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    ABSTRACT: The growth of human tumor cells transplanted into immunodeficient mice is frequently studied to gain understanding about the way potential drug treatments interfere with growth in vivo. A wide range of methods is available for learning about specific aspects of tumor cell behavior, for example, cells may be administered to follow their ability to grow close to the site of injection which may be at a generic site or one specific to that type of tumor. Some models of metastasis follow the appearance of a tumor mass after intravascular administration of tumor cells; others score remote growth after removal of a primary tumor implanted subcutaneously. Assessing metastatic growth may increasingly rely on serial observation of tumor cell numbers as seen by whole-body imaging, but the sensitivity of these methods is poor in terms of the minimum number of cells detectable, and histological follow-up to establish tumor cell numbers can be confounded by variable expression or even silencing of reporter genes. Here we describe how fluorescence in situ hybridization (FISH) using commercially available probes can very easily be used to detect even single metastatic tumor cells in mouse models, using routinely fixed and processed tissue samples, and without the tumor cell lines needing to express engineered reporter genes. The FISH protocol can be combined with other standard histological protocols to study the behavior of tumor cells and adjacent host cells to improve our understanding of tumor-stroma interactions, and is also useful for simultaneous demonstration of the cell of origin and phenotype of cells used in regenerative medicine-based applications.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1070:235-245.
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    ABSTRACT: Cryptosporidium parvum infects intestinal epithelial cells and is commonly the parasite species involved in mammalian cryptosporidiosis, a major health problem for humans and neonatal livestock. In mice, immunologically mediated elimination of C. parvum requires CD4(+) T cells and IFN-γ. However, innate immune responses also have a significant protective role in both adult and neonatal mice. NK cells and IFN-γ have been shown to be important components in immunity in T and B cell-deficient mice but IFN-γ-dependent resistance has also been demonstrated in alymphocytic mice. Epithelial cells may play a vital role in immunity since once infected these cells have increased expression of inflammatory chemokines and cytokines and demonstrate antimicrobial killing mechanisms, including production of NO and antimicrobial peptides. TLRs facilitate the establishment of immunity in mice and are involved in the development of inflammatory responses of infected epithelial cells and also dendritic cells. © 2012 Blackwell Publishing Ltd.
    Parasite Immunology 11/2012;
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    ABSTRACT: Visceral pain is studied at the level of the primary afferent fiber, spinal cord, subcortical, and cortical levels electrophysiologically and using brain imaging, which provides an objective measure of excitation at each level. However, correlation of these with actual perception of pain in conscious animal models has been problematic, and we rely on indirect measures in most preclinical research. The main method is electromyographic recording of abdominal muscle contractions in response to colorectal distension (CRD), which may reflect reflexes set up at several levels of the above pathway. Several experimental treatments for visceral pain have failed in clinical trials, possibly because of failure to translate from preclinical observations on CRD responses in animals to perception of spontaneous events in patients. Therefore, we need more objective outcomes. In this NGM issue, Hultin et al. show feasibility of routine recordings of cortical evoked electrical potentials (CEP) using implanted cranial electrodes in response to graded CRD in rats. CEP comprised three temporal components with latencies of approximately 20-50 ms, 90-180 ms, and 300 ms, which were reproducible and graded in intensity and latency with distension pressure. From this basic study it is clear that colorectal evoked potentials can be recorded reliably in awake rats and may serve as an objective marker for centrally projecting visceral sensory signals in rodents. It remains to be seen how these responses are affected by drugs under development for clinical management of visceral pain, and if there is improved translation.
    Neurogastroenterology and Motility 10/2012; 24(10):891-4.
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    ABSTRACT: Colorectal adenomas display features of senescence, but these are often lost upon progression to carcinoma, indicating that oncogene induced senescence (OIS) could be a roadblock in colorectal cancer (CRC) development. Heat shock proteins (HSPs) have been implicated in the prognosis of CRC and HSP based therapy is a current interest for drug development. Recent cell culture studies have suggested that in the absence of a TP53 mutation, OIS mediated by PI3K/AKT activation can be circumvented by high expression of HSPs. Furthermore, while PI3K/AKT activation and KRAS mutations are independent inducers of OIS, PI3K/AKT activation can suppress KRAS-induced OIS when both are present in cultured cells. As KRAS mutations, PI3K/AKT activation and TP53 mutations are all common features of CRC, it is possible that the requirement for HSP to inhibit OIS in CRC is dependent on the mutation spectrum of a tumour. However, work on HSP that utilised mutation profiled human tumour tissues has been limited. Here, we characterised the expression of two major HSP proteins (HSP27 and 72) by immunohistochemistry (IHC), the mutation status of TP53, KRAS and PIK3CA genes by direct sequencing and the activation status of AKT by IHC in a cohort of unselected primary CRC (n=74). We compare our data with findings generated from cell-based studies. Expression of HSP27 and HSP72 was correlated to clinicopathological and survival data but no significant association was found. We also established the mutation status of TP53, KRAS and PIK3CA genes and the activation status of AKT in our CRC panel. We did not detect any associations between HSP27 or HSP72 expression with TP53 mutation status. However, HSP27 expression in CRCs was strongly associated with the co-presence of wildtype KRAS and activated PI3K/AKT (p=0.004), indicating a possible role of HSP27 in overcoming PI3K/AKT induced OIS in tumours. Our studies suggest a role for using archival tissues in validating hypotheses generated from cell culture based investigations.
    Experimental and Molecular Pathology 09/2012;
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    ABSTRACT: A spectrum of enteropathies, characterized by small intestinal inflammation, reduced absorptive capacity, and increased intestinal permeability, commonly affect people in developing countries. This subclinical intestinal pathology facilitates microbial translocation across the compromised intestinal barrier, leading to chronic systemic inflammation that may adversely impact health. Environmental enteropathy (EE), ubiquitous among people living in unhygienic conditions, likely mediates two interlinked public health problems of childhood, stunting and anemia, and underlies poor oral vaccine efficacy in developing countries. Human immunodeficiency virus (HIV) enteropathy, which frequently overlaps with EE, may contribute to immune activation and modulate HIV disease progression. The interacting effects of infection and enteropathy drive a vicious cycle that can propagate severe acute malnutrition, which underlies almost half of under-5-y deaths. Enteropathies are therefore highly prevalent, interacting causes of morbidity and mortality in developing countries. Interventions to prevent or ameliorate enteropathies have potential to improve the health of millions of people in developing countries.
    The American journal of tropical medicine and hygiene 05/2012; 86(5):756-63.
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    ABSTRACT: Innate immunity against some intracellular parasitic protozoa involves interleukin 18 (IL-18)-mediated interferon γ (IFN-γ) production by natural killer (NK) cells, but the role of IL-18 in innate resistance to Cryptosporidium infection is unknown. Adult Rag2(-/-)γc(-/-) mice that lack NK cells, T cells, and B cells demonstrated resistance to Cryptosporidium parvum infection that was IFN-γ dependent. Treatment with anti-IL-18-neutralizing antibodies resulted in loss of resistance correlating with reduced intestinal IFN-γ expression. Intestinal mature IL-18 expression increased in vivo during infection and also in the intestinal epithelial cell line CMT-93 following combined IFN-γ treatment/infection. Peritoneal macrophages produced IFN-γ when stimulated with IL-18 combined with interleukin 12, and the latter was expressed in vivo during infection. Macrophage depletion in infected mice caused a rapid growth of infection with no increase in IFN-γ expression. These findings provide evidence of an NK cell-independent, IFN-γ-mediated innate immune pathway against C. parvum in which IL-18 and macrophages play prominent parts.
    The Journal of Infectious Diseases 04/2012; 206(1):117-24.
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    The Lancet 04/2012; 379(9825):1483.
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    ABSTRACT: Prospective randomized double-blind placebo-controlled crossover trial of 14 female patients (median age 52 [30-69] years) with proctographically defined evacuatory dysfunction (ED) and demonstrable rectal hyposensitivity (elevated thresholds to balloon distension in comparison with age- and sex-matched controls). Sacral nerve stimulation (SNS) is an evolving treatment for constipation. However, variable outcomes might be improved by better patient selection. Evidence that the effect of SNS may be mediated by modulation of afferent signaling promotes a role in patients with ED associated with rectal hyposensation. SNS was performed by the standard 2-stage technique (temporary then permanent implantation). During a 4-week period of temporary stimulation, patients were randomized ON-OFF/OFF-ON for two 2-week periods. Before insertion (PRE), and during each crossover period, primary (rectal sensory thresholds) and secondary (bowel diaries, constipation, and GIQoL [gastrointestinal quality of life] scores) outcome variables were blindly assessed. Thirteen patients completed the trial. Following stimulation, defecatory desire volumes to rectal balloon distension were normalized in 10 of 13 patients (PRE: mean 277 mL [234-320] vs ON: 163 mL [133-193] vs OFF: 220 mL [183-257 mL]; P = 0.006) and maximum tolerable volume in 9 of 13 (PRE: mean 350 mL [323-377] vs ON: 262 mL [219-305] vs OFF: 298 mL [256-340 mL]; P = 0.012). There was a significant increase in the percentage of successful bowel movements (PRE: median 43% [0-100] vs ON: 89% [11-100] vs OFF: 83% [11-100]; P = 0.007) and Wexner constipation scores improved (PRE: median 19 [9-26] vs ON: 10 [6-27] vs OFF: 13 [5-29]; P = 0.01). There were no significant changes in disease-specific or generic quality of life measures. Eleven patients progressed to permanent stimulation (9/11 success at 19 months). Most patients with chronic constipation secondary to ED with rectal hyposensitivity responded to temporary SNS. The physiological results presented support a mechanistic role for rectal afferent modulation.
    Annals of surgery 04/2012; 255(4):643-9.
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    ABSTRACT: Recently, a dispute has arisen concerning the operational definition and identification of epithelial stem cells. There is a current view which considers that epithelial stem cell identification depends solely on the in vitro isolation and expansion of cells from the putative stem cell. This article argues strongly that this is a perverse and erroneous view, and that lineage labelling, using genetic markers, remains the gold standard for identifying epithelial stem cells and for analysing their behaviour: in vitro methods show, at best, clonogenic potential but not fate, and constitute ancillary support for conclusions drawn from lineage analysis.
    The Journal of Pathology 03/2012; 227(3):255-66.
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    ABSTRACT: Trefoil factor family (TFF) peptides promote wound healing in the gut. Recent evidence has suggested that TFF3 may be a pancreatic mitogen, an unusual role for TFF peptides. We sought to clarify human pancreatic TFF and mucin expression and performed in vitro experiments to see how pancreatic cell lines respond to TFF3 in particular. Samples of normal and diseased pancreas (chronic pancreatitis, pancreatic intraepithelial neoplasia, neuroendocrine tumors, and pancreatic ductal adenocarcinoma [PDAC]) were studied by immunohistochemistry and in situ hybridization. Pancreatic cell lines were challenged with TFF2 and TFF3 in wound and migration assays. In normal islets, colocalization of insulin or glucagon with TFF3 was common. All TFF messenger RNAs were seen in ductal epithelium. Adenocarcinomas expressed all TFF messenger RNAs. Normal ducts were mucin free; MUC5AC was strongest in pancreatic intraepithelial neoplasia and chronic pancreatitis but was reduced in PDAC. TFF2 induced Panc-1 migration and accelerated wound closure in Capan-2 and COLO-357. Double immunohistochemistry for insulin or TFF3 and Ki67 colabeled only very rare islet cells. TFF3-positive PDAC ducts showed some Ki67 colocalization. No correlation between TFF3 or insulin and Ki67 was seen without ductal hyperplasia. TFF2 may assist pancreatic tumor cell movement, but TFF3 may not be a pancreatic mitogen.
    Pancreas 01/2012; 41(6):888-96.
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