The emergence of SARS-CoV-2 at the end of 2019 required the swift development of a vaccine to address the pandemic. Nonclinical GLP-compliant studies in Wistar Han rats were initiated to assess the ...local tolerance, systemic toxicity, and immune response to four mRNA vaccine candidates encoding immunogens derived from the spike (S) glycoprotein of SARS-CoV-2, encapsulated in lipid nanoparticles (LNPs). Vaccine candidates were administered intramuscularly once weekly for three doses at 30 and/or 100 µg followed by a 3-week recovery period. Clinical pathology findings included higher white blood cell counts and acute phase reactant concentrations, lower platelet and reticulocyte counts, and lower RBC parameters. Microscopically, there was increased cellularity (lymphocytes) in the lymph nodes and spleen, increased hematopoiesis in the bone marrow and spleen, acute inflammation and edema at the injection site, and minimal hepatocellular vacuolation. These findings were generally attributed to the anticipated immune and inflammatory responses to the vaccines, except for hepatocyte vacuolation, which was interpreted to reflect hepatocyte LNP lipid uptake, was similar between candidates and resolved or partially recovered at the end of the recovery phase. These studies demonstrated safety and tolerability in rats, supporting SARS-CoV-2 mRNA-LNP vaccine clinical development.
Microcystin (MC) exposure is an increasing concern because more geographical locations are covered with cyanobacterial blooms as eutrophication and bloom-favoring environmental factors become more ...prevalent worldwide. Acute MC exposure has been linked to gastrointestinal distress, liver toxicity, and death in extreme circumstances. The goal of this study was to provide an accurate and comprehensive description of MC-LRs impacts on liver pathology, clinical chemistry, and gap junction intercellular communication (GJIC) in CD-1 male and female mice. Mice were exposed to 0, 3000, and 5000/4000 µg/kg/day MC-LR, daily for 7 days, and were necropsied on Day 8. Blood samples for clinical chemistry analysis were processed to serum, while liver sections were fixed for histopathology or evaluated for GJIC using fluorescent cut-load dye. Results show a dose-dependent relationship with MC-LR exposure and hepatocellular hypertrophy, degradation, and necrosis. Clinical chemistry parameters alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, and cholesterol increased significantly in MC-LR exposed mice. Clinical chemistry parameter analysis showed significantly increased susceptibility to MC-LR in females compared to males. Changes in GJIC were not noted, but localization of hepatotoxicity near the central veins and midlobular areas was seen. Future toxicity studies involving MCs should consider response differences across sexes, differing MC congeners, and combinatorial exposures involving other cyanotoxins.
More than 85% of breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens, such as those in the diet, through a multistep disease process progressing from ...non-cancerous to premalignant and malignant stages. The chemical carcinogen 2-amino-1-methyl-6-phenylimidazo4,5-bpyridine (PhIP) is one of the most abundant heterocyclic amines found in high-temperature cooked meats and is recognized as a mammary carcinogen. However, the PhIP's mechanism of action in breast cell carcinogenesis is not clear. Here, we demonstrated, for the first time, that cumulative exposures to PhIP at physiologically achievable, pico to nanomolar concentrations effectively induced progressive carcinogenesis of human breast epithelial MCF10A cells from a non-cancerous stage to premalignant and malignant stages in a dose- and exposure-dependent manner. Progressive carcinogenesis was measured by increasingly- acquired cancer-associated properties of reduced dependence on growth factors, anchorage-independent growth, acinar-conformational disruption, proliferation, migration, invasion, tumorigenicity with metastasis and increased stem-like cell populations. These biological changes were accompanied by biochemical and molecular changes, including upregulated H-Ras gene expression, extracellular signal-regulated kinase (ERK) pathway activation, Nox-1 expression, reactive oxygen species (ROS) elevation, increased HIF-1α, Sp1, tumor necrosis factor-α, matrix metalloproteinase (MMP)-2, MMP-9, aldehyde dehydrogenase activity and reduced E-cadherin. The Ras-ERK-Nox-ROS pathway played an important role in not only initiation but also maintenance of cellular carcinogenesis induced by PhIP. Using biological, biochemical and molecular changes as targeted endpoints, we identified that the green tea catechin components epicatechin-3-gallate and epigallocatechin-3-gallate, at non-cytotoxic doses, were capable of suppressing PhIP-induced cellular carcinogenesis and tumorigenicity.
Purpose
Reveal mechanisms for the novel proapoptotic ability of oncogenic H-Ras to increase cell susceptibility to a histone deacetylase inhibitor (HDACI) FR901228 for inducing caspase activation and ...selective apoptosis.
Methods
Human urinary bladder cancer J82 and oncogenic H-Ras-expressing J82 cells were used to reveal differential induction of intracellular reactive oxygen species (ROS), caspase activation, and apoptosis by HDACI FR901228. ROS levels and caspase-8, -9, and -3/7 activities were measured by flow cytometry and luminescence assays, respectively. Specific inhibitors were used to suppress caspases and ROS. Western blot analysis determined modulators of caspase pathways.
Results
ROS, caspase activity, and cell death was differentially increased by FR901228 in oncogenic H-Ras-expressing J82 versus parental cells. Blocking ROS resulted in reduced FR901228-induced cell death and caspase activation. Suppression of caspase-8 resulted in reduced FR901228-activated caspase-9 and -3/7. Suppression of caspase-9 resulted in reduced FR901228-activated caspase-3/7. Although FR901228 induced an ROS-dependent increase of FasL, FasL failed to induce caspase activation and cell death.
Conclusion
Increased ROS played an important role in the activation of the extrinsic and intrinsic caspase pathways to cooperatively induce executioner caspase-3/7 through a novel FasL-independent pathway in FR901228-induced selective apoptosis of oncogenic H-Ras-expressing J82 versus parental cells.
More than 35% of human urinary bladder cancers involve oncogenic H-Ras activation. In addition to tumorigenic ability, oncogenic H-Ras possesses a novel proapoptotic ability to facilitate the ...induction of apoptosis by histone deacetylase inhibitors (HDACI). HDACIs are a new class of anticancer agents and are highly cytotoxic to transformed cells. To understand the connection between the selectivity of HDACIs on transformed cells and the proapoptotic ability of oncogenic H-Ras to facilitate HDACI-induced apoptosis, we introduced oncogenic H-Ras into urinary bladder J82 cancer cells to mimic an acquisition of the H-ras gene activation in tumor development. Expression of oncogenic H-Ras promoted J82 cells to acquire tumorigenic ability. Meanwhile, oncogenic H-Ras increased susceptibility of J82 cells to HDACIs, including FR901228 and trichostatin A, for inducing apoptosis. The caspase pathways, the B-Raf and extracellular signal-regulated kinase pathway, p21(Cip1) and p27(Kip1), and core histone contents are regulated differently by FR901228 in oncogenic H-Ras-expressed J82 cells than their counterparts in parental J82 cells, contributing to the increased susceptibility to the induction of selective apoptosis. Our results lead us to a suggestion that HDACIs activate the proapoptotic ability of oncogenic H-Ras, indicating a potential therapeutic value of this new class of anticancer agents in the control of human urinary bladder cancer that has progressed to acquire oncogenic H-Ras.
Translational models have played an important role in the rapid development of safe and effective vaccines and therapeutic agents for the ongoing coronavirus disease 2019 (COVID-19) pandemic caused ...by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Animal models recapitulating the clinical and underlying pathological manifestations of COVID-19 have been vital for identification and rational design of safe and effective vaccines and therapies. This manuscript provides an overview of commonly used COVID-19 animal models and the pathologic features of SARS-CoV-2 infection in these models in relation to their clinical presentation in humans. Also discussed are considerations for selecting appropriate animal models for infectious diseases such as COVID-19, the host determinants that can influence species-specific susceptibility to SARS-CoV-2, and the pathogenesis of COVID-19. Finally, the limitations of currently available COVID-19 animal models are highlighted.
Malignant neuroendocrine tumors were diagnosed in the stomach of two out of sixty female Sprague-Dawley rats treated for 89 weeks with a high dose of a novel, small molecule, cannabinoid-1 ...antagonist. The tumors were associated with parietal cell atrophy accompanied by foveolar hyperplasia of the glandular stomach mucosa. Parietal cell atrophy/foveolar hyperplasia was considered test article related at the high dose, given the higher incidence and severity relative to untreated controls, although the precise mechanism of the parietal cell atrophy was undetermined. Spontaneous gastric neuroendocrine tumors are very rare in rats, and the current cases were considered secondary to parietal cell atrophy causing reduced gastric acid secretion and subsequent overstimulation of gastrin release through a feedback loop.
A 2-year-old, captive, intact female African hedgehog (Atelerix albiventris) was evaluated for nonspecific clinical signs and progressive hind limb ataxia over a 1-month period. Abnormal results from ...a complete blood count included a mild regenerative anemia and an inflammatory leukogram with a degenerative left shift, toxic changes, and eosinophilia. Serum biochemistry analysis showed increased alkaline phosphatase and alanine transaminase levels. Whole-body radiograph images of the hedgehog revealed a thin body condition, oligodontia, and ventral lumbosacral spondylosis deformans. An abdominal ultrasonographic examination of the patient indicated that a hyperechoic mass with unknown origin was present in the caudal abdomen and moderate free fluid and a hyperechoic splenic nodule were present. A cytological examination of material collected through a fine needle aspirate of the abdominal mass was suggestive of carcinoma with marked eosinophilic inflammation. Exploratory surgery revealed a right ovarian mass and an abnormal spleen. An ovariohysterectomy and splenectomy were performed. Histopathology of the reproductive tract and spleen revealed an endometrial polyp in the uterus, a granulosa cell tumor in the right ovary, and a large number of immature eosinophils present throughout the spleen, consistent with eosinophilic leukemia. The hedgehog died 2 days following the surgical procedure. A postmortem examination of the patient revealed a mild bicavitary effusion, hepatomegaly, and a stomach ulcer. Histopathological examination of examined tissue indicated neoplastic eosinophils that encompassed 80% of the bone marrow and also infiltrated multiple glands, viscera, and associated blood vessels. The necropsy results supported the previous diagnosis of eosinophilic leukemia. Practitioners should be aware that similar clinical signs may be associated with eosinophilic leukemia in Atelerix albiventris.
Domestic ferrets (Mustela putorius furo) have been used in biomedical research to study influenza viruses since the early 20th century. Ferrets have continued to gain importance for the study of ...viral respiratory disease due to their disease susceptibility and anatomic
similarities to humans. Here we review features of ferret biology and management that should be considered when planning to work with this species, particularly in models of respiratory disease. We specifically discuss biosafety and husbandry, clinical and pathologic assessments, and anesthetic
considerations for ferrets with respiratory disease and systemic illness. These considerations are important for animal welfare, fidelity of the model to human disease, and ensuring accuracy and reproducibility of acquired data. Finally, we briefly review the use of ferrets to study respiratory
diseases by discussing their respiratory anatomy and 2 frequently studied viral respiratory diseases, influenza and coronavirus disease 2019 (COVID-19).
A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates ...(BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4
and IFNγ
CD8
T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA
, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).
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