Learning from our mistakes: using key opportunities to remove the perverse incentives that help drive antibiotic resistance.

Introduction: Governments need to do far more to help curb the emergence and transmission of antibiotic resistance and help protect the efficacy of any new antibiotics that come to the market. Industry is an important stakeholder that must be brought on-board such efforts given its influence on the direction and scale of antibiotic sales. Financial incentives supporting industry R&D of novel antibiotics should structurally remove the drivers of superfluous sales and encourage access to newer antibiotics where infections are otherwise resistant to treatment. Indeed, the use of public money provides an important opportunity to prioritize these public health goals within market structures such that we both adequately reward industry for their efforts and prolong antibiotic efficacy for as long as possible.Areas covered: This work discusses possible financial 'pull' incentives that fully delink the reward paid to the developer from unit sales, examining their primary advantages and limitations.Expert opinion: Pharmaceutical companies need to be rewarded generously for their efforts to develop new, badly needed antibiotics. But the current marketplace does not provide a sustained financial lure and its reliance on unit-sales for profitability jeopardizes the efficacy of antibiotics both new and old. Fully delinked models can make antibiotic R&D more financially appealing and create a market environment that is far less threatening to public health.

Endometrial Mesenchymal Stem/Stromal Cells Modulate the Macrophage Response to Implanted Polyamide/Gelatin Composite Mesh in Immunocompromised and Immunocompetent Mice.

The immunomodulatory properties of human endometrial mesenchymal stem cells (eMSC) have not been well characterised. Initial studies showed that eMSC modulated the chronic inflammatory response to a non-degradable polyamide/gelatin mesh in a xenogeneic rat skin wound repair model, but the mechanism remains unclear. In this study, we investigated the immunomodulatory effect of eMSC on the macrophage response to polyamide/gelatin composite mesh in an abdominal subcutaneous wound repair model in C57BL6 immunocompetent and NSG (NOD-Scid-IL2Rgamma null ) immunocompromised mice to determine whether responses differed in the absence of an adaptive immune system and NK cells. mCherry lentivirus-labelled eMSC persisted longer in NSG mice, inducing longer term paracrine effects. Inclusion of eMSC in the mesh reduced inflammatory cytokine (Il-1ß, Tnfα) secretion, and in C57BL6 mice reduced CCR7+ M1 macrophages surrounding the mesh on day 3 and increased M2 macrophage marker mRNA (Arg1, Mrc1, Il10) expression at days 3 and 7. In NSG mice, these effects were delayed and only observed at days 7 and 30 in comparison with controls implanted with mesh alone. These results show that the differences in the immune status in the two animals directly affect the survival of xenogeneic eMSC which leads to differences in the short-term and long-term macrophage responses to implanted meshes.

Preserving the 'commons': addressing the sustainable use of antibiotics through an economic lens.

BACKGROUND: As the growth of antibiotic resistance has resulted in large part from widespread use of antibiotics, every effort must be made to ensure their sustainable use. AIMS: This narrative review aims to assess the potential contribution of health economic analyses to sustainable use efforts. SOURCES: The work draws on existing literature and experience with health economic tools. CONTENT: The study examines some of the weaknesses in the health, regulatory, and industry arenas that could contribute to inappropriate or suboptimal prescribing of antibiotics and describes how economic analysis could be used to improve current practice by comparing both costs and health outcomes to maximize societal wellbeing over the longer-term. It finds that economic considerations underpinning current antibiotic prescribing strategies are incomplete and short-termist, with the result that they may foster suboptimal use. It also stresses that perverse incentives that drive antibiotic sales and inappropriate prescribing practices must be dis-entangled for sustainable use policies to gain traction. Finally, payment structures can be used to re-align incentives and promote optimal prescribing and sustainable use more generally. In particular, eliminating or altering reimbursement differentials could help steer clinical practice more deliberately towards the minimization of selection pressure and the resulting levels of antibiotic resistance. IMPLICATIONS: This work highlights the need for appropriately designed cost-effectiveness analyses, incentives analysis, and novel remuneration systems to underpin sustainable use policies both within and beyond the health sector.

LUCA-15-encoded sequence variants regulate CD95-mediated apoptosis.

Using an expression cloning system to discover novel genes involved in apoptosis, we identified a 326 bp bone marrow cDNA fragment (termed Je2) that suppresses, upon transfection, CD95-mediated apoptosis in Jurkat T cells. Sequence homology revealed that Je2 maps to 3p21.3, to an intronic region of the candidate TSG LUCA-15 locus. It represents, in fact, an antisense transcript to the 3'-UTR of two novel splice variants of this gene. Overexpression of sequence representing one of these splice variants (a 2.6 kb cDNA termed Clone 26), inhibited proliferation of Jurkat cells and sensitized them to CD95-mediated apoptosis. This study therefore implicates the LUCA-15 gene locus in the control of apoptosis.

Processing of radical prostatectomy specimens for correlation of data from histopathological, molecular biological, and radiological studies: a new whole organ technique.

AIMS: To develop a method of processing non-formalin fixed prostate specimens removed at radical prostatectomy to obtain fresh tissue for research and for correlating diagnostic and molecular results with preoperative imaging. METHODS/RESULTS: The method involves a prostate slicing apparatus comprising a tissue slicer with a series of juxtaposed planar stainless steel blades linked to a support, and a cradle adapted to grip the tissue sample and receive the blades. The fresh prostate gland is held in the cradle and the blades are moved through the cradle slits to produce multiple 4 mm slices of the gland in a plane perpendicular to its posterior surface. One of the resulting slices is preserved in RNAlater. The areas comprising tumour and normal glands within this preserved slice can be identified by matching it to the haematoxylin and eosin stained sections of the adjacent slices that are formalin fixed and paraffin wax embedded. Intact RNA can be extracted from the identified tumour and normal glands within the RNAlater preserved slice. Preoperative imaging studies are acquired with the angulation of axial images chosen to be similar to the slicing axis, such that stained sections from the formalin fixed, paraffin wax embedded slices match their counterparts on imaging. CONCLUSIONS: A novel method of sampling fresh prostate removed at radical prostatectomy that allows tissue samples to be used both for diagnosis and molecular analysis is described. This method also allows the integration of preoperative imaging data with histopathological and molecular data obtained from the prostate tissue slices.

Polymerase chain reaction amplification analyses of clonality in T-cell malignancy including peripheral T-cell lymphoma.

Clonality in T-cell malignancy was investigated using T-cell receptor (TcR) V beta 1-20 family primers and polymerase chain reaction amplification (PCR) of cDNA prepared from tissue biopsies and blood involved with tumour. Secondary PCR amplification of the VDJ joints of primary PCR products was performed to distinguish clonal from polyclonal products, and clonal V beta gene products were confirmed by direct PCR sequencing in the majority of cases. In eight T-cell malignancies including T-cell acute lymphoblastic leukaemia (T-ALL) and T-cell chronic lymphocytic leukaemia (T-CLL) shown to be clonal by Southern blot analysis, one or two primary PCR products were identified and shown to be clonal. In five cases of peripheral T-cell lymphoma (PTCL) all V beta 1-20 families were identified after primary PCR amplification, and clonal products were identified in two cases after secondary amplification; TcR V beta clonal families could not be demonstrated in the remaining three cases. These data were in agreement with previous Southern blot analysis of these cases, and confirmed the presence of reactive T cells in PTCL as well as providing further evidence for the genotypic heterogeneity of this entity. In the remaining case, a blood lymphocytosis, primary PCR amplification produced predominant TcR V beta 6 and V beta 12 family products, of which the V beta 6 family proved clonal after secondary PCR amplification. There was no evidence for overrepresentation of TCR V beta families by the tumour populations in this study, furthermore the data confirm the involvement of reactive cells in T-cell malignancy and the genetic heterogeneity of PTCL.

Effective interprofessional simulation training for medical and midwifery students.

Introduction: Good interprofessional teamworking is essential for high quality, efficient and safe clinical care. Undergraduate interprofessional training has been advocated for many years to improve interprofessional working. However, few successful initiatives have been reported and even fewer have formally assessed their educational impact. Methods: This was a prospective observational study of medical and midwifery students at a tertiary-level maternity unit. An interprofessional training module was developed and delivered by a multiprofessional faculty to medical and midwifery students, including short lectures, team-building exercises and practical simulation-based training for one obstetric (shoulder dystocia) and three generic emergencies (sepsis, haemorrhage, collapse). Outcome measures were interprofessional attitudes, assessed with a validated questionnaire (UWE Interprofessional Questionnaire) and clinical knowledge, measured with validated multiple-choice questions. Results: Seventy-two students participated (34 medical, 38 midwifery). Following training median interprofessional attitude scores improved in all domains (p<0.0001), and more students responded in positive categories for communication and teamwork (69-89%, p=0.004), interprofessional interaction (3-16%, p=0.012) and interprofessional relationships (74-89%, p=0.006). Scores for knowledge improved following training for medical students (65.5% (61.8-70%) to 82.3% (79.1-84.5%) (median (IQR)) p<0.0001) and student midwives (70% (64.1-76.4%) to 81.8% (79.1-86.4%) p<0.0001), and in all subject areas (p<0.0001). Conclusions: This training was associated with meaningful improvements in students' attitudes to teamwork, and knowledge acquisition. Integrating practical tasks and teamwork training, in authentic clinical settings, with matched numbers of medical and non-medical students can facilitate learning of both why and how to work together. This type of training could be adopted widely in undergraduate healthcare education.

Mutation of cytochrome bd quinol oxidase results in reduced stationary phase survival, iron deprivation, metal toxicity and oxidative stress in Azotobacter vinelandii.

Azotobacter vinelandii cydAB mutants lacking cytochrome bd lost viability in stationary phase, irrespective of temperature, but microaerobiosis or iron addition to stationary phase cultures prevented viability loss. Growth on solid medium was inhibited by a diffusible factor from neighbouring cells, and by iron chelators, In(III) or Ga(III); microaerobic growth overcame inhibition by the extracellular factor. Siderophore production and total Fe(III)-chelating activity were not markedly affected in Cyd(-) mutants, and remained responsive to iron repression. Cyd(-) mutants were hypersensitive to Cu(II), Zn(II), and compounds exerting oxidative stress. Failure to synthesise haemoproteins does not explain the complex phenotype since mutants retained significant catalase activity. We hypothesise that Cyd(-) mutants are defective in maintaining the near-anoxic cytoplasm required for reductive iron metabolism and nitrogenase activity.

Acute phase proteins in cattle: discrimination between acute and chronic inflammation.

Acute phase proteins such as serum amyloid A, haptoglobin, and alpha 1-acid glycoprotein have been identified as markers of inflammation in cattle because they are produced by the liver in response to pro-inflammatory cytokines. This study was designed to assess whether they could be used to discriminate between acute and chronic inflammation. Their concentrations were measured in serum samples from 81 cattle in which inflammation was classified by thorough clinical examination, supported by postmortem findings, as being acute in severity in 31 and chronic in 50. The classical haematological markers of inflammation were also determined in blood from the animals. Serum amyloid A had a maximum (100 per cent) clinical sensitivity in discriminating between the acute and chronic cases, and haptoglobin had the highest clinical specificity of 76 per cent; counts of neutrophils and band neutrophils had sensitivities of 71 per cent and 42 per cent and specificities of 30 per cent and 72 per cent, respectively. It was concluded that serum amyloid A and haptoglobin may be used to discriminate between acute and chronic inflammatory conditions.

Flexible intramedullary nailing of displaced diaphyseal forearm fractures in children.

INTRODUCTION: This study analyses the results of 50 displaced diaphyseal forearm fractures in children treated with flexible intramedullary nailing. METHODS: Between 1999 and 2002 we treated 50 children aged between 5 and 15 years, with diaphyseal fractures of the forearm using Flexible intramedullary nailing (FIN). Both bones were fractures in 45 patients, radius only in 4 and ulna only in 1. The indications for fixation were instability (26), re-displacement (20), and open fractures (4). RESULTS: 24 patients were reduced closed, followed by nailing, while 26 fractures required open reduction of either one bone(16 cases) or both bones(10 cases) prior to nailing. Bony union of all fractures was achieved by an average of 7 weeks (range 6 weeks to 4 months) with one delayed union. Pronation was restricted by an average of 20 degrees in 9 patients. Two patients developed post operative compartment syndrome requiring fasciotomy. Three patients were lost to follow-up. INTERPRETATION: FIN led to early bony union with acceptable bony alignment in all 47 patients available at final follow-up. We therefore recommend FIN for the treatment of unstable diaphyseal forearm fractures in children.

The value of radiography in the management of rheumatoid arthritis.

All radiographs of joints requested on 50 consecutive clinic patients with rheumatoid arthritis were reviewed; 1094 films were available. With few minor exceptions, all information relevant to diagnosis and medical management was provided by one view of each joint: postero-anterior hands, dorsi-planar feet, antero-posterior (AP) elbow, AP shoulder, AP pelvis and lateral flexion cervical spine. Norgaard's 'ballcatcher's' projection of the hands provided no extra information. Radiographs of the hands were consistently more sensitive in showing progression of erosion than those of the feet. Radiography of both hands and feet was required to avoid missing earliest erosions. Radiographs requested at times when drug treatment was under review did not consistently affect decisions, which were largely dependent on clinical findings.

Macrophage colony-stimulating factor production by murine and human keratinocytes. Enhancement by bacterial lipopolysaccharide.

CSF have a broad range of effects on differentiated cells outside the bone marrow. Site-specific elaboration of these factors may influence local immune reactions. Keratinocytes have been demonstrated to produce a number of immunoactive cytokines, including factors capable of modifying macrophage function. We have previously identified at least two products of keratinocytes that induce DNA synthesis by elicited peritoneal macrophages; one factor has been identified as granulocyte-macrophage CSF. In the present study, the second keratinocyte product has been characterized and identified as macrophage-CSF (M-CSF). Conditioned media from cultures of normal human keratinocytes and the transformed murine keratinocyte cell line PAM 212 induce formation of macrophage colonies in soft agar as well as dose-dependent proliferation of the M-CSF-dependent cell line BAC1.2F5. The bioactivity in both assays is blocked by neutralizing anti-M-CSF antibody. Western blot analysis of cell lysates from both PAM 212 and normal human keratinocytes demonstrates multiple molecular mass forms of M-CSF (45 to 98 kDa). Northern blot analysis (PAM 212 cells) and in situ hybridization (normal keratinocytes) demonstrate expression of M-CSF mRNA. Stimulation of keratinocytes with LPS increases M-CSF synthesis as measured both by bioactivity and level of mRNA expression. Thus, both murine and human keratinocytes produce M-CSF in vitro. Furthermore, production of keratinocyte-derived M-CSF is increased by bacterial LPS. CSF production by keratinocytes may play an important role in regulating the cutaneous immune response.

Immunoepidemiology of the equine tapeworm Anoplocephala perfoliata: age-intensity profile and age-dependency of antibody subtype responses.

The equine intestinal cestode Anoplocephala perfoliata has been the subject of recent epidemiological and immunological studies because of its suspected association with intestinal disease in the horse. We have previously shown that the IgG(T) subtype antibody response to the 12/13 kDa component of the parasite excretory/secretory (E/S) antigen is positively correlated with parasite intensity. In this study, we utilize that correlation to examine the changes in natural infection intensity with age. Infection intensity based on IgG(T) responses showed a triphasic age-dependency pattern with peak mean worm burden in the 6 months-2 years age group, falling to a lower plateau level from 3 to 15 years, and rising again in older age groups. Anti-E/S total IgG was found to have a convex age-dependency curve, with maximal response in the 6 months-2 years old age group. IgG(a) showed a triphasic response similar to the age-intensity profile of IgG(T); IgG(c) showed steadily increasing levels of antibody with age. The IgG(b) age-dependency profile was intermediate between IgG(a) and IgG(c). Age-specific correlation coefficients between anti-12/13 kDa IgG(T) (as a measure of infection intensity) and IgG(a) and IgG(b) revealed statistically significant values for many age groups. The relative importance of exposure to infection and the development of acquired immunity as determinants of the observed age-intensity pattern is considered.