RESUMO
The acute inflammatory response is a double-edged sword. On the one hand, it plays a key role in initial host defense, particularly against many infections. On the other hand, its aim is imprecise, and as a consequence, when it is drawn into battle, it can cause collateral damage in tissues. In situations where the inciting stimulus is sterile, the cost-benefit ratio may be high; because of this, sterile inflammation underlies the pathogenesis of a number of diseases. Although there have been major advances in our understanding of how microbes trigger inflammation, much less has been learned about this process in sterile situations. This review focuses on a subset of the many sterile stimuli that can induce inflammation-specifically dead cells and a variety of irritant particles, including crystals, minerals, and protein aggregates. Although this subset of stimuli is structurally very diverse and might appear to be unrelated, there is accumulating evidence that the innate immune system may recognize them in similar ways and stimulate the sterile inflammatory response via common pathways. Here we review established and emerging data about these responses.
Assuntos
Inflamação/imunologia , Animais , Morte Celular , Humanos , Imunidade Inata , Inflamação/metabolismo , Inflamação/patologia , Inflamação/terapia , Interleucina-1/imunologia , Interleucina-1/metabolismo , Transdução de SinaisRESUMO
Microfluidic devices support developmental and mechanobiology studies by enabling the precise control of electrical, chemical, and mechanical stimuli at the microscale. Here, we describe the fabrication of customizable microfluidic devices and demonstrate their efficacy in applying mechanical loads to micro-organs and whole organisms, such as Drosophila embryos. The fabrication technique consists in the use of xurography to define channels and chambers using thin layers of thermoplastics and glass. The superposition of layers followed by thermal lamination produces robust and reproducible devices that are easily adapted for a variety of experiments. The integration of deformable layers and glass in these devices facilitates the imaging of cellular and molecular dynamics in biological specimens under mechanical loads. The method is highly adaptable for studies in mechanobiology.
Assuntos
Embrião não Mamífero , Dispositivos Lab-On-A-Chip , Animais , Drosophila/embriologia , Fenômenos Biomecânicos , Estresse Mecânico , Drosophila melanogaster/embriologia , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Desenho de EquipamentoRESUMO
The semen-derived enhancer of viral infection (SEVI) is a positively charged amyloid fibril that is derived from a self-assembling proteolytic cleavage fragment of prostatic acid phosphatase (PAP(248-286)). SEVI efficiently facilitates HIV-1 infection in vitro, but its normal physiologic function remains unknown. In light of the fact that other amyloidogenic peptides have been shown to possess direct antibacterial activity, we investigated whether SEVI could inhibit bacterial growth. Neither SEVI fibrils nor the unassembled PAP(248-286) peptide had significant direct antibacterial activity in vitro. However, SEVI fibrils bound to both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Neisseria gonorrhoeae) bacteria, in a charge-dependent fashion. Furthermore, SEVI fibrils but not the monomeric PAP(248-286) peptide promoted bacterial aggregation and enhanced the phagocytosis of bacteria by primary human macrophages. SEVI also enhanced binding of bacteria to macrophages and the subsequent release of bacterially induced proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-1ß). Finally, SEVI fibrils inhibited murine vaginal colonization with Neisseria gonorrhoeae. These findings demonstrate that SEVI has indirect antimicrobial activity and that this activity is dependent on both the cationic charge and the fibrillar nature of SEVI.
Assuntos
Amiloide/metabolismo , Amiloide/farmacologia , Antibacterianos/farmacologia , Macrófagos/microbiologia , Fagocitose/efeitos dos fármacos , Proteínas Tirosina Fosfatases/química , Sêmen/química , Staphylococcus aureus/efeitos dos fármacos , Vaginose Bacteriana/prevenção & controle , Fosfatase Ácida , Amiloide/química , Animais , Antibacterianos/metabolismo , Citocinas/biossíntese , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Tirosina Fosfatases/metabolismo , Sêmen/metabolismo , Staphylococcus aureus/metabolismoRESUMO
BACKGROUND: Multidetector computer tomography (CT) has been used to diagnose pathologies such as osteoporosis via opportunistic screening, where the assessment of the bone structure and the measurement of bone mineral density (BMD) are of great relevance. PURPOSE: To construct reference BMD values based on the measurement of the attenuation of the L1 vertebral body by multidetector CT scan (in the soft tissue and bone windows) in adult patients and to establish normative ranges by sex and age of BMD values. MATERIALS AND METHODS: A retrospective cross-sectional study of 5080 patients who underwent multidetector CT scan between January and December 2021. Adult patients (≥18 years) with non-contrast multidetector CT scan of the abdomen or thorax-abdomen at a voltage 120 kV. The attenuation of the L1 vertebral body in Hounsfield units (HU) in both windows were compared using the Mann-Whitney U-test with α = 0.05. Additionally, the quartiles of the BMD were constructed (in both windows) grouped by sex and age. RESULTS: Only 454 (51.30 ± 15.89 years, 243 women) patients met the inclusion criteria. There is no difference in BMD values between windows (soft tissue: 163.90 ± 57.13, bone: 161.86 ± 55.80, p = 0.625), mean L1 attenuation decreased linearly with age at a rate of 2 HU per year, and the presence of BMD deficit among patients was high; 152 of 454 (33.48%) patients presented BMD values suggestive of osteoporosis, and of these, approximately half 70 of 454 (15.42%) corresponded to patients with BMD values suggestive of a high risk of osteoporotic fracture. CONCLUSIONS: From clinical practice, the bone mineral density (BMD) of a patient in either window below the first quartile for age- and sex-matched peers suggests a deficit in BMD that cannot be ignored and requires clinical management that enables identification of the etiology, its evolution, and the consequences of this alteration.
Assuntos
Densidade Óssea , Osteoporose , Humanos , Adulto , Feminino , Estudos Retrospectivos , Estudos Transversais , Absorciometria de Fóton/métodos , Tomografia Computadorizada por Raios X/métodos , Vértebras Lombares/diagnóstico por imagem , Osteoporose/diagnóstico por imagemRESUMO
BACKGROUND: Cerebral blood flow (CBF) is known to be dysregulated in persons with human immunodeficiency virus 1 (HIV-1), for uncertain reasons. This is an important issue because impaired vasoreactivity has been associated with increased risk of ischemic stroke, elevated overall cardiovascular risk and cognitive impairment. METHODS: To test whether dysregulation of CBF might be due to virally-induced neuroinflammation, we used a well-defined animal model (GFAP-driven, doxycycline-inducible HIV-1 Tat transgenic (Tat-tg) mice). We then exposed the mice to a brief hypercapnic stimulus, and assessed cerebrovascular reactivity by measuring 1) changes in cerebral blood flow, using laser Doppler flowmetry and 2) changes in vascular dilation, using in vivo two-photon imaging. RESULTS: Exposure to brief hypercapnia revealed an underlying cerebrovascular pathology in Tat-tg mice. In control animals, brief hypercapnia induced a brisk increase in cortical flow (20.8% above baseline) and vascular dilation, as measured by laser Doppler flowmetry and in vivo two-photon microscopy. These responses were significantly attenuated in Tat-tg mice (11.6% above baseline), but cortical microvascular morphology and capillary density were unaltered, suggesting that the functional pathology was not secondary to vascular remodeling. To examine the mechanistic basis for the diminished cerebrovascular response to brief hypercapnia, Tat-tg mice were treated with 1) gisadenafil, a phosphodiesterase 5 (PDE5) inhibitor and 2) tetrahydrobiopterin (BH4). Gisadenafil largely restored the normal increase in cortical flow following hypercapnia in Tat-tg mice (17.5% above baseline), whereas BH4 had little effect. Gisadenafil also restored the dilation of small (<25 µm) arterioles following hypercapnia (19.1% versus 20.6% diameter increase in control and Tat-tg plus gisadenafil, respectively), although it failed to restore full dilation of larger (>25 µm) vessels. CONCLUSIONS: Taken together, these data show that HIV-associated neuroinflammation can cause cerebrovascular pathology through effects on cyclic guanosine monophosphate (cGMP) metabolism and possibly on PDE5 metabolism.
Assuntos
Dióxido de Carbono , Sistema Cardiovascular/patologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Encefalite/complicações , Encefalite/patologia , Óxido Nítrico/metabolismo , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/metabolismo , Biopterinas/análogos & derivados , Biopterinas/farmacologia , Tempo de Circulação Sanguínea , Células COS , Dióxido de Carbono/farmacologia , Sistema Cardiovascular/virologia , Córtex Cerebral/patologia , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Chlorocebus aethiops , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Encefalite/etiologia , Encefalite/virologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Infecções por HIV/complicações , Infecções por HIV/genética , Humanos , Lectinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Tempo , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
CD8(+) T-cell responses to non-pathogen, cell-associated antigens such as minor alloantigens or peptide-pulsed dendritic cells (DC) are usually strongly dependent on help from CD4(+) T cells. However, some studies have described help-independent primary CD8(+) T-cell responses to cell-associated antigens, using immunization strategies likely to trigger natural killer (NK) cell activation and inflammatory cytokine production. We asked whether NK cell activation by MHC I-deficient cells, or administration of inflammatory cytokines, could support CD4(+) T-cell help-independent primary responses to peptide-pulsed DC. Injection of MHC I-deficient cells cross-primed CD8(+) T-cell responses to the protein antigen ovalbumin (OVA) and the male antigen HY, but did not stimulate CD8(+) T-cell responses in CD4-depleted mice; hence NK cell stimulation by MHC I-deficient cells did not replace CD4(+) T-cell help in our experiments. Dendritic cells cultured with tumour necrosis factor-α (TNF-α) or type I interferon-α (IFN-α) also failed to prime CD8(+) T-cell responses in the absence of help. Injection of TNF-α increased lymph node cellularity, but did not generate help-independent CD8(+) T-cell responses. In contrast, CD4-depleted mice injected with IFN-α made substantial primary CD8(+) T-cell responses to peptide-pulsed DC. Mice deficient for the type I IFN receptor (IFNR1) made CD8(+) T-cell responses to IFNR1-deficient, peptide-pulsed DC; hence IFN-α does not appear to be a downstream mediator of CD4(+) T-cell help. We suggest that primary CD8(+) T-cell responses will become help-independent whenever endogenous IFN-α secretion is stimulated by tissue damage, infection, or autoimmune disease.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Interferon-alfa/imunologia , Sequência de Aminoácidos , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Antígeno H-Y/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Imunização , Interferon-alfa/farmacologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.
Assuntos
Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação , Pesquisa Biomédica , Células Cultivadas , Educação , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/economia , Microfluídica/métodosRESUMO
Microfluidic devices provide a platform for analyzing both natural and synthetic multicellular systems. Currently, substantial capital investment and expertise are required for creating microfluidic devices using standard soft-lithography. These requirements present barriers to entry for many nontraditional users of microfluidics, including developmental biology laboratories. Therefore, fabrication methodologies that enable rapid device iteration and work "out-of-the-box" can accelerate the integration of microfluidics with developmental biology. Here, we have created and characterized low-cost hybrid polyethylene terephthalate laminate (PETL) microfluidic devices that are suitable for cell and micro-organ culture assays. These devices were validated with mammalian cell lines and the Drosophila wing imaginal disc as a model micro-organ. First, we developed and tested PETLs that are compatible with both long-term cultures and high-resolution imaging of cells and organs. Further, we achieved spatiotemporal control of chemical gradients across the wing discs with a multilayered microfluidic device. Finally, we created a multilayered device that enables controllable mechanical loading of micro-organs. This mechanical actuation assay was used to characterize the response of larval wing discs at different developmental stages. Interestingly, increased deformation of the older wing discs for the same mechanical loading suggests that the compliance of the organ is increased in preparation for subsequent morphogenesis. Together, these results demonstrate the applicability of hybrid PETL devices for biochemical and mechanobiology studies on micro-organs and provide new insights into the mechanics of organ development.
RESUMO
Radiation injury in the skin causes radiodermatitis, a condition in which the skin becomes inflamed and the epidermis can break down. This condition causes significant morbidity and if severe it can be an independent factor that contributes to radiation mortality. Radiodermatitis is seen in some settings of radiotherapy for cancer and is also of concern as a complication post-radiation exposure from accidents or weapons, such as a "dirty bomb". The pathogenesis of this condition is incompletely understood. Here we have developed a murine model of radiodermatitis wherein the skin is selectively injured by irradiation with high-energy electrons. Using this model we showed that the interleukin-1 (IL-1) pathway plays a significant role in the development of radiodermatitis. Mice that lack either IL-1 or the IL-1 receptor developed less inflammation and less severe pathological changes in their skin, especially at later time-points. These findings suggest that IL-1 pathway may be a potential therapeutic target for reducing the severity of radiodermatitis.
Assuntos
Interleucina-1/metabolismo , Radiodermite/metabolismo , Radiodermite/patologia , Animais , Elétrons/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Radiodermite/etiologia , Receptores de Interleucina-1/metabolismoRESUMO
Necrosis stimulates inflammation, and this response is medically relevant because it contributes to the pathogenesis of a number of diseases. It is thought that necrosis stimulates inflammation because dying cells release proinflammatory molecules that are recognized by the immune system. However, relatively little is known about the molecular identity of these molecules and their contribution to responses in vivo. Here, we investigated the role of uric acid in the inflammatory response to necrotic cells in mice. We found that dead cells not only released intracellular stores of uric acid but also produced it in large amounts postmortem as nucleic acids were degraded. Using newly developed Tg mice that have reduced levels of uric acid either intracellularly and/or extracellularly, we found that uric acid depletion substantially reduces the cell death-induced inflammatory response. Similar results were obtained with pharmacological treatments that reduced uric acid levels either by blocking its synthesis or hydrolyzing it in the extracellular fluids. Importantly, uric acid depletion selectively inhibited the inflammatory response to dying cells but not to microbial molecules or sterile irritant particles. Collectively, our data identify uric acid as a proinflammatory molecule released from dying cells that contributes significantly to the cell death-induced inflammatory responses in vivo.
Assuntos
Sistema Imunitário/metabolismo , Inflamação/metabolismo , Ácido Úrico/metabolismo , Ácido Úrico/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Células/metabolismo , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , NecroseRESUMO
CD8+ T cells are critically important for immune defense against many viral and bacterial pathogens, and are also key components of cancer immunotherapy. Help from CD4+ T cells is usually essential for optimal CD8+ T cell responses, driving the primary response, the survival of memory cells, and the generation of protective and therapeutic immunity. Understanding the mechanisms of help is thus essential for vaccine design, and for restoring protective immunity in immunosuppressed individuals. Our laboratory has developed an immunization protocol using peptide-pulsed dendritic cells to stimulate help-dependent primary, memory, and secondary CD8+ T cell responses. We have used gene-targeted and T cell receptor transgenic mice to identify two distinct pathways that generate help-dependent and help-independent CD8+ T cell responses, respectively, and are now starting to define the molecular mechanisms underlying these two pathways.