C. albicans Colonization of Human Mucosal Surfaces

Background: Candida albicans is a low level commensal organism in normal human populations with the continuous potential to expand and cause a spectrum of clinical conditions. Methodology/Principal Findings: Using ex vivo human organ cultures and populations of primary human cells, we have developed several related experimental systems to examine early-stage interactions between C. albicans and mucosal surfaces. Experiments have been conducted both with exogenously added C. albicans and with overtly normal human mucosal surfaces supporting pre-existing infections with natural isolates of Candida. Under different culture conditions, we have demonstrated the formation of C. albicans colonies on human target cells and filament formation, equivalent to tissue invasion. Conclusions/Significance: These organ culture systems provide a valuable new resource to examine the molecular and cellular basis for Candida colonization of human mucosal surfaces

Herpes Simplex Virus-Induced Epithelial Damage and Susceptibility to Human Immunodeficiency Virus Type 1 Infection in Human Cervical Organ Culture

Normal human premenopausal cervical tissue has been used to derive primary cell populations and to establish ex vivo organ culture systems to study infections with herpes simplex virus (HSV-1 or HSV-2) and human immunodeficiency virus type 1 (HIV-1). Infection with either HSV-1 or HSV-2 rapidly induced multinuclear giant cell formation and widespread damage in mucosal epithelial cells. Subsequent exposure of the damaged mucosal surfaces to HIV-1 revealed frequent co-localization of HSV and HIV-1 antigens. The short-term organ culture system provides direct experimental support for the epidemiological findings that pre-existing sexually transmitted infections, including primary and recurrent herpes virus infections at mucosal surfaces, represent major risk factors for acquisition of primary HIV-1 infection. Epithelial damage in combination with pre-existing inflammation, as described here for overtly normal human premenopausal cervix, creates a highly susceptible environment for the initiation and establishment of primary HIV-1 infection in the sub-mucosa of the cervical transformation zone

NUTRITION AND PHYSICAL ACTIVITY AMONG GRADE 6 STUDENTS IN THE PORCUPINE HEALTH UNIT AREA : a comparison with the balanced school day

This thesis / dissertation was completed and submitted at Nipissing University, and is made freely accessible through the University of Toronto’s TSpace repositoryIn recent years, childhood obesity prevalence has increased, and there are calls for action to reduce our obesogenic environment in the school, home, workplace, and community. Approximately 28% of children in the Porcupine Health Unit area are overweight or obese. This research attempts to determine local data on factors that contribute to overweight and obesity such as lack of consumption of vegetables and fruits, consumption of foods of low nutritional value, family meals, body image, and physical activity and inactivity. School boards in Ontario, including one in the Porcupine Health Unit area, are implementing the balanced school day schedule. Is there a difference in these factors among children in a balanced school day schedule compared to the traditional schedule? This research is of value for schools considering implementation of the balanced school day schedule and provides a greater understanding of local factors to generate solutions that will reduce our obesogenic environment.M.Ed

Confocal microscopy and three-dimensional image reconstructions of a <i>C. albicans</i> colony growing on human cells.

<p>A) Brightest point projection of a confocal Z series showing a <i>C. albicans</i> colony (stained blue with calcofluor) grown for 22 hours on a monolayer of primary human fibroblasts (stained red with wheat germ agglutinin). Bar = 100 µm. B) Three-dimensional reconstruction of the colony shown in panel A. This image has been rotated relative to the image shown in panel A. C) Different perspective of the three-dimensional reconstructed image shown in panel B. D) Exactly the same reconstructed view of the colony as shown in panel C but with the surface of the fibroblast monolayer now rendered semi-transparent to reveal fungal cells beneath the mammalian cell surface. Note the continuous appearance of the filamentous extension (arrowhead in D) that is not visible with the opaque monolayer surface in panel C.</p

Matched Primary Patient Isolates: Candida Strains and Proliferating Human Cell Populations.

<p>Matched Primary Patient Isolates: Candida Strains and Proliferating Human Cell Populations.</p

<i>C. albicans</i> colony formation on confluent monolayers of primary human cells.

<p>A) Direct observation of <i>C. albicans</i> colonies after 24 hours of coculture on primary tonsillar fibroblasts. B) Direct observation of <i>C. albicans</i> colonies after 48 hours of coculture on primary tonsillar fibroblasts - the same cell populations as panel A photographed 24 hours later. C) <i>C. albicans</i> colonies after 24 hours of coculture on primary tonsillar fibroblasts viewed by phase contrast microscopy. Bar = 200 µm. D) Magnified phase contrast image from (C) showing the central area between the 3 independent <i>C. albicans</i> colonies. Bar = 100 µm. E) <i>C. albicans</i> colonies after 24 hours of coculture on primary cervical fibroblasts viewed by phase contrast microscopy. Bar = 200 µm. F, G) <i>C. albicans</i> colonies after 24 hours of coculture on primary cervical epithelial cells viewed by phase contrast microscopy. Bar = 200 µm. Note that the colonies shown in panels E–G were cultured and photographed under identical conditions.</p

<i>C. albicans</i> invasion of human tissue in <i>ex vivo</i> organ culture.

<p>Freshly removed human tissue pieces were cocultured with <i>C. albicans</i> for 24 hours and then the tissue pieces were fixed in formalin and embedded in paraffin. 5 micron sections were stained with fluorescently labeled reagents and visualized by fluorescence microscopy (A–D) or stained with hematoxylin and eosin and viewed by standard bright field microscopy (E, F). These images are representative of experimental <i>C. albicans</i> colonization and invasion for five different premenopausal human cervical tissue pieces. A) <i>C. albicans</i> yeast cells and filaments (stained blue with calcofluor) invading the stratified squamous epithelium of human ectocervix (stained red with wheat germ agglutinin). Bar = 25 µm. B) <i>C. albicans</i> filaments (blue) invading the cut stromal surface of human ectocervix (red). Bar = 25 µm. C) <i>C. albicans</i> yeast cells and filaments (green: detected with a rabbit polyclonal anti-<i>C. albicans</i> antibody conjugated to FITC) invading the stratified squamous epithelium of human ectocervix (stained red with wheat germ agglutinin). Bar = 50 µm. D) <i>C. albicans</i> yeast cells and filaments (green: detected with a rabbit polyclonal anti-<i>C. albicans</i> antibody conjugated to FITC) invading a cut stromal surface of human ectocervix (stained red with wheat germ agglutinin). Bar = 50 µm. E) <i>C. albicans</i> growth on the cut stromal surface of endocervix. Note the absence of <i>C. albicans</i> growth on the columnar epithelial surface of endocervix (arrows). Bar = 50 µm. F) Magnified image of (D) showing colonization of the cut stromal surface of endocervix. Bar = 25 µm.</p

<i>C. albicans</i> outgrowth from pre-existing natural infections during <i>ex vivo</i> organ culture with human palatine tonsil.

<p>Freshly removed pieces of human palatine tonsil were cultured for 4 days in complete RPMI medium containing antibiotics and amphotericin B, fixed and processed for routine histological analysis. A, C) Hematoxylin and eosin staining of 5 micron tissue sections showing filaments (arrows) and yeast cells (arrow heads) at or near the luminal surfaces of two different palatine tonsil tissue pieces. Tonsil A: 23 year old male patient with recurrent tonsillitis. Tonsil B: 13 year old female patient with snoring problems. Bars = 50 µm. B, D) Merged fluorescence images of 5 micron sections from the corresponding tissue blocks shown in A and C respectively that were incubated with a rabbit polyclonal anti-<i>C. albicans</i> antibody conjugated to FITC (green) and then the tissues were counterstained with wheat germ agglutinin (red). Bars = 50 µm.</p

Analysis of filamentous extensions from <i>C. albicans</i> colonies growing on human cells.

<p>A) Immunocytochemical staining with a rabbit polyclonal antibody of a <i>C. albicans</i> colony grown on top of a monolayer of primary human tonsillar fibroblasts. The human cells were counterstained with hematoxylin. Bar = 200 µm. B) Immunocytochemical staining of the same <i>C. albicans</i> colony in panel A, showing a major filament extending across the surface of the primary tonsillar monolayer. Bar = 100 µm. C) Hematoxylin staining of a <i>C. albicans</i> colony growing on top of a monolayer of primary human tonsillar fibroblasts. The mammalian cell nuclei are visible directly beneath the colony structure. Bar = 100 µm.D) Enlarged image of the lower extremities of the colony shown in panel C. Bar = 25 µm.</p

Co-localization of viral antigens in multinucleated giant cells generated from cervical tissue dually-infected with HSV-1+HIV-1.

<p>Tissue pieces were infected with 1×10⁵ pfu of HSV-1, incubated for 24 hours then infected with HIV-1 (virus equivalent to 20 pg of p24gag) in normal seminal plasma and then incubated for an additional 120 hours. A, B: Giant cell aggregates visualized by confocal fluorescence microscopy; green: rabbit polyclonal anti-HSV antibody, red: mouse monoclonal anti-HIV-1 p24 gag antibody with TSA enhancement, blue: TOTO-3 nuclear stain. Coincidence of green and red signals produces a yellowish-white color. C: Control uninfected tissue section processed in parallel with the antibody combination used in A, B. Cell nuclei visualized with TOTO-3 nuclear stain. D, E: Detection of HSV-1 and HIV-1 antigens in close proximity at the ectocervical surface; green: rabbit polyclonal anti-HSV antibody, red: mouse monoclonal anti-HIV-1 p24 gag antibody with TSA enhancement, blue: TOTO-3 nuclear stain. Arrows indicate co-localization of HSV-1 and HIV-1 antigens in giant cell forms. F-G: Confocal fluorescence microscopy with normal premenopausal endocervical tissue that was cultured for 6 days prior to fixation and processing; green: rabbit polyclonal anti-cytokeratin antibody detecting epithelial cells, red: mouse monoclonal anti-CD4 antibody, with TSA enhancement, detecting a focus of CD4+ T cells located just below the epithelial surface, blue: TOTO-3 nuclear stain. Bars A = 25 µm; B = 20 µm; C = 50 µm; D = 50 µm; E = 20 µm; F = 50 µm; G = 25 µm.</p