46 results on '"Mahony, James B."'
Search Results
2. Enumeration of Viable Chlamydia from Infected Animals Using Immunofluorescent Microscopy.
- Author
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Liang S and Mahony JB
- Subjects
- Animals, Female, Humans, Mice, Microbial Viability, Vagina microbiology, Bacterial Load methods, Chlamydia Infections microbiology, Chlamydia trachomatis isolation & purification, Microscopy, Fluorescence methods
- Abstract
An appropriate means of quantitating infectious Chlamydia from infected animals is essential for the evaluation of vaccines. However, unlike methods involving culture, nonculture methods, including detection of antigen or DNA, are not able to differentiate between viable and nonviable organisms. As an obligate intracellular bacterium, Chlamydia replicates inside host cells by forming unique organelles called inclusions. Here, we describe the enumeration of viable C. trachomatis from infected mice by culturing vaginal swabs on McCoy cells and counting inclusions via immunofluorescent microscopy.
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- 2019
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3. Low-cost and versatile integration of microwire electrodes and optical waveguides into silicone elastomeric devices using modified xurographic methods.
- Author
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Liu J, Mahony JB, and Selvaganapathy PR
- Abstract
Microelectrodes are used in microfluidic devices for a variety of purposes such as heating, applying electric fields, and electrochemical sensing. However, they are still manufactured by expensive deposition techniques such as sputtering or evaporation and patterned using photolithography methods. More recently, alternate methods including nanoparticle sintering and use of liquid metal flowing through microchannels have been used to fabricate microelectrodes. These methods are limited in the material choices or require post processing to be integrated into microchannels. Here we developed a low-cost and versatile method to integrate high-quality metal microwires into polydimethylsiloxane (PDMS) using xurography. The microwire integration process includes cutting slit pattern on PDMS substrate and subsequent writing metal microwires into the slit pattern using a specialized tip. Then the microwire-integrated PDMS was sealed/bonded using uncured PDMS prepolymer. This method enables integration of metal microwires of diameter as small as 15 μm into PDMS devices. Integration of multiple microwires with minimum spacing of 150 μm has also been demonstrated. The versatility of this method is demonstrated by the fabrication of metal microwire suspended in the middle of the microchannel, which is difficult to achieve using conventional electrode fabrication methods. This low-cost method avoids expensive clean room fabrication yet producing high-quality electrodes and can be used in a variety of microfluidic and MEMS applications., Competing Interests: The authors declare no conflict of interest.
- Published
- 2017
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4. Hospital admissions for lower respiratory tract infections among infants in the Canadian Arctic: a cohort study.
- Author
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Banerji A, Panzov V, Young M, Robinson J, Lee B, Moraes T, Mamdani M, Giles BL, Jiang D, Bisson D, Dennis M, Morel J, Hall J, Hui C, Paes B, and Mahony JB
- Abstract
Background: It is unknown whether this burden of disease of lower respiratory tract infections is comparable across the Canadian Arctic. The objectives of this surveillance study were to compare the rates of hospital admission for lower respiratory tract infection and the severity of infection across Arctic Canada, and to describe the responsible viruses., Methods: We performed a prospective multicentre surveillance study of infants less than 1 year of age admitted in 2009 with lower respiratory tract infection to all hospitals (5 regional, 4 tertiary) in the Northwest Territories, Nunavut and Nunavik to assess for regional differences. Nasopharyngeal aspirates were processed by means of a polymerase chain reaction respiratory viral panel, testing for 20 respiratory viruses and influenza A (H1N1). The role of coinfection was assessed by means of regression analysis for length of stay (short: < 7 d; long: > 14 d). Outcomes compared included rates of lower respiratory tract infection, respiratory syncytial virus infection, transfer to tertiary hospital and severe lower respiratory tract infection (respiratory failure, intubation and mechanical ventilation, and/or cardiopulmonary resuscitation)., Results: There were 348 admissions for lower respiratory tract infection in the population of interest in 2009. Rates of admission per 1000 live births varied significantly, from 39 in the Northwest Territories to 456 in Nunavik ( p < 0.001). The rates of tertiary admissions and severe lower respiratory tract infection per 1000 live births in the Northwest Territories were 5.6 and 1.4, respectively, compared to 55.9 and 17.1, respectively, in Nunavut and 52.0 and 20.0, respectively, in Nunavik ( p ≤ 0.001). Respiratory syncytial virus was the most common virus identified (124 cases [41.6% of those tested]), and coinfection was detected in 51 cases (41.1%) of infection with this virus. Longer length of stay was associated with coinfection (odds ratio [OR] 2.64) and underlying risk factors (OR 4.39). Length of stay decreased by 32.2% for every 30-day increase in age (OR 0.68)., Interpretation: Nunavut and Nunavik have very elevated rates of lower respiratory tract infection, with severe outcomes. Respiratory syncytial virus was the most common virus identified, and coinfection was associated with longer length of stay. Targeted public health interventions are required to reduce the burden of disease for infants residing in these Arctic regions., Competing Interests: See the end of the article.
- Published
- 2016
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5. Immunization with chlamydial type III secretion antigens reduces vaginal shedding and prevents fallopian tube pathology following live C. muridarum challenge.
- Author
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Bulir DC, Liang S, Lee A, Chong S, Simms E, Stone C, Kaushic C, Ashkar A, and Mahony JB
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- Administration, Intranasal, Amino Acid Sequence, Animals, Antibodies, Bacterial blood, Antibodies, Neutralizing blood, Antigens, Bacterial immunology, Chlamydia muridarum, Fallopian Tubes microbiology, Female, Mice, Mice, Inbred C57BL, Neutralization Tests, Vagina microbiology, Virulence Factors immunology, Bacterial Shedding, Bacterial Vaccines immunology, Chlamydia Infections prevention & control, Fallopian Tubes pathology, Type III Secretion Systems immunology
- Abstract
Chlamydia trachomatis infections in women are often asymptomatic and if left untreated can lead to significant late sequelae including pelvic inflammatory disease and tubal factor infertility. Vaccine development efforts over the past three decades have been unproductive and there is no vaccine approved for use in humans. The existence of serologically distinct strains or serovars of C. trachomatis mandates a vaccine that will provide protection against multiple serovars. Chlamydia spp. use a highly conserved type III secretion system (T3SS) composed of both structural and effector proteins which is an essential virulence factor for infection and intracellular replication. In this study we evaluated a novel fusion protein antigen (BD584) which consists of three T3SS proteins from C. trachomatis (CopB, CopD, and CT584) as a potential chlamydial vaccine candidate. Intranasal immunization with BD584 elicited serum neutralizing antibodies that inhibited C. trachomatis infection in vitro. Following intravaginal challenge with C. muridarum, immunized mice had a 95% reduction in chlamydial shedding from the vagina at the peak of infection and cleared the infection sooner than control mice. Immunization with BD584 also reduced the rate of hydrosalpinx by 87.5% compared to control mice. Together, these results suggest that highly conserved proteins of the chlamydial T3SS may represent good candidates for a Chlamydia vaccine., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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6. Chlamydia Outer Protein (Cop) B from Chlamydia pneumoniae possesses characteristic features of a type III secretion (T3S) translocator protein.
- Author
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Bulir DC, Waltho DA, Stone CB, Liang S, Chiang CK, Mwawasi KA, Nelson JC, Zhang SW, Mihalco SP, Scinocca ZC, and Mahony JB
- Subjects
- Amino Acid Motifs, Bacterial Proteins metabolism, Binding Sites, Epithelial Cells microbiology, HeLa Cells, Host-Pathogen Interactions, Humans, Molecular Chaperones metabolism, Protein Binding, Protein Interaction Mapping, Bacterial Outer Membrane Proteins metabolism, Chlamydophila pneumoniae metabolism, Membrane Transport Proteins metabolism, Type III Secretion Systems, Virulence Factors metabolism
- Abstract
Background: Chlamydia spp. are believed to use a conserved virulence factor called type III secretion (T3S) to facilitate the delivery of effector proteins from the bacterial pathogen to the host cell. Important early effector proteins of the type III secretion system (T3SS) are a class of proteins called the translocators. The translocator proteins insert into the host cell membrane to form a pore, allowing the injectisome to dock onto the host cell to facilitate translocation of effectors. CopB is a predicted hydrophobic translocator protein within the chlamydial T3SS., Results: In this study, we identified a novel interaction between the hydrophobic translocator, CopB, and the putative filament protein, CdsF. Furthermore, we identified a conserved PxLxxP motif in CopB (amino acid residues 166-171), which is required for interaction with its cognate chaperone, LcrH_1. Using a synthetic peptide derived from the chaperone binding motif of CopB, we were able to block the LcrH_1 interaction with either CopB or CopD; this CopB peptide was capable of inhibiting C. pneumoniae infection of HeLa cells at micromolar concentrations. An antibody raised against the N-terminus of CopB was able to inhibit C. pneumoniae infection of HeLa cells., Conclusion: The inhibition of the LcrH_1:CopB interaction with a cognate peptide and subsequent inhibition of host cell infection provides strong evidence that T3S is an essential virulence factor for chlamydial infection and pathogenesis. Together, these results support that CopB plays the role of a hydrophobic translocator.
- Published
- 2015
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7. Targeting PI3K-p110α Suppresses Influenza Virus Infection in Chronic Obstructive Pulmonary Disease.
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Hsu AC, Starkey MR, Hanish I, Parsons K, Haw TJ, Howland LJ, Barr I, Mahony JB, Foster PS, Knight DA, Wark PA, and Hansbro PM
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- Adult, Aged, Animals, Bronchi drug effects, Cells, Cultured, Epithelial Cells drug effects, Female, Humans, Influenza, Human virology, Male, Mice, Middle Aged, Antiviral Agents therapeutic use, Enzyme Inhibitors therapeutic use, Influenza, Human drug therapy, Orthomyxoviridae Infections drug therapy, Phosphatidylinositol 3-Kinases drug effects, Pulmonary Disease, Chronic Obstructive drug therapy, Pulmonary Disease, Chronic Obstructive virology
- Abstract
Rationale: Chronic obstructive pulmonary disease (COPD) and influenza virus infections are major global health issues. Patients with COPD are more susceptible to infection, which exacerbates their condition and increases morbidity and mortality. The mechanisms of increased susceptibility remain poorly understood, and current preventions and treatments have substantial limitations., Objectives: To characterize the mechanisms of increased susceptibility to influenza virus infection in COPD and the potential for therapeutic targeting., Methods: We used a combination of primary bronchial epithelial cells (pBECs) from COPD and healthy control subjects, a mouse model of cigarette smoke-induced experimental COPD, and influenza infection. The role of the phosphoinositide-3-kinase (PI3K) pathway was characterized using molecular methods, and its potential for targeting assessed using inhibitors., Measurements and Main Results: COPD pBECs were susceptible to increased viral entry and replication. Infected mice with experimental COPD also had more severe infection (increased viral titer and pulmonary inflammation, and compromised lung function). These processes were associated with impaired antiviral immunity, reduced retinoic acid-inducible gene-I, and IFN/cytokine and chemokine responses. Increased PI3K-p110α levels and activity in COPD pBECs and/or mice were responsible for increased infection and reduced antiviral responses. Global PI3K, specific therapeutic p110α inhibitors, or exogenous IFN-β restored protective antiviral responses, suppressed infection, and improved lung function., Conclusions: The increased susceptibility of individuals with COPD to influenza likely results from impaired antiviral responses, which are mediated by increased PI3K-p110α activity. This pathway may be targeted therapeutically in COPD, or in healthy individuals, during seasonal or pandemic outbreaks to prevent and/or treat influenza.
- Published
- 2015
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8. Chlamydia pneumoniae CopD translocator protein plays a critical role in type III secretion (T3S) and infection.
- Author
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Bulir DC, Waltho DA, Stone CB, Mwawasi KA, Nelson JC, and Mahony JB
- Subjects
- Antibodies metabolism, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Binding Sites, Carrier Proteins genetics, Chlamydophila pneumoniae immunology, Chlamydophila pneumoniae metabolism, Computational Biology methods, Carrier Proteins chemistry, Carrier Proteins metabolism, Chlamydophila pneumoniae pathogenicity
- Abstract
Pathogenic Gram-negative bacteria use type III secretion (T3S) to inject effector proteins into the host cell to create appropriate conditions for infection and intracellular replication. Chlamydia spp. are believed to use T3S to infect their host cell, and the translocator proteins are an essential component of this system. Chlamydia pneumoniae contains genes encoding two sets of translocator proteins; CopB and CopD, and CopB2 and CopD2. In this study, we identified novel interactions between CopD and three type III secretion proteins; namely, CopN, CdsN, and CdsF. We identified a CopD putative chaperone binding motif, PxLxxP, within the N-terminal region (CopD amino acids 120-125), which was necessary for interaction with its putative chaperone LcrH_1. Using size exclusion chromatography, we showed that CopD and LcrH_1 formed higher order structures in solution with CopD and LcrH_1 binding in a ratio of 1∶1, which is unique for T3SS translocator proteins. Lastly, we showed that antibodies to CopD reduced C. pneumoniae infectivity by >95%. Collectively, this data suggests that CopD plays a critical role in pathogenesis and likely functions as a hydrophobic translocator of the type III secretion system in Chlamydia pneumoniae.
- Published
- 2014
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9. Structural characterization of a novel Chlamydia pneumoniae type III secretion-associated protein, Cpn0803.
- Author
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Stone CB, Sugiman-Marangos S, Bulir DC, Clayden RC, Leighton TL, Slootstra JW, Junop MS, and Mahony JB
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- Bacterial Proteins chemistry, Bacterial Proteins genetics, Blotting, Western, Chlamydophila pneumoniae genetics, Crystallography, X-Ray, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Protein Binding, Protein Interaction Mapping, Protein Multimerization, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Bacterial Proteins metabolism, Chlamydophila pneumoniae metabolism, Phosphatidic Acids metabolism, Phosphatidylinositols metabolism
- Abstract
Type III secretion (T3S) is an essential virulence factor used by gram-negative pathogenic bacteria to deliver effector proteins into the host cell to establish and maintain an intracellular infection. Chlamydia is known to use T3S to facilitate invasion of host cells but many proteins in the system remain uncharacterized. The C. trachomatis protein CT584 has previously been implicated in T3S. Thus, we analyzed the CT584 ortholog in C. pneumoniae (Cpn0803) and found that it associates with known T3S proteins including the needle-filament protein (CdsF), the ATPase (CdsN), and the C-ring protein (CdsQ). Using membrane lipid strips, Cpn0803 interacted with phosphatidic acid and phosphatidylinositol, suggesting that Cpn0803 may associate with host cells. Crystallographic analysis revealed a unique structure of Cpn0803 with a hydrophobic pocket buried within the dimerization interface that may be important for binding small molecules. Also, the binding domains on Cpn0803 for CdsN, CdsQ, and CdsF were identified using Pepscan epitope mapping. Collectively, these data suggest that Cpn0803 plays a role in T3S.
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- 2012
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10. Molecular diagnosis of respiratory virus infections.
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Mahony JB, Petrich A, and Smieja M
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- DNA, Viral analysis, Drug Resistance, Viral, Humans, Molecular Diagnostic Techniques, RNA, Viral analysis, Respiratory Tract Infections virology, Virus Diseases virology, Viruses genetics, Nucleic Acid Amplification Techniques, Respiratory Tract Infections diagnosis, Virus Diseases diagnosis, Viruses isolation & purification
- Abstract
The appearance of eight new respiratory viruses, including the SARS coronavirus in 2003 and swine-origin influenza A/H1N1 in 2009, in the human population in the past nine years has tested the ability of virology laboratories to develop diagnostic tests to identify these viruses. Nucleic acid based amplification tests (NATs) for respiratory viruses were first introduced two decades ago and today are utilized for the detection of both conventional and emerging viruses. These tests are more sensitive than other diagnostic approaches, including virus isolation in cell culture, shell vial culture (SVC), antigen detection by direct fluorescent antibody (DFA) staining, and rapid enzyme immunoassay (EIA), and now form the backbone of clinical virology laboratory testing around the world. NATs not only provide fast, accurate and sensitive detection of respiratory viruses in clinical specimens but also have increased our understanding of the epidemiology of both new emerging viruses such as the pandemic H1N1 influenza virus of 2009, and conventional viruses such as the common cold viruses, including rhinovirus and coronavirus. Multiplex polymerase chain reaction (PCR) assays introduced in the last five years detect up to 19 different viruses in a single test. Several multiplex PCR tests are now commercially available and tests are working their way into clinical laboratories. The final chapter in the evolution of respiratory virus diagnostics has been the addition of allelic discrimination and detection of single nucleotide polymorphisms associated with antiviral resistance. These assays are now being multiplexed with primary detection and subtyping assays, especially in the case of influenza virus. These resistance assays, together with viral load assays, will enable clinical laboratories to provide physicians with new and important information for optimal treatment of respiratory virus infections.
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- 2011
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11. Prognosis of West Nile virus associated acute flaccid paralysis: a case series.
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Johnstone J, Hanna SE, Nicolle LE, Drebot MA, Neupane B, Mahony JB, and Loeb MB
- Abstract
Introduction: Little is known about the long-term health related quality of life outcomes in patients with West Nile virus associated acute flaccid paralysis. We describe the quality of life scores of seven patients with acute flaccid paralysis who presented to hospital between 2003 and 2006, and were followed for up to two years., Case Presentations: Between 2003 and 2006, 157 symptomatic patients with West Nile virus were enrolled in a longitudinal cohort study of West Nile virus in Canada. Seven patients (4%) had acute flaccid paralysis. The first patient was a 55-year-old man who presented with left upper extremity weakness. The second patient was a 54-year-old man who presented with bilateral upper extremity weakness. The third patient was a 66-year-old woman who developed bilateral upper and lower extremity weakness. The fourth patient was a 67-year-old man who presented with right lower extremity weakness. The fifth patient was a 60-year-old woman who developed bilateral lower extremity weakness. The sixth patient was a 71-year-old man with a history of Parkinson's disease and acute onset bilateral lower extremity weakness. The seventh patient was a 52-year-old man who presented with right lower extremity weakness. All were Caucasian. Patients were followed for a mean of 1.1 years. At the end of follow-up the mean score on the Physical Component Summary of the Short-Form 36 scale had only slightly increased to 39. In contrast, mean score on the Mental Component Summary of the Short-Form 36 scale at the end of follow-up had normalized to 50., Conclusion: Despite the poor physical prognosis for patients with acute flaccid paralysis, the mental health outcomes are generally favorable.
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- 2011
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12. Evaluation and clinical validation of an alcohol-based transport medium for preservation and inactivation of respiratory viruses.
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Luinstra K, Petrich A, Castriciano S, Ackerman M, Chong S, Carruthers S, Ammons B, Mahony JB, and Smieja M
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- Alcohols pharmacology, Disinfectants pharmacology, Humans, Polymerase Chain Reaction, Respiratory Tract Diseases virology, Virus Diseases virology, Virus Inactivation, Preservation, Biological methods, Respiratory Tract Diseases diagnosis, Specimen Handling methods, Virology methods, Virus Diseases diagnosis, Viruses isolation & purification
- Abstract
The clinical and public health importance of influenza and other respiratory viruses has accelerated the development of highly sensitive molecular diagnostics, but data are limited regarding preanalytical stages of diagnostic testing. We evaluated CyMol, an alcohol-based transport medium, for its ability to maintain specimen integrity for up to 21 days of storage at various temperatures; for its ability to inactivate virus; and for its compatibility with antigen- or nucleic acid-based diagnostics for respiratory viruses in clinical samples. In mock-infected samples, both universal transport medium (UTM-RT) and CyMol maintained equivalent viral quantities for at least 14 days at room temperature or colder, whereas a dry swab collection maintained viral quantities only if refrigerated or frozen. CyMol inactivated influenza virus within 5 min of sample immersion. UTM-RT- and CyMol-collected nasal swab specimens from 73 symptomatic students attending a campus health clinic were positive for a respiratory virus in 56.2% of subjects by multiplex PCR testing, including influenza A and B viruses, rhinovirus/enteroviruses, coronaviruses, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, and adenovirus. Detection by PCR was equivalent in UTM-RT- and CyMol-collected specimens and in self- and staff-collected swabs. Direct fluorescent antibody (DFA) testing was substantially less sensitive (23.3%) than multiplex PCR, and DFA testing from UTM-RT-collected swabs was more sensitive than that from CyMol-collected swabs. These data indicate that an alcohol-based transport medium such as CyMol preserves respiratory virus integrity, rapidly inactivates viruses, and is compatible with PCR-based respiratory diagnostics.
- Published
- 2011
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13. Expectant management versus immediate treatment for low-grade cervical intraepithelial neoplasia : a randomized trial in Canada and Brazil.
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Elit L, Levine MN, Julian JA, Sellors JW, Lytwyn A, Chong S, Mahony JB, Gu C, Finch T, and Zeferino LC
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- Adolescent, Adult, Brazil, Canada, Colposcopy, Disease Progression, Female, Humans, Middle Aged, Uterine Cervical Dysplasia pathology, Uterine Cervical Dysplasia surgery, Electrosurgery methods, Watchful Waiting methods, Uterine Cervical Dysplasia therapy
- Abstract
Background: The optimal management strategy for women with low-grade biopsy-proven cervical intraepithelial neoplasia (CIN) is not clear. Our objective was to compare the effectiveness of regular colposcopic follow-up and treatment of progressive disease only versus immediate treatment., Methods: Data were accrued between November 2000 and March 2006 for a noninferiority randomized clinical trial of 415 women with biopsy-proven grade 1 CIN from 8 Canadian and 2 Brazilian colposcopy clinics. Subjects were randomly assigned to either undergo immediate treatment with a loop electrical excision procedure (LEEP) or receive regular colposcopic follow-up for 18 months. The primary outcome was progression of disease to CIN 2 to 3 was based on histology obtained during 18 months of follow-up. Treatments were compared using differences of proportion with a 9% noninferiority margin. Analysis was conducted on the basis of intention-to-treat., Results: An initial LEEP was performed on 179 women. Disease progression was found in 32. Easily controlled vaginal bleeding occurred in 16 (8.9%). During follow-up, disease progression was identified in 3 (1.7%) women in the immediate treatment arm and 9 (4.4%) in the colposcopic follow-up arm-a tolerable difference of 2.7% with 1-sided 95% confidence interval (CI) upper limit of 6.0%. Compliance with all 3 follow-up visits was 61% overall, but significantly worse in women ≤30 years of age (P < .05)., Conclusions: The risk of progression to CIN grade 2 or 3 or cancer over 18 months was similar in the 2 treatment groups. In Canada and Brazil, follow-up for 18 months is a reasonable management strategy for women with persistent low-grade cytology who are found to have grade 1 CIN on referral for colposcopy and cervical biopsy., (Copyright © 2010 American Cancer Society.)
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- 2011
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14. Chlamydia Pneumoniae CdsL Regulates CdsN ATPase Activity, and Disruption with a Peptide Mimetic Prevents Bacterial Invasion.
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Stone CB, Bulir DC, Emdin CA, Pirie RM, Porfilio EA, Slootstra JW, and Mahony JB
- Abstract
Chlamydiae are obligate intracellular pathogens that likely require type III secretion (T3S) to invade cells and replicate intracellularly within a cytoplasmic vacuole called an inclusion body. Chlamydia pneumoniae possess a YscL ortholog, CdsL, that has been shown to interact with the T3S ATPase (CdsN). In this report we demonstrate that CdsL down-regulates CdsN enzymatic activity in a dose-dependent manner. Using Pepscan epitope mapping we identified two separate binding domains to which CdsL binds viz. CdsN(221-229) and CdsN(265-270). We confirmed the binding domains using a pull-down assay and showed that GST-CdsN(221-270), which encompasses these peptides, co-purified with His-CdsL. Next, we used orthology modeling based on the crystal structure of a T3S ATPase ortholog from Escherichia coli, EscN, to map the binding domains on the predicted 3D structure of CdsN. The CdsL binding domains mapped to the catalytic domain of the ATPase, one in the central channel of the ATPase hexamer and one on the outer face. Since peptide mimetics have been used to disrupt essential protein interactions of the chlamydial T3S system and inhibit T3S-mediated invasion of HeLa cells, we hypothesized that if CdsL-CdsN binding is essential for regulating T3S then a CdsN peptide mimetic could be used to potentially block T3S and chlamydial invasion. Treatment of elementary body with a CdsN peptide mimetic inhibited C. pneumoniae invasion into HeLa cells in a dose-dependent fashion. This report represents the first use of Pepscan technology to identify binding domains for specific T3S proteins viz. CdsL on the ATPase, CdsN, and demonstrates that peptide mimetics can be used as anti-virulence factors to block bacterial invasion.
- Published
- 2011
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15. Development of a novel bead-based multiplex PCR assay for combined subtyping and oseltamivir resistance genotyping (H275Y) of seasonal and pandemic H1N1 influenza A viruses.
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Mahony JB, Chong S, Luinstra K, Petrich A, and Smieja M
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- DNA Primers pharmacology, Hemagglutinins, Viral genetics, Humans, Influenza A virus genetics, Influenza A virus isolation & purification, Influenza, Human diagnosis, Microspheres, Neuraminidase genetics, Viral Proteins genetics, Antiviral Agents pharmacology, Influenza A virus classification, Influenza A virus drug effects, Influenza, Human virology, Oseltamivir pharmacology, Polymerase Chain Reaction methods, Virology methods
- Abstract
Background: The identification of influenza A virus subtypes in clinical specimens is becoming increasingly important for clinical laboratories since seasonal H1N1, H3N2 and pandemic H1N1 influenza A viruses can have defined antiviral resistance patterns and subtyping can be used as a surrogate for antiviral resistance testing., Objectives: To develop a novel multiplex PCR (M-PCR) assay for the combined identification of influenza A subtype and oseltamivir resistance (H275Y) genotype in a combined assay format using Luminex xMAP™ technology., Study Design: The M-PCR assay employed five degenerate primers to amplify the hemagglutinin (HA) and neuraminidase (NA) genes and eight tagged primers in a target specific primer extension reaction (TSPE). Products were analysed using xTAG™ beads containing specific anti-tag oligonucleotides., Results: M-PCR correctly identified the subtype for 54/54 specimens that were influenza A positive, including 13/13 seasonal H3N2, 17/17 seasonal H1N1 and 24/24 pandemic H1N1 for both HA and NA genes. For oseltamivir resistance the M-PCR assay correctly identified 41/41 H1N1 viruses as oseltamivir sensitive (H275) or resistant (H275Y). Analysis of sequential specimens from two immunocompromised patients revealed the appearance of the H275Y allele at earlier time points after infection compared with Sanger sequencing., Conclusions: The combined M-PCR assay correctly subtyped seasonal and pandemic influenza A viruses and accurately detected the H275Y oseltamivir resistance allele. This assay should provide useful information to clinicians for appropriate patient management., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
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16. Nucleic acid amplification-based diagnosis of respiratory virus infections.
- Author
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Mahony JB
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- Drug Resistance, Viral, Humans, Influenza, Human diagnosis, Influenza, Human epidemiology, Influenza, Human virology, Pandemics, Polymerase Chain Reaction, Respiratory Tract Infections epidemiology, Viral Load, Virus Diseases epidemiology, Virus Diseases virology, Viruses drug effects, Viruses genetics, Nucleic Acid Amplification Techniques, Respiratory Tract Infections diagnosis, Respiratory Tract Infections virology, Virus Diseases diagnosis, Viruses isolation & purification
- Abstract
The appearance of eight new respiratory viruses in the human population in the past 9 years, including two new pandemics (SARS coronavirus in 2003 and swine-origin influenza A/H1N1 in 2009), has tested the ability of virology laboratories to develop diagnostic tests to identify these viruses. Nucleic acid amplification tests (NATs) that first appeared two decades ago have been developed for both conventional and emerging viruses and now form the backbone of the clinical laboratory. NATs provide fast, accurate and sensitive detection of respiratory viruses and have significantly increased our understanding of the epidemiology of these viruses. Multiplex PCR assays have been introduced recently and several commercial tests are now available. The final chapter in the evolution of respiratory virus diagnostics will be the addition of allelic discrimination and detection of single nucleotide polymorphisms associated with antiviral resistance to multiplex assays. These resistance assays together with new viral load tests will enable clinical laboratories to provide physicians with important information for optimal treatment of patients.
- Published
- 2010
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17. Development and evaluation of a flocked nasal midturbinate swab for self-collection in respiratory virus infection diagnostic testing.
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Smieja M, Castriciano S, Carruthers S, So G, Chong S, Luinstra K, Mahony JB, Petrich A, Chernesky M, Savarese M, and Triva D
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- Adult, Human Experimentation, Humans, Polymerase Chain Reaction methods, Respiratory Tract Infections virology, Sensitivity and Specificity, Virus Diseases virology, Nasal Mucosa virology, Respiratory Tract Infections diagnosis, Self Care methods, Virology methods, Virus Diseases diagnosis, Viruses isolation & purification
- Abstract
We developed and evaluated flocked nasal midturbinate swabs obtained from 55 asymptomatic and 108 symptomatic volunteers. Self-collected swabs obtained from asymptomatic volunteers yielded numbers of respiratory epithelial cells comparable to those of staff-collected nasal (n = 55) or nasopharyngeal (n = 20) swabs. Specific viruses were detected in swabs self-collected by 42/108 (38.9%) symptomatic volunteers by multiplex PCR.
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- 2010
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18. The role of cytology (Pap tests) and human papillomavirus testing in anal cancer screening.
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Salit IE, Lytwyn A, Raboud J, Sano M, Chong S, Diong C, Chapman W, Mahony JB, and Tinmouth J
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- Adult, Anal Canal cytology, Anal Canal pathology, Anus Neoplasms virology, Biopsy, Carcinoma in Situ virology, Carcinoma, Squamous Cell virology, Cross-Sectional Studies, Early Detection of Cancer, Homosexuality, Male, Humans, Male, Middle Aged, Papillomavirus Infections virology, Precancerous Conditions virology, Sensitivity and Specificity, Sexual Behavior, Specimen Handling, Anus Neoplasms pathology, Carcinoma in Situ pathology, Carcinoma, Squamous Cell pathology, Cytodiagnosis methods, Papillomavirus Infections pathology, Precancerous Conditions pathology
- Abstract
Objective: To assess anal oncogenic human papillomavirus (HPV) and anal cytology as screening tests for detecting high-grade anal intraepithelial neoplasia (AIN 2+), as this is an immediate anal cancer precursor., Design: Cross-sectional study of 401 HIV-positive men who have sex with men (MSM). The endpoint was histologically confirmed AIN 2+ obtained by high-resolution anoscopy. Cytology and biopsy specimens were assigned random numbers and independently assessed by two pathologists., Methods: We did concomitant anal cytology, anal HPV testing and HRA with directed biopsies without knowing the results of each intervention. The main outcome measures were the sensitivity, specificity, negative predictive value and positive predictive value of anal cytology and oncogenic HPV for the detection of AIN 2+., Results: Cytology was abnormal in 67% of patients: high-grade squamous intraepithelial lesion, 12%; low-grade squamous intraepithelial lesion, 43% and atypical squamous cells of undetermined significance, 12%. Biopsies were abnormal in 68% of patients: AIN 2+, 25% and AIN 1, 43%. HPV was detected in 93% with multiple HPV types in 92% and oncogenic HPV types in 88%. Test performance characteristics for the detection of AIN 2+ using any abnormality on anal cytology were: sensitivity 84%, specificity 39%, negative predictive value 88% and positive predictive value 31%; using oncogenic HPV: sensitivity 100%, specificity 16%, negative predictive value 100% and positive predictive value 28%., Conclusion: Anal cytology and HPV detection have high sensitivity but low specificity for detecting AIN 2+. HIV-positive men who have sex with men have a high prevalence of AIN 2+ and require high-resolution anoscopy for optimal detection of high-grade anal dysplasia.
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- 2010
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19. Interactions between flagellar and type III secretion proteins in Chlamydia pneumoniae.
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Stone CB, Bulir DC, Gilchrist JD, Toor RK, and Mahony JB
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- Adenosine Triphosphatases genetics, Adenosine Triphosphatases metabolism, Amino Acid Sequence, Bacterial Proteins genetics, Chlamydophila pneumoniae genetics, Glutathione, Membrane Proteins genetics, Molecular Sequence Data, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Alignment, Sequence Analysis, DNA, Two-Hybrid System Techniques, Bacterial Proteins metabolism, Chlamydophila pneumoniae enzymology, Membrane Proteins metabolism
- Abstract
Background: Flagellar secretion systems are utilized by a wide variety of bacteria to construct the flagellum, a conserved apparatus that allows for migration towards non-hostile, nutrient rich environments. Chlamydia pneumoniae is an obligate, intracellular pathogen whose genome contains at least three orthologs of flagellar proteins, namely FliI, FlhA and FliF, but the role of these proteins remains unknown., Results: Full length FliI, and fragments of FlhA, FliF, and FliI, were cloned and expressed as either GST or His tagged proteins in E. coli. The GST-tagged full length FliI protein was shown to possess ATPase activity, hydrolyzing ATP at a rate of 0.15 +/- .02 micromol min-1 mg-1 in a time- and dose-dependant manner. Using bacterial-2-hybrid and GST pull-down assays, the N-terminal domain of FliI was shown to interact with the cytoplasmic domain of FlhA, but not with FliF, and the cytoplasmic domain of FlhA was shown to interact with the C-terminus of FliF. The absence of other flagellar orthologs led us to explore cross-reaction of flagellar proteins with type III secretion proteins, and we found that FliI interacted with CdsL and CopN, while FlhA interacted with CdsL and Cpn0322 (YscU ortholog CdsU)., Conclusions: The specific interaction of the four orthologous flagellar proteins in C. pneumoniae suggests that they interact in vivo and, taken together with their conservation across members of the chlamydiae sps., and their interaction with T3S components, suggests a role in bacterial replication and/or intracellular survival.
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- 2010
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20. Difficulty with mumps diagnosis: what is the contribution of mumps mimickers?
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Hatchette TF, Mahony JB, Chong S, and LeBlanc JJ
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- Adolescent, Adult, Aged, Child, Female, Humans, Male, Middle Aged, Mumps virology, Mumps virus immunology, Nova Scotia epidemiology, Parotitis virology, Young Adult, Disease Outbreaks, Mumps diagnosis, Mumps epidemiology, Mumps virus isolation & purification, Parotitis diagnosis, Parotitis epidemiology
- Abstract
Background: Mumps is a vaccine preventable disease that typically presents with unilateral or bilateral parotitis. In February 2007, mumps re-emerged in university students in Nova Scotia. Despite highly sensitive methods for mumps virus detection, only 14% (298/2082) of cases during the peak of the outbreak were laboratory confirmed., Objectives: Due to the low positivity rate, this study investigated whether infection with other viral pathogens caused mumps-like presentations during the outbreak., Study Design: 148 buccal specimens from patients who presented with unilateral or bilateral parotitis but had negative laboratory tests for mumps virus were tested for Epstein-Barr virus (EBV) and cytomegalovirus (CMV) by quantitative PCR and 21 different viral markers using the Luminex xTAG Respiratory Virus Panel (RVP). Companion sera to each buccal specimen were available for EBV and CMV serology to differentiate acute infection from reactivation., Results: No correlation was observed since viral pathogens were detected in both the parotitis and non-parotitis groups., Conclusion: Although there was co-circulation of other viral pathogens during the mumps outbreak, no difference was observed in the prevalence between patients who presented with or without parotitis. The low positivity rate for specimens submitted for mumps diagnostics was likely the result of increased Public Health messaging and physician inexperience in recognizing mumps infection, suggesting the clinical acumen for mumps diagnosis based solely on clinical presentation is low.
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- 2009
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21. A novel inhibitor of Chlamydophila pneumoniae protein kinase D (PknD) inhibits phosphorylation of CdsD and suppresses bacterial replication.
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Johnson DL, Stone CB, Bulir DC, Coombes BK, and Mahony JB
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- Bacterial Proteins genetics, Cell Survival, Chlamydophila Infections microbiology, Chlamydophila pneumoniae genetics, Chlamydophila pneumoniae growth & development, Gene Expression Regulation, Bacterial, HeLa Cells, Humans, Oxindoles, Phosphorylation, Bacterial Proteins metabolism, Chlamydophila pneumoniae enzymology, Indoles pharmacology, Protein Kinase C antagonists & inhibitors, Protein Kinase Inhibitors pharmacology
- Abstract
Background: We have shown previously that Chlamydophila pneumoniae contains a dual-specific Ser/Thr protein kinase that phosphorylates CdsD, a structural component of the type III secretion apparatus. To further study the role of PknD in growth and development we sought to identify a PknD inhibitor to determine whether PknD activity is required for replication., Results: Using an in vitro kinase assay we screened 80 known eukaryotic protein kinase inhibitors for activity against PknD and identified a 3'-pyridyl oxindole compound that inhibited PknD autophosphorylation and phosphorylation of CdsD. The PknD inhibitor significantly retarded the growth rate of C. pneumoniae as evidenced by the presence of very small inclusions with a reduced number of bacteria as seen by electron microscopy. These inclusions contained the normal replicative forms including elementary bodies (EB), intermediate bodies (IB) and reticulate bodies (RB), but lacked persistent bodies (PB), indicating that induction of persistence was not the cause of reduced chlamydial growth. Blind passage of C. pneumoniae grown in the presence of this PknD inhibitor for 72 or 84 hr failed to produce inclusions, suggesting this compound blocks an essential step in the production of infectious chlamydial EB. The compound was not toxic to HeLa cells, did not block activation of the MEK/ERK pathway required for chlamydial invasion and did not block intracellular replication of either Chlamydia trachomatis serovar D or Salmonella enterica sv. Typhimurium suggesting that the inhibitory effect of the compound is specific for C. pneumoniae., Conclusion: We have identified a 3'-pyridyl oxindole compound that inhibits the in vitro kinase activity of C. pneumoniae PknD and inhibits the growth and production of infectious C. pneumoniae progeny in HeLa cells. Together, these results suggest that PknD may play a key role in the developmental cycle of C. pneumoniae.
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- 2009
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22. Cost analysis of multiplex PCR testing for diagnosing respiratory virus infections.
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Mahony JB, Blackhouse G, Babwah J, Smieja M, Buracond S, Chong S, Ciccotelli W, O'Shea T, Alnakhli D, Griffiths-Turner M, and Goeree R
- Subjects
- Costs and Cost Analysis, Humans, Microscopy, Fluorescence economics, Microscopy, Fluorescence methods, Ontario, Polymerase Chain Reaction methods, Virus Cultivation economics, Virus Cultivation methods, Viruses genetics, Polymerase Chain Reaction economics, Respiratory Tract Infections virology, Virus Diseases diagnosis, Viruses isolation & purification
- Abstract
We performed a cost analysis study using decision tree modeling to determine whether the use of multiplex PCR testing for respiratory viruses (xTAG RVP test) is a more or less costly strategy than the status quo testing methods used for the diagnosis of respiratory virus infections in pediatric patients. The decision tree model was constructed by using four testing strategies for respiratory virus detection, viz., direct fluorescent-antibody staining (DFA) alone, DFA plus shell vial culture (SVC), the xTAG RVP test alone, or DFA plus the xTAG RVP test. A review of the charts of 661 pediatric patients was used to determine the length of hospital stay, the number of days in isolation, antibiotic usage, and all other medical procedures performed. The cost of hospitalization by diagnostic status was determined on the basis of the average cost per patient and the number of patients in each arm of the decision tree. The cost per case was the highest for DFA plus SVC at $3,914 (in Canadian dollars), and the lowest was for the xTAG RVP test alone at $3,623, while the costs of DFA alone ($3,911) and DFA plus RVP ($3,849) were intermediate. When all four diagnostic strategies were compared, the least costly strategy was the xTAG RVP test alone when the prevalence of infection was 11% or higher and DFA alone when the prevalence was under 11%. These data indicate a savings of $291 per case investigated if the strategy of using the xTAG RVP test alone was used to replace the status quo test of DFA plus SVC, resulting in a savings of $529,620 per year in direct costs for the four Hamilton, Ontario, Canada, hospitals on the basis of the testing of specimens from 1,820 pediatric inpatients. We conclude that the use of the xTAG RVP test is the least costly strategy for the diagnosis of respiratory virus infections in children and would generate a significant savings for hospitals.
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- 2009
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23. Risk factors and viruses associated with hospitalization due to lower respiratory tract infections in Canadian Inuit children : a case-control study.
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Banerji A, Greenberg D, White LF, Macdonald WA, Saxton A, Thomas E, Sage D, Mamdani M, Lanctôt KL, Mahony JB, Dingle M, and Roberts A
- Subjects
- Breast Feeding, Canada epidemiology, Case-Control Studies, Chi-Square Distribution, Hospitalization, Humans, Infant, Logistic Models, Orthomyxoviridae isolation & purification, Respiratory Syncytial Viruses isolation & purification, Respiratory Tract Infections ethnology, Rhinovirus isolation & purification, Risk Factors, Smoking, Statistics, Nonparametric, Virus Diseases ethnology, Inuit, Respiratory Tract Infections epidemiology, Respiratory Tract Infections virology, Virus Diseases epidemiology, Virus Diseases virology
- Abstract
Objectives: To examine risk factors for lower respiratory tract infections (LRTI) hospital admission in the Canadian Arctic., Methods: This was a case-control study during a 14-month period among children less than 2 years of age. Cases were admitted to the Baffin Regional Hospital in Iqaluit, Nunavut with LRTI. Controls were age matched and came from Iqaluit and 2 communities. Odds ratios (ORs) of hospital admission for LRTI were estimated through multivariate conditional logistic regression modeling for following risk factors: smoking in pregnancy, Inuit race, prematurity, adoption status, breast-feeding, overcrowding, and residing outside of Iqaluit. Viruses in nasophayngeal aspirates were sought at the time of each hospital admission., Results: There were 101 age-matched cases and controls. The following risk factors were significantly associated with an increased risk of admission for LRTI (adjusted OR): smoking in pregnancy (OR = 4.0; 95% CI: 1.1-14.6), residence outside of Iqaluit (OR = 2.7; 95% CI: 1.0 -7.2), full Inuit race (OR = 3.8; 95% CI: 1.1-12.8), and overcrowding (OR = 2.5, 95% CI: 1.1- 6.1). Non-breast-fed children had a 3.6-fold risk of being admitted for LRTI (95% CI: 1.2-11.5) and non-breast-fed adopted children had a 4.4-fold increased risk (95% CI: 1.1-17.6) when compared with breast-fed, nonadopted children. Prematurity was not associated with an increased risk of admission. Viruses were identified in 88 (72.7%) of admissions, with respiratory syncytial virus being identified in the majority of admissions, 62 (51.2%). Multiple viruses were isolated in 19 (15.7%) admissions., Conclusions: Smoking during pregnancy, place of residence, Inuit race, lack of breast-feeding, and overcrowding were all independently associated with increased risk of hospital admission for LRTI among Inuit children less than 2 years of age. Future research on the role of adoption and genetics on the health of Inuit children are required.
- Published
- 2009
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24. Multiplex PCR tests sentinel the appearance of pandemic influenza viruses including H1N1 swine influenza.
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Mahony JB, Hatchette T, Ojkic D, Drews SJ, Gubbay J, Low DE, Petric M, Tang P, Chong S, Luinstra K, Petrich A, and Smieja M
- Subjects
- Animals, Birds, Horses, Humans, Influenza A Virus, H1N1 Subtype classification, Influenza A Virus, H1N1 Subtype genetics, Influenza A Virus, H1N1 Subtype isolation & purification, Influenza A Virus, H5N1 Subtype classification, Influenza A Virus, H5N1 Subtype genetics, Influenza A Virus, H5N1 Subtype isolation & purification, Influenza in Birds virology, Orthomyxoviridae genetics, Orthomyxoviridae Infections veterinary, Orthomyxoviridae Infections virology, Sensitivity and Specificity, Swine, Influenza, Human diagnosis, Influenza, Human virology, Orthomyxoviridae classification, Orthomyxoviridae isolation & purification, Polymerase Chain Reaction methods
- Abstract
Background: Since the turn of the century seven new respiratory viruses have infected man and two of these have resulted in worldwide epidemics. Both SARS Coronavirus which quickly spread to 29 countries in February 2003 and H1N1 swine influenza that recently spread from Mexico to 30 countries in three weeks represent major pandemic threats for mankind. Diagnostic assays are required to detect novel influenza strains with pandemic potential., Objective: In this report we evaluate the ability of a multiplex PCR test (xTAG RVP) to detect new, "non-seasonal" influenza viruses including the H1N1 swine influenza A/swine/California/04/2009., Study Design: Laboratory based study using retrospective and prospective specimens., Results: This multiplex PCR test detected the present of non-seasonal (non-H1, non-H3) influenza in 20 of 20 patients infected with H1N1 swine flu virus. In addition to detecting the current swine flu the xTAG RVP test detected the H5N1 A/Vietnam/1203/2004 high pathogenicity avian influenza virus that circulated in South East Asia in 2003 as well as 17 out of 17 influenza A viruses representing 11 HA subtypes isolated from birds, swine and horses not yet seen in the human population., Conclusion: Based on these results we believe that this molecular test can perform an important role as a sentinel test to detect novel non-seasonal influenza A viruses in patients presenting with influenza-like illness (ILI) and therefore act as an early warning system for the detection of future pandemic influenza threats., Competing Interests: All authors have agreed to the content of the manuscript and its submission to the journal. JBM is an inventor on a patent relating to the xTAG™ RVP test. All authors declare that the content of the manuscript is original and has not been published or submitted for publication to another journal.
- Published
- 2009
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25. Detection of respiratory viruses by molecular methods.
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Mahony JB
- Subjects
- Humans, Viruses genetics, Molecular Diagnostic Techniques methods, Respiratory Tract Diseases virology, Virus Diseases diagnosis, Viruses isolation & purification
- Abstract
Summary: Clinical laboratories historically diagnose seven or eight respiratory virus infections using a combination of techniques including enzyme immunoassay, direct fluorescent antibody staining, cell culture, and nucleic acid amplification tests. With the discovery of six new respiratory viruses since 2000, laboratories are faced with the challenge of detecting up to 19 different viruses that cause acute respiratory disease of both the upper and lower respiratory tracts. The application of nucleic acid amplification technology, particularly multiplex PCR coupled with fluidic or fixed microarrays, provides an important new approach for the detection of multiple respiratory viruses in a single test. These multiplex amplification tests provide a sensitive and comprehensive approach for the diagnosis of respiratory tract infections in individual hospitalized patients and the identification of the etiological agent in outbreaks of respiratory tract infection in the community. This review describes the molecular methods used to detect respiratory viruses and discusses the contribution that molecular testing, especially multiplex PCR, has made to our ability to detect respiratory viruses and to increase our understanding of the roles of various viral agents in acute respiratory disease.
- Published
- 2008
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26. Characterization of the putative type III secretion ATPase CdsN (Cpn0707) of Chlamydophila pneumoniae.
- Author
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Stone CB, Johnson DL, Bulir DC, Gilchrist JD, and Mahony JB
- Subjects
- Adenosine Diphosphate metabolism, Adenosine Triphosphatases chemistry, Adenosine Triphosphatases isolation & purification, Adenosine Triphosphate metabolism, Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins isolation & purification, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Escherichia coli genetics, Gene Expression, Kinetics, Membrane Transport Proteins chemistry, Membrane Transport Proteins isolation & purification, Molecular Sequence Data, Molecular Weight, Phosphorus metabolism, Protein Binding, Protein Interaction Mapping, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Sequence Alignment, Sequence Deletion, Adenosine Triphosphatases genetics, Adenosine Triphosphatases metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Chlamydophila pneumoniae enzymology, Chlamydophila pneumoniae genetics, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism
- Abstract
Type III secretion (T3S) is utilized by a wide range of gram-negative bacterial pathogens to allow the efficient delivery of effector proteins into the host cell cytoplasm through the use of a syringe-like injectisome. Chlamydophila pneumoniae is a gram-negative, obligate intracellular pathogen that has the structural genes coding for a T3S system, but the functionality of the system has not yet been demonstrated. T3S is dependent on ATPase activity, which catalyzes the unfolding of proteins and the secretion of effector proteins through the injectisome. CdsN (Cpn0707) is predicted to be the T3S ATPase of C. pneumoniae based on sequence similarity to other T3S ATPases. Full-length CdsN and a C-terminal truncation of CdsN were cloned as glutathione S-transferase (GST)-tagged constructs and expressed in Escherichia coli. The GST-tagged C-terminal truncation of CdsN possessed ATPase activity, catalyzing the release of ADP and P(i) from ATP at a rate of 0.55 +/- 0.07 micromol min(-1) mg(-1) in a time- and dose-dependent manner. CdsN formed oligomers and high-molecular-weight multimers, as assessed by formaldehyde fixation and nondenaturing polyacrylamide gel electrophoresis. Using bacterial two-hybrid and GST pull-down assays, CdsN was shown to interact with CdsD, CdsL, CdsQ, and CopN, four putative structural components of the C. pneumoniae T3S system. CdsN also interacted with an unannotated protein, Cpn0706, a putative CdsN chaperone. Interactions between CdsN, CdsD, and CopN represent novel interactions not previously reported for other bacterial T3S systems and may be important in the localization and/or function of the ATPase at the inner membrane of C. pneumoniae.
- Published
- 2008
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27. Asthma management by monitoring sputum neutrophil count.
- Author
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Pallan S, Mahony JB, O'Byrne PM, and Nair P
- Subjects
- Adrenal Cortex Hormones therapeutic use, Anti-Bacterial Agents therapeutic use, Anti-Infective Agents therapeutic use, Asthma microbiology, Bronchitis diagnosis, Bronchitis pathology, Chlamydophila Infections complications, Chlamydophila Infections diagnosis, Chlamydophila Infections drug therapy, Chlamydophila pneumoniae, Female, Humans, Lung microbiology, Lung pathology, Middle Aged, Recurrence, Asthma drug therapy, Asthma pathology, Neutrophils pathology, Sputum cytology
- Abstract
We report the utility of quantitative cell counts in sputum in monitoring therapy of a patient with poorly controlled asthma. Recurrent neutrophilic bronchitis without an eosinophilic bronchitis led to the identification of Chlamydophila pneumoniae as the cause of bronchitis and asthma exacerbation. Serial examination of blood and sputum by polymerase chain reaction for C pneumoniae helped to prevent exacerbations by prophylactic antibiotic therapy, reduce the dose of prednisone and inhaled corticosteroids, and improve asthma control.
- Published
- 2008
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28. Interactions between CdsD, CdsQ, and CdsL, three putative Chlamydophila pneumoniae type III secretion proteins.
- Author
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Johnson DL, Stone CB, and Mahony JB
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins isolation & purification, Blotting, Western, Carrier Proteins genetics, Carrier Proteins isolation & purification, Chlamydophila pneumoniae genetics, Cloning, Molecular, Cytoplasm chemistry, Escherichia coli genetics, Gene Expression, Inclusion Bodies chemistry, Protein Binding, Sequence Homology, Amino Acid, Bacterial Proteins metabolism, Carrier Proteins metabolism, Chlamydophila pneumoniae physiology
- Abstract
Chlamydophila pneumoniae is a gram-negative obligate intracellular bacterial pathogen that causes pneumonia and bronchitis and may contribute to atherosclerosis. The developmental cycle of C. pneumoniae includes a morphological transition from an infectious extracellular elementary body (EB) to a noninfectious intracellular reticulate body (RB) that divides by binary fission. The C. pneumoniae genome encodes a type III secretion (T3S) apparatus that may be used to infect eukaryotic cells and to evade the host immune response. In the present study, Cpn0712 (CdsD), Cpn0704 (CdsQ), and Cpn0826 (CdsL), three C. pneumoniae genes encoding yersiniae T3S YscD, YscQ, and YscL homologs, respectively, were cloned and expressed as histidine- and glutathione S-transferase (GST)-tagged proteins in Escherichia coli. Purified recombinant proteins were used to raise hyper-immune polyclonal antiserum and were used in GST pull-down and copurification assays to identify protein-protein interactions. CdsD was detected in both EB and RB lysates by Western blot analyses, and immunofluorescent staining demonstrated the presence of CdsD within inclusions. Triton X-114 solubilization and phase separation of chlamydial EB proteins indicated that CdsD partitions with cytoplasmic proteins, suggesting it is not an integral membrane protein. GST pull-down assays indicated that recombinant CdsD interacts with CdsQ and CdsL, and copurification assays with chlamydial lysates confirmed that native CdsD interacts with CdsQ and CdsL. To the best of our knowledge, this is the first report demonstrating interactions between YscD, YscQ, and YscL homologs of bacterial T3S systems. These novel protein interactions may play important roles in the assembly or function of the chlamydial T3S apparatus.
- Published
- 2008
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29. Chlamydophila pneumoniae PknD exhibits dual amino acid specificity and phosphorylates Cpn0712, a putative type III secretion YscD homolog.
- Author
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Johnson DL and Mahony JB
- Subjects
- Bacterial Proteins genetics, Chlamydophila pneumoniae genetics, Cytoplasm metabolism, Genome, Bacterial, Membrane Proteins genetics, Phosphoproteins genetics, Phosphorylation, Protein Serine-Threonine Kinases genetics, Recombinant Proteins metabolism, Substrate Specificity, Bacterial Proteins metabolism, Chlamydophila pneumoniae metabolism, Membrane Proteins metabolism, Phosphoproteins metabolism, Protein Serine-Threonine Kinases metabolism
- Abstract
Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione S-transferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.
- Published
- 2007
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30. Issues for reporting results.
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Lannigan R and Mahony JB
- Subjects
- Data Collection, Humans, Oligonucleotide Array Sequence Analysis methods, Polymerase Chain Reaction methods, Research Design, Respiratory Tract Infections diagnosis, Respiratory Tract Infections virology
- Published
- 2007
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31. The clinical need for the RVP test.
- Author
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Mahony JB
- Subjects
- Humans, Respiratory Tract Infections economics, Oligonucleotide Array Sequence Analysis methods, Polymerase Chain Reaction methods, Respiratory Tract Infections diagnosis, Respiratory Tract Infections virology
- Published
- 2007
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32. The pathogens.
- Author
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Carman WF and Mahony JB
- Subjects
- Animals, Communicable Diseases, Emerging virology, Humans, Respiratory Tract Infections epidemiology, Respiratory Tract Infections virology
- Published
- 2007
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33. Comparative evaluation of two severe acute respiratory syndrome (SARS) vaccine candidates in mice challenged with SARS coronavirus.
- Author
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See RH, Zakhartchouk AN, Petric M, Lawrence DJ, Mok CPY, Hogan RJ, Rowe T, Zitzow LA, Karunakaran KP, Hitt MM, Graham FL, Prevec L, Mahony JB, Sharon C, Auperin TC, Rini JM, Tingle AJ, Scheifele DW, Skowronski DM, Patrick DM, Voss TG, Babiuk LA, Gauldie J, Roper RL, Brunham RC, and Finlay BB
- Subjects
- Administration, Intranasal, Animals, Antibodies, Viral immunology, Antibody Specificity, Disease Models, Animal, Drug Evaluation, Preclinical, Female, Immunoglobulin A blood, Immunoglobulin A immunology, Injections, Intramuscular, Injections, Subcutaneous, Membrane Glycoproteins genetics, Membrane Glycoproteins immunology, Mice, Neutralization Tests, Nucleocapsid Proteins genetics, Severe acute respiratory syndrome-related coronavirus chemistry, Spike Glycoprotein, Coronavirus, Vaccines, DNA administration & dosage, Vaccines, DNA genetics, Viral Envelope Proteins genetics, Viral Envelope Proteins immunology, Antibodies, Viral blood, Severe acute respiratory syndrome-related coronavirus immunology, Severe Acute Respiratory Syndrome immunology, Severe Acute Respiratory Syndrome prevention & control, Vaccination, Viral Vaccines administration & dosage
- Abstract
Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated by beta-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection.
- Published
- 2006
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34. Molecular diagnosis of severe acute respiratory syndrome: the state of the art.
- Author
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Mahony JB and Richardson S
- Subjects
- Animals, Humans, RNA, Viral analysis, RNA, Viral genetics, RNA, Viral isolation & purification, Reagent Kits, Diagnostic, Reverse Transcriptase Polymerase Chain Reaction, Severe acute respiratory syndrome-related coronavirus genetics, Severe acute respiratory syndrome-related coronavirus isolation & purification, Severe Acute Respiratory Syndrome epidemiology, Specimen Handling, Severe Acute Respiratory Syndrome diagnosis, Severe Acute Respiratory Syndrome virology
- Abstract
Severe acute respiratory syndrome (SARS) first appeared in Guangdong Province, China, in November 2002. Although virus isolation and serology were useful early in the SARS outbreak for diagnosing new cases, these tests are not generally useful because virus culture requires a BSL-3 laboratory and seroconversion is often delayed until 2 to 3 weeks after infection. The first qualitative reverse transcriptase-polymerase chain reaction tests for SARS-coronavirus (CoV) were sensitive and capable of detecting 1 to 10 genome equivalents. These assays were quickly supplemented with quantitative real-time assays that helped elucidate the natural history of SARS, particularly the initial presence of low viral loads in the upper respiratory tract and high viral loads in the lower respiratory tract. The unique natural history of SARS-CoV infection dictates the testing of both respiratory and nonrespiratory specimens, the testing of multiple specimens from the same patient, and sending out positives to be confirmed by a reference laboratory. Commercially available reverse transcriptase-polymerase chain reaction tests for SARS have recently appeared; however, meaningful evaluations of these assays have not yet been performed and their true performance has not been determined. These and other issues related to diagnosis of SARS-CoV infection are discussed in this review.
- Published
- 2005
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35. Interobserver agreement in the interpretation of anal intraepithelial neoplasia.
- Author
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Lytwyn A, Salit IE, Raboud J, Chapman W, Darragh T, Winkler B, Tinmouth J, Mahony JB, and Sano M
- Subjects
- Adolescent, Biopsy, Carcinoma in Situ virology, Cytodiagnosis, HIV Seropositivity, Homosexuality, Male, Humans, Male, Anus Neoplasms pathology, Carcinoma in Situ pathology, Observer Variation
- Abstract
Background: Anal carcinoma incidence is increasing, and is highest among men with human immunodeficiency virus (HIV) infection who have sex with men. Anal carcinoma and anal intraepithelial neoplasia (AIN) are ascertained on tissue histology, but requires invasive procedures. Screening for AIN using anal cytology was suggested. The authors evaluated agreement on cytologic and biopsy specimens from HIV-positive men undergoing anal carcinoma screening., Methods: One hundred twenty-nine HIV-positive men with a history of anal-receptive intercourse underwent anal cytology, anoscopy, and biopsy. Four pathologists independently assessed cytology and biopsy specimens and reached consensus for discordant cases., Results: Each pathologist evaluated 120 cytology and 155 biopsy specimens. The weighted kappa value for overall agreement was 0.54 (95% confidence interval [CI], 0.49-0.59) for cytology specimens and 0.59 (95%CI, 0.55-0.63) for biopsy specimens. The median kappa values for pairwise agreement among pathologists and for agreement with consensus were, respectively, 0.69 and 0.77 for cytology and 0.66 and 0.75 for biopsy. At least 3 pathologists were in agreement for 92 (76.7%) cytology and 134 (86.5%) biopsy specimens. Reliability for the Bethesda classification system was at least moderate, except for the cytologic category of atypical squamous cells of undetermined significance (kappa = 0.12). Fourteen of 29 (48.3%) cytology specimens and 36 of 47 (76.6%) biopsy specimens with consensus interpretation of high-grade squamous intraepithelial lesions (HSIL) were interpreted originally as HSIL by > or = 3 pathologists. The kappa value for agreement with consensus distinguishing HSIL from non-HSIL ranged from 0.55 to 0.88 for cytology specimens and from 0.76 to 0.94 for biopsy specimens., Conclusions: Agreement for cytologic and biopsy interpretations was generally at least moderate. Nevertheless, these results supported the need for disease indicators with greater reliability., (Copyright 2005 American Cancer Society.)
- Published
- 2005
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36. Severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice.
- Author
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Zakhartchouk AN, Viswanathan S, Mahony JB, Gauldie J, and Babiuk LA
- Subjects
- Adenoviridae genetics, Adenoviridae metabolism, Animals, Antibodies, Viral biosynthesis, Drug Evaluation, Preclinical, Female, Genetic Vectors, Mice, Mice, Inbred C57BL, Molecular Weight, Nucleocapsid Proteins genetics, Nucleocapsid Proteins immunology, Phosphorylation, Recombinant Proteins biosynthesis, Severe acute respiratory syndrome-related coronavirus immunology, Severe Acute Respiratory Syndrome prevention & control, T-Lymphocytes immunology, Viral Vaccines genetics, Viral Vaccines immunology, Nucleocapsid Proteins biosynthesis, Severe acute respiratory syndrome-related coronavirus genetics, Severe Acute Respiratory Syndrome immunology, Vaccination methods, Viral Vaccines biosynthesis
- Abstract
Severe acute respiratory syndrome coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Thus, vaccination against SARS-CoV may represent an effective approach towards controlling SARS. The nucleocapsid (N) protein is thought to play a role in induction of cell-mediated immunity to SARS-CoV and thus it is important to characterize this protein. In the present study, an E1/partially E3-deleted, replication-defective human adenovirus 5 (Ad5) vector (Ad5-N-V) expressing the SARS-CoV N protein was constructed. The N protein, expressed in vitro by Ad5-N-V, was of the expected molecular mass of 50 kDa and was phosphorylated. Vaccination of C57BL/6 mice with Ad5-N-V generated potent SARS-CoV-specific humoral and T cell-mediated immune responses. These results show that Ad5-N-V may potentially be used as a SARS-CoV vaccine.
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- 2005
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37. Combining human papillomavirus testing or cervicography with cytology to detect cervical neoplasia.
- Author
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Howard M, Sellors JW, Lytwyn A, Roth P, and Mahony JB
- Subjects
- Adult, Cervix Uteri pathology, Cross-Sectional Studies, Female, Humans, ROC Curve, Randomized Controlled Trials as Topic statistics & numerical data, Reference Standards, Sensitivity and Specificity, Carcinoma, Squamous Cell diagnosis, Colposcopy methods, Cytodiagnosis methods, Papillomaviridae isolation & purification, Papillomavirus Infections diagnosis, Tumor Virus Infections diagnosis, Uterine Cervical Neoplasms diagnosis, Uterine Cervical Dysplasia diagnosis
- Abstract
Context: Cervicography and oncogenic human papillomavirus (HPV) testing have been proposed for improving the accuracy of cervical cancer screening., Objective: To examine whether cervicography and HPV testing can improve beyond chance the detection of cervical intraepithelial neoplasia (CIN) 2 or 3 in women with atypical cells of undetermined significance or low-grade squamous intraepithelial lesions on cytology., Design: Cross-sectional analysis. Oncogenic HPV testing by Hybrid Capture II assay or cervicography combined with cytology was compared with the reference standard of colposcopy with directed biopsy., Setting: Community family practices., Participants: Three hundred four women with low-grade cytologic abnormality., Main Outcome Measures: The gain in accuracy for detecting histologic CIN 2 or 3 or carcinoma. Because an adjunct test may improve sensitivity by chance alone, the sensitivity or specificity if the second test performed randomly was estimated., Results: Cervical intraepithelial neoplasia 2 or 3 was found in 11.8% (36/304) of the women and invasive squamous cell carcinoma in 0.3% (1/304). The sensitivity of cytology for detecting CIN 2 or 3 was 73.0% and increased by 21.6% to 94.6% with the addition of a cervigram showing a low-grade lesion or higher or a positive HPV test result. These gains were reduced to 8.1% and 10.8% above the sensitivities expected if the additional tests performed randomly. The corresponding specificities decreased from 49.1% to 32.2% and 33.0%. There was insufficient power to determine whether observed sensitivities were statistically significantly higher than the expected sensitivities., Conclusion: Adjunctive HPV testing or cervicography may provide similar gains in sensitivity, but they can appear misleadingly large if chance increases are not taken into account.
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- 2004
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38. Performance and Cost evaluation of one commercial and six in-house conventional and real-time reverse transcription-pcr assays for detection of severe acute respiratory syndrome coronavirus.
- Author
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Mahony JB, Petrich A, Louie L, Song X, Chong S, Smieja M, Chernesky M, Loeb M, and Richardson S
- Subjects
- Costs and Cost Analysis, Humans, RNA, Viral analysis, Reagent Kits, Diagnostic economics, Severe acute respiratory syndrome-related coronavirus genetics, Sensitivity and Specificity, Reverse Transcriptase Polymerase Chain Reaction economics, Reverse Transcriptase Polymerase Chain Reaction methods, Severe acute respiratory syndrome-related coronavirus isolation & purification, Severe Acute Respiratory Syndrome virology
- Abstract
We evaluated seven reverse transcription-PCR (RT-PCR) assays, including six in-house assays and one commercial assay for the detection of severe acute respiratory syndrome coronavirus (SARS-CoV) RNA in clinical specimens. RT-PCR assays targeted different genomic regions and included three conventional assays (one nested and two non-nested) run on a conventional heat block and four real-time assays performed in a LightCycler (LC; Roche Diagnostics). All in-house assays were optimized for assay parameters, including MgCl2, primer, and probe concentrations. The commercial assay was the RealArt HPA CoV RT-PCR assay (Artus), which was run in the LC. Testing serial dilutions of cultured SARS-CoV showed that the analytical sensitivity of the assays ranged from 10(-8) to 10(-6), corresponding to 1 and 100 copies of viral RNA, respectively. Significant differences in analytical sensitivities were observed between assays (P < 0.01, probit regression analysis for 50% sensitivity levels for the top two assays versus the others). Testing 68 clinical specimens (including 17 respiratory tract specimens, 29 urine samples, and 22 stools or rectal swabs) demonstrated that six of the seven assays detected at least 17 of 18 positives (defined as positive in at least two assays), and two of the assays had a sensitivity of 100%. There were no significant differences in sensitivity between the assays (P = 0.5 [Cochrance Q test, least sensitive 15 of 18 versus 18 of 18]). The specificities of the assays ranged from 94.0 to 100% without significant differences (P = 0.25 to 0.5 [McNemar test]). The reagent and technologist cost of performing the in-house PCR assays ranged from $5.46 to $9.81 Canadian dollars (CDN) per test. The commercial assay cost was considerably higher at $40.37 per test. The results demonstrated good performance for all assays, providing laboratories that need to do SARS RNA testing with a choice of assay formats.
- Published
- 2004
- Full Text
- View/download PDF
39. Adjunctive human papillomavirus testing in the 2-year follow-up of women with low-grade cervical cytologic abnormalities: a randomized trial and economic evaluation.
- Author
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Lytwyn A, Sellors JW, Mahony JB, Daya D, Chapman W, Howard M, Roth P, Lorincz AT, Gafni A, and Walter SD
- Subjects
- Adult, Cost-Benefit Analysis, Cytodiagnosis economics, Cytodiagnosis methods, Female, Follow-Up Studies, Humans, Papanicolaou Test, Papillomavirus Infections virology, Tumor Virus Infections virology, Uterine Cervical Neoplasms virology, Vaginal Smears economics, Vaginal Smears methods, Uterine Cervical Dysplasia virology, Papillomaviridae, Papillomavirus Infections diagnosis, Tumor Virus Infections diagnosis, Uterine Cervical Neoplasms pathology, Uterine Cervical Dysplasia pathology
- Abstract
Context: Although human papillomavirus (HPV) testing may aid in managing low-grade abnormality on screening cervical cytology, patient compliance with repeat testing programs requires consideration., Objectives: To determine effectiveness and costs of repeated Papanicolaou (Pap) test and oncogenic HPV testing for detecting cervical intraepithelial neoplasia 2 or 3., Design: We conducted a randomized controlled trial of combined Pap test and cervical HPV testing by Hybrid Capture 1 test compared with Pap test alone; tests were performed every 6 months for up to 2 years. The study end point was colposcopic examination performed on all women at 2 years, or earlier if an HPV test was positive or if a Pap test showed high-grade squamous intraepithelial lesion., Setting: Sixty-six community family practices., Participants: Two hundred fifty-seven women with atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion on screening cervical cytology., Main Outcome Measures: Detection of histologically confirmed cervical intraepithelial neoplasia 2 or 3, fully allocated costs, and loss to follow-up., Results: Combined Pap test and HPV testing detected 11 (100%) of 11 cases of cervical intraepithelial neoplasia 2/3, whereas Pap test alone detected 7 (63.6%) of these 11 cases (P =.14); corresponding specificities were 39 (46.4%) of 84 and 45 (71.4%) of 63 (P =.005). The cost-effectiveness ratio was Can $4456 per additional case of high-grade cervical intraepithelial neoplasia. Sixty-nine (26.8%) of the 257 women (24.6% combined group vs 29.1% Pap test only group, P =.41) defaulted from testing or from colposcopy when referred with an abnormal result., Conclusions: Combined testing was more costly but may detect more cases of cervical intraepithelial neoplasia 2/3 than Pap test alone. However, poor adherence limits usefulness of a management strategy that requires repeated follow-up.
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- 2003
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40. Incidence, clearance and predictors of human papillomavirus infection in women.
- Author
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Sellors JW, Karwalajtys TL, Kaczorowski J, Mahony JB, Lytwyn A, Chong S, Sparrow J, and Lorincz A
- Subjects
- Adolescent, Adult, Data Collection, Female, Follow-Up Studies, Humans, Incidence, Marital Status, Middle Aged, Multivariate Analysis, Ontario epidemiology, Predictive Value of Tests, Randomized Controlled Trials as Topic, Risk Factors, Sexual Partners, Sexually Transmitted Diseases, Viral epidemiology, Sexually Transmitted Diseases, Viral virology, Surveys and Questionnaires, Women's Health, Papillomaviridae, Papillomavirus Infections epidemiology, Papillomavirus Infections virology, Tumor Virus Infections epidemiology, Tumor Virus Infections virology
- Abstract
Background: Persistent infection with carcinogenic human papillomavirus (HPV) is linked to high-grade lesions and cervical cancer. To better understand the natural history of HPV, we sought to determine the rates of incident and cleared carcinogenic HPV infection, by age, among women aged 15-49 years and to explore risk factors for incident infection., Methods: Women enrolled in an earlier HPV prevalence survey (500 of 800 who were HPV-negative and all 121 who were HPV-positive) were invited to participate in follow-up HPV testing at their periodic health examination one year later. A cervical soft-brush specimen for HPV testing and a smear for cytologic examination were obtained, and participants completed a questionnaire on their demographic characteristics and sexual history., Results: Two hundred and fifty-three (50.6%) previously HPV-negative women and 54 (44.6%) previously HPV-positive women were retested. The mean interval between visits was 14.0 (standard deviation 2.0, median 13.5, range 9.0-21.3) months. Incident HPV infection occurred in 11.1% (28/253) of the women overall, with the highest rate, 25.0% (6/24), in the 15-19-year age group. In the univariate analyses, risk factors for incident HPV were the median number of sexual partners in the past year (< or = 1 v. > or = 2: odds ratio [OR] 8.2, 95% confidence interval [CI] 3.0-22.2; p < 0.001) and the median number of sexual partners over a lifetime (> 3 v. < or = 3: OR 3.0, 95% CI 1.2-7.2; p = 0.014). In multivariate logistic regression modelling adjusted for age, median number of sexual partners in the past year, median number of sexual partners over a lifetime, marital status, current smoking and current use of oral contraceptives, only the median number of sexual partners in the past year remained significantly associated with incidence (OR 6.2, 95% CI 1.6-24.5; p = 0.009). Of the previously HPV-positive women, 51.9% (28/54) had cleared the infection., Interpretation: Incident infection with carcinogenic HPV was highest in women aged 15-19 years, and risk factors were consistent with a sexually transmitted infection. A large proportion of the women who were HPV-positive appeared to have cleared the infection after one year.
- Published
- 2003
41. Chlamydiae host cell interactions revealed using DNA microarrays.
- Author
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Mahony JB
- Subjects
- Animals, Chlamydia Infections virology, Chlamydophila pneumoniae genetics, Chlamydophila pneumoniae physiology, Cytokines genetics, Gene Expression Profiling, Genes, Viral, Growth Substances genetics, Humans, Oligonucleotide Array Sequence Analysis, Protein Kinases genetics, Signal Transduction, Virulence genetics, Chlamydia Infections etiology, Chlamydia Infections genetics, Chlamydophila pneumoniae pathogenicity
- Abstract
Chlamydiae are obligate intracellular bacterial parasites that infect eukaryotic cells and live their entire life cycle within a cytoplasmic vacuole or inclusion. We have employed cDNA microarray and conventional biological approaches to study the pathogen-host cell interaction during C. pneumoniae infection of eukaryotic cells. Two host cell signaling pathways, MEK/ERK and PI 3-kinase/Akt, were activated within 5 and 20 minutes, respectively, following infection with chlamydiae. Pharmacological inhibition of these pathways blocked invasion of HEp2 cells indicating that activation of these pathways was required for infection. Rho family GTPase activity was essential for invasion, since the pan-Rho GTPase inhibitor, compactin, blocked infection of HEp2 cells. cDNA microarrays and reverse transcriptase PCR were used to study host cell and chlamydial gene expression during the replication cycle. Analysis of host cell gene expression following infection with C. pneumoniae indicated that genes coding for cytokines, growth factors, and signaling molecules were upregulated, as early as 2 hours postinfection. Analysis of chlamydial gene expression indicated a temporal regulation of transcription with distinct early-, mid-, and late-cycle classes of RNA transcripts. Newly discovered genes encoding three Ser/Thr protein kinases and one protein phosphatase were upregulated 6-12 hours postinfection. One protein kinase, designated CpnPK1, was first detected at 12 hours postinfection, accumulated in the inclusion throughout the replication cycle, and may be a type III effector molecule. An increased understanding of chlamydial host cell interactions, in particular the role of various chlamydial proteins in infection and identification of essential virulence factors should provide novel targets for the development of new antimicrobials.
- Published
- 2002
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42. Prevalence of infection with carcinogenic human papillomavirus among older women.
- Author
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Sellors JW, Karwalajtys TL, Kaczorowski JA, Mahony JB, Lytwyn A, Chong S, Sparrow J, and Lorincz A
- Subjects
- Adolescent, Adult, DNA, Viral analysis, Female, Genital Diseases, Female diagnosis, Genotype, Humans, Middle Aged, Ontario epidemiology, Papillomavirus Infections diagnosis, Prevalence, Risk Factors, Tumor Virus Infections diagnosis, Uterine Cervical Neoplasms virology, Uterine Cervical Dysplasia virology, Genital Diseases, Female epidemiology, Papillomaviridae genetics, Papillomavirus Infections epidemiology, Tumor Virus Infections epidemiology
- Published
- 2002
43. Influence of clarithromycin on early atherosclerotic lesions after Chlamydia pneumoniae infection in a rabbit model.
- Author
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Fong IW, Chiu B, Viira E, Jang D, and Mahony JB
- Subjects
- Animals, Anti-Bacterial Agents blood, Anti-Bacterial Agents pharmacokinetics, Aorta pathology, Arteriosclerosis pathology, Azo Compounds, Biotransformation, Chlamydia Infections pathology, Cholesterol, Dietary, Clarithromycin blood, Clarithromycin pharmacokinetics, Coloring Agents, Immunohistochemistry, Male, Rabbits, Anti-Bacterial Agents therapeutic use, Arteriosclerosis etiology, Arteriosclerosis prevention & control, Chlamydia Infections complications, Chlamydia Infections drug therapy, Chlamydophila pneumoniae, Clarithromycin therapeutic use
- Abstract
Chlamydia pneumoniae may play a role in atherogenesis and vascular diseases, and antibiotics may prove useful in these conditions. Three groups of New Zealand White rabbits (24 per group) were infected via the nasopharynx with C. pneumoniae on three separate occasions (2 weeks apart). Group I was untreated and sacrificed at 12 weeks; group II received clarithromycin at 20 mg/kg/day for 8 days, beginning 5 days after each inoculation (early treatment); and group III received a similar dose of clarithromycin starting 2 weeks after the third inoculation and continued for 6 weeks thereafter (delayed treatment). To test for a possible anti-inflammatory effect of clarithromycin, two other groups of uninfected rabbits (12 animals in each) were fed 0.5% cholesterol-enriched chow, and one of these groups was treated with clarithromycin at 30 mg/kg/day for 6 weeks. Of 23 untreated infected rabbits, 8 developed early lesions of atherosclerosis, whereas 2 of the 24 early-treated group II had similar changes (P = 0.036 [75% efficacy]). However, in the delayed-treatment group, group III, 3 of 24 rabbits developed early lesions of atherosclerosis, thus demonstrating 62.5% reduction compared to the untreated controls (P = 0.07 [trend to statistical significance]). C. pneumoniae antigen was detected in 8 of 23 group I (untreated) rabbits versus 1 of 24 of the early-treated (group II) rabbits and 4 of 24 animals in the delayed group III (P = 0.009 and 0.138, respectively). All of the untreated, cholesterol-fed rabbits had moderate to advanced atherosclerosis (grade III or IV); clarithromycin had no effect on reducing the prevalence of but did reduce the extent of atherosclerosis in the cholesterol-fed rabbits by 17% compared to untreated controls. Thus, clarithromycin administration modified C. pneumoniae-induced atherosclerotic lesions and reduced the ability to detect organism in tissue. Early treatment was more effective than delayed treatment.
- Published
- 2002
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44. Smoking, season, and detection of Chlamydia pneumoniae DNA in clinically stable COPD patients.
- Author
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Smieja M, Leigh R, Petrich A, Chong S, Kamada D, Hargreave FE, Goldsmith CH, Chernesky M, and Mahony JB
- Subjects
- Aged, Antibodies, Bacterial blood, Chlamydophila pneumoniae isolation & purification, DNA, Bacterial blood, Disease Progression, Female, Humans, Male, Nicotine blood, Nicotine metabolism, Sex Factors, Sputum chemistry, Sputum microbiology, Chlamydophila Infections microbiology, Chlamydophila pneumoniae genetics, DNA, Bacterial isolation & purification, Pulmonary Disease, Chronic Obstructive microbiology, Seasons, Smoking adverse effects
- Abstract
Background: The prevalence and role of Chlamydia pneumoniae in chronic obstructive pulmonary disease (COPD) remain unclear, and molecular methods of detection may help clarify this relationship., Methods: Consecutive clinically stable patients with smoking-related COPD attending a tertiary care outpatient clinic were enrolled in this cross-sectional study. Peripheral blood mononuclear cells were obtained from 100 patients, and induced sputum was obtained in 62 patients. C. pneumoniae DNA was detected in blood or sputum by nested polymerase chain reaction (PCR)., Results: Patients had mean age (standard deviation) of 65.8 (10.7) years, mean forced expiratory volume in one second (SD) of 1.34 (0.61) L, and 61 (61.0%) were male. C. pneumoniae nucleic acids were detected in 27 (27.0%) patients. Among 62 patients with both blood and sputum available, blood specimens were superior to induced sputum for detection of C. pneumoniae DNA (21 versus 7 detected, P=0.003). Current smoking (odds ratio [OR]=2.6, 95 % confidence interval [CI]: 1.1, 6.6, P=0.04), season (November to April) (OR=3.6, 95% CI: 1.4, 9.2, P=0.007), and chronic sputum production (OR=6.4, 95% CI: 1.8, 23.2, P=0.005) were associated with detection of C. pneumoniae DNA., Conclusions: C. pneumoniae DNA prevalence was higher among current smokers, and during winter/spring months. Prospective molecular studies are needed to examine the role of C. pneumoniae detection in COPD disease symptoms and progression.
- Published
- 2002
- Full Text
- View/download PDF
45. Identification of MEK- and phosphoinositide 3-kinase-dependent signalling as essential events during Chlamydia pneumoniae invasion of HEp2 cells.
- Author
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Coombes BK and Mahony JB
- Subjects
- Actins metabolism, Bacterial Adhesion, Cell Line, Enzyme Activation, Enzyme Inhibitors pharmacology, Focal Adhesion Kinase 1, Focal Adhesion Protein-Tyrosine Kinases, Humans, Microscopy, Electron, Scanning, Microvilli microbiology, Microvilli ultrastructure, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases metabolism, Phosphoinositide-3 Kinase Inhibitors, Phosphoproteins metabolism, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein-Tyrosine Kinases metabolism, Proteins metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-akt, Shc Signaling Adaptor Proteins, Signal Transduction, Src Homology 2 Domain-Containing, Transforming Protein 1, Tyrosine metabolism, Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport, Chlamydophila pneumoniae pathogenicity, MAP Kinase Kinase Kinase 1, Phosphatidylinositol 3-Kinases metabolism, Protein Serine-Threonine Kinases metabolism
- Abstract
The ability of Chlamydia pneumoniae to survive and cause disease is predicated on efficient invasion of cellular hosts. While it is recognized that chlamydial determinants are important for mediating attachment and uptake into non-phagocytic cells, little is known about the bacterial ligands and cellular receptors that facilitate invasion or host cell signal transduction pathways implicated in this process. We used transmission and scanning electron microscopy to demonstrate that attachment of bacteria to host cells induced the appearance of microvilli on host cell membranes. Invasion occurred 30-120 min after cell contact with the subsequent loss of membrane microvilli. Using an epithelial cell infection model, C. pneumoniae invasion caused a rapid and sustained increase in MEK-dependent phosphorylation and activation of ERK1/2, followed by PI 3-kinase-dependent phosphorylation and activation of Akt. Tyrosine phosphorylation of focal adhesion kinase (FAK) preceded its appearance in a complex with the p85 subunit of PI 3-kinase during chlamydial invasion and isoform-specific tyrosine phosphorylation of the docking protein Shc also occurred at the time of attachment and entry of bacteria. Chlamydia entry but not attachment could be abrogated with specific inhibitors of MEK, PI 3-kinase and actin polymerization, demonstrating the importance of these signalling pathways and an intact actin cytoskeleton for C. pneumoniae invasion. These results suggest that activation of specific cell signalling pathways is an essential strategy used by C. pneumoniae to invade epithelial cells.
- Published
- 2002
- Full Text
- View/download PDF
46. Chlamydia pneumoniae infection of endothelial cells induces transcriptional activation of platelet-derived growth factor-B: a potential link to intimal thickening in a rabbit model of atherosclerosis.
- Author
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Coombes BK, Chiu B, Fong IW, and Mahony JB
- Subjects
- Animals, Aorta metabolism, Aorta microbiology, Aorta pathology, Arteriosclerosis microbiology, Arteriosclerosis pathology, Arteriosclerosis physiopathology, Chlamydophila Infections pathology, Disease Models, Animal, Endothelium, Vascular pathology, Humans, Muscle, Smooth, Vascular microbiology, Muscle, Smooth, Vascular pathology, Proto-Oncogene Proteins c-sis genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Rabbits, Tumor Cells, Cultured, Tunica Intima pathology, Chlamydophila Infections metabolism, Chlamydophila Infections microbiology, Chlamydophila pneumoniae pathogenicity, Endothelium, Vascular microbiology, Proto-Oncogene Proteins c-sis metabolism, Transcriptional Activation
- Abstract
Smooth muscle cell (SMC) proliferation and intimal thickening are hallmark features of atherosclerotic disease, and Chlamydia pneumoniae may contribute to atherogenesis by imparting biological effects on SMCs. An in vitro endothelial cell model and a normocholesterolemic rabbit model were used to test the hypothesis that infection with C. pneumoniae induces SMC growth factor production, SMC proliferation, and aortic intimal thickening. Using reverse-transcriptase polymerase chain reaction, it was demonstrated that C. pneumoniae infection of endothelial cells induced platelet-derived growth factor (PDGF)-B messenger RNA expression. In C. pneumoniae-infected rabbits, maximum intimal thickness (MIT) was significantly greater than that in uninfected animals (P< .0001). MIT correlated with the presence of C. pneumoniae antigen (P= .043) and PDGF-B (P= .002) in aortic tissues, and C. pneumoniae antigen was independently correlated with the presence of PDGF-B in aortic tissues (P= .009). These results suggest that C. pneumoniae-induced SMC proliferation and intimal thickening may be mediated through PDGF-B and may be a molecular mechanism by which C. pneumoniae infection could contribute to atherogenesis.
- Published
- 2002
- Full Text
- View/download PDF
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