New drug targets in atopic dermatitis

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by eczematous skin lesions, pruritus and typical histopathological features. T cells are thought to play a key role, but B cells might also participate in the pathogenesis of AD. In two investigator-initiated pilot studies, we studied the effects of B cell depletion by monoclonal anti-CD20 antibody therapy or a reduction of activated T cells by LFA3-IgG fusion protein on moderate-to-severe AD. All patients treated with either rituximab or alefacept showed an improvement of their skin symptoms with a sustained effect after treatment. In both studies, histological alterations, such as spongiosis, acanthosis and dermal infiltrate, including T and B cell numbers, dramatically improved and the expression of IL-5 and IL-13 was reduced after therapy. Upon rituximab therapy, allergen-specific IgE levels were not altered and total serum IgE levels only slightly decreased. According to recent studies, neutralizing B and T cells products such as IgE or IL-5 might be effective in subgroups of patients with AD.

Characterization of canine dendritic cells in healthy, atopic, and non-allergic inflamed skin

Atopic dermatitis in humans and dogs is a chronic relapsing allergic skin disease. Dogs show a spontaneous disease similar to the human counterpart and represent a model to improve our understanding of the immunological mechanisms, the pathogenesis of the disease, and new therapy development. The aim of the study was to determine the frequency and phenotype of dendritic cells (DC) in the epidermis and dermis of healthy, canine atopic dermatitis lesional, and non-allergic inflammatory skin to further validate the model and to obtain insights into the contribution of DC to the pathogenesis of skin diseases in dogs. We first characterized canine skin DC using flow-cytometric analysis of isolated skin DC combined with an immunohistochemical approach. A major population of canine skin dendritic cells was identified as CD1c(+)CD11c(+)CD14(-)CD80(+)MHCII(+)MAC387(-) cells, with dermal DC but not Langerhans cells expressing CD11b. In the epidermis of lesional canine atopic dermatitis and non-allergic inflammatory skin, we found significantly more dendritic cells compared with nonlesional and control skin. Only in canine atopic dermatitis skin did we find a subset of dendritic cells positive for IgE, in the epidermis and the dermis. Under all inflammatory conditions, dermal dendritic cells expressed more CD14 and CD206. MAC387(+) putative macrophages were absent in healthy but present in inflamed skin, in particular during non-allergic diseases. This study permits a phenotypic identification and differentiation of canine skin dendritic cells and has identified markers and changes in dendritic cells and macrophage populations related to allergic and non-allergic inflammatory conditions. Our data suggest the participation of dendritic cells in the pathogenesis of canine atopic dermatitis similar to human atopic dermatitis and further validate the only non-murine spontaneous animal model for this disease.

Evaluation and quantification of spectral information in tissue by confocal microscopy

A confocal imaging and image processing scheme is introduced to visualize and evaluate the spatial distribution of spectral information in tissue. The image data are recorded using a confocal laser-scanning microscope equipped with a detection unit that provides high spectral resolution. The processing scheme is based on spectral data, is less error-prone than intensity-based visualization and evaluation methods, and provides quantitative information on the composition of the sample. The method is tested and validated in the context of the development of dermal drug delivery systems, introducing a quantitative uptake indicator to compare the performances of different delivery systems is introduced. A drug penetration study was performed in vitro. The results show that the method is able to detect, visualize and measure spectral information in tissue. In the penetration study, uptake efficiencies of different experiment setups could be discriminated and quantitatively described. The developed uptake indicator is a step towards a quantitative assessment and, in a more general view apart from pharmaceutical research, provides valuable information on tissue composition. It can potentially be used for clinical in vitro and in vivo applications.

Epidermal caspase-3 cleavage associated with interferon-gamma-expressing lymphocytes in acute atopic dermatitis lesions

Keratinocyte apoptosis mediated by Fas/Fas ligand molecular interactions and subsequent caspase activation is believed to play an important role in the pathogenesis of atopic dermatitis (AD), in particular for the formation of spongiosis. To estimate epidermal caspase activation in normal and AD skin under in vivo conditions, we analysed caspase-3 cleavage by immunohistology. In normal skin as well as non-lesional AD skin, we detected caspase-3 cleavage in single cells of the basal layer. In contrast, in acute lesional AD skin, we not only obtained evidence for increased expression of cleaved caspase-3 in keratinocytes of the basal layer but also observed caspase-3 cleavage in one or more layers of the spinous cell layer, in particular in spongiotic areas. Short-term topical treatment of the skin lesions with tacrolimus or pimecrolimus abolished the expression of cleaved caspase-3 in the spinous layer. Moreover, epidermal caspase-3 cleavage correlated with the numbers of dermal interferon-gamma (IFN-gamma)-expressing CD4+ and CD8+ lymphocytes in skin lesions of AD patients, supporting the view that IFN-gamma is important for the activation of proapoptotic pathways in keratinocytes. This is also confirmed by the observation of increased Fas expression on keratinocytes in acute AD lesions that was markedly reduced following topical calcineurin inhibitor treatment. These data suggest that caspase-3 cleavage in the spinous layer of the epidermis is a pathologic event contributing to spongiosis formation in AD, whereas cleavage of caspase-3 in basal cells might represent a physiologic mechanism within the process of epidermal renewal.

Horizontal ridge augmentation using autogenous block grafts and the guided bone regeneration technique with collagen membranes: a clinical study with 42 patients

OBJECTIVE: To analyze the clinical outcome of horizontal ridge augmentation using autogenous block grafts covered with an organic bovine bone mineral (ABBM) and a bioabsorbable collagen membrane. MATERIAL AND METHODS: In 42 patients with severe horizontal bone atrophy, a staged approach was chosen for implant placement following horizontal ridge augmentation. A block graft was harvested from the symphysis or retromolar area, and secured to the recipient site with fixation screws. The width of the ridge was measured before and after horizontal ridge augmentation. The block graft was subsequently covered with ABBM and a collagen membrane. Following a tension-free primary wound closure and a mean healing period of 5.8 months, the sites were re-entered, and the crest width was re-assessed prior to implant placement. RESULTS: Fifty-eight sites were augmented, including 41 sites located in the anterior maxilla. The mean initial crest width measured 3.06 mm. At re-entry, the mean width of the ridge was 7.66 mm, with a calculated mean gain of horizontal bone thickness of 4.6 mm (range 2-7 mm). Only minor surface resorption of 0.36 mm was observed from augmentation to re-entry. CONCLUSIONS: The presented technique of ridge augmentation using autogenous block grafts with ABBM filler and collagen membrane coverage demonstrated successful horizontal ridge augmentation with high predictability. The surgical method has been further simplified by using a resorbable membrane. The hydrophilic membrane was easy to apply, and did not cause wound infection in the rare instance of membrane exposure.

Skin rejuvenation without a scalpel. I. Fillers

Fillers are an important tool in the armamentarium of the physician combating aging phenomena. A wide variety of filler substances are now available that meet many, but by far not all, needs in aesthetic medicine. The most commonly used substances now are hyaluronic acid and collagen preparations that have slightly different indications, but collagen requires pre-use testing to rule out inflammatory complications. Poly-L-lactic acid has gained its place in the filling of adipose tissue wasting in HIV-infected patients. Autologous fat is easy to harvest and inject and has virtually no risk of adverse side effects. Permanent fillers may be of advantage but carry the risk of permanent adverse reactions. Skillful combination of different fillers as well as with botulinum toxin injections and other cosmetic procedures may give optimal results.

A study of the fate of the buccal wall of extraction sockets of teeth with prominent roots

The objective of this investigation was to determine the fate of thin buccal bone encasing the prominent roots of maxillary anterior teeth following extraction. Resorption of the buccal plate compromises the morphology of the localized edentulous ridge and makes it challenging to place an implant in the optimal position for prosthetic restoration. In addition, the use of Bio-Oss as a bone filler to maintain the form of the edentulous ridge was evaluated. Nine patients were selected for the extraction of 36 maxillary anterior teeth. Nineteen extraction sockets received Bio-Oss, and seventeen sockets received no osteogenic material. All sites were completely covered with soft tissue at the conclusion of surgery. Computerized tomographic scans were made immediately following extraction and then at 30 to 90 days after healing so as to assess the fate of the buccal plates and resultant form of the edentulous sites. The results were assessed by an independent radiologist, with a crest width of 6 mm regarded as sufficient to place an implant. Those sockets treated with Bio-Oss demonstrated a loss of less than 20% of the buccal plate in 15 of 19 test sites (79%). In contrast, 12 of 17 control sockets (71%) demonstrated a loss of more than 20% of the buccal plate. In conclusion, the Bio-Oss test sites outperformed the control sites by a significant margin. No investigator was able to predict which site would be successful without the grafting material even though all were experienced clinicians. This leads to the conclusion that a patient has a significant benefit from receiving grafting materials at the time of extraction.

A case of necrotizing fasciitis with septic shock in a cat caused by Acinetobacter baumannii

A 4-year-old, neutered female, domestic shorthair cat admitted to the animal hospital for recurrent constipation presumed to be due to post-traumatic injuries, went into shock with signs including fever and ataxia followed by stupor. On the fifth day of hospitalization, the cat developed severe, diffuse oedema of the ventral abdomen with multifocal to coalescing erythematous areas and small vesicle formation. The results of bacteriological cultures of liver, spleen and kidney specimens led to the diagnosis of Acinetobacter baumannii sepsis. Histopathological findings of skin samples taken during necropsy showed an extensive epidermal and dermal necrosis with septic vasculitis and numerous intralesional gram-negative bacteria. Detection of the bla(OXA-51-like) gene specific for A. baumannii by PCR, performed retrospectively on samples of the deep layers of the skin, confirmed the presence of A. baumannii also in the cutaneous lesions. To our knowledge this is the first report of a necrotizing fasciitis with septic shock in a cat caused by A. baumannii.

Detection of perforin and granzyme B mRNA expressing cells in lichen sclerosus

Granzyme B and perforin messenger RNA (mRNA) expression has been shown to be a specific in vivo activation marker for cytotoxic cells. The aim of this study was to assess the contribution of cell-mediated cytotoxicity in the pathogenesis of lichen sclerosus. In situ hybridization and immunohistochemistry were performed on serial tissue sections of lesional skin biopsies and normal skin as control. Immunohistochemical staining showed that the cellular infiltrate of diseased skin consisted predominantly of T cells (CD3+) and some B cells (CD20+). Among T cells CD4+ and CD8+ cells were found in about equal numbers. In normal skin samples perforin and granzyme B mRNA expressing cells were only rarely found. In contrast, in biopsies from diseased skin a high percentage of infiltrating cells expressed mRNA for perforin and granzyme B. The perforin and granzyme B expressing cells were found in the dermal infiltrate and intraepidermally in close proximity to keratinocytes suggesting in situ activation of these cells. These findings provide evidence that cell-mediated cytotoxicity plays a significant role in tissue destruction in lichen sclerosus.

Early implant placement following single-tooth extraction in the esthetic zone: biologic rationale and surgical procedures

Early implant placement is one treatment option for implant therapy following single-tooth extraction in the anterior maxilla. The surgical technique presented here is characterized by tooth extraction without flap elevation, a 4- to 8-week soft tissue healing period, implant placement in a correct three-dimensional position, simultaneous contour augmentation on the facial aspect with guided bone regeneration using a bioabsorbable collagen membrane combined with autogenous bone chips and a low-substitution bone filler, and tension-free primary wound closure. The surgical step-by-step procedure is presented with a case report. In addition, the biologic rationale is discussed.

Comparative study of biphasic calcium phosphates with different HA/TCP ratios in mandibular bone defects. A long-term histomorphometric study in minipigs

Three biphasic calcium phosphate (BCP) bone substitute materials with hydroxyapatite (HA)/tricalcium phosphate (TCP) ratios of 20/80, 60/40, and 80/20 were compared to coagulum, particulated autogenous bone, and deproteinized bovine bone mineral (DBBM) in membrane-protected bone defects. The defects were prepared in the mandibles of 24 minipigs that were divided into four groups of six with healing times of 4, 13, 26, and 52 weeks, respectively. The histologic and histomorphometric evaluation focused on differences in amount and pattern of bone formation, filler degradation, and the interface between bone and filler. Collapse of the expanded polytetrafluoroethylene barrier membrane into the coagulum defects underlined the necessity of a filler material to maintain the augmented volume. Quantitatively, BCP 20/80 showed bone formation and degradation of the filler material similar to autografts, whereas BCP 60/40 and BCP 80/20 rather equaled DBBM. Among the three BCP's, the amount of bone formation and degradation of filler material seemed to be inversely proportional to the HA/TCP ratio. The fraction of filler surface covered with bone was highest for autografts at all time points and was higher for DBBM than BCP 80/20 and 60/40 at the early healing phase. TRAP-positive multinucleated cells were identified on BCP and DBBM surfaces without showing typical signs of resorption lacunae.

Anti-CD20 (rituximab) treatment improves atopic eczema

BACKGROUND: Atopic eczema (AE) is a chronic inflammatory skin disorder characterized by eczematous skin lesions, pruritus, and typical histopathologic features. OBJECTIVE: We asked whether depletion of B cells by monoclonal anti-CD20 antibody therapy (rituximab) would improve severe AE. METHODS: Six patients (4 women and 2 men) with severe AE received 2 intravenous applications of rituximab, each 1000 mg, 2 weeks apart. To evaluate the efficacy of rituximab, we monitored clinical parameters (eczema area and severity index, pruritus), total and allergen-specific IgE levels, skin histology, and inflammatory cells and cytokine expression in the skin and peripheral blood before and after therapy. RESULTS: All patients showed an improvement of their skin symptoms within 4 to 8 weeks. The eczema area and severity index significantly decreased (before therapy, 29.4 +/- 4.3; week 8, 8.4 +/- 3.6; P < .001). Histologic alterations such as spongiosis, acanthosis, and dermal infiltrate, including T and B cell numbers, also dramatically improved. However, whereas blood B cells were below detectable levels as a consequence of rituximab administration, skin B cells were reduced by approximately 50% only. Expression of IL-5 and IL-13 was reduced after therapy. Moreover, whereas allergen-specific IgE levels were not altered, we observed a slight reduction in total IgE concentrations in blood. CONCLUSIONS: B cells play a major role in AE pathogenesis. Treatment with an anti-CD20 antibody leads to an impressive improvement of AE in patients with severe disease.

Toll-like receptor 2 is highly expressed in lesions of acne inversa and colocalizes with C-type lectin receptor

BACKGROUND: Acne inversa (hidradenitis suppurativa) is a chronic inflammatory and cicatricial disorder that affects skin areas rich in apocrine glands and terminal hairs, such as perineum and axillae. The exact pathogenesis of the disease is not well understood and the mechanisms by which bacterial superinfection contributes to the disease progression are not clear. Toll-like receptors (TLRs) expressed by inflammatory cells play a crucial role in the innate immune response to bacteria. OBJECTIVES: We sought to investigate the role of TLR2 in the pathogenesis of acne inversa. METHODS: We investigated the expression of TLR2 using real-time polymerase chain reaction analysis and immunohistochemical stainings of tissue samples from patients with acne inversa. Furthermore, we phenotypically characterized the infiltrating cells and their expression of TLR2. RESULTS: Compared with normal skin, a highly increased in situ expression of TLR2 in acne inversa skin lesions was found at both the mRNA and the protein level. The most abundant cells in the dermal infiltrate of acne inversa were CD68+ macrophages, CD209+ dendritic cells (DCs) and CD3+ T cells. CD19+ B cells and CD56+ natural killer cells were found only in small numbers. Double staining with fluorescence-labelled antibodies showed that TLR2 was expressed by infiltrating macrophages (CD68+) and DCs (CD209+). Flow cytometric analysis of isolated infiltrating cells further confirmed surface expression of TLR2 by macrophages and DCs. CONCLUSIONS: These data indicate that the enhanced expression of TLR2 by infiltrating macrophages and DCs may contribute to the pathogenesis of inflammatory lesions of acne inversa.

Human beta-defensin-2 and psoriasin are overexpressed in lesions of acne inversa

BACKGROUND: Acne inversa is a chronic inflammatory disorder of apocrine gland-bearing skin. The role of the innate immune system in the pathogenesis of the disease is controversial. OBJECTIVES: We investigated the expression of antimicrobial peptide/proteins in acne inversa. METHODS: Tissue samples were obtained from patients with acne inversa and compared with normal-appearing skin. The expression of psoriasin and human beta-defensin (hBD)-2 on messenger RNA and protein level was analyzed. RESULTS: Both messenger RNA and protein levels of psoriasin and hBD-2 were significantly increased in acne inversa. Macrophages expressing hBD-2 were found in the dermis. LIMITATIONS: Small sample size is a limitation. CONCLUSIONS: Antimicrobial peptide/proteins are overexpressed in acne inversa lesions as compared with normal-appearing skin. The site of the major expression depends on the particular antimicrobial peptide/protein. Psoriasin is overexpressed in epidermal keratinocytes whereas hBD-2 is produced mainly by dermal macrophages, leaving a relative deficiency of hBD-2 in the epidermis of acne inversa lesions.

Occupational exposure to polycyclic aromatic hydrocarbons and DNA damage by industry: a nationwide study in Germany

Exposure to polycyclic aromatic hydrocarbons (PAH) and DNA damage were analyzed in coke oven (n = 37), refractory (n = 96), graphite electrode (n = 26), and converter workers (n = 12), whereas construction workers (n = 48) served as referents. PAH exposure was assessed by personal air sampling during shift and biological monitoring in urine post shift (1-hydroxypyrene, 1-OHP and 1-, 2 + 9-, 3-, 4-hydroxyphenanthrenes, SigmaOHPHE). DNA damage was measured by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and DNA strand breaks in blood post shift. Median 1-OHP and SigmaOHPHE were highest in converter workers (13.5 and 37.2 microg/g crea). The industrial setting contributed to the metabolite concentrations rather than the air-borne concentration alone. Other routes of uptake, probably dermal, influenced associations between air-borne concentrations and levels of PAH metabolites in urine making biomonitoring results preferred parameters to assess exposure to PAH. DNA damage in terms of 8-oxo-dGuo and DNA strand breaks was higher in exposed workers compared to referents ranking highest for graphite-electrode production. The type of industry contributed to genotoxic DNA damage and DNA damage was not unequivocally associated to PAH on the individual level most likely due to potential contributions of co-exposures.