RESUMO
BACKGROUND: The large amounts of data generated by genomics, transcriptomics and proteomics have increased our understanding of the biology of Anaplasma marginale. However, these data have also led to new assumptions that require testing, ideally through classical genetic mutation. One example is the definition of genes associated with virulence. Here we describe the molecular characterization of a red fluorescent and spectinomycin and streptomycin resistant A. marginale mutant generated by Himar1 transposon mutagenesis. RESULTS: High throughput genome sequencing to determine the Himar1-A. marginale genome junctions established that the transposon sequences were integrated within the coding region of the omp10 gene. This gene is arranged within an operon with AM1225 at the 5' end and with omp9, omp8, omp7 and omp6 arranged in tandem at the 3' end. RNA analysis to determine the effects of the transposon insertion on the expression of omp10 and downstream genes revealed that the Himar1 insertion not only reduced the expression of omp10 but also that of downstream genes. Transcript expression from omp9, and omp8 dropped by more than 90% in comparison with their counterparts in wild-type A. marginale. Immunoblot analysis showed a reduction in the production of Omp9 protein in these mutants compared to wild-type A. marginale. CONCLUSIONS: These results demonstrate that transposon mutagenesis in A. marginale is possible and that this technology can be used for the creation of insertional gene knockouts that can be evaluated in natural host-vector systems.
Assuntos
Anaplasma marginale/genética , Proteínas da Membrana Bacteriana Externa/genética , Elementos de DNA Transponíveis , Óperon , Sequência de Bases , Western Blotting , Cromossomos Bacterianos , DNA Bacteriano , Técnicas de Silenciamento de Genes , Genes Bacterianos , Dados de Sequência Molecular , MutagêneseRESUMO
Philadelphia chromosome-negative myeloproliferative neoplasms, including polycythemia vera, essential thrombocytosis, and myelofibrosis, are disorders characterized by abnormal hematopoiesis. Among these myeloproliferative neoplasms, myelofibrosis has the most unfavorable prognosis. Furthermore, currently available therapies for myelofibrosis have little to no efficacy in the bone marrow and hence, are palliative. We recently developed a Janus kinase 2 (Jak2) small molecule inhibitor called G6 and found that it exhibits marked efficacy in a xenograft model of Jak2-V617F-mediated hyperplasia and a transgenic mouse model of Jak2-V617F-mediated polycythemia vera/essential thrombocytosis. However, its efficacy in Jak2-mediated myelofibrosis has not previously been examined. Here, we hypothesized that G6 would be efficacious in Jak2-V617F-mediated myelofibrosis. To test this, mice expressing the human Jak2-V617F cDNA under the control of the vav promoter were administered G6 or vehicle control solution, and efficacy was determined by measuring parameters within the peripheral blood, liver, spleen, and bone marrow. We found that G6 significantly reduced extramedullary hematopoiesis in the liver and splenomegaly. In the bone marrow, G6 significantly reduced pathogenic Jak/STAT signaling by 53%, megakaryocytic hyperplasia by 70%, and the Jak2 mutant burden by 68%. Furthermore, G6 significantly improved the myeloid to erythroid ratio and significantly reversed the myelofibrosis. Collectively, these results indicate that G6 is efficacious in Jak2-V617F-mediated myelofibrosis, and given its bone marrow efficacy, it may alter the natural history of this disease.
Assuntos
Janus Quinase 2/metabolismo , Mielofibrose Primária/tratamento farmacológico , Mielofibrose Primária/enzimologia , Inibidores de Proteínas Quinases/uso terapêutico , Bibliotecas de Moléculas Pequenas/uso terapêutico , Estilbenos/uso terapêutico , Substituição de Aminoácidos/genética , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Modelos Animais de Doenças , Hematopoese Extramedular/efeitos dos fármacos , Humanos , Hiperplasia , Janus Quinase 2/antagonistas & inibidores , Megacariócitos/efeitos dos fármacos , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Células Mieloides/efeitos dos fármacos , Células Mieloides/patologia , Fosforilação/efeitos dos fármacos , Mielofibrose Primária/sangue , Mielofibrose Primária/fisiopatologia , Inibidores de Proteínas Quinases/farmacologia , Reticulina/efeitos dos fármacos , Reticulina/metabolismo , Fator de Transcrição STAT5/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Baço/efeitos dos fármacos , Baço/patologia , Baço/fisiopatologia , Esplenomegalia/complicações , Esplenomegalia/tratamento farmacológico , Esplenomegalia/patologia , Esplenomegalia/fisiopatologia , Estilbenos/farmacologiaRESUMO
A 2-year-old, castrated male, mixed-breed dog was presented to the University of Florida Veterinary Medical Center with swelling, edema, ulceration, and draining tracts in the region surrounding the left hock. The dog had mild monocytosis and moderate hyperglobulinemia. Fine-needle aspirate specimens of the left popliteal lymph node revealed pyogranulomatous lymphadenitis with hyphal organisms. The diameters of the hyphae were variable, ranging from 11 to 22 microm. The organism was considered as most consistent with Lagenidium caninum; although Pythium insidiosum or Lagenidium karlingii were not conclusively excluded, hyphal diameter in these organisms is typically smaller (6.6-8.8 and 2.5-11 microm, respectively). A positive Western blot confirmed the presence of serum antibodies reactive against Lagenidium sp. and the absence of antibodies to P. insidoisum, Basidiobolus, and Conidiobolus antibodies. Careful assessment of hyphal diameter in cytologic specimens may be useful in differentiating L. caninum from P. insidiosum or L. karlingii.
Assuntos
Doenças do Cão/patologia , Infecções/veterinária , Lagenidium/isolamento & purificação , Linfonodos/citologia , Animais , Doenças do Cão/microbiologia , Cães , MasculinoRESUMO
Endothelial cell culture and preliminary immunofluorescent staining of Anaplasma-infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells and other potentially cryptic sites of Anaplasma sp. infection in mammalian tissues, a sensitive and specific isothermal in situ technique to detect localized Anaplasma gene sequences by using rolling-circle amplification of circularizable, linear, oligonucleotide probes (padlock probes) was developed. Cytospin preparations of uninfected or Anaplasma-infected cell cultures were examined using this technique. Via fluorescence microscopy, the technique described here, and a combination of differential interference contrast microscopy and von Willebrand factor immunofluorescence, Anaplasma phagocytophilum and Anaplasma marginale were successfully localized in situ within intact cultured mammalian cells. This work represents the first application of this in situ method for the detection of a microorganism and forms the foundation for future applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected mammalian and arthropod hosts and in cell cultures.
Assuntos
Anaplasma marginale/isolamento & purificação , Anaplasma phagocytophilum/isolamento & purificação , Hibridização In Situ/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Fator de von Willebrand/análise , Anaplasma marginale/genética , Anaplasma phagocytophilum/genética , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Células Endoteliais/microbiologia , Imunofluorescência , Humanos , Macaca mulatta , Microscopia de Fluorescência , Microscopia de Interferência , Dados de Sequência MolecularRESUMO
BACKGROUND: Previous reports suggest an association between Bartonella infection and effusions in dogs and human beings. OBJECTIVES: The aims of this study were to determine the prevalence of Bartonella infection in canine effusions and to investigate historic and clinical parameters predictive of Bartonella in dogs with effusions. METHODS: Canine cavitary effusions submitted for analysis and, if available, paired EDTA blood, were screened for Bartonella infection using the Bartonella α-proteobacteria growth medium enrichment culture/PCR diagnostic platform (Bartonella enrichment PCR or ePCR) at Galaxy Diagnostics, Inc. RESULTS: Bartonella henselaeDNA was PCR-amplified and sequenced from 15% (12/80) of sampled dogs. Enrichment culture prior to PCR testing was required for Bartonella detection in 92% (11/12) of cases. Twenty percent (4/20), 13% (8/60), and 0% (0/4) of dogs with pleural, peritoneal, and pericardial effusions, respectively, tested positive. Bartonella henselae was detected most frequently in the fall, and young and middle-aged dogs appeared to be overrepresented. Golden Retrievers and Yorkshire/Silky Terriers each comprised 25% of infected dogs (odds ratio 3.4 for Golden Retrievers). There was a weak association with hemorrhagic effusions. Fifty percent of Bartonella-positive dogs had hemorrhage as a component of their effusion compared to 37% of PCR-negative dogs (odds ratio 1.7). CONCLUSIONS: Viable B henselae organisms occur in pleural and peritoneal effusions of dogs; the clinical relevance of which remains unclear and may represent opportunistic infection. Associations found in this study included seasonal variation, age, breed, and site of effusion.
Assuntos
Angiomatose Bacilar/veterinária , Bartonella henselae , Doenças do Cão/microbiologia , Derrame Pericárdico/veterinária , Derrame Pleural/veterinária , Angiomatose Bacilar/diagnóstico , Angiomatose Bacilar/epidemiologia , Angiomatose Bacilar/patologia , Animais , Líquido Ascítico/microbiologia , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Doenças do Cão/patologia , Cães , Feminino , Masculino , Derrame Pericárdico/etiologia , Derrame Pericárdico/microbiologia , Derrame Pleural/etiologia , Derrame Pleural/microbiologia , Prevalência , Estações do AnoRESUMO
An 11-year-old intact male Green iguana (Iguana iguana) was referred for treatment of a probable iridophoroma based on previous cytopathology. A periocular mass was present near the right medial canthus. Computed tomography did not show any sign of metastasis. Clinicopathologic abnormalities included lymphopenia and hyperproteinemia. Cytologic and histologic evaluations of the mass were consistent with iridophoroma. Complete surgical excision of the mass was not possible without removal of the orbit due to local tissue involvement. Recovery and suture removal were unremarkable. Adjunctive radiation therapy was recommended, but not performed. A year later, the surgical site had healed well. To our knowledge, this is the first reported chromatophoroma cytopathology in a Green iguana. Chromatophoromas should be included in the differential diagnoses of pigmented skin tumors in reptiles. Early surgical excision is useful to limit local tissue destruction and metastatic potential.
Assuntos
Neoplasias Oculares/veterinária , Iguanas , Neoplasias Cutâneas/veterinária , Animais , Cromatóforos , Neoplasias Oculares/patologia , Masculino , Neoplasias Cutâneas/patologiaRESUMO
BACKGROUND: Anaplasma phagocytophilum (formerly known as the human granulocytic ehrlichia, Ehrlichia equi and Ehrlichia phagocytophila) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. OBJECTIVES: The objectives of this investigation were to sequence and clone the major surface protein 5 (MSP5) of A phagocytophilum and to evaluate the suitability of this antigen in the serologic diagnosis of anaplasmosis in humans and dogs. METHODS: The msp5 gene of A phagocytophilum was sequenced, cloned, and expressed in Escherichia coli. The predicted amino acid sequence homology of the various MSP5/major antigenic protein 2 orthologs was compared among various Anaplasma and Ehrlichia species. Recombinant MSP5 of A phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. RESULTS: Serum samples from 104 individuals previously diagnosed with A phagocytophilum infection, as well as samples from clinically healthy humans, were tested. In addition, multiple samples from 4 dogs experimentally infected with 2 different geographic isolates of A phagocytophilum and 5 dogs naturally infected with a Swiss isolate were tested using ELISA. Using this group of immunofluorescent antibody test-positive and immunofluorescent antibody test-negative samples, we found the overall agreement between assays to be >90%. CONCLUSIONS: These results indicate that recombinant MSP5 has potential for use as a diagnostic test antigen to detect infection with A phagocytophilum in both dogs and humans. However, sequence similarities among orthologs of MSP5 in related species of anaplasma and ehrlichia suggest that cross-reactivity among these pathogens is likely if the entire peptide is used as a test antigen.
Assuntos
Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Doenças do Cão/diagnóstico , Doenças do Cão/microbiologia , Ehrlichiose/veterinária , Sequência de Aminoácidos , Anaplasma phagocytophilum/química , Animais , Clonagem Molecular , Cães , Ehrlichiose/diagnóstico , Ehrlichiose/microbiologia , Humanos , Dados de Sequência Molecular , Especificidade da EspécieRESUMO
A 10-year-old castrated Domestic Short-Haired cat was presented to a primary care veterinarian for a wellness examination and laboratory examination for monitoring of diabetes mellitus. The CBC revealed marked thrombocytosis, leukopenia and macrocytic, normochromic anemia. The cat tested negative for FeLV and feline immunodeficiency virus, but was positive for Mycoplasma haemominutum by PCR. Hematologic abnormalities were not responsive to therapy, so a repeat CBC and a bone marrow aspiration for cytology were performed. Additional blood smear findings included anisocytosis with megaloblastic erythroid precursors, large platelets, eosinophilic myelocytes and metamyelocytes, and rare unidentified blasts. The bone marrow smear was highly cellular, and the cytologic pattern was consistent with myelodysplastic syndrome with an erythroid predominance. At that time, 15% blasts were present. The cat was treated with a vitamin K2 analog, doxycycline, and prednisolone, but without a clinical response. Within 3 months, euthanasia was elected due to declining quality of life, and a necropsy was performed. Postmortem bone marrow smears were highly cellular and dominated by monomorphic blasts of unknown line of origin (52%), persistent marked erythroid and megakaryocytic dysplasia, and ineffective erythropoiesis and granulopoiesis. Immunohistochemical, immunocytochemical, and cytochemical stains resulted in a diagnosis of acute myeloid leukemia of unclassified type. Additional histologic findings included mixed hepatitis with trematode infestation and lymphoplasmacytic interstitial nephritis with fibrosis. The marked thrombocytosis with myelodysplastic syndrome and the FeLV-negative status of this cat were unusual. The difficulty in classifying the myelodysplasia and subsequent leukemia highlights a need for further reporting and characterization of these types of disease.
Assuntos
Anemia Macrocítica/veterinária , Doenças do Gato/diagnóstico , Leucemia Mieloide/veterinária , Leucopenia/veterinária , Doenças Mieloproliferativas-Mielodisplásicas/veterinária , Trombocitose/veterinária , Anemia Macrocítica/diagnóstico , Anemia Macrocítica/patologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/patologia , Exame de Medula Óssea/veterinária , Doenças do Gato/patologia , Gatos , Complicações do Diabetes/terapia , Complicações do Diabetes/veterinária , Quimioterapia Combinada/veterinária , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/patologia , Leucopenia/diagnóstico , Leucopenia/patologia , Masculino , Mycoplasma/genética , Mycoplasma/isolamento & purificação , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/patologia , Síndromes Mielodisplásicas/veterinária , Doenças Mieloproliferativas-Mielodisplásicas/diagnóstico , Doenças Mieloproliferativas-Mielodisplásicas/patologia , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/patologia , Transtornos Mieloproliferativos/veterinária , Trombocitose/diagnóstico , Trombocitose/patologiaRESUMO
Diffuse ileal thickening and ileocecocolic lymphadenomegaly were observed during exploratory laparotomy in a 2-year-old male Japanese snow macaque (Macaca fuscata) that had flu-like signs and diarrhea. Cytologic examination of ileal biopsy imprints revealed many mature, mildly karyolytic neutrophils and fewer well-differentiated lymphocytes, eosinophils, macrophages, and plasma cells in a background containing amorphous, necrotic material. Tightly cohesive sheets of moderately pleomorphic epithelial cells also were seen. The cytologic diagnosis was chronic, active, mixed inflammation with atypical epithelial cells and necrosis. Histologically, the mucosal and crypt epithelium was moderately hyperplastic with a loss of goblet cells, increased mitoses, and frequent crypt abscesses. Within the lamina propria and extending into the submucosa was a marked neutrophilic infiltrate, with low numbers of lymphocytes, histiocytes, plasma cells, and eosinophils. The histologic diagnosis was chronic, diffuse, marked suppurative and lymphocytic ileitis. Warthin-Starry silver staining of the ileal biopsy and imprint specimens demonstrated numerous pleomorphic, curved bacilli consistent with Lawsonia intracellularis. Polymerase chain reaction (PCR) and immunohistochemistry confirmed the identity of the infectious agent. L intracellularis infection may be underdiagnosed because silver stain is required to visualize the organism with light microscopy and because the pathognomonic crypt hyperplasia may be complicated by secondary pathologic changes. Application of silver stain to cytologic specimens should be considered when distal intestinal lesions associated with hyperplastic epithelium, with or without inflammation, hemorrhage, or necrosis, are identified in animals with clinical signs of enteritis, especially in frequently affected species or in stressed or young animals.
Assuntos
Infecções por Desulfovibrionaceae/veterinária , Ileíte/veterinária , Lawsonia (Bactéria) , Macaca/microbiologia , Doenças dos Macacos/patologia , Animais , Infecções por Desulfovibrionaceae/patologia , Ileíte/microbiologia , Ileíte/patologia , Masculino , Doenças dos Macacos/microbiologiaRESUMO
OBJECTIVE: To evaluate CSF in horses with confirmed West Nile virus encephalomyelitis. DESIGN: Retrospective study. ANIMALS: 30 horses. PROCEDURE: Results of CSF analyses from horses with acute neurologic signs attributed to West Nile virus infection that was confirmed by immunoglobulin M antibody capture ELISA were reviewed and analyzed. RESULTS: Among 30 CSF samples, findings in 8 (27%) were within reference ranges and in 22 (73%) were abnormal. Among the 22 abnormal samples, mononuclear pleocytosis was found in 16 (73%) and high protein concentration with nucleated cell count within reference range was found in 6 (27%) samples. A predominance of lymphocytes was found in 11 of 16 samples with mononuclear pleocytosis, and a predominance of large mononuclear cells was found in 5 of 16 samples. Sensitivities of analyses of CSF obtained from the lumbosacral and atlanto-occipital regions of the spinal cord were 89 and 50%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that in horses with acute onset of neurologic signs caused by West Nile virus encephalomyelitis, findings in the CSF are likely to be abnormal, mononuclear pleocytosis with lymphocytic predominance may be most commonly observed, and CSF collected from the lumbosacral region may be abnormal more commonly than CSF collected from the atlanto-occipital region.
Assuntos
Líquido Cefalorraquidiano/citologia , Doenças dos Cavalos/líquido cefalorraquidiano , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/isolamento & purificação , Animais , Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/análise , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças dos Cavalos/virologia , Cavalos , Imunoglobulina M/imunologia , Contagem de Leucócitos/veterinária , Leucócitos Mononucleares , Leucocitose/líquido cefalorraquidiano , Leucocitose/veterinária , Valores de Referência , Estudos Retrospectivos , Punção Espinal/métodos , Punção Espinal/veterinária , Febre do Nilo Ocidental/líquido cefalorraquidianoRESUMO
OBJECTIVE: To determine signalment, clinical findings, results of diagnostic testing, outcome, and postmortem findings in horses with West Nile virus (WNV) encephalomyelitis. DESIGN: Retrospective study. ANIMALS: 46 horses with WNV encephalomyelitis. PROCEDURE: Clinical data were extracted from medical records of affected horses. RESULTS: On the basis of clinical signs and results of serologic testing, WNV encephalomyelitis was diagnosed in 46 of 56 horses with CNS signs. Significantly more males than females were affected. Increased rectal temperature, weakness or ataxia, and muscle fasciculations were the most common clinical signs. Paresis was more common than ataxia, although both could be asymmetrical and multifocal. Supportive treatment included anti-inflammatory medications, fluids, antimicrobials, and slinging of recumbent horses. Results of the IgM capture ELISA and the plaque reduction neutralization test provided a diagnosis in 43 horses, and only results of the plaque reduction neutralization test were positive in 3 horses. Mortality rate was 30%, and 71% of recumbent horses were euthanatized. One horse that had received 2 vaccinations for WNV developed the disease and was euthanatized. Follow-up communications with 19 owners revealed that most horses had residual deficits at 1 month after release from the hospital; abnormalities were resolved in all but 2 horses by 12 months after release. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings were similar to those of previous WNV outbreaks in horses but provided additional clinical details from monitored hospitalized horses. Diagnostic testing is essential to diagnosis, treatment is supportive, and recovery rate of discharged ambulatory horses is < 100%.
Assuntos
Doenças dos Cavalos/diagnóstico , Febre do Nilo Ocidental/veterinária , Animais , Diagnóstico Diferencial , Feminino , Doenças dos Cavalos/tratamento farmacológico , Doenças dos Cavalos/mortalidade , Cavalos , Masculino , Estudos Retrospectivos , Fatores Sexuais , Resultado do Tratamento , Febre do Nilo Ocidental/diagnóstico , Febre do Nilo Ocidental/tratamento farmacológico , Febre do Nilo Ocidental/mortalidade , Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/isolamento & purificaçãoRESUMO
BACKGROUND: Rickettsia amblyommii is a bacterium in the spotted fever group of organisms associated with the lone star tick (LST), Amblyomma americanum. The LST is the most commonly reported tick to parasitize humans in the southeastern US. Within this geographic region, there have been suspected cases of Rocky Mountain spotted fever (RMSF) where the causative agent, R. rickettsii, was not identified in the local tick population. In these areas, patients with clinical signs of RMSF had low or no detectable antibodies to R. rickettsii, resulting in an inability to confirm a diagnosis. METHODS: R. amblyommii was cultivated from host-seeking LSTs trapped in Central Florida and propagated in ISE6 (Ixodes scapularis) and AAE2 (A. americanum) cells. Quantitative PCR targeting the 17-kD gene of Rickettsia spp. identified the genus of the organism in culture. Variable regions of groEL, gtlA and rompA genes were amplified and sequenced to confirm the species. The prevalence of R. amblyommii in LSTs within the geographic region was determined by qPCR followed by conventional PCR and direct sequencing. RESULTS: Analyses of amplified sequences from the cultured organism were 100% homologous to R. amblyommii. The overall prevalence of Rickettsia spp. in the local population of LSTs was 57.1% and rompA sequence analysis identified only R. amblyommii in LSTs. CONCLUSIONS: A Florida strain of R. amblyommii was successfully cultivated in two tick cell lines. Further evaluation of the new strain and comparisons to the other geographic strains is needed. The prevalence of this SFG organism in the tick population warrants further investigation into the organism's ability to cause clinical disease in mammalian species.
Assuntos
Técnicas Bacteriológicas , Ixodidae/microbiologia , Rickettsia/classificação , Rickettsia/isolamento & purificação , Distribuição Animal , Animais , Linhagem Celular , Florida , Reação em Cadeia da Polimerase , Rickettsia/fisiologiaRESUMO
Glioblastoma multiforme (GBM) is the most common and the most aggressive form of primary brain tumor. Jak2 is a non-receptor tyrosine kinase that is involved in proliferative signaling through its association with various cell surface receptors. Hyperactive Jak2 signaling has been implicated in numerous hematological disorders as well as in various solid tumors including GBM. Our lab has developed a Jak2 small molecule inhibitor known as G6. It exhibits potent efficacy in vitro and in several in vivo models of Jak2-mediated hematological disease. Here, we hypothesized that G6 would inhibit the pathogenic growth of GBM cells expressing hyperactive Jak2. To test this, we screened several GBM cell lines and found that T98G cells express readily detectable levels of active Jak2. We found that G6 treatment of these cells reduced the phosphorylation of Jak2 and STAT3, in a dose-dependent manner. In addition, G6 treatment reduced the migratory potential, invasive potential, clonogenic growth potential, and overall viability of these cells. The effect of G6 was due to its direct suppression of Jak2 function and not via off-target kinases, as these effects were recapitulated in T98G cells that received Jak2 specific shRNA. G6 also significantly increased the levels of caspase-dependent apoptosis in T98G cells, when compared to cells that were treated with vehicle control. Lastly, when T98G cells were injected into nude mice, G6 treatment significantly reduced tumor volume and this was concomitant with significantly decreased levels of phospho-Jak2 and phospho-STAT3 within the tumors themselves. Furthermore, tumors harvested from mice that received G6 had significantly less vimentin protein levels when compared to tumors from mice that received vehicle control solution. Overall, these combined in vitro and in vivo results indicate that G6 may be a viable therapeutic option against GBM exhibiting hyperactivation of Jak2.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antineoplásicos/síntese química , Apoptose , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Feminino , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/síntese química , Carga Tumoral/efeitos dos fármacos , Vimentina/antagonistas & inibidores , Vimentina/genética , Vimentina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Germline deletion of Jak2 in mice results in embryonic lethality at E12.5 due to impaired hematopoiesis. However, the role that Jak2 might play in late gestation and postnatal life is unknown. To understand this, we utilized a conditional knockout approach that allowed for the deletion of Jak2 at various stages of prenatal and postnatal life. Specifically, Jak2 was deleted beginning at either mid/late gestation (E12.5), at postnatal day 4 (PN4), or at â¼2 months of age. Deletion of Jak2 beginning at E12.5 resulted in embryonic death characterized by a lack of hematopoiesis. Deletion beginning at PN4 was also lethal due to a lack of erythropoiesis. Deletion of Jak2 in young adults was characterized by blood cytopenias, abnormal erythrocyte morphology, decreased marrow hematopoietic potential, and splenic atrophy. However, death was observed in only 20% of the mutants. Further analysis of these mice suggested that the increased survivability was due to an incomplete deletion of Jak2 and subsequent re-population of Jak2 expressing cells, as conditional deletion in mice having one floxed Jak2 allele and one null allele resulted in a more severe phenotype and subsequent death of all animals. We found that the deletion of Jak2 in the young adults had a differential effect on hematopoietic lineages; specifically, conditional Jak2 deletion in young adults severely impaired erythropoiesis and thrombopoiesis, modestly affected granulopoiesis and monocytopoiesis, and had no effect on lymphopoiesis. Interestingly, while the hematopoietic organs of these mutant animals were severely affected by the deletion of Jak2, we found that the hearts, kidneys, lungs, and brains of these same mice were histologically normal. From this, we conclude that Jak2 plays an essential and non-redundant role in hematopoiesis during both prenatal and postnatal life and this has direct implications regarding the inhibition of Jak2 in humans.
Assuntos
Desenvolvimento Embrionário/genética , Deleção de Genes , Hematopoese/genética , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Envelhecimento/patologia , Anemia/patologia , Animais , Animais Recém-Nascidos , Contagem de Células Sanguíneas , Perda do Embrião/patologia , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Proteínas de Ligação ao GTP/metabolismo , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Fator Regulador 1 de Interferon/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Camundongos , Camundongos Knockout , Células Progenitoras Mieloides/efeitos dos fármacos , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , Especificidade de Órgãos/efeitos dos fármacos , Gravidez , Baço/patologia , Análise de Sobrevida , Tamoxifeno/farmacologiaRESUMO
Serum from 21 healthy, captive Asian elephants (Elephas maximus) was evaluated by measured and calculated osmolality. Serum osmolality results for this population of Asian elephants had a median of 261 mOsm/kg and an interquartile interval of 258-269 mOsm/kg when measured by freezing point osmometry and a median of 264 mOsm/kg and an interquartile interval of 257-269 mOsm/kg when measured by vapor pressure osmometry. These values are significantly lower than values reported in other mammalian species and have important diagnostic and therapeutic implications. Calculated osmolality produced unreliable results and needs further study to determine an appropriate formula and its clinical application in this species. A 16-hr water deprivation test in 16 Asian elephants induced a small, subclinical, but statistically significant increase in measured serum osmolality. Serum osmolality, blood urea nitrogen, and total protein by refractometer were sensitive indicators of hydration status. Serum osmolality measurement by freezing point or vapor pressure osmometry is a useful adjunct to routine clinical tests in the diagnostic evaluation of elephants.
Assuntos
Elefantes/sangue , Privação de Água/fisiologia , Animais , Feminino , Masculino , Concentração Osmolar , Osmometria/veterinária , Estatísticas não ParamétricasRESUMO
In December 2009, the American Society for Veterinary Clinical Pathology (ASVCP) Quality Assurance and Laboratory Standards committee published the updated and peer-reviewed ASVCP Quality Assurance Guidelines on the Society's website. These guidelines are intended for use by veterinary diagnostic laboratories and veterinary research laboratories that are not covered by the US Food and Drug Administration Good Laboratory Practice standards (Code of Federal Regulations Title 21, Chapter 58). The guidelines have been divided into 3 reports: (1) general analytical factors for veterinary laboratory performance and comparisons; (2) hematology, hemostasis, and crossmatching; and (3) clinical chemistry, cytology, and urinalysis. This particular report is one of 3 reports and documents recommendations for control of preanalytical, analytical, and postanalytical factors related to urinalysis, cytology, and clinical chemistry in veterinary laboratories and is adapted from sections 1.1 and 2.2 (clinical chemistry), 1.3 and 2.5 (urinalysis), 1.4 and 2.6 (cytology), and 3 (postanalytical factors important in veterinary clinical pathology) of these guidelines. These guidelines are not intended to be all-inclusive; rather, they provide minimal guidelines for quality assurance and quality control for veterinary laboratory testing and a basis for laboratories to assess their current practices, determine areas for improvement, and guide continuing professional development and education efforts.
Assuntos
Testes de Química Clínica/veterinária , Técnicas Citológicas/veterinária , Patologia Clínica/organização & administração , Manejo de Espécimes/veterinária , Urinálise/veterinária , Medicina Veterinária/organização & administração , Animais , Testes de Química Clínica/métodos , Testes de Química Clínica/normas , Técnicas Citológicas/normas , Laboratórios/normas , Patologia Clínica/normas , Garantia da Qualidade dos Cuidados de Saúde/normas , Controle de Qualidade , Sociedades Científicas/normas , Especificidade da Espécie , Manejo de Espécimes/normas , Urinálise/normas , Medicina Veterinária/normasRESUMO
CASE DESCRIPTION: A 9-year-old spayed female green iguana (Iguana iguana) was evaluated because of a distended coelom and weight loss. History included a single episode of egg binding and subsequent bilateral ovariosalpingectomy. CLINICAL FINDINGS: Physical examination revealed a mass within the coelomic cavity. Ultrasonography revealed a large, irregular mass with hypoechoic regions and coelomic effusion. Clinicopathologic derangements included heterophilia, monocytosis, lymphopenia, basophilia, hypocholesterolemia, hypoproteinemia, and hypercalcemia. Results of cytologic evaluation of the mass were suggestive of malignant epithelial neoplasia, but neoplastic cells were not found in the effusion. An ovarian tumor was suspected on the basis of clinical signs, clinicopathologic findings, and results of cytologic evaluation of the mass. TREATMENT AND OUTCOME: Surgical exploration revealed a large left ovary, a normal-appearing contralateral ovary, and a mass in the fat body, all of which were removed and submitted for histologic examination. The histologic diagnosis was granulosa cell tumor with metastasis to the fat body. The patient died 11 months after evaluation, and disseminated granulosa cell tumor was confirmed at necropsy; histologic examination at that time also identified systemic mastocytosis. CLINICAL RELEVANCE: Granulosa cell tumors are uncommon in reptiles, and this was the first granulosa cell tumor described antemortem cytologically, histologically, and ultrastructurally in an iguana. Findings in this iguana underscored concerns associated with incomplete oophorectomy of iguanas; cytologic and histopathologic findings were similar to those observed in other domestic animals. Oophorectomy should be considered as an alternative to standard ovariosalpingectomy to avoid potential complications in pet reptiles, and use of microsurgical instruments and vascular clips is advised.
Assuntos
Tumor de Células da Granulosa/veterinária , Lagartos , Ovariectomia/veterinária , Animais , Evolução Fatal , Feminino , Tumor de Células da Granulosa/patologia , Ovariectomia/efeitos adversosRESUMO
We recently developed a Janus kinase 2 (Jak2) small-molecule inhibitor called G6 and found that it inhibits Jak2-V617F-mediated pathologic cell growth in vitro, ex vivo, and in vivo. However, its ability to inhibit Jak2-V617F-mediated myeloproliferative neoplasia, with particular emphasis in the bone marrow, has not previously been examined. Here, we investigated the efficacy of G6 in a transgenic mouse model of Jak2-V617F-mediated myeloproliferative neoplasia. We found that G6 provided therapeutic benefit to the peripheral blood as determined by elimination of leukocytosis, thrombocytosis, and erythrocytosis. G6 normalized the pathologically high plasma concentrations of interleukin 6 (IL-6). In the liver, G6 eliminated Jak2-V617F-driven extramedullary hematopoiesis. With respect to the spleen, G6 significantly reduced both the splenomegaly and megakaryocytic hyperplasia. In the critically important bone marrow, G6 normalized the pathologically high levels of phospho-Jak2 and phospho-signal transducer and activator of transcription 5 (STAT5). It significantly reduced the megakaryocytic hyperplasia in the marrow and completely normalized the M/E ratio. Most importantly, G6 selectively reduced the mutant Jak2 burden by 67%on average, with virtual elimination of mutant Jak2 cells in one third of all treated mice. Lastly, clonogenic assays using marrow stem cells from the myeloproliferative neoplasm mice revealed a time-dependent elimination of the clonogenic growth potential of these cells by G6. Collectively, these data indicate that G6 exhibits exceptional efficacy in the peripheral blood, liver, spleen, and, most importantly, in the bone marrow, thereby raising the possibility that this compound may alter the natural history of Jak2-V617F-mediated myeloproliferative neoplasia.
Assuntos
Neoplasias da Medula Óssea/tratamento farmacológico , Neoplasias da Medula Óssea/genética , Transformação Celular Neoplásica/genética , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Estilbenos/uso terapêutico , Substituição de Aminoácidos/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Neoplasias da Medula Óssea/patologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Janus Quinase 2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Fenilalanina/genética , Inibidores de Proteínas Quinases/farmacologia , Estilbenos/farmacologia , Valina/genéticaRESUMO
A 14-year-old female spayed Golden Retriever was presented to the University of Florida's Veterinary Medical Center with history of lymphoplasmacytic gastroenteritis, intermittent vomiting, watery diarrhea, and weight loss for over a year. CBC, biochemical profile, and urinalysis were within reference intervals. Abdominal ultrasonographic examination revealed mesenteric and jejunal lymphadenopathy and hyperechoic hepatic nodules. Cytologic examination of the enlarged lymph nodes revealed loosely cohesive cells with moderate nuclear pleomorphism and rare punctate eosinophilic cytoplasmic granules. The cytologic interpretation was metastatic neuroendocrine neoplasia. On surgical exploration, a mass was detected in the right lobe of the pancreas. Histologic evaluation determined the mass to be an islet cell tumor. Approximately 98% of cells were positive by immunolabeling for pancreatic polypeptide (PP), and only rare cells were positive for insulin or somatostatin. All cells were negative for glucagon, gastrin, vasoactive intestinal polypeptide, protein gene product 9.5, synaptophysin, and chromogranins A and B. Pancreatic tumors that primarily produce PP are rare in dogs, and this is the first report of both the cytologic and histologic features of an islet cell tumor predominantly secreting PP. Clinical signs for these tumors are typically absent or nonspecific; signs may include watery diarrhea, as noted in this dog, although the diarrhea may have resulted from lymphoplasmacytic gastroenteritis. Additional case studies are needed to further characterize the cytomorphologic features and clinical presentation of PP-secreting islet cell tumor, or polypeptidoma, in dogs.