Tonny Karlsmark

A novel approach to the antimicrobial activity of maggot debridement therapy

Objectives Commercially produced sterile green bottle fly Lucilia sericata maggots are successfully employed by practitioners worldwide to clean a multitude of chronic necrotic wounds and reduce wound bacterial burdens during maggot debridement therapy (MDT). Secretions from the maggots exhibit antimicrobial activity along with other activities beneficial for wound healing. With the rise of multidrug-resistant bacteria, new approaches to identifying the active compounds responsible for the antimicrobial activity within this treatment are imperative. Therefore, the aim of this study was to use a novel approach to investigate the output of secreted proteins from the maggots under conditions mimicking clinical treatments. Methods cDNA libraries constructed from microdissected salivary glands and whole maggots, respectively, were treated with transposon-assisted signal trapping (TAST), a technique selecting for the identification of secreted proteins. Several putative secreted components of insect immunity were identified, including a defensin named lucifensin, which was produced recombinantly as a Trx-fusion protein in Escherichia coli, purified using immobilized metal affinity chromatography and reverse-phase HPLC, and tested in vitro against Gram-positive and Gram-negative bacterial strains. Results Lucifensin was active against Staphylococcus carnosus, Streptococcus pyogenes and Streptococcus pneumoniae (MIC 2 mg/L), as well as Staphylococcus aureus (MIC 16 mg/L). The peptide did not show antimicrobial activity towards Gram-negative bacteria. The MIC of lucifensin for the methicillin-resistant S. aureus and glycopeptide-intermediate S. aureus isolates tested ranged from 8 to >128 mg/L. Conclusions The TAST results did not reveal any highly secreted compounds with putative antimicrobial activity, implying an alternative antimicrobial activity of MDT. Lucifensin showed antimicrobial activities comparable to other defensins and could have potential as a future drug candidate scaffold, for redesign for other applications besides the topical treatment of infected wounds.

Removal of dermal edema with class I and II compression stockings in patients with lipodermatosclerosis

BACKGROUNDnLipodermatosclerosis is a sequela of deep venous insufficiency and a risk factor for the occurrence of venous leg ulceration. Medical compression stockings facilitate leg ulcer healing and prevent occurrence of ulcers resulting from removal of edema. Although the exact level of compression necessary for removal of dermal edema in patients with deep venous insufficiency has not been established, garments providing high compressive values of 30 to 40 mm Hg have been recommended. Dermal edema can be visualized by high-frequency ultrasonography.nnnOBJECTIVEnWe used ultrasound imaging to study whether a lower level of compression (class I 18 to 26 mm Hg vs class II 26 to 36 mm Hg) is effective in removal of dermal edema. This question is important because the use of hosiery with a lower compression class would enhance compliance and enable treatment of patients with mixed arteriovenous disease.nnnMETHODSnIn 11 patients skin images were obtained with 20 MHz ultrasound from the malleolar region in lipodermatosclerotic skin and corresponding normal skin of the contralateral leg. The ratio of low echogenic pixel number to total pixel number (LEP/TP), which correlates with dermal water, was measured before and after 5 days of applied compression in two treatment courses where classes of compression were switched randomly. Ankle circumference was also measured.nnnRESULTSnWe found that LEP/TP was 33% higher in lipodermatosclerotic skin than in the matched normal skin indicating presence of skin edema. Application of class I and II compressive hosiery resulted in LEP/TP decrease by 17% (95% confidence interval, 0.07 to 0.26) and 14% (95% confidence interval, 0.04 to 0.21), respectively, suggestive of dermal edema reduction. No statistically significant difference in efficacy of dermal edema removal between class I and II was found. No changes in ankle circumference after application of both classes of compression was observed.nnnCONCLUSIONnApplication of light and moderate compression results in a partial edema removal from the dermis in lipodermatosclerosis in the absence of measurable reduction in leg circumference. Class I compression is as effective as class II for elimination of dermal edema. These data indicate that light compression may be a useful modality for patients with deep venous insufficiency and lipodermatosclerosis who are not eligible for treatment with garments having higher compressive forces.

Lymphoedema of the lower extremities - background, pathophysiology and diagnostic considerations

Lymphoedema of the lower extremities is a chronic debilitating disease that is often underdiagnosed. Early diagnosis and treatment is paramount in reducing the risk of progression and complications. Lymphoedema has traditionally been defined as interstitial oedema and protein accumulation because of a defect in the lymphatic drainage; however, some findings suggest that the interstitial protein concentration may be low in some types of lymphoedema. Primary lymphoedema is caused by an inherent defect in the lymphatic vessels or lymph nodes. Secondary lymphoedema is caused by damages to the lymphatic system most often caused by cancer or its treatment. Many of the underlying pathophysiological mechanisms have yet to be elucidated. Many methods have been developed for examination of the lymphatic system. Lymphoscintigraphy is presently the preferred diagnostic modality. Lack of consensus regarding protocol and qualitative interpretation criteria results in a too observer dependent outcome. Methods for objectifying the scintigraphy through quantification have been criticized. Depot clearance rates are an alternative method of quantification of lymphatic drainage capacity. This method however has mostly been applied on upper extremity lymphoedema. The aim of this review is to provide a literature‐based overview of the aetiology and pathophysiology of lower extremity lymphoedema and to summarize the current knowledge about lymphoscintigraphy and depot clearance techniques. The abundance of factors influencing the outcome of the examination stresses the need for consensus regarding examination protocols and interpretation. Further studies are needed to improve diagnostic performance and understanding of pathophysiological mechanisms.

Quorum-sensing-regulated virulence factors in Pseudomonas aeruginosa are toxic to Lucilia sericata maggots.

Maggot debridement therapy (MDT) is widely used for debridement of chronic infected wounds; however, for wounds harbouring specific bacteria limited effect or failure of the treatment has been described. Here we studied the survival of Lucilia sericata maggots encountering Pseudomonas aeruginosa PAO1 in a simple assay with emphasis on the quorum-sensing (QS)-regulated virulence. The maggots were challenged with GFP-tagged P. aeruginosa wild-type (WT) PAO1 and a GFP-tagged P. aeruginosa ΔlasR rhlR (ΔRR) QS-deficient mutant in different concentrations. Maggots were killed in the presence of WT PAO1 whereas the challenge with the QS mutant showed a survival reduction of ∼25u200a% compared to negative controls. Furthermore, bacterial intake by the maggots was lower in the presence of WT PAO1 compared to the PAO1 ΔRR mutant. Maggot excretions/secretions (ES) were assayed for the presence of QS inhibitors; only high doses of ES showed inhibition of QS in P. aeruginosa. Thus P. aeruginosa was shown to be toxic to L. sericata maggots. This, coupled to the preferential feeding by the maggots and reduced ingestion of P. aeruginosa, could explain MDT failure in wounds colonized by P. aeruginosa. Wounds heavily colonized with P. aeruginosa should be a counterindication for MDT unless used in combination with a pre-treatment with other topical therapeutics targeting P. aeruginosa.

Effect of smoking, abstention, and nicotine patch on epidermal healing and collagenase in skin transudate.

Delayed wound healing may explain postoperative tissue and wound dehiscence in smokers, but the effects of smoking and smoking cessation on the cellular mechanisms remain unclear. Suction blisters were raised in 48 smokers and 30 never smokers. The fluid was retrieved and the epidermal roof was excised. Transepidermal water loss (TEWL) was measured after 2, 4, and 7 days. Then, the smokers were randomized to continuous smoking or abstinence with a transdermal nicotine patch or a placebo by concealed allocation. The sequence was repeated after 4, 8, and 12 weeks in all smokers and abstainers and in 6 never smokers. Matrix metalloproteinase (MMP)‐8 and MMP‐1 levels in suction blister fluid were assessed by an enzyme‐linked immunosorbent assay. Random‐effects models for repeated measurements were applied and p≤0.05 was considered significant. One week after wounding the TEWL was 17.20 (14.47–19.92) g/cm2u2003hour (mean, 95% CI) in smokers and 13.89 (9.46–18.33) in never smokers (p<0.01). In abstinent smokers TEWL was 18.95 (15.20–22.70)(p<0.01, when compared with smokers). In smokers, MMP‐8 was 36.4 (24.3–48.5)u2003ng/mL (mean, 95% CI) and 15.2 (1.4–30.2)u2003ng/mL in never smokers (p<0.01). Abstinent smokers MMP‐8 level was 21.2u2003ng/mL (6.6–43.0) (p=0.02, when compared with smokers). MMP‐1 was unaffected by smoking and abstention. Transdermal nicotine patch did not affect any parameter. We conclude that smoking attenuates epidermal healing and may enhance extracellular matrix degradation. Three months of abstinence from smoking does not restore epidermal healing, whereas 4 weeks of abstinence normalizes suction blister MMP‐8 levels. These findings suggest sustained impaired wound healing in smokers and potential reversibility of extracellular matrix degradation.

Characteristics of an autologous leukocyte and platelet‐rich fibrin patch intended for the treatment of recalcitrant wounds

We have investigated the physical, biochemical, and cellular properties of an autologous leukocyte and platelet‐rich fibrin patch. This was generated in an automated device from a sample of a patients blood at the point of care. Using microscopy, cell counting, enzyme‐linked immunosorbent assay, antibody arrays, and cell culture assays, we show that the patch is a three‐layered membrane comprising a fibrin sheet, a layer of platelets, and a layer of leukocytes. Mean recovery of platelets from the donated blood was 98% (±95%CI 0.8%). Mean levels of platelet‐derived growth factor AB, human transforming growth factor beta 1, and vascular endothelial growth factor extracted from the patch were determined as 127u2009ng (±95% CI 20), 92u2009ng (±95%CI 17), and 1.35u2009ng (±95%CI 0.37), respectively. We showed a continued release of PDGF‐AB over several days, the rate of which was increased by the addition of chronic wound fluid. By comparison with traditional platelet‐rich plasma, differences in immune components were found. The relevance of these findings was assessed by showing a mitogenic and migratory effect on cultured human dermal fibroblasts. Further, we showed that fibrocytes, a cell type important for acute wound healing, could be grown from the patch. The relevance of these findings in relation to the use of the patch for treating recalcitrant wounds is discussed.

Validity and interobserver agreement of lower extremity local tissue water measurements in healthy women using tissue dielectric constant

Tissue dielectric constant (TDC) measurement may become an important tool in the clinical evaluation of chronic lower extremity swelling in women; however, several factors are known to influence TDC measurements, and comparative data on healthy lower extremities are few.

Randomized clinical study comparing Compeed cold sore patch to acyclovir cream 5% in the treatment of herpes simplex labialis.

Backgroundu2002 Hydrocolloid technology has been proven effective in treating dermal wounds. A previous study showed that a newly developed thin hydrocolloid patch [Compeed® cold sore patch (CSP)] provided multiple wound‐healing benefits across all stages of a herpes simplex labialis (HSL) outbreak.

Good agreement between minimal erythema dose test reactions and objective measurements: an in vivo study of human skin

The erythema resulting from the minimal erythema dose (MED) test is subjectively assessed. The evaluator visually grades erythema on an ordinal scale. Both intra‐ and interobserver variation have been found for this erythema assessment. We wanted to examine if objective measurements could be used to confirm the subjective finding.

Heavy resistance training and lymphedema: Prevalence of breast cancer-related lymphedema in participants of an exercise intervention utilizing heavy load resistance training

Abstract Background. There is limited knowledge regarding progressive resistance training during adjuvant chemotherapy and the risk of developing breast cancer-related lymphedema (BCRL). Furthermore, no studies have investigated the safety of resistance training with heavy loads (> 80% 1 repetition maximum) in this population. ‘Body and Cancer’ is a six-week, nine-hour weekly, supervised, multimodal exercise intervention utilizing progressive resistance training with heavy loads for cancer patients undergoing chemotherapy. The purpose of the present study was to estimate the prevalence of BCRL in former participants, and identify associations between progressive resistance training with heavy loads, and the development of BCRL. Material and methods. This was a descriptive study. Population: Women treated for breast cancer (n = 149), who had participated in the ‘Body and Cancer’ exercise intervention between 1 January 2010 and 31 December 2011 participated in a structured telephone interview. The average follow-up time was 14 months (range 4–26). A clinical diagnosis of BCRL reported by the participant was the primary outcome. Results. A total of 27.5% reported that they had been diagnosed with BCRL by a clinician. This was true for 44.4% with axillary node dissection. No statistically significant association between strength gains during the exercise intervention, and the development of BCRL was observed, nor was self-reported participation in progressive resistance training with heavy loads up to three months post-intervention. Conclusion. The prevalence of BCRL among former “Body and Cancer” participants at follow-up was 27.5%. There appears to be no association between performing heavy resistance training during adjuvant treatment (chemotherapy/radiotherapy), and the development of BCRL. However randomized controlled trials should be performed to confirm this observation.