Spontaneous Osteoblastic Osteosarcoma in a Mongolian Gerbil (Meriones unguiculatus)

Spontaneous neoplasms in Mongolian gerbils have an incidence of 20% to 26.8%, but osteosarcomas occur at a much lower rate. Here we report a 1-y-old Mongolian gerbil with a spontaneous osteosarcoma at the level of the proximal tibia, with metastases to the pectoral muscles and lungs. Grossly, the tibial mass obliterated the tibia and adjacent muscles, and an axillary mass with a bloody, cavitary center expanded the pectoral muscles. Microscopically, the tibial mass was an infiltrative, osteoblastic mesenchymal neoplasm, and the axillary mass was an anaplastic mesenchymal neoplasm with hemorrhage. The lung contained multiple metastatic foci. Immunohistochemistry for osteonectin was strongly positive in the tibial, axillary, and pulmonary metastases. Although osteosarcoma is the most common primary malignant bone neoplasm that occurs spontaneously in all laboratory and domestic animal species and humans, it arises less frequently than does other neoplasms. The current case of spontaneous osteoblastic osteosarcoma of the proximal tibia and metastases to the pectoral muscles and lung in a Mongolian gerbil is similar in presentation, histology, and predilection site of both osteoblastic and telangiectatic osteosarcomas in humans. In addition, this case is an unusual manifestation of osteosarcoma in the appendicular skeleton of a Mongolian gerbil.Mongolian gerbils are used frequently in biologic research,^{1,2,4,9,10,12-14} particularly in oncogenic studies and filariasis research studying Brugia malayi.² There have been several reports^{1,6,10,11,13-15} of spontaneous neoplasms, particularly in gerbils 2 y of age and older, typically occurring with the highest incidences in the skin, reproductive tract, and adrenal glands; however, neoplasms have also been reported in the thyroid, thymus, liver, kidney, pancreas, and bone.^{1,6,10,11,13-15} The incidence of spontaneous neoplasms occurring in the subfamily Gerbillinae ranges from 20% to 26.8%,^{1,6,10,11,13-15} depending on the study, age, and sex of the animals.With a lower incidence than those reported for other neoplasms, osteosarcomas in gerbils have been described in the ramus of the mandible and as an extraskeletal mass throughout the peritoneum.^10,11 The usual age of onset for osteosarcomas in Mongolian gerbils is approximately 3 y (36 to 39 mo); however, no tumor type has been reported at less than 2 y of age in this species.^10,11 Here we report a spontaneous osteosarcoma that occurred at the level of the proximal tibia, with metastases to the pectoral muscles and lung, in a 1-y-old Mongolian gerbil.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

Live imaging the phagocytic activity of inner ear supporting cells in response to hair cell death

Hearing loss and balance disorders affect millions of people worldwide. Sensory transduction in the inner ear requires both mechanosensory hair cells (HCs) and surrounding glia-like supporting cells (SCs). HCs are susceptible to death from aging, noise overexposure, and treatment with therapeutic drugs that have ototoxic side effects; these ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic drug cisplatin. Although both classes of drugs are known to kill HCs, their effects on SCs are less well understood. Recent data indicate that SCs sense and respond to HC stress, and that their responses can influence HC death, survival, and phagocytosis. These responses to HC stress and death are critical to the health of the inner ear. Here we have used live confocal imaging of the adult mouse utricle, to examine the SC responses to HC death caused by aminoglycosides or cisplatin. Our data indicate that when HCs are killed by aminoglycosides, SCs efficiently remove HC corpses from the sensory epithelium in a process that includes constricting the apical portion of the HC after loss of membrane integrity. SCs then form a phagosome, which can completely engulf the remaining HC body, a phenomenon not previously reported in mammals. In contrast, cisplatin treatment results in accumulation of dead HCs in the sensory epithelium, accompanied by an increase in SC death. The surviving SCs constrict fewer HCs and display impaired phagocytosis. These data are supported by in vivo experiments, in which cochlear SCs show reduced capacity for scar formation in cisplatin-treated mice compared with those treated with aminoglycosides. Together, these data point to a broader defect in the ability of the cisplatin-treated SCs, to preserve tissue health in the mature mammalian inner ear.Hearing loss affects more than 360 million people worldwide and is often irreversible.¹ Mechanosensory hair cells (HCs), the receptor cells of hearing and balance, are not regenerated in the adult mammal and their death results in permanent hearing loss.^{2, 3} HCs are surrounded by glia-like supporting cells (SCs) that are necessary for HC survival and function (reviewed in Monzack et al.).⁴ SCs perform many functions, including providing critical trophic factors, preventing excitotoxicity, and mediating regeneration in those systems (non-mammalian vertebrates) capable of replacing lost HCs.^{5, 6, 7, 8, 9, 10, 11} When HCs die, SCs also preserve the integrity and function of the remaining tissue by forming scars and clearing dead HCs.^{2, 12, 13, 14, 15, 16, 17} Maintaining a fluid barrier at the surface of the sensory epithelium after damage is necessary to preserve the electro-chemical gradient that drives HC depolarization and therefore sensory transduction after the onset of hearing (reviewed in Wangemann).¹⁸Several major stressors cause HC death,^{19, 20, 21, 22} including aging, noise trauma, and exposure to therapeutic drugs with ototoxic side effects. When a HC is killed by noise or aminoglycoside antibiotics, surrounding SCs form a filamentous actin (F-actin) cable that constricts the HC at its apex.^{2, 12, 13, 14, 15, 16, 17} This process separates the apical portion of the cell, including the stereocilia bundle, from the HC body and preserves a sealed reticular lamina.²³ In the chick utricle, following the apical constriction of dead HCs, the SCs engulf and phagocytose the remaining HC corpse.¹⁵ Additional data from the chick indicate that the ototoxic drug cisplatin impairs some SC functions, including regeneration of HCs or clearance of HC debris.²⁴ We hypothesized that SCs would have significant phagocytic activity in the mature mammalian inner ear, and that cisplatin would impair this activity. To examine these dynamic processes, we live-imaged SC phagocytic activity in the adult mouse utricle and compared the SC responses with HC stress and death caused by aminoglycosides versus cisplatin.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

Paravascular pathways contribute to vasculitis and neuroinflammation after subarachnoid hemorrhage independently of glymphatic control

Subarachnoid hemorrhage (SAH) is a devastating disease with high mortality. The mechanisms underlying its pathological complications have not been fully identified. Here, we investigate the potential involvement of the glymphatic system in the neuropathology of SAH. We demonstrate that blood components rapidly enter the paravascular space following SAH and penetrate into the perivascular parenchyma throughout the brain, causing disastrous events such as cerebral vasospasm, delayed cerebral ischemia, microcirculation dysfunction and widespread perivascular neuroinflammation. Clearance of the paravascular pathway with tissue-type plasminogen activator ameliorates the behavioral deficits and alleviates histological injury of SAH. Interestingly, AQP4^−/− mice showed no improvements in neurological deficits and neuroinflammation at day 7 after SAH compared with WT control mice. In conclusion, our study proves that the paravascular pathway dynamically mediates the pathological complications following acute SAH independently of glymphatic control.Cerebral aneurysm rupture causes subarachnoid hemorrhage (SAH), which is associated with a high mortality due to its secondary complications, including hemorrhage, hydrocephalus and delayed cerebral ischemia (DCI).^{1, 2, 3} Therapeutic interventions against the secondary complications, especially DCI, are yet limited, as the pathological mechanism underlying that is not fully understood.^{2, 3, 4, 5, 6, 7} Current hypotheses of the development of the secondary complications mainly include cerebral vasospasm (CVS) and the microcirculation disturbance, as well as parenchymal arterial lesions, microthrombosis and neuroinflammation.^{1, 2, 4, 7, 8, 9}Previous studies have shown that the blockade of cerebral lymphatic drainage deteriorated the secondary cerebral ischemia after SAH, suggesting that the cerebral lymphatic drainage pathway could be involved in the pathological mechanism of SAH.^{10, 11} However, the central nervous system (CNS) was considered lack of a conventional lymphatic drainage system in the past. Recently, several studies have shown that the brain has in fact the proper lymphatic system, including sinus-associated lymphatic vessels and the glymphatic system (GS).^{12, 13, 14, 15} Sinus-associated lymphatic vessels express all of the molecular hallmarks of lymphatic endothelial cells, contain cerebrospinal fluid (CSF) and immune cells, and drain into the deep cervical lymph nodes.^{12, 13}There is a histologically defined space in the brain, the Virchow–Robin space, where the subarachnoid space meets the paravascular space (or perivascular space in somewhere, PVS).¹⁶ The GS is a specialized brain-wide anatomic structure locating at the PVS surrounding the brain vasculature, which is ensheathed with the astroglial endfeet and astroglial water channel aquaporin-4 (AQP4).^{14, 15} The GS facilitates the efficient lymphatic clearance of extracellular solutes and fluid in the brain through astroglial-mediated interstitial fluid bulk flow.¹⁴Impairment of GS involves neurological conditions including traumatic brain injuries,¹⁷ ischemic stroke¹⁸ and aged brain.¹⁹ Interestingly, brain imaging study with magnetic resonance imaging reported weakened GS perfusion following acute stroke or SAH.^{18, 20} However, little is known about whether the GS is involved in the secondary complications of SAH. Here, we examined the potential involvement of GS in SAH-associated pathology progression with in vivo two-photon microscopy and CLARITY technique.^{21, 22} Our data showed that subarachnoid blood flowed into the brain parenchyma rapidly through the PVS, causing CVS, vasculitis, widespread microinfraction and neuroinflammation in the animal model of SAH and SAH patients. Prevention of CVS with Fasudil²³ did not improve the neurological impairment nor alleviated the pathology, while the PVS clearance with tissue-type plasminogen activator (tPA) infusion improved the behavioral recovery and reduced neuroinflammation in the brain. Interestingly, AQP4^−/− mice showed no improvements in neurological deficits and neuroinflammation at day 7 after SAH compared with WT control mice. Our study therefore suggested that the paravacular pathway dynamically mediates the pathological complications following acute SAH independently of glymphatic control.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

Prevalence of Viremia and Oral Shedding of Rhesus Rhadinovirus and Retroperitoneal Fibromatosis Herpesvirus in Large Age-Structured Breeding Groups of Rhesus Macaques (Macaca mulatta)

We performed a cross-sectional study to estimate the prevalence of 2 gamma-2-herpesviruses, rhesus rhadinovirus (RRV) and retroperitoneal fibromatosis herpesvirus (RFHV), in breeding colonies of rhesus macaques. Of 90 animals selected for sampling, 73 (81%) were positive for RRV, which was detected only in blood in 22 (24%), only in saliva in 15 (16%), and in both blood and saliva in 36 (40%). Detection of RRV DNA in blood and saliva was significantly higher in animals younger than 2 y. In comparison, RFHV was detected in 40 (44%) of the 90 animals: only in blood in 5 (6%), only in saliva in 26 (29%), and in both blood and saliva in 9 (10%). Dual infection was detected in 38 (42%) animals; RFHV was only detected in coinfections. The mean RRV genome copy number in blood was significantly higher than that for RFHV. Age was a significant predictor of RRV copy number in blood and RFHV copy number in saliva. Of the 90 animals, 88 (98%) were positive for rhadinoviral antibodies on an immunofluorescent assay. Both RRV and RFHV are highly endemic in socially housed breeding colonies of rhesus macaques, and their patterns of infection are similar to that for the betaherpesvirus rhesus cytomegalovirus.Abbreviations: CNPRC, California National Primate Research Center; GE, genome equivalents (copy number); KSHV, Kaposi sarcoma-associated herpesvirus; RFHV, retroperitoneal fibromatosis herpesvirus; RRV, rhesus rhadinovirus; OSM, oncostatin M geneThe Rhadinovirus genus of gamma-2-herpesviruses is divided into 2 subgroups, RV1 and RV2, based on genomic sequence comparisons.^36,44 Rhadinovirus infections are generally subclinical in immunocompetent natural hosts, and overt disease is thought to arise only when hosts are immunocompromised.²⁸ In addition, the ability to establish both lytic and latent infections, a hallmark of the Herpesviridae family, occurs during rhadinovirus infections.^1,43 The RV1 subgroup includes Kaposi sarcoma-associated herpesvirus (KSHV; also referred to as human herpesvirus 8)^12,32 the causative agent of Kaposi sarcoma, an angioproliferative lesion composed of a mixed population of endothelial, inflammatory and spindle cells.^19,24 Furthermore, KSHV has been linked etiologically to 2 different B-cell lymphomas: primary effusion lymphoma and multicentric Castleman disease.¹⁷ Retroperitoneal fibromatosis herpesvirus (RFHV) is also a member of the RV1 subgroup and is thought to be the macaque homolog of KSHV.^{4,8,14,36,37,40} DNA sequences specific for RFHV have been detected in retroperitoneal fibromatosis in macaques coinfected with the potentially immunosuppressive simian betaretrovirus type 2.⁷ Histologic similarities between retroperitoneal fibromatosis and KS lesions seen in humans coinfected with KSHV and HIV have been previously described.^7,9,21,37 During outbreaks of simian betaretrovirus type 2 disease at 2 national primate research centers in the 1980s, the incidence of retroperitoneal fibromatosis was reported to be 5% to 7% for animals younger than 2 y and 1% across all age groups.^7,37,45 Since the end of these outbreaks in the late 1980s, retroperitoneal fibromatosis has occurred only rarely in primate colonies. The majority of published RFHV studies have focused on animals with recognized retroperitoneal fibromatosis lesions.^9-11 However, RFHV has proven extremely difficult to isolate and, to date, has not been propagated successfully in vitro, and only a small portion of the RFHV genome has been sequenced.^36,37,40,44 In this study we determined the prevalence of RFHV infection in nondiseased animals and address aspects of the natural history of this virus infection in captive macaque populations.Rhesus rhadinovirus (RRV) is a member of the RV2 subgroup, which naturally infects rhesus macaques.^15,38,44 RRV was isolated independently at 2 national primate research centers in the late 1990s from rhesus macaques.^15,42 Both RRV isolates were shown to have noteworthy sequence similarity to KSHV and RFHV.^2,8,15,42 Unlike RFHV, RRV can be propagated readily in vitro, thus facilitating studies of the lytic replication cycle.^5,6,16 Experimental coinfection of rhesus macaques with SIV and RRV resulted in a lymphoproliferative disease resembling multicentric Castleman disease, but variations in disease outcome between the 2 RRV isolates were noted.^30,49 More recently, RRV has been shown to be associated with nonHodgkin lymphoma and retroperitoneal fibromatosis in SIV-infected rhesus macaques.³⁴ Therefore, RRV infection in macaques is a highly useful animal model for the study of KSHV infection in humans, including studies of viral pathogenesis, factors affecting prevalence of infection, viral shedding, and transmission.^2,25,31,42 In addition, RRV is a persistent virus targeted for elimination in some specific pathogen free (SPF) macaque breeding populations. A better understanding of the natural history of RRV and RFHV infections will lead to improved characterization of host–virus interactions, contribute to the refinement of these nonhuman primate models, and allow more efficient management of SPF colonies.Here we report estimates of the prevalence of viremia and oral shedding of RRV and RFHV in large age-structured breeding colonies of rhesus macaques. Both viruses were highly endemic in the breeding populations we tested, and coinfection with both viruses was common.     

Cell cycle-dependent Cdc25C phosphatase determines cell survival by regulating apoptosis signal-regulating kinase 1

Cdc25C (cell division cycle 25C) phosphatase triggers entry into mitosis in the cell cycle by dephosphorylating cyclin B-Cdk1. Cdc25C exhibits basal phosphatase activity during interphase and then becomes activated at the G₂/M transition after hyperphosphorylation on multiple sites and dissociation from 14-3-3. Although the role of Cdc25C in mitosis has been extensively studied, its function in interphase remains elusive. Here, we show that during interphase Cdc25C suppresses apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen-activated protein (MAP) kinase kinase kinase family that mediates apoptosis. Cdc25C phosphatase dephosphorylates phospho-Thr-838 in the activation loop of ASK1 in vitro and in interphase cells. In addition, knockdown of Cdc25C increases the activity of ASK1 and ASK1 downstream targets in interphase cells, and overexpression of Cdc25C inhibits ASK1-mediated apoptosis, suggesting that Cdc25C binds to and negatively regulates ASK1. Furthermore, we showed that ASK1 kinase activity correlated with Cdc25C activation during mitotic arrest and enhanced ASK1 activity in the presence of activated Cdc25C resulted from the weak association between ASK1 and Cdc25C. In cells synchronized in mitosis following nocodazole treatment, phosphorylation of Thr-838 in the activation loop of ASK1 increased. Compared with hypophosphorylated Cdc25C, which exhibited basal phosphatase activity in interphase, hyperphosphorylated Cdc25C exhibited enhanced phosphatase activity during mitotic arrest, but had significantly reduced affinity to ASK1, suggesting that enhanced ASK1 activity in mitosis was due to reduced binding of hyperphosphorylated Cdc25C to ASK1. These findings suggest that Cdc25C negatively regulates proapoptotic ASK1 in a cell cycle-dependent manner and may play a role in G₂/M checkpoint-mediated apoptosis.Cell division cycle 25 (Cdc25) phosphatases are dual-specificity phosphatases involved in cell cycle regulation. By removing inhibitory phosphate groups from phospho-Thr and phospho-Tyr residues of cyclin-dependent kinases (CDKs),¹ Cdc25 proteins regulate cell cycle progression in S phase and mitosis. In mammals, three isoforms of Cdc25 phosphatases have been reported: Cdc25A, which controls the G₁/S transition;^{2, 3} Cdc25B, which is a mitotic starter;⁴ and Cdc25C, which controls the G₂/M phase.⁵ Overexpression of Cdc25 phosphatases is frequently associated with various cancers.⁶ Upon exposure to DNA-damaging reagents like UV radiation or free oxygen radicals, Cdc25 phosphatases are key targets of the checkpoint machinery, resulting in cell cycle arrest and apoptosis. The 14-3-3 proteins bind to phosphorylated Ser-216 of Cdc25C and induce Cdc25C export from the nucleus during interphase in response to DNA damage,^{7, 8} but they have no apparent effect on Cdc25C phosphatase activity.^{9, 10} In addition, hyperphosphorylation of Cdc25C correlates to its enhanced phosphatase activity.¹¹ Most studies with Cdc25C have focused on its role in mitotic progression. However, the role of Cdc25C is not clear when it is sequestered in the cytoplasm by binding to 14-3-3.Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAPKKK5), is a ubiquitously expressed enzyme with a molecular weight of 170 kDa. The kinase activity of ASK1 is stimulated by various cellular stresses, such as H₂O₂,^{12, 13} tumor necrosis factor-α (TNF-α),¹⁴ Fas ligand,¹⁵ serum withdrawal,¹³ and ER stress.¹⁶ Stimulated ASK1 phosphorylates and activates downstream MAP kinase kinases (MKKs) involved in c-Jun N-terminal kinase (JNK) and p38 pathways.^{17, 18, 19} Phosphorylation and activation of ASK1 can induce apoptosis, differentiation, or other cellular responses, depending on the cell type. ASK1 is regulated either positively or negatively depending on its binding proteins.^{12, 13, 15, 18, 19, 20, 21, 22, 23, 24, 25}ASK1 is regulated by phosphorylation at several Ser/Thr/Tyr residues. Phosphorylation at Thr-838 leads to activation of ASK1, whereas phosphorylation at Ser-83, Ser-967, or Ser-1034 inactivates ASK1.^{24, 26, 27, 28} ASK1 is basally phosphorylated at Ser-967 by an unidentified kinase, and 14-3-3 binds to this site to inhibit ASK1.²⁴ Phosphorylation at Ser-83 is known to be catalyzed by Akt or PIM1.^{27, 29} Oligomerization-dependent autophosphorylation at Thr-838, which is located in the activation loop of the kinase domain, is essential for ASK1 activation.^{14, 18, 30} Phosphorylation at Tyr-718 by JAK2 induces ASK1 degradation.³¹ Several phosphatases that dephosphorylate some of these sites have been identified. Serine/threonine protein phosphatase type 5 (PP5) and PP2C dephosphorylate phosphorylated (p)-Thr-838,^{28, 32} whereas PP2A and SHP2 dephosphorylate p-Ser-967 and p-Tyr-718, respectively.^{31, 33} Little is known about the kinase or phosphatase that regulates phosphorylation at Ser-1034. Although ASK1 phosphorylation is known to be involved in the regulation of apoptosis, only a few reports show that ASK1 phosphorylation or activity is dependent on the cell cycle.^{21, 34}In this study, we examined the functional relationship between Cdc25C and ASK1 and identified a novel function of Cdc25C phosphatase that can dephosphorylate and inhibit ASK1 in interphase but not in mitosis. Furthermore, we demonstrated that Cdc25C phosphorylation status plays a critical role in the interaction with and the activity of ASK1. These results reveal a novel regulatory function of Cdc25C in the ASK1-mediated apoptosis signaling pathway.     

Role of Retinoic Acid and Fibroblast Growth Factor 2 in Neural Differentiation from Cynomolgus Monkey (Macaca fascicularis) Embryonic Stem Cells

Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state. In this study, we established an FGF2 low-dose-dependent embryonic stem cell line from cynomolgus monkeys and then analyzed neural differentiation in cultures supplemented with retinoic acid and FGF2. When only retinoic acid was added to culture, neurons differentiated from FGF2 low-dose-dependent embryonic stem cells. When both retinoic acid and FGF2 were added, neurons and astrocytes differentiated from the same embryonic stem cell line. Thus, retinoic acid promotes the differentiation from embryonic stem cells to neuroectoderm. Although FGF2 seems to promote self-renewal in stem cells, its effects on the differentiation of stem cells are influenced by the presence or absence of supplemental retinoic acid.Abbreviations: EB, embryoid body; ES, embryonic stem; ESM, embryonic stem cell medium; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor; MBP, myelin basic protein; RA, retinoic acid; SSEA, stage-specific embryonic antigen; TRA, tumor-related antigenPluripotent stem cells are potential sources of material for cell replacement therapy and are useful experimental tools for in vitro models of human disease and drug screening. Embryonic stem (ES) cells are capable of extensive proliferation and multilineage differentiation, and thus ES-derived cells are suitable for use in cell-replacement therapies.^18,23 Reported ES cell characteristics including tumorigenic potential, DNA methylation status, expression of imprinted genes, and chromatin structure were elucidated by using induced pluripotent stem cells.^2,11,17 Because the social expectations of regeneration medicine are growing, we must perform basic research with ES cells, which differ from induced pluripotent stem cells in terms of origin, differentiation ability, and epigenetic status.^2,8Several advances in research have been made by using mouse ES cells. Furthermore, primate ES cell lines have been established from rhesus monkeys (Macaca mulatta),²⁴ common marmosets (Callithrix jacchus),²⁵ cynomolgus monkeys (M. fascicularis),²⁰ and African green monkeys (Chlorocebus aethiops).¹⁹ Mouse and other mammalian ES cells differ markedly in their responses to the signaling pathways that support self-renewal.^8,28 Mouse ES cells require leukemia inhibitory factor (LIF)–STAT3 signaling.¹⁴ In contrast, primate ES cells do not respond to LIF. Fibroblast growth factor 2 (FGF2) appears to be the most upstream self-renewal factor in primate ES cells. FGF2 also exerts its effects through indirect mechanisms, such as the TGFβ–Activin–Nodal signaling pathway, in primate ES cells.²¹ In addition to the biologic similarities between monkeys and humans, ES cells derived from cynomolgus monkeys or human blastocysts have extensive similarities that are not apparent in mouse ES cells.^8,14,21,28 Numerous monkey ES cell lines are now available, and cynomolgus monkeys are an efficient model for developing strategies to investigate the efficacy of ES-cell–based medical treatments in humans.Several growth factors and chemical compounds, including retinoic acid (RA),^4,9,13,22,26 FGF2,^9,10,16,22 epidermal growth factor,^9,22 SB431542,^1,4,10 dorsomorphin,^10,27 sonic hedgehog,^{12,13,16,27,29} and noggin,^1,4,9,27 are essential for the differentiation and proliferation or maintenance of neural stem cells derived from primate ES cells. Of these factors, active RA signaling suppresses a mesodermal fate by inhibiting Wnt and Nodal signaling pathways during in vitro culture and leads to neuroectoderm differentiation in ES cells.^4,13,26 RA is an indispensable factor for the specialization to neural cells. FGF2 is important during nervous system development,¹² and FGF2 and RA both are believed to influence the differentiation to neural cells. The current study was done to clarify the mechanism of RA and FGF2 in the induction of differentiation along the neural lineage.We recently established a monkey ES cell line that does not need FGF2 supplementation for maintenance of the undifferentiated state. This ES cell line allowed us to study the role of differentiation to neural cells with RA and enabled us to compare ES cell differentiation in the context of supplementation with RA or FGF2 in culture. To this end, we established a novel cynomolgus monkey cell line derived from ES cells and maintained it in an undifferentiated state in the absence of FGF2 supplementation.     

Delayed and Aberrant Presentation of VX2 Carcinoma in a Rabbit Model of Hepatic Neoplasia

A socially-housed New Zealand white rabbit presented with a large subcutaneous mass on the ventral thorax approximately 11 mo after the intrahepatic delivery of a suspension of VX2 carcinoma cells to induce hepatocellular carcinoma as part of a nanoparticle study. The mass and closely associated axillary lymph node were removed en bloc. Immunohistochemical staining identified the mass as an undifferentiated carcinoma. The rabbit demonstrated no appreciable pathology at the study end point at 16 mo after VX2 inoculation. An additional rabbit from the same VX2 injection cohort was found at necropsy to have an unanticipated intraabdominal mass, also identified as an undifferentiated carcinoma. This case report summarizes the molecular analysis of both tumors through a novel PCR assay, which identified the delayed and aberrant onset of VX2 carcinoma in an extended timeframe not previously reported.Abbreviations: CRPV, cottontail rabbit papillomavirusThe VX2 squamous cell carcinoma cell line was developed in 1938 from cells isolated from a domestic rabbit with a Shope papillomavirus-induced skin papilloma.^11,16 The inoculation of allogenic adult rabbits with the VX2 tumor cells results in a wholly anaplastic carcinoma that grows rapidly and forms frequent metastases.^7,11 Molecular analysis of the VX2 carcinoma has revealed multiple integrated copies of highly methylated cottontail rabbit papilloma virus (CRPV) genomic material.^5-7,17 For decades, the rabbit VX2 carcinoma has supported oncology research by providing a large animal model that supports the investigation of neoplasms of diverse tissues including liver, lung, pleural space, muscle, and bone.^{9,12,15,18,19} Benefits of this model include relatively simple inoculation, rapid growth with local tissue invasion, predictable metastases, and reproducibility.^3,4,13,14 In the current report, we describe the use of VX2 cells intended to induce a hepatic carcinoma model in rabbits and the unexpected outcomes associated with the model.