Application of inter-simple sequence repeat PCR to mouse models: assessment of genetic alterations in carcinogenesis

Genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been proved to be a fast and reproducible technique for quantitation of genomic instability (amplifications, deletions, translocations, and insertions) in human sporadic tumors. However, the use of inter-SSR PCR in animal models of cancer has never been described. This new technique has been adapted in our laboratory for the analysis of spontaneous and induced mouse tumors. We established the best PCR conditions for each microsatellite-anchored primer and critically evaluated the reproducibility of the band patterns. We also studied the variation of the fingerprints between and within various inbred mouse strains, including wild-derived lines. Tumor-specific alterations were detected as gains, losses, or intensity changes in bands when compared with matched normal DNA. We quantitated the extent of alterations by dividing the number of altered bands in the tumor by the total number of bands in normal DNA (instability index). By means of inter-SSR PCR, we successfully analyzed genomic alterations in various mouse tumors, including spontaneous thymic lymphomas developed in Msh2 knockout mice as well as chemically induced squamous cell carcinomas and thymic lymphomas. Instability index values ranged between 0 and 9%, the highest levels observed in N-methyl-N-nitrosourea-induced thymic lymphomas generated in Trp53 (p53) nullizygote (-/-) mice. We report here, for the first time, the use of inter-SSR PCR to detect somatic mutations in mouse tumoral DNA, including laser-capture microdissected, methanol-fixed tissues. These PCR-based fingerprints provide a novel approach to assessing the number and onset of mutational events in mouse tumors and will help to understand better the mechanisms of carcinogenesis in mouse models.     

The 16189 variant of mitochondrial DNA occurs more frequently in C282Y homozygotes with haemochromatosis than those without iron loading

Background:Patients with hereditary haemochromatosis (HH) are usually homozygous for the C282Y mutation in the HFE gene. They have variable expression of iron overload and present with a variety of complications, including liver disease, diabetes, arthropathy, fatigue, and cardiomyopathy. The mitochondrial 16189 variant is associated with diabetes, dilated cardiomyopathy, and low body fat at birth, and might contribute to genetic predisposition in further multifactorial disorders. The objective of this study was to determine the frequency of the 16189 variant in a range of patients with haemochromatosis, who had mutations in the HFE gene.

Methods:Blood DNA was analysed for the presence of the 16189 variant in British, French, and Australian C282Y homozygotes and controls, with known iron status, and in birth cohorts.

Results:The frequency of the mitochondrial 16189 variant was found to be elevated in individuals with haemochromatosis who were homozygous for the C282Y allele, compared with population controls and with C282Y homozygotes who were asymptomatic (42/292 (14.4%); 102/1186 (8.6%) (p = 0.003); and 2/64 (3.1%) (p = 0.023), respectively).

Conclusions:Iron loading in C282Y homozygotes with HH was exacerbated by the presence of the mitochondrial 16189 variant.

     

TRARESA: a tissue microarray-based hospital system for biomarker validation and discovery

Formalin fixed and paraffin embedded tissue (FFPE) collections in pathology departments are the largest resource for retrospective biomedical research studies. Based on the literature analysis of FFPE related research, as well as our own technical validation, we present the Translational Research Arrays (TRARESA), a tissue microarray centred, hospital based, translational research conceptual framework for both validation and/or discovery of novel biomarkers. TRARESA incorporates the analysis of protein, DNA and RNA in the same samples, correlating with clinical and pathological parameters from each case, and allowing (a) the confirmation of new biomarkers, disease hypotheses and drug targets, and (b) the postulation of novel hypotheses on disease mechanisms and drug targets based on known biomarkers. While presenting TRARESA, we illustrate the use of such a comprehensive approach. The conceptualisation of the role of FFPE-based studies in translational research allows the utilisation of this commodity, and adds to the hypothesis-generating armamentarium of existing high-throughput technologies.     

Inducing Humoral and Cellular Responses to Multiple Sporozoite and Liver-Stage Malaria Antigens Using Exogenous Plasmid DNA

A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4⁺ and CD8⁺ T cell compartments. Furthermore, hepatic CD8⁺ lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8⁺ granzyme B⁺ T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.     

A proteome reference map of Trichomonas vaginalis

Trichomoniasis caused by Trichomonas vaginalis is the most common sexual transmitted infection in the world. The 170-MB genome of this protozoan contains 60,000 genes, the largest number of genes ever identified in protozoan. High-throughput expression sequenced tag analysis showed that at least 4,000 genes were expressed in the trophozoite stage. In the present study, we use two-dimensional electrophoresis combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis to profile, identify, and characterize proteins expressed in the trophozoite stage of T. vaginalis. A total of 247 spots representing 164 different proteins were identified. The identified proteins with known sequence or motif/domain homologies were further classified into groups according to their biological functions. Among them, proteins related to carbohydrate metabolism represented the most abundant category in the T. vaginalis proteome. This study presented the most extensive proteomic analysis of T. vaginalis to date and provided a reference proteome database for future comparative proteomic studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Incidentally discovered mass of the adrenal gland

Increasing numbers of asymptomatic masses of the adrenal gland are being discovered by high resolution computed tomography (CT) scanning. The significance of these masses and the question of further evaluation or treatment, or both, have posed a dilemma. From 1976 to 1983, an incidental finding of adrenal gland mass was identified in 88 patients undergoing abdominal CT scanning for unrelated problems. Adrenelectomy was performed upon 23 patients (26 per cent), including three adenomas (2.5, 3.0 and 6.5 centimeters), five carcinomas of the adrenal gland (6 to 20 centimeters), two hyperplasias and three adenocarcinomas of unknown origin. Thirty-eight (43 per cent) of the nonfunctioning masses were observed with repeat scans for an average of 25.1 months (three to 60 months), revealing no change in size. Twenty-seven patients (31 per cent) were lost to follow-up study. Silent primary (nonmetastatic) masses 3.5 centimeters or less manifested benign behavior without exception. Based upon these data, we conclude that all solid metabolically inactive lesions in the adrenal glands greater than 3.5 centimeters on CT abdominal scan deserve exploration. Lesions less than 3.5 centimeters may be safely followed with serial CT scans.     

A wireless telemetry system for training users of upper limb prostheses

Telemetry from prosthetic systems is useful in aiding the users and clinical team in setting up and training and monitoring in the use of a prosthesis. The different forms are discussed and a new radio frequency based telemetry system is described. This system uses a computer to display the data and a video mixer to place the information on the same screen as the image of the user so that their actions and control signals can be simultaneously recorded for later study.