Induction of an antigen-specific CTL response by a conformationally biased agonist of human C5a anaphylatoxin as a molecular adjuvant

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSPKPMPLaR) was used as a molecular adjuvant in stimulating an Ag-specific CTL response against murine P815S target cells expressing an Ld-restricted CTL epitope of the hepatitis B surface Ag (HBsAg), Groups of BALB/c mice (H-2(d)) were immunized with aqueous solutions of the HBsAg CTL epitopes (IPQSLDSWWTSL and IPQSLDSTaVTSLRR); the C5a agonist (YSFKPMPLaR); the C5a agonist and HBsAg CTL epitopes admired (IPQSLDSWWTSL and IPQSLDSWWTSLRR + YSFKPMPLaR); the C5a-active, HBsAg CTL epitope-C5a agonist constructs (IPQSLDSWWTSLYSFKPMPLaR, IPQSLDSWWTSLRRYSFKPMPLaR, and IPQSLDSWWTSLRVRRYSFPMPLaR); a C5a-inactive, reverse-moiety construct (YSFKPMPLaRRRIPQSLDSWWTSL); and a C5a-attenuated, carboxyl-terminal-blocked construct (IPQSLDSWWTSLRRYSFKPMPLaRG). Ag-specific CD8(+) CTL responses were observed after the secondary boost in the absence of any added adjuvant only in mice that were immunized with C5a-active contructs, IPQSLDSWWTSLRRYSFKPMPLaR and IPQSLDSWWTSLRVRRYSFKPMPLaR. These two C5a-active immunogens contained potential subtilisin-sensitive linker sequences between the HBsAg CTL epitope and the C5a agonist; i.e., a double-Arg (RR) and a furin protease sensitive sequence (RVRR), The introduction of these potentially cleavable sequences may be a method of increasing the likelihood of liberating the CTL epitope from the C5a agonist by intracellular proteases, thereby facilitating entry of the epitope into Ag-processing pathways via an exogenous route.

Molecular adjuvant effects of a conformationally biased agonist elf human C5a anaphylatoxin

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSFKPMPLaR) was used as a molecular adjuvant in stimulating Ab responses against peptide epitopes derived from human MUC1 glycoprotein and the human mu and kappa opioid receptors. C57BL6 mice were immunized with the MUC1 epitope (YKQGGFLGL); the C5a agonist (YSFKPMPLaR); YSFKPMPLaR and YKQGGFLGL together, but unconjugated; a C5a-active, MUC1 epitope construct (YKQGGFLGLYSFKPMPLaR); and a C5a-inactive, reversed moiety construct (YSFKPMPLaRYKQGGFLGL). High Ab titers specific for the MUC1 epitope were observed Only in mice immunized with the C5a-active epitope construct. Similar results were obtained in BALB/c mice immunized with the C5a-active, MUC1 epitope construct, Abs from the sera of the C57BL6 mice were predominately of the IgG2a, IgC2b, and IgM isotypes and were reactive against human recombinant MUC1 and MUC1 expressed by the Panc-1 M1F.15 pancreatic cell line, When compared with the corresponding KLH-epitope conjugates in C57BL6 mice, the epitope-C5a agonist constructs produced titers of specific IgG Abs of isotypes distinct from those generated by the keyhole limpet hemocyanin-epitope conjugates, Rabbits immunized with a mu opioid receptor epitope-C5a agonist construct (GDLSDPCGNRTNLGGRDSLYSFKPMPLaR) or a kappa opioid receptor epitope-C5a agonist construct (FPGWAEPDSNGSEDAQLYSFKPMPLaR) generated high titer, epitope-specific Ab responses, Ab titers generated in response to the opioid epitope-C5a agonist constructs were comparable to those generated by the opioid KLH-epitope conjugates, The results of this study are discussed in terms of possible mechanisms by which the conformationally biased C5a agonist serves as a molecular adjuvant.

Development of response-selective agonists of human C5a anaphylatoxin: Conformational, biological, and therapeutic considerations

Numerous studies on the relationship between the structure and function of peptide agonists derived from the biologically active, C-terminal region of human C5a anaphylatoxin have been reported over the past decade. These studies have been performed with the objective of parlaying this structure-function information into the design of peptide/peptidomimetic modulators of C5a receptor (C5aR)-mediated function. In this review, we describe a rational approach for the development of conformationally biased, decapeptide agonists of C5a and described how these stabilized and specific conformational features relate to the expression of specific C5a-like activities in vitro and in vivo. The therapeutic potential of such response-selective C5a agonists is discussed and underscored by the results of one such response-selective C5a agonist that was used in vivo as an effective molecular adjuvant capable of generating antigen-specific humoral and cellular immune responses. Finally, we describe the synthesis of a new generation of highly response-selective, conformationally biased C5a agonist and discuss the in vitro and in vivo biologic results that so indicate this biologic selectivity.

Agonist-biased signaling at the sst2A receptor: the multi-somatostatin analogs KE108 and SOM230 activate and antagonize distinct signaling pathways

Somatostatin analogs that activate the somatostatin subtype 2A (sst2A) receptor are used to treat neuroendocrine cancers because they inhibit tumor secretion and growth. Recently, new analogs capable of activating multiple somatostatin receptor subtypes have been developed to increase tumor responsiveness. We tested two such multi-somatostatin analogs for functional selectivity at the sst2A receptor: SOM230, which activates sst1, sst2, sst3, and sst5 receptors, and KE108, which activates all sst receptor subtypes. Both compounds are reported to act as full agonists at their target sst receptors. In sst2A-expressing HEK293 cells, somatostatin inhibited cAMP production, stimulated intracellular calcium accumulation, and increased ERK phosphorylation. SOM230 and KE108 were also potent inhibitors of cAMP accumulation, as expected. However, they antagonized somatostatin stimulation of intracellular calcium and behaved as partial agonists/antagonists for ERK phosphorylation. In pancreatic AR42J cells, which express sst2A receptors endogenously, SOM230 and KE108 were both full agonists for cAMP inhibition. However, although somatostatin increased intracellular calcium and ERK phosphorylation, SOM230 and KE108 again antagonized these effects. Distinct mechanisms were involved in sst2A receptor signaling in AR42J cells; pertussis toxin pretreatment blocked somatostatin inhibition of cAMP accumulation but not the stimulation of intracellular calcium and ERK phosphorylation. Our results demonstrate that SOM230 and KE108 behave as agonists for inhibition of adenylyl cyclase but antagonize somatostatin's actions on intracellular calcium and ERK phosphorylation. Thus, SOM230 and KE108 are not somatostatin mimics, and their functional selectivity at sst2A receptors must be considered in clinical applications where it may have important consequences for therapy.

CB1 receptor allosteric modulators display both agonist and signaling pathway specificity

We have previously identified allosteric modulators of the cannabinoid CB1 receptor (Org 27569, PSNCBAM-1) which display a contradictory pharmacological profile: increasing the specific binding of the CB1 receptor agonist [3H]CP55940 but producing a decrease in CB1 receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signalling endpoints linked to CB1 receptor activation. We assessed the effect of these compounds on CB1 receptor agonist-induced [35S]GTPγS binding, inhibition and stimulation of forskolin stimulated cAMP production, phosphorylation of ERK, and β arrestin recruitment. We also investigated the effect of these allosteric modulators on CB1 agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent as modulators of CP55940 signalling as compared to WIN55212 and having little effect on [3H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced [35S]GTPγS binding, simulation (Gαs mediated) and inhibition (Gαi mediated) of cAMP production and β arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphoryation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high affinity CB1 agonist binding sites. The receptor conformation stabilised by the allosterics appears to induce signalling and also selectively traffics orthosteric agonist signalling via the ERK phosphorylation pathway.

The extracellular surface of the GLP-1 receptor is a molecular trigger for biased agonism

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Biased Random-key Genetic Algorithms For The Winner Determination Problem In Combinatorial Auctions.

Abstract In this paper, we address the problem of picking a subset of bids in a general combinatorial auction so as to maximize the overall profit using the first-price model. This winner determination problem assumes that a single bidding round is held to determine both the winners and prices to be paid. We introduce six variants of biased random-key genetic algorithms for this problem. Three of them use a novel initialization technique that makes use of solutions of intermediate linear programming relaxations of an exact mixed integer-linear programming model as initial chromosomes of the population. An experimental evaluation compares the effectiveness of the proposed algorithms with the standard mixed linear integer programming formulation, a specialized exact algorithm, and the best-performing heuristics proposed for this problem. The proposed algorithms are competitive and offer strong results, mainly for large-scale auctions.

Lack of Galectin-3 Drives Response to Paracoccidioides brasiliensis toward a Th2-Biased Immunity

There is recent evidence that galectin-3 participates in immunity to infections, mostly by tuning cytokine production. We studied the balance of Th1/Th2 responses to P. brasiliensis experimental infection in the absence of galectin-3. The intermediate resistance to the fungal infection presented by C57BL/6 mice, associated with the development of a mixed type of immunity, was replaced with susceptibility to infection and a Th2-polarized immune response, in galectin-3-deficient (gal3(-/-)) mice. Such a response was associated with defective inflammatory and delayed type hypersensitivity (DTH) reactions, high IL-4 and GATA-3 expression and low nitric oxide production in the organs of infected animals. Gal3(-/-) macrophages exhibited higher TLR2 transcript levels and IL-10 production compared to wild-type macrophages after stimulation with P. brasiliensis antigens. We hypothesize that, during an in vivo P. brasiliensis infection, galectin-3 exerts its tuning role on immunity by interfering with the generation of regulatory macrophages, thus hindering the consequent Th2-polarized type of response.

CONTRASTING PHYLOGEOGRAPHIC PATTERNS IN MITOCHONDRIAL DNA AND MICROSATELLITES: EVIDENCE OF FEMALE PHILOPATRY AND MALE-BIASED GENE FLOW AMONG REGIONAL POPULATIONS OF THE BLUE-AND-YELLOW MACAW (PSITTACIFORMES: ARA ARARAUNA) IN BRAZIL

Comparing the patterns of population differentiation among genetic markers with different modes of inheritance call provide insights into patterns of sex-biased dispersal and gene flow. The blue-and-yellow Macaw (Ara ararauna) is a Neotropical parrot with a broad geographic distribution ill South America. However, little is known about the natural history and current status Of remaining wild populations, including levels of genetic variability. The progressive decline and possible fragmentation of populations may endanger this species in the near future. We analyzed mitochondrial DNA (mtDNA) control-region sequences and six microsatellite 106 Of Blue-and-yellow Macaws sampled throughout their geographic range ill Brazil to describe population genetic Structure, to make inferences about historical demography and dispersal behavior, and to provide insight for conservation efforts. Analyses of population genetic structure based on mtDNA showed evidence of two major populations ill western and eastern Brazil that share a few low-frequency haplotypes. This phylogeographic pattern seems to have originated by the historical isolation of Blue-and-yellow Macaw populations similar to 374,000 years ago and has been maintained by restricted gene flow and female philopatry. By contrast, variation ill biparentally inherited microsatellites was not structured geographically, Male-biased dispersal and female philopatry best explain the different patterns observed in these two markers. Because females disperse less than males, the two regional populations with well-differentiated mtDNA haplogroups should be considered two different management units for conservation purposes. Received 4 November 2007 accepted 10 December 2008.

Transition-metal 13-atom clusters assessed with solid and surface-biased functionals

First-principles density-functional theory studies have reported open structures based on the formation of double simple-cubic (DSC) arrangements for Ru(13), Rh(13), Os(13), and Ir(13), which can be considered an unexpected result as those elements crystallize in compact bulk structures such as the face-centered cubic and hexagonal close-packed lattices. In this work, we investigated with the projected augmented wave method the dependence of the lowest-energy structure on the local and semilocal exchange-correlation (xc) energy functionals employed in density-functional theory. We found that the local-density approximation (LDA) and generalized-gradient formulations with different treatment of the electronic inhomogeneities (PBE, PBEsol, and AM05) confirm the DSC configuration as the lowest-energy structure for the studied TM(13) clusters. A good agreement in the relative total energies are obtained even for structures with small energy differences, e. g., 0.10 eV. The employed xc functionals yield the same total magnetic moment for a given structure, i.e., the differences in the bond lengths do not affect the moments, which can be attributed to the atomic character of those clusters. Thus, at least for those systems, the differences among the LDA, PBE, PBEsol, and AM05 functionals are not large enough to yield qualitatively different results. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3577999]

A TR beta-selective agonist confers resistance to diet-induced obesity

Thyroid hormone receptor beta (TR beta also listed as THRB oil the MGI Database)-selective agonists activate brown adipose tissue (BAT) thermogenesis, while only minimally affecting cardiac activity or lean body mass. Here, we tested the hypothesis that daily administration of the TR beta agonist GC-24 prevents the metabolic alterations associated with a hypercaloric diet. Rats were placed on a high-fat diet and after a month exhibited increased body weight (BW) and adiposity, fasting hyperglycemia and glucose intolerance, increased plasma levels of triglycerides, cholesterol, nonesterified Fatty acids and interleukin-6. While GC-24 administration to these animals did not affect food ingestion or modified the progression of BW gain, it did increase energy, g the increase in adiposity Without expenditure, eliminating causing cardiac hypertrophy Fasting hyperglycemia remained unchanged, but treatment with GC-24 improved glucose I tolerance by increasing insulin Sensitivity and also normalized plasma triglyceride levels. plasma cholesterol levels were only Partially normalized and liver cholesterol content remained high in the GC-24-treated animals. Gene expression in liver, skeletal muscle, and white adipose tissue was only minimally affected by treatment with GC-24, with the main target being BAT In conclusion, during high-fat feeding treatment with the TR beta-selective agonist, GC-24 only partially improves metabolic control probably as a result Of accelerating the resting metabolic rate. Journal of Endocrinology (2009) 203, 291-299

Is serum amyloid A an endogenous TLR4 agonist?

Serum amyloid A (SAA), a classical acute-phase protein, is produced predominantly by hepatocytes in response to injury, infection, and inflammation. It has been shown that SAA primes leukocytes and induces the expression and release of proinflammatory cytokines. Here, we report that SAA induces NO production by murine peritoneal macrophages. Using specific inhibitors, we showed that NO production was dependent on inducible NO synthase thorough the activation of ERK1/2 and p38 MAPKs. Moreover, SAA activity was decreased after proteolysis but not with polymyxin B, a lipid A antagonist. Finally, we found that NO production was dependent on functional TLR4, a receptor complex associated with innate immunity. Macrophages from C3H/HeJ and C57BL/10ScCr mice lacking a functional TLR4 did not respond to SAA stimulation. In conclusion, our study makes a novel observation that SAA might be an endogenous agonist for the TLR4 complex on macrophages. The contribution of this finding in amplifying innate immunity during the inflammatory process is discussed.

The influence of Lys68 in decapeptide agonists of C5a on C5a receptor binding, activation and selectivity

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a(65-74), Y65, F67, P69, P71, D-Ala73) was used as a template to gain insight into the nature and importance of lysine at position 68 in the peptide-receptor interaction. A panel of YSFKPMPLaR analogs with systematic substitutions for Lys68 was evaluated for C5a receptor (C5aR) binding affinity and activation in two well-characterized assay systems: human polymorphonuclear leukocytes (PMNs) and human fetal artery. In addition, we determined the activity of these new analogs in transfected rat basophilic leukemia (RBL) cells in which the Glu at position 199 of the C5aR (wtGlu199) was replaced by a Gin (C5aR-Gln199) or a Lys (C5aR-Lys199). Our results indicated that Lys68 in YSFKPMPLaR plays an important role in binding the C5aR expressed on PMNs and RBL cells. Furthermore, the data indicated that Lys68 interacted with Glu199 of the C5aR in PMNs and RBL cells. In human fetal artery, however, Lys68 substitutions had little or no effect on activity, which suggested that the receptor conformation may be different in this tissue. Thus, the interaction between Lys68 of the decapeptide agonist and Glu199 of the C5aR may be cell type-specific and may form the molecular basis for tissue-specific responses to C5a agonists.

Biologically active conformer of the effector region of human C5a and modulatory effects of N-terminal receptor binding determinants on activity

A conformationally biased decapeptide agonist of human C5a (C5a(65-74)Y65,F67,P69,P71,D-Ala73 or YSFKPMPLaR) was used as a functional probe of the C5a receptor (C5aR) in order to understand the conformational features in the C-terminal effector region of C5a that are important for C5aR binding and signal transduction. YSFKPMPLaR was a potent, full agonist of C5a, but at higher concentrations had a superefficacious effect compared to the natural factor. The maximal efficacy of this analogue was 216 +/- 56% that of C5a in stimulating the release of beta-glucuronidase from human neutrophils. C5aR activation and binding curves both occurred in the same concentration range with YSFKPMPLaR, characteristics not observed with natural C5a or more conformationally flexible C-terminal agonists. YSFKPMPLaR was then used as a C-terminal effector template onto which was synthesized various C5aR binding determinants from the N-terminal core domain of the natural factor. In general, the presence of N-terminal binding determinants had little effect on either potency or binding affinity when the C-terminal effector region was presented to the C5aR in this biologically active conformation. However, one peptide, C5a(12-20)-Ahx-YSFKPMPLaR, expressed a 100-fold increase in affinity for the neutrophil C5aR and a 6-fold increase in potency relative to YSFKPMPLaR. These analyses showed that the peptides used in this study have up to 25% of the potency of C5a in human fetal artery and up to 5% of the activity of C5a in the PMN enzyme release assay.