TG(mRen2)27 (Ren2) transgenic rats overexpress the mouse renin gene, manifest hypertension, and exhibit increased tissue ANG II levels and oxidative stress. Evidence indicates that elevated tissue ANG II contributes to oxidative stress, increases in glomerular macromolecular permeability, and consequent albuminuria. Furthermore, angiotensin type 1 receptor (AT1R) blockers reduce albuminuria and slow progression of renal disease. However, it is not known whether improvements in glomerular filtration barrier integrity and albuminuria during treatment are related to reductions in oxidative stress and/or kidney renin-angiotensin system (RAS) activity. To investigate the renal protective effects of AT1R blockade, we treated young (6-7 wk old) male Ren2 rats with valsartan (Ren2-V; 30 mg/kg) for 3 wk and measured urine albumin, kidney malondialdehyde (MDA), RAS component mRNAs, and NADPH oxidase subunits (gp91(phox) and Rac1) compared with age-matched untreated Ren2 and Sprague-Dawley (S-D) rats. Basement membrane thickness, slit pore diameter and number, and foot process base width were measured by transmission electron microscopy (TEM). Results indicate that AT1R blockade lowered systolic blood pressure (30%), albuminuria (91%), and kidney MDA (80%) in Ren2-V compared with untreated Ren2 rats. Increased slit pore number and diameter and reductions in basement membrane thickness and podocyte foot process base width were strongly associated with albuminuria and significantly improved following AT1R blockade. AT1R blockade was also associated with increased angiotensin-converting enzyme-2 and neprilysin expression, demonstrating a beneficial shift in balance of renal RAS. Thus reductions in blood pressure, albuminuria, and tissue oxidative stress with AT1R blockade were associated with improved indexes of glomerular filtration barrier integrity and renal RAS in Ren2 rats.

The renin-angiotensin-aldosterone system contributes to cardiac remodeling, hypertrophy, and left ventricular dysfunction. Angiotensin II and aldosterone (corticosterone in rodents) together generate reactive oxygen species (ROS) via reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which likely facilitate this hypertrophy and remodeling. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo mineralocorticoid receptor (MR) blockade in a rodent model of the chronically elevated tissue renin-angiotensin-aldosterone system, the transgenic TG (mRen2) 27 rat (Ren2). The Ren2 overexpresses the mouse renin transgene with resultant hypertension, insulin resistance, proteinuria, and cardiovascular damage. Young (6- to 7-wk-old) male Ren2 and age-matched Sprague-Dawley rats were treated with spironolactone or placebo for 3 wk. Heart tissue ROS, immunohistochemical analysis of 3-nitrotyrosine, and NADPH oxidase (NOX) subunits (gp91(phox) recently renamed NOX2, p22(phox), Rac1, NOX1, and NOX4) were measured. Structural changes were assessed with cine-magnetic resonance imaging, transmission electron microscopy, and light microscopy. Significant increases in Ren2 septal wall thickness (cine-magnetic resonance imaging) were accompanied by perivascular fibrosis, increased mitochondria, and other ultrastructural changes visible by light microscopy and transmission electron microscopy. Although there was no significant reduction in systolic blood pressure, significant improvements were seen with MR blockade on ROS formation and NOX subunits (each P < 0.05). Collectively, these data suggest that MR blockade, independent of systolic blood pressure reduction, improves cardiac oxidative stress-induced structural and functional changes, which are driven, in part, by angiotensin type 1 receptor-mediated increases in NOX.

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20-150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine-converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.

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Mass implementation of DNA vaccines is hindered by the requirement of high plasmid dosages and poor immunogenicity. We evaluated the capacity of Mannheimia haemolytica ghosts as delivery system for DNA vaccines. In vitro studies showed that bacterial ghosts loaded with a plasmid carrying the green fluorescent protein-encoding gene (pEGFP-N1) are efficiently taken up by APC, thereby leading to high transfection rates (52–60%). Vaccination studies demonstrated that ghost-mediated delivery by intradermal or i.m. route of a eukaryotic expression plasmid containing the gene coding for β-galactosidase under the control of the CMV immediate early gene promoter (pCMVβ) stimulates more efficient Ag-specific humoral and cellular (CD4+ and CD8+) immune responses than naked DNA in BALB/c mice. The use of ghosts also allows modulating the major Th response from a mixed Th1/Th2 to a more dominant Th2 pattern. Intravenous immunization with dendritic cells loaded ex vivo with pCMVβ-containing ghosts also resulted in the elicitation of β-galactosidase-specific responses. This suggests that dendritic cells play an important role in the stimulation of immune responses when bacterial ghosts are used as a DNA delivery system. Bacterial ghosts not only target the DNA vaccine construct to APC, but also provide a strong danger signal, acting as natural adjuvants, thereby promoting efficient maturation and activation of dendritic cells. Thus, bacterial ghosts constitute a promising technology platform for the development of more efficient DNA vaccines.

The glycolipid α-galactosylceramide (αGalCer) has immunomodulatory properties, which have been exploited to combat cancer, chronic inflammatory diseases, and infections. However, its poor solubility makes αGalCer a suboptimal compound for in vivo applications. In this study, a pegylated derivative of αGalCer is characterized, which exhibits improved physical and biological properties. The new compound, αGalCerMPEG, is water-soluble and retains the specificity for the CD1d receptor of αGalCer. The in vitro stimulatory properties on immune cells (e.g., dendritic cells and splenocytes) are maintained intact, even when tested at a 33-fold lower concentration of the active moiety than αGalCer. NK cells isolated from mice treated with αGalCerMPEG also had stronger cytotoxic activity on YAC-1 cells than those obtained from animals receiving either αGalCer or CpG. Intranasal immunization studies performed in mice showed that αGalCerMPEG exerts stronger adjuvant activities than the parental compound αGalCer when tested at 0.35 vs 11.7 nM/dose. Coadministration of β-galactosidase with αGalCerMPEG resulted not only in high titers of Ag-specific Abs in serum (i.e., 1:512,000), but also in the stimulation of stronger Th2 and secretory IgA responses, both at local and remote mucosal effector sites (i.e., nose, lung, and vagina). The new synthetic derivative αGalCerMPEG represents a promising tool for the development of immune interventions against infectious and noninfectious diseases.

A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell-mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV-1 Tat protein as antigen and the synthetic lipopeptide, macrophageactivating lipopeptide-2 (MALP-2), as a mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti-Tat antibody responses, and Tatspecific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freund's adjuvant. Major linear B cell epitopes mapped within aa 1-20 and 46-60, whereas T cell epitopes were identified within aa 36-50 and 56-70. These epitopes have also been described in vaccinated primates and in HIV-1-infected individuals with better prognosis. Analysis of the anti-Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP-2, as opposed to the Th2 response observed with Tat plus incomplete Freund's adjuvant. Tat-specific IFN-+ -producing cells were significantly increased only in response to Tat plus MALP-2. These data suggest that Malp-2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.

A 2-kDa synthetic derivative of the macrophage-activating lipopeptide (MALP-2) from Mycoplasma fermentans is a potent inducer of monocytes/macrophages and improves the immunogenicity of antigens co-administered by systemic and mucosal routes. Dendritic cells (DC) are the most potent antigen-presenting cells, which are able to prime naive T cells in vivo. To elucidate the underlying mechanisms of MALP-2 adjuvanticity, we analyzed its activity on bone marrow-derived murine DC. In vitro stimulation of immature murine DC with MALP-2 resulted in the induction of maturation with up-regulated expression of MHC class II, costimulatory (CD80, CD86) and adhesion (CD40, CD54) molecules. MALP-2 also enhances the secretion of cytokines (IL-1 § , IL-6 and IL-12), and increases DC stimulatory activity on naive and antigen-specific T cells. Further studies demonstrated that MALP-2 treatment of DC results in a dose-dependent shift from the protein pattern of proteasomes to immunoproteasomes (up-regulation of LMP2, LMP7 and MECL1), which correlates with an increased proteolytic activity. Thus, the adjuvanticity of MALP-2 can be mediated, at least in part, by the stimulation of DC maturation, which in turn leads to an improved antigen presentation. Therefore, MALP-2 is a promising molecule for the development of immune therapeutic or prophylactic interventions.