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A technique for quantitating bacteria in isolated pilosebaceous follicles is described. This involves microdissection of the follicles from biopsies of skin, using the method of chemical pretreatment of skin to facilitate the separation of the epidermis and epidermal appendages from the dermis. The aerobic cocci and anaerobic diphtheroids in pilosebaceous follicles in 66 biopsies of scalp and 48 biopsies of skin of the upper back were quantitated using this technique. On the back, aerobic staphylococci were very sparse in normal follicles, indicating that their primary habitat on the skin must be on the skin surface rather than within follicles. Of 138 isolated follicles from skin of the upper back, 94 contained no aerobic cocci. Anaerobic organisms were present in high numbers within normal follicles. The geometric mean density of anaerobes in 138 isolated follicles from skin of the upper back was 3.8 X 10(4) diphtheroids per follicle. Eighty-eight follicles contained more than 10(4) anaerobic diphtheroids. Using data from scalp biopsies we found that there was a correlation between the weight of sebaceous glands and the density of anaerobes within the follicles attached to these glands (coefficient of correlation = 0.6).

The effects of specific species of skin bacteria on human sebaceous gland lipids in vitro were analyzed. Isolated dissected sebaceous glands were pooled, homogenized, and sterilized, then incorporated into peptone-yeast extract medium and used as substrate for growth of Propionibacterium acnes, P. granulosum, and Staphylococcus epidermidis subgroup II. The sebaceous lipids were analyzed by thin-layer chromatography before and after bacterial growth. The most striking effect of bacteria on sebaceous gland lipid composition was the hydrolysis of sebaceous triglycerides. The degree of hydrolysis varied with bacterial strain but was most complete with P. acnes and P. granulosum. Staphylococci were not effective in hydrolyzing sebaceous triglycerides at pH 4.5 although, when the pH of the medium was raised to pH 6.4, some strains of staphylococci were as effective as the propionibacteria in hydrolyzing sebaceous triglycerides to free fatty acids. Thus minor changes in acidity may play asignificant role in controlling the lipolytic activity of staphylococci on skin. Another effect of bacterial action on sebaceous gland lipids was the esterification of sebaceous cholesterol to cholesteryl esters. Thus, bacterial action must be taken into account in evaluating studies of alterations in cutaneous cholesterol and cholesteryl esters in skin surface lipids in normal and disease states.

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Patients with severe nodulo-cystic acne are known to have elevated serum antibody levels and increased immediate hypersensitivity reactions to Propionibacterium acnes. This organism is the predominant bacterium in normal pilosebaceous follicles of human skin, and can be consistently isolated from pustular lesions in acne. Previously it had been observed that delayed cutaneous hypersensitivity reactions to P. acnes were negative in patients with acne. The present study investigated the proliferative response of lymphocytes from patients with nodulo-cystic acne to phytohaemagglutinin (PHA) and P. acnes antigen stimulation. The response to PHA stimulation was within normal limits. The response to P. acnes antigen showed a significant increase over control values obtained by testing lymphocytes from acne-free subjects. Thus cell mediated immunity to P. acnes may be present in subjects with severe inflammatory acne. These findings raise the possibility that reactions to P. acnes may contribute to intensifying the inflammatory response in acne lesions.

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