Methods for interpreting lists of affected genes obstained in a DNA microarray experiment

Background - The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence) and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding) workshop focusing on post analysis of microarray data. The participating groups were provided with identical lists of microarray probes, including test statistics for three different contrasts, and the normalised log-ratios for each array, to be used as the starting point for interpreting the affected probes. The data originated from a microarray experiment conducted to study the host reactions in broilers occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria. Results - Several conceptually different analytical approaches, using both commercial and public available software, were applied by the participating groups. The following tools were used: Ingenuity Pathway Analysis, MAPPFinder, LIMMA, GOstats, GOEAST, GOTM, Globaltest, TopGO, ArrayUnlock, Pathway Studio, GIST and AnnotationDbi. The main focus of the approaches was to utilise the relation between probes/genes and their gene ontology and pathways to interpret the affected probes/genes. The lack of a well-annotated chicken genome did though limit the possibilities to fully explore the tools. The main results from these analyses showed that the biological interpretation is highly dependent on the statistical method used but that some common biological conclusions could be reached. Conclusion - It is highly recommended to test different analytical methods on the same data set and compare the results to obtain a reliable biological interpretation of the affected genes in a DNA microarray experimen

Uncovering the multifaceted roles played by neutrophils in allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a life-saving procedure used for the treatment of selected hematological malignancies, inborn errors of metabolism, and bone marrow failures. The role of neutrophils in alloHSCT has been traditionally evaluated only in the context of their ability to act as a first line of defense against infection. However, recent evidence has highlighted neutrophils as key effectors of innate and adaptive immune responses through a wide array of newly discovered functions. Accordingly, neutrophils are emerging as highly versatile cells that are able to acquire different, often opposite, functional capacities depending on the microenvironment and their differentiation status. Herein, we review the current knowledge on the multiple functions that neutrophils exhibit through the different stages of alloHSCT, from the hematopoietic stem cell (HSC) mobilization in the donor to the immunological reconstitution that occurs in the recipient following HSC infusion. We also discuss the influence exerted on neutrophils by the immunosuppressive drugs delivered in the course of alloHSCT as part of graft-versus-host disease (GVHD) prophylaxis. Finally, the potential involvement of neutrophils in alloHSCT-related complications, such as transplant-associated thrombotic microangiopathy (TA-TMA), acute and chronic GVHD, and cytomegalovirus (CMV) reactivation, is also discussed. Based on the data reviewed herein, the role played by neutrophils in alloHSCT is far greater than a simple antimicrobial role. However, much remains to be investigated in terms of the potential functions that neutrophils might exert during a highly complex procedure such as alloHSCT

Methods for interpreting lists of affected genes obtained in a DNA microarray experiment

BACKGROUND: The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence) and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding) workshop focusing on post analysis of microarray data. The participating groups were provided with identical lists of microarray probes, including test statistics for three different contrasts, and the normalised log-ratios for each array, to be used as the starting point for interpreting the affected probes. The data originated from a microarray experiment conducted to study the host reactions in broilers occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria. RESULTS: Several conceptually different analytical approaches, using both commercial and public available software, were applied by the participating groups. The following tools were used: Ingenuity Pathway Analysis, MAPPFinder, LIMMA, GOstats, GOEAST, GOTM, Globaltest, TopGO, ArrayUnlock, Pathway Studio, GIST and AnnotationDbi. The main focus of the approaches was to utilise the relation between probes/genes and their gene ontology and pathways to interpret the affected probes/genes. The lack of a well-annotated chicken genome did though limit the possibilities to fully explore the tools. The main results from these analyses showed that the biological interpretation is highly dependent on the statistical method used but that some common biological conclusions could be reached. CONCLUSION: It is highly recommended to test different analytical methods on the same data set and compare the results to obtain a reliable biological interpretation of the affected genes in a DNA microarray experimen

Selective inhibition of IgG-mediated phagocytosis in gelsolin-deficient murine neutrophils

Phagocytosis and the microbicidal functions of neutrophils require dynamic changes of the actin cytoskeleton. We have investigated the role of gelsolin, a calcium-dependent actin severing and capping protein, in peripheral blood neutrophils from gelsolin-null (Gsn-) mice. The phagocytosis of complement opsonized yeast was only minimally affected. In contrast, phagocytosis of IgG-opsonized yeast was reduced close to background level in Gsn- neutrophils. Thus, gelsolin is essential for efficient IgG- but not complement-mediated phagocytosis. Furthermore, attachment of IgG-opsonized yeast to Gsn- neutrophils was reduced ( approximately 50%) but not to the same extent as ingestion ( approximately 73%). This was not due to reduced surface expression of the Fcgamma-receptor or its lateral mobility. This suggests that attachment and ingestion of IgG-opsonized yeast by murine neutrophils are actin-dependent and gelsolin is important for both steps in phagocytosis. We also investigated granule exocytosis and several steps in phagosome processing, namely the formation of actin around the phagosome, translocation of granules, and activation of the NADPH-oxidase. All these functions were normal in Gsn- neutrophils. Thus, the role of gelsolin is specific for IgG-mediated phagocytosis. Our data suggest that gelsolin is part of the molecular machinery that distinguishes complement and IgG-mediated phagocytosis. The latter requires a more dynamic reorganization of the cytoskeleton