Tumour-cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma.

BACKGROUND: The incidence of epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. OBJECTIVES: To study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC. METHODS: The mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real-time polymerase chain reaction. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s. RESULTS: Significantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin. Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa-associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin. Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal-related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo. CONCLUSIONS: These results provide evidence for the role of tumour-cell-derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. What's already known about this topic? The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally. Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC. What does this study add? Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC. What is the translational message? Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC.

Early dental epithelial transcription factors distinguish ameloblastoma from keratocystic odontogenic tumor.

The aim of the study was to characterize the molecular relationship between ameloblastoma and keratocystic odontogenic tumor (KCOT) by means of a genome-wide expression analysis. Total RNA from 27 fresh tumor samples of 15 solid/multicystic intraosseous ameloblastomas and 12 sporadic KCOTs was hybridized on Affymetrix whole genome arrays. Hierarchical clustering separated ameloblastomas and KCOTs into 2 distinct groups. The gene set enrichment analysis based on 303 dental genes showed a similar separation of ameloblastomas and KCOTs. Early dental epithelial markers PITX2, MSX2, DLX2, RUNX1, and ISL1 were differentially overexpressed in ameloblastoma, indicating its dental identity. Also, PTHLH, a hormone involved in tooth eruption and invasive growth, was one of the most differentially upregulated genes in ameloblastoma. The most differentially overexpressed genes in KCOT were squamous epithelial differentiation markers SPRR1A, KRTDAP, and KRT4, as well as DSG1, a component of desmosomal cell-cell junctions. Additonally, the epithelial stem cell marker SOX2 was significantly upregulated in KCOT when compared with ameloblastoma. Taken together, the gene expression profile of ameloblastoma reflects differentiation from dental lamina toward the cap/bell stage of tooth development, as indicated by dental epithelium-specific transcription factors. In contrast, gene expression of KCOT indicates differentiation toward keratinocytes.

A role for caveolin-1 in desmoglein binding and desmosome dynamics.

Desmoglein-2 (Dsg2) is a desmosomal cadherin that is aberrantly expressed in human skin carcinomas. In addition to its well-known role in mediating intercellular desmosomal adhesion, Dsg2 regulates mitogenic signaling that may promote cancer development and progression. However, the mechanisms by which Dsg2 activates these signaling pathways and the relative contribution of its signaling and adhesion functions in tumor progression are poorly understood. In this study we show that Dsg2 associates with caveolin-1 (Cav-1), the major protein of specialized membrane microdomains called caveolae, which functions in both membrane protein turnover and intracellular signaling. Sequence analysis revealed that Dsg2 contains a putative Cav-1-binding motif. A permeable competing peptide resembling the Cav-1 scaffolding domain bound to Dsg2, disrupted normal Dsg2 staining and interfered with the integrity of epithelial sheets in vitro. Additionally, we observed that Dsg2 is proteolytically processed; resulting in a 95-kDa ectodomain shed product and a 65-kDa membrane-spanning fragment, the latter of which localizes to lipid rafts along with full-length Dsg2. Disruption of lipid rafts shifted Dsg2 to the non-raft fractions, leading to the accumulation of these proteins. Interestingly, Dsg2 proteolytic products are elevated in vivo in skin tumors from transgenic mice overexpressing Dsg2. Collectively, these data are consistent with the possibility that accumulation of truncated Dsg2 protein interferes with desmosome assembly and/or maintenance to disrupt cell-cell adhesion. Furthermore, the association of Dsg2 with Cav-1 may provide a mechanism for regulating mitogenic signaling and modulating the cell-surface presentation of an important adhesion molecule, both of which could contribute to malignant transformation and tumor progression.

Complement activation in tear fluid during occupational mold challenge.

INTRODUCTION: Indoor air quality is important in occupational healthcare when evaluating the health risks of a work environment. Components of the classical and alternative complement pathways are present in ocular tissues and fluids. The authors determined the levels of complement components C1INH, C3, and C4 in sera and C3a in tear fluids of normal persons and of those who were exposed to molds. METHODS: Nine patients environmentally exposed to molds and 6 controls were selected from the Indoor Air Clinic of the Skin and Allergy Hospital. Tear fluid samples were collected from patients during the exposure to molds and after 2 weeks without mold exposure. At the same time, conjunctival cytology samples were obtained from each patient. Tear fluid was taken from 6 control subjects. All had negative skin prick tests to common environmental allergens. RESULTS: In 4 patients subjective eye symptoms and tear fluid C3a levels decreased during 2 weeks of sick leave as did conjunctival eosinophils but other inflammatory cells were unchanged. CONCLUSION: Elevated complement C3a levels in tear fluids may be influenced by environmental exposure to molds. According to the authors' clinical experience, eosinophilia is not a consistent finding in patients exposed to molds. However, molds may cause eosinophilic inflammation in the eye.

Successful liver-kidney transplantation in two children with aHUS caused by a mutation in complement factor H.

A 12-month-old boy and his 16-year-old aunt became acutely ill 6 months apart and were diagnosed to have atypical hemolytic uremic syndrome (aHUS). Genetic analysis revealed heterozygous R1215Q mutation in complement factor H (CFH) in both patients. The same mutation was found in five healthy adult relatives indicating incomplete penetrance of the disease. The patients developed terminal renal failure and experienced reversible neurological symptoms in spite of plasma exchange (PE) therapy. In both cases, liver-kidney transplantation was successfully performed 6 months after the onset of the disease. To minimize complement activation and prevent thrombotic microangiopathy or overt thrombotic events due to the malfunctioning CFH, extensive PE with fresh frozen plasma was performed pre- and perioperatively and anticoagulation was started a few hours after the operation. No circulatory complications appeared and all four grafts started to function immediately. Also, no recurrence or other major clinical setbacks have appeared during the postoperative follow-up (15 and 9 months) and the grafts show excellent function. While more experience is needed, it seems that liver-kidney transplantation combined with pre- and perioperative PE is a rational option in the management of patients with aHUS caused by CFH mutation.

The effect of combining intermittent hemodiafiltration with forced alkaline diuresis on plasma myoglobin in rhabdomyolysis.

BACKGROUND: Our aim was to examine the effect of combining intermittent hemodiafiltration (HDF) with forced alkaline diuresis on plasma myoglobin in rhabdomyolysis. METHODS: This was a prospective, randomized, controlled, cross-over study. Sixteen rhabdomyolysis patients with plasma myoglobin concentrations above 10,000 microg/l were randomized. Forced alkaline diuresis was started immediately after allocation and continued throughout the study. HDF, which lasted for 4 h, was started in group A immediately after allocation and in group B 4 h later. The primary analysis was intention-to-treat by repeated measures analysis of variance and Mann-Whitney U-test. RESULTS: The percentage elimination of myoglobin from the circulation during HDF differed significantly from that during alkaline diuresis (28.1% vs. 14.2%, respectively; P < 0.01). The mean decrease in plasma myoglobin concentration during HDF [9731 microg/l; 95% confidence interval (CI), 3672-5345 microg/l] and forced alkaline diuresis (3646 microg/l; 95% CI, 1260-6032 microg/l) did not show a statistically significant difference (P= NS). The mean total amount of myoglobin found in the ultrafiltrate was 58.4 mg. CONCLUSION: The percentage myoglobin decrease during combined HDF and forced alkaline diuresis was higher than that during forced alkaline diuresis alone. Renal replacement therapy with filtration techniques may be considered for the clearance of myoglobin from plasma when urine alkalinization is not successful.

Tight junction components occludin, ZO-1, and claudin-1, -4 and -5 in active and healing psoriasis.

BACKGROUND: Cells of the granular layer are interconnected by tight junctions (TJs) in normal epidermis. The structural proteins of epidermal TJs include occludin, ZO-1, and claudin-1 and -4. OBJECTIVES: Our aim was to correlate the expression of TJ components with keratinocyte differentiation using psoriasis as a model of premature keratinization. METHODS: The distribution of TJ proteins was evaluated in the skin of nine patients with psoriasis. Punch biopsies were taken from perilesional skin, from active psoriasis plaques, and from healed, previously lesional locations. The punch biopsies were analysed using indirect immunolabelling for ZO-1, occludin and claudin-1, -4 and -5. In addition, epidermal samples were analysed by reverse transcription-polymerase chain reaction for claudin-1, -4 and -5 mRNAs. RESULTS: Claudin-5 was localized to the granular cell layers of normal control skin as well as perilesional and lesional psoriatic epidermis. This was unexpected, as previous studies have not detected claudin-5 in the epidermis. Occludin and ZO-1 were expressed in the granular cell layer in psoriatic perilesional epidermis. In the psoriasis plaques, ZO-1 and occludin were detected in a wider zone extending from the granular layer to the middle spinous cell layers. In healed psoriasis plaques, the expression of occludin and ZO-1 resumed a normal-looking profile, being restricted to the upper epidermis only. Claudin-1 and -4 did not show marked changes in psoriasis compared with normal skin. CONCLUSIONS: The results demonstrate claudin-5 in normal epidermis and psoriatic skin, and abnormal distribution of occludin and ZO-1 in psoriasis plaques. Clinical healing of aberrant keratinization is associated with restoration of the normal distribution of occludin, ZO-1 and also involucrin.

Keratinocytes cultured from patients with Hailey-Hailey disease and Darier disease display distinct patterns of calcium regulation.

BACKGROUND: Hailey-Hailey disease (HHD) (OMIM 16960) and Darier disease (DD) (OMIM 124200) are dominantly inherited acantholytic skin diseases, respectively, caused by mutations in the genes encoding the Golgi secretory pathway Ca2+-ATPase (SPCA1, ATP2C1) and the sarco/endoplasmic reticulum Ca2+-ATPase type 2 (SERCA2, ATP2A2) genes. OBJECTIVES: To investigate calcium regulation in keratinocytes cultured from patients with HHD and DD by measuring intracellular calcium resting levels and the cellular responses to ATP and thapsigargin. METHODS: The study was carried out using keratinocyte cultures established from four patients with HHD and four with DD. Calcium concentrations were measured with fluorescence ratio imaging using fura-2 loading. RESULTS: Control and HHD keratinocytes displayed approximately the same Ca2+ levels in resting phase, while DD keratinocytes showed elevated Ca2+ levels. Application of ATP caused less pronounced elevation of intracellular calcium concentration ([Ca2+]i) in both HHD and DD keratinocytes than in control cells. HHD keratinocytes did not lower their [Ca2+]i as efficiently as control keratinocytes after treatment with thapsigargin. In addition, DD keratinocytes were practically incapable of lowering their [Ca2+]i after treatment with thapsigargin. CONCLUSIONS: The results demonstrate that the defects in SPCA1 and SERCA2 calcium ATPases result in distinct patterns of calcium metabolism. This is also supported by the different clinical features of the diseases.

Evolution and predictive power of serum cystatin C in acute renal failure.

AIMS: The serum concentration of cystatin C has recently been proposed as a better indicator of glomerular filtration rate (GFR) than plasma creatinine. Little is known about cystatin C in critical illness. We assessed serum cystatin C as a marker of renal function in acute renal failure (ARF) and its power in predicting survival of ARF patients. MATERIAL: 202 consecutive adult patients admitted into the intensive care unit (ICU) during a period of 9 months. METHOD: Serum cystatin C, plasma creatinine and plasma urea were measured on admission, daily during the first 3 days, and 5-7 times a week during the rest of the ICU stay. The patients with and without ARF were compared by the Mann-Whitney U-test. The correlation between different variables was calculated by Spearman's correlation. Forward stepwise multiple regression analysis was performed to test independent predictors of mortality. The positive predictive value of serum cystatin C and plasma creatinine for ARF and mortality was calculated by ROC analysis. RESULTS: ARF occurred in 54 patients (27%). Serum cystatin C showed excellent positive predictive value for ARF in critical illness by ROC analysis. In acute renal dysfunction, abnormal values of serum cystatin C and plasma creatinine appeared equally quickly (median 3 days). The diagnosis of ARF, the day 1 Apache II score and admission plasma creatinine appeared as independent predictors of hospital mortality. ROC analysis showed only weak predictive power for serum cystatin C and plasma creatinine regarding hospital mortality. CONCLUSIONS: Serum cystatin C was as good as plasma creatinine in detecting ARF in intensive care patients. Neither marker was clinically useful in predicting mortality.

Drug release from starch-acetate microparticles and films with and without incorporated alpha-amylase.

Acetylation of starch considerably decreases its swelling and enzymatic degradation. Thus, starch-acetate (SA) based delivery systems may be suitable for controlled drug delivery. The aim of the present study was to evaluate drug release from the SA microparticles (SA mps) and SA films. The average degree of acetyl substitution (DS) per glucose residue in the starch was either 1.9 (SA DS 1.9) or 2.6 (SA DS 2.6). Timolol (mw 332), calcein (mw 623) and bovine serum albumin (BSA, mw 68,000) were used as model drugs. A continuous timolol release from the both SA mps was observed in phosphate buffer solution (PBS) pH 7.4 (50-days incubation). The release of timolol was faster from the SA DS 1.9 mps than from the SA DS 2.6 mps. Calcein release from both SA mps was continuous in PBS pH 7.4 (5-days incubation). But, calcein release profile from the SA DS 2.6 film in PBS pH 7.4 showed discontinuities. However, the release of calcein from both SA films was continuous in human serum in vitro during the 7-day incubation, i.e. enzymes enhanced calcein release. Thus, alpha-amylase was incorporated into the SA films in order to enhance drug release from the films. However, the effects of incorporation of alpha-amylase on the model macromolecule (BSA) release from the SA films were modest. In conclusion, this study demonstrates the achievement of slow release of different molecular weight model drugs from the SA mps and films as compared to fast release from the native starch preparations. DS of SA, physicochemical properties of a drug and the presence of enzymes can all affect drug release profiles from SA based preparations.

IgE-binding components of cultured human keratinocytes in atopic eczema/dermatitis syndrome and their crossreactivity with Malassezia furfur.

BACKGROUND: Atopic eczema/dermatitis syndrome (AEDS) patients display immunoglobulin E (IgE) reactivity to several antigens, e.g. saprophytic yeasts as Malassezia furfur. AEDS patients also show IgE autoreactivity towards cells of their own tissue including epidermis. PURPOSE OF THE STUDY: The aim of this study was to investigate the IgE autoreactivity of AEDS patients to cultured keratinocytes and to reveal potential crossreacting epitopes in cultured keratinocytes and M. furfur. MATERIAL AND METHODS: Serum samples of 27 AEDS patients were analyzed, of these 13 were M. furfur radioallergosorbent test (RAST) positive and 14 negative. Four urticaria, three psoriasis, and seven nonatopic patients were included as controls. The studies were performed by using IgE immunoblotting and immunoblotting inhibition methods. RESULTS: Ten IgE-binding protein bands were detected in cultured human keratinocytes by IgE immunoblotting using sera from adult AEDS patients. Anti-keratinocyte IgE antibodies were more associated with elevated S-IgE level than M. furfur RAST. Clear crossreactivity with M. furfur could not be shown. CONCLUSIONS: The possible pathomechanism of anti-keratinocyte IgE antibodies is not due to IgE epitope mimicry of saprophytic yeast and local tissue in AEDS skin.

Immunohistological distribution of the tight junction components ZO-1 and occludin in regenerating human epidermis.

BACKGROUND: Molecular characterization of tight junction proteins during the past few years has provided novel methods for studying these specialized junctions. Tight junctions have recently been characterized in the granular cell layer of human epidermis, and the role of these junctions in the epidermal barrier is now being re-evaluated. OBJECTIVES: To investigate the expression of tight junction components during the re-epithelialization of suction blisters and the regeneration of the corneal layer after tape stripping. METHODS: Suction blisters were induced in eight healthy volunteers, and skin biopsies were taken 4 or 6 days afterwards. The restoration of epidermal barrier function was evaluated by measuring water evaporation (WE) from the wound area. Tape stripping was performed on three volunteers to remove the corneal layer. The tissues were immunolabelled using indirect immunofluorescence or the avidin-biotin method. RESULTS: Prior to the biopsies, WE from the blister wounds was markedly elevated in comparison with normal skin. In the epidermis surrounding the blister, occludin and ZO-1 were expressed in the granular cell layer only. In the hyperproliferative zone adjacent to the border of the blister, the expression of ZO-1 was redistributed into several spinous cell layers, while occludin expression was restricted to the upper epidermis. In the leading edge of migrating keratinocytes, both proteins were expressed exclusively in the most superficial layer of keratinocytes. Double labelling for ZO-1 and involucrin showed expression of both proteins in the same layers of hyperproliferative keratinocytes, while the expression patterns were clearly different in the migrating keratinocytes. CONCLUSIONS: Tight junctions of regenerating epidermis may provide a functional barrier prior to regeneration of the corneal layer.

Plasma exchange in severe postpartum HELLP syndrome.

We present a case of extremely severe postpartum HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome, associated with activation of coagulation, massive recurrent intra-abdominal bleeding requiring two laparotomies, renal failure, and central nervous system symptoms. This case underlines the complexity of pregnancy-related thrombotic microangiopathies regarding their differential diagnosis, multiple organ dysfunction, as well as management. Systemic endothelial cell injury plays a central role in the pathogenesis of thrombotic microangiopathies. Treatment of HELLP syndrome usually consists of administration of antihypertensive therapy and magnesium sulfate. No consensus exists regarding the use of plasma exchange or corticosteroids. Plasma exchange was a major part of the treatment regime in our patient. Early plasma exchange may be considered as an adjuvant therapy in severe and progressive postpartum HELLP syndrome.

Epidermal tight junctions: ZO-1 and occludin are expressed in mature, developing, and affected skin and in vitro differentiating keratinocytes.

This study demonstrates the presence of tight junction antigens in adult and developing human epidermis. Indirect immunofluorescence labeling and immunoelectron microscopy with antibodies to ZO-1 and occludin localized tight junction components ZO-1 and occludin to a narrow zone of the granular cells of adult epidermis. Double immunolabeling for tight junction components with adherens junction or desmosome proteins suggested that occludin is more specific for tight junctions than ZO-1, which may also be associated with adherens junctions. In developing skin, tight junctions interconnected the peridermal cells, and after the fetal stratification localized to the granular cell layer. Immunolabeling of psoriasis, lichen planus, and ichthyosis vulgaris, representing aberrant differentiation of the epidermis, showed that these conditions were associated with relocation of ZO-1 and occludin to the spinous cells. Cultures of epidermal keratinocytes, which offer a useful model for the formation of cellular contacts, revealed that tight junction components, ZO-1 and occludin, displayed a marked degree of colocalization relatively late during the process when the fusion zone had assumed a linear appearance. This suggests that the formation of adherens junctions and desmosomes precedes that of tight junctions. We speculate that the epidermal barrier, isolating the human body from the external environment, is in part formed by tight junctions of stratum granulosum.

Expression profiles of cell-cell and cell-matrix junction proteins in developing human epidermis.

The spatial distribution of a panel of cell junction proteins was studied in developing human epidermis by confocal laser scanning microscopy. The results demonstrated that many of the cell junction proteins were expressed in early two-layered embryonic epidermis, but their subcellular distribution displayed marked changes during development. Specifically, desmosomal proteins, desmoplakin, desmocollin and desmoglein, adherens junction components, E-cadherin, alpha-catenin and vinculin, and an actin-binding protein alpha-actinin were expressed as early as 8 weeks of estimated gestational age (EGA). Type IV collagen and beta1 and beta4 integrins were also present. At this early developmental stage, the epidermis is known to comprise two layers of cells, the basal layer and the peridermal layer. In addition to being present in cell-cell contacts, desmosomal antigens and E-cadherin were unexpectedly localized to the basal aspect of basal cells in samples at 8 weeks. On the other hand, talin, which in adult skin is localized to the dermal-epidermal junction, could not be detected until 12 weeks. These results suggest that the separation of cell membranes to the basal and apicolateral compartments does not occur before the maturation of the basement membrane zone. At 8 weeks EGA, gap junction antigen connexin 43 was expressed in scarce spots in cell-cell contacts of basal cells. In samples of 11-21 weeks EGA, the density of desmosomal and adherens junction markers as well as connexin 43 increased in cell-cell junctions, together with the appearance of the intermediate cell layer and beginning of stratification of the epidermis.

Catalytically active iron and bacterial growth in serum of haemodialysis patients after i.v. iron-saccharate administration.

BACKGROUND: I.v. iron is commonly administered to haemodialysis patients suffering from anaemia to improve their response to erythropoietin therapy. It has been unclear whether routinely used doses of i.v. iron preparations could result in iron release into plasma in amounts exceeding the iron binding capacity of transferrin. Here, we have studied the effect of 100 mg of iron saccharate given as an i.v. injection on transferrin saturation and the appearance of potentially harmful catalytically active iron. METHODS: We followed serum iron, transferrin and transferrin-saturation before and 5-210 min after administration of iron saccharate in 12 patients on chronic haemodialysis due to end-stage renal disease. We measured catalytically active iron by the bleomycin-detectable iron (BDI) assay and transferrin iron forms by urea gel electrophoresis, and studied iron-dependent growth of Staphylococcus epidermidis inoculated into the serum samples in vitro. RESULTS: The iron saccharate injection resulted in full transferrin saturation and appearance of BDI in the serum in seven out of the 12 patients. BDI appeared more often in patients with a low serum transferrin concentration, but it was not possible to identify patients at risk based on serum transferrin or ferritin level before i.v. iron. The average transferrin saturation and BDI level increased until the end of the follow-up time of 3.5 h. The appearance of BDI resulted in loss of the ability of patient serum to resist the growth of S. epidermidis, which was restored by adding iron-free apotransferrin to the serum. Iron saccharate, added to serum in vitro, released only little iron and promoted only slow bacterial growth, but caused falsely high transferrin saturation by one routinely used serum iron assay. CONCLUSIONS: The results indicate that 100 mg of iron saccharate often leads to transferrin oversaturation and the presence of catalytically active iron within 3.5 h after i.v. injection. As catalytically active iron is potentially toxic and may promote bacterial growth, it may be recommendable to use dosage regimens for i.v. iron that would not cause transferrin oversaturation.

New function for NF1 tumor suppressor.

The expression and subcellular localization of neurofibromatosis type 1 tumor suppressor was studied in keratinocytes induced to differentiate by increased Ca2+ concentration of the culture medium. Differentiating keratinocytes became intensely immunoreactive for neurofibromatosis type 1 protein, which was apparently associated with cellular fibrils. Double immunolabeling with antibodies to cytokeratin 14 and neurofibromatosis type 1 protein suggested an association of intermediate type cytoskeleton and neurofibromatosis type 1 protein. The presence of neurofibromatosis type 1 protein in cell preparations treated with cytoskeletal buffer indicated a high affinity interaction between intermediate filaments and neurofibromatosis type 1 protein. Further studies utilizing double immunolabelings revealed that the intense neurofibromatosis type 1 tumor suppressor signal on intermediate filaments was temporally limited to the period in keratinocyte differentiation in which the formation of desmosomes takes place. Keratinocytes were also cultured from nine patients with type 1 neurofibromatosis and were studied with respect to cell morphology, and association of neurofibromatosis type 1 protein with intermediate cytoskeleton. The results showed that keratinocytes cultured from patients with neurofibromatosis type 1 displayed a highly variable cell size and morphology compared to controls. The latter findings represent predicted alterations in a situation where cytoskeletal organization is disturbed. Furthermore, differentiating neurofibromatosis type 1 keratinocytes were characterized by a reduced number of cytokeratin bundles that were decorated neurofibromatosis type 1 protein. The results of this study suggest that neurofibromatosis type 1 tumor suppressor exerts its effects in part by controlling organization of cytoskeleton during the formation of cellular contacts.

A novel component of epidermal cell-matrix and cell-cell contacts: transmembrane protein type XIII collagen.

Type XIII collagen is a short chain collagen which has recently been shown to be a transmembrane protein. The purpose of this study was to elucidate the presence and localization of type XIII collagen in normal human skin and cultured keratinocytes. Expression of type XIII collagen was demonstrated in normal human skin and epidermis at the RNA level using reverse transcription followed by polymerase chain reaction and at the protein level using western blotting and indirect immunofluorescence labeling. Immunolabeling of epidermis revealed type XIII collagen both in the cell-cell contact sites and in the dermal-epidermal junction. In cultured keratinocytes type XIII collagen epitopes were detected in focal contacts and in intercellular contacts. The results of this study show very little colocalization of type XIII collagen and desmosomal components at the light microscopic level. Thus, these results suggest that type XIII collagen is unlikely to be a component of desmosomes. Instead, the punctate labeling pattern of type XIII collagen at the cell-cell contact sites and high degree of colocalization with E-cadherin suggests that type XIII collagen is very likely to be closely associated with adherens type junctions, and may, in fact, be a component of these junctions.