators, with the potential to complement antibiotic-based therapies.
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Loss of sialic acid binding domain redirects protein ?1 to enhance M cell-directed vaccination.
PLoS ONE
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Ovalbumin (OVA) genetically fused to protein sigma 1 (p?1) results in tolerance to both OVA and p?1. P?1 binds in a multi-step fashion, involving both protein- and carbohydrate-based receptors. To assess the relative p?1 components responsible for inducing tolerance and the importance of its sialic binding domain (SABD) for immunization, modified OVA-p?1, termed OVA-p?1(short), was deleted of its SABD, but with its M cell targeting moiety intact, and was found to be immunostimulatory and enhanced CD4(+) and CD8(+) T cell proliferation. When used to nasally immunize mice given with and without cholera toxin (CT) adjuvant, elevated SIgA and serum IgG responses were induced, and OVA-p?1(s) was more efficient for immunization than native OVA+CT. The immune antibodies (Abs) were derived from elevated Ab-forming cells in the upper respiratory tissues and submaxillary glands and were supported by mixed Th cell responses. Thus, these studies show that p?1(s) can be fused to vaccines to effectively elicit improved SIgA responses.
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Nasal Acai polysaccharides potentiate innate immunity to protect against pulmonary Francisella tularensis and Burkholderia pseudomallei Infections.
PLoS Pathog.
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Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-?. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-? by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-? ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-? responses by NK and ?? T cells in the lungs, while neutralization of IFN-? totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens.
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Identification and characterization of a novel class of c-Jun N-terminal kinase inhibitors.
Mol. Pharmacol.
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In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-?B/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1?, IL-1?, IL-6, IL-10, tumor necrosis factor (TNF)-?, interferon-?, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-? and IL-6 production in MonoMac-6 cells with IC(50) values of 0.25 and 0.61 ?M, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4(+) T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs.
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Bystander-mediated stimulation of proteolipid protein-specific regulatory T (Treg) cells confers protection against experimental autoimmune encephalomyelitis (EAE) via TGF-?.
J. Neuroimmunol.
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To assess the potency of regulatory T (Treg) cells induced against an irrelevant Ag, mice were orally vaccinated with Salmonella expressing Escherichia coli colonization factor antigen I fimbriae. Isolated CD25? and CD25?CD4? T cells were adoptively transferred to naive mice, and Treg cells effectively protected against experimental autoimmune encephalomyelitis (EAE), unlike Treg cells from Salmonella vector-immunized mice. This protection was abrogated upon in vivo neutralization of TGF-?, resulting in elevated IL-17 and loss of IL-4 and IL-10 production. Thus, Treg cells induced to irrelevant Ags offer a novel approach to treat autoimmune diseases independent of auto-Ag.
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Cannabinoid agonist WIN 55,212-2 prevents the development of paclitaxel-induced peripheral neuropathy in rats. Possible involvement of spinal glial cells.
Eur. J. Pharmacol.
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Spinal glial activation contributes to the development and maintenance of chronic pain states, including neuropathic pain of diverse etiologies. Cannabinoid compounds have shown antinociceptive properties in a variety of neuropathic pain models and are emerging as a promising class of drugs to treat neuropathic pain. Thus, the effects of repeated treatment with WIN 55,212-2, a synthetic cannabinoid agonist, were examined throughout the development of paclitaxel-induced peripheral neuropathy. Painful neuropathy was induced in male Wistar rats by intraperitoneal (i.p.) administration of paclitaxel (1mg/kg) on four alternate days. Paclitaxel-treated animals received WIN 55,212-2 (1mg/kg, i.p.) or minocycline (15 mg/kg, i.p.), a microglial inhibitor, daily for 14 days, simultaneous with the antineoplastic. The development of hypersensitive behaviors was assessed on days 1, 7, 14, 21 and 28 following the initial administration of drugs. Both the activation of glial cells (microglia and astrocytes) at day 29 and the time course of proinflammatory cytokine release within the spinal cord were also determined. Similar to minocycline, repeated administration of WIN 55,212-2 prevented the development of thermal hyperalgesia and mechanical allodynia in paclitaxel-treated rats. WIN 55,212-2 treatment also prevented spinal microglial and astrocytic activation evoked by paclitaxel at day 29 and attenuated the early production of spinal proinflammatory cytokines (interleukin (IL)-1?, IL-6 and tumor necrosis factor (TNF)-?). Our results confirm changes in the reactivity of glial cells during the development of peripheral neuropathy induced by paclitaxel and support a preventive effect of WIN 55,212-2, probably via glial cells reactivity inactivation, on the development of this neuropathy.
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Construction and validation of a GFP-based vector for promoter expression analysis in the fish pathogen Flavobacterium psychrophilum.
Gene
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The study of the fish pathogen Flavobacterium psychrophilum has been drastically hampered by the difficulty to perform genetic manipulation of this organism. Although recent publications described the successful transfer of genetic material into this bacterium by transformation and conjugation, additional tools are still needed. This paper reports the construction of vector pCP23-G, which permits for the first time to monitor transcriptional regulation in this pathogen by using a promoterless gfpmut3 gene as a reporter. Additionally, use of pCP23-G enabled the trancriptional analysis of three putative promoter regions of F. psychrophilum, corresponding to genes fpp2-fpp1, pdhB and gldJ, under different growth conditions. Overall, the construction of pCP23-G facilitates genetic analysis in F. psychrophilum, by enabling the determination of gene expression both in vitro and in vivo. Furthermore, this would also open the possibility for studies on the location of this bacterium in the fish tissues.
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Expression of Escherichia coli virulence usher protein attenuates wild-type Salmonella.
Virulence
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Generation of a live attenuated vaccine for bacterial pathogens often requires prior knowledge of the pathogens virulence factors. We hypothesized an alternative approach of heterologous gene expression would make a wild-type (wt) pathogen more susceptible to host cell killing, thus, resulting in immunization. As proof of concept, the heterologous expression of enterotoxigenic E. coli (ETEC) colonization factor antigen I (CFA/I) was tested to attenuate Salmonella. The overexpression of CFA/I resulted in significant attenuation of wt Salmonella. In-depth studies revealed the attenuation depended on the co-expression of chaperone (CfaA) and usher (CfaC) proteins. Remarkably, the CfaAC-attenuated Salmonella conferred protection against wt Salmonella challenge. Mechanistic study indicated CfaAC made Salmonella outer membranes permeable, causing Salmonella to be vulnerable to host destruction. Thus, enhancing bacterial permeability via CfaAC represents an alternative method to attenuate pathogens despite the presence of unknown virulence factors.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.