Design, construction, and analysis of cell line arrays and tissue microarrays for gene expression analysis

Cell line array (CMA) and tissue microarray (TMA) technologies are high-throughput methods for analysing both the abundance and distribution of gene expression in a panel of cell lines or multiple tissue specimens in an efficient and cost-effective manner. The process is based on Kononen's method of extracting a cylindrical core of paraffin-embedded donor tissue and inserting it into a recipient paraffin block. Donor tissue from surgically resected paraffin-embedded tissue blocks, frozen needle biopsies or cell line pellets can all be arrayed in the recipient block. The representative area of interest is identified and circled on a haematoxylin and eosin (H&E)-stained section of the donor block. Using a predesigned map showing a precise spacing pattern, a high density array of up to 1,000 cores of cell pellets and/or donor tissue can be embedded into the recipient block using a tissue arrayer from Beecher Instruments. Depending on the depth of the cell line/tissue removed from the donor block 100-300 consecutive sections can be cut from each CMA/TMA block. Sections can be stained for in situ detection of protein, DNA or RNA targets using immunohistochemistry (IHC), fluorescent in situ hybridisation (FISH) or mRNA in situ hybridisation (RNA-ISH), respectively. This chapter provides detailed methods for CMA/TMA design, construction and analysis with in-depth notes on all technical aspects including tips to deal with common pitfalls the user may encounter. © Springer Science+Business Media, LLC 2011.

Targeting histone deacetylases for the treatment of immune, endocrine and metabolic disorders

The 'histone code' is a well-established hypothesis describing the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as histone acetyltransferases or HATs, and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. The proinflammatory environment is increasingly being recognised as a critical element for both degenerative diseases and cancer. The present review will discuss the current knowledge surrounding the clinical potential & current development of histone deacetylases for the treatment of diseases for which a proinflammatory environment plays important roles, and the molecular mechanisms by which such inhibitors may play important functions in modulating the proinflammatory environment. © 2009 Bentham Science Publishers Ltd.

Galectin-1 : a link between tumor hypoxia and tumor immune privilege

Purpose: To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression. Methods: We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CDS (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes. Results: SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O 2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CDS staining (P = .01). Expression of galectin-1 and CDS were significant predictors for overall survival on multivariate analysis. Conclusion: Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege. © 2005 by American Society of Clinical Oncology.

A phase II study of the modulation of 5-fluorouracil and folinic acid with high-dose infusional hydroxyurea in metastatic colorectal carcinoma

Background: Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, may potentiate the activity of 5-fluorouracil (5-FU) and folinic acid (FA) by reducing the deoxyribonucleotide pool available for DNA synthesis and repair. However as HU may inhibit the formation of 5-fluoro-2-deoxyuridine-5- monophosphate (FdUMP), one of the principal active metabolites of 5-FU, the scheduling of HU may be critical. In vitro experiments suggest that administration of HU following 5-FU, maintaining the concentration in the region of I mM for six or more hours, significantly enhances the efficacy of 5-FU. Patients and methods: 5-FU/FA was given as follows: days 1 and 2 - FA 250 mg/m 2 (max. 350 mg) over two hours followed by 5-FU 400 mg/m 2 by intravenous bolus (ivb) over 15 minutes and subsequently 5-FU 400 mg/m 2 infusion (ivi) over 22 hours. HU was administered on day 3 immediately after the 5-FU with 3 g ivb over 15 minutes followed by 12 g ivi over 12 hours. Results: Thirty patients were entered into the study. Median survival was nine months (range 1-51 + months). There were eight partial responses (28%, 95% CI: 13%-47%). The median duration of response was 6.5 (range 4-9 months). Grade 3-4 toxicities included neutropenia (grade 3 in eight patients and grade 4 in five), anaemia (grade 3 in one patient) and diarrhoea (grade 3 in two patients). Neutropenia was associated with pyrexia in two patients. Phlebitis at the infusion site occurred in five patients. The treatment was complicated by pulmonary embolism in one patient and deep venous thrombosis in another. Conclusion: HU administered in this schedule is well tolerated. Based on these results and those of other phase II studies, a randomised phase III study of 5-FU, FA and HU versus 5-FU and FA using the standard de Gramont schedule is recommended.

A phase I dose-escalating study of DaunoXome, liposomal daunorubicin, in metastatic breast cancer

The aims of this phase I study were to establish the maximum tolerated dose, safety profile and activity of liposomal daunorubicin, DaunoXome (NeXstar Pharmaceuticals), in the treatment of metastatic breast cancer. DaunoXome was administered intravenously over 2 h in 21 day cycles and doses were increased from 80 to 100, 120 and 150 mg m 2. Sixteen patients were enrolled. A total of 70 cycles of DaunoXome were administered. The maximum tolerated dose was 120 mg m 2, the dose-limiting toxicity being prolonged grade 4 neutropenia or neutropenic pyrexia necessitating dose reductions at 120 and 150 mg m 2. Asymptomatic cardiotoxicity was observed in three patients: grade 1 in one treated with a cumulative dose of 800 mg m 2 and grade 2 in two, one who received a cumulative dose of 960 mg m 2 and the other a cumulative dose of 600 mg m 2 with a previous neoadjuvant doxorubicin chemotherapy of 300 mg m 2. Tumour response was evaluable in 15 patients, of whom two had objective responses, six had stable disease and seven had progressive disease. In conclusion, DaunoXome is associated with mild, manageable toxicities and has anti-tumour activity in metastatic breast cancer. The findings support further phase II evaluation of DaunoXome alone and in combination with other standard non-anthracycline cytotoxic or novel targeted agents. Although the dose-limiting toxicity for DaunoXome was febrile neutropenia at 120 mg m 2, we would recommend this dose for further evaluation, as the febrile neutropenia occurred after four or more cycles in three of the four episodes seen, was short lived and uncomplicated. © 2002 Cancer Research UK.

The role of inflammation in the pathogenesis of non-small cell lung cancer

The link between chronic immune activation and tumorigenesis is well established. Compelling evidence has accumulated that histologic assessment of infiltration patterns of different host immune response components in non-small cell lung cancer specimens helps identify different prognostic patient subgroups. This review provides an overview of recent insights gained in the understanding of the role played by chronic inflammation in lung carcinogenesis. The usefulness of quantification of different populations of lymphocytes, natural killer cells, macrophages, and mast cells within the tumor microenvironment in non-small cell lung cancer is also discussed. In particular, the importance of assessment of inflammatory cell microlocalization within both the tumor islet and surrounding stromal components is emphasized. Copyright © 2010 by the International Association for the Study of Lung Cancer.

Identifying belief targets to increase bone marrow registry participation among students who have never donated blood

New members on bone marrow registries worldwide are needed to allow sufficient diversity in the donor pool to meet patient needs. We used the theory of planned behaviour belief-basis and surveyed students who had not donated blood previously (i.e. non-donors) (N = 150) about the behavioural, normative, and control beliefs informing their intentions to join the Australian Bone Marrow Donor Registry. Key beliefs predicting non-donors’ intentions included: viewing bone marrow donation as an invasion of the body (β = −.35), normative support from parents (β = .40), anticipating pain/side effects from giving blood (β = −.27), and lack of knowledge about how to register (β  = −.30). Few non-donors endorsed these beliefs, suggesting they are ideal targets for change in strategies encouraging bone marrow donor registration.

Bioavailability study of oral and intravenous OGT 719, a novel nucleoside analogue with preferential activity in the liver

Purpose: Although oral fluoropyrimidine pro-drugs are increasingly being administered in preference to intravenous nucleoside analogues in cancer chemotherapy, their activation in malignant liver tissue may be insufficient. OGT 719 (1-galactopyranosyl-5-fluorouracil) is a novel nucleoside analogue, preferentially localized in hepatocytes and hepatoma cells via the asialoglycoprotein receptor. The aim of this study was to assess the systemic bioavailability of this rationally designed drug in 16 patients with advanced solid cancers. Method: Crossover pharmacokinetic study of oral (400 or 800 mg) and intravenous (250 mg/m 2) OGT 719. Results: Linear pharmacokinetics and oral bioavailability of approximately 25% were observed at the dose levels used in this study. Like other 5-FU prodrugs, considerable interpatient variability was observed in bioavailability following oral dosing. The mean half-life for oral doses was 4 h. OGT 719 was well tolerated. No objective tumour responses were demonstrated. Conclusion: The systemic bioavailability and half-life of oral OGT 719 are sufficient to merit dose escalation studies with frequent daily dosing. Subsequent efficacy studies should be performed in patients with primary and secondary liver malignancies.

Bioavailability study of oral and intravenous OGT 719, a novel nucleoside analogue with preferential activity in the liver

Purpose: Although oral fluoropyrimidine pro-drugs are increasingly being administered in preference to intravenous nucleoside analogues in cancer chemotherapy, their activation in malignant liver tissue may be insufficient. OGT 719 (1-galactopyranosyl-5-fluorouracil) is a novel nucleoside analogue, preferentially localized in hepatocytes and hepatoma cells via the asialoglycoprotein receptor. The aim of this study was to assess the systemic bioavailability of this rationally designed drug in 16 patients with advanced solid cancers. Method: Crossover pharmacokinetic study of oral (400 or 800 mg) and intravenous (250 mg/m 2) OGT 719. Results: Linear pharmacokinetics and oral bioavailability of approximately 25% were observed at the dose levels used in this study. Like other 5-FU prodrugs, considerable interpatient variability was observed in bioavailability following oral dosing. The mean half-life for oral doses was 4 h. OGT 719 was well tolerated. No objective tumour responses were demonstrated. Conclusion: The systemic bioavailability and half-life of oral OGT 719 are sufficient to merit dose escalation studies with frequent daily dosing. Subsequent efficacy studies should be performed in patients with primary and secondary liver malignancies.

A phase II study of Caelyx, liposomal doxorubicin : lack of activity in patients with advanced gastric cancer

Purpose A phase II study was designed to assess the efficacy and safety of Caelyx (liposomal doxorubicin) in patients with advanced or metastatic gastric cancer. Methods A total of 25 patients with gastric adenocarcinoma were treated with Caelyx 45 mg/m2 every 28 days as first-line therapy for advanced disease. Patients were treated until tumour progression or unacceptable toxicity. Results One patient was withdrawn from the study after experiencing a severe infusion reaction. Of the 24 evaluable patients, 1 had a partial response, 7 had stable disease and the others progressed. Side effects, in particular palmar-plantar erythrodysaesthesia and haematological toxicity, were minor. Conclusions We conclude that while this dose and schedule of Caelyx in this patient group is acceptable, further studies with this regimen cannot be recommended due to the lack of antitumour activity seen.

Perceptions of organ donors and willingness to donate organs upon death : a test of the prototype/willingness model

Understanding people's organ donation decisions may narrow the gap between organ supply and demand. In two studies, participants who had not recorded their posthumous organ donation decision (Study 1, N = 210; Study 2, N = 307) completed items assessing prototype/willingness model (PWM; attitude, subjective norm, donor prototype favorability and similarity, willingness) constructs. Attitude, subjective norm, and prototype similarity predicted willingness to donate. Prototype favorability and a Prototype Favorability × Similarity interaction predicted willingness (Study 2). These findings provide support for the PWM in altruistic health contexts, highlighting the importance of people's perceptions about organ donors in their donation decisions.

Biobanking of blood and bone marrow : emerging challenges for custodians of public resources

The Australian Bone Marrow Donor Registry (ABMDR) is a publicly funded company that is part of an international network that facilitates unrelated bone marrow transplantation. This role means that the ABMDR has access to a large biospecimen repository, therefore making it a highly valuable research resource. Recognising the potential value of these biospecimens for research purposes, the ABMDR is in the process of determining whether, and how, to share its biospecimens with other biobanks. While this would undoubtedly be of value to the scientific community, and ultimately to the wider community, it would also inevitably transform the role of an institution whose primary role is therapeutic, and would compromise the degree of control that a custodian has over donated material. This article describe the challenges confronting the ABMDR, and organisations like it, in balancing their duties to donors, patients, researchers and the general public. These problems have led inevitably to the use of "property" rights language in the discussion of these issues but notions of gift, ownership, trusteeship and transfer might also be considered.

Mechanism of ferromagnetism in non-magnetic ion-doped zinc oxides

Zinc oxide (ZnO) that contains non-magnetic ionic dopants, such as nitrogen (N)-doped zinc oxide (ZnO:N), has been observed to exhibit ferromagnetism. Ferromagnetism is proposed to arise from the Coulomb excitation in the localized states that is induced by the oxygen vacancy, V O. A model based on the Coulomb excitation that is associated with the electron–phonon interaction theoretically explains the ferromagnetic mechanism of ZnO:N. This study reveals that the ferromagnetism will be induced by either deep localized states with a small V O concentration or shallow localized states with a high V O concentration. Additionally, electron–phonon coupling either suppresses the ferromagnetism that is induced by the deep donor states of V O or enhances the ferromagnetism that is induced by the shallow donor states of V O.

The poor penetration of topical burn agent through burn eschar on a porcine burn model [Letter to the Editor]

We read with great interest the article entitled “Enhancing drugs absorption through third-degree burn wound eschar” by Manafi et al. [1]. The authors addressed the concern of poor penetration of topically applied anti-microbials through burn eschar and detailed the improvement of this penetration by penetration enhancers. Here, we would like to report the poor penetration of a topical agent into the viable deep dermal layer under burn eschar on a porcine burn model [2]. In burn treatment, a common practice is the topical application of either anti-microbial products or wound enhancing agents. While the activity of anti-microbial products is designed to fight against microbes on the wound surface but with the least toxicity to viable tissue, wound enhancing agents need to reach the viable tissue layer under the burn eschar. Many studies have reported the accelerated healing of superficial burn wounds and skin graft donor sites by the topical application of exogeneous growth factors [3]. It is well known that the efficacy of the penetration of a topical agent on intact skin mostly depends on the molecular size of the product [4] and [5]. While burn injury destroys this epidermal physiological barrier, the coagulated burn tissue layer on the burn wound surface makes it difficult for topical agents to reach viable tissue....