Programmed cell death of keratinocytes culminates in apoptotic secretion of a humectant upon secretagogue action of acetylcholine.

The programmed cell death of the stratified squamous epithelial cells comprising human epidermis culminates in abrupt transition of viable granular keratinocytes (KC) into dead corneocytes sloughed by the skin. The granular cell-corneocyte transition is associated with a loss in volume and dry cell weight but the mechanism for and biological significance of this form of keratinocyte apoptosis remain obscure. We show that terminally differentiated KC extrude into the intercellular spaces of living epidermis the cytoplasmic buds containing randomly congregated components of the cytosol as well as filaggrin, a precursor of the natural moisturizing factor. The discharge of secretory product is reminiscent of holocrine secretion, suggesting the term 'apoptotic secretion' for this novel, essential step in the process of cornification. The secretory product may become a part of the glycocalyx (a.k.a. 'intercellular cement substance' of epidermis) and serve as a humectant that counterbalances the osmotic pressure imposed by the natural moisturizing factor located in the stratum corneum comprised by corneocytes. The apoptotic secretion commences upon secretagouge action of acetylcholine which is synthesized and released by KC. A combination of a cholinergic nicotinic agonist and a muscarinic antagonist which increases intracellular calcium levels is required to trigger the apoptotic secretion. Analysis of the relative amounts of cholinergic enzymes and receptors expressed by KC capable of secretion and the pharmacological profiles of secretion regulation revealed an upward concentration gradient of free acetylcholine in epidermis which may provide for its unopposed secretagogue action via the m1 muscarinic and the alpha7, and alpha9 nicotinic receptor types expressed by KC at the latest stage of their development in the epidermis.

Laser resurfacing for dermal photoaging.

The skin is a highly organized system composed of resident cells, extracellular matrix, blood vessels, and circulating cells that all work together to maintain cutaneous integrity. Environmental insults, particularly sunlight, act to alter the skin permanently, producing visibly undesirable effects. By wounding the skin or inducing a healing response with minimal wounding, the repair process can be activated to return the skin to a more normal condition. Owing to the complexity of the healing response, even the most well-studied and precise laser system can result in unpredicted results when used to treat photo-damaged skin. Through continued research into the normal functioning of skin, the alterations brought about by chronic photodamage, and the repair process, an integrated approach to treatment of photoaging will evolve. Agents such as alpha-hydroxy acids, retinoids, and growth factors that impact the healing response can be combined with various lasers to optimize improvement of photo-damaged skin, while minimizing the adverse consequences of treatment.

Management of cutaneous verruciform xanthoma.

Verruciform xanthoma is an uncommon mucocutaneous condition of uncertain cause that only occasionally affects the skin. The histopathology is distinctive for the presence of foamy histiocytes present within elongated dermal papillae. Although simple excision of intraoral lesions is reportedly curative, treatment of cutaneous lesions has not been previously reported. We describe a 62-year-old man with a large lesion of verruciform xanthoma affecting both inguinal folds. Immunohistochemical staining, reverse transcriptase polymerase chain reaction for human papilloma virus, and ultrastructural analysis were performed to investigate the pathogenesis of this lesion. The results of these studies support the theory that the source of lipid in dermal histiocytes is degenerating keratinocytes. Initial treatment with wire loop electrosection, pulsed dye (585 nm) laser, and x-ray therapy of this patient proved unsuccessful. Preliminary success has been achieved using wide surgical excision with primary closure.

Pulsed dye laser therapy for sun damaged skin.

OBJECTIVE: The objective of this study was to evaluate the effectiveness of the pulsed dye laser (585 nm, 450 ms) in the treatment of sun induced wrinkles. DESIGN: Patients had one pulsed dye laser (585 nm) treatment. The treated areas were assessed by the following methods: grading of skin wrinkles at 6 weeks, 12 weeks, and 6-14 months after treatment by blinded observers and by light and electron microscopy. SETTING: An ambulatory care center at Abbott Northwestern Hospital (ANH) and the Laser & Skin Surgery Center of Northern California (LSSCNC). PATIENTS: Twenty patients were treated, half with mild to moderate and half with moderate to severe sun induced skin wrinkles. RESULTS: At last follow up 90% (9/10) of the mild to moderate wrinkles and 40% (4/10) of the treated patients with moderate to severe wrinkles had clinically observable improvement in their sun induced skin wrinkles. Histologic examinations of the treated areas showed a superficial dermal band of well organized elastin and collagen fibers replacing pre-treatment elastic tissue. Increased cellularity and mucin deposition was consistent with dermal collagen remodeling.

Ultrastructure of collagen thermally denatured by microsecond domain pulsed carbon dioxide laser.

BACKGROUND: Clinical improvement in photodamaged skin after carbon dioxide (CO2) laser resurfacing is thought to result in part from thermal collagen shrinkage. The presence of such collagen has not been unequivocally demonstrated. To identify and characterize the morphological features of collagen after CO2 laser exposure, we irradiated ex vivo human facial skin and bovine calcaneus tendon with microsecond domain pulsed CO2 laser energy and examined specimens for histopathological and ultrastructural changes in collagen. OBSERVATIONS: In dermis and tendon, 3 zones of collagen structure were apparent on electron microscopy. The first, most superficial zone demonstrated loss of collagen structure. The second zone consisted of admixed normal collagen fibers and thickened collagen fibers. Zone 3 consisted of normal-appearing collagen fibers. CONCLUSIONS: Ultrastructural examination of irradiated collagen revealed distinct morphological zones of denatured collagen fibers. Partially denatured fibers had an increased diameter consistent with lineal shrinkage. Zonal distinction was undetectable by light microscopy. Ultrastructurally, the zones of denatured collagen located above the normal fibers correlated with the zone of altered material seen on light microscopy. These findings suggest that collagen fiber shrinkage does occur after pulsed CO2 laser irradiation and that this phenomenon contributed, at least in part, to the immediate tissue contraction observed clinically.

The highly effective use of topical zinc pyrithione in the treatment of psoriasis: a case report.

Psoriasis is a common condition which, at times, can be difficult to treat. Patients with psoriasis face many social challenges and can suffer a great deal with their disease. As a result, any new topical agent is a welcomed addition to our therapeutic armamentarium. We present here a report of a case using a novel topical preparation of zinc pyrithione for the treatment of psoriasis. Topical zinc pyrithione appears to be a safe and effective treatment for psoriasis.

Teledermatology in the nursing home.

OBJECTIVE: To examine a still-image store-and-forward teledermatology system for use in the care of nursing home residents. DESIGN: Diagnosis and treatment plans made from a teledermatology system were compared with those made from an on-site dermatology consultation. SETTING: This study involved the dermatologic care of nursing home residents. PATIENTS: Dermatologic consultations sent to the senior author's office from the participating nursing home were eligible for the study. In a consecutive manner, 29 residents with a total of 30 skin conditions were enrolled. INTERVENTION: A nurse collected and sent the histories and images using the teledermatology system. A diagnosis and treatment plan was determined by examining a transmitted still image and patient history alone and in combination by 2 to 3 dermatologists independently. An independent dermatologist made an on-site dermatologic consultation within 2 days after the images had been collected. MAIN OUTCOME MEASUREMENT: The diagnosis and treatment plans made from the teledermatology system were compared with those made by the on-site dermatologist. RESULTS: Twenty-nine patients with 30 skin conditions were enrolled in the study. Correct diagnoses were made for 60 (67%) of 90, 51 (85%) of 60, and 53 (88%) of 60 patients given the history alone, image alone, and both, respectively. The correct treatment plan was seen in 63 (70%) of 90, 52 (87%) of 60, and 54 (90%) of 60 patients given the history alone, image alone, and both, respectively. No incorrect diagnoses or treatment plans would have given rise to substantial morbidity. The dermatologists felt comfortable in making a diagnosis and treatment plan in all cases in which they had access to both the image and patient history. CONCLUSION: This study provides evidence that nursing home teledermatology consults may replace some on-site consultations by offering quality care in a cost-effective manner.

Nodular basal cell carcinoma in vivo vs in vitro. Establishment of pure cell cultures, cytomorphologic characteristics, ultrastructure, immunophenotype, biosynthetic activities, and generation of antisera.

BACKGROUND AND DESIGN: In this study we developed an in vitro model of nodular basal cell carcinoma (BCC). We obtained pure cultures of BCC cells and compared the morphologic characteristics, ultrastructure, immunophenotype, and behavior of cultured tumor cells with those of their in vivo counterparts. Tumors were excised from patients undergoing Mohs micrographic surgery. We established 69 primary cell cultures from 32 patients with nodular BCC. RESULTS: Three cell types grew in primary cultures: fibroblasts, normal-appearing keratinocytes, and cells with dual (spindle and epithelioid) morphologic characteristics. Contaminating fibroblasts were removed using 0.125% trypsin-0.02% edetic acid, and normal-appearing keratinocytes were cornified and eliminated by temporarily increasing the concentration of calcium in the growth medium. The cells with dual morphologic characteristics remained intact and exhibited relentless growth in pure cultures. That these seemingly immortal cell strains represent true nodular BCC was demonstrated by (1) their biphasic morphologic characteristics and very slow cell growth rate, (2) their capability for anchorage-independent growth in soft agar, (3) their ultrastructural similarities to freshly excised nodular BCC, (4) their ability to generate antibodies selectively labeling nodular BCC tumor nests in vivo, and (5) their immunophenotypic similarities to BCC in vivo on more than 20 different cell markers. CONCLUSIONS: This study provides a simple technique for establishing pure cell cultures of nodular BCC and describes extensively the in vitro parameters of tumor cell growth. The striking differences in behavior of cultured tumor cells in the presence or absence of normal-appearing keratinocytes suggest that normal human epidermal keratinocytes can suppress the growth of BCC cells.

Clinical and histologic evaluation of psoriatic plaques treated with a flashlamp pulsed dye laser.

BACKGROUND: Psoriatic plaques can be cleared by destruction of the dermal papillae. Dilated vessels, the major component of psoriatic dermal papillae, can be selectively destroyed with yellow light lasers. Previous investigators have demonstrated partial clearing of psoriatic plaques after treatment with a pulsed dye laser (PDL) (585 nm). OBJECTIVE: This study was designed to examine the clinical and histologic events of psoriasis treated with the PDL. METHODS: Psoriatic plaques were treated with a short (450 microseconds) and long (1500 microseconds) pulse-width PDL. Photographs of the plaques were used for clinical assessment. Biopsy specimens were examined microscopically. RESULTS: Significant clinical improvement was seen, and no significant difference between the long and short pulse-width lasers was found. Patients responding to treatment with the PDL remained in remission for up to 13 months. Histologic normalization occurred after treatment. Two pretreatment vascular patterns were seen: vertically oriented vessels with few horizontal vessels and numerous tortuous vessels. Tortuous vessels were associated with poor clinical results. CONCLUSION: The PDL can induce prolonged remission in chronic plaque psoriasis. The vascular pattern may help to distinguish those patients likely to respond to this treatment.

Dermabrasive scar revision. Immunohistochemical and ultrastructural evaluation.

BACKGROUND: Dermabrasion of facial scars 4-8 weeks after injury frequently completely eliminates visible evidence of scar formation. However, efforts to define the cellular and structural mechanisms by which this phenomenon occurs have been limited in their success. OBJECTIVE: We investigated wound healing after dermabrasive scar revision. METHODS: The surgical scars of seven patients were abraded 6-8 weeks after injury. Comparative electron microscopic and immunohistochemical studies were performed on punch biopsy specimens taken before and after the dermabrasion. Ultrastructural changes in the basement membrane components and dermal structures were evaluated. Monoclonal antibody staining techniques were used to observe the presence, location, and temporal expression of tenascin, epiligrin, cadherins, and integrin subunits. RESULTS: We observed: 1) an increase in collagen bundle density and size with a tendency toward unidirectional orientation of fibers parallel to the epidermal surface, 2) an upregulation of tenascin expression throughout the papillary dermis, and 3) expression of alpha-6/beta-4 integrin subunit on the keratinocytes throughout the stratum spinosum. CONCLUSIONS: The mechanisms by which dermabrasive scar revision alters the events of primary cicatrix formation include modification of extracellular ligand expression, thereby influencing epithelial cell-cell interaction, and reorganization of connective tissue.

Keratinocyte muscarinic acetylcholine receptors: immunolocalization and partial characterization.

We have reported previously that human keratinocytes synthesize and secrete acetylcholine and that muscarinic cholinergic drugs have effects on keratinocyte proliferation, adhesion, and migration. This study defines the location of muscarinic acetylcholine receptors in human epidermis and describes some pharmacologic and molecular properties of these receptors. Confocal microscopy employing the anti-muscarinic receptor monoclonal antibody M35 visualized the receptors in the intercellular areas of normal human epidermis. Using immunoelectron microscopy, the receptors appeared to be attached to the keratinocyte plasma membranes. Functional, high-density (Bmax = 8.3 nmol/2 x 10(6) cells) and high-affinity (Kd = 21.5 nM) muscarinic receptors were demonstrated by saturable binding of the reversible radioligand [3H]quinuclidinyl benzilate to the surfaces of freshly isolated epidermal cells at 0 degrees C. Receptor proteins were separated by gel electrophoresis. An apparent isoelectric point of pH 4.3 was determined in immunoblots of sodium-cholate-solubilized receptors separated on isoelectric-focusing gels. Three protein bands, two at approximately 60 kDa and one at 95 kDa, were visualized in immunoblots of membrane-bound or solubilized receptors separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The covalent, irreversible ligand [3H]propylbenzilylcholine mustard confirmed these results. Thus, human keratinocytes express a heterogeneous population of muscarinic cholinergic receptors. Because human keratinocytes also express nicotinic cholinergic receptors, endogenously secreted acetylcholine may control different biologic processes in these cells by activating different types of their cholinergic receptors.

Ultrastructural evaluation of mid-dermal elastolysis.

Mid-dermal elastolysis is a well-defined clinical and histopathologic entity manifested by fine wrinkling of the skin and a mid-dermal loss of elastic fibers. Ultrastructural and histologic studies were performed in an attempt to better define the cause of the elastolytic process. Biopsy specimens from the lesions of 3 patients with mid-dermal elastolysis were studied at light and electron microscopic levels. Ultrastructural evidence of normal elastic fiber engulfment by activated macrophages was observed; however, some fields also demonstrated envelopment of abnormally degenerated elastic tissue. Although there are many potential causes of this degeneration, photodistribution of the lesions suggests that ultraviolet damage is a primary inciting factor.

Hidroacanthoma simplex: an ultrastructural and immunohistochemical study.

A representative case of hidroacanthoma simplex was studied with routine light microscopy, immunohistochemistry, and electron microscopy. Staining with the periodic acid-Schiff reagent and immunostaining with anti-keratin antibodies were useful in demarcating the tumor cells from adjacent normal epithelium. However, antibodies to carcinoembryonic antigen and epithelial membrane antigen did not help us to segregate or identify the neoplastic cells. Electron microscopy revealed tumor cells markedly different in appearance from luminal cells of the acrosyringium. Hidroacanthoma simplex does not appear to be derived from luminal cells of the acrosyringium. We propose criteria for the histologic diagnosis of this benign neoplasm.

T-cell receptor gene rearrangement analysis in the early diagnosis of cutaneous T-cell lymphoma.

The authors describe the techniques of T-cell gene rearrangement in the diagnosis and early detection of cutaneous T-cell lymphoma. Current and future applications of these techniques are discussed.

Clinical, histologic, and ultrastructural evaluation of tattoos treated with three laser systems.

We examined the response of tattoo pigments treated with three commercially available lasers: Q-switched ruby, Q-Switched neodynium:yttrium,aluminum,garnet (Nd:YAG), and the alexandrite. Tattoos applied to hairless guinea pigs and treated with the aforementioned lasers were evaluated clinically, histologically, and ultrastructurally. Clinical evaluation showed red brown, dark brown, and orange pigment responded best to the Nd:YAG laser (1064 nm). The alexandrite laser was most effective for removing blue and green pigment, the Q-switched ruby laser was most effective for removing purple and violet pigment, and the Nd:YAG laser (532 nm) removed red pigment the best. Black pigment was lightened equally with the Nd:YAG laser (1064 nm) and (532 nm) and the alexandrite laser (755 nm). No clinical scarring was observed; however, some colors turned black after treatment. Histologic and ultrastructural examination showed epidermal and dermal damage to be most evident after treatment with the Nd:YAG laser. Our study shows that certain tattoo pigments respond better to different laser systems.

Agarose gel keratinocyte outgrowth system as a model of skin re-epithelization: requirement of endogenous acetylcholine for outgrowth initiation.

To better understand the mechanisms of skin re-epithelization, we developed a simple technique that assays the outgrowth of human keratinocytes. Second-passage foreskin keratinocytes were inoculated at high cell density into 3-mm wells cut from agarose gels in standard 6-well tissue culture dishes. The cells settled on the dish bottom and formed a confluent colony. The cells at the periphery of the colony flattened, spread their cytoplasm, and moved away over the dish surface under the agarose gel. The morphology of migrating keratinocytes was observed microscopically through the transparent agarose, and the migration distance was measured after the gels were removed and after cells were fixed and stained. To determine which cell activities were involved in the outgrowth, the effects of cholinergic compounds on keratinocyte outgrowth were compared with their effects on keratinocyte proliferation, cell-plastic attachment, and spreading measured in separate sets of experiments. Outgrowth was inhibited by the specific inhibitor of acetylcholine synthesis bromoacetylcholine (0.05 mM) and restored by 5 mM exogenous acetylcholine. The irreversible muscarinic antagonist propylbenzilylcholine mustard (0.05 mM) abolished the restorative effects of exogenous acetylcholine, and also inhibited outgrowth of intact keratinocytes. In keratinocyte cell cultures, bromoacetylcholine stopped cell division. Propylbenzilylcholine mustard increased cell number, but interfered with cell-plastic attachment and spreading. This suggests that cell-matrix attachment, spreading, and locomotion of human keratinocytes, but not mitosis, mediate the earliest stages of skin re-epithelization, and that endogenous acetylcholine regulates these keratinocyte functions. Specifically, keratinocyte acetylcholine is required to initiate outgrowth.

Lymphoma versus pseudolymphoma of the skin: gene rearrangement study of 21 cases with clinicopathologic correlation.

BACKGROUND: Diagnosis of cutaneous lymphoma in the absence of systemic lymphoma may be difficult. Reactive lymphoid lesions can mimic lymphoma clinically and histologically and have been designated pseudolymphomas. OBJECTIVE: Our purpose was to analyze lymphoid gene rearrangements in cutaneous lymphoproliferative lesions and to correlate these findings with the histologic, immunophenotypic, and clinical profile. METHODS: We examined 21 cases of lymphoproliferative lesions that developed in skin and performed molecular rearrangement analysis of T-cell receptor and immunoglobulin genes. We examined identical tissues by histologic and immunophenotypic criteria and conducted follow-up clinical evaluation of all patients. RESULTS: Clonal rearrangements of immunoglobulin (seven cases) or T-cell receptor (two cases) gene were detected in 9 of 21 patients. No specific histologic or immunophenotypic feature was consistently associated with a clonal lymphoid gene rearrangement. Systemic lymphoma developed in one patient in whom a clonal rearrangement within the immunoglobulin gene was identified. CONCLUSION: Gene rearrangement analysis may be helpful in differentiating primary cutaneous lymphoma from pseudolymphoma. The chronic clinical course of patients with clonal lymphoid gene rearrangements supports a lack of correlation between clonality and biologic aggressiveness.

Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain.

Bullous pemphigoid (BP) and herpes gestationis (HG) are skin diseases characterized by subepidermal blisters and autoantibodies against two hemidesmosomal Ag, i.e., BP230 and BP180. Based on sequence analysis the BP180 Ag was predicted to be a transmembrane protein with a long extracellular collagenous domain. In the present investigation fusion proteins encompassing various segments of the BP180 Ag were expressed in a prokaryotic system and assayed by immunoblotting and immunoadsorption against a panel of BP, HG and control sera. One antigenic site, comprising 14 amino acids of the BP180 noncollagenous (NC) 16A domain, was shown to be recognized by 60% of BP sera and by 63% of HG sera tested. 73% (11/15) of BP sera and 100% (8/8) of HG sera reacted with at least one of three BP180 fusion proteins representing various portions of the NC16A domain. Immunoadsorption analysis identified this region of BP180 as an immunodominant site. Using an affinity purified rabbit antiserum raised against a recombinant form of BP180, this BP/HG autoantibody-reactive region was localized to the epidermal basal lamina immediately adjacent to the hemidesmosome. These findings confirmed the predicted type II transmembrane orientation of the BP180 Ag. Thus, the long, C-terminal collagenous domain of this basal keratinocyte protein projects into the basal lamina and may function as a site of interaction with an extracellular matrix component. It is proposed that autoantibodies directed against the well-defined antigenic site on the BP180 ectodomain may play an initiatory role in subepidermal blister formation in BP and HG patients.

Epidermolysis bullosa acquisita induced by GM-CSF: a role for eosinophils in treatment-related toxicity.

A patient treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) developed eosinophilia and epidermolysis bullosa acquisita. The bullae were subepidermal, and filled with an inflammatory infiltrate composed predominantly of eosinophils. Immunofluorescence studies disclosed linear deposition of IgG, IgA and C3 at the basement membrane zone and immunoelectron microscopy demonstrated antibody deposition in the lamina densa and sublamina densa region; however, the patient's serum did not contain circulating antibody to basement membrane zone antigens. Staining with monoclonal antibodies revealed dense deposits of both eosinophil peroxidase and eosinophil major basic protein at the dermal-epidermal junction. The eosinophilia and skin lesions resolved upon discontinuation of GM-CSF. This case provides evidence for two hypotheses: (1) GM-CSF induced proliferation and activation of eosinophils may contribute to some of the toxicities of GM-CSF treatment, and (2) activated granulocytes, including eosinophils, may mediate blister formation in epidermolysis bullosa acquisita.