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Title: VARIATION OF NEUTROPHIL DIAPEDESIS IN PRIMARY CELL CULTURE SYSTEMS

Author
item SMITS, ELKE
item BURVENICH, CHRISTIAN
item THAS, OLIVER
item OTTOY, JEAN-PIERRE
item Guidry, Albert

Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/12/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: Mastitis cost US dairymen $2 billion and beef cattlemen $400 million annually. Although accurate estimates cannot be made, significant losses also occur in goats, sheep and pigs. Bacteria are by far the most common cause of mastitis and these infections cause the greatest economic losses. White blood cells, namely, neutrophils are the animal's primary defense against bacteria invading the mammary gland. However, their effectiveness is decreased due to their low number in normal milk. Also, neutrphils are inhibited by the loss of energy during their migration from blood to the lumen of the gland and by the ingestion of fat and casein upon entering the gland. Antibiotics are of limited value due to the development of resistant strains and contamination of the milk supply. To circumvent use of antibiotics, researchers have attempted to increase the effectiveness of the neutrophils by enhancing their movement into the gland at the least provocation from invading bacteria. However, studying the mechanisms governing the movement of neutrophils from blood to milk has been hampered by the inability to directly observe the mechanisms involved. A three dimensional cell culture system was developed whereby these mechanisms can be observed at the cellular level. This paper describes in detail merits and limitations to this system. It is currently being used by researchers to better understand the movement of neutrophils from blood to milk and to test means of enhancing that movement to aid the cow's defense against invading bacteria. This should lead to methods that will increase the cow's defense against bacterial infection of the mammary gland, thus saving millions of dollars and a decrease in the use of antibiotics.

Technical Abstract: Polymorphonuclear leukocyte (PMN) migration from blood to the lumen of the mammary gland is a major defense against bacterial infection of the mammary gland. An in vitro model, composed of a monolayer of bovine mammary epithelial cells cultured on a porous membrane, was used to simulate diapedesis of PMN across mammary epithelium to the lumen of the mammary gland. Sources of variation due to the in vitro nature of the model and individual cow PMN were evaluated. Diapedesis of PMN from 5 cows in early lactation and 5 cows in midlactation was measured twice weekly for four weeks. Variation due to fixed factors included stage of lactation, electrical resistance of the monolayer, and concentration of PMN added to the cell culture model. The nested effect of day within cow and variation due to cell culture system were treated as random factors. Only a small portion of variation was due to the non-homogeneous population nof cows and to different concentrations of PMN added to the model. An exponential relationship was found between electrical resistance across the monolayer and amount of PMN undergoing diapedesis. Variation in electrical resistance accounted for 4.9% of variation. Variation due to the inherent biological variability of primary cell culture systems accounted for 4.2% of variance. Most of the variation (70%) was accounted for by the nested effect of day within cow. Residual variance was 20.3%. These data indicate that this model can be used to compare ability of bovine PMN from different cows, taken at various physiological states, to diapedese across an epithelial cell monolayer. It showed that using monolayers with electrical resistances greater than 1000 ohms significantly reduced variation in PMN diapedesis.