The L-arginine-NO pathway in the pathophysiology of blood vessels from solid tumours and rats in endotoxic shock

Tumour-associated arteries are a potential target for therapeutic strategies aimed at reducing or stopping tumour growth and metastases. However, the tumour vasculature exists in a state of 'near maximal vasodilation' and is markedly hyporesponsive to vasoactive agents generally. The role of nitric oxide (NO) in the growth and maintenance of solid tumours is unclear. Recent evidence suggests that it may be responsible for some of the characteristics of angiogenically- derived vessels found within the tumour and also for those of 'normal' vessels which become incorporated into the surrounding host tissue. A marked hyporesponsiveness to vasoactive agents also characterises vessels from animals in endotoxin-induced shock. One aim of the present study then was to compare the properties of arteries which formerly supplied an implanted solid tumour (tumour epigastric artery, or TEA) with arteries taken from experimental animals (rat tail artery, or RTA) injected with bacterial lipopolysaccaride (LPS). The extent of endotoxaemia resulting from LPS treatment was assessed by (a) monitoring arterial blood pressure, using the tail cuff method; (b) measuring accumulated plasma nitrite and nitrate; (c) monitoring the development of hyporesponsiveness of RTAs to phenylephrine (PE); (d) detecting expression of inducible nitric oxide synthase (iNOS) by Western blot and immunocytochemical analysis; and (e) monitoring the loss of sensitivity of pre-contracted vessels from LPS-treated rats exposed to the vasodilator S-nitroso-N-acetylpenicillamine. RTAs from LPS-treated rats and TEAs were less sensitive to PE than control arteries. This difference was abolished by non-selective NOS inhibitors (L-NAME and L-NMMA) and by the isoform-selective inhibitor aminoguanidine (AG). Furthermore, the protein synthesis inhibitor cycloheximide also reversed the hyporesponsiveness to PE of both types of vessel. The 'restoring' effect of cycloheximide was abolished when given after indomethacin, a specific cyclooxygenase inhibitor. Chronic, oral administration of L-NAME or AG to tumour-bearing rats (via the drinking water) significantly slowed tumour growth. When L-NAME treatment was halted, tumour growth resumed at pre-L-NAME (control) rates. A single i.p. injection of LPS also impaired tumour growth: this was dependent upon the time of injection, with no effect seen 24hrs prior to tumour implantation and the maximum delaying effect at 11 days post-implantation. LPS potentiated the growth retarding effects of L-NAME but abolished those of AG. These results provide evidence for the involvement of iNOS-derived NO in regulating the tone of arteries supplying a solid tumour and also of those from animals in septic shock. They suggest that NO may assist tumour growth by helping to maintain an adequate flow of blood into the tumour and they highlight the L-arginine/NO pathway as a potential target for the design of improved therapeutic interventions

Probing the surface of eukaryotic cells using combinatorial toxin libraries

AbstractThe success of proteomics hinges in part on the development of approaches able to map receptors on the surface of cells. One strategy to probe a cell surface for the presence of internalized markers is to make use of Shiga-like toxin 1 (SLT-1), a ribosome-inactivating protein that kills eukaryotic cells [1, 2]. SLT-1 binds to the glycolipid globotriaosylceramide [3, 4], which acts as a shuttle, allowing the toxin to be imported and routed near ribosomes. We investigated the use of SLT-1 as a structural template to create combinatorial libraries of toxin variants with altered receptor specificity. Since all SLT-1 variants retain their toxic function, this property served as a search engine enabling us to identify mutants from these libraries able to kill target cells expressing internalizable receptors. Random mutations were introduced in two discontinuous loop regions of the SLT-1 receptor binding subunit. Minimal searches from screening 600 bacterial colonies randomly picked from an SLT-1 library identified toxin mutants able to kill cell lines resistant to the wild-type toxin. One such mutant toxin was shown to bind to a new receptor on these cell lines by flow cytometry. Toxin libraries provide a strategy to delineate the spectrum of receptors on eukaryotic cells

Mediastinal node staging by positron emission tomography-computed tomography and selective endoscopic ultrasound with fine needle aspiration for patients with upper gastrointestinal cancer: results from a regional centre

Aim: To investigate the impact of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) and positron emission tomography-computed tomography (PET-CT) in the nodal staging of upper gastrointestinal (GI) cancer in a tertiary referral centre. Methods: We performed a retrospective review of prospectively recorded data held on all patients with a diagnosis of upper GI cancer made between January 2009 and December 2015. Only those patients who had both a PET-CT and EUS with FNA sampling of a mediastinal node distant from the primary tumour were included. Using a positive EUS-FNA result as the gold standard for lymph node involvement, the sensitivity, specificity, positive and negative predictive values (PPV and NPV) and accuracy of PET-CT in the staging of mediastinal lymph nodes were calculated. The impact on therapeutic strategy of adding EUS-FNA to PET-CT was assessed. Results: One hundred and twenty one patients were included. Sixty nine patients had a diagnosis of oesophageal adenocarcinoma (Thirty one of whom were junctional), forty eight had oesophageal squamous cell carcinoma and four had gastric adenocarcinoma. The FNA results were inadequate in eleven cases and the PET-CT findings were indeterminate in two cases, therefore thirteen patients (10.7%) were excluded from further analysis. There was concordance between PET-CT and EUS-FNA findings in seventy one of the remaining one hundred and eight patients (65.7%). The sensitivity, specificity, PPV and NPV values of PET-CT were 92.5%, 50%, 52.1% and 91.9% respectively. There was discordance between PET-CT and EUS-FNA findings in thirty seven out of one hundred and eight patients (34.3%). MDT discussion led to a radical treatment pathway in twenty seven of these cases, after the final tumour stage was altered as a direct consequence of the EUS-FNA findings. Of these patients, fourteen (51.9%) experienced clinical remission of a median of nine months (range three to forty two months). Conclusion: EUS-FNA leads to altered staging of upper GI cancer, resulting in more patients receiving radical treatment that would have been the case using PET-CT staging alone

An investigation of exoskeleton robotic systems in assisting construction tasks

Whilst industrial robots have been widely used in many industrial sectors, they are predominantly used in a structured factory environment. In recent years, off-site robotics have been investigated extensively and there are some promising candidates emerging. One such category of robots is exoskeleton robots and this paper provides an in-depth assessment of their suitability in assisting human operators in undertaking manual operations typically found in the construction industry. This work aims to objectively assess the advantages and disadvantages of these two suits and provide recommendations for further improvements of similar system designs. The paper focuses on the passive exoskeleton robotic suits which are commercially available. Three types of activities are designed and a mechatronic methodology has been designed and implemented to capture visual data in order to assess these systems in comparison with normal human operations. The study suggests that these passive suits do reduce the effort required by human operators to undertake the same construction tasks as evidenced by the results from one focused study, though a number of improvements could be made to improve their performance for wider adoption

Phototoxic aptamers selectively enter and kill epithelial cancer cells

The majority of cancers arise from malignant epithelial cells. We report the design of synthetic oligonucleotides (aptamers) that are only internalized by epithelial cancer cells and can be precisely activated by light to kill such cells. Specifically, phototoxic DNA aptamers were selected to bind to unique short O-glycan-peptide signatures on the surface of breast, colon, lung, ovarian and pancreatic cancer cells. These surface antigens are not present on normal epithelial cells but are internalized and routed through endosomal and Golgi compartments by cancer cells, thus providing a focused mechanism for their intracellular delivery. When modified at their 5′ end with the photodynamic therapy agent chlorin e6 and delivered to epithelial cancer cells, these aptamers exhibited a remarkable enhancement (>500-fold increase) in toxicity upon light activation, compared to the drug alone and were not cytotoxic towards cell types lacking such O-glycan-peptide markers. Our findings suggest that these synthetic oligonucleotide aptamers can serve as delivery vehicles in precisely routing cytotoxic cargoes to and into epithelial cancer cells

Treatment of Canine Osseous Tumors with Photodynamic Therapy: A Pilot Study

Photodynamic therapy uses nonthermal coherent light delivered via fiber optic cable to locally activate a photosensitive chemotherapeutic agent that ablates tumor tissue. Owing to the limitations of light penetration, it is unknown whether photodynamic therapy can treat large osseous tumors. We determined whether photodynamic therapy can induce necrosis in large osseous tumors, and if so, to quantify the volume of treated tissue. In a pilot study we treated seven dogs with spontaneous osteosarcomas of the distal radius. Tumors were imaged with MRI before and 48 hours after treatment, and the volumes of hypointense regions were compared. The treated limbs were amputated immediately after imaging at 48 hours and sectioned corresponding to the MR axial images. We identified tumor necrosis histologically; the regions of necrosis corresponded anatomically to hypointense tissue on MRI. The mean volume of necrotic tissue seen on MRI after photodynamic therapy was 21,305 mm3 compared with a pretreatment volume of 6108 mm3. These pilot data suggest photodynamic therapy penetrates relatively large canine osseous tumors and may be a useful adjunct for treatment of bone tumors

Photodynamic Therapy Can Induce a Protective Innate Immune Response against Murine Bacterial Arthritis via Neutrophil Accumulation

Background: Local microbial infections induced by multiple-drug-resistant bacteria in the orthopedic field can be intractable, therefore development of new therapeutic modalities is needed. Photodynamic therapy (PDT) is a promising alternative modality to antibiotics for intractable microbial infections, and we recently reported that PDT has the potential to accumulate neutrophils into the infected site which leads to resolution of the infection. PDT for cancer has long been known to be able to stimulate the innate and adaptive arms of the immune system. Methodology/Principal Findings: In the present study, a murine methicillin-resistant Staphylococcus aureus (MRSA) arthritis model using bioluminescent MRSA and polystyrene microparticles was established, and both the therapeutic (Th-PDT) and preventive (Pre-PDT) effects of PDT using methylene blue as photosensitizer were examined. Although Th-PDT could not demonstrate direct bacterial killing, neutrophils were accumulated into the infectious joint space after PDT and MRSA arthritis was reduced. With the preconditioning Pre-PDT regimen, neutrophils were quickly accumulated into the joint immediately after bacterial inoculation and bacterial growth was suppressed and the establishment of infection was inhibited. Conclusions/Significance: This is the first demonstration of a protective innate immune response against a bacterial pathogen produced by PDT.National Institutes of Health (U.S.) (Grant number R01AI050875