Integration of biochemical and topographic cues for the formation and spatial distribution of invadosomes in nasopharyngeal epithelial cells.

Invadosomes are invasive protrusions generated by cells which can secrete matrix metalloproteinases for focal digestion of extracellular matrix. They also aid invasive cancer cells in their transmigration through vascular endothelium. However, how the physical and chemical cues in a three-dimensional (3D) system signal the spatial localization of invadosomes remains largely unknown. Here we study the topographic guidance of invadosome formation in invasive nasopharyngeal cells under the stimulation of an inflammatory cytokine, TGF-ß1, using engineered gratings with different width and depth. We first report that TGF-ß1 can act as an external signal to upregulate the formation of invadosomes with a random distribution on a plane 2D surface. When the cells were seeded on parallel 3D gratings of 5 µm width and 1 µm depth, most of the invadosomes aligned to the edges of the gratings, indicating a topographic cue to the control of invadosome localization. While the number of invadosomes per cell were not upregulated when the cells were seeded on 3D topography, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges when the cells move at a lower speed and are guided along narrow gratings. The invadosomes forming at 3D edges also have a longer half-life than those forming on a plane surface. These data suggest that there are integrated biochemical and 3D geometric cues underlying the spatial regulation of invasive structures so as to elicit efficient invasion or metastasis of cells. STATEMENT OF SIGNIFICANCE: Nasopharyngeal cells were integrated with the biological cues and matrix topography to govern the activity and spatial distribution of invadosomes. The biochemical induction of invadosome formation by TGF-ß1 in nasopharyngeal cells was observed. When the cells were seeded on parallel 3D gratings, most of the invadosomes aligned to the edges of the gratings due to topographical induced invadosome localization. While the number of invadosomes per cell were not upregulated, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges with a higher stability when the cells are guided along narrow gratings. The integrated biochemical and 3D geometric cues could elicit efficient invasion or metastasis of cells.

Pyopericardium secondary to achalasia-associated squamous cell carcinoma of the oesophagus.

Patients with achalasia of the oesophagus are known to be at increased risk of oesophageal squamous cell carcinoma. To our knowledge, this is the first report of an achalasia-associated oesophageal squamous cell carcinoma presenting with acute sepsis secondary to pyopericardium.

Developing and assessing the utility of a You-Tube based clinical genetics video channel for families affected by inherited tumours.

We have designed and implemented the first worldwide You Tube channel with 22 videos covering common questions asked in familial cancer susceptibility clinics. We discuss the use of the videos including demographics of registered You Tube users, and what lessons have been learnt about how the general public uses medical information online. The most popular video on inheritance patterns has been watched on average 84 times per month. The mostly highly viewed videos include inheritance patterns, breast cancer screening and hereditary non-polyposis colorectal cancer. Registered viewers were more commonly male and the average age of the registered user was 45-54 years; similar to that seen in Genetics Clinics suggesting that age may not be a major barrier to access to this type of information for patients. The videos have been viewed in more than 140 countries confirming that there is clearly an audience for this type of information. Patient feedback questionnaires indicate that these videos provide a useful aide memoir for the clinic appointment, and most people would recommend them to others. In summary, You Tube videos are easy and cost effective to make. They have the ability to disseminate genetics education to a worldwide audience and may be a useful adjunct to clinical appointments.

Podoplanin mediates ECM degradation by squamous carcinoma cells through control of invadopodia stability.

Invadopodia are actin-rich cell membrane projections used by invasive cells to penetrate the basement membrane. Control of invadopodia stability is critical for efficient degradation of the extracellular matrix (ECM); however, the underlying molecular mechanisms remain poorly understood. Here, we uncover a new role for podoplanin, a transmembrane glycoprotein closely associated with malignant progression of squamous cell carcinomas (SCCs), in the regulation of invadopodia-mediated matrix degradation. Podoplanin downregulation in SCC cells impairs invadopodia stability, thereby reducing the efficiency of ECM degradation. We report podoplanin as a novel component of invadopodia-associated adhesion rings, where it clusters prior to matrix degradation. Early podoplanin recruitment to invadopodia is dependent on lipid rafts, whereas ezrin/moesin proteins mediate podoplanin ring assembly. Finally, we demonstrate that podoplanin regulates invadopodia maturation by acting upstream of the ROCK-LIMK-Cofilin pathway through the control of RhoC GTPase activity. Thus, podoplanin has a key role in the regulation of invadopodia function in SCC cells, controlling the initial steps of cancer cell invasion.

Imaging haematopoietic cells recruitment to an acute wound in vivo identifies a role for c-Met signalling.

We have used a direct in vivo imaging strategy to investigate the role of c-Met signalling and kinase activity during the immune response to wounding. Our assay utilizes the optical translucent properties of the zebrafish embryo and demonstrates the versatility of microscopy-based approach to the screening of compounds for inhibition of the wounding response. We have focussed on the c-Met pathway as little is known about the influence of c-Met signalling in immune responses, although it has been suggested that the c-Met tyrosine kinase receptor signalling pathway may be involved in cytokine secretion and directional migration in immune cells. Using both imaging of fixed zebrafish embryos in combination with digital time lapse microscopy of neutrophils recruited to a wound site, we find that pharmacological inhibition of c-Met, using a specific inhibitor, modulates the immune response in zebrafish embryos. We have found that inhibition of c-Met does not prevent the inflammatory response but does appear to limit recruitment and retention of immune cells at the wound site. This work demonstrates the versatility of using direct imaging assays for inhibitor studies and suggests that the HGF/c-Met signalling cascade plays an important role in the migration of haematopoietic cells in vivo.

PAK4 kinase activity and somatic mutation promote carcinoma cell motility and influence inhibitor sensitivity.

Hepatocyte growth factor (HGF) and its receptor (c-Met) are associated with cancer cell motility and invasiveness. p21-activated kinase 4 (PAK4), a potential therapeutic target, is recruited to and activated by c-Met. In response, PAK4 phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner in metastatic prostate carcinoma cells. PAK4 overexpression is known to induce increased cell migration speed but the requirement for kinase activity has not been established. We have used a panel of PAK4 truncations and mutations in a combination of overexpression and RNAi rescue experiments to determine the requirement for PAK4 kinase activity during carcinoma cell motility downstream of HGF. We find that neither the kinase domain alone nor a PAK4 mutant unable to bind Cdc42 is able to fully rescue cell motility in a PAK4-deficient background. Nevertheless, we find that PAK4 kinase activity and associated LIMK1 activity are essential for carcinoma cell motility, highlighting PAK4 as a potential anti-metastatic therapeutic target. We also show here that overexpression of PAK4 harbouring a somatic mutation, E329K, increased the HGF-driven motility of metastatic prostate carcinoma cells. E329 lies within the glycine-rich loop region of the kinase. Our data suggest that E329K mutation leads to a modest increase in kinase activity, conferring resistance to competitive ATP inhibitors in addition to promoting cell migration. The existence of such a mutation may have implications for the development of PAK4-specific competitive ATP inhibitors should PAK4 be further explored for clinical inhibition.

Increased use of laparoscopy in acute right iliac fossa pain - is it good for patients?

AIM: The use of laparoscopy, with or without appendicectomy, is becoming more common in the management of acute right iliac fossa (RIF) pain, but little is known of the 'unintended' consequences of this change. This study aimed to evaluate the impact of increased use of laparoscopy on the number and type of patients treated surgically and on the rate of negative appendicectomy. METHOD: A prospective audit was carried out of admissions to a teaching hospital over two, 3-month periods during 2007 and 2008. The management, investigations and outcome of patients presenting with RIF pain were studied. RESULTS: Admissions were stable over the two time-periods. There was a significant increase in the number of laparoscopic operations performed, from 22.5% (14/62) in 2007 to 85.7% (72/84) in 2008 (P < 0.0001), and the percentage of patients undergoing surgery rose from 55.4% (n = 62) in 2007 to 71.2% (n = 84) in 2008 (P < 0.01). In 2008, female patients were more likely to have surgery, an increase from 37.1% to 66.2% (P < 0.001), and were more likely to have a laparoscopic procedure, an increase from 50% to 98% (P < 0.0001). The rate of histologically confirmed appendicitis did not increase significantly (50/122 vs 57/118; P = 0.25), but the number of patients with a normal appendix either left in situ because it was macroscopically normal or found to be histologically normal following excision, increased significantly, from 9.01% in 2007 to 21.2% in 2008 (P < 0.01). The diagnostic value of pelvic ultrasound decreased from 75.6% of examinations in 2007 to 54.5% in 2008 (P = 0.039). CONCLUSION: An increase in laparoscopic procedures has resulted in more operations in women, an associated higher negative appendicectomy rate and decreased usefulness of pelvic ultrasound. Increased use of laparoscopy needs to be balanced against the diagnostic benefits of 'negative' laparoscopy.

Quantifying cell-matrix adhesion dynamics in living cells using interference reflection microscopy.

Focal adhesions and podosomes are integrin-mediated cell-substratum contacts that can be visualized using interference reflection microscopy (IRM). Here, we have developed automated image-processing procedures to quantify adhesion turnover from IRM images of live cells. Using time sequences of images, we produce adhesion maps that reveal the spatial changes of adhesions and contain additional information on the time sequence of these changes. Such maps were used to characterize focal adhesion dynamics in mouse embryo fibroblasts lacking one or both alleles of the vinculin gene. Loss of vinculin expression resulted in increased assembly, disassembly and/or in increased translocation of focal adhesions, suggesting that vinculin is important for stabilizing focal adhesions. This method is also useful for studying the rapid dynamics of podosomes as observed in primary mouse dendritic cells.

WASP and WIP regulate podosomes in migrating leukocytes.

Podosomes are specialized adhesion sites found in rapidly migrating and invasive cells, most notably in cells from the myeloid lineage that participate in immune surveillance and phagocyte defence mechanisms. In this review, we describe the nature of leukocyte podosomes and the regulation of their turnover during migration by the key regulatory molecules Wiskott-Aldrich syndrome protein and WASP-interacting protein.

Mechanism of Phosphodiesterase 5 inhibitor relief of prostatitis symptoms.

BACKGROUND: Chronic prostatitis is a common urological complaint without clearly defined causation or definitive treatment. HYPOTHESIS: Phosphodiesterase 5 (PDE5) Inhibitor mediated relaxation of prostatic duct smooth muscle increases washout of prostatic reflux products reducing prostatic inflammation and consequent prostatitis symptoms. RATIONALE OF HYPOTHESIS: The presence of both Nitric Oxide Synthase and Phosphodiesterase 5 in human prostatic tissue and the effect of nitric oxide donors and PDE5 inhibitors in vitro indicate PDE5 inhibitors relax prostatic smooth muscle. Significant retrograde urinary flux into prostatic ducts has been described and suggested as the mechanism of chronic prostatitis. We postulate PDE5 inhibitors alter prostatic reflux hence prostatitis symptoms. CONCLUSION: PDE5 inhibitors may represent a simple, effective treatment for chronic prostatitis.

Pharmacological rescue of noise induced hearing loss using N-acetylcysteine and acetyl-L-carnitine.

Despite the use of hearing protection devices (HPDs) and engineering changes designed to improve workspaces, noise-induced hearing loss continues to be one of the most common and expensive disabilities in the US military. Many service members suffer acoustic trauma due to improper use of HPDs, sound levels exceeding the protective capacity of the HPDs, or by unexpected, injurious exposures. In these cases, there is no definitive treatment for the hearing loss. This study investigated the use of the pharmacological agents N-acetylcysteine and acetyl-L-carnitine after acoustic trauma to treat cochlear injury. N-Acetylcysteine is an antioxidant and acetyl-L-carnitine a compound that maintains mitochondrial bio-energy and integrity. N-Acetylcysteine and acetyl-L-carnitine, respectively, significantly reduced permanent threshold shifts and hair cell loss compared to saline-treated animals when given 1 and 4 h post-noise exposure. It may be possible to obtain a greater therapeutic effect using these agents in combination or at higher doses or for a longer period of time to address the secondary oxidative events occurring 7-10 days after acute noise exposure.

Effect of wearing personal protective clothing and self-contained breathing apparatus on heart rate, temperature and oxygen consumption during stepping exercise and live fire training exercises.

Fire fighter breathing apparatus instructors (BAIs) must possess the ability to respond to both the extrinsic stress of a high temperature environment and the intrinsic stress from wearing personal protective equipment (PPE) and self-contained breathing apparatus (SCBA), repeatedly and regularly, whilst training recruits in live fire training exercises (LFTEs). There are few previous investigations on BAIs in hot environments such as LFTEs, since the main research focus has been on regular fire fighters undertaking exercises in temperate or fire conditions at a moderate to high exercise intensity. In this study, the intrinsic cardiovascular stress effects of wearing PPE + SCBA were first investigated using a step test whilst wearing gym kit (control), weighted gym kit (a rucksack weighted to the equivalent of PPE + SCBA) and full PPE + SCBA (weight plus the effects of protective clothing). The extrinsic effects of the very hot environment were investigated in BIAs in LFTEs compared to mock fire training exercises (MFTEs), where the fire was not ignited. There was an increase in heart rate due to the modest workload imposed on the BAIs through carrying out the MFTEs (25.0 (18.7)%) compared to resting. However, when exposed to fire during the LFTEs, heat storage appears to be significant as the heart rate increased by up to 39.8 (+/-20.1)% over that of the mock LFTEs at temperate conditions. Thus, being able to dissipate heat from the PPE is particularly important in reducing the cardiovascular responses for BAIs during LFTEs.

The effects of a high carbohydrate diet on cortisol and salivary immunoglobulin A (s-IgA) during a period of increase exercise workload amongst Olympic and Ironman triathletes.

The present study observed the effects of a 6-day high carbohydrate (H-CHO) diet on salivary cortisol and IgA during a period of increased exercise workload. Thirty-two competitively trained male triathletes were randomly allocated into a self-selected (SS), or an H-CHO (12 g CHO kgbm (-1) . day (-1)) dietary group. In addition to their training regimes, all subjects performed a 1-hour running exercise bout at 70 % V.O (2max) . d (-1), for six days. Saliva samples were taken pre, immediately post, and morning post-exercise bout on days 1, 4, and 6. The concentrations of s-IgA and cortisol were determined by ELISA assays. There was a significant (p < 0.001) interaction between Group x Time for cortisol, with a marked increase in concentrations occurring in the SS dietary group pre to post exercise, and pre to morning post-exercise (p < 0.01). Conversely, a significant (p = 0.009) Group x Time interaction reflected higher post exercise s-IgA concentrations (p < 0.005) than pre exercise in the H-CHO diet group. Blood glucose concentration decreased pre to post exercise in the SS diet group (p < 0.01), whilst remaining stable in the H-CHO group. It is concluded that the consumption of a high CHO diet throughout a 6-day period of overtraining had a favourable effect on markers of immune activity and thereby reduced the susceptibility of these endurance athletes to upper respiratory tract infection URTI.

Rho family GTPases are activated during HGF-stimulated prostate cancer-cell scattering.

An important process in embryogenesis and cancer-cell metastasis is the conversion of epithelial cells to a migratory phenotype, a phenomenon known as epithelial-mesenchymal transition (E-MT). To achieve E-MT, cells dissociate from neighbouring cells and adopt a migratory morphology. This transition requires remodelling of their cell shape and substratum adhesions; activities that require extensive reorganisation of the actin cytoskeleton. Hepatocyte growth factor (HGF)-induced scattering of Madin Darby canine kidney (MDCK) cells is a routinely used model of E-MT, in which actin cytoskeletal rearrangement is known to be dependent on Rho family GTPases. We have developed a novel model of HGF-induced E-MT using the human prostate cancer cell line, DU145. This model overcomes the limitation of using a canine cell line and facilitates the study of E-MT in human cancer. We demonstrate for the first time the scattering response of individual DU145 cells to HGF in real time and have characterised changes in actin cytoskeletal organisation and cell adhesions as these cells respond to HGF. HGF-induced scattering of DU145 cells is dependent on the activity of Rho family GTPases, and using this model, we are able to demonstrate for the first time that endogenous Cdc42 is activated downstream of HGF. Furthermore we have also shown that the response of DU145 cells to HGF is dependent on a phosphatidylinositide 3-kinase pathway.

A reconstructive algorithm for plastic surgery following extensive chest wall resection.

Chest wall reconstruction following extensive resection is greatly facilitated by the use of vascularised flaps and prosthetic material. Plastic surgeons are often asked to assist with coverage of large chest wall defects. However, in addition to soft tissue coverage, we need to address other important issues such as the status of the pleural cavity, and the requirement for skeletal support. The purpose of this report is to analyse our experience, provide a reconstructive algorithm following the ablative procedure and review the literature. Two hundred chest wall resections were performed from 1975 to 2000. Defect location was divided into anterior (n = 73) lateral (n = 36) anterior-lateral (n = 36) posterior-lateral (n = 19) posterior (n = 22) and forequarter (n = 14) Average number of ribs resected was four. One hundred and fifty-eight patients (79%) required chest wall reconstruction with either prosthetic material and/or flap closure. Mesh closure was required in 85 cases (43%), being highest for lateral defects (61%), and lowest for anterior defects (31%). Vascularised flaps were needed in 112 patients (56%), more common in anterior defects (79%), and less common for the posterior-lateral defects (26%). Inpatient complication rate was 27% (43/158) following reconstruction, with a mortality of 6% (10/158). Chest wall reconstruction is common following extensive resection. This includes management of the pleural cavity, skeletal support and soft tissue coverage. A better understanding of the respiratory mechanics and local thoracoabdominal anatomy is crucial for managing these complex defects. The need for skeletal support was more prevalent in lateral and posterior-lateral defects. Flap reconstruction was required more often to cover large anterior defects, with regional flaps predominating.

Opioid peptides and dipeptidyl peptidase in autism.

It has been hypothesized that autism results from an 'opioid peptide excess'. The aims of this study were to (1) confirm the presence of opioid peptides in the urine of children with autism and (2) determine whether dipeptidyl peptidase IV (DPPIV/CD26) is defective in children with autism. Opioid peptides were not detected in either the urine of children with autism (10 children; nine males, one female; age range 2 years 6 months to 10 years 1 month) or their siblings (10 children; seven males, three females; age range 2 years 3 months to 12 years 7 months) using liquid chromatography-ultraviolet-mass spectrometric analysis (LC-UV-MS). Plasma from 11 normally developing adults (25 years 5 months to 55 years 5 months) was also tested. The amount and activity of DPPIV in the plasma were quantified by an ELISA and DPPIV enzyme assay respectively; DPPIV was not found to be defective. The percentage of mononuclear cells expressing DPPIV (as CD26) was determined by flow cytometry. Children with autism had a significantly lower percentage of cells expressing CD3 and CD26, suggesting that they had lower T-cell numbers than their siblings. In conclusion, this study failed to replicate the findings of others and questions the validity of the opioid peptide excess theory for the cause of autism.

The muscle-specific marker desmin is expressed in a proportion of human dermal fibroblasts after their exposure to galectin-1.

We have previously shown that galectin-1 is a factor capable of converting mouse dermal fibroblasts to the myogenic lineage [Cell Transplant 2000;9:519]. Here, we report that human dermal fibroblasts are also capable of expressing the myogenic marker, desmin, when grown in muscle-cell-conditioned media. Furthermore, the human foetal skin cells also express this marker when grown in the presence of galectin-1. These results highlight the importance of galectin-1 in the conversion of both human and murine skin cells to a myogenic lineage. Thus galectin-1 could be an important tool for use in autologous cell therapies for the treatment of human muscular dystrophies.

Coordination of cell polarization and migration by the Rho family GTPases requires Src tyrosine kinase activity.

BACKGROUND: The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. However, it is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. RESULTS: We show here that individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively, and that the adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. CONCLUSIONS: Our findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites.

Configuration of human dendritic cell cytoskeleton by Rho GTPases, the WAS protein, and differentiation.

The cellular mechanisms that configure the cytoskeleton during migration of dendritic cells (DCs) are poorly understood. Immature DCs assemble specialized adhesion structures known as podosomes at their leading edge; these are associated with the localized recruitment of the Wiskott-Aldrich Syndrome protein (WASp) and the actin organizing actin-related protein 2/3 complex. In immature DCs lacking WASp, podosomes are absent, residual dysmorphic lamellipodia and filopodia are nonpolarized, and migration is severely compromised. Microinjection studies indicate that podosome assembly and polarization require concerted action of Cdc42, Rac, and Rho, thereby providing a link between sequential protrusive and adhesive activity. Formation of podosomes is restricted to cells with an immature phenotype, indicating a specific role for these structures during the early migratory phase. (Blood. 2001;98:1142-1149)