European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development

In situ hybridization (ISH) is used for the localization of specific nucleic acid sequences in cells or tissues by complementary binding of a nucleotide probe to a specific target nucleic acid sequence. In the last years, the specificity and sensitivity of ISH assays were improved by innovative techniques like synthetic nucleic acids and tandem oligonucleotide probes combined with signal amplification methods like branched DNA, hybridization chain reaction and tyramide signal amplification. These improvements increased the application spectrum for ISH on formalin-fixed paraffin-embedded tissues. ISH is a powerful tool to investigate DNA, mRNA transcripts, regulatory noncoding RNA, and therapeutic oligonucleotides. ISH can be used to obtain spatial information of a cell type, subcellular localization, or expression levels of targets. Since immunohistochemistry and ISH share similar workflows, their combination can address simultaneous transcriptomics and proteomics questions. The goal of this review paper is to revisit the current state of the scientific approaches in ISH and its application in drug research and development

Glomerulonephritis-induced changes in kidney gene expression in rats

We investigated a glomerulonephritis (GN) model in rats induced by nephrotoxic serum (NTS) which contains antibodies against the glomerular basement membrane (GBM). The anti-GBM GN model in rats is widely used since its biochemical and histopathological characteristics are similar to crescentic nephritis and Goodpasture's disease in humans (Pusey, 2003 [2]). Male Wistar Kyoto (WKY) and Sprague–Dawley (SD) rats were dosed once with 1, 2.5 and 5 ml/kg nephrotoxic serum (NTS) or 1.5 and 5 ml/kg NTS, respectively. GN and tubular damage were observed histopathologically in all treated rats after 14 days. To obtain insight into molecular processes during GN pathogenesis, mRNA expression was investigated in WKY and SD kidneys using Affymetrix's GeneChip Rat genome 230_2.0 arrays (GSE64265). The immunopathological processes during GN are still not fully understood and likely involve both innate and adaptive immunity. In the present study, several hundred mRNAs were found deregulated, which functionally were mostly associated with inflammation and regeneration. The β-chain of the major histocompatibility complex class II RT1.B (Rt1-Bb) and complement component 6 (C6) were identified as two mRNAs differentially expressed between WKY and SD rat strains which could be related to known different susceptibilities to NTS of different rat strains; both were increased in WKY and decreased in SD rats (Pavkovic et al., 2015 [1]). Increased Rt1-Bb expression in WKY rats could indicate a stronger and more persistent cellular reaction of the adaptive immune system in this strain, in line with findings indicating adaptive immune reactions during GN. The complement cascade is also known to be essential for GN development, especially terminal cascade products like C6

Application of a Patient Derived Xenograft Model for Predicative Study of Uterine Fibroid Disease.

Human uterine fibroids, benign tumors derived from the smooth muscle layers of the uterus, impose a major health burden to up to 50% of premenopausal women in their daily life. To improve our understanding of this disease, we developed and characterized a patient-derived xenograft model by subcutaneous transplantation of pieces of human uterine fibroid tissue into three different strains of severe combined immunodeficient mice. Engrafted uterine fibroid tissue preserved the classical morphology with interwoven bundles of smooth muscle cells and an abundant deposition of collagenous matrix, similar to uterine fibroids in situ. The grafts expressed both estrogen receptor 1 and progesterone receptor. Additionally, both receptors were up-regulated by estrogen treatment. Growth of the fibroid grafts was dependent on 17β-estradiol and progesterone supplementation at levels similar to women with the disease and was studied for up to 60 days at maximum. Co-treatment with the antiprogestin mifepristone reduced graft growth (four independent donors, p<0.0001 two-sided t-test), as did treatment with the mTOR inhibitor rapamycin (three independent donors, p<0.0001 two-sided t-test). This in vivo animal model preserves the main histological and functional characteristics of human uterine fibroids, is amenable to intervention by pharmacological treatment, and can thus serve as an adequate model for the development of novel therapies

Publication Categories in Toxicologic Pathology

Toxicologic Pathology is the official journal of the Society of Toxicologic Pathology (STP), the British Society of Toxicological Pathology, and the European STP (ESTP). Toxicologic Pathology publishes articles related to topics in various aspects of toxicologic pathology such as anatomic pathology, clinical pathology, experimental pathology, and biomarker research. Publications include society-endorsed Best Practice/Position and Points to Consider publications and ESTP Expert Workshop articles that are relevant to toxicologic pathology and scientific regulatory processes, Opinion articles under the banner of the STP Toxicologic Pathology Forum, Original Articles, Review Articles (unsolicited/contributed, mini, and invited), Brief Communications, Letters to the Editor, Meeting Reports, and Book Reviews. This article provides details on the various publication categories in Toxicologic Pathology and will serve as a reference for authors and readers

Fibroid grafts retain characteristics of human uterine fibroids.

<p>Fibroid xenografts transplanted with approx. 10mg and grown for 60d in SCID mice supplemented with 17β Estradiol and Progesterone to 31mg were stained for BrdU incorporation (left, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142429#pone.0142429.s002" target="_blank">S1 Fig</a> for higher resolution), Desmin (middle) and collagen (extracellular matrix) (right). Scale bar = 500μm.</p

17β Estradiol and Progesterone support growth of fibroid xenografts.

<p>CB17-SCID mice were ovariectomized, and either not treated or supplemented with either 17β Estradiol (0.1mg/60d release pellets) alone, or 17β Estradiol in combination with progesterone (25mg/60d release pellets). Per mouse, four small grafts of each fibroid and myometrial tissue from one donor was transplanted. Between four to six mice were transplanted per donor and treatment group in this experiment (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142429#pone.0142429.s001" target="_blank">S1 Table</a>)The weight of the grafted fibroid tissue pieces at grafting is indicated with the dotted line. Mice were sacrificed after 60d, and grafts were weighted. Symbols indicate individual graft weights, together with group means ± s.e.m.</p

Rapamycin and Mifepristone inhibit growth of fibroid xenografts in SCID mice.

<p>E2 and P supplemented SCID-beige mice were grafted with large grafts (for Rapamycin) and small grafts (for Mifepristone, SCID-beige) from fibroid tissue from three and four different patients, respectively. The dashed line indicates the average weight of the grafts at day 0. (A) Mice were treated with Mifepristone from day 0 using 10mg/60d release pellets; this is approx. 6.5mg/kg/d. (B) Mice were treated p.o. with either 15mg/kg/d Rapamycin in 2.5% PEG400 in H₂O or vehicle alone from d4 of the experiment. Mice were killed after 42–45 days and grafts were removed and weighted. Each symbol indicates the weight of an individual graft, together with group means ± s.d. Significance of difference between treatment and control groups was evaluated using a two-sided t-test.</p

Growth kinetics of human fibroid xenografts in CB17 SCID mice.

<p>17β Estradiol and Progesterone supplemented SCID mice were grafted with small (A) or large (B) grafts with fibroid tissue from four different patients. The dashed and dotted lines indicate graft weights of small and large grafts at transplantation on day 0, respectively. Mice were killed after 15, 30 and 45 days (A) or 45 days (B) and grafts were removed and weighted. Each individual graft weight is shown, together with group means ± s.d. Tissue from donors with the same numbers in panel A and B are identical. For statistical analysis, in (A) log transformed group means d15 to d30, and d30 to d45 were compared. In (B), ¹graft weights were normalized to the respective xenograft transplantation weight of 10mg for small and 40mg for large grafts, and log transformed normalized data were compared to each other. Significance of difference between treatment and control groups was evaluated using a two-sided t-test.</p