Jeni Luckett

MEK kinase activity is not necessary for Raf‐1 function

Raf‐1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf‐1 is achieved by Ras.GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a ‘knockout’ of the raf‐1 gene in mice as well as a rafFF mutant version of endogenous Raf‐1 with Y340FY341F mutations. Raf‐1−/− mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf‐1 from cells derived from raf‐1FF/FF mice has no detectable activity towards MEK in vitro, and yet raf‐1FF/FF mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf‐1−/− and raf‐1FF/FF mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf‐1 is not essential for normal mouse development and that Raf‐1 plays a key role in preventing apoptosis.

Survivin is an independent predictor of short-term survival in poor prognostic breast cancer patients

Established clinico–pathological factors can place patients with breast cancer into good and poor prognostic categories, but even within these groups behaviour and response to treatment can differ. This study examined the value of cell cycle and apoptotic regulatory proteins in predicting behaviour in a poor prognostic group. A total of 165 patients, all of whom had died of breast cancer with duration of survival 12–127 months, median 38 months, were examined using immunohistochemistry for proliferation, apoptosis, p53, phosphorylated p53, p21, checkpoint kinase 2 (Chk2), bcl-2, bax, survivin and XIAP. All had received chemotherapy and/or hormonal therapy and were predominantly T2, node positive, grade III with only half oestrogen-receptor (ER) positive. High proliferation, phosphorylated p53, Chk2 and survivin expression correlated with grade III and lack of ER, whereas low proliferation, p21 and bcl-2 related to better grade and presence of ER. On univariate analysis grade, proliferation, phosphorylated p53, bcl-2, ER and survivin related to duration of survival. In multivariate analysis, grade (P=0.001) and survivin (P=0.005) were independent prognostic factors, grade III and presence of survivin relating to shorter survival. The latter was particularly for those patients receiving neoadjuvant therapy and adjuvant chemo- and hormonal therapy. The presence of the inhibitor of apoptosis protein survivin is a highly significant independent predictor of shorter duration of survival of patients with poor prognostic features, and merits investigation as a marker in other prognostic groups.

Properdin Plays a Protective Role in Polymicrobial Septic Peritonitis

Properdin is a positive regulator of complement activation so far known to be instrumental in the survival of infections with certain serotypes of Neisseria meningitidis. We have generated a fully backcrossed properdin-deficient mouse line by conventional gene-specific targeting. In vitro, properdin-deficient serum is impaired in alternative pathway-dependent generation of complement fragment C3b when activated by Escherichia coli DH5α. Properdin-deficient mice and wild-type littermates compare in their levels of C3 and IgM. In an in vivo model of polymicrobial septic peritonitis induced by sublethal cecal ligation and puncture, properdin-deficient mice appear immunocompromised, because they are significantly impaired in their survival compared with wild-type littermates. We further show that properdin localizes to mast cells and that properdin has the ability to directly associate with E. coli DH5α. We conclude that properdin plays a significant role in the outcome of polymicrobial sepsis.

Preclinical SPECT/CT Imaging of αvβ6 Integrins for Molecular Stratification of Idiopathic Pulmonary Fibrosis

Transforming growth factor β activation by the αvβ6 integrin is central to the pathogenesis of idiopathic pulmonary fibrosis. Expression of the αvβ6 integrin is increased in fibrotic lung tissue and is a promising therapeutic target for treatment of the disease. Currently, measurement of αvβ6 integrin levels in the lung requires immunohistochemical analysis of biopsy samples. This procedure is clinically impractical for many patients with pulmonary fibrosis, and a noninvasive strategy for measuring αvβ6 integrin levels in the lungs is urgently required to facilitate monitoring of disease progression and therapeutic responses. Methods: Using a murine model of bleomycin-induced lung injury, we assessed the binding of intravenously administered 111In-labeled αvβ6-specific (diethylenetriamine pentaacetate-tetra [DTPA]-A20FMDV2) or control (DTPA-A20FMDVran) peptide by nanoSPECT/CT imaging. Development of fibrosis was assessed by lung hydroxyproline content, and αvβ6 protein and itgb6 messenger RNA were measured in the lungs. Results: Maximal binding of 111In-labeled A20FMDV2 peptide to αvβ6 integrins was detected in the lungs 1 h after intravenous administration. No significant binding was detected in mice injected with control peptide. Integrin binding was increased in the lungs of bleomycin-, compared with saline-, exposed mice and was attenuated by pretreatment with αvβ6-blocking antibodies. Levels of 111In-labeled A20FMDV2 peptide correlated positively with hydroxyproline, αvβ6 protein, and itgb6 messenger RNA levels. Conclusion: We have developed a highly sensitive, quantifiable, and noninvasive technique for measuring αvβ6 integrin levels within the lung. Measurement of αvβ6 integrins by SPECT/CT scanning has the potential for use in stratifying therapy for patients with pulmonary fibrosis.

Dual role of complement in adipose tissue.

Once thought of as purely the bodys chief energy store, adipose tissue and its constituent adipocytes have emerged as both a metabolic entity and an endocrine one. Complement is generally thought of as originating mainly from hepatic synthesis but also from synthesis by the macrophage phagocyte system. This review revisits early descriptions of adipocytic synthesis of complement components and highlights the need of a systematic analysis of the contribution of adipose tissue to systemic inflammation in order to appreciate the immunological activity of this tissue.

A novel virulence strategy for Pseudomonas aeruginosa mediated by an autotransporter with arginine-specific aminopeptidase activity

The opportunistic human pathogen, Pseudomonas aeruginosa, is a major cause of infections in chronic wounds, burns and the lungs of cystic fibrosis patients. The P. aeruginosa genome encodes at least three proteins exhibiting the characteristic three domain structure of autotransporters, but much remains to be understood about the functions of these three proteins and their role in pathogenicity. Autotransporters are the largest family of secreted proteins in Gram-negative bacteria, and those characterised are virulence factors. Here, we demonstrate that the PA0328 autotransporter is a cell-surface tethered, arginine-specific aminopeptidase, and have defined its active site by site directed mutagenesis. Hence, we have assigned PA0328 with the name AaaA, for arginine-specific autotransporter of P. aeruginosa. We show that AaaA provides a fitness advantage in environments where the sole source of nitrogen is peptides with an aminoterminal arginine, and that this could be important for establishing an infection, as the lack of AaaA led to attenuation in a mouse chronic wound infection which correlated with lower levels of the cytokines TNFα, IL-1α, KC and COX-2. Consequently AaaA is an important virulence factor playing a significant role in the successful establishment of P. aeruginosa infections.

Properdin Deficiency in Murine Models of Nonseptic Shock

Hereditary properdin deficiency is linked to susceptibility to meningococcal disease (Neisseria meningitidis serotypes Y and W-135) with high mortality. Its relative contribution toward the outcome of nonseptic shock has not been investigated. Using properdin-deficient C57BL/6 mice and their littermates, this study examines their survival of zymosan-induced and LPS-induced shock. Properdin-deficient mice were more resistant to zymosan shock compared with wild-type mice, which showed greater impairment of end-organ function 24 h after zymosan injection, higher TNF-α production by alveolar and peritoneal macrophages, higher TNF-α, and, inversely, lower IL-10 levels in peritoneal lavage and circulation and higher plasma C5a levels. Properdin-deficient mice showed significantly higher mortality in LPS shock, elevated TNF-α, and, inversely, reduced IL-10 production by peritoneal macrophages as well as lower plasma C5a levels compared with wild-type littermates. NO production by peritoneal macrophages and plasma α1-antitrypsin levels at 24 h after the injection of LPS or zymosan were decreased in properdin-deficient mice in both models, and fewer histopathologic changes in liver were observed in properdin-deficient animals. This study provides evidence that properdin deficiency attenuates zymosan-induced shock and exacerbates LPS-induced shock.

Ex vivo cancer chemoprevention research possibilities

The concept of cancer prevention with naturally occurring or synthetic compounds is rapidly gaining momentum as a key field in cancer research. The availability of good models for the determination of the molecular mechanisms of these agents, which frequently have multiple sites of action within a cell, is key to the progression of the field. In this review, we concentrate on the emergence of several in vitro techniques that have significant advantages over more traditional monolayer cell culture, and/or in vivo models. In particular, we focus on the potential of 3D multicellular spheroid models as versatile intermediates between monolayer culture and tumours in situ. In these models, cell-cell interactions and cell-extracellular matrix interactions can closely mimic the environment to which tumour cells would be exposed in vivo, while maintaining the advantages of ease of manipulation of an in vitro system. The in vitro tube formation assay for the study of angiogenesis, the availability of human tissues for research, and the sophisticated technology surrounding DNA microarray and proteomics are also briefly discussed.

Antibody-Mediated Inhibition of the FGFR1c Isoform Induces a Catabolic Lean State in Siberian Hamsters

Hypothalamic tanycytes are considered to function as sensors of peripheral metabolism. To facilitate this role, they express a wide range of receptors, including fibroblast growth factor receptor 1 (FGFR1). Using a monoclonal antibody (IMC-H7) that selectively antagonizes the FGFR1c isoform, we investigated possible actions of FGFR1c in a natural animal model of adiposity, the Siberian hamster. Infusion of IMC-H7 into the third ventricle suppressed appetite and increased energy expenditure. Likewise, peripheral treatment with IMC-H7 decreased appetite and body weight and increased energy expenditure and fat oxidation. A greater reduction in body weight and caloric intake was observed in response to IMC-H7 during the long-day fat state as compared to the short-day lean state. This enhanced response to IMC-H7 was also observed in calorically restricted hamsters maintained in long days, suggesting that it is the central photoperiodic state rather than the peripheral adiposity that determines the response to FGFR1c antagonism. Hypothalamic thyroid hormone availability is controlled by deiodinase enzymes (DIO2 and DIO3) expressed in tanycytes and is the key regulator of seasonal cycles of energy balance. Therefore, we determined the effect of IMC-H7 on hypothalamic expression of these deiodinase enzymes. The reductions in food intake and body weight were always associated with decreased expression of DIO2 in the hypothalamic ependymal cell layer containing tanycytes. These data provide further support for the notion the tanycytes are an important component of the mechanism by which the hypothalamus integrates central and peripheral signals to regulate energy intake and expenditure.

Impact of an educational intervention upon the hand hygiene compliance of children

BACKGROUND Hand hygiene compliance is the single most effective way to reduce healthcare-associated infections. Children are notoriously vulnerable to infection as well as acting as conduits to transmission. Based on these observations, the authors formulated the hypothesis that behavioural change which improved childrens hand hygiene compliance would decrease the spread of infectious diseases. AIM To create an educational intervention to induce long-term behavioural change culminating in increased hand hygiene compliance of children, and thus a decrease in the rate of infections. METHODS Focus groups conducted during interactive teaching sessions identified what children felt would help them to increase their hand hygiene compliance. This informed the design of an educational device that was subsequently trialled to measure its effectiveness in increasing hand hygiene compliance. Initial developmental stages were conducted in two schools in the East Midlands with study participants aged 5-8 years; the device was subsequently used in a healthcare setting to assess deployment flexibility. FINDINGS Focus groups indicated that children enjoyed interactive learning, developed knowledge about cross-transmission of infections, and became motivated to encourage others to improve hand hygiene compliance. Microbiological swabbing verified the presence of pathogens on childrens hands and environmental surfaces that could serve as reservoirs of infection, and questionnaires indicated an increase in handwashing following the intervention. CONCLUSION Educational interventions have the potential to increase hand hygiene and reduce the transmission of infections.