mRNA-based approach to monitor recombinant gamma-interferon restoration of LPS-induced endotoxin tolerance

Introduction: It is now well accepted that sepsis is associated with the development of a pronounced immunosuppressive state, characterized by severe immune alterations (e.g. reduced proliferative capacity, endotoxin tolerance, apoptosis) participating in increased mortality and susceptibility to nosocomial infections. Efforts are currently aimed at restoring a functional immune response in septic patients. Successful therapydepends on the identification of appropriate immunostimulatory drugs and on the development of suitable biomarkers that could be used to stratify patients and to follow response to treatment.Methods: In this study, we evaluated the ex vivo effect of recombinant interferon gamma (rIFN-g) in restoring monocyte functionality (endotoxin-induced Tumor Necrosis Factor-a production) in a two-hit model of endotoxin tolerance (ET) with peripheral blood mononuclear cells from healthy volunteers and in whole blood of septic shockpatients. Importantly, we used quantitative-reverse transcription polymerase-chain reaction to monitor the effect of rIFN-g on the expression of seven genes known to participate in ET (TNF-a, IL-10, HLA-DRA, CIITA, IRAK-M, ABIN-3 and LY64).Results: Expression analysis of those genes confirmed the presence of an immunosuppression state and the ex vivo restoration of immune functions by rIFN-g. We show for the first time that rIFN-g is able to bypass, at the mRNA level, the effect of negative regulators of the LPS signalling pathway such as IRAK-M, ABIN-3 and LY64.Conclusions: Overall, mRNA expressions of a panel of genes could represent promising candidates for the ex vivo evaluation of rIFN-g effect on monocyte functionality. This ex vivo translational research study demonstrates the potential of a mRNA-based approach to successfully monitor drug efficacy

TLR3 modulates immunopathology during a Schistosoma mansoni egg-driven Th2 response in the lung

We examined the role of TLR3 in Th2-driven pulmonary granulomatous disease, using wildtype (TLR3 +/+ ) and TLR3 gene-deficient (TLR3 −/− ) mice in a well-established model of Schistosoma mansoni egg-induced pulmonary granuloma. The intravenous bolus injection of S. mansoni eggs into S. mansoni -sensitized TLR3 +/+ mice was associated with an increase in TLR3 transcript expression in alveolar macrophages and ex vivo spleen and lung cultures at day 8 after egg injection. Lungs from TLR3 −/− mice showed an increase in granuloma size, greater collagen deposition around the granuloma, and increased Th2 cytokine and chemokine levels compared with similarly sensitized and challenged TLR3 +/+ mice. Macrophages from TLR3 −/− mice exhibited an M2 phenotype characterized by increased arginase and CCL2 expression. Significantly greater numbers of CD4 + CD25 + T cells were present in the lungs of TLR3 −/− mice compared with TLR3 +/+ mice at day 8 after egg embolization. Cells derived from granulomatous lung and lung draining lymph nodes of TLR3 −/− mice released significantly higher levels of IL-17 levels relative to TLR3 +/+ cells. Thus, our data suggest that TLR3 has a major regulatory role during a Th2-driven granulomatous response as its absence enhanced immunopathology.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61443/1/3436_ftp.pd

Serological Profiling of a Candida albicans Protein Microarray Reveals Permanent Host-Pathogen Interplay and Stage-Specific Responses during Candidemia

Candida albicans in the immunocompetent host is a benign member of the human microbiota. Though, when host physiology is disrupted, this commensal-host interaction can degenerate and lead to an opportunistic infection. Relatively little is known regarding the dynamics of C. albicans colonization and pathogenesis. We developed a C. albicans cell surface protein microarray to profile the immunoglobulin G response during commensal colonization and candidemia. The antibody response from the sera of patients with candidemia and our negative control groups indicate that the immunocompetent host exists in permanent host-pathogen interplay with commensal C. albicans. This report also identifies cell surface antigens that are specific to different phases (i.e. acute, early and mid convalescence) of candidemia. We identified a set of thirteen cell surface antigens capable of distinguishing acute candidemia from healthy individuals and uninfected hospital patients with commensal colonization. Interestingly, a large proportion of these cell surface antigens are involved in either oxidative stress or drug resistance. In addition, we identified 33 antigenic proteins that are enriched in convalescent sera of the candidemia patients. Intriguingly, we found within this subset an increase in antigens associated with heme-associated iron acquisition. These findings have important implications for the mechanisms of C. albicans colonization as well as the development of systemic infection

Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii

We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii. For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip. We could also show that mitotic division proceeds at a constant rate of 0. 64 microm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae. Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration. For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e. generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae. This phenotype supported the view that dynein is important for the movement of nuclei towards the tip. In A. gossypii the opposite seems to be the case. A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches. Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules. The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network