The precise pathological processes of comedo formation are not well understood. In patients with acne, there are changes in the process of cornification within the sebaceous follicle. It is thought that an increase in proliferating keratinocytes [1, 2] and their subsequent retention are factors which contribute to this process. In order to investigate the early events involved in the initiation of comedogenesis, normal follicles from acne patients have been labelled using immunohistochemistry with antibodies to Ki-67 and keratin 16 (K16). Ki-67 is a non-histone protein which labels all cycling cells except those in G₀, whereas K16 is a protein which is up-regulated in all abnormally differentiating and hyperproliferating suprabasal keratinocytes.4-mm punch biopsies were taken from the backs of acne patients using a local anaesthetic. Approval was obtained from the Leeds General Infirmary Ethical Committee in addition to the informed consent from the patient. Patients were between the ages of 17 and 25 years and most were not on treatment. Patients on treatment had shown no response to mega dose antibiotic and non-retinoid treatment and were thus prescribed oral isotretinoin. However, no patient of this study had received oral isotretinoin 12 months prior to the start of the project. Biopsies were serially sectioned to a thickness of 6 µm using an Anglia scientific cryostat. Immunohistochemistry was then performed by incubating the sections with antibodies to K16 and Ki-67 for 1 h at room temperature. Localization of antigen was achieved using the standard avidin-biotin-immunoperoxidase method with a diaminobenzidine chromagen. Sections were then counterstained with methyl green. Negative controls were set up by omitting the primary antibody.Ki-67 labelled the nuclei of basal cells, so results were quantified by counting positive Ki-67 nuclei and expressing these as a fraction of the total number of basal cells in each area. Several microscopic anatomical sites were counted: three areas from the interfollicular epidermis, three areas from the perifollicular epidermis and three areas from the follicular duct. Growth fractions obtained from each area were then added to give an overall Ki-67 value per follicle. K16 labelled the cytoplasm of cells and therefore labelling was graded according to the number of anatomical areas in which the antibody was located (fig. 1).It was observed that many of the follicles biopsied demonstrated microcomedonal characteristics which were not evident clinically. Some follicles demonstrated high numbers of Ki-67-positive cells whereas others did not. In the same way some follicles demonstrated high amounts of K16 whereas others did not. In total, 24 clinically normal follicles from acne patients were investigated (table 1). All of these demonstrated a wide variety of Ki-67 positivity, the lowest Ki-67 value obtained being 0.07 and the highest being 0.92. The majority of follicles investigated demonstrated a minimal amount of K16 labelling (stage 1 type classification); however, 3 follicles showed an increased amount of K16 labelling. Histological examination of these follicles confirmed microcomedonal features even though clinically they were classified as normal follicles. It was established that follicles could fall into one of the four following groups: group 1 = low Ki-67 expression, low K16 expression; group 2 = high Ki-67 expression, low K16 expression; group 3 = high Ki-67 expression, high K16 expression; group 4 = low Ki-67 expression, high K16 expression.This study is based on the assessment of two features of keratinocyte proliferation. Ki-67 shows the number of cells in the growth fraction and K16 is a measure of both epidermal hyperproliferation and abnormal differentiation. There are previous studies demonstrating results using either Ki-67 or K16, but this is the first time that comparisons with both markers have been made on the same sample. In conclusion we found that some clinically normal follicles demonstrated definite microcomedonal features when examined histologically. In addition there were differences in the type and amount of Ki-67 and K16 labelling patterns within these normal follicles. Thus, we propose that the four different groups of follicles correspond to four different stages of development of the normal pilosebaceous follicle. Further work is required in order to increase our understanding of proliferation and differentiation of ductal keratinocytes in health and disease.
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Aldana et al. (1998) studied this question.