Utilizing Whole Slide Images for the Primary Evaluation and Peer Review of a GLP-Compliant Rodent Toxicology Study.

The approach undertaken to deliver a Good Laboratory Practice (GLP) validation of whole slide images (WSIs) and the associated workflow for the digital primary evaluation and peer review of a GLP-compliant rodent inhalation toxicity study is described. The contract research organization (CRO) undertook validation of the slide scanner, scanner software, and associated database software. This provided a GLP validated environment within the database software for the primary histopathologic evaluation using WSI and viewed with the database software web viewer. The CRO also validated a cloud-based digital pathology platform that supported the upload and transfer of WSI and metadata to a cache within the sponsor's local area network. The sponsor undertook a separate GLP validation of the same cloud-based digital pathology platform to cover the download and review of the WSI. The establishment of a fit-for-purpose GLP-compliant workflow for WSI and successful deployment for the digital primary evaluation and peer review of a large GLP toxicology study enabled flexibility in accelerated global working and potential future reuse of digitized data for advanced artificial intelligence and machine learning image analysis.

Inhaled nintedanib is well-tolerated and delivers key pharmacokinetic parameters required to treat bleomycin-induced pulmonary fibrosis.

Oral nintedanib is marketed for the treatment of idiopathic pulmonary fibrosis (IPF), Systemic Sclerosis-Associated Interstitial Lung Disease and Chronic Fibrosing Interstitial Lung Diseases with a Progressive Phenotype. While effective at slowing fibrosis progression, as an oral medicine nintedanib has limitations. To reduce side effects and maximize efficacy, nintedanib was reformulated as a solution for nebulization and inhaled administration. To predict effectiveness treating IPF, inhalation was used as a tool to dissect the pharmacokinetic components required for nintedanib pulmonary anti-fibrotic activity. Following oral administration, nintedanib extensively partitioned into tissue and exhibited flip-flop pharmacokinetics, whereby resulting lung Cmax and AUC were substantially higher than plasma. By comparison, inhaled nintedanib was capable of delivering an oral-equivalent lung Cmax with lower local and systemic AUC. Using a multi-challenge bleomycin rat model, this distinct inhaled pharmacokinetic profile was dose responsive (0.05, 0.25 and 0.375 mg/kg), delivering oral-superior pulmonary anti-fibrotic activity with an equivalent delivered lung Cmax (QD inhaled 0.375 mg/kg versus BID oral 60 mg/kg). Possibly assisting this improvement, the infrequent high inhaled dose also improved bleomycin-challenged animal weight gain to levels equivalent to sham. By comparison, BID oral weight gain was substantially less than controls, suggesting a negative health impact on oral administered animals combating fibrosis. Both oral and inhaled administration exhibited anti-inflammatory activity, with oral achieving significance. In summary, inhalation (short-duration nintedanib lung Cmax without high local or systemic AUC) was well-tolerated and was effective reducing bleomycin-induced pulmonary fibrosis.

Historical control background incidence of spontaneous pituitary gland lesions of Han-Wistar and Sprague-Dawley rats and CD-1 mice used in 104-week carcinogenicity studies.

The aim of this study was to determine the range and incidences of spontaneous microscopic lesions of the pituitary gland in control Han-Wistar and Sprague-Dawley rats and CD-1 mice from 104-week carcinogenicity studies carried out between 1998 and 2010 at Charles River Edinburgh. In both strains of rats and in CD-1 mice, non-proliferative lesions of the pituitary gland were generally uncommon, excluding cysts/pseudocysts (6.42% in Han-Wistar rats, 5.85% in Sprague-Dawley rats, and 2.08% in CD-1 mice). Primary proliferative lesions were most frequently found in the pars distalis of the pituitary gland. Adenomas and carcinomas of the pars distalis were more common in Sprague-Dawley rats (49.33% and 2.85%, respectively) than in Han-Wistar rats (27.29% and 0.21%, respectively), and adenomas in both strains of rats and CD-1 mice exhibited a marked sex predisposition, with females more commonly affected.

Human myocardial pericytes: multipotent mesodermal precursors exhibiting cardiac specificity.

Perivascular mesenchymal precursor cells (i.e., pericytes) reside in skeletal muscle where they contribute to myofiber regeneration; however, the existence of similar microvessel-associated regenerative precursor cells in cardiac muscle has not yet been documented. We tested whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts. Fetal and adult human heart pericytes (hHPs) express canonical pericyte markers in situ, including CD146, NG2, platelet-derived growth factor receptor (PDGFR) ß, PDGFRα, alpha-smooth muscle actin, and smooth muscle myosin heavy chain, but not CD117, CD133, and desmin, nor endothelial cell (EC) markers. hHPs were prospectively purified to homogeneity from ventricular myocardium by flow cytometry, based on a combination of positive- (CD146) and negative-selection (CD34, CD45, CD56, and CD117) cell lineage markers. Purified hHPs expanded in vitro were phenotypically similar to human skeletal muscle-derived pericytes (hSkMPs). hHPs express mesenchymal stem/stromal cell markers in situ and exhibited osteo-, chondro-, and adipogenic potentials but, importantly, no ability for skeletal myogenesis, diverging from pericytes of all other origins. hHPs supported network formation with/without ECs in Matrigel cultures; hHPs further stimulated angiogenic responses under hypoxia, markedly different from hSkMPs. The cardiomyogenic potential of hHPs was examined following 5-azacytidine treatment and neonatal cardiomyocyte coculture in vitro, and intramyocardial transplantation in vivo. Results indicated cardiomyocytic differentiation in a small fraction of hHPs. In conclusion, human myocardial pericytes share certain phenotypic and developmental similarities with their skeletal muscle homologs, yet exhibit different antigenic, myogenic, and angiogenic properties. This is the first example of an anatomical restriction in the developmental potential of pericytes as native mesenchymal stem cells.

Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia.

BACKGROUND: The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine γ-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. METHODS AND RESULTS: Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8-12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. CONCLUSIONS: These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia.

Kynurenine monooxygenase regulates inflammation during critical illness and recovery in experimental acute pancreatitis.

Kynurenine monooxygenase (KMO) blockade protects against multiple organ failure caused by acute pancreatitis (AP), but the link between KMO and systemic inflammation has eluded discovery until now. Here, we show that the KMO product 3-hydroxykynurenine primes innate immune signaling to exacerbate systemic inflammation during experimental AP. We find a tissue-specific role for KMO, where mice lacking Kmo solely in hepatocytes have elevated plasma 3-hydroxykynurenine levels that prime inflammatory gene transcription. 3-Hydroxykynurenine synergizes with interleukin-1ß to cause cellular apoptosis. Critically, mice with elevated 3-hydroxykynurenine succumb fatally earlier and more readily to experimental AP. Therapeutically, blockade with the highly selective KMO inhibitor GSK898 rescues the phenotype, reducing 3-hydroxykynurenine and protecting against critical illness and death. Together, our findings establish KMO and 3-hydroxykynurenine as regulators of inflammation and the innate immune response to sterile inflammation. During critical illness, excess morbidity and death from multiple organ failure can be rescued by systemic KMO blockade.

Transplantation of Purified Human Pericytes for Myocardial Repair in SCID-hu Mice.

The physiological, pathological, and regenerative roles of pericytes as microvascular mural cells and multipotent precursors have gained significant attention. The capacity to prospectively purify pericytes from multiple organs enables the investigation of their tissue-specific regenerative capabilities. Here, we describe the application of purified human pericytes for cardiac regeneration post-infarct in an immunodeficient mouse model. This protocol includes experimental details of pericyte isolation from both human skeletal and cardiac muscle, an immunodeficient mouse model of acute myocardial infarction, and xenogeneic pericyte transplantation.

Epithelial-mesenchymal transition (EMT) of renal tubular cells in canine glomerulonephritis.

Tubulo-interstitial fibrosis in dogs may result from primary injury to the interstitium or develop secondary to other renal diseases. As in human renal pathology, tubular epithelial cells (TEC) are believed to actively participate in the mechanisms of renal fibrosis. In this study, we examined the changes in the tubular epithelial component in two specific canine diseases. Immunohistochemistry showed the expression of the epithelial marker cytokeratin, the smooth muscle marker alpha-SMA, the mesenchymal marker vimentin and PCNA in 20 dogs with membranous glomerulonephritis and membrano-proliferative glomerulonephritis. Results showed that the loss of the epithelial marker in TEC was directly correlated to the grade of tubulo-interstitial disease present and independent of the type of glomerulonephritis. Varying degrees of vimentin positivity were detected in tubular epithelium in areas of inflammation, and low numbers of scattered alpha-SMA-positive cells were also observed. Immunohistochemistry showed that epithelial tubular cells lose their cytokeratin staining characteristics and transdifferentiate into cells exhibiting key mesenchymal immunophenotypic feature of vimentin-positive staining in both diseases investigated. The integrity of the tubular basement membrane is likely to be fundamental in maintaining the epithelial phenotype of TEC. Animal models provide opportunities for investigating the pathogenesis of renal fibrosis in humans.

Skeletal and cardiac muscle pericytes: Functions and therapeutic potential.

Pericytes are periendothelial mesenchymal cells residing within the microvasculature. Skeletal muscle and cardiac pericytes are now recognized to fulfill an increasing number of functions in normal tissue homeostasis, including contributing to microvascular function by maintaining vessel stability and regulating capillary flow. In the setting of muscle injury, pericytes contribute to a regenerative microenvironment through release of trophic factors and by modulating local immune responses. In skeletal muscle, pericytes also directly enhance tissue healing by differentiating into myofibers. Conversely, pericytes have also been implicated in the development of disease states, including fibrosis, heterotopic ossication and calcification, atherosclerosis, and tumor angiogenesis. Despite increased recognition of pericyte heterogeneity, it is not yet clear whether specific subsets of pericytes are responsible for individual functions in skeletal and cardiac muscle homeostasis and disease.

Perivascular Mesenchymal Stem Cells in Sheep: Characterization and Autologous Transplantation in a Model of Articular Cartilage Repair.

Previous research has indicated that purified perivascular stem cells (PSCs) have increased chondrogenic potential compared to conventional mesenchymal stem cells (MSCs) derived in culture. This study aimed to develop an autologous large animal model for PSC transplantation and to specifically determine if implanted cells are retained in articular cartilage defects. Immunohistochemistry and fluorescence-activated cell sorting were used to ascertain the reactivity of anti-human and anti-ovine antibodies, which were combined and used to identify and isolate pericytes (CD34-CD45-CD146+) and adventitial cells (CD34+CD45-CD146-). The purified cells demonstrated osteogenic, adipogenic, and chondrogenic potential in culture. Autologous ovine PSCs (oPSCs) were isolated, cultured, and efficiently transfected using a green fluorescence protein (GFP) encoding lentivirus. The cells were implanted into articular cartilage defects on the medial femoral condyle using hydrogel and collagen membranes. Four weeks following implantation, the condyle was explanted and confocal laser scanning microscopy demonstrated the presence of oPSCs in the defect repaired with the hydrogel. These data suggest the testability in a large animal of native MSC autologous grafting, thus avoiding possible biases associated with xenotransplantation. Such a setting will be used in priority for indications in orthopedics, at first to model articular cartilage repair.

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Kynurenine-3-monooxygenase inhibition prevents multiple organ failure in rodent models of acute pancreatitis.

Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death. Acute mortality from AP-MODS exceeds 20% (ref. 3), and the lifespans of those who survive the initial episode are typically shorter than those of the general population. There are no specific therapies available to protect individuals from AP-MODS. Here we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism, is central to the pathogenesis of AP-MODS. We created a mouse strain that is deficient for Kmo (encoding KMO) and that has a robust biochemical phenotype that protects against extrapancreatic tissue injury to the lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of the oxazolidinone GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 Å resolution. Treatment with GSK180 resulted in rapid changes in the levels of kynurenine pathway metabolites in vivo, and it afforded therapeutic protection against MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS, and they open up a new area for drug discovery in critical illness.

Deducing the stage of origin of Wilms' tumours from a developmental series of Wt1-mutant mice.

Wilms' tumours, paediatric kidney cancers, are the archetypal example of tumours caused through the disruption of normal development. The genetically best-defined subgroup of Wilms' tumours is the group caused by biallelic loss of the WT1 tumour suppressor gene. Here, we describe a developmental series of mouse models with conditional loss of Wt1 in different stages of nephron development before and after the mesenchymal-to-epithelial transition (MET). We demonstrate that Wt1 is essential for normal development at all kidney developmental stages under study. Comparison of genome-wide expression data from the mutant mouse models with human tumour material of mutant or wild-type WT1 datasets identified the stage of origin of human WT1-mutant tumours, and emphasizes fundamental differences between the two human tumour groups due to different developmental stages of origin.

Heart on a plate: histological and functional assessment of isolated adult zebrafish hearts maintained in culture.

The zebrafish is increasingly used for cardiovascular genetic and functional studies. We present a novel protocol to maintain and monitor whole isolated beating adult zebrafish hearts in culture for long-term experiments. Excised whole adult zebrafish hearts were transferred directly into culture dishes containing optimized L-15 Leibovitz growth medium and maintained for 5 days. Hearts were assessed daily using video-edge analysis of ventricle function using low power microscopy images. High-throughput histology techniques were used to assess changes in myocardial architecture and cell viability. Mean spontaneous Heart rate (HR, min(-1)) declined significantly between day 0 and day 1 in culture (96.7 ± 19.5 to 45.2 ± 8.2 min-1, mean ± SD, p = 0.001), and thereafter declined more slowly to 27.6 ± 7.2 min(-1) on day 5. Ventricle wall motion amplitude (WMA) did not change until day 4 in culture (day 0, 46.7 ± 13.0 µm vs day 4, 16.9 ± 1.9 µm, p = 0.08). Contraction velocity (CV) declined between day 0 and day 3 (35.6 ± 14.8 vs 15.2 ± 5.3 µms(-1), respectively, p = 0.012) while relaxation velocity (RV) declined quite rapidly (day 0, 72.5 ± 11.9 vs day 1, 29.5 ± 5.8 µms(-1), p = 0.03). HR and WMA responded consistently to isoproterenol from day 0 to day 5 in culture while CV and RV showed less consistent responses to beta-agonist. Cellular architecture and cross-striation pattern of cardiomyocytes remained unchanged up to day 3 in culture and thereafter showed significant deterioration with loss of striation pattern, pyknotic nuclei and cell swelling. Apoptotic markers within the myocardium became increasingly frequent by day 3 in culture. Whole adult zebrafish hearts can be maintained in culture-medium for up to 3 days. However, after day-3 there is significant deterioration in ventricle function and heart rate accompanied by significant histological changes consistent with cell death and loss of cardiomyocyte cell integrity. Further studies are needed to assess whether this preparation can be optimised for longer term survival.

Laser-targeted ablation of the zebrafish embryonic ventricle: a novel model of cardiac injury and repair.

BACKGROUND: While the adult zebrafish (Danio rerio) heart demonstrates a remarkable capacity for self-renewal following apical resection little is known about the response to injury in the embryonic heart. METHODS: Injury to the beating zebrafish embryo heart was induced by laser using a transgenic zebrafish expressing cardiomyocyte specific green fluorescent protein. Changes in ejection fraction (EF), heart rate (HR), and caudal vein blood flow (CVBF) assessed by video capture techniques were assessed at 2, 24 and 48 h post-laser. Change in total and mitotic ventricular cardiomyocyte number following laser injury was also assessed by counting respectively DAPI (VCt) and Phospho-histone H3 (VCm) positive nuclei in isolated hearts using confocal microscopy. RESULTS: Laser injury to the ventricle resulted in bradycardia and mild bleeding into the pericardium. At 2 h post-laser injury, there was a significant reduction in cardiac performance in lasered-hearts compared with controls (HR 117 ± 11 vs 167 ± 9 bpm, p ≤ 0.001; EF 14.1 ± 1.8 vs 20.1 ± 1.3%, p ≤ 0.001; CVBF 103 ± 15 vs 316 ± 13 µms(-1), p ≤ 0.001, respectively). Isolated hearts showed a significant reduction in VCt at 2 h post-laser compared to controls (195 ± 15 vs 238 ± 15, p ≤ 0.05). Histology showed necrosis and apoptosis (TUNEL assay) at the site of laser injury. At 24 h post-laser cardiac performance and VCt had recovered fully to control levels. Pretreatment with the cell-cycle inhibitor, aphidicolin, significantly inhibited functional recovery of the ventricle accompanied by a significant inhibition of cardiomyocyte proliferation. CONCLUSIONS: Laser-targeted injury of the zebrafish embryonic heart is a novel and reproducible model of cardiac injury and repair suitable for pharmacological and molecular studies.

Molecular detection of Chlamydia-like organisms in cattle drinking water.

A substantial proportion of the causes of infectious bovine abortion remain largely undiagnosed, potentially due to the presence of previously unrecognised infectious agents. Recently, several reports have demonstrated the presence of Parachlamydia sp. in placental and foetal tissues derived from bovine abortions of unknown aetiology but the route of transmission remains undefined. The drinking water from one such recent case study was analysed for the presence of Parachlamydia sp. as a potential source of infection. Chlamydiales sp. 16S rRNA genes were PCR-amplified from the drinking water and a 16S rRNA gene clone library constructed. DNA sequencing of thirty-one clones indicated the presence of organisms belonging to the Parachlamydiaceae, specifically the genera Parachlamydia and Neochlamydia. Seven 16S rRNA gene sequences were identical to a Parachlamydia sp. sequence obtained from placental tissue from an abortion case originating from the same farm. These results raise the possibility that the drinking water is a source of Parachlamydia, which may play a role in infectious bovine abortion.