The use of chromogenic bacteria as coloured substitutes for pathogens: a simple strategy during design and development of a new method for sample pretreatment.

AIMS: In the present study, chromogenic (red) bacteria were used to simulate actual target bacteria during set-up and optimization of an isolation process of bacteria, designed for food samples. Isolation of bacteria from food in the context of molecular biological detection of food pathogens is a multistep process. Development of such a separation method requires continuous monitoring of the location of the presumable targets in the sample tubes. Therefore, red-coloured pigmented bacteria were used as substitutes for the actual target bacteria, during the establishment of a new sample preparation technique. METHODS AND RESULTS: The chromogenic bacteria Micrococcus roseus and Serratia marcescens were confirmed to withstand the physical (e.g. centrifugal forces) and chemical (e.g. lysis buffer composition) conditions required during establishment of the new technique. Furthermore, the suitability of these model bacteria to substitute for the actual target pathogens (Salmonella enterica subsp. enterica serovar Typhimurium and Listeria monocytogenes) was assured by testing the physical properties of the model bacteria with respect to the proposed separation methods. CONCLUSION: Visibility of the pigmented bacteria within the complex sample matrices served to allocate bacterial content during the various steps necessary for finalization of the method protocol. The presumptive bacterial targets can be allocated simply by visualization of their bright red colour silhouetted against the background sample matrix. SIGNIFICANCE AND IMPACT OF STUDY: The use of pigmented bacteria as substitutes for actual colourless target bacteria during design and development of a bacterial isolation method is a simple and inexpensive application. It saves a huge amount of time and resources, as the proof of principle of new methods is possible in rapid succession.

Enumeration of Enterobacteriaceae in various foods with a new automated most-probable-number method compared with petrifilm and international organization for standardization procedures.

An automated most-probable-number (MPN) system (TEMPO, bioMérieux, Marcy l'Etoile, France) for enumeration of Enterobacteriaceae (EB) was compared with methods involving violet red bile glucose agar (VRBG) (International Organization for Standardization [ISO] method 21528-2) (ISO-VRBG) and Petrifilm (PF-EB). The MPN partitioning (three different volumes with 16 replicates of each) is done automatically in a disposable card. Bacterial growth is indicated by acid production from sugars, lowering the pH of the medium, and quenching the fluorescence of 4-methylumbelliferone. After incubation, the number of nonfluorescent wells is read in a separate device, and the MPN is calculated automatically. A total of 411 naturally contaminated foods were tested, and 190 were in the detection range for all methods. For these results, the mean (+/- standard deviation) counts were 2.540 +/- 1.026, 2.547 +/- 0.995, and 2.456 +/- 1.014 log CFU/g for the ISO-VRBG, PF-EB, and automated MPN methods, respectively. Mean differences were -0.084 +/- 0.460 log units for the automated MPN results compared with the ISO-VRBG and 0.007 +/- 0.450 for the PF-EB results compared with the ISO-VRBG results. The automated MPN method tends to yield lower numbers and the PF-EB method tends to yield higher numbers than does the ISO-VRBG method (difference not significant; Kruskal-Wallis test, P = 0.102). Thus, the average difference was highest between the automated MPN method and the PF-EB method (-0.091 +/- 0.512 log units). Differences between the automated MPN and ISO-VRBG results of > 1 log unit were detected in 3.4% of all samples. For 3.9% of the samples, one comparison yielded differences of < 1 log CFU/g and the other yielded > 1 but < 2 log CFU/g, which means that the differences are possibly > 1 log CFU/g. For the ISO-VRBG method, confirmation of isolates was necessary to avoid a bias due to the presence of oxidase-positive glucose-fermenting colonies. The automated MPN system yielded results comparable to those of the confirmed Enterobacteriaceae ISO-VRBG method but required only 24 h of analysis time.

Enumeration of total aerobic bacteria and Escherichia coli in minced meat and on carcass surface samples with an automated most-probable-number method compared with colony count protocols.

An automated most-probable-number (MPN) system for the enumeration of total bacterial flora and Escherichia coli was compared with plate count agar and tryptone-bile-glucuronide (TBX) and ColiID (in-house method) agar methodology. The MPN partitioning of sample aliquots was done automatically on a disposable card containing 48 wells of 3 different volumes, i.e., 16 replicates per volume. Bacterial growth was detected by the formation of fluorescent 4-methylumbilliferone. After incubation, the number of fluorescent wells was read with a separate device, and the MPN was calculated automatically. A total of 180 naturally contaminated samples were tested (pig and cattle carcass surfaces, n = 63; frozen minced meat, n = 62; and refrigerated minced meat, n = 55). Plate count agar results and MPN were highly correlated (r = 0.99), with log MPN = -0.25 + 1.05 x log CFU (plate count agar) (n = 163; range, 2.2 to 7.5 log CFU/g or cm2). Only a few discrepancies were recorded. In two samples (1.1%), the differences were > or = 1.0 log; in three samples (1.7%), the differences were > or = 0.5 log. For E. coli, regression analysis was done for all three methods for 80 minced meat samples, which were above the limit of detection (1.0 log CFU/g): log MPN = 0.18 + 0.98 x log CFU (TBX), r = 0.96, and log MPN = -0.02 + 0.99 x log CFU (ColiID), r = 0.99 (range, 1.0 to 4.2 log CFU/g). Four discrepant results were recorded, with differences of > 0.5 but < 1.0 log unit. These results suggest that the automated MPN method described is a suitable and labor-saving alternative to colony count techniques for total bacterial flora and E. coli determination in minced meat or on carcass surfaces.

Treatment of therapy-refractive ulcera cruris of various origins with autologous keratinocytes in fibrin sealant.

BACKGROUND: Evaluation of the effects of cultivated, subconfluent, autologous keratinocytes in fibrin sealant (BioSeed-S) on the healing of therapy-refractive chronic wounds. PATIENTS AND METHODS: Open observational study in 60 patients with chronic leg ulcers and impaired wound healing of various origins. After whole-skin excision and cultivation of the autologous keratinocytes, the suspended cells were applied to the preconditioned wound in fibrin sealant. Wound epithelization and wound size were recorded at defined times. RESULTS: Fifty-two of the 60 participating patients could be evaluated. After 6 weeks, 29 ulcers (55.8%) were healed. The mean epithelization increased between the 8th and 42nd postoperative day from 23% to 62.5%. In 50.0% of the patients, global assessment of the wound showed a high degree of epithelization or healing after 42 days. In 32.6% of treated patients, improvement was observed, while no healing tendency was to be found in 17.4%. CONCLUSION: The present observational study indicates that the transplantation of autologous keratinocytes suspended in fibrin sealant could be of advantage in the treatment of refractive leg ulcers.

[St. John's wort (Hypericum perforatum L.). A plant with relevance for dermatology]. / Johanniskraut (Hypericum perforatum L.). Eine Pflanze mit Relevanz für die Dermatologie.

Saint John's wort (Hypericum perforatum L.) is a herbal remedy that is effective in the treatment of mild to moderate depression. In traditional folk medicine, oily extracts of St. John's wort are used for topical treatment of wounds, burns and myalgia. The lipophilic phloroglucin-derivative hyperforin has antibacterial and antiinflammatory effects. These effects could be of relevance in topical treatment of infected wounds and other dermatoses, but no studies have been conducted so far. The naphtodianthrone hypericin is a photodtodynamic active substance that kills tumor cells via the induction of apoptosis. Hypericin also displays antiviral activity in vitro. In vivo, intravenous or oral treatment with hypericin of HIV-infected subjects did not result in a reduction of the virus load. Most of the patients treated with hypericin experienced phototoxicity. Similar phototoxic symptoms ("hypericism") have been observed in grazing animals ingesting large amounts of St. John's wort. In contrast, antidepressant medication with St. John's wort usually does not produce phototoxic symptoms. Recent pharmacokinetic studies suggest that the phototoxic threshold level of hypericin is not reached with dosages used for the oral treatment of depression. However, very recent reports demonstrated interactions of St. John's wort with other drugs such as digoxin, indinavir and cyclosporin. Blood levels of these drugs were dramatically decreased by St. John's wort. This should be considered in the treatment of skin conditions with antiviral drugs or cyclosporin.

[Plant-induced toxic and allergic dermatitis (phytodermatitis)]. / Durch Pflanzen ausgelöste toxische und allergische Dermatitis (Phytodermatitis).

Herbal products are being used increasingly for medical or cosmetic purposes. Many cosmetics contain plant extracts for fragrance. Sensitizing plants in cosmetics are tea tree oil, arnica, chamomile, yarrow, citrus extracts, common ivy, aloe, lavender, peppermint, and others. However, the sensitizing potential of these plants varies. Most of the sensitizing substances are sesquiterpene lactones or terpenes. The present paper reviews the various forms of phytodermatitis, including irritant plant dermatitis, phototoxic and photo-allergic dermatitis, allergic dermatitis, and airborne contact dermatitis.

Long-term efficacy and safety of tacalcitol ointment in patients with chronic plaque psoriasis.

BACKGROUND: As psoriasis patients often require continuous treatment optimal therapy has to provide efficacy and a good safety profile over the long term. OBJECTIVES: The aim of this multicentre study was to assess the efficacy, safety and tolerability of tacalcitol (4 microg g(-1)) ointment (Curatoderm, Hermal, Reinbek, Germany) applied once daily over a treatment period of 18 months. PATIENTS AND METHODS: Efficacy parameters were Psoriasis Area Severity Index (PASI), based on summed scores of erythema, infiltration and scaling and total body surface involvement (TBI). Safety assessment included serum levels of calcium, parathyroid hormone, calcitonin, 1,25-dihydroxy vitamin D3 (calcitriol); urinary calcium, creatinine, calcium/creatinine ratio in spot and 24-h urine and urinary alpha(1)-microglobulin. A group of 304 patients with chronic plaque psoriasis, covering between 7% and 20% of the body surface area was included for the initial treatment phase of 3 months. Of the 257 patients who completed the initial 3 months, 197 patients continued in a second treatment phase of 15 months. RESULTS: Tacalcitol treatment proved to be effective in reducing the severity of psoriasis and maintained therapeutic response over the study period. The median PASI fell from 9.5 to 4 .6 at month 3 and to 3.25 at month 18 (P < 0.0001). The median improvement in TBI was 30% at month 3 and 50% at month 18. In no patient was there any relevant disturbance of calcium homeostasis. There were no significant changes in mean values of serum calcium, parathyroid hormone and calcitriol. Additionally no significant changes in 24-h urinary excretion evaluation were observed. There was no correlation between levels of serum calcium or urinary calcium and amount of tacalcitol ointment used, even in the patients requiring the largest amounts of ointment (up to 13 g day(-1) and up to 20% of body area affected). Treatment was generally well tolerated and there were no serious or unexpected adverse events reported. However, discontinuation of treatment as a result of skin irritation was seen in 5.9% of patients. The greatest frequency of cutaneous side-effects occurred during initial treatment and the incidence decreased markedly as the treatment was well-tolerated with continued use. CONCLUSIONS: Tacalcitol ointment once daily was demonstrated to be efficacious, safe and well tolerated in the long-term control of plaque psoriasis in patients with up to 20% body surface involvement.

The role of the urokinase-type plasminogen activator (uPA) and its receptor (CD87) in lipodermatosclerosis.

BACKGROUND: Lipodermatosclerosis refers to a sclerosing panniculitis and dermopathy of the lower extremities sometimes seen in association with venous ulceration. Matrix metalloproteinases are implicated in the pathogenesis of venous leg ulcers and the in vitro activation of recombinant MMP-2 is controlled by the plasminogen activation system. To better understand the role of plasminogen activation in the pathogenesis of venous leg ulcers we investigated fibrinolytic factors and their inhibitors in tissue samples of lipodermatolsclerosis. METHODS: The expression and the functional state of the urokinase-type plasminogen activator (uPA), the tissue-type plasminogen activator (tPA), the urokinase receptor (CD87), the plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) were assayed using reverse transcription polymerase chain reaction, Western blot, fibrin zymography and immunohistochemistry analyses in tissue samples of lipodermatosclerosis. RESULTS: Our results provide direct evidence of elevated expression of uPA (p<0.01) and CD87 (p<0.01) mRNA and protein level in lipodermatosclerosis in comparison with healthy skin. By immunohistochemistry, elevated expression of uPA and CD87 could be detected. Fibrin zymography showed significantly elevated endogenous uPA activity (p<0.01) in liposclerotic lesions compared to healthy controls. CONCLUSION: Our findings indicate that elevated plasminogen activation in lipodermatosclerotic tissue may play a crucial role in the pathogenesis of venous leg ulceration.

[UVA1 phototherapy. Pilot study of dose finding in acute exacerbated atopic dermatitis]. / UVA1-Phototherapie. Pilotstudie zur Dosisfindung bei der bei akut exazerbierten atopischen Dermatitis.

BACKGROUND AND OBJECTIVE: UVA1 phototherapy is an new effective treatment modality for acute atopic dermatitis (AD). However there is still some controversy about the optimal UVA1 single and cumulative dose. PATIENTS/METHODS: We compared in a randomized, controlled, prospective pilot study the efficacy of a therapy with 15 treatments of a "high dose" (max. single dose of 130 J/cm2, max. cumulative dose 1840 J/cm2), "medium dose" (max. single dose of 65 J/cm2, max. cumulative dose 975 J/cm2) or "low dose" (max. single dose of 20 J/cm2, max. cumulative dose 300 J/cm2) UVA1 in patients with acutely exacerbated atopic dermatitis (SCORAD > 30). After determination of the IPD, patients randomized into one of the three treatment arms. The patients received 15 treatments (5 times per week) without any additional therapy except for topical skin care. RESULTS: After 15 treatments the "high dose" and "medium dose" groups showed a statistically significant reduction of the SCORAD. No significant reduction of the SCORAD was observed in the "low dose" group. All three treatment arms displayed no statistically significant changes in the IgE and ECP levels and in the number of eosinophils in the peripheral blood. The UVA1 therapy was well tolerated by all patients. No side effects were observed. CONCLUSIONS: This study suggests that both the "high dose" and the "medium dose" regimens are effective in the treatment of patients with acutely exacerbated atopic dermatitis.

Calcitriol 3 microg g-1 ointment in combination with ultraviolet B phototherapy for the treatment of plaque psoriasis: results of a comparative study.

BACKGROUND: Combinations of topical treatments and ultraviolet (UV) B phototherapy for plaque psoriasis may be more beneficial than either type of treatment used alone. OBJECTIVES: To determine the efficacy of calcitriol 3 microg g-1 ointment in combination with UVB phototherapy in treating plaque psoriasis. METHODS: Calcitriol ointment with UVB was compared with vehicle plus UVB in a randomized, double-blind study in 104 patients. RESULTS: Mean global improvement scores for both groups increased over the 8-week study period; there was a statistically significant difference (P < 0.05) in favour of the calcitriol/UVB combination from week 1. At end-point, 45% of the calcitriol/UVB group showed considerable improvement or clearing of psoriasis, compared with 21% of the control group. The superiority of calcitriol plus UVB was also reflected in the global severity and Psoriasis Area and Severity Index (PASI) scores; at end-point the mean percentage decrease in PASI score was 65% for the calcitriol/UVB group and 43% for vehicle/UVB (P = 0.0014). The incidence of skin-related adverse events was low (< 12%) and similar in the two treatment groups. No clinically significant changes in blood chemistry, in particular calcium levels, occurred. The greater efficacy of combined calcitriol and phototherapy allowed a 34% decrease in total UVB exposure. CONCLUSIONS: Calcitriol 3 microg g-1 ointment and UVB phototherapy in combination provides a promising therapy for managing chronic plaque psoriasis.

Inflammation in stasis dermatitis upregulates MMP-1, MMP-2 and MMP-13 expression.

Stasis dermatitis is a common disorder, which is a consequence of impaired venous drainage of the legs. It is characterized histologically by proliferation of small blood vessels in the papillary dermis. This neovascularization may lead occasionally to the formation of discrete papules due to inflammatory processes. In order to evaluate the role of matrix metalloproteinases (MMPs) in the acute phase of chronic venous insufficiency, we examined the production of MMP-1, -2, -13 and tissue inhibitors of metalloproteinase (TIMP)-1 and -2 in lesional skin of stasis dermatitis. A total of 19 patients affected by stasis dermatitis were included in this experimental study. Polymerase chain reaction, western blot and immunohistochemical studies on tissue specimen were performed. In lesional skin of stasis dermatitis, there was elevated gene expression and immunoreactivity for MMP-1, -2 and -13 in comparison to healthy controls. In contrast, genexpression and immunoreactivity for TIMP-1 and -2 were diminished in stasis dermatitis in comparison with healthy controls. Overexpression and production of MMP-1, -2 and -13 without inhibitory effects could be the result of cytokine mediated induction. Matrix metalloproteinases (MMPs) may play an important role in the remodeling of lesional skin in stasis dermatitis.

[Reproducible drug exanthema to terbinafine with characteristic distribution of baboon syndrome]. / Fixes Arzneimittelexanthem auf Terbinafin mit charakteristischem Verteilungsmuster eines Baboon-Syndroms.

A 26 year old patient developed a fixed drug eruption located on his hands, inguinal and gluteal areas following oral treatment of onychomycosis with terbinafine. The rash showed the characteristic distribution of the "baboon-syndrome", so-named because of the red perianal region of the baboon. Although epicutaneous testing revealed no positive reaction, the rash could be induced in identical sites by oral administration of terbinafine. As the underlying pathomechanism for the "baboon-syndrome" a systemically induced allergic contact dermatitis has been suggested. In addition to the described substances, e.g. mercury, amoxicillin, ampicillin, heparin and nickel, this is the first report of "baboon syndrome" induced by terbinafine.

Recent advances in phototherapy.

This synopsis reviews recent developments in dermatological phototherapy. UVA1 phototherapy (340-400 nm) is effective in the treatment of inflammatory skin diseases such as acutely exacerbated atopic dermatitis, localized scleroderma, urticaria pigmentosa and disseminated granuloma annulare. Narrowband UVB radiation (311-313 nm) is used successfully as monotherapy or combined with dithranol, oral retinoids or 8-MOP in psoriasis, atopic dermatitis (AD) or photosensitivity disorders such as polymorphic light eruption. Bath water delivery of 8-methoxypsoralen and subsequent UVA-irradiation (PUVA bath therapy) for the treatment of psoriasis as well as for mycosis fungoides, localized scleroderma, urticaria pigmentosa or lichen planus is an effective alternative to its systemic application. The combination of salt water brine baths in different concentrations and subsequent UVA/B irradiation is used increasingly for the treatment of psoriasis or AD. Extracorporeal photopheresis (ECP) has proven to be a very effective treatment modality for cutaneous T cell lymphoma, chronic graft-versus-host disease and certain autoimmune diseases such as systemic scleroderma or pemphigus. However, despite the documented benefits of these new treatment modalities, little data exist as of yet on potential long-term side effects, thus the indications for these therapies should be considered carefully and patients should be followed up at regular intervals.

Plasminogen activation in venous leg ulcers.

BACKGROUND: Venous leg ulceration results from chronic venous insufficiency of the lower extremities. We recently showed that matrix metalloproteinase (MMP) -2 plays a major part in the pathogenesis of venous leg ulcers. In vitro activation of recombinant MMP-2 is controlled by the activity of the urokinase-type plasminogen activator (uPA), which acts as a fibrin-independent plasminogen activator. The activity of MMP-2 is potentiated by binding of uPA to the uPA receptor (uPAR). OBJECTIVES: We aimed to clarify the role of plasminogen activation in venous leg ulcers. METHODS: The expression of uPA, uPAR, the tissue-type plasminogen activator, and plasminogen activator inhibitor (PAI) -1 and PAI-2 was investigated using reverse transcription followed by polymerase chain reaction and Western blotting. RESULTS: These provided direct evidence of elevated expression of uPA and uPAR at the mRNA and protein levels in venous leg ulcers, in comparison with healthy skin. By immunohistochemistry, elevated expression of uPA and uPAR was detected. Fibrin zymography showed significantly elevated endogenous uPA activity in venous leg ulcers in comparison with healthy controls. CONCLUSIONS: Our findings indicate venous leg ulcers to be characterized by elevated plasminogen activation, suggesting that this enzyme cascade plays a crucial part in maintaining proteolytic activity in venous leg ulcers.

Chronic urticaria: diagnostic recommendations.

A group of European recognized dermatologists - the European Working Group on the Diagnosis of Chronic Urticaria - met on 12 March 1999 to discuss best practice for assessing and diagnosing patients with chronic urticaria. These are their recommendations. The recommendations will be valuable for the majority of cases, but are not exhaustive and may not include every possible precipitating factor.

Autologous platelet-derived wound healing factor promotes angiogenesis via alphavbeta3-integrin expression in chronic wounds.

Healing of venous leg ulcers depends on the adhesive interaction and formation of new vascular cells. Angiogenesis on the surface of angiogenic blood vessels requires the vascular integrin alphavbeta3 also known as the vitronectin receptor. Autologous platelet-derived wound healing factor (autologous PDWHF) has been described to regulate the wound healing process by forming granulation tissue in the early healing phase. Here we analysed the influence of autologous PDWHF on the expression of the alphavbeta3 integrin in tissue specimen of venous leg ulcers in comparison with placebo treated controls by using reverse transcriptase-polymerase chain reaction and immunohistochemistry. Our investigations provide evidence that mRNA and protein expression of alphavbeta3 were significantly increased in healing venous leg ulcers after 96 h treatment (p<0.05), whereas the total amount of alphavbeta3 mRNA and protein was not altered in placebo treated patients. In healing leg ulcers the alphavbeta3 integrin was predominantly localized around capillary vessels preferentially at sites of newly formed granulation tissue. Placebo controlled patients displayed no altered expression of the alphavbeta3 integrin in biopsy specimen. These findings suggest that topical autologous platelet-derived wound healing factor influences the process of angiogenesis/revascularization via alphavbeta3 integrin-expression hereby promoting granulation tissue formation in healing leg ulcers.

Magnesium ions inhibit the antigen-presenting function of human epidermal Langerhans cells in vivo and in vitro. Involvement of ATPase, HLA-DR, B7 molecules, and cytokines.

The combination of seawater baths and solar radiation at the Dead Sea is known as an effective treatment for patients with psoriasis and atopic dermatitis. Dead Sea water is particularly rich in magnesium ions. In this study we wished to determine the effects of magnesium ions on the capacity of human epidermal Langerhans cells to stimulate the proliferation of alloreactive T cells. Twelve subjects were exposed on four subsequent days on the volar aspects of their forearms to 5% MgCl2, 5% NaCl, ultraviolet B (1 minimal erythemal dose), MgCl2 + ultraviolet B, and NaCl + ultraviolet B. Epidermal sheets were prepared from punch biopsies and were stained for ATPase and HLA-DR. Compared with untreated skin, the number of ATPase+/HLA-DR+ Langerhans cells was significantly reduced after treatment with MgCl2 (p = 0.0063) or ultraviolet B (p = 0.0005), but not after NaCl (p = 0.7744). We next questioned whether this reduced expression of ATPase and HLA-DR on Langerhans cells bears a functional relevance. Six subjects were treated on four subsequent days with 5% MgCl2, ultraviolet B (1 minimal erythemal dose), and MgCl2 + ultraviolet B. Epidermal cell suspensions from treated and untreated skin were assessed for their antigen-presenting capacity in a mixed epidermal lymphocyte reaction with allogeneic naive resting T cells as responder cells. Treatment with MgCl2, similarly to ultraviolet B, significantly reduced the capacity of epidermal cells to activate allogeneic T cells (p = 0.0356). Magnesium ions also suppressed Langerhans cells function when added to epidermal cell suspensions in vitro. The reduced antigen-presenting capacity of Langerhans cells after treatment with MgCl2 was associated with a reduced expression by Langerhans cells of HLA-DR and costimulatory B7 molecules, and with a suppression of the constitutive tumor necrosis factor-alpha production by epidermal cells in vitro. These findings demonstrate that magnesium ions specifically inhibit the antigen-presenting capacity of Langerhans cells and may thus contribute to the efficacy of Dead Sea water in the treatment of inflammatory skin diseases.

[Therapy of lichen sclerosus et atrophicus vulvae with the CO2 silk touch laser]. / Therapie des Lichen sclerosus et atrophicus vulvae mit dem CO2-Silk-touch-Laser.

A 50 year old woman with distinct lichen sclerosus et atrophicus was suffering from severe genital itching, dyspareunia and increasing urinary burning. Therapy attempts with topical glucosteroids and estrogens had been without effort. Treatment with CO2 laser in silk touch mode under insufflation anesthesia to an improvement of her skin lesions and a nearly complete remission of her symptoms.