ABSTRACT
Nischarin (Nisch) is a key protein functioning as a molecular scaffold and thereby hosting interactions with several protein partners. To explore the physiological importance of Nisch, here we generated Nisch loss-of-function mutant mice and analyzed their metabolic phenotype. Nisch-mutant embryos exhibited delayed development, characterized by small size and attenuated weight gain. We uncovered the reason for this phenotype by showing that Nisch binds to and inhibits the activity of AMP-activated protein kinase (AMPK), which regulates energy homeostasis by suppressing anabolic and activating catabolic processes. The Nisch mutations enhanced AMPK activation and inhibited mechanistic target of rapamycin signaling in mouse embryonic fibroblasts as well as in muscle and liver tissues of mutant mice. Nisch-mutant mice also exhibited increased rates of glucose oxidation with increased energy expenditure, despite reduced overall food intake. Moreover, the Nisch-mutant mice had reduced expression of liver markers of gluconeogenesis associated with increased glucose tolerance. As a result, these mice displayed decreased growth and body weight. Taken together, our results indicate that Nisch is an important AMPK inhibitor and a critical regulator of energy homeostasis, including lipid and glucose metabolism.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Energy Metabolism , Gluconeogenesis , Intracellular Signaling Peptides and Proteins/metabolism , Lipid Metabolism , Liver/metabolism , AMP-Activated Protein Kinases/genetics , Animals , Cell Line , Glucose/genetics , Glucose/metabolism , Humans , Imidazoline Receptors , Intracellular Signaling Peptides and Proteins/genetics , Liver/pathology , Mice , Mice, Mutant Strains , Mutation , Oxidation-Reduction , Protein BindingABSTRACT
BACKGROUND: Of all human cancers, gastric carcinoma is the one of the leading causes of death. Helicobacter pylori is considered a major etiologic agent of this disease. Spontaneously occurring gastric carcinoma is a rare diagnosis in nonhuman primates. A 2011 case report documented a high incidence of gastric adenocarcinoma in a closed colony of captive sooty mangabeys (Cercebus atys). However, H. pylori infection was not detected in these animals. MATERIALS AND METHODS: In this study, using archived formalin-fixed, paraffin-embedded stomach sections of these animals alternative methodologies were used to identify H. pylori and other non-H. pylori Helicobacter species. In addition, two additional cases of sooty mangabeys with metastatic gastric carcinoma are characterized. RESULTS: Using fluorescent in situ hybridization, we identified gastric H. suis in 75% of archived and new gastric carcinoma cases. In the two newly reported cases, H. suis and a novel Helicobacter species were detected via PCR and sequence analysis of the 16S rRNA gene. H. pylori was not identified in any of the gastric carcinoma cases via FISH and/or PCR and sequence analysis of Helicobacter spp. in DNA from of available tissues. CONCLUSIONS: This report is the first to characterize Helicobacter species infection in spontaneous gastric carcinoma with metastatic potential in nonhuman primates.
Subject(s)
Adenocarcinoma/veterinary , Cercocebus atys/microbiology , Helicobacter Infections/veterinary , Helicobacter/isolation & purification , Monkey Diseases/diagnosis , Stomach Neoplasms/veterinary , Adenocarcinoma/microbiology , Adenocarcinoma/pathology , Animals , Female , Helicobacter/classification , Helicobacter/genetics , Helicobacter Infections/diagnosis , Helicobacter Infections/microbiology , In Situ Hybridization, Fluorescence/veterinary , Male , Monkey Diseases/microbiology , Monkey Diseases/pathology , Phylogeny , Polymerase Chain Reaction/veterinary , Sequence Analysis, DNA/veterinary , Stomach/microbiology , Stomach/pathology , Stomach Neoplasms/microbiology , Stomach Neoplasms/pathologyABSTRACT
Severe malaria, a leading cause of mortality among children and nonimmune adults, is a multisystemic disorder characterized by complex clinical syndromes that are mechanistically poorly understood. The interplay of various parasite and host factors is critical in the pathophysiology of severe malaria. However, knowledge regarding the pathophysiological mechanisms and pathways leading to the multisystemic disorders of severe malaria in humans is limited. Here, we systematically investigate infections with Plasmodium coatneyi, a simian malaria parasite that closely mimics the biological characteristics of P. falciparum, and develop baseline data and protocols for studying erythrocyte turnover and severe malaria in greater depth. We show that rhesus macaques (Macaca mulatta) experimentally infected with P. coatneyi develop anemia, coagulopathy, and renal and metabolic dysfunction. The clinical course of acute infections required suppressive antimalaria chemotherapy, fluid support, and whole-blood transfusion, mimicking the standard of care for the management of severe malaria cases in humans. Subsequent infections in the same animals progressed with a mild illness in comparison, suggesting that immunity played a role in reducing the severity of the disease. Our results demonstrate that P. coatneyi infection in rhesus macaques can serve as a highly relevant model to investigate the physiological pathways and molecular mechanisms of malaria pathogenesis in naïve and immune individuals. Together with high-throughput postgenomic technologies, such investigations hold promise for the identification of new clinical interventions and adjunctive therapies.
Subject(s)
Macaca mulatta/parasitology , Malaria/veterinary , Monkey Diseases/blood , Plasmodium/classification , Anemia/immunology , Anemia/parasitology , Anemia/veterinary , Animals , Blood Coagulation , Bone Marrow Diseases/blood , Bone Marrow Diseases/parasitology , Bone Marrow Diseases/veterinary , Disease Models, Animal , Erythropoiesis , Erythropoietin/blood , Malaria/blood , Malaria/immunology , Malaria/parasitology , Male , Monkey Diseases/immunology , Monkey Diseases/parasitology , Random Allocation , Time FactorsABSTRACT
One common sign of human cytomegalovirus infection is altered liver function. Murine cytomegalovirus strain v70 induces a rapid and severe hepatitis in immunocompetent mice that requires the presence of T cells in order to develop. v70 exhibits approximately 10-fold-greater virulence than the commonly used strain K181, resulting in a more severe, sustained, and lethal hepatitis but not dramatically higher viral replication levels. Hepatitis and death are markedly delayed in immunodeficient SCID compared to immunocompetent BALB/c mice. Transfer of BALB/c splenocytes to SCID mice conferred rapid disease following infection, and depletion of either CD4 or CD8 T cells in BALB/c mice reduced virus-induced hepatitis. The frequency of CD8 T cells producing gamma interferon and tumor necrosis factor in response to viral antigen was higher in settings where more severe disease occurred. Thus, virus-specific effector CD8 T cells appear to contribute to lethal virus-induced hepatitis, contrasting their protective role during sublethal infection. This study reveals how protection and disease during cytomegalovirus infection depend on viral strain and dose, as well as the quality of the T cell response.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cytomegalovirus Infections/complications , Hepatitis, Viral, Animal/immunology , Hepatitis, Viral, Animal/virology , Immunity, Cellular/immunology , Muromegalovirus/pathogenicity , Adoptive Transfer , Animals , CD8-Positive T-Lymphocytes/metabolism , Flow Cytometry , Fluorescence , Hepatitis, Viral, Animal/etiology , Histological Techniques , Interferon-gamma/metabolism , Mice , Mice, Inbred BALB C , Mice, SCID , Statistics, Nonparametric , Tumor Necrosis Factor-alpha/metabolismABSTRACT
We have developed a major histocompatibility complex-defined primate model of graft-versus-host disease (GVHD) and have determined the effect that CD28/CD40-directed costimulation blockade and sirolimus have on this disease. Severe GVHD developed after haploidentical transplantation without prophylaxis, characterized by rapid clinical decline and widespread T-cell infiltration and organ damage. Mechanistic analysis showed activation and possible counter-regulation, with rapid T-cell expansion and accumulation of CD8(+) and CD4(+) granzyme B(+) effector cells and FoxP3(pos)/CD27(high)/CD25(pos)/CD127(low) CD4(+) T cells. CD8(+) cells down-regulated CD127 and BCl-2 and up-regulated Ki-67, consistent with a highly activated, proliferative profile. A cytokine storm also occurred, with GVHD-specific secretion of interleukin-1 receptor antagonist (IL-1Ra), IL-18, and CCL4. Costimulation Blockade and Sirolimus (CoBS) resulted in striking protection against GVHD. At the 30-day primary endpoint, CoBS-treated recipients showed 100% survival compared with no survival in untreated recipients. CoBS treatment resulted in survival, increasing from 11.6 to 62 days (P < .01) with blunting of T-cell expansion and activation. Some CoBS-treated animals did eventually develop GVHD, with both clinical and histopathologic evidence of smoldering disease. The reservoir of CoBS-resistant breakthrough immune activation included secretion of interferon-γ, IL-2, monocyte chemotactic protein-1, and IL-12/IL-23 and proliferation of cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin-resistant CD28(-) CD8(+) T cells, suggesting adjuvant treatments targeting this subpopulation will be needed for full disease control.
Subject(s)
CD28 Antigens , CD8-Positive T-Lymphocytes/immunology , Graft vs Host Disease/prevention & control , Immunosuppression Therapy/methods , Sirolimus/therapeutic use , Animals , Cell Proliferation , Graft vs Host Disease/etiology , Graft vs Host Disease/pathology , Haplotypes , Hematopoietic Stem Cell Transplantation/adverse effects , Lymphocyte Activation , Macaca mulatta , Sirolimus/immunologyABSTRACT
Withaferin A (WFA) is purified from the plant Withania somnifera and inhibits the vimentin cytoskeleton. Vimentin overexpression in cancer correlates with metastatic disease, induction of epithelial to mesenchymal transition and reduced patient survival. As vimentin functions in cell motility, we wanted to test the hypothesis that WFA inhibits cancer metastasis by disrupting vimentin function. These data showed that WFA had weak cytotoxic and apoptotic activity at concentrations less than or equal to 500 nM, but retained potent anti-invasive activity at these low doses. Imaging of breast cancer cell lines revealed that WFA induces perinuclear vimentin accumulation followed by rapid vimentin depolymerization. A concomitant induction of vimentin ser56 phosphorylation was observed, which is consistent with vimentin disassembly. Structure activity relationships were established using a set of chemically modified WFA analogs and showed that the predicted vimentin-binding region of WFA is necessary to induce vimentin ser56 phosphorylation and for its anti-invasive activity. Pharmacokinetic studies in mice revealed that WFA reaches peak concentrations up to 2 µM in plasma with a half-life of 1.36 hr following a single 4 mg/kg dose. In a breast cancer metastasis mouse model, WFA showed dose-dependent inhibition of metastatic lung nodules and induced vimentin ser56 phosphorylation, with minimal toxicity to lung tissue. Based upon these studies, we conclude that WFA is a potent breast cancer anti-metastatic agent and the anti-metastatic activity of WFA is, at least in part, mediated through its effects on vimentin and vimentin ser56 phosphorylation.
Subject(s)
Breast Neoplasms/drug therapy , Cell Movement/drug effects , Liver Neoplasms/drug therapy , Lung Neoplasms/drug therapy , Serine/chemistry , Vimentin/metabolism , Withanolides/therapeutic use , Animals , Antineoplastic Agents/therapeutic use , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Differentiation/drug effects , Cell Line, Tumor , Female , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/secondary , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Mice , Mice, Inbred BALB C , Neoplasm Invasiveness , Phosphorylation/drug effects , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A Chinese rhesus macaque infected with the pathogenic CCR5-tropic clade C simian-human immunodeficiency virus, SHIV-1157ipd3N4, had persistent viremia, depletion of CD4(+) T cells to <200 cells/µl, opportunistic infections, coagulopathy, and gradual development of bilateral blindness. MRI revealed marked thickening of both optic nerves. Histopathological evaluation showed diffuse cellular infiltration at necropsy and a focus of SHIV-infected cells. This is the first report of CNS pathology following chronic infection with an obligate R5 SHIV.
Subject(s)
HIV-1/genetics , Macaca mulatta , Monkey Diseases/virology , Optic Neuritis/veterinary , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/genetics , AIDS-Related Opportunistic Infections/veterinary , AIDS-Related Opportunistic Infections/virology , Animals , Female , Optic Neuritis/virology , ViremiaABSTRACT
Chronic malaria is a major public health problem and significant challenge for disease eradication efforts. Despite its importance, the biological factors underpinning chronic malaria are not fully understood. Recent studies have shown that host metabolic state can influence malaria pathogenesis and transmission, but its role in chronicity is not known. Here, with the goal of identifying distinct modifications in the metabolite profiles of acute versus chronic malaria, metabolomics was performed on plasma from Plasmodium-infected humans and nonhuman primates with a range of parasitemias and clinical signs. In rhesus macaques infected with Plasmodium coatneyi, significant alterations in amines, carnitines, and lipids were detected during a high parasitemic acute phase and many of these reverted to baseline levels once a low parasitemic chronic phase was established. Plasmodium gene expression, studied in parallel in the macaques, revealed transcriptional changes in amine, fatty acid, lipid and energy metabolism genes, as well as variant antigen genes. Furthermore, a common set of amines, carnitines, and lipids distinguished acute from chronic malaria in plasma from human Plasmodium falciparum cases. In summary, distinct host-parasite metabolic environments have been uncovered that characterize acute versus chronic malaria, providing insights into the underlying host-parasite biology of malaria disease progression.
Subject(s)
Amino Acids/blood , Amino Acids/metabolism , Lipid Metabolism , Lipids/blood , Malaria/metabolism , Adolescent , Adult , Aged , Animals , Disease Models, Animal , Fatty Acids/blood , Fatty Acids/metabolism , Female , Gene Expression , Glycerophospholipids/blood , Glycerophospholipids/metabolism , Host-Parasite Interactions/physiology , Humans , Macaca mulatta , Malaria/genetics , Male , Metabolome , Middle Aged , Parasitemia , Plasmodium , Plasmodium falciparum , Young AdultABSTRACT
Controlling graft-versus-host disease (GVHD) remains a major unmet need in stem cell transplantation, and new, targeted therapies are being actively developed. CD28-CD80/86 costimulation blockade represents a promising strategy, but targeting CD80/CD86 with CTLA4-Ig may be associated with undesired blockade of coinhibitory pathways. In contrast, targeted blockade of CD28 exclusively inhibits T cell costimulation and may more potently prevent GVHD. Here, we investigated FR104, an antagonistic CD28-specific pegylated-Fab', in the nonhuman primate (NHP) GVHD model and completed a multiparameter interrogation comparing it with CTLA4-Ig, with and without sirolimus, including clinical, histopathologic, flow cytometric, and transcriptomic analyses. We document that FR104 monoprophylaxis and combined prophylaxis with FR104/sirolimus led to enhanced control of effector T cell proliferation and activation compared with the use of CTLA4-Ig or CTLA4-Ig/sirolimus. Importantly, FR104/sirolimus did not lead to a beneficial impact on Treg reconstitution or homeostasis, consistent with control of conventional T cell activation and IL-2 production needed to support Tregs. While FR104/sirolimus had a salutary effect on GVHD-free survival, overall survival was not improved, due to death in the absence of GVHD in several FR104/sirolimus recipients in the setting of sepsis and a paralyzed INF-γ response. These results therefore suggest that effectively deploying CD28 in the clinic will require close scrutiny of both the benefits and risks of extensively abrogating conventional T cell activation after transplant.
Subject(s)
CD28 Antigens/antagonists & inhibitors , Graft vs Host Disease/prevention & control , T-Lymphocytes/immunology , Abatacept/administration & dosage , Animals , Antibodies, Monoclonal/administration & dosage , Disease Models, Animal , Graft vs Host Disease/immunology , Hematopoietic Stem Cell Transplantation/adverse effects , Lymphocyte Activation , Macaca mulatta , Sirolimus/administration & dosage , Systems BiologyABSTRACT
We report the first case of disseminated intravascular coagulation (DIC) complicated by peripheral gangrene induced by Plasmodium coatneyi in rhesus monkeys. Ten days after experimental challenge, numerous petechiae were noted over the trunk and extremities, with polychromasia, severe anemia, thrombocytopenia, and moderate parasitemia. These changes were accompanied by elevated serum activity of blood urea nitrogen, creatinine, transaminases, and creatinine phosphokinase. The animal received intravenous fluid support, artemether, and blood transfusion. Three days after treatment, the platelet counts returned to normal, and parasitemia was abated. However, several areas of skin discoloration with gangrenous tissue in the hands and the tail were observed. Coagulation profile showed elevated D-dimers and elevated levels of fibrinogen/fibrin degradation products with low levels of protein S functional activity. DIC with peripheral gangrene is very rare in Plasmodium-infected individuals. Our results indicate that the experimental model of P. coatneyi infection of rhesus monkeys is important for studies of malarial anemia and coagulopathy.
Subject(s)
Disseminated Intravascular Coagulation/complications , Gangrene/complications , Malaria/complications , Plasmodium/pathogenicity , Animals , Disease Models, Animal , Disseminated Intravascular Coagulation/pathology , Gangrene/pathology , Macaca mulatta , Malaria/parasitology , Malaria/pathology , MaleABSTRACT
Histopathological data collected from patients with severe malaria have been instrumental for studying malaria pathogenesis. Animal models of malaria are critical to complement such studies. Here, the histopathological changes observed in a rhesus macaque with severe and complicated Plasmodium cynomolgi malaria are reported. The animal presented with thrombocytopenia, severe anemia, and hyperparasitemia during the acute infection. The macaque was given subcurative antimalarial treatment, fluid support, and a blood transfusion to treat the clinical complications, but at the time of transfusion, kidney function was compromised. These interventions did not restore kidney function, and the animal was euthanized due to irreversible renal failure. Gross pathological and histological examinations revealed that the lungs, kidneys, liver, spleen, and bone marrow exhibited abnormalities similar to those described in patients with malaria. Overall, this case report illustrates the similarities in the pathophysiological complications that can occur in human malaria and cynomolgi malaria in rhesus macaques.
Subject(s)
Macaca mulatta/parasitology , Malaria/complications , Malaria/parasitology , Plasmodium cynomolgi/isolation & purification , Plasmodium cynomolgi/parasitology , Plasmodium cynomolgi/pathogenicity , Animals , Bone Marrow/anatomy & histology , Disease Models, Animal , Humans , Kidney/cytology , Liver/cytology , Lung/cytology , Malaria/pathology , Spleen/cytologyABSTRACT
Generating tier 2 HIV-neutralizing antibody (nAb) responses by immunization remains a challenging problem, and the immunological barriers to induction of such responses with Env immunogens remain unclear. Here, some rhesus monkeys developed autologous tier 2 nAbs upon HIV Env trimer immunization (SOSIP.v5.2) whereas others did not. This was not because HIV Env trimers were immunologically silent because all monkeys made similar ELISA-binding antibody responses; the key difference was nAb versus non-nAb responses. We explored the immunological barriers to HIV nAb responses by combining a suite of techniques, including longitudinal lymph node fine needle aspirates. Unexpectedly, nAb development best correlated with booster immunization GC B cell magnitude and Tfh characteristics of the Env-specific CD4 T cells. Notably, these factors distinguished between successful and unsuccessful antibody responses because GC B cell frequencies and stoichiometry to GC Tfh cells correlated with nAb development, but did not correlate with total Env Ab binding titers.
Subject(s)
Antibodies, Neutralizing/immunology , Antibody Formation/immunology , Germinal Center/immunology , HIV-1/immunology , Protein Multimerization , env Gene Products, Human Immunodeficiency Virus/immunology , Animals , B-Lymphocytes/immunology , Biopsy, Fine-Needle , Cell Lineage , Clone Cells , Immunization , Macaca mulatta , Protein Binding , T-Lymphocytes, Helper-Inducer/immunologyABSTRACT
Ten veterinary pathologists at 1 veterinary institution independently assigned histologic grades to the same 60 canine cutaneous mast cell tumors (MCTs). There was significant variation among pathologists in grading the MCTs (P < 0.001). The probability of assigning a low grade was significantly higher for the pathologists in this study who use a published reference for histologic grading of canine cutaneous MCTs that allows subcutaneous MCTs or MCTs with mitotic figures to be included in the low-grade category (P < 0.0001 and P < 0.0001, respectively).
Subject(s)
Dog Diseases/pathology , Mast-Cell Sarcoma/veterinary , Skin Neoplasms/veterinary , Animals , Dogs , Mast-Cell Sarcoma/pathology , Observer Variation , Pathology, Veterinary/standards , Skin Neoplasms/pathologyABSTRACT
Idiopathic pulmonary fibrosis (IPF), one of the most severe interstitial lung diseases, is a progressive fibrotic disorder of unknown etiology. However, there is growing appreciation for the role of viral infection in disease induction and/or progression. A small animal model of multi-organ fibrosis, which involves murine gammaherpesvirus (MHV68) infection of interferon gamma receptor deficient (IFNγR-/-) mice, has been utilized to model the association of gammaherpesvirus infections and lung fibrosis. Notably, several MHV68 mutants which fail to induce fibrosis have been identified. Our current study aimed to better define the role of the unique MHV68 gene, M1, in development of pulmonary fibrosis. We have previously shown that the M1 gene encodes a secreted protein which possesses superantigen-like function to drive the expansion and activation of Vß4+ CD8+ T cells. Here we show that M1-dependent fibrosis is correlated with heightened levels of inflammation in the lung. We observe an M1-dependent cellular infiltrate of innate immune cells with most striking differences at 28 days-post infection. Furthermore, in the absence of M1 protein expression we observed reduced CD8+ T cells and MHV68 epitope specific CD8+ T cells to the lungs-despite equivalent levels of viral replication between M1 null and wild type MHV68. Notably, backcrossing the IFNγR-/- onto the Balb/c background, which has previously been shown to exhibit weak MHV68-driven Vß4+ CD8+ T cell expansion, eliminated MHV68-induced fibrosis-further implicating the activated Vß4+ CD8+ T cell population in the induction of fibrosis. We further addressed the role that CD8+ T cells play in the induction of fibrosis by depleting CD8+ T cells, which protected the mice from fibrotic disease. Taken together these findings are consistent with the hypothesized role of Vß4+ CD8+ T cells as mediators of fibrotic disease in IFNγR-/- mice.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Herpesviridae Infections/immunology , Idiopathic Pulmonary Fibrosis/immunology , Receptors, Interferon/metabolism , Animals , Female , Herpesviridae Infections/complications , Idiopathic Pulmonary Fibrosis/etiology , Immunity, Innate , Inflammation/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Interferon/deficiency , Receptors, Interferon/genetics , Interferon gamma ReceptorABSTRACT
The competence of cellular immunity depends on a diverse T-cell receptor (TCR) repertoire arising from thymic output. Normal thymopoiesis arises from marrow-derived CD3(-)CD4(-)CD8(-) triple-negative T-cell progenitors (TN), which develop into mature single-positive (SP) CD4 or CD8 T cells after expressing both CD4 and CD8 (double-positive, DP) transiently, leading to de novo T-cell production. Interleukin-7 (IL7) is a singularly important common γ-chain IL involved in normal thymic development. Our previous work has demonstrated that γc cytokines fused with granulocyte-macrophage colony stimulating factor (GMCSF) at the N-terminus acquire unheralded biological properties. Therefore, to enhance thymopoiesis, we developed a novel biopharmaceutical based on the fusion of GMCSF and IL7, hereafter GIFT7. Systemic administration of GIFT7 leads to cortical thymic hyperplasia including the specific expansion of CD44(int)CD25(-) double-negative 1 (DN1) thymic progenitors. During murine cytomegalovirus (mCMV) infection of aged animals, GIFT7-mediated neo-thymopoiesis led to increased absolute numbers of viral-specific CD8(+) T cell. Our work demonstrated that thymic precursors can be therapeutically repopulated and its reconstitution leads to meaningful central and peripheral T-cell neogenesis, correcting immune dysfunction arising from age-associated thymic atrophy.
ABSTRACT
OBJECTIVE: To assess effects of zoledronic acid on biomarkers, radiographic scores, and gross articular cartilage changes in dogs with induced osteoarthritis. ANIMALS: 21 purpose-bred hound-type dogs. PROCEDURES: The left stifle joint of each dog was examined arthroscopically to determine initial articular cartilage status, which was followed by cranial cruciate ligament (CrCL) transection to induce osteoarthritis. Dogs were assigned to 3 groups (control group, low dose [10 µg of zoledronic acid/kg], or high dose [25 µg of zoledronic acid/kg). Treatments were administered SC every 3 months for 1 year beginning the day after CrCL transection. Serum and synovial fluid samples and radiographs were obtained 0, 1, 3, 6, 9, and 12 months after transection. At 12 months, each joint was scored for cartilage defects. Serum and synovial fluid biomarkers of bone and cartilage turnover (bone-specific alkaline phosphatase, type I and II collagen, carboxy-propeptide of type II collagen, and chondroitin sulfate 846) were analyzed with ELISAs. RESULTS: The high-dose group had fewer total articular defects and lower severity scores in CrCL-transected stifle joints than did the control group. In addition, the high-dose group had significantly less change in collagenase cleavage of type I or II collagen in the synovial fluid at 1 and 3 months after CrCL transection than did the control group and also had greater changes in bone-specific alkaline phosphatase in synovial fluid at 3 months after CrCL transection than did the control group. CONCLUSIONS AND CLINICAL RELEVANCE: Zoledronic acid had a chondroprotective effect in dogs with a transected CrCL.
Subject(s)
Bone Density Conservation Agents/therapeutic use , Cartilage, Articular/drug effects , Diphosphonates/therapeutic use , Dog Diseases/prevention & control , Imidazoles/therapeutic use , Osteoarthritis/veterinary , Alkaline Phosphatase/analysis , Animals , Bone Density Conservation Agents/pharmacology , Cartilage, Articular/pathology , Collagen/blood , Diphosphonates/pharmacology , Dog Diseases/drug therapy , Dog Diseases/pathology , Dogs , Enzyme-Linked Immunosorbent Assay , Imidazoles/pharmacology , Male , Osteoarthritis/drug therapy , Osteoarthritis/pathology , Osteoarthritis/prevention & control , Radiography , Random Allocation , Stifle/diagnostic imaging , Stifle/drug effects , Stifle/pathology , Synovial Fluid/enzymology , Zoledronic AcidABSTRACT
Though clinicians can predict which patients are at risk for developing metastases, traditional therapies often prove ineffective and metastatic disease is the primary cause of cancer patient death; therefore, there is a need to develop anti-metastatic therapies that can be administered over long durations to specifically inhibit the motility of cancer cells. Withaniasomnifera root extracts (WRE) have anti-proliferative activity and the active component, Withaferin A, inhibits the pro-metastatic protein, vimentin. Vimentin is an intermediate filament protein and is part of the epithelial to mesenchymal transition (EMT) program to promote metastasis. Here, we determined whether WRE standardized to Withaferin A (sWRE) possesses anti-metastatic activity and whether it inhibits cancer motility via inhibition of vimentin and the EMT program. Several formulations of sWRE were created to enrich for Withaferin A and a stock solution of sWRE in EtOH could recover over 90% of the Withaferin A found in the original extract powder. This sWRE formulation inhibited breast cancer cell motility and invasion at concentrations less than 1µM while having negligible cytotoxicity at this dose. sWRE treatment disrupted vimentin morphology in cell lines, confirming its vimentin inhibitory activity. To determine if sWRE inhibited EMT, TGF-ß was used to induce EMT in MCF10A human mammary epithelial cells. In this case, sWRE prevented EMT induction and inhibited 3-D spheroid invasion. These studies were taken into a human xenograft and mouse mammary carcinoma model. In both models, sWRE and Withaferin A showed dose-dependent inhibition of tumor growth and metastatic lung nodule formation with minimal systemic toxicity. Taken together, these data support the hypothesis that low concentrations of sWRE inhibit cancer metastasis potentially through EMT inhibition. Moreover, these doses of sWRE have nearly no toxicity in normal mouse organs, suggesting the potential for clinical use of orally administered WRE capsules.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/pathology , Epithelial-Mesenchymal Transition/drug effects , Plant Extracts/pharmacology , Plant Roots/chemistry , Withania/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cell Movement/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Humans , Inhibitory Concentration 50 , Mice , Neoplasm Metastasis , Plant Extracts/chemistry , Plant Extracts/toxicity , Solubility , Transforming Growth Factor beta/pharmacology , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
A 3-y-old male rhesus macaque (Macaca mulatta) was noticed to be lethargic in the compound. Physical exam revealed cyanotic mucous membranes, dyspnea, bilateral harsh lung sounds, wheezing on expiration, and a firm mass possibly associated with the liver. Radiographs revealed bilateral soft tissue opacities in the thorax. Due to poor prognosis, the rhesus was euthanized, and a necropsy was performed. Both right and left lung lobes were consolidated and had multifocal white-tan masses. On cut section, the masses were firm, had areas of necrosis, hemorrhage, and often contained a tenacious exudate. Masses were identified in the liver and both kidneys. Given the morphologic features of the neoplasm, a diagnosis of squamous cell carcinoma was made. Immunohistochemistry staining for thyroid transcription factor, a nuclear transcription factor normally found in lung, thyroid, and tumors arising from either of those tissues, confirmed that the masses originated from the lung. Malignant primary lung tumors are divided into 8 main histologic subtypes: squamous cell carcinoma, small-cell carcinoma, large-cell carcinoma, adenocarcinoma, adenosquamous carcinoma, sarcomatoid carcinoma, carcinoid tumor, and salivary gland tumors. Clinical signs associated with lung tumors include, but are not limited to, dyspnea, coughing, hemoptysis, lethargy, anorexia, and weight loss. Although squamous cell carcinoma will be low on the differential list for these clinical signs, we encourage clinicians and researchers to not rule it out solely based on incidence and age of the animal.
Subject(s)
Carcinoma, Squamous Cell/veterinary , Lung Neoplasms/veterinary , Monkey Diseases/diagnosis , Animals , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/pathology , Kidney Neoplasms/diagnosis , Kidney Neoplasms/secondary , Kidney Neoplasms/veterinary , Liver Neoplasms/diagnosis , Liver Neoplasms/secondary , Liver Neoplasms/veterinary , Lung Neoplasms/diagnosis , Lung Neoplasms/pathology , Macaca mulatta , Male , Monkey Diseases/metabolism , Monkey Diseases/pathology , Nuclear Proteins/metabolism , Radiography, Thoracic , Thyroid Nuclear Factor 1 , Transcription Factors/metabolismABSTRACT
An 8.5-mo-old female rhesus macaque was examined for an apparent lump on the right arm, below the elbow. The macaque showed no signs of pain or discomfort. Examination revealed that the lump was actually a bend in the forearm. Radiography demonstrated that some of the long bones of the animal were bowed. Differential diagnoses included rickets, hyperparathyroidism, pseudohyperparathyroidism, and a growth dysplasia. No other similar abnormalities in animals from that cage or any other enclosure in our large colony were observed. Blood chemistries and a complete hemogram were within normal limits for a macaque of this age. Serum was submitted for a vitamin D profile that included assays for parathyroid hormone, 25-hydroxyvitamin D, and ionized calcium. Serum samples from sex- and age-matched normal controls were sent for comparison and to establish a baseline profile. The affected animal had vitamin D levels comparable to unaffected controls. Bone biopsies appeared normal for a macaque of this age. Fluorine levels in the drinking water supply were within acceptable limits. Consistent with the information available, a diagnosis of idiopathic camptomelia, or bowing of the long bones, was made. In humans, developmental camptomelia is associated with several bone dysplasias in infants and children. These conditions are thought to be caused by genetic mutations in enzymes or transcription factors that control development of the epiphyses and are almost always associated with other lethal and nonlethal developmental abnormalities.