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1.
Clin Exp Immunol ; 163(2): 199-206, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21166665

ABSTRACT

A wide variety of the host immune elements play an influential role in the defence against cytomegalovirus (CMV) infection. However, the role of complement in the clearance of CMV infection is less well studied. Decay accelerating factor (DAF/CD55) is a membrane-bound complement regulatory protein that inhibits the formation and accelerates the decay of C3-convertase. Here we hypothesize that murine CMV (MCMV) utilizes DAF as an immunoevasive strategy through down-regulation of host adaptive responses against the virus. To test our hypothesis, DAF knock-out (DAF KO) C57BL/6 mice and wild-type (WT) littermates were infected with a sublethal dose of MCMV, and their immune responses were compared. WT mice lost 7·8% of their initial weight within the first 4 days after infection and quickly began to recover. This is in contrast to the DAF KO mice, that lost a total of 19·4% of their initial weight and did not start recovery until 6 days post-infection. Flow cytometric analysis of lung digests revealed that infected DAF KO mice had a significantly increased infiltration of inflammatory cells, the majority being CD8(+) T lymphocytes. Serum levels of tumour necrosis factor (TNF)-α and interferon (IFN)-γ were also increased markedly in the DAF KO mice compared to the infected WT mice. More interestingly, increased viral genome copies (DNA) in the splenocytes of DAF KO mice was accompanied with mRNA transcripts in the DAF KO mice, an indication of active viral replication. These data suggest an intriguing effect of reduced DAF expression on host responses following in vivo MCMV infection.


Subject(s)
CD55 Antigens/immunology , Herpesviridae Infections/immunology , Host-Pathogen Interactions/immunology , Muromegalovirus/immunology , Animals , CD55 Antigens/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , DNA, Viral/immunology , Gene Expression Regulation, Viral/immunology , Interferon-gamma/blood , Interferon-gamma/immunology , Lung Diseases/immunology , Lung Diseases/virology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muromegalovirus/genetics , Spleen/immunology , Spleen/virology , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/immunology
2.
Clin Exp Immunol ; 162(2): 379-91, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20840652

ABSTRACT

Infection with cytomegalovirus (CMV) remains a significant cause of morbidity and mortality following allogeneic bone marrow transplantation (allo-BMT). The manifestations of CMV infection can range from neurological and haematological abnormalities to diminished graft survival and, in extreme cases, death. Many clinical studies have shown a direct correlation between cytomegalovirus infection and increased morbidity and mortality post allo-BMT, yet the exact mechanism is not well understood. Although driven primarily by T cell responses, the role of complement activation in acute and chronic graft-versus-host disease (GVHD) has also become more evident in recent years. The present studies were performed to examine the effects of murine cytomegalovirus (MCMV) infection on decay accelerating factor (DAF) and MCMVs role in exacerbating morbidity and mortality post-allo-BMT. Mice infected previously with a sublethal dose of MCMV (1 × 105 plaque-forming units) have reduced expression of DAF on lung tissues and lymphocytes following allo-BMT. More importantly, mortality rates post-allo-BMT in recipient DAF knock-out mice receiving wild-type bone marrow are increased, similar to wild-type MCMV-infected recipient mice. Similarly, DAF knock-out mice showed greater intracellular interferon (IFN)-γ production by lung CD8 T cells, and infection with MCMV further exacerbated both intracellular IFN-γ production by CD8 T cells and mortality rates post-allo-BMT. Together, these data support the hypothesis that MCMV infection augments morbidity and mortality post-allo-BMT by reducing surface DAF expression.


Subject(s)
Bone Marrow Transplantation/mortality , CD55 Antigens/metabolism , Cytomegalovirus Infections/metabolism , Animals , Body Weight/immunology , Bone Marrow Transplantation/immunology , Bone Marrow Transplantation/pathology , CD4-Positive T-Lymphocytes/pathology , CD55 Antigens/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Cell Count , Complement Activation/immunology , Complement C3a/metabolism , Complement C3d/metabolism , Cytomegalovirus Infections/immunology , Female , Immediate-Early Proteins/immunology , Intercellular Adhesion Molecule-1/metabolism , Interferon-gamma/metabolism , Lung/metabolism , Lung/pathology , Lung/virology , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Lymphocytes/immunology , Lymphocytes/metabolism , Lymphocytes/pathology , Mice , Mice, Inbred BALB C , Mice, Knockout , Survival Analysis , Transplantation, Homologous , Vascular Cell Adhesion Molecule-1/metabolism
3.
Anaerobe ; 12(1): 17-22, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16701608

ABSTRACT

Bacteroides fragilis is an important anaerobic pathogen accounting for up to 10% of bacteremias in adult patients. Enterotoxin producing B. fragilis (ETBF) strains have been identified as enteric pathogens of children and adults. In order to further characterize the B. fragilis pathogenicity island (BfPAI) and using PCR assays for bft- and mpII-metalloprotease genes, we determined the frequency of B. fragilis strains with pattern I (containing the BfPAI and its flanking region), pattern II (lacking both the BfPAI and the flanking region), and pattern III (lacking the BfPAI but containing the flanking region) in 63 blood culture isolates. The results were compared to 197 B. fragilis isolates from different clinical sources. We found 19% of blood culture isolates were pattern I (ETBF), 43% were pattern II (NTBF) and 38% were pattern III (NTBF). Comparatively, B. fragilis isolates from other clinical sources were 10% pattern I, 47% pattern II and 43% pattern III. This suggests that the pathogenicity island and the flanking elements may be general virulence factors of B. fragilis.


Subject(s)
Bacteremia/microbiology , Bacteroides Infections/microbiology , Bacteroides fragilis/genetics , Bacteroides fragilis/pathogenicity , Genomic Islands/genetics , Bacterial Toxins/genetics , Bacteroides fragilis/classification , Bacteroides fragilis/isolation & purification , Base Sequence/genetics , DNA Fingerprinting , DNA, Bacterial/chemistry , Deoxyribonucleases/chemistry , Humans , Metalloendopeptidases/genetics , Metalloproteases/genetics , Molecular Sequence Data , Polymerase Chain Reaction/methods , Sequence Analysis, DNA
4.
Mol Cell Probes ; 18(4): 271-4, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15271388

ABSTRACT

Toxigenic Clostridium difficile isolates harbor a 19 kb pathogenicity locus that encodes the genes for toxins A and B. Toxins A and B are among the largest known bacterial toxins expressing potent cytotoxicity and enterotoxicity, and thus the major virulence factors in C. difficile associated diarrhea. Cloning and sequencing of toxin genes is of interest for studies of molecular pathogenesis. We report the amplification and cloning of the complete toxin A gene into an Escherichia coli expression vector. Ten clones analyzed contained the complete toxin A gene. Four of these clones showed cytotoxic activity in cell culture, and were positive for toxin A as determined by ELISA. Toxin A expression was confirmed by Western immunoblot analysis. The presence of cytotoxic activity in cell culture suggests that toxin A activity is independent of other genes in the pathogenicity locus.


Subject(s)
Bacterial Toxins/genetics , Clostridioides difficile/genetics , Enterotoxins/genetics , Escherichia coli/genetics , Blotting, Western , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Polymerase Chain Reaction
5.
Clin Microbiol Infect ; 9(6): 526-30, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12848728

ABSTRACT

OBJECTIVE: Moxifloxacin is characterized by high activity against Gram-positive cocci and some Gram-positive and -negative anaerobes, including Clostridium difficile. This study investigates the role of prior quinolone use in relation to patterns of susceptibility of C. difficile to moxifloxacin. METHODS: Sixty-three clinical isolates of C. difficile were investigated for toxigenicity, susceptibility to moxifloxacin, and mutations in the DNA gyrase gene. The medical histories for 50 of these patients were available and used to identify previous fluoroquinolone use. RESULTS: Thirty-three (52.4%) strains showed resistance to moxifloxacin (MICs > or = 16 mg/L). All moxifloxacin-resistant strains harbored a mutation at amino acid codon Ser-83 of gyrA. Forty-five isolates (71.4%) were toxigenic; all moxifloxacin-resistant strains were in this group. Resistance to moxifloxacin was associated with prior use of fluoroquinolones (P-value 0.009, chi-square). CONCLUSIONS: Although the use of moxifloxacin to treat C. difficile-associated diarrhea is not likely to be common, these data show a relationship between antecedent fluoroquinolone use and resistance to moxifloxacin in C. difficile isolates, and raise questions regarding selection pressure for resistance placed on colonizing bacteria exposed to fluoroquinolones. Mutations in gyrA are involved in moxifloxacin resistance.


Subject(s)
Anti-Infective Agents/pharmacology , Aza Compounds , Clostridioides difficile/drug effects , Drug Resistance, Bacterial/physiology , Fluoroquinolones/pharmacology , Quinolines , Clostridioides difficile/genetics , Drug Resistance, Bacterial/genetics , Enterocolitis, Pseudomembranous/drug therapy , Fluoroquinolones/adverse effects , Humans , Moxifloxacin , Polymerase Chain Reaction
6.
Mol Cell Probes ; 16(3): 179-83, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12144768

ABSTRACT

The toxin genes of Clostridium difficile have been previously cloned by reconstructing the entire gene in a series of steps in sequence using several cloned fragments. Amplification of a 7.9 kb fragment corresponding to the toxin B gene (tcdB) was obtained with EXPAND Long Template PCR system. The amplified fragment was inserted into the E. coli expression vector pBAD and cloned into competent E. coli TOP 10 cells. tcdB gene sequences representing the complete toxin gene were detected in 3/120 (2.5%) clones analyzed. Culture filtrates of 2/3 clones were found to have cytotoxic activity in human lung fibroblasts. The recombinant protein expressed in E. coli was identified as toxin B by Western immunoblot analysis using C. sordellii antitoxin. This rapid cloning method may be useful in determining the role that individual genes in the pathogenicity locus (PaLoc) play in the virulence of C. difficile. Our results also suggest that the activity of toxin B is independent of other genes in the PaLoc.


Subject(s)
Bacterial Proteins , Bacterial Toxins/genetics , Cloning, Molecular/methods , Bacterial Toxins/biosynthesis , Bacterial Toxins/pharmacology , Cell Death/drug effects , Clostridioides difficile/genetics , Clostridioides difficile/pathogenicity , Escherichia coli/genetics , Fibroblasts/drug effects , Humans , Lung/cytology , Polymerase Chain Reaction , Virulence/genetics
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