Uta-Christina Hipler

Identification of dermatophyte species causing onychomycosis and tinea pedis by MALDI-TOF mass spectrometry

Abstract:  Identification of dermatophytes is currently performed based on morphological criteria and is increasingly supported by genomic sequence comparison. The present study evaluates an alternative based on the analysis of clinical fungal isolates by mass spectrometry. Samples originating from skin and nail were characterized morphologically and by sequencing the internal transcribed spacer 1 (ITS1), ITS2 and the 5.8S rDNA regions of the rDNA clusters. In a blind comparative study, samples were analyzed by matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF MS). The mass spectra were compared to a database comprising of the spectral data of reference strains by applying the saramis software package. All fungal isolates belonging to the taxa Trichophyton rubrum, T. interdigitale, T. tonsurans, Arthroderma benhamiae and Microsporum canis were correctly identified, irrespective of host origin and pathology. To test the robustness of the approach, four isolates were grown on five different media and analyzed. Although the resulting mass spectra varied in detail, a sufficient number of signals were conserved resulting in data sets exploitable for unequivocal species identification. Taken together, the usually widespread dermatophytes can be identified rapidly and reliably by mass spectrometry. Starting from pure cultures, MALDI‐TOF MS analysis uses very simple sample preparation procedures, and a single analysis is performed within minutes. Costs for consumables as well as preparation time are considerably lower than for PCR analysis.

Comparative in vitro study on cytotoxicity, antimicrobial activity, and binding capacity for pathophysiological factors in chronic wounds of alginate and silver-containing alginate

Chronic wounds contain elevated levels of proteases, proinflammatory cytokines, and free radicals. The presence of bacteria further exaggerates the tissue‐damaging processes. For successful treatment, the wound dressing needs to manage wound exudates, create a moist environment, inhibit infection, bind pathophysiological factors that are detrimental to wound healing, and provide thermal isolation. Furthermore, it has to relieve pain, be easy to use, show no allergic potency, and not release toxic residues. The present study suggests a comprehensive in vitro approach to enable the assessment of wound dressings to support optimal conditions for wound healing. Three alginate‐based wound dressings: alginate alone, alginate containing ionic silver, and alginate with nanocrystalline silver, were tested for biocompatibility, antimicrobial activity, and influence on chronic wound parameters such as elastase, matrix metalloproteases‐2, tumor necrosis factor‐α, interleukin‐8, and free radical formation. Alginate was found to bind considerable amounts of elastase, reduce the concentration of proinflammatory cytokines and inhibit the formation of free radicals. Furthermore, alginate showed antibacterial activity and high biocompatibility. Incorporation of silver into alginate fibers increased antimicrobial activity and improved the binding affinity for elastase, matrix metalloproteases‐2, and the proinflammatory cytokines tested. Addition of silver also enhanced the antioxidant capacity. However, a distinct negative effect of silver‐containing alginates on human HaCaT keratinocytes was noted in vitro.

The immune system in healthy adults and patients with atopic dermatitis seems to be affected differently by a probiotic intervention

Background Probiotic bacteria are proposed to alleviate atopic dermatitis (AD) in infants. There are few indications about the effect of probiotics on AD in adults.

Active wound dressings based on bacterial nanocellulose as drug delivery system for octenidine

Although bacterial nanocellulose (BNC) may serve as an ideal wound dressing, it exhibits no antibacterial properties by itself. Therefore, in the present study BNC was functionalized with the antiseptic drug octenidine. Drug loading and release, mechanical characteristics, biocompatibility, and antimicrobial efficacy were investigated. Octenidine release was based on diffusion and swelling according to the Ritger-Peppas equation and characterized by a time dependent biphasic release profile, with a rapid release in the first 8h, followed by a slower release rate up to 96 h. The comparison between lab-scale and up-scale BNC identified thickness, water content, and the surface area to volume ratio as parameters which have an impact on the control of the release characteristics. Compression and tensile strength remained unchanged upon incorporation of octenidine in BNC. In biological assays, drug-loaded BNC demonstrated high biocompatibility in human keratinocytes and antimicrobial activity against Staphylococcus aureus. In a long-term storage test, the octenidine loaded in BNC was found to be stable, releasable, and biologically active over a period of 6 months without changes. In conclusion, octenidine loaded BNC presents a ready-to-use wound dressing for the treatment of infected wounds that can be stored over 6 months without losing its antibacterial activity.

Protease and pro-inflammatory cytokine concentrations are elevated in chronic compared to acute wounds and can be modulated by collagen type I in vitro

Physiological wound repair is a highly regulated, complex process, which leads to formation of new tissue after injury. However, the healing process is not perfect and healing impairments can occur. Delayed healing and formation of chronic wounds has been linked to the excessive production of proteolytic enzymes leading to reduced amounts of growth factors and successive destruction of the extracellular matrix. It has been implied that there is an alteration in the normal control mechanisms regulating the levels of these enzymes. The study presented provides data on the concentration of proteases and cytokines in wound fluid from chronic when compared with acute wounds. Levels of proteases such as PMN elastase, matrix metalloproteinases-2 (MMP-2), and MMP-13 were found to be profoundly elevated in chronic when compared with acute wound fluids. In addition, concentrations of IL-1β, IL-6, and IL-8 were shown to be significantly higher in chronic than in acute wounds. Furthermore, the ability of a wound dressing, consisting of bovine collagen type I, to bind pro-inflammatory cytokines was investigated. Collagen type I was able to bind significant amounts of the pro-inflammatory cytokines in vitro. Thus, it should be able to establish a more physiological wound milieu in vivo and promote healing.

Melatonin suppresses reactive oxygen species induced by UV irradiation in leukocytes

Abstract  An investigation of the antioxidative UV protective effect of melatonin was performed in an in vitro irradiation model with leukocytes. Leukocytes were isolated from EDTA‐treated whole blood and taken up in phosphate‐buffered saline (PBS). Five of 10 aliquots were incubated with 2 mmol/L melatonin and 5 with PBS as a control. The samples were irradiated by UV light (280–360 nm, max: 310 nm) at doses between 75 and 300 mJ/cm2 or left unirradiated. Radical formation was measured using the chemiluminescence technique. Staining with trypan blue was performed to assess cell viability. Melatonin significantly suppressed radical formation in cell solutions irradiated from 75 to 300 mJ/cm2 (P ≤ 0.001). Controls showed an increase of reactive oxygen species (ROS) formation as a sign of oxidative stress when irradiated with increasing UV doses and a maximum ROS formation under 300 mJ/cm2 UV light. The cytotoxicity of UV light was reduced by melatonin up to a UV dose of 1.5 J/cm2. Leukocytes were suitable cells for the evaluation of the efficacy of melatonin as a radical scavenger under UV light. The results confirm that the clinically observed UV protective effects of melatonin may be at least partially based on its radical scavenging properties.

Melatonin reduces UV-induced reactive oxygen species in a dose-dependent manner in IL-3-stimulated leukocytes

Reactive oxygen species (ROS) are presumed to be involved in inflammatory UV reactions of the skin. This in vitro study was performed to investigate the suppressive effect of melatonin in interleukin‐3 (IL‐3) stimulated leukocytes. Neutrophilic granulocytes were isolated from EDTA‐treated whole blood and placed in a phosphate‐buffered saline (PBS) containing IL‐3. Cell suspensions were either treated with PBS (control) or with increasing doses of melatonin (0.1, 0.5, 1, 2, 3, 5, 7.5, 10 mmol). One PBS solution was left unirradiated and the other nine solutions (PBS and melatonin) were irradiated with 750 mJ/cm2 UVB light (280–360 nm, max: 310 nm). Radical formation was measured by the chemiluminescence technique. UV‐irradiated leukocytes showed a 5‐fold higher radical formation than unirradiated leukocytes. Melatonin, in increasing doses in powers of ten, led to a maximum suppression of free radicals at 10 nmol (P=0.01) and 1 mmol melatonin (P=0.001), showing a biphasic, non‐linear, dose–response relationship. Melatonin, given in amounts of 0.1–10 mmol, led to a direct dose‐dependent suppression of ROS. Radical formation was suppressed significantly in a range from 0.5 to 10 mmol (P=0.001). Melatonin is known to function as a radical scavenger and antioxidant; some of these melatonin effects may be receptor independent, while others may be receptor dependent.

HaCaT keratinocytes in co-culture with Staphylococcus aureus can be protected from bacterial damage by polihexanide

Wound healing is compromised by critical colonization and infection with bacteria. Hence, antimicrobial agents are used clinically to decrease the bacterial load and promote wound healing. Polihexanide (PHMB) has been found to be effective against a broad spectrum of micro‐organisms and is increasingly utilized in rinsing solutions or in combination with wound dressings because of its good biocompatibility. In the present study, a co‐culture of human keratinocytes and Staphylococcus aureus was established to serve as an in vitro model for infected wounds. Incubation of keratinocytes with increasing concentrations of S. aureus led to a dose‐dependent decline of cell viability and proliferation. Lactate dehydrogenase release and interleukin‐8 liberation were found to be elevated under these conditions. Polihexanide dose‐dependently was able to protect keratinocytes from bacterial damage and re‐establish normal human cell proliferation in vitro. Furthermore, a dressing consisting of biocellulose derived from Acetobacter xylinum with the addition of polihexanide was adept to safeguard keratinocytes against S. aureus. In conclusion, the co‐culture system presented embodies a valuable tool as a model system for infected cells in a non‐healing wound. Furthermore, the results obtained support the favorable function of polihexanide in the treatment of infected chronic wounds.

Effect of the sterilization method on the performance of collagen type I on chronic wound parameters in vitro

In the treatment of chronic wounds, it is necessary to establish a physiological wound milieu to improve healing. Application of collagen as wound dressing has been described as beneficial as it possesses the ability to reduce elevated levels of proteases, cytokines, and free radicals. Consequently, a wide range of wound dressings based on collagen have been developed. Native collagen is susceptible to alterations because of influences during the production process; to minimize effects on the molecule itself collagen wound dressings are usually aseptically produced. Common sterilization methods (autoclaving, irradiation, and ethylene oxide (EtO) treatment) can induce changes in the protein chemistry and physical properties, potentially affecting the absorption rate, mechanical strength, or performance. In this study, we have evaluated the influence of gamma- and beta-irradiation as well as EtO sterilization on the binding capacity of collagen type I for selected proteases and cytokines associated with nonhealing wounds. Although a pronounced effect on the physical properties of the collagen was found, there was no significant loss in the binding affinity for polymorphonuclear elastase, matrix metalloproteinase-2, and interleukin-1beta, or in the antioxidant capacity.

The use of ITS DNA sequence analysis and MALDI-TOF mass spectrometry in diagnosing an infection with Fusarium proliferatum

Abstract:  Although mycoses are among the most common diseases worldwide, infections with Fusarium spp. occur only rarely. Mostly patients suffering from underlying immune deficiency are infected with this mould, resulting in a considerably decreasing prognosis. In immunocompromised patients, cutaneous manifestations are more often associated with Fusarium sp. than with Candida sp. or Aspergillus sp. We describe one patient with acute lymphoblastic leukaemia, who was first treated with chemotherapy after GMALL protocol 07/03. After relapse, the patient was successfully transplanted in second remission with a human leukocyte antigen (HLA)‐matched unrelated peripheral blood stem cell graft. Ten months later, the patient died from respiratory insufficiency and recurrence of leukaemia. Previously, Aspergillus antigen was detected in blood. In the latter course, disseminated papules appeared. One of these was examined histologically and mycologically. Conventional cultural diagnostics led to the diagnosis of a fusariosis, further supported by internal transcribed spacer (ITS) sequencing and matrix assisted laser desorption/ionisation–time‐of‐flight mass spectrometry (MALDI‐TOF) mass spectrometry, both determining the isolated strain as Fusarium proliferatum, which is a very infrequent pathogen within this genus. Our investigations underline the potential of MALDI‐TOF MS based identification of Fusarium species as an innovative, time and cost efficient alternative to ITS sequencing.