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Summary Background Discoid lupus erythematosus (DLE) is a scarring disease. Although the scarring and deformity may affect any part of the body, such changes have been reported to be most obvious on the face and scalp. The pathogenesis behind this scarring process is not well understood. Once lesions have scarred, recurrent disease tends to occur at the edge of the scarred lesions but not within them. Objectives The fact that inflammation in DLE generally involves the bulge area of the follicles raises the possibility that damage to the stem cells of the bulge region may be one process leading to the permanent loss of follicles. The aim of this study was to investigate the role of the hair follicle stem cells which reside in the bulge region in the scarring process in cutaneous lupus erythematosus (CLE). Methods We studied the reactivity of an antibody to the CD8 antigen (C8/144B), which recognizes cytokeratin (CK) 15 and preferentially immunostains hair follicle stem cells without staining the remaining hair follicle, on skin biopsies (scalp and body lesions) from patients with CLE (36 with discoid lesions and 10 with subacute lesions). Normal scalp and body biopsy specimens served as controls. The correlation between the extent of the cytotoxic inflammatory cell infiltrate (CD8+) and the presence of stem cells was investigated. Results were analysed semiquantitatively. Results The expression of CK15 in hair follicle stem cells was variable in the DLE lesions; there was normal to moderate CK15 expression at the bulge region of hair follicles when surrounded by mild or moderate inflammatory infiltrate (CD8+), but in cases of severe inflammation, CK15 expression was weak or absent. Conclusions The bulge region appears to be involved in this disease as part of a broader involvement of the hair follicles; it is secondarily affected by the surrounding inflammatory cell infiltrate. Expression of C8/144B diminished and was then absent, indicating either damage to stem cells or differentiation to help in the repair process. Damage to follicular stem cells may help to explain the irreversible alopecia and the scarring process which characterize this disease.

This was a preliminary investigation to define the conditions of colonization of a human skin equivalent (SE) model with cutaneous microorganisms. SEs of 24 mm diameter were constructed with a dermal matrix of fibrin containing fibroblasts and a stratified epidermis. Microbial colonization of the SEs was carried out in a dry environment, comparable to 'in vivo' skin, using a blotting technique to remove inoculation fluid. The microbial communities were sampled by scrub washing and viable cells enumerated on selective growth medium. Staphylococcus epidermidis, Propionibacterium acnes and Malassezia furfur (human skin commensals) and Staphylococcus aureus (transient pathogen) were colonized at inoculum densities of 10(2)-10(6) CFU SE(-1) on the surface of replicate SEs. Growth of all species was supported for upto 72-120 h, with recovery densities of between 10(4)-10(9) CFU SE(-1). A novel, real-time growth monitoring method was also developed, using S. aureus containing a lux cassette. Light output increased from 20 to 95 h, and colonization increased from 10(2) to 10(8) CFU SE(-1), as confirmed by conventional recovery. Thus, the SE model has potential to investigate interactions between resident and transient microbial communities with themselves and their habitat, and for testing treatments to control pathogen colonization of human skin.

Summary Background Propionibacterium acnes has been strongly implicated in inflammatory acne. However, its role in the disease is unclear. It has been hypothesized that an immune response to P. acnes and/or P. acnes heat shock proteins (HSPs) may play a role in the pathogenesis of inflammatory acne. Objectives To compare the cell-mediated immune response to P. acnes and HSPs in acne patients, nonacne controls and individuals with resolved acne. Methods The proliferative response of peripheral blood mononuclear cells (PBMC) from acne patients, resolved acne donors and healthy controls to P. acnes, P. acnes HSP60 and HSP70, and mycobacterial HSPs was assessed by lymphocyte transformation assay (LTA). The proliferative response of purified CD4+ T cells was further analysed by limiting dilution analysis (LDA). Contingency tables (G-test) were used to analyse the proportion of individuals in each group showing a positive proliferative response for LTA or data fitting single-hit kinetics for LDA. Results Analysis of stimulation of PBMC with P. acnes, P. acnes HSP60 and HSP70 in the LTA showed the proportion of positive responders to be independent of subject group. However, the proportion of acne patients with a positive response to mycobacterial HSPs was significantly higher than those for the other subject groups. Analysis of LDA data showed the proportion of resolved donors with responses to P. acnes fitting the single-hit kinetics model to be significantly lower than those of the other groups. There were no significant differences in responses to other antigens. Conclusions The significantly lower proportion of resolved donors demonstrating a single-hit kinetics response to P. acnes by LDA may represent negative regulation of the CD4+ T-cell response to P. acnes in these subjects.

SUMMARY Correlation matrices were constructed for various human skin variables (propionibacteria, micrococcaceae, sebum excretion rate, percentage of free fatty acids and free fatty acid production rate). Two independent populations were studied (n= 87 and n= 103), and a subpopulation (n= 30) was investigated on three occasions at intervals of 1 month. In this way consistently high correlations could be differentiated from ‘fortuitous’ correlations. Propionibacteria versus micrococcaceae showed the highest correlation coefficient and propionibacteria versus percentage free fatty acids was also highly correlated. A scatter diagram indicated a relationship between the lower threshold of microbial density and sebum excretion rate. An explanation based on relative duct and gland size is presented.

Objectives The mechanisms by which antibiotics induce a post-antibiotic effect in susceptible bacteria are poorly understood. To explore the mechanisms more fully we examined the recovery of macromolecular synthesis in Escherichia coli during gentamicin- and rifampicin-induced post-antibiotic effects. Methods E. coli ATCC 25922 was exposed to rifampicin and to gentamicin at 5x MIC for 60 min to induce post-antibiotic effects. The antibiotics were then removed from the culture medium by washing the cells. The rates of DNA, RNA and protein synthesis during the post-antibiotic effect and recovery periods were subsequently determined by measuring the incorporation of radiolabelled uridine, thymidine and leucine into trichloroacetic acid precipitable material. Results Recovery of E. coli ATCC 25922 from the rifampicin-induced post-antibiotic effect coincided with the recovery of RNA and protein synthesis. Recovery from the gentamicin-induced post-antibiotic effect coincided with the recovery of protein synthesis. Conclusions These data support the hypothesis that antibiotic molecules retained in the cell mediate the post-antibiotic effect by suppressing the biochemical activity of their molecular targets.

BACKGROUND: Keratinocytes form the first line of defence in the skin and alert the host to danger by the production of a number of cytokines and chemokines. However, the interaction of commensal microorganisms with keratinocytes has not been well studied. OBJECTIVES: To investigate the effect of viable and nonviable cells of Propionibacterium acnes in both exponential and stationary growth phases, and of P. acnes GroEL on cytokine production by human primary keratinocytes. METHODS: Actively proliferating or contact-inhibited keratinocytes were cocultured with viable or formaldehyde-killed P. acnes cells in either the exponential or stationary phase of growth. Culture supernatants were assayed by enzyme-linked immunosorbent assay for the cytokines interleukin (IL)-1alpha, tumour necrosis factor (TNF)-alpha and granulocyte/macrophage colony-stimulating factor (GM-CSF). Keratinocytes were also stimulated with different concentrations of P. acnes GroEL and supernatants assayed for cytokines. RESULTS: Viable P. acnes in the stationary phase of growth stimulated keratinocyte monolayers to produce significantly higher amounts of IL-1alpha, TNF-alpha and GM-CSF than unstimulated keratinocytes. Viable exponential-phase bacteria stimulated production of significantly higher amounts of TNF-alpha and GM-CSF but these levels were significantly lower than those for stimulation with stationary-phase bacteria. Nonviable P. acnes from either growth phase was not able to stimulate cytokine production. P. acnes GroEL at concentrations in the range 0.05-1.0 micro g mL(-1) was able to induce increased production of cytokines by keratinocytes in a dose-dependent manner. This was analogous to stimulation with Escherichia coli GroEL. CONCLUSIONS: Stimulation of cytokine production by P. acnes and P. acnes GroEL may be important in the pathogenesis of inflammatory acne vulgaris and may have wider implications for the immunomodulation of the human immune system by commensal skin microorganisms.

To study the pathogenicity of Malassezia, the agent of pityriasis versicolor, it is necessary to obtain the mycelial form in vitro. A range of different components and conditions were tested to induce yeast cells of the organism to produce mycelia in vitro using different culture media. A mycelial culture medium was developed that consisted of bacteriological peptone, glucose, yeast extract, ox bile, glycerol, glycerol monostearate, Tween 80, squalene, glycine, potassium nitrate, sodium chloride, ferrous sulphate and magnesium sulphate with or without agar. The liquid and solid medium had a pH of 5.6 and the temperature of incubation was 30 degrees C. Cultures were incubated in air. This medium was able to induce some strains of Malassezia to produce up to 40% mycelium in vitro. In total, 33 different strains of Malassezia obtained from the skin of the healthy individuals and patients with pityriasis versicolor were tested for mycelium production. The strains of Malassezia capable of producing mycelium in vitro all possessed the serovar A antigen.

Malassezia spp., the causative agents of pityriasis versicolor, are members of the normal human cutaneous microflora. Utilizing a combination of both enzyme-linked immunosorbent assay (ELISA) and bioassay, we have investigated the ability of both formalin-preserved and viable Malassezia (serovars A, B and C) to modulate pro-inflammatory cytokine (IL-6, IL-1beta and TNF-alpha) release by human peripheral blood mononuclear cells (PBMNC) in vitro, over a 48-h co-incubation period. The results demonstrated that formalin-preserved Malassezia (serovars A, B and C) at mid-exponential phase were generally able to induce a pro-inflammatory cytokine response at a yeast cell to PBMNC ratio of 1:1. In addition, the results consistently demonstrated that at a yeast cell to PBMNC ratio of 20:1, formalin-preserved Malassezia, irrespective of serovar, growth phase or PBMNC donor, were capable of significantly (P

IgG subclasses specific to Staphylococcus epidermidis and Propionibacterium acnes were determined in sera from patients with mild, moderate or severe acne and from a control group. Titres specific to S. epidermidis were all within the same range and did not differ between groups. The titres of IgG subclasses to specific to P. acnes did vary between groups. IgG1 and IgG3 were significantly higher in severe acne patients compared with moderate acne patients, while IgG2 was significantly higher in moderate and severe patients compared with controls. Titres of IgG4 did not differ between groups. The pattern of titres observed suggests that, while the antibody response to S. epidermidis is relatively harmless, antibodies to P. acnes may be involved in the pathogenesis of acne vulgaris.

A transferable solid-phase (TSP) ELISA was developed for the determination of antibody titres specific to Malassezia furfur serovars A, B and C in human sera. A survey of levels of class-specific antibodies (IgM, IgG and IgA) to M. furfur serovars A, B and C in relation to age (2-64 years; 60 individuals) demonstrated that individuals had immunity to M. furfur by the age of 2-3 years. There was no difference in either IgM or IgG levels into adulthood. The only age-related differences were lower IgM titres to the three serovars in the 60-64 year age-group compared with younger individuals. There was, however, a difference between titres of antibody specific to the three serovars. The mean reciprocal log2 IgM titre to serovar A (6.9) was significantly higher (P < 0.05) than that to serovar B (mean reciprocal log2 titre of 5.8), but not to serovar C (6.1). In contrast, the mean reciprocal log2 IgG titre to serovar A (6.5) was significantly lower (P < 0.05) than those to serovars B and C (mean reciprocal log2 titre of 8.9 in both cases).

The effects of minocycline and tetracycline on the mitotic response of human peripheral blood lymphocytes was investigated in vitro. The effects of the antibiotics on the mitotic response of purified lymphocytes stimulated with Interleukin-1 beta varied according to the individual from whom the lymphocytes were obtained. At concentrations above those reported to be present in serum during conventional therapy (2-8 mg/l), there was a tendency for both minocycline and tetracycline to suppress the mitotic response. Minocycline was superior to tetracycline in this respect. However, at physiological concentrations the antibiotics either had no significant effect, suppressed the mitotic response (minocycline at 2 mg/l with one of six donors), or enhanced the mitotic response (tetracycline at 2 and 8 mg/l with four of six donors). The stimulatory effect of tetracycline was not demonstrated when lymphocytes were cultured in whole blood for up to seven days with the antibiotic alone. Similar effects of the antibiotics were observed when mononuclear cell fractions isolated from six donors were stimulated with an optimal concentration of phytohaemagglutinin (PHA). Stimulation of lymphocytes in whole blood cultures with PHA in the presence of minocycline and tetracycline revealed that, under these culture conditions, the antibiotics could suppress the mitotic response of lymphocytes at physiological doses with cells from a majority of donors.

Attempts were made to produce antisera to purified preparations of Propionibacterium acnes lipase, hyaluronate lyase and acid phosphatase in rabbits. Antiserum to lipase (neutralizing titre I:32) was produced using conventional methods. Lipase (30 micrograms) in Freund's complete adjuvant (FCA) was injected into multiple sites thrice at weekly intervals. Antibody levels were boosted by i.v. injections of 30 micrograms in saline at 2-weekly intervals for 2 months. Such regimes failed to raise antibodies to hyaluronate lyase and acid phosphatase. In order to produce antiserum to hyaluronate lyase, rabbit mononuclear cells were incubated with 45 micrograms of hyaluronate lyase in vitro (I h at 37 degrees C) prior to injection subcutaneously above the rabbit ankle joint. Simultaneous injections of hyaluronate lyase in FCA (45 micrograms) were administered into the footpad and thigh. Three treatments at weekly intervals produced an antiserum with a neutralizing titre of I:256. A similar regime failed to raise antibodies to acid phosphatase. Antibodies to P. acnes lipase and hyaluronate lyase also neutralize the enzymes produced by P. granulosum. The neutralization curves suggest that the enzymes produced by both species are related antigenically but not identical.

Total serum IgM and IgG agglutinins to P. acnes and neutralizing antibodies to P. acnes lipase, hyaluronate lyase and acid phosphatase were measured in normal individuals of different age groups. Agglutinins to P. acnes were detected in infants at 4 months of age and were present at a high level throughout life. A switch from predominantly IgM agglutinins in children, to IgG agglutinins in adults, occurred during adolescence. Anti-P. acnes lipase antibodies were present in 20% of teenagers and 17-42% of adults. Anti-P. acnes hyaluronate lyase antibodies were found in adults only (4-17%). Antibodies to acid phosphatase were not detected. Agglutinins to P. acnes were measured in individuals with mild, moderate and severe acne, and in normal controls. Only patients with severe acne had significantly higher titres than the controls. IgM and IgG agglutinins were determined in 13-14-year-olds with mild, moderate and severe acne, and in normal controls. Thirty-three per cent, 60% and 100% of the acne patients, respectively, but none of the normal controls, had predominantly IgG agglutinins. No difference in the prevalence or titre of antibodies to P. acnes exocellular enzymes was observed when patients with severe acne were compared with normal controls. There was no evidence to suggest a role for antibodies to P. acnes exocellular enzymes in the initiation of inflammatory acne.