Influence of solution viscosity and injection protocol on distribution patterns of jet injectors: Application to photodynamic tumour targeting

Photodynamic therapy of deep or nodular skin tumours is currently limited by the poor tissue penetration of the porphyrin precursor 5-aminolevulinic acid (ALA) and preformed photosensitisers. In this study, we investigated the potential of jet injection to deliver both ALA and a preformed photosensitiser (meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate, TMP) into a defined volume of skin. Initial studies using a model hydrogel showed that as standoff distance is increased, injection depth decreases. As the ejected volume is increased, injection depth increases. It was also shown, for the first time, that, as injection solution viscosity was increased, for a given injection setting and standoff distance, both total depth of jet penetration, L-t, and depth at which the maximum width of the penetration pattern occurred, L-m, decreased progressively. For a standoff distance of zero, the maximum width of the penetration pattern, L-w, increased progressively with increasing viscosity at each of the injection settings. Conversely, when the standoff distance was 2.5 mm, L-w decreased progressively with increasing viscosity. Studies with neonate porcine skin revealed that an injection protocol comprising an 8.98 mPas solution, an arbitrary injection setting of 8 and a standoff distance of zero was capable of delivering photosensitisers to a volume of tissue (L-t of 2.91 mm, L-m of 2.14 mm, L-w of 5. 10 mm) comparable to that occupied by a typical nodular basal cell carcinoma. Both ALA and TMP were successfully delivered using jet injection, with peak tissue concentrations (67.3 mg cm(-3) and 5.6 mg cm(-3), respectively) achieved at a depth of around 1.0 mm and substantial reductions in drug concentration seen at depths below 3.0 mm. Consequently, jet injection may be suitable for selective targeting of ALA or preformed photosensitisers to skin tumours. (c) 2007 Elsevier B.V. All rights reserved.

Hexyl Aminolaevulinate Is a More Effective Topical Photosensitiser Precursor than Methyl Aminolaevulinate and 5-Aminolaevulinic Acids When Applied in Equimolar Doses

Aminolaevulinic acid (ALA) is known to poorly penetrate into thick lesions, such as nodular basal cell carcinomas Short chain ALA esters, possessing increased lipophilicity relative to their hydrophilic parent, have previously been shown to be highly efficient at inducing protoporphyrin IX (PpIX) production in cell culture, at equimolar concentrations. In contrast, in vitro skin permeation and in vivo animal studies, which up to now have compared prodrugs on a % w/vv basis, have failed to demonstrate such benefits For the first time, equimolar concentrations of ALA, methyl-ALA (m-ALA) and hexyl-ALA (h-ALA) have been incorporated into an o/w cream preparation. In vitro penetration studies into excised porcine skin revealed that increased levels of h-ALA, compared to ALA and m-ALA were found in the upper skin layers, at all drug loadings studied. Topical application of the formulations to nude murine skin in vivo, revealed that creams containing h-ALA induced significantly higher levels of peak PpIX fluorescence (F-max = 289.0) at low concentrations compared to m-ALA (F-max = 159.2) and ALA (F-max = 191 9). Importantly, this study indicates that when compared on an equimolar basis, h-ALA has improved skin penetration, leading to enhanced PpIX production compared to the parent drug and m-ALA (C) 2010 Wiley-Liss, Inc and the American Pharmacists Association J Pharm Sci 99 3486-3498, 2010

Influence of penetration enhancers on topical delivery of 5-aminolevulinic acid from bioadhesive patches

Objectives The inclusion 01 chemical penetration enhancers in a novel patch-based system for the delivery of 5-aminolevulinic acid (ALA) was examined in vitro and in vivo. Poor penetration of ALA has been implicated as the primary factor for low response rates achieved with topical ALA-based photodynamic therapy of thicker neoplastic lesions. such as nodular basal cell carcinomas.

Fluorescence (FISH) and chromogenic (CISH) in situ hybridisation in prostate carcinoma cell lines: comparison and use of virtual microscopy

Chromogenic in situ hybridisation (CISH) has become an attractive alternative to fluorescence in situ hybridisation (FISH) due to its permanent stain which is more familiar to pathologists and because it can be viewed using light microscopy, The aim of the present study is to examine reproducibility in the assessment of abnormal chromosome number by CISH in comparison to FISH. Using three prostate cell lines - PNTIA (derived from normal epithelium), LNCAP and DU145 (derived from prostatic carcinoma), chromosomes 7 and 8 were counted in 40 nuclei in FISH preparations (x100 oil immersion) and 100 nuclei in CISH preparations (x40) by two independent observers. The CISH slides were examined using standard fight microscopy and virtual microscopy. Reproducibitity was examined using paired Student's t-test (P

Effects of IL-13 on Mucociliary Differentiation of Pediatric Asthmatic Bronchial Epithelial Cells

Goblet cell hyperplasia (GCH) and decreased ciliated cells are characteristic of asthma. We examined the effects of IL-13 (2 and 20 ng/mL) on in vitro mucociliary differentiation in pediatric bronchial epithelial cells (PBECs) of normal PBEC [PBEC(N)] and asthmatic PBEC [PBEC(A)] children. Markers of differentiation, real-time PCR for MUC5AC, MUC5AC ELISA, and transepithelial electrical resistance (TEER) were assessed. Stimulation with 20 ng/mL IL-13 in PBEC(N) resulted in GCH [20 ng/mL IL-13: mean, 33.8% (SD, 7.2) versus unstimulated: mean, 18.9% (SD, 5.0); p < 0.0001] and decreased ciliated cell number [20 ng/mL IL-13: mean, 8% (SD, 5.6) versus unstimulated: mean, 22.7% (SD,7.6); p < 0.01]. PBEC(N) stimulated with 20 ng/mL IL-13 resulted in >5-fold (SD, 3.2) increase in MUC5AC mRNA expression, p < 0.001, compared with unstimulated PBEC(N). In PBEC(A), GCH was also seen [20 ng/mL IL-13: mean, 44.7% (SD, 16.4) versus unstimulated: mean, 30.4% (SD, 13.9); p < 0.05] with a decreased ciliated cell number [20 ng/mL IL-13: mean, 8.8% (SD, 7.5) versus unstimulated: mean, 16.3% (SD, 4.2); p < 0.001]. We also observed an increase in MUC5AC mRNA expression with 20 ng/mL IL-13 in PBEC(A), p < 0.05. IL-13 drives PBEC(N) toward an asthmatic phenotype and worsens the phenotype in PBEC(A) with reduced ciliated cell numbers and increased goblet cells.

The RET mutation E768D confers a late-onset Familial medullary thyroid carcinoma - only phenotype with incomplete penetrance: Implications for screening and management of carrier status

Objective: We describe a 4-generation family with familial medullary thyroid carcinoma (FMTC) - a variant of multiple endocrine neoplasia type 2 (MEN 2) without extra-thyroid features. RET mutation analysis confirmed an E768D mutation in exon 13 in 8 family members, 3 affected with medullary thyroid cancer alone while the other 5 were detected to be mutation carriers. This mutation has been described in very few families worldwide and the spectrum of disease and natural history is unclear. Results: Three affected members had medullary thyroid cancer (MTC) confirmed histologically at ages 25, 50 and 56 years, respectively. The E768D mutation appears to have a less aggressive clinical course compared to other high risk RET mutations with no evidence of clinical recurrence up to I I years after initial therapy. Of five gene carriers identified, two are asymptomatic at the age of 70 and 61, and three had raised calcitonin levels at 46, 39, and 45 years. Following total thyroidectomy, one gene carrier had a histologically normal thyroid at age 46, following a mildly elevated calcitonin, one had C-cell hyperplasia at the age of 39, and one had a frank focus of carcinoma in the left thyroid lobe at the age of 45. No members had evidence of phaeochromocytoma or parathyroid disease on screening. Conclusion: The RET E768D mutation is associated with MTC with a later age at presentation, incomplete penetrance and less aggressive course compared with other high risk RET mutations. To date in this family the E768D mutation has not been associated with either phaeochromocytoma or hyperparathyroidism. The appropriate screening strategy for and management of E768D carriers is difficult reflecting the phenotypic heterogeneity.

Synovial membrane immunohistology in early untreated juvenile idiopathic arthritis: differences between clinical subgroups

Abstract: Objective Juvenile idiopathic arthritis (JIA) consists of a heterogeneous group of inflammatory disorders, within which there are a number of clinical subgroups. Diagnosis and assignment to a particular subgroup can be problematical and more concise methods of subgroup classification are required. This study of the synovial membrane characterises the immunohistochemical features in early untreated, newly diagnosed JIA and compares findings with disease subgroup at 2 years.

Methods: 42 patients with newly diagnosed untreated JIA underwent synovial biopsy before the administration of steroids or disease-modifying antirheumatic drugs. Patients were classified as either polyarticular, persistent oligoarticular or extended-to-be oligoarticular. The location and semiquantitative analysis of T-cell subsets, B cells, macrophages and blood vessels were determined using immunohistochemistry.

Results: Synovial hyperplasia varied significantly between the three groups
(p<0.0001). There was a significant difference in the CD3 T-cell population between the three groups (p=0.004) and between the extended-to-be and persistent group (p=0.032). CD4 expression was significantly higher in the poly and extended-to-be oligo groups (p=0.002), again the extended-to-be group had more CD4 T cells than the persistent group (p=0.008). B-cell infiltrates were more marked in the polyarticular group and were significantly higher in the extended-to-be group compared with the persistent group (p=0.005). Vascularisation was more pronounced in the polyarticular and extended-to-be oligoarticular groups, the extended-to-be group had significantly more vascularisation than the persistent group (p=0.0002).

Conclusions: There are significant differences in the histomorphometric features of synovial tissue between patient subgroups. Immunohistological examination of synovial membrane biopsies may provide further insight into early disease processes in JIA.

Deposit-feeding mechanisms and resource partitioning in tropical holothurians

Aspidochirote holothurians found on tropical reef flats feed on particulate deposits which form a variety of substrata. The synaptid holothurian Opheodesoma grisea (Semper) feeds in a similar manner by scraping deposits from the surfaces of sea grasses. Distributional and gut content analyses showed that species partitioning is on the basis of substratum and particle size preference. Scanning electron microscopy revealed that the tentacles of aspidochirotes have a nodular surface while those of O. grisea have a tessellated surface structure. The twelve different species examined were shown to have different tentacular surface textures which bore an apparent relationship with the mean particle sizes selected by the different species. Light microscope studies of tentacle sections confirmed earlier observations on the extent of the water vascular system in aspidochirote and pinnate tentacles. From these observations a functional interpretation is proposed for tentacular operation and the means of particle selection in such holothurians.

In Silico Prediction of the Mechanobiological Response of Arterial Tissue: Application to Angioplasty and Stenting

One way to restore physiological blood flow to occluded arteries involves the deformation of plaque using an intravascular balloon and preventing elastic recoil using a stent. Angioplasty and stent implantation cause unphysiological loading of the arterial tissue, which may lead to tissue in-growth and reblockage; termed “restenosis.” In this paper, a computational methodology for predicting the time-course of restenosis is presented. Stress-induced damage, computed using a remaining life approach, stimulates inflammation (production of matrix degrading factors and growth stimuli). This, in turn, induces a change in smooth muscle cell phenotype from contractile (as exists in the quiescent tissue) to synthetic (as exists in the growing tissue). In this paper, smooth muscle cell activity (migration, proliferation, and differentiation) is simulated in a lattice using a stochastic approach to model individual cell activity. The inflammation equations are examined under simplified loading cases. The mechanobiological parameters of the model were estimated by calibrating the model response to the results of a balloon angioplasty study in humans. The simulation method was then used to simulate restenosis in a two dimensional model of a stented artery. Cell activity predictions were similar to those observed during neointimal hyperplasia, culminating in the growth of restenosis. Similar to experiment, the amount of neointima produced increased with the degree of expansion of the stent, and this relationship was found to be highly dependant on the prescribed inflammatory response. It was found that the duration of inflammation affected the amount of restenosis produced, and that this effect was most pronounced with large stent expansions. In conclusion, the paper shows that the arterial tissue response to mechanical stimulation can be predicted using a stochastic cell modeling approach, and that the simulation captures features of restenosis development observed with real stents. The modeling approach is proposed for application in three dimensional models of cardiovascular stenting procedures.

In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo

Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.

Evaluation of c-FLIP in castrate-resistant prostate cancer antagonizes therapeutic response to androgen receptor-targeted therapy

Purpose: To characterize the importance of cellular Fas-associated death domain (FADD)–like interleukin 1ß-converting enzyme (FLICE) inhibitory protein (c-FLIP), a key regulator of caspase-8 (FLICE)–promoted apoptosis, in modulating the response of prostate cancer cells to androgen receptor (AR)–targeted therapy.

Experimental Design: c-FLIP expression was characterized by immunohistochemical analysis of prostatectomy tissue. The functional importance of c-FLIP to survival and modulating response to bicalutamide was studied by molecular and pharmacologic interventions.

Results: c-FLIP expression was increased in high-grade prostatic intraepithelial neoplasia and prostate cancer tissue relative to normal prostate epithelium (P < 0.001). Maximal c-FLIP expression was detected in castrate-resistant prostate cancer (CRPC; P < 0.001). In vitro, silencing of c-FLIP induced spontaneous apoptosis and increased 22Rv1 and LNCaP cell sensitivity to bicalutamide, determined by flow cytometry, PARP cleavage, and caspase activity assays. The histone deacetylase inhibitors (HDACi), droxinostat and SAHA, also downregulated c-FLIP expression, induced caspase-8- and caspase-3/7–mediated apoptosis, and increased apoptosis in bicalutamide-treated cells. Conversely, the elevated expression of c-FLIP detected in the CRPC cell line VCaP underpinned their insensitivity to bicalutamide and SAHA in vitro. However, knockdown of c-FLIP induced spontaneous apoptosis in VCaP cells, indicating its relevance to cell survival and therapeutic resistance.

Conclusion: c-FLIP reduces the efficacy of AR-targeted therapy and maintains the viability of prostate cancer cells. A combination of HDACi with androgen deprivation therapy may be effective in early-stage disease, using c-FLIP expression as a predictive biomarker of sensitivity. Direct targeting of c-FLIP, however, may be relevant to enhance the response of existing and novel therapeutics in CRPC. Clin Cancer Res; 18(14); 3822–33.

Plasma concentrations of glucagon-like peptide-2 in adult patients with treated and untreated coeliac disease

Coeliac disease is a common chronic inflammatory enteropathy characterized by villous atrophy and crypt hyperplasia in the small intestine. The mechanism of the intestinal damage in coeliac disease remains unclear. Glucagon-like peptide (GLP)-2 is an enterotrophic peptide that causes crypt hyperplasia and intestinal cell proliferation. We postulate that GLP-2 may be involved in the mucosal changes found in coeliac disease.