The Pseudomonas toxin pyocyanin inhibits the dual oxidase-based antimicrobial system as it imposes oxidative stress on airway epithelial cells

The dual oxidase-thiocyanate-lactoperoxidase (Duox/SCN(-)/LPO) system generates the microbicidal oxidant hypothiocyanite in the airway surface liquid by using LPO, thiocyanate, and Duox-derived hydrogen peroxide released from the apical surface of the airway epithelium. This system is effective against several microorganisms that infect airways of cystic fibrosis and other immunocompromised patients. We show herein that exposure of airway epithelial cells to Pseudomonas aeruginosa obtained from long-term cultures inhibits Duox1-dependent hydrogen peroxide release, suggesting that some microbial factor suppresses Duox activity. These inhibitory effects are not seen with the pyocyanin-deficient P. aeruginosa strain PA14 Phz1/2. We show that purified pyocyanin, a redox-active virulence factor produced by P. aeruginosa, inhibits human airway cell Duox activity by depleting intracellular stores of NADPH, as it generates intracellular superoxide. Long-term exposure of human airway (primary normal human bronchial and NCI-H292) cells to pyocyanin also blocks induction of Duox1 by Th2 cytokines (IL-4, IL-13), which was prevented by the antioxidants glutathione and N-acetylcysteine. Furthermore, we showed that low concentrations of pyocyanin blocked killing of wild-type P. aeruginosa by the Duox/SCN(-)/LPO system on primary normal human bronchial epithelial cells. Thus, pyocyanin can subvert Pseudomonas killing by the Duox-based system as it imposes oxidative stress on the host. We also show that lactoperoxidase can oxidize pyocyanin, thereby diminishing its cytotoxicity. These data establish a novel role for pyocyanin in the survival of P. aeruginosa in human airways through competitive redox-based reactions between the pathogen and host.     

Adaptable TCR avidity thresholds for negative selection

Central tolerance plays a significant role in preventing autoimmune diseases by eliminating T cells with high and intermediate avidity for self. To determine the manner of setting the threshold for deletion, we created a unique transgenic mouse strain with a diverse T cell population and globally increased TCR avidity for self-peptide/MHC complexes. Despite the adaptations aimed at reducing T cell reactivity (reduced TCR levels and increased levels of TCR signaling inhibitor CD5), transgenic mice displayed more severe experimental allergic encephalomyelitis and lupus. The numbers and activity of natural (CD4(+)CD25(+)) regulatory T cells were not altered. These findings demonstrate that the threshold for deletion is adaptable, allowing survival of T cells with higher avidity when TCR avidity is globally increased.     

A novel role for non-neutralizing antibodies against nucleoprotein in facilitating resistance to influenza virus

Current influenza vaccines elicit Abs to the hemagglutinin and neuraminidase envelope proteins. Due to antigenic drift, these vaccines must be reformulated annually to include the envelope proteins predicted to dominate in the following season. By contrast, vaccination with the conserved nucleoprotein (NP) elicits immunity against multiple serotypes (heterosubtypic immunity). NP vaccination is generally thought to convey protection primarily via CD8 effector mechanisms. However, significant titers of anti-NP Abs are also induced, yet the involvement of Abs in protection has largely been disregarded. To investigate how Ab responses might contribute to heterosubtypic immunity, we vaccinated C57BL/6 mice with soluble rNP. This approach induced high titers of NP-specific serum Ab, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization significantly reduced morbidity and viral titers after influenza challenge. Importantly, Ab-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer protection to naive hosts in an Ab-dependent manner. Therefore, Ab to conserved, internal viral proteins, such as NP, provides an unexpected, yet important mechanism of protection against influenza. These results suggest that vaccines designed to elicit optimal heterosubtypic immunity to influenza should promote both Ab and T cell responses to conserved internal proteins.     

Multivariate classification analysis of metabolomic data for candidate biomarker discovery in type 2 diabetes mellitus

Recent advances in genomics, metabolomics and proteomics have made it possible to interrogate disease pathophysiology and drug response on a systems level. The analysis and interpretation of the complex data obtained using these techniques is potentially fertile but equally challenging. We conducted a small clinical trial to explore the application of metabolomics data in candidate biomarker discovery. Specifically, serum and urine samples from patients with type 2 diabetes mellitus (T2DM) were profiled on metabolomics platforms before and after 8 weeks of treatment with one of three commonly used oral antidiabetic agents, the sulfonyurea glyburide, the biguanide metformin, or the thiazolidinedione rosiglitazone. Multivariate classification techniques were used to detect serum or urine analytes, obtained at baseline (pre-treatment) that could predict a significant treatment response after 8 weeks. Using this approach, we identified three analytes, measured at baseline, that were associated with response to a thiazolidinedione after 8 weeks of treatment. Although larger and longer-term studies are required to validate any of the candidate biomarkers, pharmacometabolomic profiling, in combination with multivariate classification, is worthy of further exploration as an adjunct to clinical decision making regarding treatment selection and for patient stratification within clinical trials. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cleavage of the signaling mucin Msb2 by the aspartyl protease Yps1 is required for MAPK activation in yeast

Signaling mucins are cell adhesion molecules that activate RAS/RHO guanosine triphosphatases and their effector mitogen-activated protein kinase (MAPK) pathways. We found that the Saccharomyces cerevisiae mucin Msb2p, which functions at the head of the Cdc42p-dependent MAPK pathway that controls filamentous growth, is processed into secreted and cell-associated forms. Cleavage of the extracellular inhibitory domain of Msb2p by the aspartyl protease Yps1p generated the active form of the protein by a mechanism incorporating cellular nutritional status. Activated Msb2p functioned through the tetraspan protein Sho1p to induce MAPK activation as well as cell polarization, which involved the Cdc42p guanine nucleotide exchange factor Cdc24p. We postulate that cleavage-dependent activation is a general feature of signaling mucins, which brings to light a novel regulatory aspect of this class of signaling adhesion molecule.     

FGF-23-Klotho signaling stimulates proliferation and prevents vitamin D-induced apoptosis

Fibroblast growth factor 23 (FGF-23) and Klotho are secretory proteins that regulate mineral-ion metabolism. Fgf-23(-/-) or Klotho(-/-) knockout mice exhibit several pathophysiological processes consistent with premature aging including severe atrophy of tissues. We show that the signal transduction pathways initiated by FGF-23-Klotho prevent tissue atrophy by stimulating proliferation and preventing apoptosis caused by excessive systemic vitamin D. Because serum levels of active vitamin D are greatly increased upon genetic ablation of Fgf-23 or Klotho, we find that these molecules have a dual role in suppression of apoptotic actions of vitamin D through both negative regulation of 1alpha-hydroxylase expression and phosphoinositide-3 kinase-dependent inhibition of caspase activity. These data provide new insights into the physiological roles of FGF-23 and Klotho.     

Fhit proteins can also recognize substrates other than dinucleoside polyphosphates

We show here that Fhit proteins, in addition to their function as dinucleoside triphosphate hydrolases, act similarly to adenylylsulfatases and nucleoside phosphoramidases, liberating nucleoside 5'-monophosphates from such natural metabolites as adenosine 5'-phosphosulfate and adenosine 5'-phosphoramidate. Moreover, Fhits recognize synthetic nucleotides, such as adenosine 5'-O-phosphorofluoridate and adenosine 5'-O-(gamma-fluorotriphosphate), and release AMP from them. With respect to the former, Fhits behave like a phosphodiesterase I concomitant with cleavage of the P-F bond. Some kinetic parameters and implications of the novel reactions catalyzed by the human and plant (Arabidopsis thaliana) Fhit proteins are presented.     

Hypoxic osteocytes recruit human MSCs through an OPN/CD44-mediated pathway

Little is known about the role or identity of signaling molecules released by osteocytes to recruit MSCs to areas of matrix damage. Vascular disruption at fracture sites results in hypoxia which is known to up-regulate genes involved in cell migration including osteopontin (OPN). We examined the effect of conditioned media from hypoxic osteocytes on MSC migration. Hypoxic osteocyte media significantly increased MSC migration and expression of OPN was significantly increased in hypoxic osteocytes. OPN and CD44 neutralizing antibodies significantly reduced MSC migration. Further, recombinant OPN significantly increased MSC migration in a dose-dependent manner. Our data support the hypothesis that hypoxia at a fracture site stimulates the release of chemotactic factors, such as OPN, from osteocytes, that induce MSC migration to aid in fracture repair. To our knowledge, these are the first data to suggest a role for osteocytes and OPN in the recruitment of MSCs to aid in fracture repair.