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1.
Mol Pharm ; 15(3): 1215-1225, 2018 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-29421865

RESUMO

The nontuberculous mycobacterial (NTM) pathogens, M. avium complex (MAC) and M. abscessus, can result in severe pulmonary infections. Current antibiotics confront significant challenges for treatment of these NTM infections due to emerging multidrug-resistance. Thus, development of new antibiotics targeted against these agents is needed. We examined the inhibitory activities of Ga(NO3)3, GaCl3, gallium meso-tetraphenylporphyrine (GaTP), and gallium nanoparticles (GaNP) against intra- and extracellular M. avium and M. abscessus. GaTP, an analogue of natural heme, inhibited growth of both M. avium and M. abscessus with MICs in Fe-free 7H9 media of 0.5 and 2 µg/mL, respectively. GaTP was more active than Ga(NO3)3 and GaCl3. Ga(NO3)3 and GaCl3 were not as active in Fe-rich media compared to Fe-free media. However, GaTP was much less impacted by exogenous Fe, with MICs against M. avium and M. abscessus of 2 and 4 µg/mL, respectively, in 7H9 OADC media (Fe rich). Confocal microscopy showed that GaNP penetrates the M. avium cell wall. As assessed by determining colony forming units, GaNP inhibited the growth of NTM growing in THP-1 macrophages up to 15 days after drug-loading of the cells, confirming a prolonged growth inhibitory activity of the GaNP. Biodistribution studies of GaNP conducted in mice showed that intraperitoneal injection is more effective than intramuscular injection in delivering Ga(III) into lung tissue. GaTP exhibits potential as a lead compound for development of anti-NTM agents that target heme-bound iron uptake mechanisms by mycobacteria and inhibit growth by disrupting mycobacterial iron acquisition/utilization.


Assuntos
Antibacterianos/farmacologia , Gálio/farmacologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium avium/efeitos dos fármacos , Infecções Respiratórias/tratamento farmacológico , Animais , Antibacterianos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Gálio/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Modelos Animais , Infecções por Mycobacterium não Tuberculosas/microbiologia , Nanopartículas/química , Porfirinas/química , Infecções Respiratórias/microbiologia , Distribuição Tecidual
2.
Infect Immun ; 85(9)2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28630072

RESUMO

Pseudomonasaeruginosa causes lung infections in patients with cystic fibrosis (CF). The Pseudomonas quinolone signal (PQS) compound is a secreted P. aeruginosa virulence factor that contributes to the pathogenicity of P. aeruginosa We were able to detect PQS in sputum samples from CF patients infected with P. aeruginosa but not in samples from uninfected patients. We then tested the hypothesis that PQS induces oxidative stress in host cells by determining the ability of PQS to induce the production of reactive oxygen species (ROS) in lung epithelial cells (A549 and primary normal human bronchial epithelial [NHBE]) cells and macrophages (J774A.1 and THP-1). ROS production induced by PQS was detected with fluorescent probes (dichlorodihydrofluorescein diacetate, dihydroethidium, and MitoSOX Red) in conjunction with confocal microscopy and flow cytometry. PQS induced ROS production in lung epithelial (A549 and NHBE) cells and macrophages (J774A.1 and THP-1 cells). NHBE cells were sensitive to PQS concentrations as low as 500 ng/ml. PQS significantly induced early apoptosis (P < 0.05, n = 6) in lung epithelial cells, as measured by annexin/propidium iodide detection by flow cytometry. However, no change in apoptosis upon PQS treatment was seen in J774A.1 cells. Heme oxygenase-1 (HO-1) protein is an antioxidant enzyme usually induced by oxidative stress. Interestingly, incubation with PQS significantly reduced HO-1 and NrF2 expression in A549 and NHBE cells but increased HO-1 expression in J774A.1 cells (P < 0.05, n = 3), as determined by immunoblotting and densitometry. These PQS effects on host cells could play an important role in the pathogenicity of P. aeruginosa infections.


Assuntos
Inibidores Enzimáticos/metabolismo , Células Epiteliais/efeitos dos fármacos , Heme Oxigenase-1/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Estresse Oxidativo , Quinolonas/metabolismo , Animais , Linhagem Celular , Células Epiteliais/química , Células Epiteliais/enzimologia , Citometria de Fluxo , Humanos , Macrófagos/química , Macrófagos/enzimologia , Camundongos , Microscopia Confocal , Espécies Reativas de Oxigênio/análise
3.
Cancer Immunol Immunother ; 59(1): 47-62, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19449184

RESUMO

Female mice transgenic for the rat proto-oncogene c-erb-B2, under control of the mouse mammary tumor virus (MMTV) promoter (neuN), spontaneously develop metastatic mammary carcinomas. The development of these mammary tumors is associated with increased number of GR-1(+)CD11b(+) myeloid derived suppressor cells (MDSCs) in the peripheral blood (PB), spleen and tumor. We report a complex relationship between tumor growth, MDSCs and immune regulatory molecules in non-mutated neu transgenic mice on a FVB background (FVB-neuN). The first and second tumors in FVB-neuN mice develop at a median of 265 (147-579) and 329 (161-523) days, respectively, resulting in a median survival time (MST) of 432 (201 to >500) days. During tumor growth, significantly increased number of MDSCs is observed in the PB and spleen, as well as, in infiltrating the mammary tumors. Our results demonstrate a direct correlation between tumor size and the number of MDSCs infiltrating the tumor and an inverse relationship between the frequency of CD4(+) T-cells and MDSCs in the spleen. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assessment of enzyme and cytokine transcript levels in the spleen, tumor, tumor-infiltrating non-parenchymal cells (NPCs) and mammary glands revealed a significant increase in transcript levels from grossly normal mammary glands and tumor-infiltrating NPCs during tumor progression. Tumor NPCs, as compared to spleen cells from wild-type (w/t) mice, expressed significantly higher levels of arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor (VEGF-A) and significantly lower levels of interferon (IFN)-gamma, interleukin (IL)-2 and fms-like tyrosine kinase-3 ligand (Flt3L) transcript levels. Transcript levels in the spleens of tumor-bearing (TB) mice also differed from normal mice, although to a lesser extent than transcript levels from tumor-infiltrating NPCs. Furthermore, both spleen cells and NPCs from TB mice, but not control mice, suppressed alloantigen responses by syngeneic control spleen cells. Correlative studies revealed that the number of MDSCs in the spleen was directly associated with granulocyte colony stimulating factor (G-CSF) transcript levels in the spleen; while the number of MDSCs in the tumors was directly correlated with splenic granulocyte macrophage stimulating factor (GM-CSF) transcript levels, tumor volume and tumor cell number. Together our results support a role for MDSCs in tumor initiation and progressive, T-cell depression and loss of function provide evidence which support multiple mechanisms of MDSC expansion in a site-dependent manner.


Assuntos
Neoplasias Mamárias Experimentais/imunologia , Células Mieloides/fisiologia , Animais , Células Cultivadas , Feminino , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células Mieloides/imunologia , Esplenomegalia/imunologia , Esplenomegalia/patologia
4.
Aging (Albany NY) ; 8(9): 2153-2181, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27689748

RESUMO

We describe age-related molecular and neuronal changes that disrupt mobility or energy balance based on brain region and genetic background. Compared to young mice, aged C57BL/6 mice exhibit marked locomotor (but not energy balance) impairments. In contrast, aged BALB mice exhibit marked energy balance (but not locomotor) impairments. Age-related changes in cerebellar or hypothalamic gene expression accompany these phenotypes. Aging evokes upregulation of immune pattern recognition receptors and cell adhesion molecules. However, these changes do not localize to microglia, the major CNS immunocyte. Consistent with a neuronal role, there is a marked age-related increase in excitatory synapses over the cerebellum and hypothalamus. Functional imaging of these regions is consistent with age-related synaptic impairments. These studies suggest that aging reactivates a developmental program employed during embryogenesis where immune molecules guide synapse formation and pruning. Renewed activity in this program may disrupt excitatory neurotransmission, causing significant behavioral deficits.


Assuntos
Envelhecimento/fisiologia , Cerebelo/fisiologia , Aminoácidos Excitatórios/fisiologia , Hipotálamo/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Envelhecimento/genética , Envelhecimento/imunologia , Animais , Metabolismo Energético/fisiologia , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia
5.
Artigo em Inglês | MEDLINE | ID: mdl-23366406

RESUMO

Inexpensive, high-throughput, low maintenance systems for precise temporal and spatial measurement of mouse home cage behavior (including movement, feeding, and drinking) are required to evaluate products from large scale pharmaceutical design and genetic lesion programs. These measurements are also required to interpret results from more focused behavioral assays. We describe the design and validation of a highly-scalable, reliable mouse home cage behavioral monitoring system modeled on a previously described, one-of-a-kind system. Mouse position was determined by solving static equilibrium equations describing the force and torques acting on the system strain gauges; feeding events were detected by a photobeam across the food hopper, and drinking events were detected by a capacitive lick sensor. Validation studies show excellent agreement between mouse position and drinking events measured by the system compared with video-based observation--a gold standard in neuroscience.


Assuntos
Actigrafia/instrumentação , Comportamento Animal/fisiologia , Ecossistema , Abrigo para Animais , Monitorização Ambulatorial/instrumentação , Fotometria/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Camundongos
6.
Int Immunopharmacol ; 11(7): 816-26, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21376153

RESUMO

Myeloid-derived suppressor cells (MDSCs) increase during tumor growth and following cytoreductive therapy resulting in immune dysfunction and tumor escape from host control. We report organ- and tumor-specific expansion of MDSCs, differences in their molecular and membrane phenotypes and T-cell suppressive activity. A significant increase in MDSCs was observed within the spleen, peripheral blood (PB), bone marrow (BM), lungs, and livers of mice bearing orthotopic 4T1, but not CI66 mammary tumors. The PB of 4T1 TB mice had the highest frequency of MDSCs (78.6±2.1%). Similarly, the non-parenchymal cells (NPCs) in the tumor tissue, livers and lungs of 4T1 tumor-bearing (TB) mice had an increased MDSCs frequency. Studies into Gr-1 and Ly-6C staining of MDSCs revealed significant increases in CD11b+Gr-1(dull)Ly-6C(high) and CD11b+Gr-1(bright)Ly-6C(low) subsets. The frequency of MDSCs inversely correlated with the CD3+ T-cell frequency in the spleen, and blood of 4T1 TB mice and was associated with a significant decrease in splenic and NPCs IFN-γ and IL-12 transcript levels, as well as significantly increased levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), interleukin-10 (IL-10), interleukin-13 (IL-13), arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor-A (VEGF-A) transcripts. In summary, MDSCs are significantly increased not only in lymphoid organs, but also in parenchymal organs including lungs and livers of TB mice, where they may facilitate metastasis to these organ sites.


Assuntos
Adenocarcinoma/imunologia , Fígado/patologia , Pulmão/patologia , Neoplasias Mamárias Animais/imunologia , Células Mieloides/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/imunologia , Feminino , Hematopoese Extramedular , Terapia de Imunossupressão , Leucocitose , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/imunologia , Células Mieloides/patologia , Metástase Neoplásica , Transplante de Neoplasias , Especificidade de Órgãos , Comunicação Parácrina , Microambiente Tumoral/imunologia
7.
Int Immunopharmacol ; 10(1): 140-5, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19833232

RESUMO

Mouse mammary tumor virus-Neu (MMTV/neu) transgenic mice on an FVB-background (FVB-neuN) have increased numbers of myeloid derived suppressor cells (MDSCs) and regulatory T-cells (T-regs) in the spleen during mammary tumor induction and progression. Using this transgenic tumor model, we assessed the therapeutic activity of sunitinib, a multi-targeted, tyrosine kinase (TK) inhibitor and its effects on immune-regulatory cells. Our preliminary results show that sunitinib at 40mg/kg/day, p.o. (per os), delayed the time to tumor induction and reduced the incidence and growth of tumors in FVB-neuN mice. In association with its therapeutic activity, sunitinib reduced the absolute number of splenic T-reg cells (CD4(+)CD25(+)CD62L(+)) and MDSCs (CD11b(+)Gr1(+)) that were increased during tumor progression with less activity in mice with gross tumors. A significant decrease in the absolute number of splenic T-regs, dendritic cells (DCs), MDSCs and hematopoietic progenitors (Lin(-)Sca1(+)CD90(dull)) was observed following sunitinib treatment. The frequency of splenic T-regs and hematopoietic progenitors, but not MDSCs was also reduced by sunitinib treatment. Additionally immune-regulatory cytokines and enzymes were down regulated by sunitinib treatment, including TGFbeta and NOS2 in the spleen cells of sunitinib treated mice as compared to untreated tumor bearing (TB) mice. We conclude that sunitinib has therapeutic activity, in association with the down regulation of MDSCs and T-regs and has a trend towards the normalization of the inflammatory cytokine levels induced by tumor progression and growth. Based on these results, we suggest that sunitinib reduction of immune suppressive cells is a critical part of its adjuvant immune therapeutic activity.


Assuntos
Antineoplásicos/administração & dosagem , Indóis/administração & dosagem , Neoplasias Mamárias Animais/tratamento farmacológico , Pirróis/administração & dosagem , Receptor ErbB-2/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Antígenos CD/biossíntese , Antineoplásicos/efeitos adversos , Progressão da Doença , Feminino , Evasão da Resposta Imune/efeitos dos fármacos , Evasão da Resposta Imune/genética , Indóis/efeitos adversos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/imunologia , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Animais/fisiopatologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Pirróis/efeitos adversos , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Sunitinibe , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia
8.
Int Immunopharmacol ; 9(7-8): 937-48, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19362167

RESUMO

The tumor microenvironment is heterogeneous for the expansion and infiltration by myeloid derived suppressor cells (MDSCs) which has been hypothesized to be dependent on tumor burden. We report a relationships between tumor size, MDSCs and T-cells; using four murine mammary tumors to assess tumor growth, infiltration and gene expression. Our analysis of cellular infiltration into tumors and gene expression used collagenase dissociated tumors and density gradient isolation of non-parenchymal cells (NPCs). The frequency of splenic and peripheral blood (PB) MDSCs was tumor dependent resulting in a significantly increased number of MDSCs. The MDSC frequency inversely correlated with the frequency of CD3+ lymphocytes in the spleen, independent of the tumor studied and directly correlated with tumor burden. Tumor growth up-regulated cyclooxygenase-2 (COX-2), vascular endothelial growth factor-A (VEGF-A), granulocyte (G-) and granulocyte-monocyte-colony stimulating factor (GM-CSF), arginase-1 (ARG-1), and nitric oxide synthase-2 (NOS-2) transcription in the tumor and spleens (not VEGF-A). The frequency of splenic MDSCs directly correlated with splenic COX-2, NOS-2, and ARG-1 message levels, while COX-2 and NOS-2 transcript levels inversely correlated with splenic CD3+ cell frequency. COX-2 mRNA levels also directly correlated with the ARG-1 and NOS-2 transcript levels from tumor-infiltrating leukocytic cells, supporting prostaglandin E2 as a regulator of ARG-1 and NOS-2 transcription. In summary, MDSC numbers in the spleen and tumor microenvironment are tumor dependent, directly correlating with tumor size and inversely correlating with T-cell number. MDSCs are also directly associated with VEGF-A and G-CSF transcript levels suggesting multiple mechanisms for MDSC regulation and COX-2, NOS-2 and ARG-1 supporting multiple mechanisms of T-cell suppression.


Assuntos
Linfócitos do Interstício Tumoral/metabolismo , Neoplasias Mamárias Animais/imunologia , Células Mieloides/metabolismo , Baço/metabolismo , Linfócitos T/metabolismo , Animais , Arginase/genética , Arginase/imunologia , Arginase/metabolismo , Complexo CD3 , Contagem de Células , Processos de Crescimento Celular/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/imunologia , Dinoprostona/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Tolerância Imunológica , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Neoplasias Mamárias Animais/enzimologia , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/imunologia , Células Mieloides/patologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Baço/imunologia , Baço/patologia , Linfócitos T/imunologia , Linfócitos T/patologia , Carga Tumoral/imunologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
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