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      Transcriptomic Analysis of the Host Response to Giardia duodenalis Infection Reveals Redundant Mechanisms for Parasite Control

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          ABSTRACT

          The immune system has numerous mechanisms that it can use to combat pathogens and eliminate infections. Nevertheless, studies of immune responses often focus on single pathways required for protective responses. We applied microarray analysis of RNA in order to investigate the types of immune responses produced against infection with the intestinal pathogen Giardia duodenalis. Infection with G. duodenalis is one of the most common causes of diarrheal disease in the world. While several potential antiparasitic effector mechanisms, including complement lysis, nitric oxide (NO), and α-defensin peptides, have been shown to inhibit parasite growth or kill Giardia in vitro, studies in vivo have thus far shown clear roles only for antibody and mast cell responses in parasite control. A total of 96 transcripts were identified as being upregulated or repressed more than 2-fold in the small intestine 10 days following infection. Microarray data were validated using quantitative PCR. The most abundant category of transcripts was antibody genes, while the most highly induced transcripts were all mast cell proteases. Among the other induced transcripts was matrix metalloprotease 7 (Mmp7), the protease responsible for production of mature α-defensins in mice. While infections in Mmp7-deficient mice showed only a small increase in parasite numbers, combined genetic deletion of Mmp7 and inducible nitric oxide synthase (iNOS, Nos2) or pharmacological blockade of iNOS in Mmp7-deficient mice resulted in significant increases in parasite loads following infection. Thus, α-defensins and NO are redundant mechanisms for control of Giardia infections in vivo.

          IMPORTANCE

          The immune system has multiple weapons which it uses to help control infections. Many infections result in activation of several of these response mechanisms, but it is not always clear which responses actually contribute to control of the pathogen and which are bystander effects. This study used the intestinal parasite Giardia duodenalis to examine the redundancy in immune responses during infections in mice. Our results showed that at least four distinct mechanisms are activated following infections. Furthermore, by blocking two pathways at the same time, we showed that both mechanisms contribute to control of the infection, whereas blocking single responses showed no or minimal effect in these cases.

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          Most cited references 27

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          Biology of Giardia lamblia.

           Lorne Adam (2001)
          Giardia lamblia is a common cause of diarrhea in humans and other mammals throughout the world. It can be distinguished from other Giardia species by light or electron microscopy. The two major genotypes of G. lamblia that infect humans are so different genetically and biologically that they may warrant separate species or subspecies designations. Trophozoites have nuclei and a well-developed cytoskeleton but lack mitochondria, peroxisomes, and the components of oxidative phosphorylation. They have an endomembrane system with at least some characteristics of the Golgi complex and encoplasmic reticulum, which becomes more extensive in encysting organisms. The primitive nature of the organelles and metabolism, as well as small-subunit rRNA phylogeny, has led to the proposal that Giardia spp. are among the most primitive eukaryotes. G. lamblia probably has a ploidy of 4 and a genome size of approximately 10 to 12 Mb divided among five chromosomes. Most genes have short 5' and 3' untranslated regions and promoter regions that are near the initiation codon. Trophozoites exhibit antigenic variation of an extensive repertoire of cysteine-rich variant-specific surface proteins. Expression is allele specific, and changes in expression from one vsp gene to another have not been associated with sequence alterations or gene rearrangements. The Giardia genome project promises to greatly increase our understanding of this interesting and enigmatic organism.
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            Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense.

            Precursors of alpha-defensin peptides require activation for bactericidal activity. In mouse small intestine, matrilysin colocalized with alpha-defensins (cryptdins) in Paneth cell granules, and in vitro it cleaved the pro segment from cryptdin precursors. Matrilysin-deficient (MAT-/-) mice lacked mature cryptdins and accumulated precursor molecules. Intestinal peptide preparations from MAT-/- mice had decreased antimicrobial activity. Orally administered bacteria survived in greater numbers and were more virulent in MAT-/- mice than in MAT+/+ mice. Thus, matrilysin functions in intestinal mucosal defense by regulating the activity of defensins, which may be a common role for this metalloproteinase in its numerous epithelial sites of expression.
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              Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR.

              Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the exponential phase of the PCR reaction using a dual-labelled fluorogenic probe, has been developed. This method allows direct detection of PCR-product formation by measuring the increase in fluorescent emission continuously during the PCR reaction. Here we present data validating this PCR-method for the quantification of murine cytokines and other factors playing a role in immune regulation (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-gammaTNF-alphaTGF-beta and iNOS). For each substance of interest, a set of primers and internal probe was designed, which specifically amplify the target cDNA, not co-amplifying contaminating genomic DNA. Furthermore, a corresponding reference plasmid cDNA clone was constructed, allowing direct quantification. Additionally, normalization to the housekeeping genes beta-actin or GAPDH was performed. The assay is very sensitive and accurate. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations and decreasing the potential risk of PCR contamination. Copyright 1999 Academic Press.
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                Author and article information

                Journal
                mBio
                MBio
                mbio
                mbio
                mBio
                mBio
                American Society of Microbiology (1752 N St., N.W., Washington, DC )
                2150-7511
                5 November 2013
                Nov-Dec 2013
                : 4
                : 6
                Affiliations
                Department of Biology, Georgetown University, Washington, DC, USA [ a ]
                Agilent Technologies, Santa Clara, California, USA [ b ]
                Author notes
                Address correspondence to Steven M. Singer, sms3@ 123456georgetown.edu .
                [*]

                Present address: Ernest A. Tako, Department of Obstetrics and Gynecology, Bronx Lebanon Hospital Center, Bronx, New York, USA; Maryam F. Hassimi, Genomics Core Laboratory, Sloan-Kettering Institute, New York, New York, USA.

                Editor Louis Miller, NIAID/NIH

                Article
                mBio00660-13
                10.1128/mBio.00660-13
                3892777
                24194537
                Copyright © 2013 Tako et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 7
                Categories
                Research Article
                Custom metadata
                November/December 2013

                Life sciences

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