Safe ablation of the anal mucosa and perianal skin in rats using Photodynamic Therapy--a promising approach for treating Anal Intraepithelial Neoplasia.

BACKGROUND: Anal Intraepithelial Neoplasia (AIN), 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 200mg/kg 5-aminolaevulinic acid (ALA) and killed 1-8h 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 3h later, laser light (635 nm) was delivered. Anal canal: 50-150 J/cm using 1cm 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 3h, 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. Peri-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 peri-anal skin with safe healing and no underlying damage. However, over treatment can damage the sphincters. This technique is ready to undergo clinical trials.

Influence of zinc and zinc chelator on HT-29 colorectal cell line.

Trace elements are involved in many key pathways involving cell cycle control. The influence of zinc and zinc chelator (TPEN) on transcription levels of the main zinc transporters (ZnT1 and ZIP1) in the HT-29 colorectal cell line has not been reported. Proliferation of HT-29 cells was measured using the methylene blue assay after exposure to zinc (two concentrations), TPEN (two concentrations), or a combination of zinc and TPEN (simultaneously and sequentially) for 4 h, 8 h, and 24 h. The transcription levels of ZnT1, ZIP1, vascular endothelial growth factor (VEGF), and caspase-3 were determined using reverse transcriptase real-time polymerase chain reaction (RT-PCR) after exposure of cells to zinc and TPEN. The zinc content in the substrate (medium used for culture) was determined using atomic absorption spectrometry. TPEN decreased cellular proliferation causing complete cell death by 8 h. Zinc had a protective effect against short periods of exposure to TPEN. There was no correlation between the transcripts of main zinc transporters and the zinc content in the substrate. The zinc content in the substrate remained constant after varying periods of cell culture. TPEN decreased the transcript levels of caspase-3 and VEGF, which are surrogate markers for apoptosis and angiogenesis. Zinc chelation of HT-29 cells causes cell death. Zinc appears to be protective for short periods of exposure to TPEN but has no protective effect on prolonged exposure. HT-29 cells are not able to counteract the effect of intracellular chelation of zinc by altering zinc transport. Further research into the mechanisms of these findings is necessary and may lead to novel therapeutic options.

Photochemical internalisation of chemotherapy potentiates killing of multidrug-resistant breast and bladder cancer cells.

Multidrug resistance (MDR) is the major confounding factor in adjuvant solid tumour chemotherapy. Increasing intracellular amounts of chemotherapeutics to circumvent MDR may be achieved by a novel delivery method, photochemical internalisation (PCI). PCI consists of the co-administration of drug and photosensitiser; upon light activation the latter induces intracellular release of organelle-bound drug. We investigated whether co-administration of hypericin (photosensitiser) with mitoxantrone (MTZ, chemotherapeutic) plus illumination potentiates cytotoxicity in MDR cancer cells. We mapped the extent of intracellular co-localisation of drug/photosensitiser. We determined whether PCI altered drug-excreting efflux pump P-glycoprotein (Pgp) expression or function in MDR cells. Bladder and breast cancer cells and their Pgp-overexpressing MDR subclones (MGHU1, MGHU1/R, MCF-7, MCF-7/R) were given hypericin/MTZ combinations, with/without blue-light illumination. Pilot experiments determined appropriate sublethal doses for each. Viability was determined by the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide assay. Intracellular localisation was mapped by confocal microscopy. Pgp expression was detected by immunofluorescence and Pgp function investigated by Rhodamine123 efflux on confocal microscopy. MTZ alone (0.1-0.2 microg ml(-1)) killed up to 89% of drug-sensitive cells; MDR cells exhibited less cytotoxicity (6-28%). Hypericin (0.1-0.2 microM) effects were similar for all cells; light illumination caused none or minimal toxicity. In combination, MTZ /hypericin plus illumination, potentiated MDR cell killing, vs hypericin or MTZ alone. (MGHU1/R: 38.65 and 36.63% increase, P<0.05; MCF-7/R: 80.2 and 46.1% increase, P<0.001). Illumination of combined MTZ/hypericin increased killing by 28.15% (P<0.05 MGHU1/R) compared to dark controls. Intracytoplasmic vesicular co-localisation of MTZ/hypericin was evident before illumination and at serial times post-illumination. MTZ was always found in sensitive cell nuclei, but not in dark resistant cell nuclei. In illuminated resistant cells there was some mobilisation of MTZ into the nucleus. Pgp expression remained unchanged, regardless of drug exposure. Pgp efflux was blocked by the Pgp inhibitor verapamil (positive control) but not impeded by hypericin. The increased killing of MDR cancer cells demonstrated is consistent with PCI. PCI is a promising technique for enhancing treatment efficacy.

Aspergillus fumigatus strains recovered from immunocompromised patients (ICP): subtyping of strains by RAPD analysis.

Invasive pulmonary aspergillosis in immunocompromised patients (ICP) is the second most frequent opportunistic fungal infection. The causative organism includes 16 species of Aspergillus, of which A. fumigatus dominates the ubiquitous incidence of invasive or allergic broncho-pulmonary aspergillosis (ABPA). The definitive diagnosis of invasive aspergillosis is difficult. We have analyzed 24 strains of A. fumigatus recovered from ICP using the RAPD technique. The profiles generated with the 20 primers tested were mostly unique. These results may have a profound impact on the management of aspergillosis, especially in the ICP.

RAPD profile variation amongst provenances of neem.

Neem, described as a tree for solving global problems, is an evergreen, long-lived, multipurpose tree of the tropics with a wide distribution range in India. It is believed to be highly cross-pollinated. Inter-provenance variations have been reported in neem in case of morphological and physiological characters. Yet no reports about the genetic determinism for these variations are available to our knowledge. In order to have an idea about the extent and/or nature of genetic (DNA) variation in neem, the powerful RAPD technique has been employed. RAPD profiles of 34 accessions/provenances of neem were generated with 200 decamer random primers, of which the data from the 49 primers, that resulted in reproducible amplification products, were considered for analysis. Based on the presence/absence of bands, a similarity matrix was computed. Dendrogram was constructed by UPGMA method based on the pairwise similarities amongst the RAPD profiles. The similarities in RAPD profiles amongst the different DNAs was more than that expected due to the cross-pollinated nature of the tree and furthermore, these more-than-expected similarities were not due to random chance. These results suggest that neem may have a narrow genetic base.

RAPD analysis of Candida albicans strains recovered from different immunocompromised patients (ICP) reveals an apparently non-random infectivity of the strains.

The opportunistic imperfect fungus Candida albicans causing life-threatening infections in immunocompromised patients (ICP), especially in HIV-positive cases, is recognized to be one of the most important nosocomial pathogens in the recent decades. The extent of strain-to-strain variation within a species and its relationship to the ability of the organism to colonize or invade a specific group of patients or even a body site is, however, not well known. We have analysed 19 strains of C. albicans recovered from ICP at different locales and times, employing the RAPD technique. No two strains generated identical RAPD profiles with any of the 21 primers tested. Further, the UPGMA clustering of the strains seemingly reflected a certain relationship or nonrandomness in the infection of the patients with the strain of C. albicans vis-a-vis the immunocompromised status due to underlying disease such as diabetes, cancer, asthma and meningitis. These results may have a profound impact on the management of candidiasis, especially in the ICP.