TROPHY registry - status report.

INTRODUCTION: The TROPHY registry has been established to conduct an international multicenter prospective data collection on the surgical management of neonatal intraventricular hemorrhage (IVH)-related hydrocephalus to possibly contribute to future guidelines. The registry allows comparing the techniques established to treat hydrocephalus, such as external ventricular drainage (EVD), ventricular access device (VAD), ventricular subgaleal shunt (VSGS), and neuroendoscopic lavage (NEL). This first status report of the registry presents the results of the standard of care survey of participating centers assessed upon online registration. METHODS: On the standard of treatment forms, each center indicated the institutional protocol of interventions performed for neonatal post-hemorrhagic hydrocephalus (nPHH) for a time period of 2 years (Y1 and Y2) before starting the active participation in the registry. In addition, the amount of patients enrolled so far and allocated to a treatment approach are reported. RESULTS: According to the standard of treatment forms completed by 56 registered centers, fewer EVDs (Y1 55% Y2 46%) were used while more centers have implemented NEL (Y1 39%; Y2 52%) to treat nPHH. VAD (Y1 66%; Y2 66%) and VSGS (Y1 42%; Y2 41%) were used at a consistent rate during the 2 years. The majority of the centers used at least two different techniques to treat nPHH (43%), while 27% used only one technique, 21% used three, and 7% used even four different techniques. Patient data of 110 infants treated surgically between 9/2018 and 2/2021 (13% EVD, 15% VAD, 30% VSGS, and 43% NEL) were contributed by 29 centers. CONCLUSIONS: Our results emphasize the varying strategies used for the treatment of nPHH. The international TROPHY registry has entered into a phase of growing patient recruitment. Further evaluation will be performed and published according to the registry protocol.

[Renaissance of mouse favus : Retrospective analysis of Trichophyton quinckeanum infections at Jena University Hospital in the period 2015-2020]. / Renaissance des Mäusefavus : Retrospektive Analyse der Trichophyton-quinckeanum-Infektionen am Universitätsklinikum Jena im Zeitraum 2015 bis 2020.

The number of Trichophyton quinckeanum infections has increased significantly in recent years. In 2020 in particular, the number of cases increased fivefold compared to 2015. Infections multiplied, especially in the second half of the year, which correlated with the upsurge in field mouse populations. Typical vectors are mice and rats as well as dogs and cats, which hunt the rodents. The animals are usually asymptomatic. In humans, on the other hand, the course is usually more inflammatory corresponding to other zoophilic mycoses. Typical clinical manifestations of the infections are tinea corporis and tinea capitis. Treatment of T. quinckeanum infections is similar to other dermatophyte infections, depending on the severity, location and age of the patient as well as the immune status, previous illnesses and medication. The duration of local therapy should be at least 4 weeks and continued for up to 14 days after the normalization of the skin presentation. Systemic treatment should take place with terbinafine 250 mg once a day orally (in adults). Alternatives are itraconazole, fluconazole and griseofulvin. Only the preparation griseofulvin, which is no longer available in Germany, is approved for children. Alternatively, terbinafine, itraconazole or fluconazole can also be used in children as an "off-label" treatment in an individual healing attempt.

Mating analyses of Trichophyton benhamiae offspring reveals linkage of genetic markers used in taxonomy.

Mating experiments were conducted with four clinical Trichophyton benhamiae isolates, genetically similar to the Trichophyton benhamiae CBS 112371, featuring the plus mating type and with two minus type strains. One minus type strain belonged to the white subgroup, and the other minus type strain, DSM 6916, showed genetic kinship to the yellow subgroup. Only two plus type strains were able to form mature, pigmented gymnothecia with DSM 6916. These two plus type strains demonstrated dark pigmentation and powdery mycelium on Takashio agar, whereas the other three strains exhibited a low degree of pigmentation on the same medium. All five plus strains were able to mate with the minus type strain of their own white subgroup. Cultures from single ascospore isolates showed highly variable morphology and pigmentation. Three genetic markers (ITS, mating type, EF1 alpha) were analyzed in polymerase chain reaction (PCR) experiments with optimized primers and PCR conditions to discriminate between subgroups. Furthermore, RAPD-PCR was used to generate a DSM 6916-specific DNA-fragment which served as an additional genetic marker. Assessing the isolates with recombinant genotypes, it was found that three genetic markers behave like linked genes. The recombination of plus mating type went together with ITS, EF1 alpha and RAPD marker of the DSM 6916 parental strain and was most frequently isolated, whereas plus types recombinants in this case were completely missing. This shows a high imbalance in mating type distribution of recombinants.

Innate immune response of human epidermal keratinocytes and dermal fibroblasts to in vitro incubation of Trichophyton benhamiae DSM 6916.

BACKGROUND: Superficial cutaneous infection caused by the zoophilic dermatophyte Trichophyton benhamiae is often associated with a highly inflammatory immune response. As non-professional immune cells, epidermal keratinocytes and dermal fibroblasts contribute to the first line of defence by producing pro-inflammatory cytokines and antimicrobial peptides (AMP). OBJECTIVE: Purpose of this study was to gain a deeper understanding of the pathogenesis and the fungal-host interaction as not much is known about the innate immune response of these cutaneous cells against T. benhamiae. METHODS: Using a dermatophytosis model of fibroblasts and keratinocytes incubated with T. benhamiae DSM 6916, analyses included determination of cell viability and cytotoxicity, effects on the innate immune response including expression and secretion of pro-inflammatory cytokines/chemokines and expression of AMP, as well as alterations of genes involved in cell adhesion. RESULTS: Trichophyton benhamiae DSM 6916 infection led to severe cell damage and direct induction of a broad spectrum of pro-inflammatory cytokines and chemokines in both cutaneous cells. Only keratinocytes differentially up-regulated AMP genes expression after T. benhamiae DSM 6916 infection. Expression of AMPs in fibroblasts was not inducible by fungal infection, whereas their absences potentially contributed to a continuous increase in the fungal biomass on fibroblasts, which in turn was reduced in keratinocytes possibly due to the antimicrobial actions of induced AMPs. On mRNA level, T. benhamiae DSM 6916 infection altered cell-cell contact proteins in keratinocytes, indicating that targeting specific cell-cell adhesion proteins might be part of dermatophytes' virulence strategy. CONCLUSION: This study showed that in addition to immune cells, keratinocytes and fibroblasts could participate in antimicrobial defence against an exemplary infection with T. benhamiae DSM 6916.

[Dermatophytosis caused by rare anthropophilic and zoophilic agents]. / Dermatophytosen, verursacht durch seltene anthropophile und zoophile Erreger.

The basis for effective treatment of any dermatomycosis is the correct and timely identification of the pathogen, which allows the targeted choice of the most suitable antimycotic and is important for the prevention of repeated infections. In recent years, infections with dermatophytes seem to have increased. In fact, from 2007 to 2018, there was an increase in the number of samples processed in the Mycology Laboratory of the Department of Dermatology at the University Hospital Jena. The most common isolated dermatophytes between 2007 and 2018 were Trichophyton (T.) rubrum, T. interdigitale, Microsporum (M.) canis and T. benhamiae. However, dermatophytoses may also be caused by rare anthropophilic agents such as Epidermophyton floccosum, zoophiles such as T. verrucosum, T. quinckeanum or Nannizzia (N.) persicolor as well as by geophiles such as N. gypsea. Therefore, these dermatophytes should at least be known, so that in case of unusual observations investigations can be performed accordingly. Changes in the pathogen spectrum of dermatophytoses have taken place over time and it is expected that the occurrence of dermatophytes will be subject of continuous fluctuations, which may mean that the incidence of some of these "rare" dermatophytes, as described here in five clinical examples, may be changing.

CALCIUM HYDROXYLAPATITE MICROSPHERES - BIOCOMPATIBILITY AND CLINICAL EFFECTS.

Soft tissue fillers are used in aesthetic medicine for volumizing and for contouring. Calcium hydroxylapatite (CaHA) is a fully biodegradable biostimulatory filler. The study presents results of in vitro biocompatibility and cytotoxicity investigations performed on CaHA and illustrates its clinical effects. We used a human cell culture model for in vitro studies with HaCaT keratinocytes and human dermal fibroblasts, known to be a sensitive cell type for biocompatibility and cytotoxicity. Cells were exposed to different concentrations of CaHA (Radiesse®). Cell proliferation was calculated by luminometric adenosine triphosphate (ATP) measurement using the ATPLiteTM-M Assay. Possible cytotoxic effects were detected by the calorimetric Cytotoxicity Detection Kit. Clinical data were obtained from our own treatment files. CaHA did neither inhibit cell proliferation nor cause cytotoxicity. Clinical data suggest an excellent tolerability and long-term efficacy superior to hyaluronic acid-based fillers. CaHA is a versatile and well-tolerated biodegradable and biostimulatory filler.

Cold atmospheric pressure plasmas exhibit antimicrobial properties against critical bacteria and yeast species.

OBJECTIVE: Cold atmospheric pressure plasmas (CAPPs) have been used to sterilise implant materials and other thermally unstable medical products and to modify chemical surfaces. This study investigates the antimicrobial effect of the gas and input power used to generate CAPPs on microorganisms causing skin infections, such as Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Malassezia pachydermatis. METHOD: Microorganisms were cultivated on Mueller Hinton 2 (MH2) agar plates. CAPP treatment was performed using the Plasma BLASTER MEF. To investigate the antimicrobial effects the following CAPP parameters were varied: the gas used, input power, as well as number of treatments and treatment time. RESULTS: The antimicrobial efficacy of the CAPPs was found to increase with increasing input power and treatment time (or cycles). Furthermore the plasma generated from nitrogen is more effective than from air. CONCLUSION: The study showed that CAPPs demonstrate strong bactericidal and fungicidal properties in vitro. The selective application of CAPPs for the treatment of wound infections may offer a promising supplementary tool alongside current therapies.

A novel native collagen dressing with advantageous properties to promote physiological wound healing.

OBJECTIVE: Chronic hard-to-heal wounds generate high costs and resource use in western health systems and are the focus of intense efforts to improve healing outcomes. Here, we introduce a novel native collagen (90 %):alginate (10 %) wound dressing and compare it with the established oxidised dressings Method: Matrices were analysed by atomic force microscopy (AMF), scanning electron microscopy (SEM), and immunoelectron microscopy for collagen types I, III and V. Viability assays were performed with NIH-3T3 fibroblasts. Matrix metalloproteinase (MMP) binding was analysed, and the effect of the wound dressings on platelet-derived growth factor B homodimer (PDGF-BB) was investigated. RESULTS: Unlike oxidised regenerated cellulose (ORC)/collagen matrix and ovine forestomach matrix (OFM), the three-dimensional structure of the native collagen matrix (NCM) was found to be analogous to intact, native, dermal collagen. Fibroblasts seeded on the NCM showed exponential growth whereas in ORC/collagen matrix or OFM, very low rates of proliferation were observed after 7 days. MMP sequestration was effective and significant in the NCM. In addition, the NCM was able to significantly stabilise PDGF-BB in vitro. CONCLUSION: We hypothesise that the observed microstructure of the NCM allows for an effective binding of MMPs and a stabilisation and protection of growth factors and also promotes the ingrowth of dermal fibroblasts, potentially supporting the re commencement of healing in previously recalcitrant wounds. DECLARATION OF INTEREST: This work was supported by BSN Medical, Hamburg, Germany.

[Trichophyton species of Arthroderma benhamiae : Clinical therapeutic aspects of a new pathogen in dermatology]. / Trichophyton Spezies von Arthroderma benhamiae : Klinisch therapeutische Aspekte eines neuen Erregers in der Dermatologie.

Cutaneous infections with Trichophyton species of Arthroderma (A.) benhamiae are increasingly being detected in Germany. This dermatophyte typically causes tinea corporis, tinea faciei or tinea capitis with in part heavy clinical manifestation like kerion celsi. In special cases diagnosis and therapy can be difficult. In this article, four clinical cases are presented, whereby attention is given to special clinical situations and therapeutic aspects with regard to Trichophyton species of A. benhamiae: Case 1: Kerion celsi by in a 6-year-old boy; Case 2: Deep trichophytia at the mons pubis in a 32-year-old man working in a pet shop and his 27-year-old female partner; Case 3: Tinea manuum in a 7-year-old girl; Case 4: Tinea corporis in an 8­year-old girl.

[Trichophyton violaceum : Main cause of tinea capitis in children at Mbarara Regional Referral Hospital in Uganda]. / Trichophyton violaceum : Haupterreger der Tinea capitis bei Kindern im Mbarara Regional Referral Hospital in Uganda.

BACKGROUND: Tinea capitis is caused by anthropophilic, zoophilic or geophilic dermatophytes of the genera Microsporum or Trichophyton. OBJECTIVE: The aim of this study was to analyze the clinical presentation of tinea capitis among children in western Uganda. PATIENTS AND METHODS: From February to June 2012, skin and hair samples were obtained from 115 patients aged from 1 to 16 years presenting at Mbarara Regional Referral Hospital (MUSC) with clinically suspected tinea capitis. Conventional mycological diagnostics comprised Blancophor preparation and cultivation of fungi for species identification. RESULTS: Tinea capitis among the children included in the MUSC study was mainly noninflammatory showing mostly a seborrhoeic pattern or "black dot" and "gray patch" form and highly inflammatory kerion celsi. Blancophor preparation identified 82.6 % positive and 17.4 % negative samples. Cultural species differentiation showed Trichophyton (T.) violaceum as the causative agent for tinea capitis in 56.6 % of the patients. In 13 %, Microsporum (M.) audouinii was isolated followed by T. soudanense (2.6 %), and T. rubrum (1.7 %). In addition, moulds (contamination?) such as Scopulariopsis brevicaulis, Aspergillus niger, and Fusarium oxysporum were found as well as mixed infections. CONCLUSION: The anthropophilic dermatophyte T. violaceum represents the most frequent cause of tinea capitis in western Uganda. For successful management oral antifungal therapy is necessary together with supportive topical treatment.

Antimicrobial impact of cold atmospheric pressure plasma on medical critical yeasts and bacteria cultures.

OBJECTIVES: Plasma medicine focuses on the application of cold atmospheric pressure plasmas (CAPs) in or on the human body. So far, plasmas have been used to sterilize implant materials or other thermally unstable medical products and have been applied for chemical surface modifications. This study investigates the antimicrobial effect of physical plasmas on microorganisms which cause skin infections, such as Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, depending on the plasma source and the kind of plasma excitation used. MATERIALS: Microorganisms were plated onto MH2 agar plates. Plasma treatment was performed using the plasma sources BLASTER MEF and kinpen 09. To investigate the antimicrobial effects, the following plasma parameters have been varied: working gas, distance from nozzle to surface, electrical power, grid spacing of treatment lines, number of treatments and work piece velocity. RESULTS: The generated plasmas had an antimicrobial effect that depended on the chosen plasma parameters, in particular on the process gas used, the plasma power and the number of treatments performed. Thus, different reactive species were observed by optical emission spectroscopy measurement in the generated plasmas. CONCLUSIONS: The study showed that CAPs exhibit profound bactericidal and fungicidal properties in vitro. However, an important factor for the antimicrobial efficacy is the composition of the 'chemical soup' supplied by the CAP system which can be regulated by the process gases used.

Binding and inhibition of protease enzymes, including MMPs, by a superabsorbent dressing in vitro.

OBJECTIVE: To demonstrate the binding and inactivation action of a superabsorbent dressing on proteolytic enzymes MMP-2, MMP-9 and collagenase using an established methodology. METHOD: An in vitro assay of MMP binding and collagenase inactivation has been conducted using the superabsorbent wound dressing (Eclypse; Advancis Medical UK). Dressings in this category, and other absorbents, have been claimed to possess MMP-binding characteristics; however, for most there is no published evidence as yet. In this series of experiments, we have used validated experimental techniques to evaluate such activity. RESULTS: Results show that the superabsorbent dressing does have a statistically-significant effect in binding two of the most important MMPs, MMP-2 and MMP-9, as well as inhibiting collagenase. CONCLUSION: These results support this activity for the superabsorbent dressing and indicate a probable beneficial clinical action in reducing the influence of these enzymes in delayed wound healing.

In vitro studies on the beneficial effect of a hydrokinetic fiber dressing on wound healing by reduction of protease activity.

OBJECTIVE: To test the binding capacity of a hydrokinetic fiber dressing for PMN elastase, MMP-2 and MMP-9 in vitro, and to determine whether testing of dressing material samples in vitro is sufficient to predict the performance of the whole dressing. METHOD: In vitro protease binding assays for PMN elastase, MMP-2 and MMP-9 were used to evaluate the protease modulating capacity of sorbion sachet EXTRA, a superabsorbant dressing that consists of special hydrokinetic fibers, which are formed from cellulose and sodium polyacrylate in a mechanical process without any bonding agents or adhesives. The ability of the hydrokinetic fiber dressing to inhibit elastase and collagenase activity was also tested at 0%, 50% and 100% saturation volume. RESULTS: The hydrokinetic fiber wound dressing was able to bind considerable amounts of elastase, reducing elastase activity by approximately 84%. Moreover, it significantly decreased MMP-2 and MMP-9 concentrations in vitro and was able to completely inhibit collagenase activity. CONCLUSION: In summary, the hydrokinetic fiber dressing sorbion sachet EXTRA was able to significantly reduce the concentration and activity of proteolytic enzymes in vitro. These results suggest that sorbion sachet EXTRA should have a beneficial action by reducing the detrimental effects of proteolytic enzymes in vivo. DECLARATION OF INTEREST: This work was supported by Sorbion GmbH & Co. KG, Senden, Germany.

SAP-containing dressings exhibit sustained antimicrobial effects over 7 days in vitro.

OBJECTIVE: To investigate the antimicrobial activity of SAP-containing wound dressings in vitro over a prolonged period of time (7 days) and to assess their ability to sustain the antimicrobial effect. METHOD: SAP dressings were tested according to the JIS L 1902:2002 against the pathogens Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Candida albicans.Additionally, effect on S. aureus and P. aeruginosa growth was investigated after a prolonged incubation period of 7 days. Furthermore, both SAP dressings were repeatedly inoculated with P. aeruginosa suspension and, after 7 days, microbial growth under the dressings was evaluated. RESULTS: Both SAP-containing wound dressings tested exhibited a significant to strong antimicrobial activity against Staphylococcus aureus, MRSA, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Candida albicans in vitro. Moreover, it could be shown that they were able to sustain antibacterial efficacy over a prolonged period of time. Using a direct incubation method with repeated re-inoculation of the dressing samples, it could be shown that growth of P. aeruginosa was reduced after 4 days of treatment and completely inhibited after 7 days. No significant differences were observed between the two SAP-dressings tested. CONCLUSION: These in vitro experiments impressively demonstrated the antimicrobial mechanism of SAP-containing wound dressings: rapid up-take of fluid, binding of microorganisms to the SAP-core, and retention of the bacteria inside the dressing. Moreover, it could be shown that they are able to exhibit their antimicrobial activity over a prolonged period of time unless the amount of fluid present exceeds their fluid-handling capacity.

Analysis of the adaptation capacity of Staphylococcus aureus to commonly used antiseptics by microplate laser nephelometry.

BACKGROUND: Bacterial colonization and infection are important factors in compromised wound healing. Antiseptics have become an alternative for antimicrobial applications as antibiotic resistance is increasing; they have multiple targets with a broad spectrum of activity. Hence, the risk for developing resistance should be low. However, concerns have been raised that their growing use may result in bacteria that are less susceptible. METHODS: The capacity of common antiseptics such as silver nitrate, polihexanide, octenidine, chlorhexidine and polyvinylpyrrolidone (PVP)-iodine to induce adaptation in a Staphylococcus aureus strain was analyzed in vitro using microplate laser nephelometry. S. aureus was repeatedly incubated with the respective half maximal inhibitory concentration (IC(50)) over a time period of 100 days. The influence of the continued treatment was determined by in situ monitoring of changes in the dose-response curves and calculation of the current IC(50) values for the substances tested. RESULTS: During the experiment, S. aureus quickly adapted to high concentrations of the antibiotic mupirocin during repeated treatment. Moreover, a significant increase of the IC(50) for silver nitrate was observed over time. On the other hand, no significant difference was observed for polihexanide or chlorhexidine. While the IC(50) for octenidine was also found to increase significantly, although the change was only marginal, reiterated incubation with PVP-iodine led to a decrease in the IC(50). CONCLUSION: Repeated treatment of S. aureus with polihexanide, chlorhexidine, octenidine and PVP-iodine did not trigger bacterial adaptation to these substances.

Typha capensis (Rohrb.)N.E.Br. (bulrush) extract scavenges free radicals, inhibits collagenase activity and affects human sperm motility and mitochondrial membrane potential in vitro: a pilot study.

The biodiversity in South Africa provides more than 30,000 higher plants, of which more than 3000 are used by traditional healers to treat diseases. Typha capensis (bulrush) is one of the medicinal plants used in South Africa to treat male fertility problems. Considering that South African traditional healers have been recognised by Law and the health benefits of T. capensis have not been scientifically investigated yet, this study aimed at investigating the in vitro effects of aqueous extracts from this plant on male reproductive functions. Both leaves and rhizomes of T. capensis were dried, infused with distilled water and freeze-dried. Motile sperm from 50 men were isolated by swim-up and incubated with 1 µg ml(-1) aqueous extract of Typha rhizome for 1 h at 37 °C. Vitality, motility, sperm production of reactive oxygen species and mitochondrial membrane potential were analysed in the test sample, a control and in the pellet from the swim-up. Results showed that the rhizome extract had significant (P < 0.0001) negative effects on all parameters. The extracts from the leaves and rhizomes revealed dose-dependent inhibitory activity for collagenase and free radical formation. No inhibitory activity for elastase was found. The inhibitory activity for collagenase might indicate possible anti-cancer effects.

Superabsorbent polymer-containing wound dressings have a beneficial effect on wound healing by reducing PMN elastase concentration and inhibiting microbial growth.

A comprehensive in vitro approach was used to assess the effects of superabsorbent polymer (SAP) containing wound dressings in treatment of non-healing wounds. A slight negative effect on HaCaT cells was noted in vitro which is most likely due to the Ca(2+) deprivation of the medium by binding to the SAP. It could be shown that SAP wound dressings are able to bind considerable amounts of elastase reducing enzyme activity significantly. Furthermore, SAP's inhibit the formation of free radicals. The SAP-containing wound dressings tested also exhibited a significant to strong antimicrobial activity effectively impeding the growth of gram-negative and gram-positive bacteria as well as yeasts. In conclusion, in vitro data confirm the positive effect of SAP wound dressings observed in vivo and suggest that they should be specifically useful for wound cleansing.

Redox Enzymes of the Thioredoxin Family as Potential and Novel Markers in Pemphigus.

Pemphigus vulgaris (PV) is a severe autoimmune blistering disease affecting both skin and mucous membranes. Its pathogenesis is related to IgG autoantibodies primarily targeting the cellular adhesion protein desmoglein (Dsg) 3, one of the major desmosome components. Impaired redox regulation is considered a major player in the pathogenesis of autoimmune diseases such as pemphigus by enhancing inflammation and breakdown of immunological tolerance by structural protein modifications. Despite many recent advances, local and systemic redox profiles that characterize the immune response in pemphigus are virtually unknown but potentially crucial in further advancing our understanding of redox-dependent modifications that eventually lead to clinical manifestation. Here, we have analyzed the individual expression pattern of four major redox enzymes that are members of the thioredoxin (Trx) fold superfamily (peroxiredoxins (Prxs) 1 and 4, glutaredoxin (Grx) 2, and Trx1) in serum and PBMCs as well as their distribution in the skin of pemphigus patients compared to healthy controls. We show that in groups of five pemphigus patients, Prx1 is upregulated in both serum and PBMCs, while its epithelial distribution remains within the spinous epithelial layer. Expression of Grx2 and Prx4 is both reduced in serum and PBMCs, while their distinct and similar expression in the skin changes from an even distribution throughout the basal layer (healthy) to ubiquitous nuclear localization in pemphigus patients. In PV patients, Trx1 is secreted into serum, and cellular distribution appears membrane-bound and cytosolic compared to healthy controls. We furthermore showed that a 3D ex vivo human skin model can indeed be used to reproduce similar changes in the protein levels and distribution of redox enzymes by application of cold atmospheric plasma. Deciphering the relationship between redox enzyme expression and autoimmunity in the context of pemphigus could be critical in elucidating key pathogenic mechanisms and developing novel interventions for clinical management.

Both copy number and sequence variations affect expression of human DEFB4.

Copy number variations (CNVs) were found to contribute massively to the variability of genomes. One of the best studied CNV region is the beta-defensin cluster (DEFB) on 8p23.1. Individual DEFFB copy numbers (CNs) between 2 and 12 were found, whereas low CNs predispose for Crohn's disease. A further level of complexity is represented by sequence variations between copies (multisite variations, MSVs). To address the relation of DEFB CN and MSV to the expression of beta-defensin genes, we analyzed DEFB4 expression in B-lymphoblastoid cell lines (LCLs) and primary keratinocytes (normal human epidermal keratinocyte, NHEK) before and after stimulation with lipopolysaccharide, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Moreover, we quantified one DEFB4 MSV in DNA and mRNA as a marker for variant-specific expression (VSE) and resequenced a region of approximately 2 kb upstream of DEFB4 in LCLs. We found a strong correlation of DEFB CN and DEFB4 expression in 16 LCLs, although several LCLs with very different CNs exhibit similar expression levels. Quantification of the MSV revealed VSE with consistently lower expression of one variant. Costimulation of NHEKs with TNF-alpha/IFN-gamma leads to a synergistic increase in total DEFB4 expression and suppresses VSE. Analysis of the DEFB4 promoter region showed remarkably high density of sequence variabilities (approximately 1 MSV/41 bp).

Molecular-weight-dependent toxic effects of chitosans on the human keratinocyte cell line HaCaT.

The cationic polysaccharide chitosan possesses bioactive properties such as antimicrobial activity, antitumor effects, hemostatic assets and positive effects on wound healing. The influence of polycations like chitosan on human cells has been reported to depend on their molecular weight. However, the mechanism of cytotoxicity caused by polycations is not yet fully understood. In the study presented, the influence of two chitosans with a similar degree of deacetylation but different molecular weight, chitosan 1130 (120 kDa) and chitosan oligosaccharide (5 kDa), on the human keratinocyte cell line HaCaT was analyzed. The results obtained indicate that chitosans exhibit a molecular-weight-dependent negative effect on HaCaT cell viability and proliferation in vitro. The chitosans tested also stimulated the release of inflammatory cytokines by HaCaT cells depending on incubation time and concentration. Chitosan 1130 and chitosan oligosaccharide induced apoptotic cell death which was mediated by activation of the effector caspases 3/7. At least for chitosan 1130, the involvement of both, extrinsic and intrinsic signal pathways, was shown by activation of caspases 8 and 9.