P. Louise Coletta

Localization of cyclooxygenase-2 in human sporadic colorectal adenomas.

A putative target for the anti-colorectal cancer action of nonsteroidal anti-inflammatory drugs is the inducible isoform of cyclooxygenase (COX), COX-2. COX-2 is expressed within intestinal adenomas in murine polyposis models, but expression has been poorly characterized in human colorectal neoplasms. Therefore, we investigated the localization of the COX-2 protein in human sporadic colorectal adenomas. Immunohistochemistry for COX-2 and CD68 (a tissue macrophage marker) was performed on formalin-fixed, paraffin-embedded (n = 52) and frozen, acetone-fixed (n = 6) sections of human sporadic colorectal adenomas. Forty of 52 (77%) formalin-fixed adenomas expressed immunoreactive COX-2. COX-2 was localized to superficial interstitial macrophages in 39 cases (75%) and to deep interstitial macrophages in 9 cases (17%). COX-2 staining of dysplastic epithelial cells was observed in 15 cases (29%). A logistic regression analysis identified the adenoma site (P = 0.012) and histological type (P = 0.001) as independent predictors of superficial macrophage COX-2 expression. There was no relationship between the number of macrophages within an adenoma and macrophage COX-2 expression. These results indicate that COX-2 is expressed predominantly by interstitial macrophages within human sporadic colorectal adenomas. If COX-2 does indeed play a role in the early stages of colorectal carcinogenesis in man, these data suggest COX-2-mediated paracrine signaling between the macrophages and epithelial cells within adenomas.

Interstitial cell cyclooxygenase-2 expression is associated with increased angiogenesis in human sporadic colorectal adenomas

Cyclooxygenase (COX)‐2 plays an important role in intestinal tumorigenesis and angiogenesis in animal models. In superficial areas of human sporadic colorectal adenomas, COX‐2 is expressed predominantly by interstitial macrophages, in close proximity to microvessels. The aim of this study was to investigate the association between microvessel density (MVD) and COX‐2 expression in human sporadic colorectal adenomas. Immunohistochemistry and immunofluorescence for CD31 and COX‐2 were performed on a well‐characterized series of human sporadic colorectal adenomas (n = 37). The mean MVD and COX‐2 expression level (scored 0–3) in superficial and deep interstitial cells of adenomas were assessed by two independent observers. Superficial MVD was increased in COX‐2‐positive adenomas, compared with COX‐2‐negative adenomas (p = 0.037). There was a significant correlation between superficial MVD and increasing superficial interstitial cell COX‐2 expression score (p = 0.048). COX‐2‐expressing interstitial cells aggregated in areas of high MVD. No relationship was evident between MVD and COX‐2 expression in either deep interstitial cells or epithelial cells. Multivariate analysis demonstrated that only adenoma size (p = 0.005) was a significant independent predictor of MVD. COX‐2 protein expression by superficial interstitial cells in human sporadic colorectal adenomas is associated with increased angiogenesis. Promotion of angiogenesis may play a role in the pro‐tumourigenic activity of COX‐2 during growth of human colorectal adenomas. Copyright

Expanding 3D geometry for enhanced on-chip microbubble production and single step formation of liposome modified microbubbles

Micron sized, lipid stabilized bubbles of gas are of interest as contrast agents for ultra-sound (US) imaging and increasingly as delivery vehicles for targeted, triggered, therapeutic delivery. Microfluidics provides a reproducible means for microbubble production and surface functionalisation. In this study, microbubbles are generated on chip using flow-focussing microfluidic devices that combine streams of gas and liquid through a nozzle a few microns wide and then subjecting the two phases to a downstream pressure drop. While microfluidics has successfully demonstrated the generation of monodisperse bubble populations, these approaches inherently produce low bubble counts. We introduce a new micro-spray flow regime that generates consistently high bubble concentrations that are more clinically relevant compared to traditional monodisperse bubble populations. Final bubble concentrations produced by the micro-spray regime were up to 10(10) bubbles mL(-1). The technique is shown to be highly reproducible and by using multiplexed chip arrays, the time taken to produce one millilitre of sample containing 10(10) bubbles mL(-1) was ∼10 min. Further, we also demonstrate that it is possible to attach liposomes, loaded with quantum dots (QDs) or fluorescein, in a single step during MBs formation.

Paracrine cyclooxygenase-2-mediated signalling by macrophages promotes tumorigenic progression of intestinal epithelial cells

In human colorectal adenomas or polyps, cyclooxygenase-2 is expressed predominantly by stromal (or interstitial) macrophages. Therefore, we tested the hypothesis that macrophage cyclooxygenase-2 has paracrine pro-tumorigenic activity using in vitro models of macrophage-epithelial cell interactions. We report that macrophages can promote tumorigenic progression of intestinal epithelial cells (evidenced by decreased cell–cell contact inhibition, increased proliferation and apoptosis, gain of anchorage-independent growth capability, decreased membranous E-cadherin expression, up-regulation of cyclooxygenase-2 expression, down-regulation of transforming growth factor-β type II receptor expression and resistance to the anti-proliferative activity of transforming growth factor-β1) in a paracrine, cyclooxygenase-2-dependent manner. Pharmacologically relevant concentrations (1–2 μM) of a selective cyclooxygenase-2 inhibitor had no detectable, direct effect on intestinal epithelial cells but inhibited the macrophage-epithelial cell signal mediating tumorigenic progression. Cyclooxygenase-2-mediated stromal-epithelial cell signalling during the early stages of intestinal tumorigenesis provides a novel target for chemoprevention of colorectal cancer (and other gastro-intestinal epithelial malignancies, which arise on a background of chronic inflammation, such as gastric cancer) and may explain the discrepancy between the concentrations of cyclooxygenase inhibitors required to produce anti-neoplastic effects in vitro and in vivo.

Molecular analysis of the presenilin 1 (S182) gene in "sporadic" cases of Alzheimer's disease: identification and characterisation of unusual splice variants.

Abstract: Mutations of the presenilin 1 (PS‐1) gene at the Alzheimers disease (AD) FAD3 locus on chromosome 14q24.3 are responsible for the majority of familial early‐onset AD. As genes responsible for familial forms of AD are obvious candidates for further investigation in “sporadic” disease, we performed a molecular analysis of PS‐1 transcripts extracted from brain tissues of a series of histologically confirmed cases of “sporadic” AD (n = 10) and also from histologically “normal” (non‐Alzheimer) age‐matched brain controls (n = 5). No sequence changes in the PS‐1 coding sequence were detected after analysis by reverse transcription‐PCR. This suggests that the frequency of mutations in the PS‐1 (S182) coding region in “sporadic” Alzheimers disease is very low. However, we demonstrated that the PS‐1 gene is highly variably spliced. One splice variant involves the 5′ untranslated region of the PS‐1 gene only and hence encodes for normal PS‐1. Six further splice variants involve coding regions of the PS‐1 gene and result in truncated proteins lacking specific transmembrane domains. Most of these variants do not coincide with recognized sites of introns in the PS‐1 gene. One of these variants, resulting in the loss of transmembrane domain TM‐VII, was found only in an AD patient.

Increasing the sonoporation efficiency of targeted polydisperse microbubble populations using chirp excitation

The therapeutic use of microbubbles for targeted drug or gene delivery is a highly active area of research. Phospholipid-encapsulated microbubbles typically have a polydisperse size distribution over the 1 to 10 μm range and can be functionalized for molecular targeting and loaded with drug-carrying liposomes. Sonoporation through the generation of shear stress on the cell membrane by microbubble oscillations is one mechanism that results in pore formation in the cell membrane and can improve drug delivery. A microbubble oscillating at its resonant frequency would generate maximum shear stress on a membrane. However, because of the polydisperse nature of phospholipid microbubbles, a range of resonant frequencies would exist in a single population. In this study, the use of linear chirp excitations was compared with equivalent duration and acoustic pressure tone excitations when measuring the sonoporation efficiency of targeted microbubbles on human colorectal cancer cells. A 3 to 7 MHz chirp had the greatest sonoporation efficiency of 26.9 ± 5.6%, compared with 16.4 ± 1.1% for the 1.32 to 3.08 MHz chirp. The equivalent 2.2- and 5-MHz tone excitations have efficiencies of 12.8 ± 2.1% and 15.6 ± 1.1%, respectively, which were all above the efficiency of 4.1 ± 3.1% from the control exposure.

Analysis of cyclooxygenase expression in human colorectal adenomas

AbstractINTRODUCTION: Evidence from rodent intestinal tumorigenesis models suggests that both cyclooxygenase-1 and cyclooxygenase-2 may play important roles in the development and progression of human sporadic colorectal adenomas. However, previous studies of cyclooxygenase isoform expression in human colorectal adenomas have produced conflicting data. Cyclooxygenase-1 expression has been poorly studied, and cyclooxygenase-2 positivity of adenomas has been variable depending on the detection technique used. It also remains unclear whether villous adenomas express cyclooxygenase-2. METHODS: Cyclooxygenase isoform expression in human sporadic colorectal adenomas was analyzed by reverse transcription–polymerase chain reaction, Western blot analysis, and immunohistochemistry. RESULTS: Variable cyclooxygenase-1 expression was detected in all adenomas (n = 9) by both reverse transcription–polymerase chain reaction and Western blot analysis. Cyclooxygenase-2 expression was detected in eight (89 percent) of nine adenomas by reverse transcription–polymerase chain reaction and immunohistochemistry. Cyclooxygenase-2 protein was not detected by Western blot analysis in any adenoma. Cyclooxygenase-2 was expressed by all histopathologic types of adenoma and localized predominantly to superficial interstitial cells, in which it was associated with increased adenoma size. CONCLUSION: Cyclooxygenase-1 is expressed at variable levels by all adenomas. Cyclooxygenase-2 is expressed by the majority of adenomas, including those of the villous type, at levels below the sensitivity of Western blot analysis.

CEA-targeted nanoparticles allow specific in vivo fluorescent imaging of colorectal cancer models

Fluorescent imaging of colorectal tumor cells would improve tumor localization and allow intra-operative staging, facilitating stratification of surgical resections thereby improving patient outcomes. We aimed to develop and test fluorescent nanoparticles capable of allowing this in vivo. Dye-doped silica nanoparticles were synthesized. Anti-CEA (carcinoembryonic antigen) or control IgGs were conjugated to nanoparticles using various chemical strategies. Binding of CEA-targeted or control nanoparticles to colorectal cancer cells was quantified in vitro, and in vivo after systemic-delivery to murine xenografts. CEA-targeted, polyamidoamine dendrimer-conjugated, nanoparticles, but not control nanoparticles, allowed strong tumor-specific imaging. We are the first to demonstrate live, specific, in vivo imaging of colorectal cancer cells using antibody-targeted fluorescent nanoparticles. These nanoparticles have potential to allow intra-operative fluorescent visualization of tumor cells.

Efficient infection and persistence of a herpesvirus saimiri-based gene delivery vector into human tumor xenografts and multicellular spheroid cultures

Herpesvirus saimiri (HVS) has the ability to infect a variety of human cell lines and establish a persistent infection by virtue of episomal maintenance. Moreover, the viral episome provides sustained expression of a heterologous transgene. HVS-based vectors can also persist for a long term in tumor xenografts generated from HVS-infected human carcinoma cell lines. The viral episome remains latent within the xenograft allowing long-term transgene expression. These properties, in addition to its ability to incorporate large amounts of heterologous DNA, make HVS an attractive potential gene delivery vector. Here we report on the further evaluation of such HVS-based vectors. We demonstrate for the first time that HVS can efficiently infect solid tumor xenografts derived from a variety of human carcinoma cells via direct intratumoral injections. Furthermore, HVS can efficiently infect spheroid cultures, a three-dimensional cell culture system that closely resembles a tumor. Upon infection of both the tumor xenografts and spheroid cultures, HVS-based vectors can establish a persistent episomal infection within the tumor xenograft allowing expression of a heterologous transgene. These results suggest that HVS-based vectors may be suitable for cancer gene therapy applications.

The herpesvirus saimiri ORF 73 regulatory region provides long-term transgene expression in human carcinoma cell lines.

Herpesvirus saimiri (HVS) is capable of establishing a persistent infection in a variety of human carcinoma cell lines, by virtue of episomal maintenance. Moreover, the viral episome provides expression of a transgene in both in vitro and in vivo environments. At present, HVS vectors utilize heterologous promoters such as the IE hCMV promoter. However, this promoter maybe unsuitable for long-term expression in vivo, as promoter silencing has been observed in this and other herpesvirus-based vector systems. Ideal regulatory regions would be functional when the herpesvirus genome is maintained as a latent episome. We have previously shown that gene expression in an HVS-persistently-infected human carcinoma cell line is limited to an adjacent set of genes encoding ORFs 71–73. These genes are transcribed as a polycistronic mRNA species from a common regulatory region upstream of the ORF 73 gene. In this report, we assess the potential of the ORF 73 regulatory region to provide heterologous gene expression in a wide variety of human cancer cell lines. We demonstrate, utilizing transient transfection assays, that the ORF 73 regulatory region can provide transgene expression in a variety of human carcinoma cell lines, although levels of transgene expression are not as high as achieved under the control of heterologous promoters such as the IE hCMV promoter. Furthermore, incorporation of the minimal ORF 73 regulatory region in a recombinant HVS-based vector provides sustained expression of the green fluorescent protein in both in vitro and in vivo environments. These results suggest that the ORF 73 regulatory region may be suitable for use in HVS-based cancer gene therapy applications.