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

Interleukin-17A blockade with secukinumab results in decreased neutrophil infiltration in psoriasis: minimally-invasive measurement by tape stripping

Psoriasis is a well characterized interleukin (IL)-17A-driven skin disease with neutrophil infiltration and epidermal hyperkeratosis. Several biomarkers, most prominently β-defensin-2 (BD-2), have been identified using local and systemic invasive measurements as responsive markers of IL-17A-driven skin pathology. We sought to determine whether measurements of epidermal proteins by tape stripping could offer a minimally-invasive method to assess treatment responses. We compared the expression of 170 proteins in the epidermis (tape stripping) and dermis (open flow microperfusion) of 8 psoriatic subjects before and after administration of a single dose of subcutaneous (s.c.) antiIL-17A mAb secukinumab. Proteomic analyses of tape strips revealed a >3-fold decrease in 32 epidermal and inflammatory cell proteins in response to secukinumab. The epidermal proteins with the largest (>10-fold) decreases were: matrix metalloproteinase-8 (MMP-8, 15.68-fold, p<0.05); myeloperoxidase (MPO, 14.72-fold, p<0.005); IL-8 (11.93-fold, p<0.05); MMP-9 (10.81-fold, p<0.005); and IL-1β (10.35-fold, p<0.05). For these proteins, greater-fold protein changes were detected in the epidermis compared to dermis. Immunohistochemical analysis confirmed that neutrophils are the predominant cell type in psoriatic skin lesions that express MPO, MMP-8 and MMP-9, and that secukinumab treatment dramatically decreases neutrophil accumulation. Thus, tape stripping may be used to assess epidermal neutrophils, and protein biomarker responses to anti-IL-17A therapy in psoriasis

Differential promotion of hematopoietic chimerism and inhibition of alloreactive T cell proliferation by combinations of anti-CD40Ligand, anti-LFA-1, everolimus, and deoxyspergualin.

Allogeneic bone marrow (BM) engraftment for chimerism and transplantation tolerance may be promoted by combinations of costimulation blocking biologics and small molecular weight inhibitors. We showed previously in a mouse model that anti-CD40Ligand (anti-CD40L, CD154) combined with anti-LFA-1 or everolimus (40-O-(2-hydroxyethyl)-rapamycin) resulted in stable chimerism in almost all BM recipients, whereas anti-LFA-1 plus everolimus conferred approximately 50% chimerism stability. Here, we investigated whether this lower incidence could be increased with deoxyspergualin (DSG) in place of or in addition to everolimus. However, DSG and everolimus were similarly synergistic with costimulation blockade for stable hematopoietic chimerism. This correlated with allospecific T cell depletion and inhibition of acute but not chronic skin allograft rejection. Different treatments were also compared for their inhibition of alloreactive T cell proliferation in vivo. While anti-CD40L did not impair T cell proliferation, anti-LFA-1 reduced both CD4 and CD8 T cell proliferation, and combining anti-LFA-1 with everolimus or DSG had an additive inhibitory effect on CD4 T cell proliferation. Thus, despite their strong inhibition of alloreactive T cell proliferation, combinations of anti-LFA-1 with everolimus or DSG did not reach the unique potency of anti-CD40L-based combinations to support stable hematopoietic chimerism in this system

Spontaneous Metastatic Angiosarcoma of the Tongue in a Wistar Rat: Morphological and Immunohistochemical Characterisation

A primary angiosarcoma was found in the tongue of a 6 weeks old female Wistar rat, sacrificed for humane reasons during the course of a 4-week toxicology study. At necropsy, a nodule protruding from the dorsal part of the tongue was found and displayed microscopically, irregularly shaped vascular spaces separated by collagenous stroma. The spindle-shaped endothelial cells showed pleomorphysm, hyperchromatism and a low mitotic activity; large nuclei with one or more nucleoli were present. Multiple metastases were found in the lungs and the morphology of the cells resembled that of the primary tumour. Immunohistochemically, the primary tumour and the lung metastases were positive for von Willebrand factor and vimentin. The diagnosis of tongue angiosarcoma metastasizing to the lungs was made on the basis of microscopic and immunohistochemical findings

Investigation of serotonin type 4 receptor expression in human and non-human primate gastrointestinal samples.

BACKGROUND: The serotonin type 4 (5-HT4) receptor has been associated with functions of the gastrointestinal tract such as modulation of the peristaltic reflex, smooth muscle tone, intestinal secretion and visceral sensitivity. The activation of peripheral 5-HT4 receptors with agonists such as tegaserod has been shown to accelerate gastric emptying and improve symptoms of constipation in animals and humans. However, detailed data on the expression profile and on the localization of this receptor subtype are lacking so far. OBJECTIVE: To study the pattern and expression levels of 5-HT4 receptor messenger RNA expression in the gut. METHOD: Normal tissue samples were collected from the whole gastrointestinal tract of patients undergoing abdominal surgery and, in addition, of monkeys. We performed a comprehensive analysis of 5-HT4 receptor expression by quantitative reverse transcription-polymerase chain reaction, using human and non-human primate tissues from the oesophagus to the rectum. In addition, the brain and heart of non-human primates were analysed. RESULTS: Significantly higher levels of 5-HT4 receptor mRNA were measured in the human stomach, duodenum, jejunum, ileum and caecum and also in the corresponding non-human primate gut segments, ranging from 2- to 12-fold compared with the liver. No differences were found between females and males of both human and non-human primates. CONCLUSIONS: These results show 5-HT4 receptor mRNA expression throughout the gastrointestinal tract in humans and primates, and also support the preclinical and clinical findings of 5-HT4 receptors ligands exhibiting multiple effects throughout the gastrointestinal tract

Vav1 GEF activity is required for T cell mediated allograft rejection

The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Disruption of Vav1 GEF activity towards RhoGTPases is thus an attractive approach for immunosuppressive therapy. However, in addition to its GEF activity, Vav1 has been shown to transduce signals independent of its GEF function downstream of the TCR. The contribution of Vav1 GEF-dependent and –independent functions for allogeneic T cell activation is not clear. To address this question, we used knock-in mice containing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, Vav1 GEF activity strongly contributed to in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings indicate an unexpectedly strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone can induce immunosuppression

Combinations of anti-LFA-1, everolimus, anti-CD40 ligand, and allogeneic bone marrow induce central transplantation tolerance through hemopoietic chimerism, including protection from chronic heart allograft rejection.

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vbeta11(+) T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening ("chronic rejection")

Combinations of Anti-LFA-1, Everolimus, Anti-CD40 Ligand, and Allogeneic Bone Marrow Induce Central Transplantation Tolerance through Hemopoietic Chimerism, Including Protection from Chronic Heart Allograft Rejection

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vbeta11(+) T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening ("chronic rejection")