Development of a Quantitative Assay for the Characterization of Human Collectin-11 (CL-11, CL-K1)

Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway of complement with diverse functions spanning from host defense to embryonic development. CL-11 is found in the circulation in heterocomplexes with the homologous collectin-10 (CL-10). Abnormal CL-11 plasma levels are associated with the presence of disseminated intravascular coagulation, urinary schistosomiasis, and congenital disorders. Although there has been a marked development in the characterization of CL-11 there is still a scarcity of clinical tools for its analysis. Thus, we generated monoclonal antibodies and developed a quantitative ELISA to measure CL-11 in the circulation. The antibodies were screened against recombinant CL-11 and validated by ELISA and immunoprecipitation of serum and plasma. The best candidates were pairwise compared to develop a quantitative ELISA. The assay was validated regarding its sensitivity, reproducibility, and dilution linearity, demonstrating a satisfactory variability over a working range of 0.29–18.75 ng/ml. The mean plasma concentration of CL-11 in healthy controls was determined to be 289.4 ng/ml (range 143.2–459.4 ng/ml), highly correlated to the levels of CL/10/11 complexes (r = 0.729). Plasma CL-11 and CL-10/11 co-migrated in size exclusion chromatography as two major complexes of ~400 and >600 kDa. Furthermore, we observed a significant decrease at admission in CL-11 plasma levels in patients admitted to intensive care with systemic inflammatory response syndrome. By using the in-house antibodies and recombinant CL-11, we found that CL-11 can bind to zymosan independently of calcium by a separate site from the carbohydrate-binding region. Finally, we showed that CL-11/MASP-2 complexes trigger C4b deposition on zymosan. In conclusion, we have developed a specific and sensitive ELISA to investigate the ever-expanding roles of CL-11 in health and disease and shown a novel interaction between CL-11 and zymosan

Success Rate of Split-Thickness Skin Grafting of Chronic Venous Leg Ulcers Depends on the Presence of Pseudomonas aeruginosa: A Retrospective Study

The last years of research have proposed that bacteria might be involved in and contribute to the lack of healing of chronic wounds. Especially it seems that Pseudomonas aeruginosa play a crucial role in the healing. At Copenhagen Wound Healing Centre it was for many years clinical suspected that once chronic venous leg ulcers were colonized (weeks or months preoperatively) by P. aeruginosa, the success rate of skin grafting deteriorated despite aggressive treatment. To investigate this, a retrospective study was performed on the clinical outcome of 82 consecutive patients with chronic venous leg ulcers on 91 extremities, from the 1st of March 2005 until the 31st of August 2006. This was achieved by analysing the microbiology, demographic data, smoking and drinking habits, diabetes, renal impairment, co-morbidities, approximated size and age of the wounds, immunosuppressive treatment and complicating factors on the clinical outcome of each patient. The results were evaluated using a Student T-test for continuous parameters, chi-square test for categorical parameters and a logistic regression analysis to predict healing after 12 weeks. The analysis revealed that only 33,3% of ulcers with P. aeruginosa, isolated at least once from 12 weeks prior, to or during surgery, were healed (98% or more) by week 12 follow-up, while 73,1% of ulcers without P. aeruginosa were so by the same time (p = 0,001). Smoking also significantly suppressed the outcome at the 12-week follow-up. Subsequently, a logistic regression analysis was carried out leaving P. aeruginosa as the only predictor left in the model (p = 0,001). This study supports our hypothesis that P. aeruginosa in chronic venous leg ulcers, despite treatment, has considerable impact on partial take or rejection of split-thickness skin grafts

An in vitro collagen perfusion wound biofilm model; with applications for antimicrobial studies and microbial metabolomics

BackgroundThe majority of in vitro studies of medically relevant biofilms involve the development of biofilm on an inanimate solid surface. However, infection in vivo consists of biofilm growth on, or suspended within, the semi-solid matrix of the tissue, whereby current models do not effectively simulate the nature of the in vivo environment. This paper describes development of an in vitro method for culturing wound associated microorganisms in a system that combines a semi-solid collagen gel matrix with continuous flow of simulated wound fluid. This enables culture of wound associated reproducible steady state biofilms under conditions that more closely simulate the dynamic wound environment. To demonstrate the use of this model the antimicrobial kinetics of ceftazidime, against both mature and developing Pseudomonas aeruginosa biofilms, was assessed. In addition, we have shown the potential application of this model system for investigating microbial metabolomics by employing selected ion flow tube mass spectrometry (SIFT-MS) to monitor ammonia and hydrogen cyanide production by Pseudomonas aeruginosa biofilms in real-time. ResultsThe collagen wound biofilm model facilitates growth of steady-state reproducible Pseudomonas aeruginosa biofilms under wound like conditions. A maximum biofilm density of 1010 cfu slide-1 was achieved by 30 hours of continuous culture and maintained throughout the remainder of the experiment. Treatment with ceftazidime at a clinically relevant dose resulted in a 1.2 – 1.6 log reduction in biofilm density at 72 hours compared to untreated controls. Treatment resulted in loss of complex biofilm architecture and morphological changes to bacterial cells, visualised using confocal microscopy. When monitoring the biofilms using SIFT-MS, ammonia and hydrogen cyanide levels peaked at 12 hours at 2273 ppb (±826.4) and 138 ppb (±49.1) respectively and were detectable throughout experimentation. ConclusionsThe collagen wound biofilm model has been developed to facilitate growth of reproducible biofilms under wound-like conditions. We have successfully used this method to: (1) evaluate antimicrobial efficacy and kinetics, clearly demonstrating the development of antimicrobial tolerance in biofilm cultures; (2) characterise volatile metabolite production by P. aeruginosa biofilms, demonstrating the potential use of this method in metabolomics studies

The detrimental impact of extracellular bacterial proteases on wound healing

In addition to clinical signs of infection (e.g. inflammation, purulence and pain), a microbial count of ≥105 colony‐forming units/g has historically been used to define wound infection. However, it is increasingly recognised that, rather than a high bioburden level alone being detrimental to wound healing, it is the virulence of the invading microorganism and the host's immune status that can affect clinical outcomes. Bacteria, such as Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis, have developed a range of virulence factors to help them overcome host defences and proliferate within the underlying soft tissue. More specifically, bacterial proteases are one such virulence factor that has been implicated in promoting the invasion and destruction of the host tissue. Because of the complexities of microorganisms, the proteases can negatively impact the wound environment, leading to delayed wound healing. The aim of the present paper is to describe various extracellular bacterial proteases; review the impact they have on the wound environment, the host immune response and biofilms; and discuss potential wound management strategies against them. The evidence discussed suggests that proteases may play a profound role in wound infections, contribute to the development of an inflammatory response and impede wound healing

Advanced therapeutic dressings for effective wound healing

Advanced therapeutic dressings that take active part in wound healing to achieve rapid and complete healing of chronic wounds is of current research interest. There is a desire for novel strategies to achieve expeditious wound healing due to the enormous financial burden worldwide. This paper reviews the current state of wound healing and wound management products, with emphasis on the demand for more advanced forms of wound therapy and some of the current challenges and driving forces behind this demand. The paper reviews information mainly from peer reviewed literature and other publicly available sources such as the FDA. A major focus is the treatment of chronic wounds including amputations, diabetic and leg ulcers, pressure sores, surgical and traumatic wounds (e.g. accidents and burns) where patient immunity is low and the risk of infections and complications are high. The main dressings include medicated moist dressings, tissue engineered substitutes, biomaterials based biological dressings, biological and naturally derived dressings, medicated sutures and various combinations of the above classes. Finally, the review briefly discusses possible prospects of advanced wound healing including some of the emerging approaches such as hyperbaric oxygen, negative pressure wound therapy and laser wound healing, in routine clinical care