Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157:H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice

Human infections with EHEC such as O157:H7 have been a great concern for worldwide food-industry surveillance. This pathogen is commonly associated with bloody diarrhea that can evolve to the life-threatening hemolytic uremic syndrome. Animals are the natural reservoir where this pathogen remains asymptomatically, in steps of ingestion and colonization of the bowel. The bacterium is shed in the feces, contaminating the surroundings, including water and food that are directed for human consumption. A major player in this colonization process is intimin, an outer membrane adhesion molecule encoded by the E. coli attachment and effacement (eae) gene that has been shown to be essential for intimate bacterial attachment to eukaryotic host cells. In an attempt to reduce the colonization of animal reservoirs with EHEC O157:H7, we designed a vaccine model to induce an immune response against intimin gamma. The model is based on its recombinant expression in attenuated Salmonella, used as a suitable vaccine vector because of its recognized ability to deliver recombinant antigens and to elicit all forms of immunity: mucosal, systemic, and humoral responses. To test this model, mice were orally immunized with a S. enterica serovar Typhimurium strain carrying the pYA3137eaeA vector, and challenged with E. coli O157:H7. Here we show that immunization induced the production of high levels of specific IgG and IgA antibodies and promoted reduction in the fecal shedding of EHEC after challenge. The live recombinant vaccine reported herein may contribute to the efforts of reducing animal intestinal mucosa colonization.

Lactobacillus sakei 2a and its Concentrated Acid Extract on Inhibition of Food-Borne Salmonella strains

Lactic acid bacteria are used in food production to provide desirable organoleptic characteristics, and can also act as biopreservatives, controlling the growth of undesirable microorganisms. In this study, we examined the antimicrobial action of Lactobacillus sakei 2a and its concentrated acid extract against food-borne Salmonella spp. The extract was obtained by acid extraction from culture broth of L. sakei 2a and was designated extract 2a. We determined that extract 2a had significant activity (approximately 500 AU ml(-1)). We used different antimicrobial substances alone or in combination with extract 2a to evaluate the inhibitory activity of the various treatments on a pool of five Salmonella strains. The pathogen Listeria monocytogenes Scott A Cm-r Em(r) was used as an indicator strain of inhibitory activity. In summary, all antimicrobials substances that were tested showed an inhibitory effect against the growth of Salmonella, andthis action was enhanced in the presence of extract 2a. Moreover, among the treatments applied, the combination of extract 2a and 0.1% lactic acid exhibited the most potent inhibitory effect towards the pool of Salmonella strains. Our findings indicate that L. sakei 2a and extract 2a, especially in combination with other antimicrobials, present potential technological application in the control of salmonellae in foods.

Release of NO from a nitrosyl ruthenium complex through oxidation of mitochondrial NADH and effects on mitochondria

Nitrosyl ruthenium complexes are promising NO donor agents with numerous advantages for the biologic applications of NO. We have characterized the NO release from the nitrosyl ruthenium complex [Ru(NO2)(bpy)(2)(4-pic)](+) (I) and the reactive oxygen/nitrogen species (ROS/RNS)-mediated NO actions on isolated rat liver mitochondria. The results indicated that oxidation of mitochondrial NADH promotes NO release from (I) in a manner mediated by NO2 formation (at neutral pH) as in mammalian cells, followed by an oxygen atom transfer mechanism (OAT). The NO released from (I) uncoupled mitochondria at low concentrations/incubation times and inhibited the respiratory chain at high concentrations/incubation times. In the presence of ROS generated by mitochondria NO gave rise to peroxynitrite, which, in turn, inhibited the respiratory chain and oxidized membrane protein-thiols to elicit a Ca2+-independent mitochondrial permeability transition; this process was only partially inhibited by cyclosporine-A, almost fully inhibited by the thiol reagent N-ethylmaleimide (NEM) and fully inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,45,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). These actions correlated with the release of cytochrome c from isolated mitochondria as detected by Western blotting analysis. These events, typically involved in cell necrosis and/or apoptosis denote a potential specific action of (I) and analogs against tumor cells via mitochondria-mediated processes. (C) 2012 Elsevier Inc. All rights reserved.

Maternal LAMP/p55gagHIV-1 DNA Immunization Induces In Utero Priming and a Long-Lasting Immune Response in Vaccinated Neonates

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+ CD25+ Foxp3+ T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-gamma-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods.

Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip

The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5 gamma 2 chain as well as alpha 3, beta 1 subunits of alpha 3 beta 1 heterodimer and beta 4 subunit of integrin alpha 6 beta 4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of beta 1, beta 4 and alpha 3 integrins, and SISCCs lacked beta 1, beta 4 and alpha 3 integrins in the invasive front. AC cases were negative for the Ln-5 gamma 2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5 gamma 2 chain expression in the invasive front of the tumor. Integrin beta 1, beta 4 and alpha 3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5 gamma 2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype. (C) 2012 Elsevier GmbH. All rights reserved.

Elevated tissue omega-3 fatty acid status prevents age-related glucose intolerance in fat-1 transgenic mice

The objective of this study was to investigate the impact of elevated tissue omega-3 (n-3) polyunsaturated fatty acids (PUFA) status on age-related glucose intolerance utilizing the fat-1 transgenic mouse model, which can endogenously synthesize n-3 PUFA from omega-6 (n-6) PUFA. Fat-1 and wild-type mice, maintained on the same dietary regime of a 10% corn oil diet, were tested at two different ages (2months old and 8months old) for various glucose homeostasis parameters and related gene expression. The older wild-type mice exhibited significantly increased levels of blood insulin, fasting blood glucose, liver triglycerides, and glucose intolerance, compared to the younger mice, indicating an age-related impairment of glucose homeostasis. In contrast, these age-related changes in glucose metabolism were largely prevented in the older fat-1 mice. Compared to the older wild-type mice, the older fat-1 mice also displayed a lower capacity for gluconeogenesis, as measured by pyruvate tolerance testing (PTT) and hepatic gene expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase (G6Pase). Furthermore, the older fat-1 mice showed a significant decrease in body weight, epididymal fat mass, inflammatory activity (NFκ-B and p-IκB expression), and hepatic lipogenesis (acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) expression), as well as increased peroxisomal activity (70-kDa peroxisomal membrane protein (PMP70) and acyl-CoA oxidase1 (ACOX1) expression). Altogether, the older fat-1 mice exhibit improved glucose homeostasis in comparison to the older wild-type mice. These findings support the beneficial effects of elevated tissue n-3 fatty acid status in the prevention and treatment of age-related chronic metabolic diseases

Movement of NH3 through the human urea transporter B: a new gas channel

Aquaporins and Rh proteins can function as gas (CO2 and NH3) channels. The present study explores the urea, H2O, CO2, and NH3 permeability of the human urea transporter B (UT-B) (SLC14A1), expressed in Xenopus oocytes. We monitored urea uptake using [14C]urea and measured osmotic water permeability (Pf) using video microscopy. To obtain a semiquantitative measure of gas permeability, we used microelectrodes to record the maximum transient change in surface pH (∆pHS) caused by exposing oocytes to 5% CO2/33 mM HCO3- (pHS increase) or 0.5 mM NH3/NH4+ (pHS decrease). UT-B expression increased oocyte permeability to urea by >20-fold, and Pf by 8-fold vs. H2O-injected control oocytes. UT-B expression had no effect on the CO2-induced ∆pHS but doubled the NH3-induced ∆pHS. Phloretin reduced UT-B-dependent urea uptake (Jurea * ) by 45%, Pf * by 50%, and (- ∆pHS * )NH3 by 70%. p-Chloromercuribenzene sulfonate reduced Jurea * by 25%, Pf * by 30%, and (∆pHS * )NH3 by 100%. Molecular dynamics (MD) simulations of membrane-embedded models of UT-B identified the monomeric UT-B pores as the main conduction pathway for both H2O and NH3 and characterized the energetics associated with permeation of these species through the channel. Mutating each of two conserved threonines lining the monomeric urea pores reduced H2O and NH3 permeability. Our data confirm that UT-B has significant H2O permeability and for the first time demonstrate significant NH3 permeability. Thus the UTs become the third family of gas channels. Inhibitor and mutagenesis studies and results of MD simulations suggest that NH3 and H2O pass through the three monomeric urea channels in UT-B.

Actions of translocator protein ligands on neutrophil adhesion and motility induced by G-protein coupled receptor signaling

The 18 kDa translocator protein (TSPO) also known as the peripheral benzodiazepine receptor (PBR), mediates the transportation of cholesterol and anions from the outer to the inner mitochondrial membrane in different cells types. Although recent evidences indicate a potential role for TSPO in the development of inflammatory processes, the mechanisms involved have not been elucidated. The present study investigated the ability of the specific TSPO ligands, the isoquinoline carboxamide PK11195 and benzodiazepine Ro5-4864, on neutrophil recruitment promoted by the N-formylmethionyl-leucyl-phenylalanine peptide (fMLP), an agonist of G-protein coupled receptor (GPCR). Pre-treatment with Ro5-4864 abrograted fMLP-induced leukocyte-endothelial interactions in mesenteric postcapillary venules in vivo. Moreover, in vitro Ro5-4864 treatment prevented fMLP-induced: (i) L-selectin shedding and overexpression of PECAM-1 on the neutrophil cell surface; (ii) neutrophil chemotaxis and (iii) enhancement of intracellular calcium cations (iCa(+2)). Intriguingly, the two latter effects were augmented by cell treatment with PK11195. An allosteric agonist/antagonist relation may be suggested, as the effects of Ro5-4864 on fMLP-stimulated neutrophils were reverted by simultaneous treatment with PK11195. Taken together, these data highlight TSPO as a modulator of pathways of neutrophil adhesion and locomotion induced by GPCR, connecting TSPO actions and the onset of an innate inflammatory response. (C) 2011 Elsevier Inc. All rights reserved.