A next-generation antimicrobial wound dressing: a real-life clinical evaluation in the UK and Ireland.

OBJECTIVE: To assess the effectiveness of a new, next-generation antimicrobial dressing (NGAD; AQUACEL Ag+ EXTRA dressing) in managing wound exudate, infection and biofilm, and facilitating progression toward healing. METHOD: Clinicians from the UK and Ireland selected stalled or deteriorating wounds that were considered to be compromised by infection and/or biofilm. Only the primary dressing was replaced by the NGAD, for up to 4 weeks or as deemed clinically appropriate; otherwise, standard protocols of care were used. Evaluation forms captured the baseline and final assessment characteristics of wound status, exudate levels, skin health, wound bed appearance, signs of infection and biofilm, and wound dimensions. RESULTS: In all, 29 wounds were suitable for inclusion in the final analysis. Following the NGAD evaluation, wound statuses were shifted from stagnant/deteriorating to mainly improved, exudate levels were shifted from moderate/high to moderate/low, and skin health was improved in 20 wounds (69%). Wound bed tissue types were shifted from largely suspected biofilm/sloughy tissue (76%) to largely granulation tissue (53%). All signs of clinical infection were reduced in average frequency, with biofilm suspicion falling from 76% to 45% of the cases. The median management period with the NGAD was 4.5 weeks, after which 26 wounds (90%) became smaller in size and 10 wounds (34%) completely healed. CONCLUSION: This real-life clinical evaluation of the NGAD suggests that its successful management of exudate, infection and biofilm is generally accompanied by notable improvements in wound health and size, and in some cases, complete healing. DECLARATION OF INTEREST: The authors are all employed by ConvaTec Ltd. but have no other conflict of interest to declare. Dressings were provided to the clinicians free of charge.

A clinical algorithm for wound biofilm identification.

Recognition of the existence of biofilm in chronic wounds is increasing among wound care practitioners, and a growing body of evidence indicates that biofilm contributes significantly to wound recalcitrance. While clinical guidelines regarding the involvement of biofilm in human bacterial infections have been proposed, there remains uncertainty and lack of guidance towards biofilm presence in wounds. The intention of this report is to collate knowledge and evidence of the visual and indirect clinical indicators of wound biofilm, and propose an algorithm designed to facilitate clinical recognition of biofilm and subsequent wound management practices.

A real-life clinical evaluation of a next-generation antimicrobial dressing on acute and chronic wounds.

OBJECTIVE: To assess the effectiveness of a new, next-generation antimicrobial dressing (AQUACEL Ag+ dressing) in facilitating healing in a variety of hard-to-heal wounds that may have been compromised by infection and/or biofilm. METHOD: This was an international, multi-centred, real-life, non-randomised evaluation involving patients with a wide variety of slow-, non-healing or deteriorating chronic and acute wounds. There were no strict inclusion or exclusion criteria and the clinicians were asked to use their discretion in the selection of patients. The clinicians continued to use their standard protocol of care but replaced their existing primary wound-contact dressing with the next-generation antimicrobial dressing (NGAD) for up to 4 weeks. Clinicians could extend the treatment period if this was deemed clinically appropriate. Baseline assessments included wound bed characteristics, exudate level, indicators of wound biofilm, and signs and symptoms of infection. At the final assessment, the investigators reported the wound size, wound bed characteristics, and exudate level. RESULTS: A total of 121 patients were recruited into the original evaluation, of which eight were excluded for incomplete data sets. Most wounds (73; 64%) were either venous leg ulcers (59; 52%) or diabetic foot ulcers (14; 12%). At baseline, the wounds of (26; 23%) patients were slowly improving, 65 were stagnant (58%) and 22 (19%) were deteriorating. Just under three-quarters (74%) of the wounds had suspected biofilm (criteria including failure of a wound to heal, lack of response to topical and systemic antimicrobial agents, or the presence of slimy substances on the wound surface). Following the evaluations, the average wound closure achieved for all wounds was 72.6%, 19 (17%) wounds healed, 47 (42%) achieved at least 90% wound closure, and 71 (63%) achieved at least 75% closure. The average treatment period was 4.1 weeks; 35 wounds were treated with the dressing for more than 4 weeks. Cost analysis indicated that potential antimicrobial dressing cost reductions of approximately 30% were realised using the NGAD. CONCLUSION: This real-life, non-randomised evaluation provides encouraging evidence that the NGAD may have a role to play in facilitating wound progression towards healing by helping to eliminate the biofilm barrier. DECLARATION OF INTEREST: M. Walker, D. Metcalf, D. Parsons and P. Bowler are all employees of ConvaTec Ltd. Aysha Mendes da Mata is an independent writer and Annemarie Brown is an independent clinician, both received a fee and support from MA Healthcare to write up the evaluation using data supplied by ConvaTec.

A clinical algorithm for wound biofilm identification.

Recognition of the existence of biofilm in chronic wounds is increasing among wound care practitioners, and a growing body of evidence indicates that biofilm contributes significantly to wound recalcitrance. While clinical guidelines regarding the involvement of biofilm in human bacterial infections have been proposed, there remains uncertainty and lack of guidance towards biofilm presence in wounds. The intention of this report is to collate knowledge and evidence of the visual and indirect clinical indicators of wound biofilm, and propose an algorithm designed to facilitate clinical recognition of biofilm and subsequent wound management practices.

Two distinct types of murine blast colony-forming cells are multipotential hematopoietic precursors.

Two types of blast colonies can be stimulated to develop in semisolid agar cultures of murine bone marrow cells. Typically, these are either multicentric colonies stimulated by stem cell factor (SCF) plus interleukin-6 (IL-6) or dispersed colonies stimulated by Flt3 ligand (FL) plus IL-6. Both types of blast colony-forming cells (BL-CFCs) can generate large numbers of lineage-committed granulocyte-macrophage progenitor cells and exhibit some capacity for self-generation and the formation of eosinophil and megakaryocyte progenitor cells. However, the two populations of BL-CFCs are largely distinct and partially separable by fluorescence-activated cell sorting and are distinguished by differing capacity to form granulocyte-committed progeny. Both types of BL-CFCs can generate dendritic cells and small numbers of lymphocytes but the FL-responsive BL-CFCs have a greater capacity to form both B and T lymphocytes. Both types of blast colonies offer remarkable opportunities to analyze multilineage commitment at a clonal level in vitro.

The excess numbers of peritoneal macrophages in granulocyte-macrophage colony-stimulating factor transgenic mice are generated by local proliferation.

Mice transgenic for the hemopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit a sustained elevation of GM-CSF levels and a 50-100-fold elevation of peritoneal macrophage cell numbers. The excess cell numbers were found to be generated in pre-adult life, with numbers remaining relatively constant thereafter. In the pre-adult period, no abnormalities were noted in the number or composition of blood, bone marrow, or spleen cells, the type or number of GM progenitor cells in the marrow or spleen, or the rate of appearance of newly formed monocytes in the peripheral blood. Peritoneal macrophages in pre-adult transgenic mice exhibited elevated mitotic activity and, after tritiated thymidine labeling, a more rapid accumulation of labeled progeny. The increase in peritoneal macrophage cell numbers appears, therefore, to be based on a GM-CSF-induced increase in local proliferative activity by peritoneal macrophages. This increased activity declined at the age of 8-10 wk, in parallel with a change in the morphology of the transgenic macrophages and an increase in binucleate and multinucleate macrophages arising by cell fusion. This change in macrophage phenotype was restricted to the transgenic mice and may therefore be a consequence of continued overstimulation by GM-CSF.

Analysis of rat hemopoietic cells on the fluorescence-activated cell sorter. I. Isolation of pluripotent hemopoietic stem cells and granulocyte-macrophage progenitor cells.

A scheme is presented whereby pluripotent hemopoietic stem cells (PHSC) from rat bone marrow can be enriched 320-fold with the aid of the fluorescence- activated cell sorter. This scheme is based on the observations that PHSC are strongly positive for Thy-1 antigen (upper 10th percentile); have light- scattering properties (size distribution) between those of bone marrow lymphocytes and myeloid progenitor cells; and are relatively resistant to cortisone. It is estimated that PHSC may constitute 80 percent of the cells isolated according to these parameters. Candidate PHSC are described at the light and electron microscopic levels. At least two populations of accessory cells appear to influence the number and/or the nature of the hemopoietic colonies that form in the in vivo spleen colony-forming unit assay. Putative amplifier cells are strongly Thy-1(+) and cortisone sensitive; putative suppressor cells are weakly Thy-1(+) and cortisone resistant. Three subsets of granulocyte (G) -macrophage (M) progenitor cells (in vitro colony-forming cells [CFC]) are identified on the basis of relative fluorescence intensity for Thy-1 antigen: G-CFC are strongly Thy-l(+); M-CFC are weakly Thy-l(+); and cells that produce mixed G and M CFC have intermediate levels of Thy-1. GM-cluster-forming cells and mature G and M are Thy-1(-). The results suggest that G-CFC are bipotential cells that give rise to G and M-CFC; and that the latter produce mature M through a cluster- forming cell intermediate. Thy-1 antigen is also demonstrated on members of the eosinophil, megakaryocyte, erythrocyte, and lymphocyte cell series in rat bone marrow. In each instance, the relative concentration of Thy-1 antigen is inversely related to the state of cellular differentiation.

Autoradiographic analysis of lymphopoiesis and lymphocyte migration in mice bearing multiple thymus grafts.

Lymphopoiesis was studied in 3-month-old normal C57Bl mice and in 3-month-old C57Bl mice carrying from 12 to 48 C57Bl thymus grafts using tritiated thymidine labeling. Thymus graft lymphopoiesis was found to be identical with that of normal thymus tissue and the presence of thymus grafts was found to have no influence on host thymus lymphopoiesis. No evidence was found that the massive amounts of thymus graft tissue in the mice affected any parameter of host lymph node lymphopoiesis nor was any evidence detected for the migration of thymic lymphocytes from these massive deposits of thymus graft tissue either to host lymph nodes and blood or to other organs in the host animal. It is concluded that the majority of small lymphocytes produced in the thymus and thymus graft tissue do not migrate from these tissues but die locally at the end of their intrathymic life span of 3 to 4 days.

Induction of bone marrow colony-stimulating activity by a filterable agent in leukemic and normal mouse serum.

1. Leukemic Swiss mice of ICR/Ha strain which had been injected at birth with a lymphoid-leukemia-inducing virus preparation yielded sera which produced elevations of serum colony-stimulating activity within 16 hr and significant plasma-LDH-enzyme elevation at 4 days when injected intraperitoneally into normal ICR/Ha Swiss mice. Colony-stimulating activity was assayed in vitro by the stimulation of hemopoietic colony formation by DBA/1 bone marrow cells. 2. The inducing agent in leukemic serum was passageable, filterable, sedimentable, and heat-, ether-, and UV-labile. 3. A similar agent was recovered from normal Swiss serum after blind serial passages through normal mice. 4. LDH elevating virus induced a similar elevation of serum colony-stimulating activity when injected at high titers, and cross-resistance was demonstrated between LDH virus and the passaged leukemic serum agent.

Growth of factor-dependent hemopoietic precursor cell lines.

Cell lines have been produced from long-term cultures of mouse bone marrow that require a factor, present in WEHI-3 conditioned medium (CM) or in spleen CM, for their sustained growth. The cell lines were obtained from nonvirus-treated cultures, are nonleukemic, maintain a normal karyotype, and form colonies showing granulocyte maturation when plated in soft agar. Granulocyte/macrophage (GM) colony-stimulating factor is not the inductive moiety involved in the maintenance of proliferation of these cells. It is suggested that the cell lines represent a self-renewing population of cells ancestral to GM colony-forming cells, which may be responding to a hitherto unrecognized regulator.

Analysis of rat hemopoietic cells on the fluorescence-activated cell sorter. II. Isolation of terminal deoxynucleotidyl transferase-positive cells.

A method is described by which highly enriched populations of viable terminal deoxynucleotidyl transferase-positive (TdT+) cells can be isolated from rat bone marrow by use of the fluorescence-activated cell sorter. Such cells have been postulated to be progenitors of thymocytes and, possibly, of B lymphocytes, and may serve as the targets of neoplastic transformation in acute lymphoblastic leukemia. The separation procedure is based on differences in relative low-angle light scatter and relative fluorescence intensity for Thy-1 antigen between TdT+ cells and other lymphohemopoietic cell populations in bone marrow. Simultaneous sorting of bone marrow cells according to these two parameters resulted in a mean 87% purification of TdT+ cells. The morphological characteristics of the isolated TdT+ cells are described at the light and electron miscroscopic levels.

Enhancement of human blood eosinophil cytotoxicity by semi-purified eosinophil colony-stimulating factor(s).

Purified human blood eosinophils, when incubated in human placental conditioned medium (a source of colony-stimulating factors) [CSF]) demonstrate an enhanced ability to damage antibody- or complement-coated schistosomula. This enhancement represents a 4- to 10-fold increase of eosinophil schistosomicidal ability and a 10-fold lowering of the threshold for antibody or complement required in the killing reaction. The activity that enhances eosinophil cytotoxicity and the eosinophil colony-stimulating activity in the placental conditioned medium are eluted in the same fraction (CSF-alpha) after chromatography on Sephadex G-100 and phenyl-Sepharose columns, suggesting that these two activities might be associated with the same molecule. CSF-alpha enhances the adherence step of the killing reaction: antibody-coated larvae were frequently found covered by several layers of eosinophils in tubes containing CSF-alpha. Such a degree of adherence was rarely seen in control tubes lacking CSF-alpha. This enhancement of the eosinophil adherence is detectable 45-60 min after addition of CSF-alpha to the culture. It is not affected by washing the cells after a short time of preincubation with CSF-alpha, and it occurs in the absence of protein synthesis, whereas colony-stimulating activity requires continuous protein synthesis and ceases when CSF is removed from the culture. Finally, CSF-alpha enhances the temperature-dependent reaction that insures the irreversibility of eosinophil attachment to schistosomula. These observations suggest that eosinopoietic factors could be responsible for some of the modified properties of blood eosinophils in eosinophilic individuals.

Membrane receptors of mouse leukocytes. II. Sequential expression of membrane receptors and phagocytic capacity during leukocyte differentiation.

Analysis of four mature cell markers on mouse bone marrow leukocytes grown in vitro, demonstrated a distinct sequence of marker appearance during the terminal phases of granulocytic cell differentiation. A similar pattern of marker expression was also suggested by analysis of mature neutrophils and macrophages isolated from normal tissues. Among cultured neutrophils, receptors for the Fc portion of IgG (FcR) were first expressed on myelocytes and metamyelocytes, and then subsequently on more mature cells. Morphologically mature colony neutrophils (polymorphs) from agar cultures contained only FcR and complement receptor type two (CR(2)) (C3d receptor), and lacked both complement receptor type one (CR(1)) (C3b receptor) and the capacity to ingest latex, bacteria, or iron particles. Neutrophils from 2 and 3 wk liquid media cultures of marrow cells differed from agar grown neutrophils in that they had phagocytic capacity (particle ingestion) [Pi] in addition to FcR and CR(2). Furthermore, in the 4th and 5th wk of these continuous liquid cultures, CR(1) was also expressed, completing the surface marker profile of normal blood neutrophils. Based on these studies, the following order of appearance of these four markers on cells from the myelocytic series was proposed: FcR {arrow} FcR CR(2) {arrow} FcR CR(2) Pi {arrow} FcR CR(2) Pi CR(1). Differential studies of tissue leukocytes containing these same markers revealed that a heterogeneity existed among morphologically mature neutrophils. Even though 95 percent of blood polymorphs contained all four markers, the same was true of only half of spleen polymorphs and only 20 percent of bone marrow polymorphs. Cells of the monocyte-macrophage series were studies in parallel with neutrophils. Cultured marrow monocytes acquired the four mature cell markers so rapidly that the order of receptor appearance could not be determined. However, it was found that CR2 was lost during the terminal phase of monocyte maturation into activated macrophages.

Growth of B-lymphocyte colonies in vitro.

In semisolid agar cultures containing mercaptoethanol, cells from the spleen, lymph nodes, marrow, peritoneal cavity, thoracic duct, and blood of normal mice generated clusters and colonies of up to 3,000 cells. Colony numbers and growth were markedly enhanced by the addition of sheep red cells. The frequency of colony-forming cells in the spleen or lymph nodes was 0.5-2.0%, and cluster forming cells were approximately five times more numerous. The mononuclear cells comprising these colonies had the electronmicroscopic morphology of immature lymphoid and plasma cells. The majority of the cells possessed Fc receptors, 61-69% reacted with anti-mu-serum and 4-11% with anti-gamma2-serum. Analysis of single cells from individual colonies indicated a higher frequency of the cells with membrane immunoglobulin and a clonal pattern of anti-mu or anti-gamma-reactivity. The clonal nature of colonies was supported by an analysis of NIP-binding cells in colonies grown from CBA spleen cells enriched for NIP-binding cells. Mass-harvested colony cells synthesized immunoglobulin in short-term liquid cultures. It is concluded that the colonies are clones of functionally active B-lymphoid cells.

Characters of aerated compost tea from immature compost that limit colonization of bean leaflets by Botrytis cinerea.

AIMS: The aim was to produce and characterize an aerated compost tea (ACT) that suppressed growth of the plant pathogen Botrytis cinerea. METHODS AND RESULTS: Three different open-windrow composts were sampled weekly from the early secondary mesophilic stage until maturity. Each 10kg of compost sample was extracted in 30 l of aerated water for 24, 48 or 72h. Relative to water, all batches of ACT applied to detached bean leaflets reduced lesion development following single-point inoculations of B. cinerea. There was a significant linear, inverse relationship between the internal windrow temperature of compost (≤51°C) used to prepare ACT and the extent of lesion development. Bacterial diversity in ACTs from one windrow was highest using compost sampled at 48°C. The compost weight-to-water volume ratios of 1:3, 1:10 or 1:30, using compost sampled from a fourth windrow at 50°C, also produced ACTs that reduced the growth of B. cinerea on bean leaflets. The '1 : 3' ACT, and to a lesser degree the same ACT filtered to remove micro-organisms, inhibited the germination of B. cinerea conidia. CONCLUSIONS: ACT produced using the methods reported here suppressed the growth of B. cinerea on bean leaflets, with an abundant and diverse microbial community likely to contribute to pathogen suppression. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the use of immature compost to produce a pathogen-suppressive ACT, suggesting that compost stage is an important production variable.

Clostridium difficile in vegetables, Canada.

BACKGROUND: Clostridium difficile is an important gastrointestinal pathogen of humans and animals. It has been isolated from various foods, including meat and ready-to-eat salads, and concern has been expressed regarding food as a possible source of human C. difficile infection (CDI). AIMS: We sought to isolate C. difficile from a variety of vegetables obtained from local grocery stores and to characterize these isolates. MATERIALS AND METHODS: Vegetables were purchased from 11 different grocery stores in Guelph, Ontario, Canada between May and August 2009. Enrichment culture was performed and isolates were characterized by ribotyping, PFGE, toxinotyping and PCR detection of toxin genes. RESULTS: Clostridium difficile was isolated from 4.5% (5/111) of retail vegetables. Two different ribotypes and two different toxinotypes were identified. Three isolates were ribotype 078/NAP 7/toxinotype V, possessing all three toxin genes. The other two isolates shared a ribotype with a toxigenic strain previously found in humans with CDI in this region. DISCUSSION: Contamination of vegetables was found at relatively low levels, however, all isolates were toxigenic and belonging to ribotypes previously associated with CDI. CONCLUSIONS: Contamination of vegetables with CDI-associated isolates can occur and although the implications for food safety practices remain elusive, the presence of toxigenic isolates suggests vegetables could be a source of C. difficile in humans.

Control of granulocytes and macrophages: molecular, cellular, and clinical aspects.

The production and functional activity of two important white blood cells, the granulocytes and macrophages, are regulated mainly by a group of glycoprotein colony-stimulating factors. The colony-stimulating factors have been mass-produced with recombinant technology and are now proving of value in preventing or suppressing infections in a variety of individuals with subnormal or defective formation of blood cells.

The granulocyte-macrophage colony-stimulating factors.

The granulocyte-macrophage colony-stimulating factors are well-characterized specific glycoproteins that interact to control the production, differentiation, and function of two related white cell populations of the blood, the granulocytes and monocyte-macrophages. Widely produced in the body, these regulators probably play an important role in resistance to infections. The proliferation of myeloid leukemia cells remains dependent on stimulation by colony-stimulating factors, although one of them also has the ability to suppress leukemic populations by inducing terminal differentiation.

Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity.

Apoptosis can be triggered by members of the Bcl-2 protein family, such as Bim, that share only the BH3 domain with this family. Gene targeting in mice revealed important physiological roles for Bim. Lymphoid and myeloid cells accumulated, T cell development was perturbed, and most older mice accumulated plasma cells and succumbed to autoimmune kidney disease. Lymphocytes were refractory to apoptotic stimuli such as cytokine deprivation, calcium ion flux, and microtubule perturbation but not to others. Thus, Bim is required for hematopoietic homeostasis and as a barrier to autoimmunity. Moreover, particular death stimuli appear to activate apoptosis through distinct BH3-only proteins.

Hemopoietic regulators.

The production and maturation of blood cells from the eight major blood cell lineages is a complex and continuous process, which is largely controlled by specific glycoprotein hemopoietic regulators. These regulators also control the functional activity of the blood cells through eliciting a diverse set of intracellular responses initiated by a regulator-specific membrane receptor. Twenty of these regulators have now been characterized, and their mass production has led to four already being licensed for clinical use in disease states involving subnormal blood cell formation.