A gastrin transcript expressed in gastrointestinal cancer cells contains an internal ribosome entry site.

As the hormone gastrin promotes gastrointestinal (GI) cancer progression by triggering survival pathways, regulation of gastrin expression at the translational level was explored. Sequence within the 5' untranslated region of a gastrin transcript expressed in GI cancer cells was investigated, then cloned into a bicistronic vector upstream of firefly luciferase and transfected into a series of GI cancer cell lines. Firefly luciferase activity was measured relative to that of a cap-dependent Renilla luciferase. A gastrin transcript that was different from that described in Ensembl was expressed in GI cancer cells. Its transcription appears to be initiated within the region designated as the gene's first intron. In GI cancer cells transfected with the bicistronic construct, firefly luciferase activity increased 8-15-fold compared with the control vector, and there was a further induction of the signal (up to 25-fold) following exposure of the cells to genotoxic stress or hypoxia, suggesting that the sequence acts as an internal ribosome entry site. These data suggest that the gastrin transcript within GI cancer cells contains an internal ribosome entry site that may allow continued expression of gastrin peptides when normal translational mechanisms are inactive, such as in hypoxia, thereby promoting cancer cell survival.

Pre-clinical evaluation of a new orally-active CCK-2R antagonist, Z-360, in gastrointestinal cancer models.

BACKGROUND: Gastrin has a role in gastrointestinal (GI) malignancy. This study provides pre-clinical evaluation of a novel, orally-active gastrin/cholecystokinin-2 receptor (CCK-2R) antagonist, Z-360. METHODS: (125)I gastrin-17 (G17) displacement and G17-stimulated calcium assays were used in classical CCK-2R-transfected cell lines. Akt phosphorylation was assessed by Western blotting. Z-360 efficacy in vivo was evaluated in three human xenograft models, and microvessel density and apoptosis in these models were investigated by immunohistochemistry. RESULTS: Z-360 inhibited (125)I G17 binding to cells expressing CCK-2R, and G17-stimulated signalling. Reduced Akt phosphorylation in an oesophageal cell-line treated with Z-360 was reversed by co-treatment with G17. Z-360 increased survival in a gastric ascites model (p=0.011) and decreased tumour growth in a hepatic metastasis model (81%, p=0.02). In an orthotopic pancreatic model, Z-360 combined with gemcitabine decreased final tumour weight compared to single agents (84%, p=0.002) and there was increased apoptosis and decreased microvessel density in ex vivo tumour tissue. CONCLUSIONS: These results show that the orally-active CCK-2R antagonist, Z-360 has high sub-nM affinity for classical CCK-2R, is well tolerated in vivo and exerts an anti-tumour effect.

In Vitro Tissue Microarrays for Quick and Efficient Spheroid Characterization.

Three-dimensional (3D) in vitro microphysiological cultures, such as spheroids and organoids, promise increased patient relevance and therapeutic predictivity compared with reductionist cell monolayers. However, high-throughput characterization techniques for 3D models are currently limited to simplistic live/dead assays. By sectioning and staining in vitro microtissues, researchers can examine their structure; detect DNA, RNA, and protein targets; and visualize them at the level of single cells. The morphological examination and immunochemistry staining for in vitro cultures has historically been done in a laborious manner involving testing one set of cultures at a time. We have developed a technology to rapidly screen spheroid phenotype and protein expression by arranging 66 spheroids in a gel array for paraffin embedding, sectioning, and immunohistochemsitry. The process is quick, mostly automatable, and uses 11 times less reagents than conventional techniques. Here we showcase the capabilities of the technique in an array made up of 11 different cell lines stained in conventional hematoxylin and eosin (H&E) staining, as well as immunohistochemistry staining for estrogen (ER), progesterone (PR), and human epidermal growth factor (Her-2) receptors, and TP53. This new methodology can be used in optimizing stem cell-based models of disease and development, for tissue engineering, safety screening, and efficacy screens in cancer research.

Stable DHLA-PEG capped PbS quantum dots: from synthesis to near-infrared biomedical imaging.

The short shelf-life of water-soluble quantum dots (QDs) due to colloidal instability represents a major drawback to their exploitation. This work examines the colloidal stability of PbS nanoparticles capped with dihydrolipoic acid-polyethylene glycol (DHLA-PEG) ligands terminated with functional groups such as -NH2, -COOH, OMe and -N3. and their application for in vivo imaging. We prove a mechanism of colloidal instability and develop a strategy to produce for the first time stable PEG-capped PbS quantum dots with high quantum yield and optical emission in the first and the second near-infrared (NIR) windows of low absorption of biological tissues. The NIR imaging of in vivo biodistribution is demonstrated at wavelengths >1000 nm, with benefits of reduced tissue absorption and light scattering. The stability, biocompatibility and potential for further QD functionalization open up realistic prospects for non-invasive bioimaging applications.

Androgen receptors may act in a paracrine manner to regulate oesophageal adenocarcinoma growth.

BACKGROUND: The role of androgen receptors (ARs) in tumorigenesis, including transcription of fibroblast growth factors (FGFs), is established in prostate cancer. This study examined the role of ARs and FGFs in oesophageal adenocarcinoma (EAC), where tumour incidence in males is higher. METHODS: AR gene expression was analysed using quantitative RT-PCR; AR, fibroblast growth factor receptor-1 (FGFR-1) and fibroblast growth factor-8 isoform b (FGF-8b) protein by immunohistochemistry; and serum steroid levels (testosterone, progesterone, luteinising hormone and follicle stimulating hormone (FSH)) by immunoassay. A human oesophageal adenocarcinoma cell line was grown subcutaneously in nude mice. RESULTS: AR gene expression was of significantly higher levels than oesophageal adenocarcinomas (n=21, p=0.002) and in the squamous carcinoma line (OE21) compared with the adenocarcinoma lines (OE33 and OE19). Median serum testosterone levels in oesophageal carcinoma patients were higher than in age-matched controls (p=0.01) and reduced postoperatively, in patients undergoing curative resection (p=0.006). No significant differences were observed in hormones except FSH, where preoperative levels were significantly higher in the EAC group. AR protein was expressed in normal oesophageal squamous epithelial cells and also in the stroma of 18/23 EAC samples. FGFR-1 protein was expressed in malignant epithelium of 23/23 tumour samples. OE19 xenografts grew faster in male versus female mice (tumour weight at day 21, 1.14 g and 0.28 g, respectively, p=0.005) and had elevated FGF receptor expression. CONCLUSIONS: AR expressed in the stroma of oesophageal adenocarcinomas may induce paracrine effects following stimulation by androgens (including tumour-derived), possibly via FGFs, including FGF-8b.

Data on the number and frequency of scientific literature citations for established medulloblastoma cell lines.

This article collates information about the number of scientific articles mentioning each of the established medulloblastoma cell lines, derived through a systematic search of Web of Science, Scopus and Google Scholar in 2016. The data for each cell line have been presented as raw number of citations, percentage share of the total citations for each search engine and as an average percentage between the three search engines. In order to correct for the time since each cell line has been in use, the raw citation data have also been divided by the number of years since the derivation of each cell line. This is a supporting article for a review of in vitro models of medulloblastoma published in "in vitro models of medulloblastoma: choosing the right tool for the job" (D.P. Ivanov, D.A. Walker, B. Coyle, A.M. Grabowska, 2016) [1].

Development of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA.

The recent development of tagged RT-PCR and rTth RT-PCR has greatly improved strand-specific detection of hepatitis C virus (HCV) RNA but these assays are still prone to some false detection of the incorrect strand of RNA. In this study we aimed to address additional factors which contribute towards false detection of HCV RNA. Firstly the benefits of both tagged primers and the thermostable reverse transcriptase rTth during cDNA synthesis were combined and it was found that strand specificity was greatly improved without compromising sensitivity. The reliability of the assay was then optimised by addressing the following issues: control synthetic transcripts should be free of contaminating plasmid DNA, residual RT activity should be minimised in the presence of PCR primers and cDNA should be free of unincorporated tagged RT primer prior to PCR amplification. The alterations made to the assay eliminated completely false detection of the incorrect strand of RNA in the control assay whilst the correct strand was consistently detected at a cDNA dilution of 10(-3)-10(-4). Negative strand was not detected in RNA isolated from serum but was detected, at a ten-fold lower level than positive strand, in RNA isolated from liver tissue.

Use of interfering RNA to investigate the role of endogenous gastrin in the survival of gastrointestinal cancer cells.

Gastrin isoforms, acting through a variety of receptors, have proliferative and anti-apoptotic effects on gastrointestinal (GI) cancers. A small interfering RNA (siRNA) targeting the gastrin gene was used to investigate the role of endogenous gastrin in GI cancer cell survival. Downregulation of the gastrin gene in siRNA-transfected cells was measured using real-time reverse transcriptase-PCR. The most effective siRNA was tested in a panel of GI cancer cell lines at various concentrations and time points, and the effect on cell survival and apoptosis was measured using methyl thiazoyl tetrazolium (MTT) and caspase 3 activation assays. Gastrin siRNA reduced gene expression by more than 90% in a range of GI cancer cell lines. Downregulation of the gastrin gene was dose-dependent and effective over approximately 1 week in vitro. However, downregulation at the protein level was delayed by 3-4 days. Gastrin siRNA-transfected cells showed up to a 60% reduction in growth and up to a 50% increase in apoptosis compared with control siRNA-transfected cells. The effects were most marked in the cell line with the highest constitutive level of gastrin gene expression (human metastatic colon, C170HM2) and in epidermal growth factor (EGF)-treated cells as the gastrin promoter contains an EGF-response element, gERE. The ability of the siRNAs to reduce survival of these GI cell lines is further evidence of the importance of autocrine and/or intracrine gastrin loops in GI cancer, where expression of the gastrin gene and autonomous gastrin appears widespread.

PTHrP increases transcriptional activity of the integrin subunit alpha5.

Increasing evidence is emerging highlighting the role of parathyroid hormone-related protein (PTHrP) during metastasis by regulating cell adhesion. The current study demonstrated that modulation of PTHrP expression by PTHrP overexpression and small interfering RNA-induced silencing resulted in changes in cell adhesion and integrin expression. RNA interference of endogenous PTHrP caused a significant reduction in cell adhesion of a breast cancer cell line to collagen type I, fibronectin and laminin (P<0.05) and of a colon cancer cell to collagen type I and fibronectin (P<0.05). Overexpression of PTHrP induced a significant increase in cell adhesion of colon (P<0.0001) and breast (P<0.05) cancer cells to the same extracellular matrix proteins. These PTHrP-mediated effects were attributed to changes in integrin expression as the differences in adhesion profile correlated with the integrin expression profile. In an attempt to elucidate the mechanism whereby PTHrP regulates integrin expression, promoter activity of the integrin alpha5 subunit was analysed and significant increases in transcriptional activity were observed in PTHrP overexpressing cells (P<0.0001), which was dependent on nuclear localisation. These results indicate that modulation of cell adhesion is a normal physiological action of PTHrP, mediated by increasing integrin gene transcription.

De-regulation of the sonic hedgehog pathway in the InsGas mouse model of gastric carcinogenesis.

This study investigated sonic hedgehog (Shh) signalling in gastric metaplasia in the insulin-gastrin (InsGas) hypergastrinaemic mouse +/- Helicobacter felis (H. felis) infection. Sonic hedgehog gene and protein expression was reduced in pre-metaplastic lesions from non-infected mice (90% gene reduction, P<0.01) compared to normal mucosa. Sonic hedgehog was reactivated in gastric metaplasia of H. felis-infected mice (3.5-fold increase, P<0.01) compared to pre-metaplastic lesions. Additionally, the Shh target gene, glioma-associated oncogene (Gli)-1, was significantly reduced in the gastric glands of InsGas mice (75% reduction, P<0.05) and reactivated with H. felis infection (P<0.05, base of glands, P<0.01 stroma of metaplastic glands). The ability of H. felis to activate the Shh pathway was investigated by measuring the effect of target cytokine, interleukin-8 (IL-8), on Shh expression in AGS and MGLVA1 cells, which was shown to induce Shh expression at physiological concentrations. H. felis induced the expression of NF-kappaB in inflammatory infiltrates in vivo, and the expression of the IL-8 mouse homologue, protein KC, in inflammatory infiltrates and metaplastic lesions. Sonic hedgehog pathway reactivation was paralleled with an increase in proliferation of metaplastic lesions (15.75 vs 4.39% in infected vs non-infected mice, respectively, P<0.001). Furthermore, Shh overexpression increased the growth rate of the gastric cancer cell line, AGS. The antiapoptotic protein, bcl-2, was expressed in the stroma of infected mice, along with a second Shh target gene, patched-1 (P=0.0001, stroma of metaplastic gland). This study provides evidence suggesting reactivation of Shh signalling from pre-metaplastic to advanced metaplastic lesions of the stomach and outlines the importance of the Shh pathway as a potential chemoprophylactic target for gastric carcinogenesis.

Right hemisphere superiority in the perception of different kinds of non-verbal material.

Hemispheric asymmetries in different perceptual functions have been tested using the method of lateral presentation of stimuli i.e. in the left or right visual field. The first two experiments showed the right hemisphere advantage in the accuracy of detection of stereoscopic depth and in the strength of the tilt after-effect, i.e. in such phenomena which seem to be produced in the striate cortex. The third experiment, in which evoked potentials from point 01 and 02 during face perception were recorded, revealed the existence of right hemisphere superiority as early as 150 ms after stimulus presentation. These results indicate that the two hemispheres can already differ in their functions at the sensory level of processing.

Immunisation with phage displaying peptides representing single epitopes of the glycoprotein G can give rise to partial protective immunity to HSV-2.

Filamentous phage displaying peptides representing single epitopes of the glycoprotein G of HSV-2 (gG2) were used as immunogens via the subcutaneous route in Balb/c mice without additional adjuvant. The phage were isolated from a random phage peptide display library and contain 15-mer peptide inserts that mimic epitopes of gG2. In each case, an antibody response to gG2 was generated that was dependent on the dose of phage administered and on the presence of the peptide insert. Phage displaying epitopes of gG2, which map to amino acids 551-570, were the most immunogenic; interestingly, this region of gG2 is frequently recognised by patients infected with HSV-2. The data also provide interesting information as regards choice of peptide mimics for use as immunogens because, surprisingly, the most antigenic of the individual clones was the least immunogenic. In two of the experiments, mice immunised with phage displaying a single epitope of gG2 were protected against challenge with a lethal dose of whole HSV-2. This suggests a possible role for phage-displayed peptides in inducing protective immunity against pathogens and provides a model system for investigating the underlying mechanisms.

Human antibody responses to Schistosoma mansoni: the influence of epitopes shared between different life-cycle stages on the response to the schistosomulum.

Sera from 120 Kenyan schoolchildren who were infected with S. mansoni were individually examined, using an enzyme-linked immunoabsorbent assay (ELISA), for the presence of IgG and IgM antibodies reactive with antigens derived from adult worms, the outer membrane of the schistosomulum or from the parasite egg. In addition, antibodies against more purified egg antigens, an egg stage-specific glycoprotein preparation and a polysaccharide egg antigen known to share epitopes with the schistosomular surface were measured in ELISA, as were antibodies reactive with trichloroacetic acid-soluble and periodate-insensitive antigens derived from the outer membrane of schistosomulum and antigens shed when schistosomula were cultured in vitro. IgG subclass responses to the unfractionated egg antigen were also measured. The results from each of these assays were compared with the results of each other assay and with the number of parasite eggs excreted by each child, using Spearmans rank correlations. These comparisons revealed a number of statistically significant positive correlations. IgG4 anti-egg antibodies correlated better with intensity of infection than did other IgG subclasses. Total IgG responses against polysaccharide antigens did not correlate with intensity of infection as well as IgG responses against other antigens; epitopes shared between the schistosomulum surface and the adult worm were different to those shared with the parasite egg; and, there was antigen-directed restriction of IgG subclass responses to some egg and adult worm antigens which carried these shared epitopes. It is argued that this might have a qualitative effect on the nature of the antibodies directed against the schistosomulum by infected individuals and therefore have important consequences for the outcome of a subsequent exposure to infection with the same parasite.

TT virus sequence heterogeneity in vivo: evidence for co-infection with multiple genetic types.

TT virus (TTV) is a newly described DNA virus of humans that exhibits an unusually high degree of genetic heterogeneity. We have performed extensive analysis of the TTV populations present in samples, taken over a period of 2 to 6 years, from three individuals with persistent TTV infection. TTV DNA titres estimated for sequential samples were found to be quite stable over the entire study period in two patients, but fluctuated considerably in the third. DNA sequence analysis revealed different genetic diversity among TTV populations from samples from the three patients. In one case, absolute sequence homogeneity was observed among samples over a 3 year period. In a second, a limited amount of heterogeneity was found, including one sequence exhibiting G-->A hypermutation. TTV DNA sequences from the third patient exhibited quite remarkable genetic heterogeneity: evidence was found of seven distinct infecting viruses, representing four of the six TTV genotypes that have been described. In addition, minor variants of three of these seven sequences were observed. The heterogeneity of the viral population in this individual declined steadily over a 6 year period. This patient infected with a genetically diverse TTV population had the highest viral DNA titre.

TT virus infection in patients with hepatitis C: frequency, persistence, and sequence heterogeneity.

TT virus (TTV) was recently identified in the serum of a patient with hepatitis. The role of TTV in liver disease has not been established. Three polymerase chain reaction (PCR) protocols were used to detect TTV DNA in sera of persons infected with hepatitis C virus (HCV) and in blood donors. Sera from 11.5% of HCV-infected patients and 7.7% of blood donors were positive by protocols 1 or 2. In contrast, 48.7% and 57.7% of sera, respectively, were positive when tested by protocol 3. There was no difference in the severity of hepatitis in persons coinfected with TTV and HCV when compared with those infected with HCV alone, regardless of which TTV PCR protocol was used. TTV DNA persisted in serum samples taken up to 6 years apart in individual patients. Sequence analysis indicated that most viral sequences were distinct between patients, and there was evidence of genetic heterogeneity and viral evolution within individuals.

Direct ex vivo comparison of the breadth and specificity of the T cells in the liver and peripheral blood of patients with chronic HCV infection.

The role of intrahepatic lymphocytes in the control of hepatitis C virus (HCV) infection and the pathology associated with it is not understood; most studies of the immunology of this infection use peripheral blood lymphocyte populations. To address this further, we examined in detail the IHL from HCV-infected patients and controls, focusing on the antigen-specific CD8(+) T lymphocyte component. Individual T cells from needle liver biopsies and peripheral blood were isolated from patients with chronic HCV infection and examined directly ex vivo. We used RT-PCR spectratyping to compare the breadth of the T cell receptor usage in the liver in comparison with the peripheral blood, and applied MHC class I tetramer technology to investigate the numbers of HCV-specific CD8(+) cells in the two compartments. T cell receptor usage in the liver of HCV-infected patients was broad, comparable with that in the peripheral blood of the same patients. A much higher proportion of liver CD8(+) cells expressed receptors specific for HCV antigens compared with paired peripheral blood CD8(+) cells. A greater proportion of the liver tetramer-positive cells expressed the activation marker CD69, compared with those in the periphery or other CD8(+) cells in the liver. In the course of chronic HCV infection, HCV-specific CD8 cells, which have been recently activated, appear to accumulate specifically in the livers of infected patients but are present in much lower numbers in the peripheral circulation. Further studies are needed to determine the function of these cells and their role in protection and immunopathology.

GBV-C/HGV coinfection in HIV-1-positive men: frequent detection of viral RNA in blood plasma but absence from seminal fluid plasma.

Sequential paired samples of blood and seminal fluid were obtained from a cohort of 54 HIV-1-infected homosexual males. The prevalence of GBV-C/HGV RNA in the cell-free fractions of some of these patients was determined using reverse-transcription polymerase chain reaction (RT-PCR). To assess the effects of HIV-1 and HCV infection upon GBV-C/HGV RNA status, blood CD4 cell counts, HCV RNA status, and HIV-1 proviral DNA and viral RNA titres were also determined. GBV-C/HGV RNA was detected in 8/30 (27%) of the blood plasma samples obtained at the start of the study, and was present at a frequency of 14/64 (22%) in all the blood plasma samples tested. By contrast, GBV-C/HGV RNA was not detected in the 26 seminal fluid samples obtained at the start of the study, including 8 samples obtained from patients for which GBV-C/HGV RNA was detected in the corresponding blood sample. Of the samples tested for the presence of both GBV-C/HGV and HCV RNA, there was no evidence of coinfection. Although GBV-C/HGV RNA detection rates were significantly higher in individuals with blood CD4 cell counts greater than 200 cells per microlitre, there were no significant differences in the median blood CD4 cell counts or HIV-1 proviral DNA or viral RNA titres observed between the GBV-C/HGV-positive and -negative individuals. The failure to detect GBV-C/HGV RNA in seminal fluid samples obtained from this cohort would suggest that further studies need to be carried out to determine the roles of sexual transmission and of seminal fluid in GBV-C/HGV infection.