The microanatomy of Bowman's layer in the cornea of the pig: Changes in collagen fibril architecture at the corneoscleral limbus

In most animals, Bowman's layer is a feature of the cornea of the eye, and lies between the sur-face epithelium and the stromal extracellular matrix that makes up the bulk of the cornea. It is comprised of a condensation of disorganised collagen fibrils. However, it has been conjectured that not all species possess Bowman’s layer, and pigs are a species that has classically been stated to lack this anatomical structure, although there is disagreement in the published literature. Here, we studied the porcine cornea using transmission and scanning electron microscopy (TEM and SEM) to ascertain whether Bowman’s layer existed. TEM identified a thin band of disorganised collagen fibrils between the epithelial basement membrane and corneal stroma. SEM images of the central and peripheral corneal surfaces, following removal of the corneal epithelium by cell maceration, revealed a disorganised meshwork of collagen fibrils, with a highly aligned annulus of collagen at the limbus. In between the peripheral cornea and limbus, a "transition zone" is observed where collagenfibrils start to align. Quantification of fibril alignment demonstrates a significant increase in collagen alignment from 0.08 ± 0.04 to 0.33 ± 0.07 (p < 0.001; n = 60; 0 = no alignment, 1 = full alignment) with increasing distance from the corneal centre. These data together lead us to conclude that the porcine cornea does include Bowman's layer, though it is thin (contributing roughly 0.2% of corneal thickness), and thus, reaffirms the porcine cornea's similarity to its human counterpart and usefulness as a model system

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Chondroitin Sulfate as a Potential Modulator of the Stem Cell Niche in Cornea.

Chondroitin sulfate (CS) is an important component of the extracellular matrix in multiple biological tissues. In cornea, the CS glycosaminoglycan (GAG) exists in hybrid form, whereby some of the repeating disaccharides are dermatan sulfate (DS). These CS/DS GAGs in cornea, through their presence on the proteoglycans, decorin and biglycan, help control collagen fibrillogenesis and organization. CS also acts as a regulatory ligand for a spectrum of signaling molecules, including morphogens, cytokines, chemokines, and enzymes during corneal growth and development. There is a growing body of evidence that precise expression of CS or CS/DS with specific sulfation motifs helps define the local extracellular compartment that contributes to maintenance of the stem cell phenotype. Indeed, recent evidence shows that CS sulfation motifs recognized by antibodies 4C3, 7D4, and 3B3 identify stem cell populations and their niches, along with activated progenitor cells and transitional areas of tissue development in the fetal human elbow. Various sulfation motifs identified by some CS antibodies are also specifically located in the limbal region at the edge of the mature cornea, which is widely accepted to represent the corneal epithelial stem cell niche. Emerging data also implicate developmental changes in the distribution of CS during corneal morphogenesis. This article will reflect upon the potential roles of CS and CS/DS in maintenance of the stem cell niche in cornea, and will contemplate the possible involvement of CS in the generation of eye-like tissues from human iPS (induced pluripotent stem) cells.

Recovering vision in corneal epithelial stem cell deficient eyes.

A healthy corneal epithelium, which is essential for proper vision and protection from external pathogens, is continuously replenished throughout life by stem cells located at the limbus. In diseased or injured eyes, however, in which stem cells are deficient, severe ocular problems manifest themselves. These are notoriously difficult to manage and as a result the last 20 or so years has seen a number of therapeutic strategies emerge that aim to recover the ocular surface and restore vision in limbal stem cell deficient eyes. The dominant concept involves the generation of laboratory cultivated epithelial cell sheets expanded from small biopsies of the epithelial limbus (for patient or donors) or another non-corneal epithelial tissue such as the oral mucosa. Typically, cells are grown on sterilised human amniotic membrane as a substrate, which then forms part of the graft, or specially formulated plastic culture dishes from which cells sheets can be released by lowering the temperature, and thus the adherence of the plastic to the cells. Overall, clinical results are promising, as is discussed, with new cultivation methodologies and different cell lineages currently being investigated to augment the treatment options for visual disturbance caused by a corneal epithelial limbal stem cell deficiency.

Multidrug-resistant North American pulsotype 2 Clostridium difficile was the predominant toxigenic hospital-acquired strain in the province of Manitoba, Canada, in 2006-2007.

The objective of the current study was to determine whether the antimicrobial susceptibility profile or genotype of hospital-acquired isolates of Clostridium difficile differed from isolates causing community-acquired disease. Five hundred diarrhoeal stool samples (one >2 ml sample per patient) from patients across Manitoba, Canada, in 2006-2007 that were reported as C. difficile toxin positive were cultured, resulting in 432 isolates of toxin-positive C. difficile for analysis. Of these 432 isolates, acquisition status could be determined for 235 (54.4%); 182 (77.4%) isolates were hospital acquired and 53 (22.6%) were community acquired. North American pulsotype (NAP) designations based on SmaI PFGE could be defined for 52.3% of the 432 isolates, with NAP2 (n=122) being the most common. Ninety-one per cent (71/78) of NAP2 isolates were recovered from patients with hospital-acquired C. difficile disease. Other NAP types and isolates with non-NAP-type PFGE patterns were less frequently associated with hospital-acquired disease. Community-acquired disease (35.3% of isolates) was associated with a wide variety of NAP types. NAP2 isolates were homogeneous (85.5% had SmaI PFGE pattern 0003) and demonstrated low susceptibility to moxifloxacin (6.6%) and clindamycin (1.6%) compared with non-NAP2 isolates (64.1-93.2% moxifloxacin susceptible; 14.1-28.2% clindamycin susceptible). All isolates of C. difficile in Manitoba were susceptible to metronidazole, piperacillin-tazobactam, amoxicillin-clavulanate and meropenem. NAP2 isolates of toxigenic C. difficile were approximately three times more common than NAP1 isolates (28.2 vs 9.1%) in Manitoba in 2006-2007, and these isolates demonstrated high levels of clonality and multidrug resistance, and were associated with hospital acquisition.

The responses of Aboriginal Canadians to adjuvanted pandemic (H1N1) 2009 influenza vaccine.

BACKGROUND: Because many Aboriginal Canadians had severe cases of pandemic (H1N1) 2009 influenza, they were given priority access to vaccine. However, it was not known if the single recommended dose would adequately protect people at high risk, prompting our study to assess responses to the vaccine among Aboriginal Canadians. METHODS: We enrolled First Nations and Métis adults aged 20-59 years in our prospective cohort study. Participants were given one 0.5-mL dose of ASO3-adjuvanted pandemic (H1N1) 2009 vaccine (Arepanrix, GlaxoSmithKline Canada). Blood samples were taken at baseline and 21-28 days after vaccination. Paired sera were tested for hemagglutination-inhibiting antibodies at a reference laboratory. To assess vaccine safety, we monitored the injection site symptoms of each participant for seven days. We also monitored patients for general symptoms within 7 days of vaccination and any use of the health care system for 21-28 days after vaccination. RESULTS: We enrolled 138 participants in the study (95 First Nations, 43 Métis), 137 of whom provided all safety data and 136 of whom provided both blood samples. First Nations and Métis participants had similar characteristics, including high rates of chronic health conditions (74.4%-76.8%). Pre-existing antibody to the virus was detected in 34.3% of the participants, all of whom boosted strongly with vaccination (seroprotection rate [titre ≥ 40] 100%, geometric mean titre 531-667). Participants with no pre-existing antibody also responded well. Fifty-eight of 59 (98.3%) First Nations participants showed seroprotection and a geometric mean titre of 353.6; all 30 Métis participants with no pre-existing antibody showed seroprotection and a geometric mean titre of 376.2. Pain at the injection site and general symptoms frequently occurred but were short-lived and generally not severe, although three participants (2.2%) sought medical attention for general symptoms. INTERPRETATION: First Nations and Métis adults responded robustly to ASO3-adjuvanted pandemic (H1N1) 2009 vaccine. Virtually all participants showed protective titres, including those with chronic health conditions.

Population-based surveillance of Clostridium difficile infection in Manitoba, Canada, by using interim surveillance definitions.

OBJECTIVE: To apply interim surveillance definitions of Clostridium difficile infection (CDI) cases to 1 year of data from the provincewide surveillance system of Manitoba, Canada, to determine the epidemiology of CDI incident cases in a population. METHODS: CDI cases were categorized with interim surveillance definitions developed by an ad hoc C. difficile surveillance working group. Incident cases recorded in the provincial CDI database between July 2005 and June 2006 were linked to the provincial hospitalization and nursing home databases and analyzed. RESULTS: One thousand six incident cases were identified over 1 year. Five hundred fifteen (51%) cases were associated with and began in a healthcare facility (HCF), whereas 275 (27%) were associated with and began in the community. An additional 131 (13%) cases were HCF associated but began in the community, while 85 (8%) were of indeterminate origin. Cases of HCF-associated CDI occurred in patients who were older than did cases of community-associated CDI (P < .0001). The provincial rate of community-onset cases was 23.4 per 100,000 person-years, and rates varied among geographic areas. HCF-associated CDI rates among the 10 largest hospitals varied from 0.5 to 8.4 per 10,000 patient-days. The time to CDI onset after hospital admission indicated that 25% of nosocomial cases began by the 8th day, and 50% began by the 17th day. CONCLUSIONS: Although the majority of CDI cases were associated with exposure to a HCF, 40% of incident CDI began in the community. Populations with HCF- and community-associated CDI demonstrated significantly different age distributions. The wide variation of rates among HCFs requires explanation. The high percentage of incident cases in the community warrants increased study.

Clinical characteristics and outcomes in patients with pulmonary blastomycosis.

BACKGROUND: Blastomycosis is an uncommon granulomatous infection caused by the thermally dimorphic fungus Blastomyces dermatitidis. The most frequent clinical infections involve the lung, skin, and bone. Pulmonary manifestations range from asymptomatic self-limited infection to severe diffuse pneumonia causing respiratory failure. OBJECTIVES: To establish the clinical characteristics and outcomes of patients with pulmonary blastomycosis diagnosed at hospitals in Manitoba and northwestern Ontario, Canada. METHODS: A retrospective review of medical records was done for 318 patients with blastomycosis in these regions. RESULTS: The majority of patients were Caucasian (198 (62.5%) patients), male (193 (61%) patients), and residents of Ontario (209 (65.7%) patients). Most patients were treated in an inpatient hospital ward (266 (84%) patients) and survived (294 (92%) patients). Pulmonary involvement, either alone or associated with other sites, was present in 296 (93%) of the 318 patients; 22 (7%) patients had no evidence of pulmonary blastomycosis. The majority of patients had localized lung disease (1-3 quadrants on chest radiograph involved; 225 (82%) patients). Of 294 (92%) patients requiring hospitalization, 266 (90%) patients received all inpatient care on a general medical ward; 28 (10%) patients received some care in the intensive care unit (ICU). Factors associated with ICU admission included diffuse pulmonary disease (four quadrants involved on chest radiograph), diabetes, and prior use of antimicrobial therapy. Twenty-four (8%) patients died, and multivariate analysis showed that older age and Aboriginal ethnicity were the significant risk factors for death from blastomycosis. CONCLUSION: Blastomycosis is a cause of serious, potentially life-threatening pulmonary infection in this geographic region.

Risk factors for acquisition of endemic blastomycosis.

BACKGROUND: Blastomycosis is potentially fatal, but environmental risk factors for acquiring blastomycosis are not well established. METHOD: Matched cross-sectional questionnaire of 112 patients with history of blastomycosis and 118 control subjects in Manitoba and northwestern Ontario. RESULTS: The most common tissues involved with blastomycosis were pulmonary, skin and soft tissues, and bone. A significantly greater proportion of patients with blastomycosis than control subjects were involved in outdoor occupations. A significantly greater percentage of patients with blastomycosis were immunosuppressed either from collagen vascular disease or immunosuppressive therapy, or had hypothyroidism. A significant association between canine and human blastomycosis was not observed. CONCLUSIONS: Independent risk factors for development of blastomycosis included immunosuppression for any reason (including drugs or disease), collagen vascular disease, being an outdoor worker, and having a coworker with blastomycosis. Canine blastomycosis was not a risk factor for human disease in dog owners.

Seasonal variations in the clinical presentation of pulmonary and extrapulmonary blastomycosis.

Blastomycosis is a granulomatous infection caused by the thermally dimorphic fungus, Blastomyces dermatitidis, for which seasonal variation has been proposed. We conducted a retrospective review of medical records of 324 patients with blastomycosis in Manitoba and northwestern Ontario. The average age of patients at the time of diagnosis was 39+/-20 (range, 0-85) years. Symptoms referable to blastomycosis were first noted in the autumn and winter (September to February) by 63% of the patients. The seasonal distribution of cases was different for localized pulmonary infection than the disseminated disease (P<0.0001). For localized lung disease, the peak incidence of symptom onset occurred in the autumn, and lowest incidence in the spring; one half (50%) of the patients with diffuse lung disease had onset of symptoms in the spring months and a few (11%) cases occurred during the summer. We noted a distinct seasonal variation in the clinical presentation of blastomycosis. The observed pattern suggests that summer environmental exposure and acquisition of the infection results in an early (1-6 months) localized pneumonia in the majority of cases, followed by later (4-9 months) reactivation or slow progression of asymptomatic infection resulting in isolated extrapulmonary or disseminated hematogenous disease in the minority.

Key research issues in Clostridium difficile.

Clostridium difficile is an emerging pathogen that causes C difficile-associated diarrhea, an important nosocomial infection. Control of this infection remains a challenge, and much needs to be determined about the antimicrobial resistance of the organism, antibiotic stewardship, contamination of the patient environment, and various host factors that determine susceptibility or resistance to infection. A national symposium focusing on C difficile infections, the Clostridium difficile Symposium on Emerging Issues and Research, was hosted on November 23, 2004, by the Department of Medical Microbiology and Infectious Diseases at the University of Manitoba, Winnipeg, Manitoba, in partnership with the Canadian Institutes of Health Research. This symposium, which aimed to summarize key research issues regarding C difficile infections in Canada, had the following objectives: to provide a forum for learning and discussion about C difficile and its impact on the health of Canadians; to identify the key research issues that should be addressed; and to explore potential research funding opportunities and collaboration. The present report summarizes key research issues identified for C difficile infections in Canada by addressing four major themes: diagnosis and surveillance, infection prevention and control, antibiotic stewardship, and clinical management.

Canadian Association for Immunization Research and Evaluation (CAIRE) guidelines for industry-sponsored clinical trial and epidemiology contract research.

In response to concerns about interactions of academic and public health investigators with industry, the Canadian Association for Immunization Research and Evaluation (CAIRE), in collaboration with six major vaccine manufacturers, developed guidelines for participation in industry-sponsored clinical trial and epidemiology contract research within Canada. Topics addressed include definition of investigators, data ownership, protocol development, data management, data analysis, producing a study report and publication of the results of the study.

Group-theoretic structure in the sequential flow of sensory experiences.

The hypothesis presented here is that in a given person, the overall configuration of sensory variables at any given moment, i.e. everything that the person is seeing, hearing, etc., can be treated as belonging to an infinite sequence of such momentary configurations, with the sequence being generated by a particular mathematical group. The ideas behind this are as follows. If a person's various moment-to-moment sensory experiences can all be described within the same coordinate system, then they can be treated as objects that sequentially transform into one another. If they are also encoded by brain events, then the person's own encounters with this encoding, e.g. when participating in brain-stimulation experiments, amount to instances where a given sensory experience inevitably follows other sensory experiences occurring in a specific sequence. For example, a person's visual encounters with certain equipment-settings might always be followed by a particular visual hallucination. If there exist sequences like this for any given momentary sensory experience, then these collectively form the person's sensory code, as it would be encountered from the person's own perspective. The person's ongoing flow of sensory experiences may then be treated as part of the infinite sequence implied by the various code-conserving ways in which sensory experiences can flow from one to the next. If this sequence can be generated by a finite number of code-conserving segments that transform into each other and link together end to end, then a person's full range of sensory experiences can be defined in group-theoretic terms.