Immunogold labeling of Pneumocystis carinii for Electron microscopy

1991 ◽  
Vol 49 ◽  
pp. 270-271
Author(s):  
Michael P. Goheen ◽  
Marilyn S. Bartlett ◽  
James W. Smith
Keyword(s):  
Electron Microscopy ◽  
Pneumocystis Carinii ◽  
Severe Pneumonia ◽  
Culture Fluid ◽  
Immunogold Labeling ◽  
Antigenic Sites ◽  
Transmission Electron ◽  
Response To Chemotherapy ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome

Studies of the biology of Pneumocystis carinii (PC) are of increasing importance because this extracellular pathogen is a frequent source of severe pneumonia in patients with acquired immunodeficiency syndrome (AIDS) and is a leading cause of mortality in these patients. Immunoelectron microscopic localization of antigenic sites on the surface of PC would improve the understanding of these sites and their role in pathenogenisis of the disease and response to chemotherapy. The purpose of this study was to develop a methodology for visualizing immunoreactive sites on PC with transmission electron microscopy (TEM) using immunogold labeled probes.Trophozoites of PC were added to spinner flask cultures and allowed to grow for 7 days, then aliquots of tissue culture fluid were centrifuged at 12,000 RPM for 30 sec. Pellets of organisims were fixed in either 1% glutaraldehyde, 0.1% glutaraldehyde-4% paraformaldehyde, or 4% paraformaldehyde for 4h. All fixatives were buffered with 0.1M Na cacodylate and the pH adjusted to 7.1. After fixation the pellets were rinsed in 0.1M Na cacodylate (3X), dehydrated with ethanol, and immersed in a 1:1 mixture of 95% ethanol and LR White resin.

Effects of Atovaquone on the Ultrastructural Morphology of Pneumocystis Carinii

1997 ◽  
Vol 3 (S2) ◽  
pp. 81-82
Author(s):  
M.P. Goheen ◽  
M.S. Bartlett ◽  
M.M. Shaw ◽  
S.R. Meshnick ◽  
J.W. Smith
Keyword(s):  
Adverse Reactions ◽  
Pneumocystis Carinii ◽  
Spinner Flask ◽  
Short Term ◽  
Human Embryonic Lung ◽  
Transmission Electron ◽  
Significant Incidence ◽  
Wide Range ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome

Pneumocystis carinii pneumonia (PCP) occurs at some time in most patients with acquired immunodeficiency syndrome (AIDS). Trimethoprim/sulfamethoxazole or pentamidine isothionate are the traditional modes of therapy for treatment and prophylaxis of PCP. Unfortunately these drugs are associated with a significant incidence of adverse side effects particularly in patients with AIDS. Toxicity and a growing concern that P. carinii strains are becoming resistant to these compounds is providing the impetus for the search for additional drugs to combat P. carinii. Atovaquone, developed as an antimalarial agent, has activity against a wide range of other organisms, including Toxoplasma sp. and P. carinii, with a lower incidence of adverse reactions during clinical trials. Atovaquone inhibits mitochondrial respiration in P. falciparum and P. carinii. In this study transmission electron microscopy (TEM) was used to observe the effects of atovaquone on P. carinii organisms in short term spinner flask culture.Spinner flask cultures of human embryonic lung cells were inoculated with P. carinii from infected rat lung.

Enhancement of the fine structure of Pneumocystis carinii using potassium ferrocyanide and tannic acid

1989 ◽  
Vol 47 ◽  
pp. 984-985 ◽  
Author(s):  
M.P. Goheen ◽  
P. Blumershine ◽  
M.T. Hull ◽  
M.S. Bartlett ◽  
J.W. Smith
Keyword(s):  
Tannic Acid ◽  
Pneumocystis Carinii ◽  
Calf Serum ◽  
Severe Pneumonia ◽  
Culture Fluid ◽  
Lung Fibroblasts ◽  
Human Embryonic Lung ◽  
Ultrastructural Studies ◽  
Cacodylate Buffer ◽  
Immunodeficiency Syndrome

Pneumocystis carinii (PC) is an obligate parasite found in the lungs of many vertebrates including man. A recent upsurge of interest in this organism is due to its role in producing severe pneumonia in over two thirds of individuals with acquired immunodeficiency syndrome (AIDS). Many studies have been published of the ultra- structure of PC in steroid treated rats or immunocompromised patients. Several laboratories have reported growing PC in culture, but few ultrastructural studies have resulted. Additionally, PC from culture or vertebrates has been difficult to fix in a fashion which would preserve the fine structural detail. This study reports that post-fixation with tannic acid-osmium and ferrocyanide-osmium mixtures greatly improves the ultrastructural morphology of PC from culture.Human embryonic lung fibroblasts were cultured in minimal essential medium with 10% fetal calf serum. The cultures were inoculated with viable trophozoites obtained from steroid treated rat lung and incubated in 5% O2 10% CO2, balance N2 at 35°C for seven days. At this time three aliquots of tissue culture fluid were centrifuged to obtain pellets of PC. The supernatants were decanted and replaced with Karnovsky's fixative, and the pellets allowed to fix for two hours at 20°C. Following a rinse in cacodylate buffer two of the specimens were post-fixed in 1% buffered osmium, and the third specimen was immersed in a mixture of 1.5% OSO4 and 2.5%

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

Role of Flexible Bronchoscopy and Bronchoalveolar Lavage in the Diagnosis of Pediatric Acquired Immunodeficiency Syndrome-Related Pulmonary Disease

PEDIATRICS ◽  
1991 ◽  
Vol 87 (6) ◽  
pp. 897-899
Author(s):  
Jose A. Birriel ◽  
Jose A. Adams ◽  
Kunjana Mavunda ◽  
Sue Goldfinger ◽  
Donald Vernon ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Acquired Immunodeficiency Syndrome ◽  
Pneumocystis Carinii ◽  
Fiberoptic Bronchoscopy ◽  
Relative Safety ◽  
Flexible Fiberoptic Bronchoscopy ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome ◽  
Parenchymal Lung Disease

Flexible fiberoptic bronchoscopy with bronchoalveolar lavage was performed in 16 pediatric patients with the acquired immunodeficiency syndrome (AIDS) and deterioration in pulmonary function suggestive of opportunistic infection. In 62% of the patients Pneumocystis carinii was identified. Culture results showed a pure growth of Pseudomonas aeruginosa for one patient in addition to the Pneumocystis carinii. Bronchoscopy with lavage was well tolerated, with few complications even among patients with significant tachypnea and hypoxia. Because of its relative safety and effectiveness, this procedure should be considered the first invasive measurement used for evaluation of parenchymal lung disease in this population of patients.

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