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1.  18β-Glycyrrhetinic Acid Delivered Orally Induces Isolated Lymphoid Follicle Maturation at the Intestinal Mucosa and Attenuates Rotavirus Shedding 
PLoS ONE  2012;7(11):e49491.
Glycyrrhizin, an abundant bioactive component of the medicinal licorice root is rapidly metabolized by gut commensal bacteria into 18β-glycyrrhetinic acid (GRA). Either or both of these compounds have been shown to have antiviral, anti-hepatotoxic, anti-ulcerative, anti-tumor, anti-allergenic and anti-inflammatory activity in vitro or in vivo. In this study, the ability of GRA to modulate immune responses at the small intestinal mucosa when delivered orally was investigated. Analysis of cytokine transcription in duodenal and ileal tissue in response to GRA treatment revealed a pattern of chemokine and chemokine receptor gene expression predictive of B cell recruitment to the gut. Consistent with this finding, GRA induced increases in CD19+ B cells in the lamina propria and B220+ B cell aggregates framed by CD11c+ dendritic cells in structures resembling isolated lymphoid follicles (ILF). Using a mouse model of rotavirus infection, GRA reduced the duration of viral antigen shedding, and endpoint serum antibody titers were higher in GRA-treated animals. Together the data suggest GRA delivered orally augments lymphocyte recruitment to the intestinal mucosa and induces maturation of B cell-rich ILF independently of ectopic antigenic stimulus. These results provide further support a role for dietary ligands in modulation of dynamic intestinal lymphoid tissue.
PMCID: PMC3496704  PMID: 23152913
2.  Differential Induction of Isolated Lymphoid Follicles in the Gut by 18β-Glycyrrhetinic Acid 
PLoS ONE  2014;9(7):e100878.
18β-glycyrrhetinic acid (GRA) is a pharmacologically active component of licorice root with documented immunomodulatory properties. We reported that GRA administered orally to mice induces B cell recruitment to isolated lymphoid follicles (ILF) in the small intestine and shortens the duration of rotavirus antigen shedding. ILF are dynamic lymphoid tissues in the gut acquired post-natally upon colonization with commensal bacteria and mature through B cell recruitment to the follicles, resulting in up-regulation of IgA synthesis in response to changes in the composition of microbiota. In this study, we investigated potential mechanisms by which GRA induces ILF maturation in the ileum and the colon using mice depleted of enteric bacteria and a select group of mice genetically deficient in pattern recognition receptors. The data show GRA was unable to induce ILF maturation in ileums of mice devoid of commensal bacteria, MyD88−/− or NOD2−/− mice, but differentially induced ILF in colons. Increased expression of chemokine and chemokine receptor genes that modulate B and T cell recruitment to the mucosa were in part dependent on NOD2, TLR, and signaling adaptor protein MyD88. Together the results suggest GRA induces ILF through cooperative signals provided by bacterial ligands under normal conditions to induce B cell recruitment to ILF to the gut, but that the relative contribution of these signals differ between ileum and colon.
PMCID: PMC4081046  PMID: 24992099
3.  Granzyme B (GraB) Autonomously Crosses the Cell Membrane and Perforin Initiates Apoptosis and GraB Nuclear Localization 
Granzyme B (GraB) induces apoptosis in the presence of perforin. Perforin polymerizes in the cell membrane to form a nonspecific ion pore, but it is not known where GraB acts to initiate the events that ultimately lead to apoptosis. It has been hypothesized that GraB enters the target cell through a perforin channel and then initiates apoptosis by cleaving and activating members of the ICE/Ced-3 family of cell death proteases. To determine if GraB can enter the cell, we treated YAC-1 or HeLa cells with FITC-labeled GraB and measured intracellular fluorescence with a high sensitivity CCD camera and image analyzer. GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min. Uptake was inhibited at low temperature (4°C) and by pretreatment with metabolic inhibitors, NaF and DNP, or cytochalasin B, a drug that both blocks microfilament formation, and FITC-GraB remained on the cell membrane localized in patches. With the simultaneous addition of perforin and FITC-GraB, no significant increase in cytoplasmic fluorescence was observed over that found in cells treated only with FITC-GraB. However, FITC-GraB was now detected in the nucleus of apoptotic cells labeling apoptotic bodies and localized areas within and along the nuclear membrane. The ability of GraB to enter cells in the absence of perforin was reexamined using anti-GraB antibody immunogold staining of ultrathin cryosections of cells incubated with GraB. Within 15 min, gold particles were detected both on the plasma membrane and in the cytoplasm of cells with some gold staining adjacent to the nuclear envelope but not in the nucleus. Cells internalizing GraB in the absence of perforin appeared morphologically normal by Hoechst staining and electron microscopy. GraB directly microinjected into the cytoplasm of B16 melanoma cells induced transient plasma membrane blebbing and nuclear coarsening but the cells did not become frankly apoptotic unless perforin was added. We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus.
PMCID: PMC2196167  PMID: 9120391
4.  Curative Effect of 18β-Glycyrrhetinic Acid in Experimental Visceral Leishmaniasis Depends on Phosphatase-Dependent Modulation of Cellular MAP Kinases 
PLoS ONE  2011;6(12):e29062.
We earlier showed that 18β-glycyrrhetinic acid (GRA), a pentacyclic triterpenoid from licorice root, could completely cure visceral leishmaniasis in BALB/c mouse model. This was associated with induction of nitric oxide and proinflammatory cytokine production through the up regulation of NF-κB. In the present study we tried to decipher the underlying cellular mechanisms of the curative effect of GRA. Analysis of MAP kinase pathways revealed that GRA caused strong activation of p38 and to a lesser extent, ERK in bone marrow-derived macrophages (BMDM). Almost complete abrogation of GRA-induced cytokine production in presence of specific inhibitors of p38 and ERK1/2 confirmed the involvement of these MAP kinases in GRA-mediated responses. GRA induced mitogen- and stress-activated protein kinase (MSK1) activity in a time-dependent manner suggested that GRA-mediated NF-κB transactivation is mediated by p38, ERK and MSK1 pathway. As kinase/phosphatase balance plays an important role in modulating infection, the effect of GRA on MAPK directed phosphatases (MKP) was studied. GRA markedly reduced the expression and activities of three phosphatases, MKP1, MKP3 and protein phosphatase 2A (PP2A) along with a substantial reduction of p38 and ERK dephosphorylation in infected BMDM. Similarly in the in vivo situation, GRA treatment of L. donovani-infected BALB/c mice caused marked reduction of spleen parasite burden associated with concomitant decrease of individual phosphatase levels. However, activation of kinases also played an important role as the protective effect of GRA was significantly abrogated by pharmacological inhibition of p38 and ERK pathway. Curative effect of GRA may, therefore, be associated with restoration of proper cellular kinase/phosphatase balance, rather than modulation of either kinases or phosphatases.
PMCID: PMC3237588  PMID: 22194991
5.  18β-Glycyrrhetinic Acid Inhibits Methicillin-Resistant Staphylococcus aureus Survival and Attenuates Virulence Gene Expression 
Methicillin-resistant Staphylococcus aureus (MRSA) has become a major source of infection in hospitals and in the community. Increasing antibiotic resistance in S. aureus strains has created a need for alternative therapies to treat disease. A component of the licorice root Glycyrrhiza spp., 18β-glycyrrhetinic acid (GRA), has been shown to have antiviral, antitumor, and antibacterial activity. This investigation explores the in vitro and in vivo effects of GRA on MRSA pulsed-field gel electrophoresis (PFGE) type USA300. GRA exhibited bactericidal activity at concentrations exceeding 0.223 μM. Upon exposure of S. aureus to sublytic concentrations of GRA, we observed a reduction in expression of key virulence genes, including saeR and hla. In murine models of skin and soft tissue infection, topical GRA treatment significantly reduced skin lesion size and decreased the expression of saeR and hla genes. Our investigation demonstrates that at high concentrations GRA is bactericidal to MRSA and at sublethal doses it reduces virulence gene expression in S. aureus both in vitro and in vivo.
PMCID: PMC3535912  PMID: 23114775
6.  Biochemical and Genetic Analysis of the γ-Resorcylate (2,6-Dihydroxybenzoate) Catabolic Pathway in Rhizobium sp. Strain MTP-10005: Identification and Functional Analysis of Its Gene Cluster▿  
Journal of Bacteriology  2006;189(5):1573-1581.
We identified a gene cluster that is involved in the γ-resorcylate (2,6-dihydroxybenzoate) catabolism of the aerobic bacterium Rhizobium sp. strain MTP-10005. The cluster consists of the graRDAFCBEK genes, and graA, graB, graC, and graD were heterologously expressed in Escherichia coli. Enzymological studies showed that graD, graA, graC, and graB encode the reductase (GraD) and oxygenase (GraA) components of a resorcinol hydroxylase (EC 1.14.13.x), a maleylacetate reductase (GraC) (EC, and a hydroxyquinol 1,2-dioxygenase (GraB) (EC Bioinformatic analyses suggested that graE, graR, and graK encode a protein with an unknown function (GraE), a MarR-type transcriptional regulator (GraR), and a benzoate transporter (GraK). Quantitative reverse transcription-PCR of graF, which encodes γ-resorcylate decarboxylase, revealed that the maximum relative mRNA expression level ([5.93 ± 0.82] × 10−4) of graF was detected in the total RNA of the cells after one hour of cultivation when γ-resorcylate was used as the sole carbon source. Reverse transcription-PCR of graDAFCBE showed that these genes are transcribed as a single mRNA and that the transcription of the gene cluster is induced by γ-resorcylate. These results suggested that the graDAFCBE genes are responsible as an operon for the growth of Rhizobium sp. strain MTP-10005 on γ-resorcylate and are probably regulated by GraR at the transcriptional level. This is the first report of the γ-resorcylate catabolic pathway in an aerobic bacterium.
PMCID: PMC1855702  PMID: 17158677
7.  A validation study of the psychometric properties of the Groningen Reflection Ability Scale 
BMC Medical Education  2014;14(1):214.
Reflection, the ability to examine critically one’s own learning and functioning, is considered important for ‘the good doctor’. The Groningen Reflection Ability Scale (GRAS) is an instrument measuring student reflection, which has not yet been validated beyond the original Dutch study. The aim of this study was to adapt GRAS for use in a Danish setting and to investigate the psychometric properties of GRAS-DK.
We performed a cross-cultural adaptation of GRAS from Dutch to Danish. Next, we collected primary data online, performed a retest, analysed data descriptively, estimated measurement error, performed an exploratory and a confirmatory factor analysis to test the proposed three-factor structure.
361 (69%) of 523 invited students completed GRAS-DK. Their mean score was 88 (SD = 11.42; scale maximum 115). Scores were approximately normally distributed. Measurement error and test-retest score differences were acceptable, apart from a few extreme outliers. However, the confirmatory factor analysis did not replicate the original three-factor model and neither could a one-dimensional structure be confirmed.
GRAS is already in use, however we advise that use of GRAS-DK for effect measurements and group comparison awaits further review and validation studies. Our negative finding might be explained by a weak conceptualisation of personal reflection.
PMCID: PMC4286925  PMID: 25304774
Assessment; Instrument; Reflection; Undergraduate medical education; Validation
8.  Molecular Basis of Resistance to Muramidase and Cationic Antimicrobial Peptide Activity of Lysozyme in Staphylococci 
PLoS Pathogens  2007;3(7):e102.
It has been shown recently that modification of peptidoglycan by O-acetylation renders pathogenic staphylococci resistant to the muramidase activity of lysozyme. Here, we show that a Staphylococcus aureus double mutant defective in O-acetyltransferase A (OatA), and the glycopeptide resistance-associated two-component system, GraRS, is much more sensitive to lysozyme than S. aureus with the oatA mutation alone. The graRS single mutant was resistant to the muramidase activity of lysozyme, but was sensitive to cationic antimicrobial peptides (CAMPs) such as the human lysozyme-derived peptide 107R-A-W-V-A-W-R-N-R115 (LP9), polymyxin B, or gallidermin. A comparative transcriptome analysis of wild type and the graRS mutant revealed that GraRS controls 248 genes. It up-regulates global regulators (rot, sarS, or mgrA), various colonization factors, and exotoxin-encoding genes, as well as the ica and dlt operons. A pronounced decrease in the expression of the latter two operons explains why the graRS mutant is also biofilm-negative. The decrease of the dlt transcript in the graRS mutant correlates with a 46.7% decrease in the content of esterified d-alanyl groups in teichoic acids. The oatA/dltA double mutant showed the highest sensitivity to lysozyme; this mutant completely lacks teichoic acid–bound d-alanine esters, which are responsible for the increased susceptibility to CAMPs and peptidoglycan O-acetylation. Our results demonstrate that resistance to lysozyme can be dissected into genes mediating resistance to its muramidase activity (oatA) and genes mediating resistance to CAMPs (graRS and dlt). The two lysozyme activities act synergistically, as the oatA/dltA or oatA/graRS double mutants are much more susceptible to lysozyme than each of the single mutants.
Author Summary
In humans, lysozyme plays an important role in the suppression of bacterial infections. However, some bacterial pathogens, such as Staphylococcus aureus, are completely resistant to lysozyme. Here we demonstrate that lysozyme acts on S. aureus in two ways: as a muramidase (cell wall lytic enzyme) and as a cationic antimicrobial peptide (CAMP). S. aureus has developed resistance mechanisms against both activities by modifying distinct cell wall structures. Modification of the peptidoglycan by O-acetylation (OatA) renders the cells resistant to the muramidase activity. Modification of teichoic acids by d-alanine esterification (Dlt) renders the cells resistant to lysozyme's CAMPs and other CAMPs. Transcriptome analysis of the glycopeptide resistance-associated (GraRS) two-component system revealed that this global regulator controls 248 genes such as other global regulators, colonization factors, or exotoxin-encoding genes. Since GraRS also upregulates the dlt operon, it was not surprising that in the graRS mutant teichoic acid d-alanylation is markedly decreased, which explains its increased sensitivity to CAMPs. By comparative analysis of mutants we were able to dissect genes that were responsive to the dual activities of lysozyme. Here we show how efficiently S. aureus is protected from the human defense system, which enables this pathogen to cause persistent infections.
PMCID: PMC1933452  PMID: 17676995
9.  Rotavirus Disrupts Calcium Homeostasis by NSP4 Viroporin Activity 
mBio  2010;1(5):e00265-10.
Many viruses alter intracellular calcium homeostasis. The rotavirus nonstructural protein 4 (NSP4), an endoplasmic reticulum (ER) transmembrane glycoprotein, increases intracellular levels of cytoplasmic Ca2+ ([Ca2+]cyto) through a phospholipase C-independent pathway, which is required for virus replication and morphogenesis. However, the NSP4 domain and mechanism that increases [Ca2+]cyto are unknown. We identified an NSP4 domain (amino acids [aa] 47 to 90) that inserts into membranes and has structural characteristics of viroporins, a class of small hydrophobic viral proteins that disrupt membrane integrity and ion homeostasis to facilitate virus entry, assembly, or release. Mutational analysis showed that NSP4 viroporin activity was mediated by an amphipathic α-helical domain downstream of a conserved lysine cluster. The lysine cluster directed integral membrane insertion of the viroporin domain and was critical for viroporin activity. In epithelial cells, expression of wild-type NSP4 increased the levels of free cytoplasmic Ca2+ by 3.7-fold, but NSP4 viroporin mutants maintained low levels of [Ca2+]cyto, were retained in the ER, and failed to form cytoplasmic vesicular structures, called puncta, which surround viral replication and assembly sites in rotavirus-infected cells. When [Ca2+]cyto was increased pharmacologically with thapsigargin, viroporin mutants formed puncta, showing that elevation of calcium levels and puncta formation are distinct functions of NSP4 and indicating that NSP4 directly or indirectly responds to elevated cytoplasmic calcium levels. NSP4 viroporin activity establishes the mechanism for NSP4-mediated elevation of [Ca2+]cyto, a critical event that regulates rotavirus replication and virion assembly.
Rotavirus is the leading cause of viral gastroenteritis in children and young animals. Rotavirus infection and expression of nonstructural protein 4 (NSP4) alone dramatically increase cytosolic calcium, which is essential for replication and assembly of infectious virions. This work identifies the intracellular mechanism by which NSP4 disrupts calcium homeostasis by showing that NSP4 is a viroporin, a class of virus-encoded transmembrane pores. Mutational analyses identified residues critical for viroporin activity. Viroporin mutants did not elevate the levels of cytoplasmic calcium in mammalian cells and were maintained in the endoplasmic reticulum rather than forming punctate vesicular structures that are critical for virus replication and morphogenesis. Pharmacological elevation of cytoplasmic calcium levels rescued puncta formation in viroporin mutants, demonstrating that elevation of calcium levels and puncta formation are distinct NSP4 functions. While viroporins typically function in virus entry or release, elevation of calcium levels by NSP4 viroporin activity may serve as a regulatory function to facilitate virus replication and assembly.
PMCID: PMC2999940  PMID: 21151776
10.  Toxoplasma gondii sporozoites form a transient parasitophorous vacuole that is impermeable and contains only a subset of dense-granule proteins. 
Infection and Immunity  1997;65(11):4598-4605.
Toxoplasma gondii sporozoites form two parasitophorous vacuoles during development within host cells, the first (PV1) during host cell invasion and the second (PV2) 18 to 24 h postinoculation. PV1 is structurally distinctive due to its large size, yet it lacks a tubulovesicular network (C. A. Speer, M. Tilley, M. Temple, J. A. Blixt, J. P. Dubey, and M. W. White, Mol. Biochem. Parasitol. 75:75-86, 1995). Confirming the finding that sporozoites have a different electron-dense-granule composition, we have now found that sporozoites within oocysts lack the mRNAs encoding the 5' nucleoside triphosphate hydrolases (NTPase). NTPase first appears 12 h postinfection. Other tachyzoite dense-granule proteins, GRA1, GRA2, GRA4, GRA5, and GRA6, were detected in oocyst extracts, and antibodies against these proteins stained granules in the sporozoite cytoplasm. In contrast to tachyzoite invasion of host cells, however, sporozoites did not exocytose the dense-granule proteins GRA1, GRA2, or GRA4 during PV1 formation. Even after NTPase induction, these proteins were retained within cytoplasmic granules rather than being secreted into PV1. Only GRA5 was secreted by the sporozoite during host cell invasion, becoming associated with the membrane surrounding PV1. Microinjection of sporozoite-infected cells with fluorescent dyes showed that PV1 is impermeable to fluorescent dyes with molecular masses as small as 330 Da, indicating that PV1 lacks channels through which molecules can pass from the host cytoplasm into the vacuole. By contrast, lucifer yellow rapidly diffused into PV2, demonstrating the presence of molecular channels. These studies indicate that PV1 and PV2 are morphologically, immunologically, and functionally distinct, and that PV2 appears to be identical to the tachyzoite vacuole. The inaccessibility of PV1 to host cell nutrients may explain why parasite replication does not occur in this vacuole.
PMCID: PMC175660  PMID: 9353039
11.  GraXSR Proteins Interact with the VraFG ABC Transporter To Form a Five-Component System Required for Cationic Antimicrobial Peptide Sensing and Resistance in Staphylococcus aureus 
The GraSR two-component system (TCS) controls cationic antimicrobial peptide (CAMP) resistance in Staphylococcus aureus through the synthesis of enzymes that increase bacterial cell surface positive charges, by d-alanylation of teichoic acids and lysylination of phosphatidylglycerol, leading to electrostatic repulsion of CAMPs. The GraS histidine kinase belongs to the “intramembrane-sensing kinases” subfamily, with a structure featuring a short amino-terminal sensing domain, and two transmembrane helices separated only by a short loop, thought to be buried in the cytoplasmic membrane. The GraSR TCS is in fact a multicomponent system, requiring at least one accessory protein, GraX, in order to function, which, as we show here, acts by signaling through the GraS kinase. The graXRS genes are located immediately upstream from genes encoding an ABC transporter, vraFG, whose expression is controlled by GraSR. We demonstrated that the VraFG transporter does not act as a detoxification module, as it cannot confer resistance when produced on its own, but instead plays an essential role by sensing the presence of CAMPs and signaling through GraS to activate GraR-dependent transcription. A bacterial two-hybrid approach, designed to identify interactions between the GraXSR and VraFG proteins, was carried out in order to understand how they act in detecting and signaling the presence of CAMPs. We identified many interactions between these protein pairs, notably between the GraS kinase and both GraX and the VraG permease, indicating the existence of an original five-component system involved in CAMP sensing and signal transduction to promote S. aureus resistance.
PMCID: PMC3264281  PMID: 22123691
12.  Genomic Analysis Reveals a Point Mutation in the Two-Component Sensor Gene graS That Leads to Intermediate Vancomycin Resistance in Clinical Staphylococcus aureus▿  
Antimicrobial Agents and Chemotherapy  2008;52(10):3755-3762.
Methicillin-resistant Staphylococcus aureus (MRSA), once restricted to hospitals, is spreading rapidly through the wider community. Resistance to vancomycin, the principal drug used to treat MRSA infections, has only recently emerged, is mainly low level, and characteristically appears during vancomycin therapy (vancomycin-intermediate S. aureus [VISA] and hetero-resistant VISA). This phenomenon suggests the adaptation of MRSA through mutation, although defining the mutations leading to resistance in clinical isolates has been difficult. We studied a vancomycin-susceptible clinical MRSA isolate (MIC of 1 μg/ml) and compared it with an isogenic blood culture isolate from the same patient, despite 42 days of vancomycin treatment (MIC of 4 μg/ml). A whole-genome sequencing approach allowed the nearly complete assembly of the genome sequences of the two isolates and revealed only six nucleotide substitutions in the VISA strain compared with the parent strain. One mutation occurred in graS, encoding a putative two-component regulatory sensor, leading to a change from a polar to a nonpolar amino acid (T136I) in the conserved histidine region of the predicted protein. Replacing the graS allele of the vancomycin-susceptible parent strain with the graS allele from the VISA derivative resulted in increased vancomycin resistance at a level between those of the vancomycin-susceptible S. aureus and VISA clinical isolates, confirming a role for graRS in VISA. Our study suggests that MRSA is able to develop clinically significant vancomycin resistance via a single point mutation, and the two-component regulatory system graRS is a key mediator of this resistance. However, additional mutations are likely required to express the full VISA phenotype.
PMCID: PMC2565880  PMID: 18644967
13.  Interaction between Parasitophorous Vacuolar Membrane-associated GRA3 and Calcium Modulating Ligand of Host Cell Endoplasmic Reticulum in the Parasitism of Toxoplasma gondii 
A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.
PMCID: PMC2612604  PMID: 19127325
Toxoplasma gondii; GRA3; cDNA sequence; yeast two-hybrid; CAMLG; PVM-ER interaction
14.  Rotavirus Antigenemia in Children Is Associated with Viremia 
PLoS Medicine  2007;4(4):e121.
Antigenemia is commonly detected in rotavirus-infected children. Although rotavirus RNA has been detected in serum, definitive proof of rotavirus viremia has not been shown. We aimed to analyze a defined patient population to determine if infectious virus could be detected in sera from children with rotavirus antigenemia.
Methods and Findings
Serum samples obtained upon hospitalization from children with gastroenteritis (57 stool rotavirus-positive and 41 rotavirus-negative), children with diagnosed bronchiolitis of known (n = 58) or unknown (n = 17) viral etiology, children with noninfectious, nonchronic conditions (n = 17), and healthy adults (n = 28) were tested for rotavirus antigen by enzyme immunoassay (EIA). Results of serum antigen testing were assessed for association with clinical and immunological attributes of the children. Rotavirus antigenemia was detected in 90% (51/57) of children with rotavirus-positive stools, in 89% (8/9) of children without diarrhea but with rotavirus-positive stools, in 12% (2/17) of children with bronchiolitis of unknown etiology without gastroenteritis, and in 12% (5/41) of children with gastroenteritis but with rotavirus-negative stools. Antigenemia was not detected in sera from children with noninfectious nonchronic conditions, children with bronchiolitis of known etiology and no gastroenteritis, or healthy adults. Neither age nor timing of serum collection within eight days after onset of gastroenteritis significantly affected levels of antigenemia, and there was no correlation between antigenemia and viral genotype. However, there was a negative correlation between serum rotavirus antigen and acute rotavirus-specific serum IgA (r = −0.44, p = 0.025) and IgG (r = −0.40, p = 0.01) titers. We examined 11 antigen-positive and nine antigen-negative sera for infectious virus after three blind serial passages in HT-29 cells using immunofluorescence staining for rotavirus structural and nonstructural proteins. Infectious virus was detected in 11/11 (100%) sera from serum antigen-positive children and in two out of nine (22%) sera samples from antigen-negative children (p = 0.002).
Most children infected with rotavirus are viremic. The presence of viremia is directly related to the detection of antigenemia and is independent of the presence of diarrhea. Antigenemia load is inversely related to the titer of antirotavirus antibody in the serum. The finding of infectious rotavirus in the blood suggests extraintestinal involvement in rotavirus pathogenesis; however, the impact of rotavirus viremia on clinical manifestations of infection is unknown.
A study of 57 children with rotavirus-positive stools found that most were viremic, and that the presence of viremia was directly related to antigenemia and independent of the presence of diarrhea.
Editors' Summary
Rotavirus is a type of virus that is the commonest cause of severe diarrhea among children worldwide. It is passed from one person to another when virus present in the stool of an infected person is swallowed by another individual. The infection causes vomiting, watery diarrhea, and fever; many children need to be hospitalized as a result and globally more than 600,000 children are thought to die as a result of rotavirus infections per year. Evidence from single case descriptions of infected children have suggested that rotavirus might also cause symptoms outside of the gut—for example, in the lungs or brain. Previous studies have found fragments of rotavirus, for example RNA or parts of virus protein, in tissues outside of the gut such as liver, kidney, blood, and heart. However, simply finding fragments such as RNA or protein does not necessarily mean that rotavirus infects these tissues.
Why Was This Study Done?
These researchers wanted to find out whether rotavirus was present in the blood of infected children. If evidence of rotavirus in the blood was found, this might help explain why some children infected with rotavirus have symptoms affecting organs other than the gut.
What Did the Researchers Do and Find?
In this study, five groups of patients were recruited and tests were done on each to find out whether infectious rotavirus was present in their bloodstream, and also whether the researchers could detect rotavirus components in blood using antibodies against particular parts of the rotavirus particle. The five groups of patients that were compared included children hospitalized with gastroenteritis; children hospitalized with noninfectious conditions; healthy adult laboratory workers; children with lung infections from known viruses; and finally children with lung infections of unknown cause. The researchers found that among the children with gastroenteritis who had rotavirus in their stool, 90% also had evidence of rotavirus particles in their bloodstream. By contrast, control individuals (either children who were hospitalized with noninfectious conditions or healthy adults) did not have rotavirus particles in blood. A small proportion of children with gastroenteritis but no rotavirus in their stool did have rotavirus particles in blood. Interestingly, a small proportion of the children who had lung infections (but in whom no known virus had been identified as the cause) showed evidence of rotavirus in their bloodstreams. Finally, in a group of 11 children with evidence of rotavirus particles in their bloodstreams, all were found to also have infectious virus present in the blood.
What Do These Findings Mean?
These results show that rotavirus is able to spread beyond the gut and into the bloodstream. The finding that rotavirus can spread into the bloodstream may explain some earlier suggestions that rotavirus is responsible for symptoms outside of the gut. However, it is not yet clear how commonly children with rotavirus have other symptoms resulting from the virus spreading into their bloodstream.
Additional Information.
Please access these Web sites via the online version of this summary at
Read the related PLoS Medicine Perspective article by David Candy
Information from the World Health Organization Initiative for Vaccine Research on rotavirus disease burden; see also the Rotavirus Vaccine Program, a partnership that aims to develop rotavirus vaccines appropriate for use in developing countries
Information from the US Centers for Disease Control and Prevention about rotavirus
Health Encyclopedia entry from the UK's NHS Direct on Rotavirus
PMCID: PMC1852122  PMID: 17439294
15.  Selection of Polymorphic Peptides from GRA6 and GRA7 Sequences of Toxoplasma gondii Strains To Be Used in Serotyping▿  
The evaluation of Toxoplasma gondii isolates obtained from geographical environments other than Europe and North America revealed the existence of atypical strains that are not included in the three archetypal clonal lineages (lineages I, II, and III). GRA6 and GRA7 are polymorphic genes that have been used for the genotyping of Toxoplasma. The coding regions of GRA6 and GRA7 from 49 nonarchetypal strains were sequenced and compared with the sequences of type I, II, and III reference strains. Eighteen and 10 different amino acid sequences were found for GRA6 and GRA7, respectively. The polymorphisms found between the different sequences were analyzed, with the objective of defining peptides to be used for the serotyping of Toxoplasma infections. Two peptides specific for clonal lineages I and III (peptides GRA7I and GRA7III, respectively) were selected from the GRA7 locus. Three peptides specific for some atypical strains (peptides Am6, Af6, and Am7) were selected from both the GRA6 and the GRA7 loci. Serum samples from humans infected with Toxoplasma strains of known genotypes were serotyped with the selected peptides. Peptide GRA7III seems to be a good candidate for the serotyping of infections caused by type III strains. Peptide GRA7I had a very low sensitivity. Peptides Am6 and Af6 had low specificities, since they reacted with serum samples from patients infected with strains belonging to the three archetypal lineages. Although peptide Am7 was specific, it had low sensitivity.
PMCID: PMC2725539  PMID: 19494084
16.  Nucleolar translocalization of GRA10 of Toxoplasma gondii transfectionally expressed in HeLa cells 
Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.
PMCID: PMC2526324  PMID: 17876161
Toxoplasma gondii; GRA10; transfection; nucleolar localization; NoLS; TAF1B; rRNA synthesis
17.  Chemoinformatic Analysis of GRAS (Generally Recognized as Safe) Flavor Chemicals and Natural Products 
PLoS ONE  2012;7(11):e50798.
Food materials designated as “Generally Recognized as Safe” (GRAS) are attracting the attention of researchers in their attempts to systematically identify compounds with putative health-related benefits. In particular, there is currently a great deal of interest in exploring possible secondary benefits of flavor ingredients, such as those relating to health and wellness. One step in this direction is the comprehensive characterization of the chemical structures contained in databases of flavoring substances. Herein, we report a comprehensive analysis of the recently updated FEMA GRAS list of flavoring substances (discrete chemical entities only). Databases of natural products, approved drugs and a large set of commercial molecules were used as references. Remarkably, natural products continue to be an important source of bioactive compounds for drug discovery and nutraceutical purposes. The comparison of five collections of compounds of interest was performed using molecular properties, rings, atom counts and structural fingerprints. It was found that the molecular size of the GRAS flavoring substances is, in general, smaller cf. members of the other databases analyzed. The lipophilicity profile of the GRAS database, a key property to predict human bioavailability, is similar to approved drugs. Several GRAS chemicals overlap to a broad region of the property space occupied by drugs. The GRAS list analyzed in this work has high structural diversity, comparable to approved drugs, natural products and libraries of screening compounds. This study represents one step towards the use of the distinctive features of the flavoring chemicals contained in the GRAS list and natural products to systematically search for compounds with potential health-related benefits.
PMCID: PMC3511266  PMID: 23226386
18.  Anti-Diabetic Efficacy and Impact on Amino Acid Metabolism of GRA1, a Novel Small-Molecule Glucagon Receptor Antagonist 
PLoS ONE  2012;7(11):e49572.
Hyperglucagonemia is implicated in the pathophysiology of hyperglycemia. Antagonism of the glucagon receptor (GCGR) thus represents a potential approach to diabetes treatment. Herein we report the characterization of GRA1, a novel small-molecule GCGR antagonist that blocks glucagon binding to the human GCGR (hGCGR) and antagonizes glucagon-induced intracellular accumulation of cAMP with nanomolar potency. GRA1 inhibited glycogenolysis dose-dependently in primary human hepatocytes and in perfused liver from hGCGR mice, a transgenic line of mouse that expresses the hGCGR instead of the murine GCGR. When administered orally to hGCGR mice and rhesus monkeys, GRA1 blocked hyperglycemic responses to exogenous glucagon. In several murine models of diabetes, acute and chronic dosing with GRA1 significantly reduced blood glucose concentrations and moderately increased plasma glucagon and glucagon-like peptide-1. Combination of GRA1 with a dipeptidyl peptidase-4 inhibitor had an additive antihyperglycemic effect in diabetic mice. Hepatic gene-expression profiling in monkeys treated with GRA1 revealed down-regulation of numerous genes involved in amino acid catabolism, an effect that was paralleled by increased amino acid levels in the circulation. In summary, GRA1 is a potent glucagon receptor antagonist with strong antihyperglycemic efficacy in preclinical models and prominent effects on hepatic gene-expression related to amino acid metabolism.
PMCID: PMC3501516  PMID: 23185367
19.  Effects of specific monoclonal antibodies to dense granular proteins on the invasion of Toxoplasma gondii in vitro and in vivo 
Although some reports have been published on the protective effect of antibodies to Toxoplasma gondii surface membrane proteins, few address the inhibitory activity of antibodies to dense granular proteins (GRA proteins). Therefore, we performed a series of experiments to evaluate the inhibitory effects of monoclonal antibodies (mAbs) to GRA proteins (GRA2, 28 kDa; GRA6, 32 kDa) and surface membrane protein (SAG1, 30 kDa) on the invasion of T. gondii tachyzoites. Passive immunization of mice with one of three mAbs following challenge with a lethal dose of tachyzoites significantly increased survival compared with results for mice treated with control ascites. The survival times of mice challenged with tachyzoites pretreated with anti-GRA6 or anti-SAG1 mAb were significantly increased. Mice that received tachyzoites pretreated with both mAb and complement had longer survival times than those that received tachyzoites pretreated with mAb alone. Invasion of tachyzoites into fibroblasts and macrophages was significantly inhibited in the anti-GRA2, anti-GRA6 or anti-SAG1 mAb pretreated group. Pretreatment with mAb and complement inhibited invasion of tachyzoites in both fibroblasts and macrophages. These results suggest that specific antibodies to dense-granule molecules may be useful for controlling infection with T. gondii.
PMCID: PMC2721072  PMID: 11590913
Toxoplasma gondii; monoclonal antibody; dense-granule molecules; complement; major surface protein; host cell invasion
20.  Natural products that reduce rotavirus infectivity identified by a cell-based moderate-throughput screening assay 
Virology Journal  2006;3:68.
There is widespread interest in the use of innate immune modulators as a defense strategy against infectious pathogens. Using rotavirus as a model system, we developed a cell-based, moderate-throughput screening (MTS) assay to identify compounds that reduce rotavirus infectivity in vitro, toward a long-term goal of discovering immunomodulatory agents that enhance innate responses to viral infection.
A natural product library consisting of 280 compounds was screened in the assay and 15 compounds that significantly reduced infectivity without cytotoxicity were identified. Time course analysis of four compounds with previously characterized effects on inflammatory gene expression inhibited replication with pre-treatment times as minimal as 2 hours. Two of these four compounds, α-mangostin and 18-β-glycyrrhetinic acid, activated NFκB and induced IL-8 secretion. The assay is adaptable to other virus systems, and amenable to full automation and adaptation to a high-throughput format.
Identification of several compounds with known effects on inflammatory and antiviral gene expression that confer resistance to rotavirus infection in vitro suggests the assay is an appropriate platform for discovery of compounds with potential to amplify innate antiviral responses.
PMCID: PMC1564392  PMID: 16948846
21.  Expression and Single-step Purification of GRA8 Antigen of Toxoplasma gondii in Escherichia coli  
Diagnosis of Toxoplasma gondii (T.gondii) infection is of great medical importance especially for pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several toxoplasma antigens, including dense granule antigens (GRAs) has a great potential as diagnostic reagents. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags. In the present study, we produced truncated GRA8 (GRA8), excluded from the signal peptide and C-terminal transmembrane domain, with a short fusion tag in Escherichia coli (E.coli). GRA8 was purified using an optimized single-step Immobilized Metal ion Affinity Chromatography (IMAC). The purity and yield of GRA8 was highest at pH = 9.25. At this pH, 13.6 mg of GRA8 was obtained with the purity of 97.97%. Immunogenicity of the protein was evaluated in Western blot analysis showing the serum sample from a rabbit immunized with GRA8 recognized a single antigen of T.gondii tachyzoite at the expected molecular weight of native GRA8. To diagnosis acute toxoplasma infection in pregnant women, an indirect immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was developed using GRA8 resulting in a test specificity and sensitivity of 97.1% and 60.6%, respectively. These results demonstrated that immunogenic GRA8 can be produced in fusion with a short tag and purified near to homogeneity using an optimized IMAC. GRA8-IgM-ELISA was useful for detection of acute toxoplasma infection.
PMCID: PMC3558177  PMID: 23407862
Enzyme-linked immunosorbent assay; Gene expression; GRA8 protein; Immunoglobulin M; Toxoplasma gondii
22.  Limited Value of Assays Using Detection of Immunoglobulin G Antibodies to the Two Recombinant Dense Granule Antigens, GRA1 and GRA6 Nt of Toxoplasma gondii, for Distinguishing between Acute and Chronic Infections in Pregnant Women 
An enzyme-linked immunosorbent assay (ELISA) using two recombinant antigens of Toxoplasma gondii (GRA1 and GRA6 Nt) was developed in order to differentiate between pregnant women with a serological profile of recently acquired infection and those with chronic infection. Both proteins were expressed in Escherichia coli as glutathione S-transferase fusion proteins. Thirty-two serum samples from subjects who presented seroconversion within 3 months before sampling (group 1; acute profile), 46 serum samples from women who had a positive serology at least 1 year before sampling (group 2; chronic profile), and 100 serum samples from pregnant women who were not infected by T. gondii (group 3) were examined for immunoglobulin G (IgG) reactivity. For both antigens, the specificity reached 98%. In both groups of infected patients, the overall sensitivity scored was 60% for GRA1 and 83% for GRA6 Nt. In group 1, 34% of sera reacted with GRA1 whereas 84% of sera reacted with GRA6 Nt; in group 2, however, sensitivities were 78.2 and 82.6%, respectively. Combination of the readings obtained with both antigens yielded a sensitivity of 91%. A serological follow-up of 10 women who seroconverted during pregnancy displayed three different serological patterns: (i) a GRA profile paralleling the IgG curve, as detected by the commercial kit, (ii) a GRA1 profile, or (iii) GRA1 and GRA6 Nt profiles remaining negative for at least 8 weeks after the reference test gave positive results. Taken together, these results suggest that neither GRA1 nor GRA6 Nt is sensitive enough to be used routinely to differentiate between acute and chronic toxoplasmic infections.
PMCID: PMC524737  PMID: 15539499
23.  Thiazolides, a New Class of Antiviral Agents Effective against Rotavirus Infection, Target Viral Morphogenesis, Inhibiting Viroplasm Formation 
Journal of Virology  2013;87(20):11096-11106.
Rotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented double-stranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC50s) ranging from 0.3 to 2 μg/ml and 50% cytotoxic concentrations (CC50s) higher than 50 μg/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against rotavirus gastroenteritis.
PMCID: PMC3807293  PMID: 23926336
24.  Cellular Immune Responses to Recombinant Antigens in Pregnant Women Chronically Infected with Toxoplasma gondii 
The parasite Toxoplasma gondii can infect most mammals and birds, sometimes causing severe pathology. Primary infection during pregnancy can result in abortion or fetal defects. Host immunity, particularly cellular immunity towards antigenic peptides, can control infection, but an efficient vaccine is not yet available. We have evaluated T-cell responses to a crude soluble toxoplasma antigen (ST-Ag) and to five recombinant peptide antigens of cells in whole-blood cultures from 22 pregnant women with preexisting infections and from 7 pregnant negative controls. Cells from all infected patients but from none of the controls responded specifically to ST-Ag by expressing surface CD25 on culture. Responses to the recombinant antigens showed considerable variation between individuals. rGRA1 elicited a response in 16 of the 22 samples (73%), rSAG1 in 13, rGRA7 in 9, rGRA6-CT in 4, and rGRA6-NT in only 1. Most responding cells were CD4+. Cells from infected subjects cultured with ST-Ag all released high levels of gamma interferon (IFN-γ) into the culture supernatant (4,343 ± 2,536 pg/ml). Cells from 12 patients released IFN-γ after culture with rGRA1 (130 ± 98 pg/ml), those from 10 patients released it after culture with rSAG1 (183 ± 128 pg/ml), and those from 4 patients released it after culture with rGRA7 (324 ± 374 pg/ml). Intensity of IFN-γ production in response to the latter two recombinant antigens correlated with responses to ST-Ag (r = 0.61 and 0.53, respectively; P < 0.01). Interleukin-4 was always absent from supernatants of cells stimulated with toxoplasma antigens. The heterogeneity of human responses to individual recombinant toxoplasma antigens should be considered in the design of potential vaccines.
PMCID: PMC120002  PMID: 11986281
25.  Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms 
Journal of Virology  2006;80(12):6061-6071.
Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.
PMCID: PMC1472611  PMID: 16731945

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