Background: Anal Intraepithelial Neoplasia (AIM), a pre-cursor of anal squamous carcinoma, is increasingly detected in individuals with impaired immune function. However, choices for effective, low morbidity treatment are limited. Photodynamic Therapy (PDT) is promising as it is known to ablate more proximal gastrointestinal mucosa with safe healing, without damage to underlying muscle. It can also ablate skin with safe healing and minimal scarring.Methods: Pharmacokinetics: Normal rats were sensitised with 200 mg/kg 5-aminolaevulinic acid (ALA) and killed 1-8 h later. Anal tissues were examined by fluorescence microscopy to quantify the concentration of PPIX (protoporphyrin IX, the active derivative of ALA) in anal mucosa and in the underlying sphincter.PDT: Normal rats were sensitised similarly 3 h later, laser light (635 nm) was delivered. Anal canal: 50-150 J/cm using 1 cm diffuser fibre; for peri-anal skin, 50-200 J/cm(2), using microlens fibre. In each group, 2 rats were killed 3, 7, 14 and 28 days later and the anal region removed for histological examination.Results: Pharmacokinetics: Peak concentration of PPIX in mucosa was at 3 h, peak ratio mucosa: muscle, 6, seen at same time.PDT. Anal canal 50 J/cm: complete mucosal ablation by 3 days, complete regeneration by 28 days. Higher energies caused muscle damage with scarring. Peni-anal skin: 200 J/cm(2); complete ablation of skin, including appendages, complete healing by 28 days. Minimal effect with lower energy.Conclusion: ALA-PDT can ablate anal mucosa and pen-anal skin with safe healing and no underlying damage. However, over treatment can damage the sphincters. This technique is ready to undergo clinical trials. (C) 2013 Elsevier B.V. All rights reserved.

Photochemical internalisation (PCI) is a site-specific technique for improving cellular delivery of macromolecular drugs. In this study, a cell penetrating peptide, containing the core HIV-1 Tat 48­ 57 sequence, conjugated with a porphyrin photosensitiser has been shown to be effective for PCI. Herein we report an investigation of the photophysical and photobiological properties of a water soluble bioconjugate of the cationic Tat peptide with a hydrophobic tetraphenylporphyrin derivative. The cellular uptake and localisation of the amphiphilic bioconjugate was examined in the HN5 human head and neck squamous cell carcinoma cell line. Efficient cellular uptake and localisation in endo/lysosomal vesicles was found using fluorescence detection, and lightinduced,rupture of the vesicles resulting in a more diffuse intracellular fluorescence distribution was observed. Conjugation of the Tat sequence with a hydrophobic porphyrin thus enables cellular delivery of an amphiphilic photosensitiser which can then localise in endo/lysosomal membranes, as required for effective PCI treatment. PCI efficacy was tested in combination with a protein toxin, saporin, and a significant reduction in cell viability was measured versus saporin or photosensitiser treatment alone. This study demonstrates that the cell penetrating peptide­ photosensitiser bioconjugation strategy is a promising and versatile approach for enhancing the therapeutic potential of bioactive agents through photochemical internalisation.

Taiwan had been declared rabies-free in humans and domestic animals for five decades until July 2013, when surprisingly, three Formosan ferret badgers (FB) were diagnosed with rabies. Since then, a variety of wild carnivores and other wildlife species have been found dead, neurologically ill, or exhibiting aggressive behaviors around the island. To determine the affected animal species, geographic areas, and environments, animal bodies were examined for rabies by direct fluorescent antibody test (FAT). The viral genomes from the brains of selected rabid animals were sequenced for the phylogeny of rabies viruses (RABV). Out of a total of 1016 wild carnivores, 276/831 (33.2%) Formosan FBs were FAT positive, with occasional biting incidents in 1 dog and suspected spillover in 1 house shrew. All other animals tested, including dogs, cats, bats, mice, house shrews, and squirrels, were rabies-negative. The rabies was badger-associated and confined to nine counties/cities in sylvatic environments. Phylogeny of nucleoprotein and glycoprotein genes from 59 Formosan FB-associated RABV revealed them to be clustered in two distinct groups, TWI and TWII, consistent with the geographic segregation into western and eastern Taiwan provided by the Central Mountain Range and into northern rabies-free and central-southern rabies-affected regions by a river bisecting western Taiwan. The unique features of geographic and genetic segregation, sylvatic enzooticity, and FB-association of RABV suggest a logical strategy for the control of rabies in this nation.

Photodynamic therapy (PDT), also known as photoradiation therapy, phototherapy, or photochemo therapy, involves the use of a photoactive dye (photosensitizer) that is activated by exposure to light of a specific wavelength in the presence of oxygen. The transfer of energy from the activated photosensitizer to available oxygen results in the formation of toxic oxygen species, such as singlet oxygen and free radicals. These very reactive chemical species can damage proteins, lipids, nucleic acids, and other cellular components. Applications of PDT in dentistry are growing rapidly: the treatment of oral cancer, bacterial and fungal infection therapies, and the photodynamic diagnosis (PDD) of the malignant transformation of oral lesions. PDT has shown potential in the treatment of oral leukoplakia, oral lichen planus, and head and neck cancer. Photodynamic antimicrobial chemotherapy (PACT) has been efficacious in the treatment of bacterial, fungal, parasitic, and viral infections. The absence of genotoxic and mutagenic effects of PDT is an important factor for long-term safety during treatment. PDT also represents a novel therapeutic approach in the management of oral biofilms. Disruption of plaque structure has important consequences for homeostasis within the biofilm. Studies are now leading toward selective photosensitizers, since killing the entire flora leaves patients open to opportunistic infections. Dentists deal with oral infections on a regular basis. The oral cavity is especially suitable for PACT, because it is relatively accessible to illumination. Anti-microbial photodynamic therapy: useful in the future?

Fewer nerve-related side effects have been noted after treating head and neck cancer with photodynamic therapy (PDT) compared to conventional cancer therapy. Our aim is to investigate the biological basis for any such nerve-sparing effect. In this study the intracellular localisation and effect on cell viability of the photosensitiser meta-tetrahydroxylphenylchlorin (mTHPC) was investigated in cell culture models using peripheral nerve cells. Primary cells from adult rat dorsal root ganglia (containing both neurons and glia) were used in these experiments. Localisation of mTHPC was detected using fluorescence and confocal microscopy. Levels of mTHPC fluorescence were quantified using digital image analysis. Immunocytochemistry with anti-?-III-tubulin and anti-S100 was used to distinguish neuronal and glial cell populations respectively. A cell-death assay using propidium iodide was used to evaluate neural cell susceptibility to PDT following incubation with mTHPC. The results showed that mTHPC was localised in cytoplasmic regions of neurons and glia, but was not detected in neuronal axons. Necrotic cell death was detected after PDT in these neural cell types. These results suggest that the cells of the peripheral nervous system are susceptible to PDT-mediated necrosis, but that the sparing of nerves observed during clinical PDT may be related to the heterogeneous distribution of mTHPC within neurons.

Photodynamic therapy (PDT) is a tool for the treatment of certain cancerous and pre-cancerous conditions. The natural precursor of porphyrins 5-aminolevulinic acid (ALA) has been extensively used as a pro-photosensitiser in PDT. ALA’s poor permeability has been enhanced by chemical esterification with aliphatic alcohols. Some of the ALA esters proved to be more efficient than ALA for porphyrin synthesis. In the present work we studied the nature of porphyrin synthesis regulation from the ALA esters Hexyl-ALA (He-ALA) and R,S-ALA-2-(hydroxymethyl)tetrahydropyranyl ester (THP-ALA) in an adenocarcinoma cell line. We found that He-ALA is incorporated into the cells at a higher rate, followed by THP-ALA and ALA, whereas ALA and ALA esters efflux at the same rate mediated by passive diffusion. Although ALA entrance to the cell might be regulatory at low concentrations, ALA derivative uptake is not a limiting factor. At high concentrations, the regulation of ALA conversion into porphyrins is driven by the enzyme porphobilinogenase, whereas ALA esters hydrolysis is regulated by esterases. The key conclusion of this contribution is that the use of ALA esters has to be limited to low concentrations where no regulation on porphyrin synthesis takes place.

s from the 6th Biennial Meeting of the Leeds Castle Polyposis Group (LCPG) May 23-25, 1995; Toronto, Canada The 6th Biennia l Meet ing of the Leeds Castle Polyposis Group was held from the 23 25 May 1995 in Toronto, Canada, under the Chai rmanship of Professor Hart ley Stern, assisted by Professor Zane Cohen and Ms Terri Berk. There were 65 delegates from 16 countries. A total of 28 papers were given on the fol lowing main topics: the upper gastrointest inal tract (6); desmoid disease (3); surgery (5); molecular genetics (10); psychology (2); and pathology (2). The David Jagelman Memoria l Lecture 'Other Polyposis Syndromes ' was given by Robin Phillips, the Sir Ian Todd Lecture 'An Overview Of Famil ia l Adenomatous Polyposis Where We Have Been And Where We Are Going ' by Stanley Hamil ton, the HJR Bussey Lecture 'Ethical Issues ' by Dr Bernard Dickens, and the Eldon Gardner Debate 'Predict ive Testing: Panacea Or Pandora ' s Box' was co-chaired by Terri Berk and Pam Chapman. The Group heard with regret of the sad premature death of Lemuel Herrera who did so much for polyposis and the LCPG in his lifetime. It was decided that future meet ings would accept abstracts on Peutz-Jeghers syndrome, juven i l e polyposis, and other rare polyposis syndromes. The next meet ing will be held at Nordwijk Aarl Zee, The Netherlands, under the Chai rmanship of Dr. Hans Vasen during the first week in June 1997.

Analysis of the formation and organization of new connective tissue formed in tissue-engineered constructs is a major requirement for tissue bioreactor technology. We have analyzed early-stage responses in collagen lattices, using elastic scattering spectroscopy to assess its potential to monitor tissue structural changes in structures up to 3 mm thick, under normal culture conditions. The method is based on an optical system in which an optical fiber delivers white light onto the tissue and the back-scattered light is collected for spectroscopy by another optical fiber. Results show correlation between changes in the spectral signatures with changes in the collagen gel contraction or internal organization in all three models of collagen construct analyzed. Therefore elastic scattering spectroscopy is a promising tool to monitor tissue-engineered constructs or early repair in collagenous tissues.

ABSTRACTWe have previously demonstrated thatPorphyromonas gingivalisis susceptible to killing by toluidine blue O (TBO) when irradiated with light from a helium-neon (HeNe) laser. The aim of this study was to determine whether a TBO-antibody conjugate (Ab-TBO) could be used to specifically targetP. gingivalisto lethal photosensitization in the presence ofStreptococcus sanguisor human gingival fibroblasts (HGFs). When a mixture ofP. gingivalisandS. sanguiswas exposed to 4 μg of TBO/ml and irradiated with HeNe laser light, there were 1.5- and 4.0-log10-unit reductions in the viable counts, respectively. In contrast, when TBO was conjugated with a murine monoclonal antibody againstP. gingivalislipopolysaccharide, the reductions in viable counts ofP. gingivalisandS. sanguisamounted to 5.0 and 0.1 log10units, respectively. Lethal photosensitization ofP. gingivalisin the presence of HGFs using unconjugated TBO resulted in a 0.7-log10-unit reduction inP. gingivalisviable counts and a 99% reduction in the incorporation of tritiated thymidine ([3H]Tdr) by the HGFs. In contrast, when the Ab-TBO conjugate was used, there was a 100% reduction inP. gingivalisviable counts but no significant reduction in the incorporation of [3H]Tdr by HGFs. These results demonstrate that specific targeting ofP. gingivaliscan be achieved using TBO conjugated to a monoclonal antibody raised against a cell surface component of this organism.

A novel technique was investigated for the use of photodynamic therapy (PDT) for regulation of fibroblast contraction of three-dimensional (3D) collagen constructs. The optimal minimally lethal dose of the photosensitizing agent selected was established using the MTT assay on both cell suspensions and plated cells on the basis of a less than 35% cell death effect. The results showed that PDT played an important role in cell attachment/contraction. Initial dose response and cell force generation findings indicate that fibroblast contraction is dramatically reduced far in excess of that on cell viability.