Histone H2AX phosphorylation as a molecular pharmacological marker for DNA interstrand crosslink cancer chemotherapy

The aims of this study were to investigate mechanisms of action involved in H2AX phosphorylation by DNA interstrand crosslinking (ICL) agents and determine whether gamma H2AX could be a suitable pharmacological marker for identifying potential ICL cellular chemosensitivity. In normal human fibroblasts, after treatment with nitrogen mustard (HN2) or cisplatin, the peak gamma H2AX response was detected 2-3 h after the peak of DNA ICLs measured using the comet assay, a validated method for detecting ICLs in vitro or in clinical samples. Detection of gamma H2AX foci by immunofluorescence microscopy could be routinely detected with 6-10 times lower concentrations of both drugs compared to detection of ICLs using the comet assay. A major pathway for repairing DNA ICLs is the initial unhooking of the ICL by the ERCC1-XPF endonuclease followed by homologous recombination. HN2 or cisplatin-induced gamma H2AX foci persisted significantly longer in both, ERCC1 or XRCC3 (homologous recombination) defective Chinese hamster cells that are highly sensitive to cell killing by ICL agents compared to wild type or ionising radiation sensitive XRCC5 cells. An advantage of using gamma H2AX immunofluorescence over the comet assay is that it appears to detect ICL chemosensitivity in both ERCC1 and HR defective cells. With HN2 and cisplatin, gamma H2AX foci also persisted in chemosensitive human ovarian cancer cells (A2780) compared to chemoresistant (A2780cisR) cells. These results show that gamma H2AX can act as a highly sensitive and general marker of DNA damage induced by HN2 or cisplatin and shows promise for predicting potential cellular chemosensitivity to ICL agents. (c) 2008 Elsevier Inc. All rights reserved.

Drug delivery strategies for photodynamic antimicrobial chemotherapy: From benchtop to clinical practice

Light and photosensitizer-mediated killing of many pathogens, termed photodynamic antimicrobial chemotherapy (PACT), has been extensively investigated in vitro. A wide range of organisms from the Gram-positive Staphylococcus aureus to the Gram-negative Pseudomonas aeruginosa have been proven to be susceptible to PACT. Multidrug-resistant strains are just as susceptible to this treatment as their naive counterparts. Both enveloped and non-enveloped viruses have demonstrated susceptibility in vitro, in addition to fungi and protozoa. Significantly, however, no clinical treatments based on PACT are currently licensed. This paper provides a comprehensive review of work carried out to date on delivery of photosensitizers for use in PACT, including topical, intranasal and oral/buccal delivery, as well as targeted delivery. We have also reviewed photo-antimicrobial surfaces. It is hoped that, through a rational approach to formulation design and subsequent success in small-scale clinical trials, more widespread use will be made of PACT in the clinic, to the benefit of patients worldwide. (C) 2009 Elsevier B.V. All rights reserved.

Anti-adherent and antifungal activities of surfactant-coated poly (ethylcyanoacrylate) nanoparticles

Application of non-drug-loaded poly(ethylcyanoacrylate) nanoparticles (NP) to buccal epithelial cells (BEC) imparted both anti-adherent and antifungal effects. NP prepared using emulsion polymerisation and stabilised using cationic, anionic and non-ionic surfactants decreased Candida albicans blastospore adhesion, an effect attributable to the peripheral coating of surfactant. Cetrimide and Pluronic (R) P 123 were shown to be most effective, producing mean percentage reductions in blastospore adherence of 52.7 and 37.0, respectively. Resultant zeta potential matched the polarity of the surfactant, with those stabilised using cetrimide being especially positive (+31.3 mV). Preparation using anionic surfactants was shown to be problematic, with low yield and wide particle size distribution. Evaluation of the antifungal effect of the peripheral coat was evaluated using zones of inhibition and viable counts assays. The former test revealed poor surfactant diffusion through agar, but did show evidence of limited kill. However, the latter method showed that cationic surfactants associated with NP produced high levels of kill, in contrast to those coated with anionic surfactants, where kill was not evident. Non-ionic surfactant-coated NP produced intermediate kill rates. Results demonstrate that surfactant-coated NP, particularly the cationic types, form the possible basis of a prophylactic formulation that primes the candidal target (BEC) against fungal adhesion and infection. (c) 2007 Elsevier B.V. All rights reserved.

Metabolic and structural properties of human obestatin {1-23} and two fragment peptides

Obestatin is a peptide produced in the oxyntic mucosa of the stomach and co-localizes with ghrelin on the periphery of pancreatic islets. Several studies demonstrate that obestatin reduces food and water intake, decreases body weight gain, inhibits gastrointestinal motility, and modulates glucose-induced insulin secretion. In this study we evaluated the acute metabolic effects of human obestatin {1-23} and fragment peptides {1-10} or {11-23} in high-fat fed mice, and then investigated their solution structure by NMR spectroscopy and molecular modelling. Obestatins {1-23} and {11-23} significantly reduced food intake (86% and 90% respectively) and lowered glucose responses to feeding, whilst leaving insulin responses unchanged. No metabolic changes could be detected following the administration of obestatin (1-10). In aqueous solution none of the obestatin peptides possessed secondary structural features. However, in a 2,2,2-trifluoroethanol (TFE-d(3))-H2O solvent mixture, the structure of obestatin {1-23} was characterized by an a-helix followed by a single turn helix conformation between residues Pro(4) and Gln(15) and His(19) and Ala(22) respectively. Obestatin {1-10} showed no structural components whereas {11-23} contained an a-helix between residues Val(14) and Ser(20) in a mixed solvent. These studies are the first to elucidate the structure of human obestatin and provide clear evidence that the observed a-helical structures are critical for in vivo activity. Future structure/function studies may facilitate the design of novel therapeutic agents based on the obestatin peptide structure. (C) 2010 Elsevier Inc. All rights reserved.

Measuring the state of consciousness in a free-living diving sea turtle

We report on results from two types of data-logger attached to hawksbill turtles (Eretmochelys coriacea) in the breeding season at the Seychelles, Indian Ocean. Conventional time-depth recorders (TDRs) showed prolonged bouts of long dives to the seabed, consistent with benthic resting. This behaviour has been widely reported in sea turtles and appears to be a common feature for energy conservation. An Inter-Mandibular Angle Sensor (IMASEN) recorded mouth opening and buccal pumping by one turtle for 2.5 days. Buccal pumping occurred widely while the turtle was submerged, consistent with a function of olfactory sensory perception of the turtle's environment. However, buccal pumping stopped during the middle of long benthic dives consistent with the turtle entering a phase of sleep. It therefore appears that by recording buccal oscillations, it is possible to assess the state of consciousness of turtles allowing the eco-physiology of diving to be more fully explored. (C) 2007 Elsevier B.V. All rights reserved.

Cofilin immunolabelling correlates with depth of invasion in gastrointestinal endocrine cell tumors

Gastrointestinal endocrine cell tumors are a heterogeneous population of lesions believed to arise from neuroendocrine cells of the gastrointestinal mucosa. The current classification of these tumors is based on tumor size, microscopic features and clinical evidence of metastasis. Although diagnostic categories generally correlate with prognosis, molecular prognostic markers will be clinically useful adjuncts. Cofilin has been implicated in tumor invasion, and its immunolocalisation was studied in gastrointestinal endocrine cell tumors. The immunolocalisation of cofilin was studied by immunohistochemistry in 34 formalin-fixed, paraffin wax-embedded gastrointestinal endocrine cell tumors using a tissue microarray platform. A significant correlation was found between high cofilin immunolabelling and the depth of invasion (p

RUNX3 Inactivation in Colorectal Polyps Arising Through Different Pathways of Colonic Carcinogenesis

OBJECTIVES: We hypothesized that RUNX3 inactivation by promoter hypermethylation in colorectal polyps is an early molecular event in colorectal carcinogenesis.
METHODS: RUNX3 protein expression was analyzed immunohistochemically in 50 sporadic colorectal polyps comprising 19 hyperplastic polyps (HPs), 14 traditional serrated adenomas (TSAs), and 17 sporadic traditional adenomas (sTAs) as well as in 19 familial adenomatous polyposis (FAP) samples from 10 patients showing aberrant crypt foci (ACF) (n=91), small adenomas (SmAds) (n=40), and large adenomas (LAds) (n=13). In addition, we assessed the frequency of promoter hypermethylation of RUNX3 by methylation-specific PCR (MSP) in all the 50 sporadic polyps as well as 38 microdissected FAP polyps comprising ACF, SmAds, and LAds obtained from 7 FAP samples. A total of 12 normal colon samples were also included for RUNX3 MSP analysis.
RESULTS: Compared to normal colon (2 of 12, 16%) and sTAs (3 of 17, 18%), HPs (15 of 19, 79%) and TSAs (8 of 14, 57%) displayed significant inactivation of RUNX3 (P<0.05). In FAP, RUNX3 inactivation was more frequently seen in ACF (78 of 91, 86%), SmAds (25 of 40, 62%), and LAds (6 of 13, 46%) compared to normal mucosa (0 of 19, 0%) in the same samples (all P<0.05). Promoter hypermethylation of RUNX3 was significantly higher in colorectal polyps (64 of 87, 74%) compared to normal colon (2 of 12, 16%) (P=0.001). Serrated polyps such as HPs (17 of 19, 89%) and TSAs (12 of 14, 86%) were significantly more methylated than sTAs (7 of 17, 44%) (P=0.004). RUNX3 hypermethylation was observed in 28 of the total 38 (74%) FAP polyps. Overall, RUNX3 promoter methylation correlated with inactivation of RUNX3 expression in sporadic (27 of 36, 75%) (P=0.022) and FAP (21 of 28, 75%) (P=0.021) polyps.
CONCLUSIONS: Our data suggest that RUNX3 inactivation due to promoter hypermethylation in colorectal polyps represents an early event in colorectal cancer (CRC) progression. In addition, epigenetic RUNX3 inactivation is a frequent event in the serrated colonic polyps as well as in the ACF of FAP polyps.

RUNX3, a novel tumor suppressor, is frequently inactivated in gastric cancer by protein mislocalization

Loss of RUNX3 expression is suggested to be causally related to gastric cancer as 45% to 60% of gastric cancers do not express RUNX3 mainly due to hypermethylation of the RUNX3 promoter. Here, we examined for other defects in the properties of RUNX3 in gastric cancers that express RUNX3. Ninety-seven gastric cancer tumor specimens and 21 gastric cancer cell lines were examined by immunohistochemistry using novel anti-RUNX3 monoclonal antibodies. In normal gastric mucosa, RUNX3 was expressed most strongly in the nuclei of chief cells as well as in surface epithelial cells. In chief cells, a significant portion of the protein was also found in the cytoplasm. RUNX3 was not detectable in 43 of 97 (44%) cases of gastric cancers tested and a further 38% showed exclusive cytoplasmic localization, whereas only 18% showed nuclear localization. Evidence is presented suggesting that transforming growth factor-beta is an inducer of nuclear translocation of RUNX3, and RUNX3 in the cytoplasm of cancer cells is inactive as a tumor suppressor. RUNX3 was found to be inactive in 82% of gastric cancers through either gene silencing or protein mislocalization to the cytoplasm. In addition to the deregulation of mechanisms controlling gene expression, there would also seem to be at least one other mechanism controlling nuclear translocation of RUNX3 that is impaired frequently in gastric cancer.

What is the significance of muciphages in colorectal biopsies?

Muciphages (mucin-containing macrophages), first described in 1966 by Azzopardi & Evans, are a common feature of biopsies of large intestinal mucosa, even in the absence of other abnormalities such as active inflammation or evidence of chronic inflammatory bowel disease. Should they be mentioned in diagnostic reports? Do muciphages reliably indicate previous mucosal disease, now quiescent? In the following articles, Salto-Tellez & Price review what is known about muciphages and conclude that they reflect previous occult and clinically unimportant mucosal damage and that, in an otherwise normal colorectal mucosa, they have no diagnostic significance; and Shepherd draws attention to a wide range of clinically much more significant mucosal infiltrates that could be mistakenly regarded as muciphages and thus overlooked.

Llama antibody fragments have good potential for application as HIV-1 topical microbicides

There is an urgent global need for preventative strategies against HIV-1 infections. Llama heavy-chain antibody fragments (VHH) are a class of molecules recently described as potent cross-clade HIV-1 entry inhibitors. We studied the potential of a VHH-based microbicide in an application-oriented fashion. We show that VHH can be inexpensively produced in high amounts in the GRAS organism S. cerevisiae, resulting in very pure, and endotoxin free product. VHH are very stable under conditions they might encounter during transport, storage or use by women. We developed active formulations of VHH in aqueous gel and compressed and lyophilized tablets for controlled release from an intra vaginal device. The release profile of the VHH from e.g. a vaginal ring suggests sufficient bioavailability and protective concentration of the molecule at the mucosal site at the moment of the infection. The ex vivo penetration kinetics through human tissues show that the VHH diffuse into the mucosal layer and open the possibility to create a second defense layer either by blocking the HIV receptor binding sites or by blocking the receptors of immune cells in the mucosa. In conclusion, our data show that VHH have

Molecular investigations into vaginal immunization with HIV gp41 antigenic construct H4A in a quick release solid dosage form

A robust vaginal immune response is considered essential for an effective prophylactic vaccine that prevents transmission of HIV and other sexually acquired diseases. Considerable attention has recently focused on the potential of vaginally administered vaccines as a means to induce such local immunity. However, the potential for vaccination at this site remains in doubt as the vaginal mucosa is generally considered to have low immune inductive potential. In the current study, we explored for the first time the use of a quick release, freeze-dried, solid dosage system for practical vaginal administration of a protein antigen. These solid dosage forms overcome the common problem associated with leakage and poor retention of vaginally administered antigen solutions. Mice were immunized vaginally with H4A, an HIV gp41 envelope based recombinant protein, using quick release, freeze-dried solid rods, and the immune responses compared to a control group immunized via subcutaneous H4A injection. Vaginally immunized mice failed to elicit robust immune responses. Our detailed investigations, involving cytokine analysis, the stability of H4A in mouse cervicovaginal lavage, and elucidation of the state of H4A protein in the immediate-release dosage form, revealed that antigen instability in vaginal fluid, the state of the antigen in the dosage form, and the cytokine profile induced are all likely to have contributed to the observed lack of immunogenicity. These are important factors affecting vaginal immunization and provide a rational basis for explaining the typically poor and variable elicitation of immunity at this site, despite the presence of immune responsive cells within the vaginal mucosae. In future mucosal vaccine studies, a more explicit focus on antigen stability in the dosage form and the immune potential of available antigen-responsive cells is recommended. © 2012 Elsevier Ltd. All rights reserved.

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis:in vitro evidence in cultured epithelial cells

BACKGROUND:
Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR.
OBJECTIVES:
The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects.
METHODS:
Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function.
RESULTS:
Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway.
CONCLUSIONS:
This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.

Local delivery of chlorhexidine using a tooth-bonded delivery system

Films containing 20% w/w chlorhexidine base (particle size 63-125 mu m) in poly(epsilon-caprolactone), MW 35 000-45 000, were prepared by solvent evaporation and sections attached to the mesio-lingual and mesio-buccal surfaces of the lower first molar in healthy volunteers. Saliva (

PRELIMINARY INVESTIGATIONS CONCERNING THE ANTI-ADHERENCE PROPERTIES OF POLYHEXAMETHYLENEBIGUANIDE (VANTOCIL IB(TM))

The effect of polyhexamethylenebiguanide (PHMB) on adherence of Candida albicans blastospores to human buccal epithelial cells (BEG) was examined in vitro. Treatments of either blastospores or BEC with PHMB (50 and 1000 mu g ml(-1)) significantly reduced the number of adherent blastospores per BEC and increased the number of BEC devoid of blastospores.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF NEUROPEPTIDES IN THE PERIPHERAL NERVOUS-SYSTEM OF THE ROUNDWORM ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localisation and distribution of neuropeptides in the peripheral nervous system of the pig roundworm Ascaris suum have been determined by an indirect immunofluorescence technique in conjunction with confocal microscopy. Of the 31 antisera tested, immunostaining was obtained only with antisera to peptide YY (PYY), pancreatic polypeptide (PP) and FMRFamide. Immunostaining for PYY and FMRFamide was evident in the amphidial and papillary ganglia associated with the anterior nerve ring and in the nerves from these ganglia that terminated in sensory receptors within the buccal lips of the parasite. The only peptide immunoreactivity (IR) observed in the reproductive system of either sex was that evident in the nerve supply to the distal region of the vagina in the female worm. It took the form of a well-developed plexus of parallel nerve fibres, cross-connectives and looped commissures. The nerve net diminished in the more proximal region of the vagina. PP-IR was less intense than that for PYY and FMRFamide and was more restricted in distribution, being confined to a small number of nerve fibres in the nerve supply to the vagina; it did not occur in the nerves supplying the anterior sensory receptors. The possible roles of neuropeptides in the sensory and reproductive biology of nematodes are discussed.