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In order to overcome all encapsulation variations during a complex coacervation process, the replacement of gelatin cationic polymer has been performed using p(AMA). The synthesis of p(AMA) was realized through a random radical methodology. Under these conditions a polymer with 18,600 g/mol was found appropriate for optimal capsule yield and physico-chemical properties. Turbidity measurements performed during the coacervation reactions with different ratios of both CMC and p(AMA) allowed optimizing coacervation conditions. Coacervates characterizations particularly demonstrate the stability of the capsules exhibiting a break strength over 3 N/m2.

The significance of Coenzyme Q10 (CoQ10) as an anti-oxidant barrier of the skin, as well as a key component in anti-aging strategies for skin care products, has been firmly established. Biosynthesis of CoQ10 in the mitochondria is well known, but there is only limited information on the non-mitochondrial synthesis of CoQ10 in the skin. Recent findings in zebrafish identified that a tumor suppressor, Ubiad1, is also a key enzyme in the non-mitochondrial synthesis of CoQ10. The purpose of this study was to investigate expression of Ubiad1 in human skin, and its implication in the skin’s cutaneous response to oxidative stress. We observed Ubiad1 localization in the epidermis, particularly a subcellular localization in the Golgi apparatus. Ubiad1 modulation by a pentapeptide was associated with an observed reduction in ROS/RNS stresses (−44%/−19% respectively), lipid peroxidation (−25%) and preservation of membrane fluidity under stress conditions. Electron microscopy of keratinocytes revealed a significant degree of stimulation of the Golgi complex, as well as significantly improved mitochondrial morphology. Given the importance of CoQ10 in mitigating the visible signs of skin aging, our findings identify Ubiad1 as an essential component of the defensive barriers of the epidermis.

Skin aging is a process of structural and compositional remodeling that can be manifested by wrinkling and sagging. Remarkably, the dermis plays a dominant role in the aging process. Recent studies suggest that microRNAs are implicated in the regulation of gene expression during aging. However, studies about age-related microRNAs and how they modulate skin aging remain limited. In the present work, a complex of hydrolyzed natural yeast proteins (Saccharomyces cerevisiae) and hydrolyzed natural soya bean was developed and showed the ability to modulate the expression of telomere-binding protein TRF2, which is a key factor for telomere protection and to prevent cellular senescence in vitro and DNA damage. The aim of the study was to identify microRNAs specifically modulated after application of the ingredient complex to cultured fibroblasts, and their possible involvement in remodeling of the human extracellular matrix and fibroblast senescence. Consequently, human skin fibroblasts were cultured and treated with 1% of the ingredient complex for 48 h before analyzing microRNA modulation by RT-qPCR. The use of bioinformatics allowed us to predict the target genes for modulated microRNAs. Results show that the ingredient complex modulated a pattern of microRNAs including the down-regulation of miR-29a-3p, miR-30a-5p and miR-34a-5p, which are associated with fibroblast senescence and remodeling of the human dermal extracellular matrix. In conclusion, our results indicate that miR-29a-3p, miR-30a-5p and miR-34a-5p possibly represent key microRNAs that impact human fibroblast senescence and remodeling of the dermal extracellular matrix.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy is classically used to monitor homopolymerizations. In this article, this analytical technique was extended to monitor the synthesis of VIVIPRINT 300, which is a copolymer of 2-hydroxyethyl methacrylate (HEMA) and N-[3-dimethylaminopropyl]methacrylamide (DMAPMA). The calibration curves devised for this study were based on the two homopolymers P(HEMA) and P(DMAPMA). A good correlation was realized between the FTIR absorbance intensities observed respectively at 1300 cm−1 (polymerized C-O ester bond) and 1230 cm−1 (polymerized C-N amide bond) and the level of residual HEMA and DMAPMA monomers determined by GC. Application of these calibration curves to the copolymerization also exhibited a good correlation of data relating to residual monomer determination by FTIR and GC, validating the success of this spectroscopic in situ technique.

Summary Telomere shortening is considered as one of the main characteristics of cellular aging by limiting cellular division. Besides the fundamental advances through the discoveries of telomere and telomerase, which were recognized by a Nobel Prize, telomere protection remains an essential area of research. Recently, it was evidenced that studying the cross-talks between the proteins associated with telomere should provide a better understanding of the mechanistic basis for telomere-associated aging phenotypes. In this review, we discuss the current knowledge on telomere shortening, telomerase activity, and the essential role of telomere binding proteins in telomere stabilization and telomere-end protection. This review highlights the capacity of telomere binding proteins to limit cellular senescence and to maintain skin tissue homeostasis, which is of key importance to reduce accelerated tissue aging. Future studies addressing telomere protection and limitation of DNA damage response in human skin should include investigations on telomere binding proteins. As little is known about the expression of telomere binding proteins in human skin and modulation of their expression with aging, it remains an interesting field of skin research and a key area for future skin protection and anti-aging developments.

Summary Caspase-14, a cysteine endoproteinase belonging to the conserved family of aspartate-specific proteinases, was shown to play an important role in the terminal differentiation of keratinocytes and barrier function of the skin. In the present study, we developed a biofunctional compound that we described as a modulator of caspase-14 expression. Using normal human keratinocytes (NHK) in culture and human skin biopsies, this compound was shown to increase caspase-14 expression and partially reverse the effect of caspase-14-specific siRNA on NHK. Moreover, the increase in filaggrin expression visualized on skin biopsies and the recovery of the barrier structure after tape-stripping indicated that this compound could exhibit a beneficial effect on the skin barrier function. Considering the possible link between caspase-14 and the barrier function, a UVB irradiation on NHK and skin biopsies previously treated with the caspase-14 inducer, was performed. Results indicated that pretreated skin biopsies exhibited less signs of UV damage such as active caspase-3 and cyclobutane pyrimidine dimers (CPDs). Likewise, pretreated NHK were protected from UV-induced genomic DNA damage, as revealed by the Comet Assay. Finally, a clinical test showed a reduction of transepidermal water loss (TEWL) on the treated skin compared with placebo, under UV stress condition, confirming a protecting effect. Taken together, these results strongly suggest that, by increasing caspase-14 expression, the biofunctional compound could exhibit a protective effect on the skin barrier function, especially in case of barrier damage and UV irradiation.

Objective Human epidermis provides the body a barrier against environmental assaults. To assume this function, the epidermis needs the renewal of keratinocytes allowed by constant mitosis, which replace the exfoliating corneocytes. Keratinocyte stem cells (KSCs) located in the basal epidermis are mitotically active, self-renewing and govern the epithelial stratification by producing renewed source of keratinocytes. Protein complex such as the chromosomal passenger complex (CPC) allows the correct development of this process. The CPC is composed of four members: INCENP, survivin, borealin and aurora kinase B, and the disruption of the CPC during cell division induces mitotic spindle defects and improper repartition of chromosomes. The aim of our study was to investigate the implication of CRM1 and survivin in the progress of mitosis in skin keratinocytes. Methods Cultured human keratinocytes and skin biopsies were used in this study. KSCs-enriched population of keratinocytes was isolated from total keratinocytes by differential attachment to a type IV collagen matrix. Survivin and CRM1 expression levels were assessed by immunofluorescence and immunoblotting. Specific siRNAs for each CPC member and for CRM1 were used to determine the relationship between these proteins. Survivin-specific siRNA was used to induce the apparition of mitotic abnormalities in cultured keratinocytes. Results We demonstrated the ability of our compound 'IV08.009' to modulate the expression level of survivin and CRM1 in keratinocytes and in skin biopsies. We observed that members of the CPC are interdependent: siRNA-induced inhibition of one component caused a decrease in the expression of all other CPC members. Downregulation of survivin or CRM1 induced mitotic abnormalities in keratinocytes. However, decreased number of mitotic abnormalities was observed in keratinocytes after 'IV08.009' application. Conclusion Basal keratinocytes may divide frequently during skin lifespan, and signs of deterioration could appear such as loss of protein factors required for correct mitosis. Our findings suggest that mitotic abnormalities can be prevented by the modulation of CRM1 and survivin. We demonstrated the ability of compound 'IV08.009' to efficiently protect cultured keratinocytes from mitotic abnormalities.

Introduction Le derme humain est une structure fondamentale de soutien de la peau comportant des cellules souches multipotentes nommees SKin derived Precursors (SKPs). Ce sont des cellules ayant une capacite d’auto-renouvelement, formant des dermospheres en culture et exprimant des marqueurs de cellules souches comme sox2, oct4 ou nestine. Les SKPs peuvent proliferer et se differencier en differents types cellulaires comme les fibroblastes, les neurones et les adipocytes. Cette capacite leur confere donc la possibilite de regenerer le derme et donc, a terme, d’ameliorer la peau. In vitro, les SKPs peuvent etre isoles directement a partir de derme ou generes a partir de fibroblastes apres un stress au froid. Ces SKPs-like gardent le meme potentiel multipotent que les SKPs et expriment les memes marqueurs. Dans cette etude, nous avons caracterise un extrait derive de jasmin comme un nouvel activateur des SKPs. Les impacts biologiques et physiques des traitements ont ete evalues sur cellules et sur un modele de derme equivalent. Materiel et methodes Premierement, l’expression de sox2, oct4 et nestine a ete observee apres un traitement de 48 heures avec l’extrait, par immunomarquage sur SKPs-like et qPCR sur des fibroblastes. La capacite des fibroblastes a generer des SKPs-like a ensuite ete evaluee par numeration des spheres obtenues 5 jours apres l’application du stress au froid. Enfin, nous avons etudie la morphologie, les fibres de tropo-elastine ainsi que l’expression du collagene III sur un modele de derme equivalent contenant des SKPs-like. Les effets sur la contractilite ont ete visualises par une mesure des diametres au cours du temps. Resultats Une augmentation de l’expression de sox2, oct4 et nestine a ete observee sur SKPs-like et fibroblastes apres traitement avec l’extrait. De plus, une augmentation significative du nombre de spheres a ete comptee dans la condition traitee, indiquant une meilleure capacite a produire de nouvelles cellules souches. Enfin, une amelioration globale de la morphologie a ete observee apres traitement des dermes equivalents contenant des SKPs-like. De plus, une augmentation des fibres de tropo-elastine et d’expression du collagene III ont ete visualisees. En outre, une reduction significative du diametre des dermes a ete mesuree apres 4 jours de traitement, suggerant une acceleration de la contraction. Discussion Dans cette etude, nous avons montre le potentiel in vitro du traitement a augmenter la production de SKPs-like et des marqueurs de multipotence associes. En parallele, nous avons montre qu’en activant ces cellules souches incluses dans un modele de derme reconstruit, les proteines de la matrice extracellulaire ainsi que la contractilite ont ete significativement ameliorees. Conclusion Finalement, nous pouvons conclure que nous avons developpe avec succes un extrait derive de jasmin ciblant les SKPs qui peut aider a promouvoir un derme sain et donc, a terme, limiter le vieillissement cutane.

Mitochondria, long considered to have the primary role in cellular energetic, have been the center of much research interest in the recent past. Technological advances in microscopy and development of new and specific fluorescent dyes for visualization of mitochondrial dynamics in living cells have facilitated the newfound interest in these fascinating organelles, which are now implicated in diverse cellular functions crucial in health and disease. Mitochondria play crucial roles in several age-related diseases, and in the physiology of normal aging. In this review, we discuss the structural and functional aspects of mitochondria and their implications to the aging process, as well as its significance to skin aging. Available information on active molecules that can impact the mitochondrial functions, and their potential use in skin care products is also discussed, highlighting these organelles as a new focus for anti-aging strategies in personal care.

SummaryBackground The chromosomal passenger complex (CPC) is an assembly made of four interacting proteins: survivin, borealin, INCENP, and aurora kinase B. CPC is the key regulatory complex responsible for the correct development of cellular mitosis, accompanying each step of the chromosomal segregation. This control of mitosis is particularly important in undifferentiated cells that must renew themselves and also further differentiate and specialize. The epidermis is a self-renewing tissue that needs to continuously generate new cells through proliferation and differentiation of progenitor cells. Both the mitosis supervision by the CPC and a correct extracellular environment are physiologically required for the homeostasis of the adult keratinocyte stem cells (KSCs) of the epidermis. KSCs are mainly found in the basal layer of the epidermis and are responsible for the replenishment and maintenance of the tissue, by compensating for the loss of terminally differentiated cells called corneocytes, especially during aging. Aim The aim of our study was to investigate the implication of survivin in epidermal renewal and the relationships between survivin expression and UVB-induced DNA damage levels in cultured human keratinocytes and in skin biopsies. In parallel, the effects of a treatment by compound IV08.009 were studied. Material and methods Cultured human keratinocytes and skin biopsies were used in this study. KSCs-enriched fractions of keratinocytes were isolated from total keratinocytes by differential attachment to a type IV collagen matrix. Survivin expression levels were assessed by immunoblotting in cultured keratinocytes, and α6-integrin, β1-integrin, keratin 15, and survivin were observed after immunodetection in skin biopsies cross sections. Comet assay, immunodetection of CPDs and of cleaved-caspase 3, and electron microscopy were used to characterize UVB-induced DNA damage. Results We demonstrated the ability of compound IV08.009 to efficiently protect ex vivo skin against basal UVB-induced damage. Moreover, comet assay studies demonstrated the efficacy of IV08.009 in protecting DNA damage from UVB stress. We found that IV08.009 protects skin from apoptosis induced by oxidative stress, ex vivo. Electron microscopy confirmed the protective efficiency of IV08.009 on cell ultrastructural damage induced by UVB exposure. Conclusion Compound IV08.009 demonstrated to be effective in regulating survivin expression and in preserving the basal epidermis from stresses such as UVB and H2O2. These results suggest a protective activity of IV08.009 on the essential renewing potential of KSCs.

Synopsis Objective Monitoring the chronic and subacute toxicity is essential in the development of new cosmetic ingredients. In response to the present lack of validated alternative methods, we developed an in vitro model for repeated dose cytotoxicity on THP-1 cells. Methods Cultured in suspension, cells were treated with chemicals for 14 days with a frequency of three applications per week, and cell viability was determined by MTT assay. We first investigated the long-term effects of chemicals that induce different kinds of cytotoxicity: Paraquat (PQ), 3-Nitropropanoic acid (3-NPA) and sodium dodecyl sulphate (SDS). From acute studies, doses between 1 and 10 μg ml−1 were chosen to perform our subacute cytotoxicity assay. Comparative genotoxicity evaluations were made with H2O2 or Paraquat treated TPH-1 cells. Comet assays were performed at 1 h (4°C); after a 24-h recovery period (37°C); and finally, after a long-term period of treatment (14 days, 37°C).Once adapted to plant extracts or highly diluted molecules, some of our cosmetic compounds were tested with this model. Results As expected, after 14 days of treatment with Paraquat, cell viability rates dramatically decreased for doses as low as 3 μg ml−1, whereas 10 μg ml−1 of 3-NPA and SDS did not induce more than 44% of cell death. Surprisingly, after subacute treatment, comet assay results revealed a dose-dependent increase in tail moments for Paraquat, whereas those of H2O2 remained low. Moreover, all our compounds tested at 0.5–5 μg ml−1 were classified as safe, even with a cut-off at 90% of cell viability. Conclusion In conclusion, this assay could be of interest for subacute cytotoxicity and genotoxic assessment of daily and topically applied products and suggests that PQ is a choice worthy positive control. Resume Objectifs La surveillance de la toxicite subaigue et chronique est essentielle lors du developpement de nouvelles molecules cosmetiques. Compte tenu du manque de methodes alternatives validees, nous avons developpe un modele de cytotoxicite a doses reiterees in vitro sur cellules TPH-1. Methodes Cultivees en suspension, les cellules ont ete traitees avec des produits chimiques pendant 14 jours, a raison de 3 applications par semaine, et le taux de viabilite cellulaire a ete determine par le test MTT. Nous avons commence par etudier les effets a long terme de produits chimiques induisant differents degres de cytotoxicite : le Paraquat, le 3-acide nitropropanoique (3-NPA) et le dodecyl sulfate de sodium (SDS). A partir d'une etude de cytotoxicite aigue, des doses comprises entre 1 et 10 μg ml−1 ont donc ete choisis pour realiser notre etude de cytotoxicite subaigue. Une etude comparative du potentiel genotoxique du Paraquat et du peroxyde d'hydrogene (H2O2) a ete menee sur les cellules TPH-1. Plusieurs tests des cometes ont ete realises : a 1 h (4°C), apres une periode de recuperation de 24 h (37°C) et apres un traitement de 14 jours (37°C). Une fois adapte pour tester des extraits vegetaux ou de molecules fortement diluees, certains de nos produits cosmetiques ont ete teste avec ce model. Resultats Comme pressentie, apres 14 jours de traitement au Paraquat, le taux de viabilite cellulaire a fortement diminue pour des doses superieures a 3 μg ml−1, alors que 10 μg ml−1 de 3¬NPA ou de SDS n'induisaient pas plus de 44 % de mortalite cellulaire. Etonnamment, les resultats des tests des cometes ont montres que les « tails moments » dus a l'H2O2 restent stables, alors que ceux lies au Paraquat augmentent de maniere «dose-dependante. D'autre part tous les composes testes entre 0.5 et 5 μg ml−1 ont ete classes comme inoffensifs et ceci meme avec un seuil place a 90% de viabilite cellulaire. Conclusions En conclusion, ce test pourrait presenter un interet pour l'etude de cytotoxicite subaigue et genotoxique des produits appliques quotidiennement et suggere que le Paraquat en serait un controle positif de choix.

Telomere shortening is considered as one of the main characteristics of cellular aging by limiting cellular division. Besides the fundamental advances through the discoveries of telomere and telomerase, which were recognized by a Nobel Prize, telomere protection remains an essential area of research. Recently, it was evidenced that studying the cross-talks between the proteins associated with telomere should provide a better understanding of the mechanistic basis for telomere-associated aging phenotypes. In this review, we discuss the current knowledge on telomere shortening, telomerase activity, and the essential role of telomere binding proteins in telomere stabilization and telomere-end protection. This review highlights the capacity of telomere binding proteins to limit cellular senescence and to maintain skin tissue homeostasis, which is of key importance to reduce accelerated tissue aging. Future studies addressing telomere protection and limitation of DNA damage response in human skin should include investigations on telomere binding proteins. As little is known about the expression of telomere binding proteins in human skin and modulation of their expression with aging, it remains an interesting field of skin research and a key area for future skin protection and anti-aging developments.

Caspase-14, a cysteine endoproteinase belonging to the conserved family of aspartate-specific proteinases, was shown to play an important role in the terminal differentiation of keratinocytes and barrier function of the skin. In the present study, we developed a biofunctional compound that we described as a modulator of caspase-14 expression. Using normal human keratinocytes (NHK) in culture and human skin biopsies, this compound was shown to increase caspase-14 expression and partially reverse the effect of caspase-14-specific siRNA on NHK. Moreover, the increase in filaggrin expression visualized on skin biopsies and the recovery of the barrier structure after tape-stripping indicated that this compound could exhibit a beneficial effect on the skin barrier function. Considering the possible link between caspase-14 and the barrier function, a UVB irradiation on NHK and skin biopsies previously treated with the caspase-14 inducer, was performed. Results indicated that pretreated skin biopsies exhibited less signs of UV damage such as active caspase-3 and cyclobutane pyrimidine dimers (CPDs). Likewise, pretreated NHK were protected from UV-induced genomic DNA damage, as revealed by the Comet Assay. Finally, a clinical test showed a reduction of transepidermal water loss (TEWL) on the treated skin compared with placebo, under UV stress condition, confirming a protecting effect. Taken together, these results strongly suggest that, by increasing caspase-14 expression, the biofunctional compound could exhibit a protective effect on the skin barrier function, especially in case of barrier damage and UV irradiation.

Cutaneous aging is a complex biological phenomenon affecting the different constituents of the skin. Two independent processes, clinical and biological, affect the skin during aging. The first is called “chronological aging” and the second one, “extrinsic aging” or “photoaging”, which is the result of out-door exposure (such as solar irradiation). In this study, we mainly investigated the features of “chronological aging”.

Patients with anti-epiligrin cicatricial pemphigoid have anti-basement membrane autoantibodies that immunoprecipitate a set of disulfide-linked human keratinocyte polypeptides that co-migrate in sodium dodecyl sulfate polyacrylamide gel electrophoresis with the same complex identified by monoclonal anti-epiligrin (P1E1) and monoclonal anti-nicein/kalinin (GB3) antibodies. In an attempt to further compare the reactivity of patient autoantibodies, P1E1 and GB3, these reagents were tested against the tumor nest basement membranes of 7 papulonodular basal cell carcinomas. These studies found that all of these reagents showed markedly decreased or no reactivity against this substrate. Though their concordant lack of reactivity failed to distinguish these antibodies, these studies did identify a significant defect in papulonodular basal cell carcinoma tumor nest basement membranes. Similarly, integrin subunits alpha 6, beta 4, alpha 3, and alpha 2 as well as bullous pemphigoid antigens 1 and 2 (all potential receptors for the extracellular matrix ligands epiligrin and nicein/kalinin) were also reduced in these tumor nest basement membranes. These findings signify an extensive impairment in the lamina lucida of this neoplasm's basement membrane. Related comparative studies of normal human keratinocytes and transformed human epithelial cell lines (specifically, A-431 and HaCat cells) showed that epiligrin production is markedly decreased in the latter. Decreased expression of epiligrin and nicein/kalinin in papulonodular basal cell carcinoma tumor nest basement membranes in vivo and transformed epithelial cells in vitro indicate that this complex is a transformation-sensitive cell adhesion ligand.

Indirect immunogold electron microscopy studies of cryofixed, freeze-substituted, and post-embedded normal human skin were performed to localize precisely the ultrastructural binding site of circulating autoantibodies from two groups of patients with cicatricial pemphigoid. One group of patients had circulating IgG autoantibodies that bound the dermal side of 1 M NaC1-split skin and immunoprecipitated epiligrin. The other group of patients had circulating IgG autoantibodies directed against the epidermal side of 1 M NaCl-split skin and showed no specific reactivity to any keratinocyte polypeptide by iminunoprecipitation. IgG autoantibodies from all patients with anti-epiligrin cicatricial pemphigoid bound the lowermost aspect of the lamina lucida at its interface with the lamina densa; the greatest staining was seen beneath and beside hemidesinosomes, In contrast, IgG from cicatricial pemphigoid patients whose autoantibodies bound the epidermal side of 1 M NaCl- split skin localized to hemidesmosomes and the junction between hemidesmosoines and the plasma membranes of basal keratinocytes. Although the latter staining pattern is similar to that observed with anti-BPAG2 autoantibodies, sera from our patients with cicatricial pemphigoid did not bind BPAG2 in immunoprecipitation studies of radiolabeled human keratinocyte extracts or show immunoblot reactivity to a fusion protein corresponding to the immunodominant epitope of this polypeptide. These studies demonstrate the following: 1) Autoantibodies from patients with anti-epiligrin cicatricial pemphigoid consistently bind the lower lamina lucida at its interface with the lamina densa; and 2) other patients with the same phenotype may have IgG autoantibodies against yet-unknown epitopes in basal keratinocytes.

Background: Epiligrin is a glycoprotein complex deposited in extracellular matrix by cultured human keratinocytes that serves as the major integrin ligand of these cells. In human skin, epiligrin is found at the interface of the lamina lucida and lamina densa in epidermal basement membrane where it is believed to be associated with anchoring filaments and plays an important role in keratinocyte adhesion. Methods and Results: We have identified six patients with a subepithelial bullous disorder of mucous membranes and skin who have IgG anti-basement membrane autoantibodies that immunoprecipitate epiligrin from human keratinocyte extracts and culture media. These patients' IgG autoantibodies also bind epiligrin in human keratinocyte extracellular matrix and epidermal basement membrane as determined by immunofluorescence and immunoelectron microscopy. Studies of 10 patients who are clinically indistinguishable from subjects with antiepiligrin autoantibodies (ie, cicatricial pemphigoid pa- tients) found that while seven had anti-basement membrane autoantibodies, the latter are directed exclusively against a region of epidermal basement membrane that does not contain epiligrin, are present in low titer (ie, ≤1:10), do not react with keratinocyte extracellular matrix, and do not bind epiligrin (or any other specific antigen) in immunoprecipitation studies of human keratinocyte extracts or media. Antiepiligrin autoantibodies were also not detected in studies of 36 additional patients with bullous diseases or six normal volunteers. Conclusions: Cicatricial pemphigoid is a disease phenotype in which patients' autoantibodies may target different constituents of epidermal basement membrane. Antiepiligrin autoantibodies are a specific immunologic marker for a group of patients with a disease entity that we propose to designate antiepiligrin cicatricial pemphigoid. (Arch Dermatol. 1994;130:1521-1529)

REPORT OF A CASE A 23-month-old white boy was referred to us for evaluation of a vesiculobullous eruption of 7 months' duration involving the scalp, perioral surface, chin, neck, umbilicus, suprapubic surface, scrotum, and perianal surface. Before his referral, the child had been treated with multiple courses of antibiotics for bullous impetigo; no improvement was seen. The child was at the appropriate developmental stage and was in excellent general health. The physical examination revealed an alert, happy child with perioral vesicles, pustules, and erosions. In addition, he had a bullous eruption on a slightly erythematous base with erosions and crusts involving the scalp, chin, umbilicus, suprapubic surface (Fig 1), groin, and scrotum (Fig 2). Some of the lesions had a serpiginous border. No oral, ocular, hand, or foot lesions were present. The following laboratory results were normal or negative: complete blood cell count, automated seven-channel analysis System, renal panel, hepatic panel

• Background.— We describe a patient with a subepidermal bullous skin disease associated with autoantibodies recognizing separate epitopes in 1 mol/L sodium chloride (NaCl) split skin. Observations.— Direct immunofluorescence microscopy showed deposits of immunoglobulins and C3 in a continuous pattern in the patient's epidermal basement membrane zone. Direct immunoelectron microscopy demonstrated thick deposits of IgG overlying the lamina lucida and the lamina densa in a unique pattern. The patient had circulating IgG anti— basement membrane zone antibodies that bound both sides of 1 mol/L NaCl split skin, exhibited at least a fourfold-higher titer against the dermal side of this test substrate, and bound basal keratinocyte hemidesmosomes as well as focal sites along the superior portion of the lamina densa on indirect immunoelectron microscopy. Affinity purification of anti—base-ment membrane zone antibodies against epidermal or dermal strips of 1 mol/L NaCl split skin yielded IgG that only bound the side of split skin from which it was eluted. The patient's serum contained IgG that immunoprecipitated and immunoblotted the 230- and 170-kd bullous pemphigoid antigens. Affinity purification of patient antibody against bullous pemphigoid antigen immobilized on nitrocellulose paper yielded IgG that bound only the epidermal side of 1 mol/L NaCl split skin. The patient showed no evidence of reactivity against type VII collagen by direct immunoelectron microscopy, indirect immunoelectron microscopy, or immunoblot. Conclusions.— This patient's bullous skin disease is associated with IgG anti—basement membrane zone antibodies with two specificities: one recognizing the bullous pemphigoid antigen in the epidermal side of 1 mol/L NaCl split skin, and another binding a distinct, yet presently unidentified, epitope in the superior aspect of the lamina densa. ( Arch Dermatol. 1992;128:1096-1101)

• Background.— Childhood localized vulvar pemphigoid has been recently reported in four girls. A fifth child with this proposed rare variant of bullous pemphigoid is described. Moreover, findings in the various immunopathologic studies we performed establish this entity as a true morphologic variant of bullous pemphigoid. Observations.— In situ deposits of IgG in this patient's epidermal basement membrane zone localized to the epidermal side of 1 mol/L of saline-split skin. Moreover, the patient had circulating IgG autoantibodies that bound the epidermal side of 1 mol/L of saline-split skin in indirect immunofluorescence microscopy and immunoprecipitated the 230-kd bullous pemphigoid antigen from biosynthetically radiolabeled human keratinocyte extracts. These laboratory findings are identical to those documented in patients with the generalized "classic" form of bullous pemphigoid. Conclusions.— This study demonstrates that a child with clinical, histopathologic, and immunopathologic features of localized vulvar pemphigoid had circulating autoantibodies that identify a specific keratinocyte antigen, the bullous pemphigoid antigen, which may serve as a molecular marker for this disease. (Arch Dermatol.1992;128:807-810)

• Background.— Waldenstrom macroglobulinemia, a lymphoplasmacytoid cell malignant neoplasm associated with a monoclonal IgM paraprotein, has been associated with a number of cutaneous manifestations. On rare occasions, IgM deposits have been demonstrated in the epidermal basement zone of patients with WM. Observations.— We report the case of a patient with Waldenstrom macroglobulinemia and IgM-κ paraprotein who had development of an eruption of pruritic papules and demonstrated the following unusual immunopathologic findings: (1) deposits of IgM-κ in the epidermal basement membrane zone of lesional and nonlesional skin; (2) a circulating IgM-κ antiepidermal basement membrane zone antibody; and (3) binding of this circulating IgM-κ antiepidermal basement membrane zone antibody to both sides of 1 mol/L sodium chloride split skin. The cutaneous eruption cleared completely with oral psoralen with long-wave UV radiation in the A range (PUVA) therapy. Conclusions.— We present a patient with Waldenstrom macroglobulinemia who had a distinctive papular eruption and immunopathologic findings suggesting that his paraprotein has specificity for the epidermal basement membrane zone. (Arch Dermatol.1992;128:372-376)

Two patients demonstrating the typical clinical, histologic, and immunopathologic features of nonscarring localized bullous pemphigoid are described. These patients possess circulating IgG autoantibodies that bind the epidermal side of 1.0-mol/L sodium chloride-split human skin in indirect immunofluorescence microscopy. Immunnoprecipitation studies demonstrate that these patients have circulating autoantibodies that immunoprecipitate the same 230-kd bullous pemphigoid antigen that is precipitated by autoantibodies from patients with generalized bullous pemphigoid. These findings indicate that localized bullous pemphigoid is a true clinical variant of generalized pemphigoid rather than a separate nosologic entity.

Biosynthetic radiolabeling studies demonstrate that human keratinocytes and A-431 cells, a human epidermoid carcinoma cell line, synthesize and secrete factor B as a monomeric 105-kD protein. Epithelial cell-derived factor B comigrates in SDS-PAGE with that produced by HepG2 cells, a human hepatoma cell line traditionally employed in studies of complement component biosynthesis. Comparative pulse-chase studies in A-431 and HepG2 cells show that this alternative pathway complement component is produced as co-migrating 100-kD intracellular proteins that are processed in both cell types to 105-kD extracellular factor B. Quantitatively, immunoprecipitable factor B accounts for 0.05% of radiolabeled proteins in A-431 cell culture media. Treatment of biosynthetically radiolabeled A-431 cell culture media with cobra venom factor and factor D for 60 min at 37 degrees C produces the specific factor B cleavage products Ba and Bb. These fragments are not identifiable in control culture media subjected to similar treatment in the absence of alternative pathway activators. Northern blot analysis of total cellular RNA from human keratinocytes, A-431 cells, and HepG2 cells reveals qualitative identity of a 2.8-kb factor B mRNA species in these three cell types. The relative level of factor B mRNA expression in these cells parallels their level of factor B protein synthesis (i.e., HepG2 cells greater than A-431 cells greater than human keratinocytes). Epithelial cell-derived factor B may play an important role in local inflammatory reactions and also directly interact with epithelial cell derived C3--a key classical and alternative pathway complement component recently shown to be produced by human keratinocytes.