RESUMO
BACKGROUND: The utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the vascularization and subsequent maturation of human pluripotent stem cell-derived kidney organoids after renal subcapsular transplantation. This raises the question of whether the glomeruli also become functional upon transplantation. METHODS: We transplanted kidney organoids under the renal capsule of the left kidney in immunodeficient mice followed by the implantation of a titanium imaging window on top of the kidney organoid. To assess glomerular function in the transplanted human pluripotent stem cell-derived kidney tissue 1, 2, and 3 weeks after transplantation, we applied high-resolution intravital multiphoton imaging through the imaging window during intravenous infusion of fluorescently labeled low and high molecular mass dextran molecules or albumin. RESULTS: After vascularization, glomerular structures in the organoid displayed dextran and albumin size selectivity across their glomerular filtration barrier. We also observed evidence of proximal tubular dextran reuptake. CONCLUSIONS: Our results demonstrate that human pluripotent stem cell-derived glomeruli can develop an appropriate barrier function and discriminate between molecules of varying size. These characteristics together with tubular presence of low molecular mass dextran provide clear evidence of functional filtration. This approach to visualizing glomerular filtration function will be instrumental for translation of organoid technology for clinical applications as well as for disease modeling.
Assuntos
Células-Tronco Pluripotentes Induzidas/transplante , Glomérulos Renais/metabolismo , Organoides/transplante , Albuminas/metabolismo , Animais , Dextranos/metabolismo , Genes Reporter , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia Intravital/métodos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia de Fluorescência por Excitação Multifotônica , Organoides/irrigação sanguínea , Organoides/metabolismo , Tamanho da Partícula , Técnica de Janela Cutânea , Imagem com Lapso de Tempo/métodosRESUMO
Chronic electroencephalography (EEG) is a widely used tool for monitoring cortical electrical activity in experimental animals. Although chronic implants allow for high-quality, long-term recordings in preclinical studies, the electrodes are foreign objects and might therefore be expected to induce a local inflammatory response. We here analyzed the effects of chronic cranial electrode implantation on glymphatic fluid transport and in provoking structural changes in the meninges and cerebral cortex of male and female mice. Immunohistochemical analysis of brain tissue and dura revealed reactive gliosis in the cortex underlying the electrodes and extensive meningeal lymphangiogenesis in the surrounding dura. Meningeal lymphangiogenesis was also evident in mice prepared with the commonly used chronic cranial window. Glymphatic influx of a CSF tracer was significantly enhanced at 30 d postsurgery in both awake and ketamine-xylazine anesthetized mice with electrodes, supporting the concept that glymphatic influx and intracranial lymphatic drainage are interconnected. Altogether, the experimental results provide clear evidence that chronic implantation of EEG electrodes is associated with significant changes in the brain's fluid transport system. Future studies involving EEG recordings and chronic cranial windows must consider the physiological consequences of cranial implants, which include glial scarring, meningeal lymphangiogenesis, and increased glymphatic activity.SIGNIFICANCE STATEMENT This study shows that implantation of extradural electrodes provokes meningeal lymphangiogenesis, enhanced glymphatic influx of CSF, and reactive gliosis. The analysis based on CSF tracer injection in combination with immunohistochemistry showed that chronically implanted electroencephalography electrodes were surrounded by lymphatic sprouts originating from lymphatic vasculature along the dural sinuses and the middle meningeal artery. Likewise, chronic cranial windows provoked lymphatic sprouting. Tracer influx assessed in coronal slices was increased in agreement with previous reports identifying a close association between glymphatic activity and the meningeal lymphatic vasculature. Lymphangiogenesis in the meninges and altered glymphatic fluid transport after electrode implantation have not previously been described and adds new insights to the foreign body response of the CNS.
Assuntos
Dura-Máter/metabolismo , Eletrodos Implantados/efeitos adversos , Reação a Corpo Estranho/etiologia , Gliose/etiologia , Sistema Glinfático/fisiologia , Linfangiogênese , Animais , Astrócitos/fisiologia , Córtex Cerebral/patologia , Líquido Cefalorraquidiano/fisiologia , Dura-Máter/patologia , Eletroencefalografia/instrumentação , Feminino , Reação a Corpo Estranho/metabolismo , Gliose/metabolismo , Gliose/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/fisiologia , Técnica de Janela Cutânea , Fases do Sono/fisiologiaRESUMO
Sensory cortex exhibits receptive field plasticity throughout life in response to changes in sensory experience and offers the experimental possibility of aligning functional changes in receptive field properties with underpinning structural changes in synapses. We looked at the effects on structural plasticity of two different patterns of whisker deprivation in male and female mice: chessboard deprivation, which causes functional plasticity; and all deprived, which does not. Using 2-photon microscopy and chronic imaging through a cranial window over the barrel cortex, we found that layer 2/3 neurones exhibit robust structural plasticity, but only in response to whisker deprivation patterns that cause functional plasticity. Chessboard pattern deprivation caused dual-component plasticity in layer 2/3 by (1) increasing production of new spines that subsequently persisted for weeks and (2) enlarging spine head sizes in the preexisting stable spine population. Structural plasticity occurred on basal dendrites, but not apical dendrites. Both components of plasticity were absent in αCaMKII-T286A mutants that lack LTP and experience-dependent potentiation in barrel cortex, implying that αCaMKII autophosphorylation is not only important for stabilization and enlargement of spines, but also for new spine production. These studies therefore reveal the relationship between spared whisker potentiation in layer 2/3 neurones and the form and mechanisms of structural plasticity processes that underlie them.SIGNIFICANCE STATEMENT This study provides a missing link in a chain of reasoning that connects LTP to experience-dependent functional plasticity in vivo We found that increases in dendritic spine formation and spine enlargement (both of which are characteristic of LTP) only occurred in barrel cortex during sensory deprivation that produced potentiation of sensory responses. Furthermore, the dendritic spine plasticity did not occur during sensory deprivation in mice lacking LTP and experience-dependent potentiation (αCaMKII autophosphorylation mutants). We also found that the dual-component dendritic spine plasticity only occurred on basal dendrites and not on apical dendrites, thereby resolving a paradox in the literature suggesting that layer 2/3 neurones lack structural plasticity in response to sensory deprivation.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Espinhas Dendríticas/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/enzimologia , Privação Sensorial/fisiologia , Córtex Somatossensorial/fisiopatologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Tamanho Celular , Espinhas Dendríticas/ultraestrutura , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/ultraestrutura , Fosforilação , Processamento de Proteína Pós-Traducional , Técnica de Janela Cutânea , Córtex Somatossensorial/citologia , Distúrbios Somatossensoriais/fisiopatologia , Vibrissas/lesões , Vibrissas/inervaçãoRESUMO
Human skin graft mouse models are widely used to investigate and develop therapeutic strategies for the severe generalized form of recessive dystrophic epidermolysis bullosa (RDEB), which is caused by biallelic null mutations in COL7A1 and the complete absence of type VII collagen (C7). Most therapeutic approaches are focused on reintroducing C7. Therefore, C7 and anchoring fibrils are widely used as readouts in therapeutic research with skin graft models. In this study, we investigated the expression pattern of human and murine C7 in a grafting model, in which human skin is reconstituted out of in vitro cultured keratinocytes and fibroblasts. The model revealed that murine C7 was deposited in both human healthy control and RDEB skin grafts. Moreover, we found that murine C7 is able to form anchoring fibrils in human grafts. Therefore, we advocate the use of human-specific antibodies when assessing the reintroduction of C7 using RDEB skin graft mouse models.
Assuntos
Colágeno Tipo VII/biossíntese , Colágeno Tipo VII/metabolismo , Epidermólise Bolhosa Distrófica/patologia , Fibroblastos/metabolismo , Queratinócitos/metabolismo , Transplante de Pele , Animais , Anticorpos Heterófilos/imunologia , Membrana Basal/metabolismo , Células Cultivadas , Colágeno Tipo VII/deficiência , Colágeno Tipo VII/genética , Colágeno Tipo VII/imunologia , Derme/patologia , Epidermólise Bolhosa Distrófica/imunologia , Feminino , Fibroblastos/transplante , Expressão Gênica , Xenoenxertos , Humanos , Queratinócitos/transplante , Masculino , Camundongos , Camundongos SCID , Modelos Animais , Técnica de Janela CutâneaRESUMO
BACKGROUND: Lack of data on skin whitening (SW) among Cameroonian female university students prompted us to undertake the present study which aimed at assessing the knowledge, attitudes, practices and motivations of female university students vis-a-vis SW. METHODS: This was a cross-sectional study conducted from January to April 2013 in 4 university campuses of Yaoundé, Cameroon. Any female student regularly registered in one of the study sites, who was present at the campus when the investigator visited and volunteered to participate in the study was enrolled. RESULTS: Overall, we recruited 620 female students, their ages ranging from 16 to 46 years with a mean of 21.3 ± 2.9 years. Only 87 participants (14%) found that SW was a good practice. One hundred and sixty nine respondents (27.3%) were currently practicing SW with no age difference when compared to their counterparts (p = 0.09). The desire to have a uniform body skin color was the prevailing reason motivating the practice of SW (39.1%), followed by the need to have a soft skin (29%). Assessment of levels of knowledge regarding advantages of the black skin and deleterious effects of SW showed excellent scores (≥75% of good answers) only in 6.1 and 0.5% of cases respectively, with no difference between those practicing SW or not (all p values > 0.05). CONCLUSION: The practice of SW is common among Cameroonian female university students who should therefore be educated on the advantages of the black skin and the harmful effects of SW.
Assuntos
Conhecimentos, Atitudes e Prática em Saúde , Estudantes/psicologia , Adolescente , Adulto , Camarões , Distribuição de Qui-Quadrado , Estudos Transversais , Feminino , Humanos , Pessoa de Meia-Idade , Motivação , Técnica de Janela Cutânea , Universidades/organização & administraçãoRESUMO
Cell-responsive hydrogels hold tremendous potential as cell delivery devices in regenerative medicine. In this study, we developed a hydrogel-based cell delivery vehicle, in which the encapsulated cell cargo control its own release from the vehicle in a protease-independent manner. Specifically, we have synthesized a modified poly(ethylene glycol) (PEG) hydrogel that undergoes degradation responding to cell-secreted molecules by incorporating disulfide moieties onto the backbone of the hydrogel precursor. Our results show the disulfide-modified PEG hydrogels disintegrate seamlessly into solution in presence of cells without any external stimuli. The rate of hydrogel degradation, which ranges from hours to months, is found to be dependent upon the type of encapsulated cells, cell number, and fraction of disulfide moieties present in the hydrogel backbone. The differentiation potential of human mesenchymal stem cells released from the hydrogels is maintained in vitro. The in vivo analysis of these cell-laden hydrogels, through a dorsal window chamber and intramuscular implantation, demonstrated autonomous release of cells to the host environment. The hydrogel-mediated implantation of cells resulted in higher cell retention within the host tissue when compared to that without a biomaterial support. Biomaterials that function as a shield to protect cell cargos and assist their delivery in response to signals from the encapsulated cells could have a wide utility in cell transplantation and could improve the therapeutic outcomes of cell-based therapies.
Assuntos
Materiais Biocompatíveis/administração & dosagem , Transplante de Células/métodos , Hidrogéis/administração & dosagem , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Polietilenoglicóis/administração & dosagem , Medicina Regenerativa/métodos , Transplantes/metabolismo , Implantes Absorvíveis , Adipogenia/efeitos dos fármacos , Animais , Materiais Biocompatíveis/metabolismo , Sobrevivência Celular , Células Cultivadas , Dissulfetos/administração & dosagem , Dissulfetos/metabolismo , Sobrevivência de Enxerto , Xenoenxertos , Humanos , Hidrogéis/metabolismo , Injeções Intramusculares , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Osteogênese/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Polietilenoglicóis/síntese química , Polietilenoglicóis/metabolismo , Técnica de Janela Cutânea , SolubilidadeRESUMO
Tissue repair processes are characterized by the biphasic recruitment of distinct subpopulations of blood monocytes, including classical ("inflammatory") monocytes (IMs, Ly6C(hi)Gr1(+)CX3CR1(lo)) and non-classical anti-inflammatory monocytes (AMs, Ly6C(lo)Gr1(-)CX3CR1(hi)). Drug-eluting biomaterial implants can be used to tune the endogenous repair process by the preferential recruitment of pro-regenerative cells. To enhance recruitment of AMs during inflammatory injury, a novel N-desulfated heparin-containing poly(ethylene glycol) diacrylate (PEG-DA) hydrogel was engineered to deliver exogenous stromal derived factor-1α (SDF-1α), utilizing the natural capacity of heparin to sequester and release growth factors. SDF-1α released from the hydrogels maintained its bioactivity and stimulated chemotaxis of bone marrow cells in vitro. Intravital microscopy and flow cytometry demonstrated that SDF-1α hydrogels implanted in a murine dorsal skinfold window chamber promoted spatially-localized recruitment of AMs relative to unloaded internal control hydrogels. SDF-1α delivery stimulated arteriolar remodeling that was correlated with AM enrichment in the injury niche. SDF-1α, but not unloaded control hydrogels, supported sustained arteriogenesis and microvascular network growth through 7 days. The recruitment of AMs correlated with parameters of vascular remodeling suggesting that tuning the innate immune response by biomaterial SDF-1α release is a promising strategy for promoting vascular remodeling in a spatially controlled manner.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Microvasos/fisiologia , Monócitos/efeitos dos fármacos , Animais , Arteríolas/crescimento & desenvolvimento , Arteríolas/ultraestrutura , Células da Medula Óssea/fisiologia , Células Cultivadas , Quimiocina CXCL12/administração & dosagem , Citometria de Fluxo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Heparina , Hidrogéis , Inflamação , Microscopia Intravital , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Polietilenoglicóis , Quimera por Radiação , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Técnica de Janela Cutânea , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologiaRESUMO
Myeloid-derived CD11b(+)Gr1(+) suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are considered a major obstacle for effective adoptive T cell therapy. Myeloid cells suppress naive T cell proliferation ex vivo and can prevent the generation of T cell responses in vivo. We find, however, that adoptively transferred immune T cells eradicate well-established tumors in the presence of MDSCs and TAMs, which are strongly immunosuppressive ex vivo. These MDSCs and TAMs were comparable in numbers and immunosuppressive capacity among different tumor models. Longitudinal microscopy of tumors in vivo revealed that after T cell transfer, tumor vasculature and cancer cells disappeared simultaneously. During T cell-mediated tumor destruction, the tumor stroma contained abundant myeloid cells (mainly TAMs) that retained their suppressive properties. Preimmunized but not naive mice resisted immune suppression caused by an unrelated tumor burden, supporting the idea that in vivo, myeloid immunosuppressive cells can suppress naive but not memory T cell responses.
Assuntos
Imunoterapia Adotiva , Macrófagos/imunologia , Células Mieloides/imunologia , Neoplasias Experimentais/terapia , Subpopulações de Linfócitos T/transplante , Evasão Tumoral/imunologia , Animais , Antígeno CD11b/análise , Vacinas Anticâncer/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Homeodomínio/genética , Imunização , Memória Imunológica , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/terapia , Receptores de Quimiocinas/análise , Técnica de Janela Cutânea , Subpopulações de Linfócitos T/imunologia , Carga TumoralRESUMO
Optical imaging techniques have shown tremendous potential for assessing cutaneous microcirculation, but the imaging depth and contrast is limited by the strong scattering of skin. Current skin windows have to be fulfilled by surgical operation and suffer from some side effects. In this study, a switchable skin window was developed by topical application of an optical clearing agent (OCA) and saline on rat skin in vivo. The validity of the skin window was evaluated by the laser speckle contrast imaging technique, and the safety of OCA to the body was tested through histologic examinations. The results indicated that administration of OCA or saline on rat skin in vivo can open or close the window of skin repeatedly for three days. With the repair effect of hyaluronic acid and Vaseline, it is able to repeatedly visualize the dermal blood vessels and flow distribution. Long-term observation shows that there is no abnormal reflection in micro-structure, body weight, organ coefficients, histopathologic lesions, or toxic reactions compared with a control group. This switchable window will provide an effective tool not only for cutaneous microcirculation with laser speckle contrast imaging, but also for diagnosis and treatment of peripheral vascular diseases, including tumor research with various optical imaging techniques.
Assuntos
Microvasos/anatomia & histologia , Imagem Óptica/métodos , Técnica de Janela Cutânea/métodos , Pele/irrigação sanguínea , Administração Tópica , Análise de Variância , Animais , Histocitoquímica , Ácido Hialurônico/administração & dosagem , Rim/citologia , Rim/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Vaselina/administração & dosagem , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/farmacologia , Ratos , Ratos Sprague-Dawley , Pele/química , Pele/efeitos dos fármacos , Tiadiazinas/administração & dosagem , Tiadiazinas/efeitos adversos , Tiadiazinas/farmacologiaRESUMO
CONCLUSION: The study showed the value of using intravital microscopy (IVM) analysis for the study of neoangiogenesis. It demonstrated that the model and the analytical methodology could be used to evaluate in detail the effects of treatment strategies for solid tumours. OBJECTIVES: Neoangiogenesis is a key component of tumour progression, invasion and metastasis. In clinical trials monoclonal antibodies specific for vascular endothelial growth factor - VEGF (bevacizumab) - have been shown to significantly affect tumour progression when given in combination with standard chemotherapy, and also to improve the overall survival of patients. For squamous cell carcinoma of the head and neck (HNSCC), we still await definitive evidence of the effect of such treatment. The present study was designed to investigate the anti-angiogenesis effect of beviacizumab in green fluorescent protein (GFP)-labelled HNSCC xenografts using IVM technology. METHODS: We performed IVM and used image analysis for quantification of angiogenesis and of effects of bevacizumab on cell viability, combined with histochemical and immunohistochemical analysis to standardize the digital analysis of changes in tumour vascularization and cell viability. RESULTS: We found significant effects of bevacizumab on angiogenesis and cancer cell survival in HNSCC. Repeated injections of bevacizumab were found to provide the greatest effects.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Carcinoma de Células Escamosas/irrigação sanguínea , Dermoscopia/métodos , Modelos Animais de Doenças , Transplante de Neoplasias/patologia , Neovascularização Patológica/patologia , Neoplasias Otorrinolaringológicas/irrigação sanguínea , Técnica de Janela Cutânea , Animais , Bevacizumab , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Transplante HeterólogoRESUMO
Recombinant tissue plasminogen activator (r-tPA) is the drug of choice for thrombolysis, but it is associated with a significant risk of bleeding and is not always successful. By cleaving von Willebrand factor (VWF), the metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) down-regulates thrombus formation in injured vessels. We investigated whether recombinant ADAMTS13 (r-ADAMTS13) induces thrombolysis in vivo in mice. Thrombosis was produced by ferric chloride-induced (FeCl(3)) injury in the venules of a dorsal skinfold chamber. Phosphate-buffered saline (PBS, vehicle), r-tPA or r-ADAMTS13, supplemented with hirudin (to stop on-going thrombin generation), was directly applied onto the occluded vessel, and thrombus dissolution was evaluated by intravital microscopy. The incidence of blood flow restoration significantly increased 30 minutes (min) after r-ADAMTS13 vs. PBS treatment (60% vs. 0%, p<0.05) and 60 min after r-tPA treatment (75% vs. 17%, p<0.05). Both r-tPA and r-ADAMTS13 significantly reduced thrombus size 60 min after their superfusion (53.2% and 62.3% of the initial thrombus size, p<0.05 and p<0.01, respectively). Bleeding occurred in all r-tPA-treated chambers, while it was absent in mice treated with r-ADAMTS13 or PBS. We observed that, similar to r-tPA, r-ADAMTS13 can dissolve occlusive thrombi induced by FeCl(3) injury in venules. In contrast to r-tPA, the in vivo thrombolytic effect of ADAMTS13 was not associated with any signs of haemorrhage. ADAMTS13 could represent a new therapeutic option for thrombolysis.
Assuntos
Proteínas ADAM/uso terapêutico , Fibrinolíticos/uso terapêutico , Terapia Trombolítica , Trombose Venosa/tratamento farmacológico , Proteínas ADAM/farmacologia , Proteínas ADAM/toxicidade , Proteína ADAMTS13 , Animais , Cloretos/toxicidade , Sistemas Computacionais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Compostos Férricos/toxicidade , Fibrinolíticos/farmacologia , Fibrinolíticos/toxicidade , Hemorragia/induzido quimicamente , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação/efeitos dos fármacos , Microscopia/métodos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Proteínas Recombinantes/toxicidade , Técnica de Janela Cutânea , Terapia Trombolítica/efeitos adversos , Ativador de Plasminogênio Tecidual/farmacologia , Ativador de Plasminogênio Tecidual/uso terapêutico , Trombose Venosa/induzido quimicamente , VênulasRESUMO
Ratiometric fluorescence-imaging technique is commonly used to measure extracellular pH in tumors and surrounding tissue within a dorsal skin-fold window chamber. Using a pH-sensitive fluorophore such as carboxy SNARF-1 one can measure pH distributions with high precision. However, it is often observed that the measured pH is lower than expected, with a bias that varies from one image to another. A comprehensive analysis of possible error sources is presented. These error sources include photon noise, estimator bias, instrument errors, temperature, and calibration errors from biological factors.
Assuntos
Microambiente Celular/fisiologia , Espaço Extracelular/química , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Técnica de Janela Cutânea/métodos , Animais , Benzopiranos/análise , Benzopiranos/química , Calibragem , Linhagem Celular Tumoral , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Camundongos , Naftóis/análise , Naftóis/química , Neoplasias Experimentais/química , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Distribuição de Poisson , Rodaminas/análise , Rodaminas/químicaRESUMO
Alpha-1-antitrypsin (A1AT) is an important inhibitor of neutrophil proteases including elastase, cathepsin G, and proteinase 3. Transcription profiling data suggest that A1AT is expressed by human neutrophil granulocytes during all developmental stages. A1AT has hitherto only been found associated with azurophile granules in neutrophils indicative of A1AT expression being restricted to the promyelocyte stage. We examined the localization and production of A1AT in healthy donor neutrophils and found A1AT to be a constituent of all granule subtypes and to be released from neutrophils following stimulation. A1AT is produced at all stages of myeloid maturation in the bone marrow. The production increases as neutrophils enter circulation and increases further upon migration to tissues as observed in skin windows and when blood neutrophils are incubated with granulocyte colony-stimulating factor. Neutrophils from patients with A1AT-deficiency carrying the (PI)ZZ mutation in the A1AT gene appeared structurally and functionally normal, but A1AT produced in leukocytes of these patients lacked the ability to bind proteases efficiently. We conclude that A1AT generation and release from neutrophils add significantly to the antiprotease levels in tissues during inflammation. Impaired binding of neutrophil A1AT to serine proteases in patients with (PI)ZZ mutations may enhance their susceptibility to the development of emphysema.
Assuntos
Neutrófilos/metabolismo , alfa 1-Antitripsina/biossíntese , Estudos de Casos e Controles , Degranulação Celular/efeitos dos fármacos , Diferenciação Celular , Grânulos Citoplasmáticos/metabolismo , Eosinófilos/enzimologia , Exocitose/efeitos dos fármacos , Genótipo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Técnicas In Vitro , Transplante de Fígado , Transplante de Pulmão , Microscopia Eletrônica de Transmissão , Mutação , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Técnica de Janela Cutânea , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/enzimologia , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/patologia , Deficiência de alfa 1-Antitripsina/cirurgiaRESUMO
BACKGROUND & AIMS: Deranged neutrophil function in alcoholic hepatitis has been shown to be transmissible to normal neutrophils by patient plasma. The aims of this study were (i) to evaluate whether patients with stable cirrhosis have a similar transmissible neutrophil defect and (ii) to explore the possible mechanisms. METHODS: Plasma samples from 108 stable cirrhotic patients (Child A or B: 58; Child C: 50) and matched controls were incubated with normal neutrophils. Neutrophil resting respiratory burst, phagocytosis, and toll-like receptors 2, 4, and 9 expressions as well as plasma endotoxin, bacterial DNA, and cytokines were measured. In a separate study, eight patients and five controls were studied using a novel 'skin-window' technique to evaluate neutrophil function in an area of induced sterile inflammation. RESULTS: Patient plasma induced neutrophil phagocytic dysfunction was greater in patients with more severe disease and was associated with increased expression of toll-like receptors 2 and 4. An increased resting respiratory burst was observed in a subset of patients, showing higher levels of inflammatory cytokines and more pronounced phagocytic impairment. No correlation was found with endotoxemia or bacterial DNA. In patients with compensated cirrhosis and apparently normal neutrophil function, the 'skin-window' study disclosed a severe phagocytic defect at the site of inflammation. Significantly higher levels of neutrophil elastase and IL-8 were found in the blister fluid. CONCLUSIONS: Stable cirrhosis is characterized by neutrophil phagocytic dysfunction which may be subtle and only revealed in inflamed peripheral tissues where excessive inflammatory mediators continue to be released.
Assuntos
Cirrose Hepática/imunologia , Neutrófilos/fisiologia , Idoso , Células Cultivadas , Citocinas/sangue , DNA Bacteriano/sangue , Endotoxinas/sangue , Feminino , Humanos , Inflamação/imunologia , Cirrose Hepática/sangue , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Neutrófilos/metabolismo , Elastase Pancreática/metabolismo , Fagocitose/fisiologia , Explosão Respiratória/fisiologia , Técnica de Janela Cutânea , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismoRESUMO
Alterations in neutrophil extravasation are seen in disease states and in response to therapeutics. To investigate neutrophil extravasation during the acute inflammatory response, a skin-window technique used in humans was adapted for use in cynomolgus macaques (Macaca fasicularis). Modulation of neutrophil extravasation was attempted with systemic methotrexate and local application of the anaphylatoxin recombinant C5a (rC5a). On day 1, skin windows were created in 4 ketamine-anesthetized monkeys on both forearms by mildly abrading the skin and then overlaying the abrasions with filter paper either saturated in saline or rC5a for 6 h. At 2.5 h prior to generation of new skin windows on day 2, the monkeys received 4.5 mg methotrexate IM, and skin windows and treatment with saline or rC5a were repeated on new forearm sites on each monkey. All papers were analyzed for albumin, neutrophil number, and the neutrophil chemoattractant IL8. Day 1 albumin levels did not differ between groups, indicating consistent abrasion. Methotrexate given prior to the day 2 abrasions reduced neutrophil extravasation and IL8 levels compared with those on day 1. rC5a partially abrogated the methotrexate-induced reduction in neutrophil extravasation and IL8 production. The skin-window technique was well tolerated by the monkeys and successfully accommodated measurement of changes in neutrophil extravasation in response to inflammatory modulators.
Assuntos
Macaca fascicularis , Neutrófilos/patologia , Técnica de Janela Cutânea/veterinária , Animais , Antígeno CD11a/metabolismo , Metotrexato/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Técnica de Janela Cutânea/efeitos adversos , Técnica de Janela Cutânea/métodosRESUMO
This study tested the hypothesis that TRAIL could play a role in regulating monocyte migration. TRAIL has been widely studied for its anti-tumor function and signaling mechanisms. Using chemotaxis and mouse air-pouch model analyses, we determined that TRAIL-induced chemotactic migration of THP-1 human leukemia and LPS-primed primary human monocytes as well as LPS-stimulated BALB/c mouse monocytes in vivo. To expand the understanding of the TRAIL signaling pathway in this process, we found that the TRAIL receptor DR4 was highly expressed in THP-1 and LPS-primed primary monocytes but not in the non-primed primary monocytes. DR4 neutralization antibody specifically suppressed TRAIL-induced migration of the monocytes. Furthermore, PI3K, Rho GTPase and its downstream effectors, MLC and Pak1, were activated during cell migration. PI3K inhibitors and dominant negative mutants of RhoGTPase blocked monocyte migration toward TRAIL, indicating that PI3K and RhoGTPases were involved in the migration signaling. The DR4 neutralization antibody blocked the activation of PI3K and Rho GTPase effectors in the cells. Thus, these data support the hypothesis that TRAIL induces monocyte migration mediated by TRAIL receptor DR4 via the RhoGTPase signaling pathway. This study is expected to provide novel evidence of the non-apoptotic function of TRAIL in immune defense.
Assuntos
Quimiotaxia/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Linhagem Celular Tumoral/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células HL-60/efeitos dos fármacos , Humanos , Imunidade Inata , Rim , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Distribuição Aleatória , Ratos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Proteínas Recombinantes/farmacologia , Técnica de Janela Cutânea , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
OBJECTIVES: The purpose was to evaluate inflammatory and microcirculatory reactions after implantation of various calcium phosphate bone substitutes in an in vivo model. METHODS: Calcium phosphate-based bone substitutes were implanted in dorsal skinfold chambers of mice. Intravital fluorescence microscopy was performed to measure inflammatory and microcirculatory reactions based on functional vessel density (FVD), capillary leakage, and relative white blood cell velocity (rWBCV). RESULTS: An increase of FVD was observed in all groups and the capillary leakage grew with a level of significance (p < 0.001). The fraction of rolling and sticking leukocytes (rWBCV) was highest at the beginning of the trial and decreased during the course. CONCLUSIONS: There are differences in microvascular soft tissue reactions between various calcium phosphate bone substitutes, but inflammatory reactions were moderate, and the results revealed no reasons which explain the sporadic failure of the tested substances under clinical conditions.
Assuntos
Substitutos Ósseos , Fosfatos de Cálcio , Síndrome de Vazamento Capilar/patologia , Reação a Corpo Estranho/patologia , Microcirculação/fisiologia , Microvasos/patologia , Animais , Velocidade do Fluxo Sanguíneo , Adesão Celular/fisiologia , Feminino , Contagem de Leucócitos , Migração e Rolagem de Leucócitos , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica/fisiologia , Pele/irrigação sanguínea , Técnica de Janela CutâneaRESUMO
Abnormal microvascular physiology and function is common in many diseases. Numerous pathologies include hypervascularity, aberrant angiogenesis, or abnormal vascular remodeling among the characteristic features of the disease, and quantitative imaging and measurement of microvessel function can be important to increase understanding of these diseases. Several optical techniques are useful for direct imaging of microvascular function. Spectral imaging is one such technique that can be used to assess microvascular oxygen transport function with high spatial and temporal resolution in microvessel networks through measurements of hemoglobin saturation. We highlight novel observation made with our intravital microscopy spectral imaging system employed with mouse dorsal skin-fold window chambers for imaging hemoglobin saturation in microvessel networks. Specifically, we image acute oxygenation fluctuations in a tumor microvessel network, the development of arteriovenous malformations in a mouse model of hereditary hemorrhagic telangiectasia, and the formation of spontaneous and induced microvascular thromboses and occlusions.
Assuntos
Anastomose Arteriovenosa/fisiopatologia , Análise Espectral/métodos , Trombose/fisiopatologia , Animais , Neoplasias da Mama , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Hemoglobinas/metabolismo , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Nus , Microvasos/fisiologia , Microvasos/fisiopatologia , Transplante de Neoplasias , Neovascularização Patológica/fisiopatologia , Oxigênio/metabolismo , Técnica de Janela Cutânea , Telangiectasia Hemorrágica HereditáriaRESUMO
BACKGROUND: Diclofenac, like selective cyclooxygenase-2 inhibitors, which alter vascular levels of platelet active prostaglandins, has been reported to increase rates of acute myocardial infarction. OBJECTIVE: The study was performed to investigate, in an animal model of arterial thrombosis in vivo, whether diclofenac differentially influences platelet activation and thrombosis in vessels under non-stimulated conditions or during acute systemic inflammation, such as induced by tumor necrosis factor-alpha (TNF-alpha). METHODS: Platelet-vessel wall interaction (PVWI), firm platelet adhesion and arterial thrombosis following vessel injury were analyzed by intravital microscopy in arterioles of hamsters in the dorsal skinfold chamber model. Prostacyclin [prostaglandin I(2) (PGI(2))] and thromboxane A(2) (TxA(2)) metabolites were measured. In vitro, endothelial adhesion molecule expression in cultured human microvascular endothelial cells was analyzed. RESULTS: Under non-stimulated conditions, diclofenac (1 mg kg(-1)) enhanced PVWI, which was not mediated by increased adhesion molecule expression, but by decreased systemic PGI(2) levels. Following ferric chloride-induced endothelial injury, diclofenac accelerated thrombotic vessel occlusion time, an effect that was reversed by the stable PGI(2) analog iloprost. TNF-alpha, through induction of endothelial adhesion molecule expression, also enhanced PVWI, firm adhesion, and arterial thrombosis, but simultaneous treatment with TNF-alpha and diclofenac did not have an additive effect. CONCLUSIONS: By decreasing levels of PGI(2) without, at the same time, altering prothrombotic TxA(2) levels, diclofenac can exert prothrombotic effects. However, this is not the case when an inflammatory situation is created by TNF-alpha treatment. These data may explain the enhanced risk of acute myocardial infarction observed in patients taking diclofenac.
Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Inibidores de Ciclo-Oxigenase/toxicidade , Diclofenaco/toxicidade , Ativação Plaquetária/efeitos dos fármacos , Trombose/induzido quimicamente , 6-Cetoprostaglandina F1 alfa/sangue , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Arteríolas/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Cloretos , Cricetinae , Inibidores de Ciclo-Oxigenase/farmacologia , Diclofenaco/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Compostos Férricos/toxicidade , Humanos , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Adesividade Plaquetária/efeitos dos fármacos , Técnica de Janela Cutânea , Tromboplastina/análise , Trombose/sangue , Tromboxano B2/sangue , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
INTRODUCTION: Recent anticancer drugs have been made larger to pass selectively through tumor vessels and stay in the interstitium. Understanding drug movement in association with its size at the single-molecule level and estimating the time needed to reach the targeted organ is indispensable for optimizing drug delivery because single cell-targeted therapy is the ongoing paradigm. This report describes the tracking of single solid nanoparticles in tumor xenografts and the estimation of arrival time. METHODS: Different-sized nanoparticles measuring 20, 40, and 100 nm were injected into the tail vein of the female Balb/c nu/nu mice bearing human breast cancer on their backs. The movements of the nanoparticles were visualized through the dorsal skin-fold chamber with the high-speed confocal microscopy that we manufactured. RESULTS: An analysis of the particle trajectories revealed diffusion to be inversely related to the particle size and position in the tumor, whereas the velocity of the directed movement was related to the position. The difference in the velocity was the greatest for 40-nm particles in the perivascular to the intercellular region: difference = 5.8 nm/s. The arrival time of individual nanoparticles at tumor cells was simulated. The estimated times for the 20-, 40-, and 100-nm particles to reach the tumor cells were 158.0, 218.5, and 389.4 minutes, respectively, after extravasation. CONCLUSIONS: This result suggests that the particle size can be individually designed for each goal. These data and methods are also important for understanding drug pharmacokinetics. Although this method may be subject to interference by surface molecules attached on the particles, it has the potential to elucidate the pharmacokinetics involved in constructing novel drug-delivery systems involving cell-targeted therapy.