ACKR3 Antagonism Enhances the Repair of Demyelinated Lesions Through Both Immunomodulatory and Remyelinating Effects.

Addressing inflammation, demyelination, and associated neurodegeneration in inflammatory demyelinating diseases like multiple sclerosis (MS) remains challenging. ACT-1004-1239, a first-in-class and potent ACKR3 antagonist, currently undergoing clinical development, showed promise in preclinical MS models, reducing neuroinflammation and demyelination. However, its effectiveness in treating established disease and impact on remyelination after the occurrence of demyelinated lesions remain unexplored. This study assessed the therapeutic effect of ACT-1004-1239 in two demyelinating disease models. In the proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) model, ACT-1004-1239 administered upon the detection of the first signs of paralysis, resulted in a dose-dependent reduction in EAE disease severity, concomitant with diminished immune cell infiltrates in the CNS and reduced demyelination. Notably, efficacy correlated with elevated plasma concentrations of CXCL11 and CXCL12, two pharmacodynamic biomarkers of ACKR3 antagonism. Combining ACT-1004-1239 with siponimod, an approved immunomodulatory treatment for MS, synergistically reduced EAE severity. In the cuprizone-induced demyelination model, ACT-1004-1239 administered after 5 weeks of cuprizone exposure, significantly accelerated remyelination, already quantifiable one week after cuprizone withdrawal. Additionally, ACT-1004-1239 penetrated the CNS, elevating brain CXCL12 concentrations. These results demonstrate that ACKR3 antagonism significantly reduces the severity of experimental demyelinating diseases, even when treatment is initiated therapeutically, after the occurrence of lesions. It confirms the dual mode of action of ACT-1004-1239, exhibiting both immunomodulatory effects by reducing neuroinflammation and promyelinating effects by accelerating myelin repair. The results further strengthen the rationale for evaluating ACT-1004-1239 in clinical trials for patients with demyelinating diseases.

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.

Results of the European Society of Toxicologic Pathology Survey on the Use of Artificial Intelligence in Toxicologic Pathology.

The European Society of Toxicologic Pathology (ESTP) initiated a survey through its Pathology 2.0 workstream in partnership with sister professional societies in Europe and North America to generate a snapshot of artificial intelligence (AI) usage in the field of toxicologic pathology. In addition to demographic information, some general questions explored AI relative to (1) the current status of adoption across organizations; (2) technical and methodological aspects; (3) perceived business value and finally; and (4) roadblocks and perspectives. AI has become increasingly established in toxicologic pathology with most pathologists being supportive of its development despite some areas of uncertainty. A salient feature consisted of the variability of AI awareness and adoption among the responders, as the spectrum extended from pathologists having developed familiarity and technical skills in AI, to colleagues who had no interest in AI as a tool in toxicologic pathology. Despite a general enthusiasm for these techniques, the overall understanding and trust in AI algorithms as well as their added value in toxicologic pathology were generally low, suggesting room for the need for increased awareness and education. This survey will serve as a basis to evaluate the evolution of AI penetration and acceptance in this domain.

ACT-1004-1239, a first-in-class CXCR7 antagonist with both immunomodulatory and promyelinating effects for the treatment of inflammatory demyelinating diseases.

Current strategies for the treatment of demyelinating diseases such as multiple sclerosis (MS) are based on anti-inflammatory or immunomodulatory drugs. Those drugs have the potential to reduce the frequency of new lesions but do not directly promote remyelination in the damaged central nervous system (CNS). Targeting CXCR7 (ACKR3) has been postulated as a potential therapeutic approach in demyelinating diseases, leading to both immunomodulation by reducing leukocyte infiltrates and promyelination by enhancing myelin repair. ACT-1004-1239 is a potent, selective, insurmountable, and orally available first-in-class CXCR7 receptor antagonist. The effect of ACT-1004-1239 was evaluated in the myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) and the cuprizone-induced demyelination mouse models. In addition, ACT-1004-1239 was assessed in a rat oligodendrocyte precursor cell (OPC) differentiation assay in vitro. In the MOG-induced EAE model, ACT-1004-1239 treatment (10-100 mg/kg, twice daily, orally) showed a significant dose-dependent reduction in disease clinical scores, resulting in increased survival. At the highest dose tested (100 mg/kg, twice daily), ACT-1004-1239 delayed disease onset and significantly reduced immune cell infiltrates into the CNS and plasma neurofilament light chain concentration. Treatment with ACT-1004-1239 dose-dependently increased plasma CXCL12 concentration, which correlated with a reduction of the cumulative disease score. Furthermore, in the cuprizone model, ACT-1004-1239 treatment significantly increased the number of mature myelinating oligodendrocytes and enhanced myelination in vivo. In vitro, ACT-1004-1239 promoted the maturation of OPCs into myelinating oligodendrocytes. These results provide evidence that ACT-1004-1239 both reduces neuroinflammation and enhances myelin repair substantiating the rationale to explore its therapeutic potential in a clinical setting.

Orbit Image Analysis: An open-source whole slide image analysis tool.

We describe Orbit Image Analysis, an open-source whole slide image analysis tool. The tool consists of a generic tile-processing engine which allows the execution of various image analysis algorithms provided by either Orbit itself or from other open-source platforms using a tile-based map-reduce execution framework. Orbit Image Analysis is capable of sophisticated whole slide imaging analyses due to several key features. First, Orbit has machine-learning capabilities. This deep learning segmentation can be integrated with complex object detection for analysis of intricate tissues. In addition, Orbit can run locally as standalone or connect to the open-source image server OMERO. Another important characteristic is its scale-out functionality, using the Apache Spark framework for distributed computing. In this paper, we describe the use of Orbit in three different real-world applications: quantification of idiopathic lung fibrosis, nerve fibre density quantification, and glomeruli detection in the kidney.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Dog.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/inhand.asp) is a joint initiative of the societies of toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in most tissues and organs from the dog used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions, lesions induced by exposure to test materials, and relevant infectious and parasitic lesions. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Pharmacological Characterization of Aprocitentan, a Dual Endothelin Receptor Antagonist, Alone and in Combination with Blockers of the Renin Angiotensin System, in Two Models of Experimental Hypertension.

The endothelin (ET) system has emerged as a novel target for hypertension treatment where a medical need persists despite availability of several pharmacological classes, including renin angiotensin system (RAS) blockers. ET receptor antagonism has demonstrated efficacy in preclinical models of hypertension, especially under low-renin conditions and in hypertensive patients. We investigated the pharmacology of aprocitentan (N-[5-(4-bromophenyl)-6-[2-[(5-bromo-2-pyrimidinyl)oxy]ethoxy]-4-pyrimidinyl]-sulfamide), a potent dual ETA/ETB receptor antagonist, on blood pressure (BP) in two models of experimental hypertension: deoxycorticosterone acetate (DOCA)-salt rats (low-renin model) and spontaneously hypertensive rats [(SHR), normal renin model]. We also compared the effect of its combination with RAS blockers (valsartan and enalapril) with that of the combination of the mineraloreceptor antagonist spironolactone with the same RAS blockers on BP and renal function in hypertensive rats. Aprocitentan was more potent and efficacious in lowering BP in conscious DOCA-salt rats than in SHRs. In DOCA-salt rats, single oral doses of aprocitentan induced a dose-dependent and long-lasting BP decrease and 4-week administration of aprocitentan dose dependently decreased BP (statistically significant) and renal vascular resistance, and reduced left ventricle hypertrophy (nonsignificant). Aprocitentan was synergistic with valsartan and enalapril in decreasing BP in DOCA-salt rats and SHRs while spironolactone demonstrated additive effects with these RAS blockers. In hypertensive rats under sodium restriction and enalapril, addition of aprocitentan further decreased BP without causing renal impairment, in contrast to spironolactone. In conclusion, ETA/ETB receptor antagonism represents a promising therapeutic approach to hypertension, especially with low-renin characteristics, and could be used in combination with RAS blockers, without increasing the risk of renal impairment.

Comparison of Macitentan and Bosentan on Right Ventricular Remodeling in a Rat Model of Non-vasoreactive Pulmonary Hypertension.

AIMS: We compared the efficacy of macitentan, a novel dual endothelin A/endothelin B receptor antagonist, with that of another dual endothelin receptor antagonist, bosentan, in a rat model of non-vasoreactive pulmonary hypertension (PH) with particular emphasis on right ventricular (RV) remodeling. METHODS AND RESULTS: Unlike monocrotaline or hypoxic/sugen rats, bleomycin-treated rats presented a non-vasoreactive PH characterized by the absence of pulmonary dilatation to adenosine. We therefore chose the bleomycin rat model to compare the effects of the maximally effective doses of macitentan and bosentan on pulmonary vascular and RV remodeling. Macitentan (100 mg·kg(-1)·d(-1)), but not bosentan (300 mg·kg(-1)·d(-1)), significantly prevented pulmonary vascular remodeling, RV hypertrophy, and cardiomyocyte diameter increase. Cardiac protection by macitentan was associated with a significant attenuation of genes related to cell hypertrophy and extracellular matrix remodeling. Microautoradiography and high performance liquid chromatography analysis showed greater distribution of macitentan than bosentan in the RV and pulmonary tissue. CONCLUSIONS: Macitentan was more efficacious than bosentan in preventing the development of pulmonary and RV hypertrophies in a model of non-vasoreactive PH. Greater ability to distribute into the tissue could contribute to the greater structural improvement by macitentan compared with bosentan.

Spontaneous glomerulonephritis in Göttingen minipigs.

The Göttingen minipig is one of the nonrodent species recommended by various regulatory authorities for safety assessment of drugs in preclinical studies. In such studies, knowledge of background pathology is critical in order to evaluate the potential renal toxicity. In the present study, the authors report 4 cases of glomerulonephritis out of 154 microbiologically defined Göttingen minipigs microscopically evaluated in preclinical studies. One animal required early sacrifice because of general poor health, and an additional animal died spontaneously. Histopathological evaluation revealed renal lesions in all 4 animals, exhibiting membranous or membranoproliferative glomerulonephritis at different stages, accompanied by secondary tubulo-interstitial damage. The renal changes observed were considered spontaneous in origin and of unknown etiology. Development of this condition in this strain should be considered in future studies.

Cenerimod, a selective S1P1 receptor modulator, improves organ-specific disease outcomes in animal models of Sjögren's syndrome.

BACKGROUND: Sjögren's syndrome is a systemic autoimmune disease characterized by immune cells predominantly infiltrating the exocrine glands and frequently forming ectopic lymphoid structures. These structures drive a local functional immune response culminating in autoantibody production and tissue damage, associated with severe dryness of mucosal surfaces and salivary gland hypofunction. Cenerimod, a potent, selective and orally active sphingosine-1-phosphate receptor 1 modulator, inhibits the egress of lymphocytes into the circulation. Based on the mechanism of action of cenerimod, its efficacy was evaluated in two mouse models of Sjögren's syndrome. METHODS: Cenerimod was administered in two established models of Sjögren's syndrome; firstly, in an inducible acute viral sialadenitis model in C57BL/6 mice, and, secondly, in the spontaneous chronic sialadenitis MRL/lpr mouse model. The effects of cenerimod treatment were then evaluated by flow cytometry, immunohistochemistry, histopathology and immunoassays. Comparisons between groups were made using a Mann-Whitney test. RESULTS: In the viral sialadenitis model, cenerimod treatment reduced salivary gland immune infiltrates, leading to the disaggregation of ectopic lymphoid structures, reduced salivary gland inflammation and preserved organ function. In the MRL/lpr mouse model, cenerimod treatment decreased salivary gland inflammation and reduced T cells and proliferating plasma cells within salivary gland ectopic lymphoid structures, resulting in diminished disease-relevant autoantibodies within the salivary glands. CONCLUSIONS: Taken together, these results suggest that cenerimod can reduce the overall autoimmune response and improve clinical parameters in the salivary glands in models of Sjögren's syndrome and consequently may reduce histological and clinical parameters associated with the disease in patients.

Spontaneous aortitis in the Balb/c mouse.

We examined whether high incidence rates (18%-56%) of inflammation in the root of the aorta detected in a Balb/c mouse model for hind limb ischemia were related to the surgical procedure. Twenty mice underwent ligation of the femoral artery; incidences of aortic root inflammation were compared to those observed in controls. We used a multiple-section sampling method to increase the sensitivity of the diagnostic rates. Although a cumulative incidence of 12.5% was found, no difference was seen in the overall incidence rates between the control and the surgically treated groups. Evaluation of blood levels of inflammatory cytokines showed that ligation of the femoral artery produced higher levels of interleukin-6 in the surgically transected group of mice. The development of spontaneous arteritis in this strain must be considered in future studies.

Feline intestinal T-cell lymphoma: assessment of morphologic and kinetic features in 30 cases.

In this study, 30 feline intestinal T-cell lymphomas (ITCLs) from 77 cats with gastrointestinal lymphoma were evaluated. Neoplastic lesions were composed predominantly of small (n = 21) or medium to large (n = 9) anaplastic cells. Different patterns of tumor growth were observed. A starry-sky pattern was evident in 7 cases (23.3%), fibrosis in 18 cases (60%), and neovascularization in 19 cases (63.3%). The cell proliferation index (assessed by MIB1 [mindbomb homolog 1] immunohistochemistry) ranged from 0.21% to 66.91%. Mean MIB1 index was 3.49% within the first 33rd percentile, 18.31% within the second 33rd percentile, and 40.16% within the third 33rd percentile (P < 0.000001). Microscopic and kinetic features provided evidence that ITCL in cats exhibits a spectrum in cytological composition and growth patterns that could putatively reflect differences in biologic behavior.

A fully automated image analysis method to quantify lung fibrosis in the bleomycin-induced rat model.

Intratracheal administration of bleomycin induces fibrosis in the lung, which is mainly assessed by histopathological grading that is subjective. Current literature highlights the need of reproducible and quantitative pulmonary fibrosis analysis. If some quantitative studies looked at fibrosis parameters separately, none of them quantitatively assessed both aspects: lung tissue remodeling and collagenization. To ensure reliable quantification, support vector machine learning was used on digitalized images to design a fully automated method that analyzes two important aspects of lung fibrosis: (i) areas having substantial tissue remodeling with appearance of dense fibrotic masses and (ii) collagen deposition. Fibrotic masses were identified on low magnification images and collagen detection was performed at high magnification. To insure a fully automated application the tissue classifier was trained on several independent studies that were performed over a period of four years. The detection method generates two different values that can be used to quantify lung fibrosis development: (i) percent area of fibrotic masses and (ii) percent of alveolar collagen. These two parameters were validated using independent studies from bleomycin- and saline-treated animals. A significant change of these lung fibrosis quantification parameters- increased amount of fibrotic masses and increased collagen deposition- were observed upon intratracheal administration of bleomycin and subsequent significant beneficial treatments effects were observed with BIBF-1120 and pirfenidone.

Clinicopathologic features and outcome predictors of Leptospira interrogans Australis serogroup infection in dogs: a retrospective study of 20 cases (2001-2004).

BACKGROUND AND HYPOTHESIS: We retrospectively evaluated the clinicopathologic findings and outcome predictors in dogs with Leptospira interrogans Australis serogroup infections. ANIMALS AND METHODS: The medical records of 159 dogs that had a leptospiral microscopic agglutination test (MAT) performed between 2001 and 2004 were reviewed. RESULTS: Twenty dogs met serologic criteria for either symptomatic (16 dogs) or asymptomatic (4 dogs) infection caused by Leptospira interrogans Australis serogroup. Seven of 16 symptomatic dogs died or were euthanized and 9/16 recovered. Systemic inflammatory response syndrome (SIRS) was observed in 9/16 dogs. The presence of SIRS did not affect prognosis (P = .357). C-reactive protein (CRP) and haptoglobin (Hpt) concentrations were altered in all symptomatic dogs, but results did not differ significantly between survivors and nonsurvivors (P = .08 and P = .055, respectively). Conversely, the CRP to Hpt ratio (CRP/Hpt) was significantly increased in nonsurvivors. Disseminated intravascular coagulation (DIC) was diagnosed in 7/16 dogs. DIC did not significantly affect outcome (P = .126). Multiple organ involvement was present with renal failure in 16/16, liver damage in 12/16, cardiac damage in 11/16, and muscular damage in 8/16 dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Among the evaluated clinicopathologic biomarkers, serum albumin, cardiac troponin I, CRP/Hpt, urinary albumin, and urinary total protein to creatinine ratio were found to predict outcome and warrant evaluation in larger prospective studies.

Cenerimod, a novel selective S1P1 receptor modulator with unique signaling properties.

Sphingosine-1-phosphate receptor 1 (S1P1 ) modulators sequester circulating lymphocytes within lymph nodes, thereby preventing potentially pathogenic autoimmune cells from exiting into the blood stream and reaching inflamed tissues. S1P1 receptor modulation may thus offer potential to treat various autoimmune diseases. The first nonselective S1P1-5 receptor modulator FTY720/fingolimod/Gilenya® has successfully demonstrated clinical efficacy in relapsing forms of multiple sclerosis. However, cardiovascular, hepatic, and respiratory side-effects were reported and there is a need for novel S1P1 receptor modulators with better safety profiles. Here, we describe the discovery of cenerimod, a novel, potent and selective S1P1 receptor modulator with unique S1P1 receptor signaling properties and absence of broncho- and vasoconstrictor effects ex vivo and in vivo. Cenerimod dose-dependently lowered circulating lymphocyte counts in rats and mice after oral administration and effectively attenuated disease parameters in a mouse experimental autoimmune encephalitis (EAE) model. Cenerimod has potential as novel therapy with improved safety profile for autoimmune diseases with high unmet medical need.