A cynomolgus monkey E. coli urinary tract infection model confirms efficacy of new FimH vaccine candidates.

The increase in urinary tract infections (UTI) caused by antibiotic-resistant Escherichia coli requires the development of new therapeutic agents and prophylactic vaccines. To evaluate the efficacy of new lead candidates, we implemented a cynomolgus macaque UTI challenge model that mimics human uncomplicated cystitis in response to transurethral challenge with a multidrug-resistant (MDR) E. coli serotype O25b ST131 isolate. E. coli fimbrial adhesin FimH and O-antigens are separately under clinical evaluation by others as vaccine candidates to prevent UTI and invasive urosepsis disease, respectively. Accordingly, we assessed the protective efficacy of three 50-µg intramuscular doses of a novel recombinant FimH antigen adjuvanted with liposomal QS21/MPLA compared with saline placebo in groups of nine animals. A third group was vaccinated with this FimH formulation in combination with 1 µg each of a four-valent mixture of serotype O1a, O2, O6, and O25b O-antigen CRM197 lattice glycoconjugates. Both vaccines elicited high levels of serum FimH IgG and adhesin blocking antibodies at the time of bacterial challenge and, for the combination group, O-antigen-specific antibodies. Following bacterial challenge, both vaccinated groups showed >200- and >700-fold reduction in bacteriuria at day 2 and day 7 post-infection compared with placebo, respectively. In parallel, both vaccines significantly reduced levels of inflammatory biomarkers IL-8 and myeloperoxidase in the urine at day 2 post-infection relative to placebo. Results provide preclinical proof-of-concept for the prevention of an MDR UTI infection by these new vaccine formulations.

Semisynthetic Glycoconjugate Vaccine Candidates against Escherichia coli O25B Induce Functional IgG Antibodies in Mice.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a major health concern due to emerging antibiotic resistance. Along with O1A, O2, and O6A, E. coli O25B is a major serotype within the ExPEC group, which expresses a unique O-antigen. Clinical studies with a glycoconjugate vaccine of the above-mentioned O-types revealed O25B as the least immunogenic component, inducing relatively weak IgG titers. To evaluate the immunological properties of semisynthetic glycoconjugate vaccine candidates against E. coli O25B, we here report the chemical synthesis of an initial set of five O25B glycan antigens differing in length, from one to three repeat units, and frameshifts of the repeat unit. The oligosaccharide antigens were conjugated to the carrier protein CRM197. The resulting semisynthetic glycoconjugates induced functional IgG antibodies in mice with opsonophagocytic activity against E. coli O25B. Three of the oligosaccharide-CRM197 conjugates elicited functional IgGs in the same order of magnitude as a conventional CRM197 glycoconjugate prepared with native O25B O-antigen and therefore represent promising vaccine candidates for further investigation. Binding studies with two monoclonal antibodies (mAbs) revealed nanomolar anti-O25B IgG responses with nanomolar K D values and with varying binding epitopes. The immunogenicity and mAb binding data now allow for the rational design of additional synthetic antigens for future preclinical studies, with expected further improvements in the functional antibody responses. Moreover, acetylation of a rhamnose residue was shown to be likely dispensable for immunogenicity, as a deacylated antigen was able to elicit strong functional IgG responses. Our findings strongly support the feasibility of a semisynthetic glycoconjugate vaccine against E. coli O25B.

MicroRNAs play an essential role in the development of cardiac hypertrophy.

MicroRNAs are naturally existing, small, noncoding RNA molecules that downregulate posttranscriptional gene expression. Their expression pattern and function in the heart remain unknown. Here we report an array of microRNAs that are differentially and temporally regulated during cardiac hypertrophy. Significantly, the muscle-specific microRNA-1 (miR-1) was singularly downregulated as early as day 1 (0.56+/-0.036), persisting through day 7 (0.29+/-0.14), after aortic constriction-induced hypertrophy in a mouse model. Overexpression experiments showed that miR-1 inhibited its in silico-predicted, growth-related targets, including Ras GTPase-activating protein (RasGAP), cyclin-dependent kinase 9 (Cdk9), fibronectin, and Ras homolog enriched in brain (Rheb), in addition to protein synthesis and cell size. Thus, we propose that microRNAs play an essential regulatory role in the development of cardiac hypertrophy, wherein downregulation of miR-1 is necessary for the relief of growth-related target genes from its repressive influence and induction of hypertrophy.

Backbone flexibility of CDR3 and immune recognition of antigens.

Conformational entropy is an important component of protein-protein interactions; however, there is no reliable method for computing this parameter. We have developed a statistical measure of residual backbone entropy in folded proteins by using the ϕ-ψ distributions of the 20 amino acids in common secondary structures. The backbone entropy patterns of amino acids within helix, sheet or coil form clusters that recapitulate the branching and hydrogen bonding properties of the side chains in the secondary structure type. The same types of residues in coil and sheet have identical backbone entropies, while helix residues have much smaller conformational entropies. We estimated the backbone entropy change for immunoglobulin complementarity-determining regions (CDRs) from the crystal structures of 34 low-affinity T-cell receptors and 40 high-affinity Fabs as a result of the formation of protein complexes. Surprisingly, we discovered that the computed backbone entropy loss of only the CDR3, but not all CDRs, correlated significantly with the kinetic and affinity constants of the 74 selected complexes. Consequently, we propose a simple algorithm to introduce proline mutations that restrict the conformational flexibility of CDRs and enhance the kinetics and affinity of immunoglobulin interactions. Combining the proline mutations with rationally designed mutants from a previous study led to 2400-fold increase in the affinity of the A6 T-cell receptor for Tax-HLAA2. However, this mutational scheme failed to induce significant binding changes in the already-high-affinity C225-Fab/huEGFR interface. Our results will serve as a roadmap to formulate more effective target functions to design immune complexes with improved biological functions.

MicroRNA-21 targets Sprouty2 and promotes cellular outgrowths.

The posttranscriptional regulator, microRNA-21 (miR-21), is up-regulated in many forms of cancer, as well as during cardiac hypertrophic growth. To understand its role, we overexpressed it in cardiocytes where it revealed a unique type of cell-to-cell "linker" in the form of long slender outgrowths and branches. We subsequently confirmed that miR-21 directly targets and down-regulates the expression of Sprouty2 (SPRY2), an inhibitor of branching morphogenesis and neurite outgrowths. We found that beta-adrenergic receptor (betaAR) stimulation induces up-regulation of miR-21 and down-regulation of SPRY2 and is, likewise, associated with connecting cell branches. Knockdown of SPRY2 reproduced the branching morphology in cardiocytes, and vice versa, knockdown of miR-21 using a specific 'miRNA eraser' or overexpression of SPRY2 inhibited betaAR-induced cellular outgrowths. These structures enclose sarcomeres and connect adjacent cardiocytes through functional gap junctions. To determine how this aspect of miR-21 function translates in cancer cells, we knocked it down in colon cancer SW480 cells. This resulted in disappearance of their microvillus-like protrusions accompanied by SPRY2-dependent inhibition of cell migration. Thus, we propose that an increase in miR-21 enhances the formation of various types of cellular protrusions through directly targeting and down-regulating SPRY2.

Histone H2A.z is essential for cardiac myocyte hypertrophy but opposed by silent information regulator 2alpha.

In this study we have shown that the histone variant H2A.z is up-regulated during cardiac hypertrophy. Upon its knock-down with RNA interference, hypertrophy and the underlying increase in growth-related genes, protein synthesis, and cell size were down-regulated. During attempts to understand the mode of regulation of H2A.z, we found that overexpression of silent information regulator 2alpha (Sir2alpha) specifically induced down-regulation of H2A.z via NAD-dependent activity. This effect was reversed by the proteasome inhibitor epoxomicin, suggesting a Sir2alpha-mediated ubiquitin/proteasome-dependent mechanism for degradation of H2A.z. An increase in Sir2alpha also resulted in a dose-dependent reduction of the response to hypertrophic stimuli, whereas its inhibition resulted in enhanced hypertrophy and apoptosis. We have shown that Sir2alpha directly deacetylates H2A.z. Mutagenesis proved that lysines 4, 7, 11, and 13 do not play a role in the stability of H2A.z, whereas Lys-15 was indispensable. Meanwhile, Lys-115 and conserved, ubiquitinatable Lys-121 are critical for Sir2alpha-mediated degradation. Fusion of the C terminus of H2A.z (amino acids 115-127) to H2A.x or green fluorescence protein conferred Sir2alpha-inducible degradation to the former protein only. Because H2A.x and H2A.z have conserved N-tails, this implied that both the C and N termini are critical for mediating the effect of Sir2alpha. In short, the results suggest that H2A.z is required for cardiac hypertrophy, where its stability and the extent of cell growth and apoptosis are moderated by Sir2alpha. We also propose that Sir2alpha is involved in deacetylation of H2A.z, which results in ubiquitination of Lys-115 and Lys-121 and its degradation via a ubiquitin/proteasome-dependent pathway.

An alliance between Ras GTPase-activating protein, filamin C, and Ras GTPase-activating protein SH3 domain-binding protein regulates myocyte growth.

We have previously reported that Ras GTPase-activating protein (RasGAP) is involved in a pathway that regulates total cellular mRNA and protein synthesis in cardiac myocytes. A yeast two-hybrid screen resulted in identification of filamin C (FLN-C) as one of its targets. Knockdown of RasGAP or FLN-C, or severing their interaction, resulted in down-regulation of the RNA polymerase II kinase, cyclin-dependent kinase 7 (Cdk7). This appeared to be provoked by the release of cdk7 mRNA from RasGAP SH3 domain-binding protein, G3BP, and its subsequent degradation. In parallel, myocyte growth was also inhibited. On the other hand, overexpression of RasGAP induced a Cdk7- and FLN-C-dependent growth. Thus, we propose that the physical interaction between RasGAP and FLN-C facilitates an interaction between G3BP and cdk7 mRNA. This results in stabilization of cdk7 mRNA, an increase in its protein, which is required for cell growth.