Langerhans cells integrated into human reconstructed epidermis respond to known sensitizers and ultraviolet exposure.
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Epidermal Cells↗
Biomedical subjects
Rainer Schmidt
Publications and source records attributed to Rainer Schmidt.
A constitutive NADPH oxidase-like system containing gp91phox homologs in human keratinocytes.
In non-phagocytic cells, superoxide has been implicated in physiological and pathological cellular functions in the skin and mucosa, such as, host defense, mitogenic responses, and malignant conversion. Here, we identify a constitutively expressed heme-flavoprotein NADPH oxidase (Nox) system as a source of superoxide in human skin (HaCaT) and gingival mucosal (GM16) keratinocyte cell lines. Western blot analysis showed that both cell lines expressed the phagocyte oxidase (phox) cytosolic proteins Rac1, p40phox, and p67phox. With respect to the catalytic flavoheme protein subunit, HaCaT membranes, which expressed p22phox, showed an absorbance peak at 558 nm indicative of a b-type cytochrome. At mRNA levels, both GM16 and HaCaT cells expressed gp91phox homologs Nox1, Nox2, and Nox4, however, HaCaT cells expressed very low levels of Nox1 mRNA. At protein levels, Nox1 was readily detected in HaCaT but was nearly undetectable in GM16 cells. Consistently, Nox activity of HaCaT membranes was demonstrated by electron paramagnetic resonance spin-trapping and cytochrome c reduction, and the activity was sensitive to the flavoprotein inhibitor diphenylene iodonium. V(max) values were 20-fold lower than those reported for phagocytic oxidase. In conclusion, keratinocytes expressed a Nox distinct from the phox isoform of phagocytes providing molecular evidence for a source of superoxide that may regulate cell proliferation and host defense in skin and oral mucosa.
Cell Line↗
Degradation of corneodesmosome proteins by two serine proteases of the kallikrein family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7.
Corneodesmosin (CDSN), desmoglein 1 (DSG1), and desmocollin 1 (DSC1) are adhesive proteins of the extracellular part of the corneodesmosomes, the junctional structures that mediate corneocyte cohesion. The degradation of these proteins at the epidermis surface is necessary for desquamation. Two serine proteases of the kallikrein family synthesized as inactive precursors have been implicated in this process: the stratum corneum chymotryptic enzyme (SCCE/KLK7/hK7) and the stratum corneum tryptic enzyme (SCTE/KLK5/hK5). Here, we analyzed the capacity of these enzymes to cleave DSG1, DSC1, and epidermal or recombinant forms of CDSN, at an acidic pH close to that of the stratum corneum. SCCE directly cleaved CDSN and DSC1 but was unable to degrade DSG1. But incubation with SCTE induced degradation of the three corneodesmosomal components. Using the recombinant form of CDSN, either with its N-glycan chain or enzymatically deglycosylated, we also demonstrated that oligosaccharide residues do not protect CDSN against proteolysis by SCCE. Moreover, our results suggest that SCTE is able to activate the proform of SCCE. These results strongly suggest that the two kalikreins are involved in desquamation. A model is proposed for desquamation that could be regulated by a precisely controlled protease-protease inhibitor balance.
Antibody Specificity↗
Increased incidence of squamous cell carcinomas in Mastomys natalensis papillomavirus E6 transgenic mice during two-stage skin carcinogenesis.
Papillomaviruses cause certain forms of human cancers, most notably carcinomas of the uterine cervix. In contrast to the well-established involvement of papillomavirus infection in the etiology of cervical carcinomas and in carcinomas of a rare hereditary condition, epidermodysplasia verruciformis, a causative role for cutaneous human papillomavirus types in the development of nonmelanoma skin cancer has not been proven. In order to better understand the functions of individual genes of cutaneous papillomavirus types, we generated transgenic mice carrying oncogene E6 of the Mastomys natalensis papillomavirus (MnPV), which causes keratoacanthomas of the skin in its natural host. In the present study, we demonstrate that under conditions of experimental two-stage skin carcinogenesis, fast-paced squamous cell carcinomas develop in nearly 100% of MnPV E6 transgenic mice in comparison to 10% in their nontransgenic littermates (log rank test; P < 0.0001). Therefore, we conclude that the MnPV E6 transgene favors the malignant progression of chemically induced tumors. Whereas an activating H-ras mutation is a consistent feature in benign and malignant tumors in wild-type mice, the majority of papillomas and keratoacanthomas and all squamous cell carcinomas obtained in MnPV E6 transgenic mice contain nonmutated ras alleles. These results indicate that the development of squamous cell carcinomas in MnPV E6 transgenic mice does not depend on an activated H-ras oncogene.
9,10-Dimethyl-1,2-benzanthracene↗
Osteodensitometry after total hip replacement with uncemented taper-design stem.
We followed 24 patients (26 hips) with uncemented total hip replacement for a minimum of 3 years. Patient mean age was 52 (40-69) years. The aim was to evaluate femoral periprosthetic bone mineral density (BMD) using quantitative computed tomography osteodensitometry. At 3-years' follow-up, 25 hips were clinically rated good or excellent. The mean Harris hip score rose from 58 (49-68) pre-operatively to 94 (81-98) at the last follow-up. All hips were radiologically stable. Mean decrease of the overall BMD in the femoral metaphysis was 14.3%, and mean decrease of cortical BMD was 17.3%. In the diaphysis, mean decrease of overall BMD was 5.5% and mean decrease of cortical BMD was 4.5%. Observed loss was markedly lower than in comparable series with uncemented stems.
Adult↗
Long-term expansion of human functional epidermal precursor cells: promotion of extensive amplification by low TGF-beta1 concentrations.
We have previously introduced the concept of high proliferative potential-quiescent (HPP-Q) cells to refer to primitive human hematopoietic progenitors, on which transforming growth factor-beta1 (TGF-beta1) exerts a pleiotropic effect. TGF-beta1 confers to these slow-dividing cells a mitogenic receptor(low) phenotype and maintains immature properties by preventing differentiation and apoptosis. However, the effect of TGF-beta1 on long-term expansion has not yet been clearly demonstrated. Here, we describe the characterization of a human skin keratinocyte subpopulation, highly enriched for primitive epidermal precursors, on the basis of high adhesion capacity (Adh+++) and low expression of the epidermal growth factor receptor (Adh+++EGF-Rlow). In our standard culture condition without feeder cells, the mean estimated output for cells from an unfractionated population of primary foreskin keratinocytes was 10(7)-10(8), increasing to 10(12)-10(13) in cultures initiated with selected Adh+++EGF-Rlow precursors. Characterization of these cells revealed a hitherto unknown property of TGF-beta1: its addition at a very low concentration (10 pg/ml) in long-term cultures induces a very significant additional increase of expansion. In this optimized system, outputs obtained in cultures initiated with Adh+++EGF-Rlow cells repeatedly reached 10(16)-10(17) ( approximately 60 population doublings, approximately 4 x 10(18) keratinocytes produced per clonogenic cell present in the initial population). At the molecular level, this effect is associated with an increase in Smad1, Smad2 and Smad3 phosphorylation and an increase in alpha6 and beta1 integrin expression. No such effect could be observed on mature keratinocytes with low adhesion capacity (Adh-/+). We finally demonstrated that the progeny of Adh+++EGF-Rlow precursors after long-term expansion is still capable of generating a pluristratified epidermis in a model for skin reconstruction. In conclusion, after further characterizing the phenotype of primitive epidermal precursors, we demonstrated a new function of TGF-beta1, which is to promote undifferentiated keratinocyte amplification.
Cell Adhesion↗
Association of gp91phox homolog Nox1 with anchorage-independent growth and MAP kinase-activation of transformed human keratinocytes.
Among five members of the NADPH oxidase (Nox) family, Nox1 confers mitogenic properties and is implicated to participate in the process of cell transformation. We have established two phenotypes of carcinogenesis model by ethanol treatment of human gingival keratinocytes immortalized with E6/E7 oncogenes of human papillomavirus type16: immortalized (EPI) nontransformed cells with epithelium-like morphology and more advanced transformed (FIB) cells with spindle fibroblastic-shape morphology. FIB membranes possessed a 63-kDa Nox1 protein at higher levels and exhibited 2.8-fold higher capability for superoxide and hydroxyl radical generation, compared with EPI membranes. Both EPI and FIB cells expressed more abundant Nox1 protein at a proliferating stage than that at a quiescent confluent phase. Immunofluorescence staining with an anti-Nox1 antibody showed that immunoreactive materials were distributed in the whole interior of both types of cells, while they were preferentially localized in the nuclei of FIB cells. Nuclei isolated from EPI and FIB cells contained a 63 kDa-Nox1 protein. Compared with EPI cells, FIB cells expressed elevated levels of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase proteins. Furthermore, JNK2 was constitutively phosphorylated in FIB cells. Together, our data strongly implicate Nox1 in redox-mediated signaling related to cellular activation of human keratinocytes at a more advanced stage of transformation.
Cell Division↗
Clinical outcome and periprosthetic bone remodelling of an uncemented femoral component with taper design.
We followed 48 consecutive patients (50 hips) with osteoarthritis who received a primary total hip replacement using a tapered stem with hydroxylapatite coating (Cerafit Multicone H-A.C.) and a press-fit cup. Mean follow-up was 3 years, mean patient age was 54.7 years, and mean preoperative Harris hip score was 57. Quantitative evaluation of periprosthetic bone remodelling was assessed 1 year after the index operation using computed tomography (CT). Clinical and radiological follow-up was obtained in all hips. The mean Harris hip score at follow-up was 96. Forty-nine hips were clinically rated good or excellent. No thigh pain was reported. Radiographs showed stable fixation by bone ingrowth in all hips. Fifteen hips were eligible for CT. Three years after operation, mean decrease of overall bone mineral density (BMD) was 14.2% and mean decrease of cortical BMD 15.5% in the metaphyseal portion. In the diaphyseal portion, mean decrease of overall BMD was 10.0% and mean decrease of cortical BMD 7.7%. Minimal changes were observed at the level of the tip of the stem. Clinical and radiological outcome using a tapered femoral stem with hydroxylapatite coating compares favourably with other reports. Osteodensitometry shows limited proximal femoral bone resorption.
Arthroplasty, Replacement, Hip↗
Uncemented acetabular components with polyethylene or alumina liners.
Sixty consecutive patients (60 hips) were allocated in two matched-pair groups. Patients were matched for age, gender, body mass index, level of activity, and bone stock. Both groups received an uncemented total hip arthroplasty with a modular press-fit cup and an alumina femoral head. In the first group, an acetabular liner made of polyethylene was used; in the second group, an alumina liner. Both groups were followed for a minimum of 5 years. No hip required revision, and there was no radiographic evidence of aseptic loosening in either group. Mean Harris hip score was 94.7 (87.1-99) points in the polyethylene group and 93 (88.5-100) points in the alumina group.
Acetabulum↗
Methods, architecture, evaluation and usability of a case-based antibiotics advisor.
In this paper, we discuss the usability of an antibiotics therapy adviser, with a broad, complex spectrum of functions which we have developed within the ICONS project. We present the architecture of the system, case-based reasoning methods used, steps and results of medical evaluations, which are concerning the quality of the recommended therapies, the user friendliness of the system and the interpretation of laboratory results. Furthermore, we discuss problems of transferability of such a system from one site to another as well as problems of local susceptibility patterns and individual dose regimens.
Anti-Bacterial Agents↗
The use of reconstructed human skin to evaluate UV-induced modifications and sunscreen efficacy.
Biological and clinical effects of sun exposures are characterized by short-term reactions, i.e. sunburn reaction and suntan, as well as long-term consequences corresponding to photoaging and photocancers. We have developed several human in vitro three-dimensional models in order to assess both the photodamage and the photoprotection afforded by sunscreens. Using a full thickness reconstructed skin comprising a differentiated epidermis and a living dermal equivalent, UVB- and UVA-induced biological markers could be found at both the keratinocyte and the fibroblast level. Typical markers of the sunburn reaction could be reproduced in that model as well as dermal damages related to the photoaging process. Another model of reconstructed epidermis, comprising keratinocytes but also melanocytes and Langerhans cells, has been developed. The study of the UV-induced pigmentation as possible using the pigmented reconstructed epidermis and allowing to reproduce the epidermal melanin unit. The assessment of cellular parameters related to UV-induced immunosuppression could be performed using the reconstructed epidermis containing Langerhans cells. Exposure to solar-simulated radiation provokes morphological alterations and the reduction in numbers of Langerhans cells within the exposed epidermis. Using all these models, the efficiency of sunscreens could be envisaged after topical application. The results showed that appropriate sunscreens could efficiently prevent the damage described above.
Cells, Cultured↗
Carbohydrate expression and modification during keratinocyte differentiation in normal human and reconstructed epidermis.
Using fluorescein isothiocyanate (FITC)-labeled lectins we were able to demonstrate the presence of specific carbohydrate moieties in normal human and reconstructed epidermis. Evidence is provided that in both cases the strongly reduced lectin staining at the level of the stratum corneum is the result of a hindered accessibility of the lectins in this lipid-rich hydrophobic environment. Isolated corneocytes and purified cornified envelopes (CEs) exhibited clearly glycosylated structures reacting with distinct lectins. The presence of glycosidase activity, particularly in the upper layers of the epidermis characterized by an acidic environment (pH 5.5), indicates that modifications of the sugar residues might be important in epidermal homeostasis, barrier behavior and desquamation. Absent or strongly reduced glycosidase activity in the stratum corneum of reconstructed epidermis with an impaired pH gradient could be in part responsible for the reduced barrier function and the lack of desquamation in this model.
Carbohydrate Metabolism↗
Epithelium and fibroblast-like phenotypes derived from HPV16 E6/E7-immortalized human gingival keratinocytes following chronic ethanol treatment.
Epithelial-mesenchymal transition (EMT) may be critical for neoplastic progression and its eventual tumorigenicity of epithelia. In this context, we investigated whether EMT and EMT-associated features occurred after chronic ethanol treatment of human gingival keratinocytes immortalized with the E6/E7 oncogenes of human papillomavirus (HPV) type 16. Following a nine-week treatment of cells with 30 mM ethanol in keratinocyte growth medium, they were cultured in normal DMEM with 10% serum. These cell populations were able to proliferate in this medium gradually exhibiting elongated morphology indicating that these cells underwent EMT. Control cells without ethanol treatment did not survive subcultures in DMEM. Upon long-term subcultures of ethanol-treated cells, two phenotypes were obtained exhibiting epithelium-like and spindle-shape fibroblast-like morphology (respectively, termed as EPI and FIB cells), the latter indicating EMT. In comparison to EPI cells, the phenotypic transition to FIB cells was concomitant with a decrease in the expression of keratins, desmoplakins and a complete loss of K14. Moreover, FIB cell transition strongly correlates with an increase in the expression of vimentin and simple epithelial keratin K18. These alterations in FIB cells were associated with the ability of these cells to exhibit anchorage-independent growth, while EPI cells exhibited anchorage-dependent growth. Concerning the transformation stage, FIB cells represent a progressively more advanced transformed phenotype which may reflect an early step during HPV- and ethanol-dependent multi-step carcinogenesis.
Animals↗
CT-based preoperative analysis of scapula morphology and glenohumeral joint geometry.
OBJECTIVE: To investigate the geometry of the glenohumeral joint using three-dimensional (3D) models; define landmarks, planes, angles and regions of interest; and analyze the exact morphology of the scapula. MATERIALS AND METHODS: We reconstructed 3D scapula models based on computed tomography (CT) data sets of 12 healthy controls. Three-dimensional models were reconstructed using the 3D Slicer2 (Surgical Planning Lab, Boston, MA), which provides interactive measurement. The 3D model and measuring tools can be freely shifted and rotated in all planes. RESULTS: The average length of the scapulas was 209.58 +/- 26.72 mm (left) and 223.68 +/- 47.74 mm (right); width was 124.03 +/- 13.28 mm (left) and 141.44 +/- 27.78 mm (right); and volume was 121.38 +/- 12.02 ml (left) and 130.24 +/- 20.86 ml (right). The glenoid dimensions were 34.34 +/- 5.62 mm (left) and 36.44 +/- 7.36 mm (right) (anteroposterior); and 49.16 +/- 7.68 mm (left), 51.46 +/- 10.07 mm (right) (superoinferior). For all average values, scapulas from male controls were significantly larger than those from females, and right sides were larger than left sides (p < 0.05). The glenoid version was nearly the same for male/female and left/right (left = 9.02 +/- 3.89 degrees retroversion; right = 8.26 +/- 3.72 degrees retroversion) (p > or = 0.05). CONCLUSION: The 3D measurement of scapula morphology compared with geometry of the glenohumeral joint leads to a more precise planning of shoulder surgery. As new shoulder prostheses are intended to reconstruct the normal anatomy as closely as possible, such exact 3D measurements may be used for optimization.
Computer Simulation↗
A prognostic model for temporal courses that combines temporal abstraction and case-based reasoning.
Since clinical management of patients and clinical research are essentially time-oriented endeavours, reasoning about time has recently become a hot topic in medical informatics. Here we present a method for prognosis of temporal courses, which combines temporal abstractions with Case-based Reasoning. We have applied our method in two programs. The first one deals with multiparametric time course prognosis of the kidney function. The intention of the second program is to compute early warnings against approaching infectious diseases like influenza.
Disease Progression↗
Cation- and peptide-binding properties of human calmodulin-like skin protein.
Human CLSP, a new Ca(2+)-binding protein specifically expressed in differentiated keratinocytes, is a 15.9 kDa, four EF-hand containing protein with 52% sequence identity to calmodulin (CaM). The protein binds four Ca(2+) ions at two pairs of sites with [Ca(2+)](0.5) values of 1.2 and 150 microM, respectively. Mg(2+) at millimolar concentrations strongly decreases the affinity for Ca(2+) of the two high-affinity sites, but has no effect on the low-affinity sites. The protein can also bind two Mg(2+) ([Mg(2+)](0.5) = 57 microM) at the sites of high Ca(2+) affinity. Thus, as fast skeletal muscle troponin C (TnC), CLSP possesses two high-affinity Ca(2+)-Mg(2+) mixed sites and two low-affinity Ca(2+)-specific sites. Studies on the isolated recombinant N- (N-CLSP) and C-terminal half domains of CLSP (C-CLSP) revealed that, in contrast to the case of TNC, the high-affinity Ca(2+)-Mg(2+) mixed sites reside in the N-terminal half. The binding of cations modifies the intrinsic fluorescence of the two Tyr residues. Upon Ca(2+) binding, hydrophobicity is exposed at the protein surface that can be monitored with a fluorescent probe. The Ca(2+)-dependency of the two conformational changes is biphasic in the absence of Mg(2+), but monophasic in the presence of 2 mM Mg(2+), both corresponding closely to direct binding of Ca(2+) to CLSP. In the presence of Ca(2+), human CLSP forms a high-affinity 1:1 complex with melittin, a natural peptide considered to be a model for the interaction of CaM with its targets. In the complex, CLSP binds Ca(2+) with high affinity to all four binding sites. Isolated N- and C-CLSP show only a weak interaction with melittin, which is enhanced when both halves are simultaneously presented to the model peptide.
Binding Sites↗
[Medical radiation physics of the "Wurzburg School"].
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Germany↗
Keratinocytes control the pheo/eumelanin ratio in cultured normal human melanocytes.
The pheo/eumelanin ratio of cultured normal human melanocytes is distinct from the ratio observed for the same cells in vivo where they are in close contact with keratinocytes. To study the possible involvement of keratinocytes in the control of melanogenesis, we compared quantitatively and qualitatively the melanin production in melanocyte mono-cultures, in melanocyte-keratinocyte co-cultures and in pigmented reconstructed epidermis. Pheomelanin and eumelanin contents were assessed by high-performance liquid chromatography with electrochemical and fluorometric detection of their specific degradation products and revealed striking differences in the presence of keratinocytes. In the absence of keratinocytes (melanocyte mono-cultures), we observed a very limited eumelanin production and a very high pheomelanin synthesis. The pheo/eumelanin ratio in mono-cultures could be slightly influenced by changing the composition of the culture medium, however, the very strong imbalance in favor of pheomelanin remained unchanged. An induction of eumelanin synthesis accompanied by an important reduction of pheomelanin formation was only observed in the presence of keratinocytes. The pheo/eumelanin ratio in melanocyte mono-culture dropped from 1043 down to about 25 in the presence of keratinocytes (co-cultures). The same observations were made when the melanocytes were integrated into a reconstructed human epidermis. Interestingly, under co-culture conditions resulting in only a partial contact between melanocytes and keratinocytes, the reduction of the pheo/eumelanin ratio were less pronounced. From these results we conclude that keratinocytes play an important role in the melanin production, affecting the melanogenic pathways.
Cell Communication↗