Craig Hammerberg

Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.

Flow cytometric analysis of primary ex vivo keratinocyte cultures demonstrated that stem cells, (beta 1 integrin+, keratin 1/keratin 10 [K1/K10-], proliferating cell nuclear antigen [PCNA-] [Bata-Csorgo, Zs., C. Hammerberg, J. J. Voorhees, and K. D. Cooper. 1993. J. Exp. Med. 178:1271-1281]) establish such cultures. This methodology also enabled the quantitation of synchronized recruitment of these cells from G0 into G1 of the cell cycle (PCNA expression), which preceded bright beta 1 integrin expression. (beta 1 integrinbright expression has been shown to be a characteristic feature of keratinocyte stem cells in culture (Jones, P. H., and F. M. Watt. 1993. Cell. 73:713-724). Using the above assay, we determined whether lesional T lymphocytes in psoriasis could be directly responsible for the induction of the stem cell hyperproliferation that is characteristic of this disease. Indeed, CD4+ T lymphocytes, cloned from lesional psoriatic skin and stimulated by immobilized anti-CD3 plus fibronectin, promoted psoriatic uninvolved keratinocyte stem cell proliferation via soluble factors. This induction appeared to be through accelerated recruitment of stem cells from their quiescent state (G0) into cell cycle. By contrast, normal keratinocyte stem cells exhibited no such growth stimulation. Supernatants exhibiting growth induction all contained high levels of GM-CSF and gamma-IFN, low IL-3 and TNF-alpha, and variable IL-4. Only anti-gamma-IFN antibody was able to neutralize growth stimulatory activity of the supernatants on psoriatic uninvolved keratinocyte stem cells. However, because recombinant gamma-IFN alone inhibited growth in this assay, these data suggest that, in psoriasis, gamma-IFN acts cooperatively with other growth factors in the immune induction of cell cycle progression by the normally quiescent stem cell keratinocytes.

Inflammation and Extracellular Matrix Degradation Mediated by Activated Transcription Factors Nuclear Factor-κB and Activator Protein-1 in Inflammatory Acne Lesions in Vivo

Acne is the most common skin disease, causing significant psychosocial problems for those afflicted. Currently available agents for acne treatment, such as oral antibiotics and isotretinoin (Accutane), have limited use. Thus, development of novel agents to treat this disease is needed. However, the pathophysiology of acne inflammation is poorly understood. Before new therapeutic strategies can be devised, knowledge regarding molecular mechanisms of acne inflammation is required. We report here that transcription factors nuclear factor-kappaB and activator protein-1 are activated in acne lesions with consequent elevated expression of their target gene products, inflammatory cytokines and matrix-degrading metalloproteinases, respectively. These elevated gene products are molecular mediators of inflammation and collagen degradation in acne lesions in vivo. This new knowledge enables a rational strategy for development of pharmacological agents that can target the inflammation and matrix remodeling that occurs in severe acne.

Interleukin-1 receptor antagonist in normal and psoriatic epidermis.

The objective of these studies was to characterize the IL-1 inhibitory activity present in normal and psoriatic epidermis from clinically stable lesions. Fractionation of normal epidermal cytosol on a molecular sizing column failed to reveal the presence of IL-1 inhibitory bioactivity. However, specific ELISAs indicated that both the IL-1 receptor antagonist (IL-1ra) and IL-1 alpha were present in overlapping peaks. Further fractionation of the normal epidermal cytosol by anion exchange chromatography separated these two molecules, revealing the IL-1 inhibitory bioactivity of the IL-1ra molecule. Similar studies on psoriatic epidermal cytosol indicated the presence of IL-1 inhibitory bioactivity and IL-1ra protein. The IL-1 inhibitory bioactivity of both normal and psoriatic cytosol was neutralized by a mAb specific for IL-1ra. The ratio of IL-1ra to IL-1 alpha proteins was significantly increased in involved psoriatic skin compared with normal skin. By Western blot analysis this IL-1ra was approximately 20 kD, slightly larger than monocyte-derived IL-1ra and equivalent to an intracellular variant of IL-1ra expressed by keratinocytes. Polymerase chain reaction indicated the presence of mRNA for both forms of IL-1ra in normal epidermis, with both forms increased in psoriatic-involved skin. Immunofluorescence studies revealed the IL-1ra protein to be concentrated in the stratum granulosum of normal skin and in the basal-midbasal layers of psoriatic epidermis. These results suggest that the balance between intracellular IL-1ra and IL-1 alpha may be an important influence on keratinocyte growth and/or differentiation, as well as on the inflammatory potential of IL-1 in injured skin.

Immune-Mediated Subepithelial Blistering Diseases of Mucous Membranes: Pure Ocular Cicatricial Pemphigoid Is a Unique Clinical and Immunopathological Entity Distinct From Bullous Pemphigoid and Other Subsets Identified by Antigenic Specificity of Autoantibodies

BACKGROUND AND DESIGN There is much confusion in the clinical classification of immune-mediated subepithelial blistering diseases of mucous membranes. We conducted a 6-year comprehensive study to better classify this heterogeneous disease group. Indirect immunofluorescence was performed on a salt-split-skin substrate to detect circulating antibasement membrane antibodies (n = 47). Serologic reactivity against cultured keratinocyte antigens was examined by immunoblots (n = 38) and immunoprecipitation (n = 15). The results were correlated with the clinical features and direct immunofluorescence data of the entire patient group (n = 87) without preassignment of clinical diagnoses. chi 2 Statistical analyses compared these results with those of the classic bullous pemphigoid group (n = 36). RESULTS When compared with the bullous pemphigoid patients, a subset of patients with combined oral mucosal and skin lesions demonstrated marked similarity in direct and indirect immunofluorescence findings and in serologic reactivity to bullous pemphigoid antigens. By contrast, a subset of patients with only ocular lesions exhibited significantly lower in vivo deposits of IgG and C3, higher deposits of fibrin, virtual absence of circulating antibodies, and negative serologic reactivity to bullous pemphigoid antigens. CONCLUSIONS Ocular patients without skin or mouth lesions, in particular those with negative indirect immunofluorescence, should be distinctively classified as ocular cicatricial pemphigoid, a unique clinical and immunopathologic entity. Patients with mucous membrane involvement who also demonstrate skin lesions and antibodies to the root of salt-split-skin substrate should be classified as anti-BP Ag mucosal pemphigoid, even though they may exhibit severe oral and/or ocular diseases. The remaining mucous membrane patients are heterogeneous. Some can be classified on the basis of autoantibodies to other basement membrane determinants, or if serum autoantibody negative, on the basis of clinical features (ie, pure oral mucosal pemphigoid or overlapping mucosal involvement).

Linkage disequilibrium between H-2 and t complexes in chromosome 17 of the mouse.

Mice with t factors belonging to the same complementation group carry similar, if not identical, H–2 haplotypes although these factors were derived from widely separated geographical areas. This association between t and H–2 complexes suggests more than a casual relationship between the two complexes, at least at the population level.

Molecular Effects of Photodynamic Therapy for Photoaging

OBJECTIVE To quantitatively examine the epidermal and dermal cellular and molecular changes that occur after photodynamic therapy of photodamaged human skin. DESIGN Serial in vivo biochemical and immunohistochemical analyses after photodynamic therapy using topical 5-aminolevulinic acid (5-ALA) and pulsed-dye laser treatment. SETTING Academic referral center, Department of Dermatology, University of Michigan, Ann Arbor. PATIENTS A volunteer sample of 25 adults, 54 to 83 years old, with clinically apparent photodamage of the forearm skin. INTERVENTIONS Three-hour application of 5-ALA followed by pulsed-dye laser therapy using non-purpura-inducing settings to focal areas of photodamaged forearms and serial biopsy specimens taken at baseline and various times after treatment. MAIN OUTCOME MEASURES Immunohistochemical analysis was used to assess levels of markers of epidermal proliferation (Ki67), epidermal injury (cytokeratin 16), and photodamage (p53), as well as various markers of dermal collagen production (including prolyl 4-hydroxylase and heat shock protein 47, and type I procollagen). Real-time reverse transcriptase-polymerase chain reaction technology was used to quantify type I and type III collagen. Type I procollagen protein was quantified with enzyme-linked immunosorbent assay. RESULTS Epidermal proliferation was stimulated as demonstrated by increases in Ki67 (more than a 5-fold increase; P < .05) and epidermal thickness (more than a 1.4-fold increase; P < .05). Epidermal injury was produced with increased cytokeratin 16 levels demonstrated (to nearly 70-fold of baseline levels; P < .05). Upregulation of collagen production was demonstrated with increases in procollagen I messenger RNA (2.65-fold; P < .05), procollagen III messenger RNA (3.32-fold; P < .05), and procollagen I protein (2.42-fold; P < .05) levels detected. The baseline epidermal p53 level correlated with cytokeratin 16 levels at acute time points, and the latter were found to correlate with peak collagen production. CONCLUSIONS Photodynamic therapy with the specific treatment regimen employed produces statistically significant quantitative cutaneous molecular changes (eg, production of types I and III collagen) that are associated with improved appearance of the skin. Baseline epidermal p53 immunostaining levels may be predictive of dermal responses to this therapy. Comparison with historical data using pulsed-dye laser therapy alone suggests that use of the photosensitizer may enhance dermal remodeling. The quantitative in vivo molecular data presented herein are in keeping with an evolving model to potentially predict the efficacy of new techniques for the treatment of photoaging.

Fibronectin and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased fibronectin potentiation of T cell lymphokine-driven keratinocyte hyperproliferation in psoriasis.

In addition to being T lymphocyte-driven, psoriasis may be due in part to abnormal integrin expression. Normal-appearing (uninvolved) skin from psoriatic patients was examined to determine whether altered fibronectin or its receptor expression is detectable before development of psoriatic lesions. In contrast to skin from normal subjects, we detect by immunofluorescence the abnormal presence of plasma fibronectin in the basal cell layer of the epidermis of psoriatic uninvolved skin. Furthermore, increased fibronectin exposure superinduces the in vitro cell cycle induction and expansion of psoriatic nonlesional keratinocytes in response to a cocktail of T cell lymphokines. Fibronectin alone also appeared to increase cell cycle entry among uninvolved but not normal keratinocytes. Concordantly, the alpha5 integrin fibronectin receptor, but not alpha2 or alpha3, is overexpressed in the in vivo nonlesional psoriatic epidermis. The involvement of alpha5beta1 in the early outgrowth of clonogenic keratinocytes in the ex vivo culture was demonstrated by the ability of anti-alpha5 mAb to inhibit keratinocyte growth on fibronectin. Thus, the fibronectin receptor appears to be one of the components required for the development of the hyperresponsiveness of psoriatic keratinocytes to signals for proliferation provided by lymphokines produced by intralesional T lymphocytes in psoriasis.

Long-term efficacy and safety of tretinoin emollient cream 0.05% in the treatment of photodamaged facial skin : A two-year, randomized, placebo-controlled trial

AbstractBackground: Long-term (>1 year) placebo-controlled studies of tretinoin in the treatment of photodamaged skin have not been conducted. Recently, we conducted a 2-year placebo-controlled study of tretinoin emollient cream 0.05%, including histopathologic assessment of safety and analysis of markers of collagen deposition. Objective: The objective of the study was to determine the long-term safety and efficacy of tretinoin emollient cream 0.05% in the treatment of moderate to severe facial photodamage. Methods: A total of 204 subjects were treated with tretinoin or placebo (vehicle emollient cream) applied to the entire face once a day for up to 2 years. Clinical and histologic effects were assessed at regularly scheduled clinic visits. Results: Treatment with tretinoin resulted in significantly greater improvement relative to placebo in clinical signs of photodamage (fine and coarse wrinkling, mottled hyperpigmentation, lentigines, and sallowness), overall photodamage severity, and investigator’s global assessment of clinical response (p < 0.05). Histologic evaluation showed no increase in keratinocytic or melanocytic atypia, dermal elastosis, or untoward effects on stratum corneum following treatment with tretinoin compared with placebo. Immunohistochemistry studies, conducted at three study centers, showed a significant increase relative to placebo in facial procollagen 1C terminal, a marker for procollagen synthesis, at month 12 (p = 0.0074). Conclusion: Long-term treatment with tretinoin emollient cream 0.05% is safe and effective in subjects with moderate to severe facial photodamage.

Etanercept suppresses regenerative hyperplasia in psoriasis by acutely downregulating epidermal expression of interleukin (IL)-19, IL-20 and IL-24

Background Psoriasis is a Th17/Th1‐mediated skin disease that often responds to antitumour necrosis factor (TNF)‐α therapies, such as etanercept.

Effect of Increased Pigmentation on the Antifibrotic Response of Human Skin to UV-A1 Phototherapy

OBJECTIVE To investigate the efficacy, potential limitations, and biological mechanisms of UV-A1 phototherapy for skin sclerosis due to collagen deposition disorders. DESIGN Before-and-after trial of UV-A1 irradiation of sclerotic skin; in vivo biochemical analyses after UV-A1 irradiation of normal skin. SETTING Academic referral center. PARTICIPANTS Patients with morphea/scleroderma or sclerodermoid graft-vs-host disease and volunteers without skin disease. Intervention Sclerotic skin was treated with high-dose (130 J/cm(2); n = 12) or medium-dose (65 J/cm(2); n = 6) UV-A1 phototherapy 3 times per week for 14 weeks; normal skin was treated with UV-A1 irradiation at various doses and frequencies, with biopsies performed afterwards. MAIN OUTCOME MEASURES In sclerotic skin, induration was clinically assessed using a scoring scale. In normal skin, quantitative polymerase chain reaction was used to assess antifibrotic responses, defined as decreased type I and type III procollagen and increased matrix metalloproteinase levels. RESULTS In patients with sclerotic skin treated with high-dose UV-A1 irradiation, clinical scores for induration modestly decreased. To investigate what factors prevented further improvement (ie, complete clearance), normal skin with light pigmentation was exposed to UV-A1 irradiation (70-150 J/cm(2)) and was assessed for antifibrotic responses. A single high-dose exposure (110-150 J/cm(2)) elicited substantial antifibrotic responses and induced skin darkening. This skin darkening attenuated responses to subsequent UV-A1 exposures and was dose dependent. Thus, to minimize skin darkening, additional patients with sclerotic skin were treated with medium-dose UV-A1 phototherapy, which was no less effective than high-dose therapy. CONCLUSION Clinical responses of sclerotic skin to UV-A1 phototherapy were modest because of UV-A1-induced skin darkening, which is photoprotective and attenuates antifibrotic responses. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00129415.