BIRN - Burns Injury Research Network
BIRN - Burns Injury Research Network
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Resources & Facilities

Introduction to the St. Andrew's, Broomfield Burn Service

The St Andrews Centre for Plastic Surgery and Burns was established at St Andrew's Hospital, Billericay in 1973 as the regional and supra-regional centre for specialised Plastic Surgery and Burn Treatment. It has been a pioneer in burn treatment since its inception, and has gained a national and international reputation. The Centre was relocated in April 1998 into Broomfield Hospital, Chelmsford, and the concept of the Centre is that of a Supra-Regional Unit within a hospital to maintain clinical excellence. The purpose built Burn Unit was opened in 1998. There are 20 beds. These comprise of 4 large intensive care rooms and 4 high dependency rooms for acute care. There are 12 low dependency beds for patients with smaller injuries. There is an integral operating theatre with anaesthetic room and recovery area, a purpose built admissions room, and laboratories for clinical investigation and research purposes including skin culture, and support areas. Adjacent to the unit is a specialist Burns Outpatient facility. Burn care is delivered by a multidisciplinary team, including Surgeons, Anaesthetists, Intensivists, Paediatricians, Microbiologists, Specialist Nurses (including intensive care, burn, paediatric), Physiotherapists, Occupational therapists, Dieticians, Pharmacists, and Psychotherapists. This burn team works together closely to optimise acute and rehabilitative care in terms of survival, function, aesthetics and psychosocial outcome. The philosophy of the unit is to endeavour to restore the burn victim as much as possible to their pre-injury activities and environment.

Burned patients are referred from Essex and East Anglia, London, the South East England and beyond.   Patients are referred for assessment, resuscitation, intensive care, surgical treatment, and rehabilitation. Over 700 patients are admitted to the unit each year. Over a hundred of these patients have severe or life threatening burn injuries. St Andrews burn centre admits nearly 10% of all patients admitted with burn injury in the UK. In addition to this approximately 400 new patients receive their care on an outpatient basis.

The National Burn Care Review has recognised the unit as a burns centre. The St Andrews Burn Centre is unique in the UK in having three dedicated burn surgeons delivering a comprehensive clinical service on a daily basis. They are supported by a team of dedicated anaesthetists and intensive care specialists.

Collaborations in burns research between CCR and St Andrew's

Collaborative research between St Andrew's Billericay and CCR started soon after the inception of the laboratory with the personal support of Mr Mike Hackett who saw the potential of such collaboration and funded both the laboratory development and many of the first Clinical Research Fellows who worked in the laboratory until his early death. Additionally, close research relationships were developed with Prof Roy Sanders in the development of RAFT at Mount Vernon Hospital, and for animal work with Prof Colin Green at Northwick Park. A research collaboration was also established with Robin Martin and the Blond-McIndoe Centre, East Grinstead. One of the earliest appointments to the group was a keratinocyte biologist, Harshad Navsaria, who provided support for early collaborations and has developed an independent programme of tissue engineering (see publication list). The collaboration has been particularly successful in sponsoring research fellows in plastic surgery training, many of whom are now established consultants in the UK including Bruce Philp and Simon Myers himself. Tissue engineering research is currently jointly providing a producer unit for cultured keratinocytes to Broomfield Hospital and research activity in tissue engineering.

Existing research programmes

Externally funded programmes each led by a senior academic and employing over 80 researchers

These research teams are :

  1. Cell immortalisation and senescence (David Beach)
  2. Barrier function (Carolyn Byrne)
  3. Genetically inherited skin disease (David Kelsell)
  4. Epidermal Stem Cells (Ian Mackenzie)
  5. Tissue engineering (Harshad Navsaria)
  6. Keratinocyte migration and invasion (Edel O'Toole)
  7. Hair biology (Mike Philpott)
  8. Clinical cancer programme (Charlotte Proby)
  9. Prostate cancer (David Prowse)
  10. Epidermolysis Bullosa (Andy South)
  11. HPV and cancer (Alan Storey)

Existing clinical research projects in wound healing and tissue engineering

1. Wound Healing (Simon Myers) (Lon.), PhD 1999 1 Wound healing is a complex biological process involving the coordinated response of multiple cell types: keratinocytes, fibroblasts, endothelial cells and immune cells in the processes of cell proliferation, migration, angiogenesis and scar matrix remodelling. The unique nature of burn injury causes deregulation of many of these processes with resultant delay in healing, hypertrophic scarring and poor function and cosmesis. Studies in progress:

  1. Growth factor profiles have been established in a suction blister model – a controlled human model of superficial partial-thickness cutaneous insult, and are being extended to follow gene expression longitudinally in wounds of different depths in porcine models and human wounds This work forms a sound base for specific investigation of factors, and assessment of their therapeutic benefit (see proposal in supplementary papers)

  2. Antimicrobial peptides Using RT-PCR and in situ hybridization (ISH), human beta defensin-1 and -2 transcripts have been localised to keratinocytes within interfollicular skin and the hair follicle This work was extended to gastric mucosa where HBD-2 was markedly upregulated in a dose and time dependent manner following H pylori and proinflammatory stimuli 2-4

  3. Hypertrophic scarring and keloids Hypertrophic scarring most commonly occurs when epithelialization has been delayed during, for example, the healing of deep dermal burn wounds, and is considered a dermal pathology in which the epidermis has only a passive role. However, in a previous study we showed activation of the epidermis of hypertrophic wounds and keloids with expression of hyperproliferative keratins K6, K16 and K17 by immunochemistry, and ISH 5. These studies are being pursued in a dedicated nurse-led keloid clinic with a clinical research fellow (E Anthony)

  4. Psychotherapy & Counselling Research (Nancy Cohen) Under the supervision of a Consultant Psychotherapist, body image, mood and quality of life have been investigated in young people burned in childhood in a doctorate thesis. The experiences of senior medical staff of different professional groups have been explored in an MSc

2. Tissue Engineering/Somatic Stem Cell Therapy & Ex Vivo Gene Therapy (Harshad Navsaria)

Over the last twenty years, our work has focused on the biology of cultured keratinocytes using complex three-dimensional organo-typical cultures, preclinical animal models in rodents and pigs, and their clinical application to acute and chronic wounds in humans.   Investigations into the use of delivery systems, dermal matrices, hair follicle transplantation, and the biology of transplantation of allogeneic grafts have led to further clinical refinement.

The CCR is a thus  provider unit for cultured keratinocytes to patients with large burn injuries at The Chelsea & Westminster burn service, and Broomfield burn service, using a variety of delivery systems. This programme currently involves three Clinical Research Fellows who are Plastic Surgeons: Mobin Syed [PhD student], Edwin Anthony [MD student], and Partha Vaiude. Ongoing research projects in this area include studies to improve graft structure, identify derived therapeutic agents, and investigate both somatic and embryonic stem cell grafts. Ex vivo gene therapy to treat patients with RDEB is also under way.

  1. Development and application of new dermal templates New dermal templates are currently being evaluated in acute burns at Broomfield. The application of cultured epidermal autografts and allografts is improved by the use of dermal templates including Alloderm and Integra. However results are still suboptimal and new dermal agents are needed, which requires a fundamental understanding of matrix expression and organisation during normal wound healing, and of epidermal-mesenchymal interactions in vitro and in vivo

  2. Improved cultured epidermal autografting (CEA) Research on CEA has been a traditional line of collaborative investigation between Broomfield and CCR and is currently addressing the synthesis of dermo-epidermal junction proteins and the long-term viability of the grafts. The development of composite dermo-epidermal grafts in the laboratory to create more sophisticated skin replacements are being evaluated including the inclusion of skin adenexae and melanocytes

  3. Allogenic transplantation Adequate autologous skin is often not available in major burns, so the use of allogeneic materials and tissue banking has been intimately linked with burn care. The advent of new anti-rejection therapies raises the possibility of allogenic skin and composite graft transplantation. Reports of hand transplantation have shown that full thickness skin transplantation is possible with immunosuppression

  4. Ex vivo gene therapy In autosomal recessive skin diseases marked by absence of type VII collagen and laminin 5 (RDEB and JEB), keratinocytes have been transduced in vitro to express the missing proteins both in vitro and in transplants onto SCID mice. Clinical trials of ex vivo gene therapy using corrected keratinocytes are therefore in preparation and require the technologies developed for burns patients in CEA grafting

  5. Bronchial epithelium Human bronchial epithelial cells have been cultured from bronchial brushings, which retain their in vivo phenotype, and immortal cell lines using HPV constructs are being developed to model inhalational injury and to study in-vitro toxicology on bronchial epithelium and for tissue engineering

References

  1. Keratinocyte growth and differentiation in cutaneous wound healing and cultured keratinocyte grafting
  2. Ali RS et al J Invest Dermatol 2001;117; 106-11
  3. Bajaj-Elliot M, et al. Gut 2002 51:356-61
  4. Chronnell CM, et al J Invest Dermatol. 2001 Nov;117(5):1120-5
  5. Machesney M, et al Am J Pathol. 1998 May;152(5):1133-41

Postgraduate Clinical Research Training

The following plastic surgical research fellows have trained for higher degrees with in the CCR (MS, MD and PhD).
B=Billericay/Broomfield; R=RAFT funded. CRF=Clinical Research Fellow

Kevin Hancock (B)
Culturing keratinocyte for clinical application CRF

Anne Brain (B)
Allogenic keratinocyte survival CRF

Nigel Carver (R)
Allogenic keratinocyte grafting (MS 1992)

Loshan Kangesu (R)
Significance of dermis for epidermal grafting (MS 1994)

Simon Myers (R)
Cytokines in wound healing (PhD 1999)

Satvinder Muddan
Essential fatty acids and keratinocytes(MD 1999)

Bruce Philps (R)
Models of dermal replacement (RAFT)

Paul Harris
Epithelial-Mesenchymal interactions (MD 1999)

Mark Ho-Asjoe (B)
Fas ligand and cell immunity

Mike Machesney (R)
Hypertropic Scarring (MD 2004)

Rosina Ali (B)
Role of Defensins (MD 2004)

Richard Price (B)
Hyaluronic acid-Clinical application (MD 2003)

Christian Duncan
Keratinocyte delivery systems (MPhil 2002)

Matthew Griffiths (B)
Apligraf clinical studies (MD 2004)

Mobin Syed (B)
Defensins in wound healing (PhD 2006)

Communication strategy

As the network involves many sites in the Southeast, thought has been given to the need for a formal communication structure, in addition to mutual recognition of key investigators by Honorary academic and NHS consultant contracts. Thus a dedicated web site has already been established (www.burnshealing.com) and a WebCT teaching site has been developed for undergraduate and postgraduate teaching. Web cameras have been purchased to enable video conferencing.

Education strategy

In addition to substantial undergraduate teaching, and a large number of PhD students, the CCR has been developing a series of distance learning, now WebCT based e-learning, educational courses. A postgraduate diploma in Clinical Dermatology (Director Vicky Jolliffe) has been in place for over 5 years and provides a year long, 30 module course in the basics of clinical dermatology for general practitioners wishing to develop a special interest (GPSIs). This has become available to overseas students from May 2006 (Director Ginny Hubbard). A postgraduate diploma is aesthetic surgery commenced in October 2006. There is also significant infrastructure to deliver educational programmes (3 educational administrators), to allow the development and delivery of short courses, a postgraduate diploma in burn care, and a modular based MSc in Plastic surgery in the near future.

Commercialisation

Through its Code of Practice for the Exploitation of Intellectual Property (IP), QMUL actively encourages the translation of academic research into commercial outputs. The IP exploitation process is implemented by "Innovation and Enterprise", the technology transfer office at QMUL, consisting of a team with appropriate business and technology transfer experience and expertise. Innovation and Enterprise is active in developing commercial opportunities for a wide range of QMUL technologies. It has been responsible for negotiating and completing over 150 commercial and research contracts, including recent licensing deals with Biotech and Pharma. Where appropriate, QMUL also encourages IP exploitation with a number of existing spin-out companies that have been successful in attracting investment. In developing its companies, Innovation and Enterprise has adopted an approach which involves building a close working relationship between the founders, funders and technology transfer personnel.

Research Proposals The Science Case for Clinical Research Director and Non-clinical Chair of Burns Research

Phase I : Components of Proposed Programme

This integrated research proposal encompasses acute care, wound healing and rehabilitation and aims to bring the laboratory to the bedside to improve care for burn patients.

Clinical Research Network

The Burn Injury research Network is located within the University of London in facilities provided by Barts and the London School of Medicine and Dentistry, Queen Mary University of London. It is housed within the new research laboratories provided by the Institute of Cell and Molecular Science (ICMS) as a new team within the Centre for Cutaneous Research, led by nomination to the position of the Clinical Research Director of Mr Simon Myers, an experienced burn surgeon, who has recently been recruited to the CCR as a Clinical Senior Lecturer. However the clinical burns research network requires satellite clinical research centres and use of existing satellite laboratories within partner NHS Trusts, and dedicated research staff on those sites together with a strategy to maintain the links between the sites. The research will be directed by the Clinical Research Director in collaboration with the current consultant staff who have been nominated for honorary Clinical Senior Lecturerships, and executed by Clinical Research Fellows and Research Nurses.

Clinical Research Programme directed by Simon Myers

The scope of the clinical studies that could be produced over a 10 year period from the clinical team (Clinical Director, Senior lecturers/consultants, clinical research fellows and research nurses) are so extensive that they are described in brief. The studies will have collaborations with work going on in the CCR already. They fall into three categories:

Mechanisms of burn wound healing

There is a need for a more complete understanding of the mechanisms of burn wound extension & burn wound healing, particularly the cellular interactions and signalling pathways activated in wound healing following burn injury. These studies can be performed in surgical specimens removed as part of patient care following suitable LREC approval and informed consent - for example:

The expression of growth factors and cytokines in the burn wound: a spatiotemporal study using micro-array and proteomics approaches

A detailed study of the cytokine and growth factor expression will be undertaken with biopsies from burn wound base and edge at different time points following injury. Studies by immunochemistry and ISH will be extended from the suction blister model to include modern investigative techniques: expression arrays and proteomics using high density 2D gel maps and mass spectrometry. All these facilities are available as core facilities in the ICMS.

Analysis of the mechanisms of burn wound extension

Following burn injury a zone of stasis appears around the edge with progressive microvascular deterioration, mediated in part by the local inflammatory response, release of prostaglandins/thromboxanes, free radical production by histamine activation of the xanthine oxidase pathway, hypernatraemic effects on neutrophil function and thrombomodulin shedding. Cell signalling pathways involved in wound extension will be studied in human burn wound material, and animal models will be established.

How are stem cells replaced in burn wounds?

The identification of stem cells in vivo has been assisted by the identification of novel stem cell markers which will be applied to burn edges including the patent protected MCSP marker. In addition, generative potential will be assessed by colony forming assays and the development of holoclones, meroclones and paraclones from isolated keratinocyes in the wound edge.

The complications of burn injury:
These include both local and systemic effects

The role of fibroblast senescence in the development of hypertrophic scarring

Studies have shown that abnormalities in hypertrophic scarring affect epidermal-mesenchymal interactions. The lack of a suitable animal model means that many studies have been performed in vitro. Normal fibroblasts undergo senescence and we hypothesise that the hypercellularity of the hypertrophic scar could result from failure of these mechanisms. Understanding the regulation of this process could lead to therapeutic interventions

The response of the bone marrow to major cutaneous injury

Thermal injury quantitatively and qualitatively alters hematopoiesis. Granulocyte and macrophage production after burn injury or burn wound infection is significantly reduced, and further compromised by endotoxin. Hematopoietic stem cells normally proliferate and mature into lymphoid, erythroid, and myeloid precursor cells, but the balance of these cell populations is modulated by major thermal injury, with or without sepsis favouring the monocyte/macrophage lineage. Granulocyte colony-stimulating factor-treated animals exhibit significantly less bone marrow suppression although, G-CSF levels in burn injury are elevated, and neutropenia and myeloid maturation arrest correlate with reduction in bone marrow G-CSF receptor expression. Prostaglandin PGE2 can, in part, also restore the balance in bone marrow granulocyte and monocyte production. Understanding the dynamics of the different precursor pools could be used to identify patients at greater risk for systemic inflammatory sequelae following major thermal injury.

Metabolic Consequencies of Major Cutaneous loss, Burn Sepsis, and Multi-Organ Failure: Modulation of the Cytokine Storm

The metabolic response to large burn injury is complex, including skeletal muscle proteolysis, lipolysis, gluconeogenesis, increased metabolic rate, and a severe systemic inflammatory response induced by the injury, infections, and surgical procedures. Hypermetabolism is mediated by hormones – catecholamines, glucagon, and cortisol – and by cytokine and lipid mediators – inlerleukin-1, interleukin-6, tumour necrosis factor. These responses are interlinked, since cytokines activate the hypothalamo-adrenal axis. The systemic "cytokine storm" and inflammation may develop into shock and multiple organ failure if the primary insult is overwhelming or a second inflammatory insult such as sepsis triggers exaggerated inflammation – the two-hit phenomenon.

Pharmacological modulation of the exaggerated inflammatory response in burn primed patients may prevent organ dysfunction. Corticosteroid treatment and administration of cytokine synthesis inhibitors are potential approaches.

Therapeutic Interventions in Burn Injury

Opportunities will arise not only as a direct effect of the research in CCR but also from advances in the medical and surgical management of burns including:

Skin Engineering – Developments involving Integra and Recell

Integra dermal regeneration template has become an accepted reconstructive option in burn care providing wound closure post-early near-total wound excision with the potential to limit late scar morbidity. The Recell technique provides a suspension of autograft keratinocytes without the delay of cell culture and by, for example, applying keratinocytes suspended in fibrin glue beneath Integra an improved autologous permanent wound closure for massive burn injuries may be achieved.

Embryonic Signalling Pathways and the Stem Cell Niche

Whilst the environmental stimuli and the specific cues that direct keratinocyte stem cell lineage and commitment are not well understood, a number of important developmental, molecular signalling pathways appear to play a major role in directing epidermal stem cell fate 6,7. Wnt/ß-catenin/Lef-1 constitutes one such pathway, and activation of this pathway appears to direct keratinocyte stem cells to follow hair follicle differentiation pathways 8-11. The Sonic Hedgehog (Shh) signalling pathway regulates proliferation of cells that undergo differentiation into hair follicle epithelium 12-13. However, de-regulation of the Shh pathway also leads to tumour development demonstrating the importance of tightly controlled pathways.

Antimicrobial Peptides in the Treatment of Burn Wounds

Human defensins have specific spectrums, but synergistic effects are achieved by various combinations. The induction of human defensins could provide a completely new strategy for the treatment of infections, perhaps avoiding the problems of acquired resistance seen with current antibiotics particularly in burn care. The production of human defensins via genetic engineering is the preferred method, either by cell-based or cell-free protein expression systems. Understanding the post-translational modification of these, and other, antimicrobial peptides may allow their incorporation and active release from biological dressing systems, dermal regeneration templates, and cultured cell delivery systems. It will also be important to understand the in vivo consequencies of upregulation, or modification of expression profiles.

Hepatocyte Growth Factor Expression in Burns: the Effect of Exogenous Application

This Study is Currently able to be Pursued as soon as a Clinical Fellow in Recruited.

References

  1. Kaufman CK, Zhou P, Pasolli HA, Rendl M, Bolotin D, Lim KC, Dai X, Alegre ML, Fuchs E. GATA-3: an unexpected regulator of cell lineage determination in skin. Genes Dev 2003; 17: 2108-22

  2. Jamora C, DasGupta R, Kocieniewski P, Fuchs E. Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 2003; 422: 317-22

  3. Zhou P, Byrne C, Jacobs J, Fuchs E. Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate. Genes Dev 1995; 9: 700-13

  4. Huelsken J, Vogel R, Erdmann B, Cotsarelis G, Birchmeier W. beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 2001; 105: 533-45

  5. Merrill BJ, Gat U, DasGupta R, Fuchs E. Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin. Genes Dev 2001; 15: 1688-705

  6. DasGupta R, Rhee H, Fuchs E. A developmental conundrum: a stabilized form of beta-catenin lacking the transcriptional activation domain triggers features of hair cell fate in epidermal cells and epidermal cell fate in hair follicle cells. J Cell Biol 2002; 158: 331-44

  7. St-Jacques B, Dassule HR, Karavanova I, Botchkarev VA, Li J, Danielian PS, McMahon JA, Lewis PM, Paus R, McMahon AP. Sonic hedgehog signaling is essential for hair development. Curr Biol 1998; 8: 1058-68

  8. Wang LC, Liu ZY, Gambardella L, Delacour A, Shapiro R, Yang J, Sizing I, Rayhorn P, Garber EA, Benjamin CD, Williams KP, Taylor FR, Barrandon Y, Ling L, Burkly LC. Regular articles: conditional disruption of hedgehog signaling pathway defines its critical role in hair development and regeneration. J Invest Dermatol 2000; 114: 901-8

The Non-Clinical Professor of Burn Injury Study: Potential Research Themes

The major funding component of phase I will consist of a Non-Clinical Professor to investigate specific areas of infection and/or immunity in burns patients.

Burn Infection (Collaboration with M Curtis, D Wareham, Centre for Infectious Disease, ICMS)

Introduction

Under normal circumstances, a highly effective panel of integrated mechanical, chemical and biological systems protect the host from infections through the skin. The breakdown of this barrier function following major thermal injury coupled with the physical environment of abundant moisture, nutrients and gaseous supply provide the perfect setting for bacterial growth and rapid proliferation. Secondary infection of burn wound thus still constitutes one of the greatest threats to burns patients, who have survived the acute episode.

Current standard care involves systemic antibiotics (ß-lactams and aminoglycosides) or topical antimicrobial agents such as silver sulfadiazine. Whilst this approach has limitations in terms of penetration of partial and full thickness burns, limited efficacy against both Gram-negatives and Gram-positives and potential toxicity to human cells, the major obstacle to overcome in the management of burns wounds is treatment of infection by multiply antibiotic resistant bacteria: the most common pathogens now isolated from burn wounds are methicillin resistant Staphylococcus aureus (MRSA), multiply drug resistant Pseudomonas aeruginosa and Acinetobacter sp and increasingly vancomycin resistant enterococci (VRE). The problem of ongoing outbreaks with such resistant organisms has attracted considerable public and professional concern and in 2005 a working party under the auspices of the Health Protection Agency was set up to address the problem of multi-drug resistant Acinetobacters (Health Protection Agency, 2005).

The key target of preventing bacteraemia and sepsis due to bacterial infections of burn wounds remains a fundamental issue in burns care. Hence a major goal of the research lead by the Non Clinical Professor of burns injury will involve analysis of the bacterial determinants of pathogenesis in burns infections and, in the context of the wider interdisciplinary team, the identification of novel, candidate therapeutic strategies based on an improved understanding of the patho-physiology of the infected wound.

Background to Bacterial Research in Epithelial Pathogens Group, ICMS

Bacterial research in the ICMS is focused on the molecular basis of pathogenicity of Gram-negative bacteria at epithelial surfaces. The Centre of Infectious Disease has considerable expertise in the characterisation of bacterial virulence factors through both reverse genetic and directed mutagenesis strategies. This approach combines in-silico analyses of the genomes of diverse strains with the genes required for full virulence in model systems. This provides the perfect partnership through the ICMS for the development of microbial research in burns patients.

Microbial Virulence

The identification and characterisation of the major virulence determinants of Gram-negative organisms and from this the development of novel targeted therapeutic approaches is a major theme. A breach in the normal defensive barriers of the host, through for example thermal injury or co-infection by another organism, can dramatically influence the virulence of a given organism. Two examples of microbial virulence research currently underway are given below.

Degradative Enzymes

Extracellular and cell associated hydrolytic enzymes are considered to play an important role in the infection process not only by the generation of nutrients for bacterial growth but also by inactivation/degradation of host macromolecules involved in tissue homeostasis and host defence. Hence these enzymes represent potential targets for the development of novel treatment approaches. Proof of principle studies in P. gingivalis,   led to a larger applied genomics programme in the identification and validation of protease targets in other Gram negative bacteria including P. aeruginosa.   Less information is available with regard to the degradative capabilities of Acinetobacter sp. However, we have hypothesized that a protease of A. baumanii is able to degrade insulin leading to the development of glucose intolerance in burns patients 14, and an extracellular lipase produced by clinical isolates of this organism may cause   extensive damage to fat deposits seen in infected burns patients.

Type III Secretion Systems

Type III secretion systems (TTSS) are devices used by many Gram-negative pathogens to subvert the function of epithelial and immune cells and/or to facilitate invasion. A "molecular syringe" protrudes from the bacterial cell surface and inserts into the plasma membrane of the target cell transporting TTSS effector proteins into the cytoplasm where they interact with specific components. P. aeruginosa possesses a type III secretion system which enables delivery of at least four effector (Exo) proteins (ExoS, T, U and Y) directly into the cytoplasm of epithelial cells. Inactivation of Rho and Rac by the Rho-GAP activity of ExoS and ExoT leads to inhibition of both wound healing and tissue regeneration. Active or passive immunisation with recombinant PcrV, a component of the TTSS apparatus essential for secretion, was found to be entirely protective in a mouse burn model of P. aeruginosa infection. Similarly, administration of anti-PcrV monoclonal antibodies reduced mortality, inflammation and injury in an acute lung infection model 15. Further characterisation of the role and nature of the TTSS in burn wound isolates may enable similar approaches to be implemented in patients with infected burns

Serological Identification of Novel Virulence Determinants

Analysis of the human serum antibody response developed during an episode of infection provides a well established means of identifying gene products likely to have a role in the infection process. In-vivo induced antigen technology (IVIAT) uses sera from infected patients to probe an expression library for genes specifically expressed in-vivo. Using sera from patients who have recovered from A. baumannii infection, and comparing them with the sera of those who have simply been colonised with the organism, it would be possible to define those antigens that are expressed in-vivo during the establishment and progression of A. baumannii infection.

Regulation of Virulence

Pseudomonas aeruginosa can switch between a free-living (planktonic) or biofilm lifestyle, a versatility that enables it to thrive in many different environments contributing to its success as a human pathogen. The production of many bacterial virulence factors relies on a cell density dependent signalling system known as quorum sensing (QS) whereby bacteria secrete LMW molecules into the environment which are detected by the surrounding bacteria. The signalling molecules are often homoserine lactone substances, which act as autoinducers driving transcriptional regulation of QS responsive genes. Disruption of the P. aeruginosa QS system has been shown to influence both local and systemic spread of  P. aeruginosa in thermally injured skin. A number of interrelated systems have been identified in P. aeruginosa and S. aureus and may also be present in A. baumannii.   Characterisation of these systems may therefore help elucidate common mechanisms of pathogenesis, which could be open to modification with novel inhibitors of QS systems.

Proposed Areas of Study

Genomic Analysis of Burn Pathogens

Molecular analysis of the interaction of a bacterial pathogen with the human host using modern technologies is now fundamentally dependent on the complete genome sequence of that pathogen. This information enables rapid identification of genes from protein analyses, transcriptome analyses using microarrays, proteomic analysis via 2D gels and related separation techniques and description of co-ordinatedly regulated networks of genes involved in pathogenesis. The genome sequences of representatives of the predominant isolates from burns infections are already available but there are still significant gaps in our knowledge base. In particular, the genome sequence of Acinetobacter sp is not yet publicly available. As part of the broader initiative in burns microbiology, consideration will therefore be given to develop funding applications to work with the Sanger Centre to fully sequence a clinically relevant multi-drug resistant form of this species and other unsequenced pathogens relevant to burns microbiology.

Epidemiology and infection outcome Although genomic analyses of sequenced strains may give important information on the bacterial determinants involved in burn wound infection, clinical isolates often differ markedly from those chosen for sequencing. Acquisition of foreign DNA in the form of plasmids, transposons and integrons may lead to increased virulence as well as additional antimicrobial resistance. In order to develop better infection control and treatment strategies there is a need to understand where and why virulent and resistant strains evolve and the dynamics of transmission. Multi-locus sequence typing (MLST) which relies on DNA sequence comparison of fragments of protein encoding housekeeping genes has become the gold standard typing technique and is being applied to multi drug resistant isolates of A. baumannii. Correlation of the molecular epidemiology of burn wound pathogens with the type and severity of human infection may help to identify virulent clones, distinguish between colonisation and infection and target or rationalise antimicrobial therapy.

Model System Development for the Analysis of Pathogen/Epithelium Interactions

More sophisticated models, employing higher organisms, attempt to reproduce the complex interaction of bacterial virulence factors with the host immune defence. Such an approach has been used by us to identify the importance of P. aeruginosa virulence factors in-vivo using the nematode C. elegans. This model has additional advantages enabling gene expression in-vivo to be studied using green fluorescent protein (GFP) or RT-PCR reporter assays. A collection of C. elegans innate immunity mutants are also available enabling bacterial pathogenicity to be studied in an immunocompromised background. As C. elegans is susceptible to most burn wound pathogens such as P. aeruginosa, S. aureus and more recently A. baumannii 16 it could be used for high throughput screening of clinical isolates to identify virulent strains.

References

  1. D. Furniss, S. Gore, B. Azadian, SR Myers.  JBCR. 2005:26(5):405-408

  2. 15. Sawa T, Yahr TL, Ohara M, Kurahashi K, Gropper MA, Wiener-Kronish JP, Frank DW. Nat Med.1999 Apr;5(4):378-9

  3. 16. Smith MG, Des Etages SG, Snyder M. Mol Cell Biol. 2004 May;24(9):3874-84

B. Burn Care and Immunity (Collaboration with Dan Pennington, Immunology, ICMS)

Innate Immune Response

Human immunological defences consist of two immunological subsystems - innate and adaptive. The innate immune response is a collection of host defences from barrier function to highly selective recognition of pathogens; which give together a rapid and blunt response to infection and tissue destruction. The adaptive immune system, in contrast uses antigen receptor genes encoding receptors to any antigen, but is slower to respond and requires instruction from the innate immune response.

It is recognized that extensively burned patients have an increased susceptibility to infection and often succumb to multiple organ failure related to sepsis. Numerous investigations performed over many years have demonstrated immunologic abnormalities in burn patients many of them in the innate responses eg. involving opsonin levels , the complement system , phagocyte and neutrophil function . More recently it has become clear that the innate immune system of the skin (dendritic as well as epithelial cell mediated) recognises pathogen associated molecular patterns (PAMP) using pathogen recognition receptors (PRP). The toll-like receptors (TLR) have 10 family members specific for microbial ligands, which direct pathogen killing and the release of proinflammatory cytokines and antimicrobial peptides or host defence peptides. Amongst the functions of these peptides are: activation and attraction of antigen presenting cells and lymphocytes, endotoxin neutralisation, promotion of healing and angiogenesis, modulation of the adaptive immune responses, inactivation of microbes through multiple effects at their membranes, and simultaneous attack of both internal and external targets in some groups. There are hundreds of receptors involved in innate immune recognition of PAMPs such as: bacterial lipopolysaccharide (LPS), peptidoglycan (PGN), lipoteichoic acids (LTA), etc. PAMPs have in common that they are produced by the pathogen not host, they are necessary for pathogen survival and pathogenicity, and they are shared by the pathogen class – so that all gram negative bacteria will be recognised by a PRP to LPS. The TLRs are an important group of PRPs, and link the innate with adaptive immune systems via activation of the NF- kB pathway.

In the skin, Langherhan's cells, monocytes and macrophages respond to bacterial LPS via TLR4 to produce IL-1, which in turn stimulates beta defensin-2 (HBD-2) synthesis in neighbouring keratinocytes 17. Burn injury primes the innate immune system for enhanced TLR2- and TLR4-mediated responses. Augmented TLR reactivity might contribute to the development of heightened systemic inflammation following severe injury 18. A previously unsuspected role for CD4(+)CD25(+) T regulatory cells in controlling host inflammatory responses via TLR after injury has been suggested 19.

Effector Molecules

Defensins are effector molecules of the innate host defense system with antimicrobial activity against a variety of pathogens, including microorganisms commonly found in burn units. beta-Defensins are variably expressed in the epithelia of skin and other organs.

Syndecan 1 is a major heparan sulfate proteoglycan present on many host cells involved in thermal injury. Syndecan 1 cleavage results in the release of intact, soluble proteoglycan ectodomains that have diverse roles in innate immunity.

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