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Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade increasing evidence from preclinical models suggests that cells, which are not normally resident in the lung can be utilized to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathologic remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "-omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.
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Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best prevention, 50% of lung cancer diagnoses occur in people who have quit smoking. Research into treatment options for high-risk patients is constrained to rodent models, which are time-consuming, expensive, and require large cohorts. Embedding precision-cut lung slices (PCLS) within an engineered hydrogel and exposing this tissue to vinyl carbamate, a carcinogen from cigarette smoke, creates an in vitro model of lung cancer premalignancy. Hydrogel formulations are selected to promote early lung cancer cellular phenotypes and extend PCLS viability to six weeks. Hydrogel-embedded PCLS are exposed to vinyl carbamate, which induces adenocarcinoma in mice. Analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content after six weeks reveals that vinyl carbamate induces premalignant lesions with a mixed adenoma/squamous phenotype. Putative chemoprevention agents diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue are validated with hydrogel-embedded human PCLS and results show increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the foundation for more sophisticated ex vivo models that enable the study of carcinogenesis and chemoprevention strategies.
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Neoplasias Pulmonares , Lesões Pré-Cancerosas , Humanos , Camundongos , Animais , Hidrogéis , Neoplasias Pulmonares/patologia , Pulmão/patologia , UretanaRESUMO
Most human killer cell immunoglobulin-like receptors (KIR) are expressed by natural killer (NK) cells and recognize HLA class I molecules as ligands. KIR3DL3 is a conserved but polymorphic inhibitory KIR recognizing a B7 family ligand, HHLA2, and is implicated for immune checkpoint targeting. The expression profile and biological function of KIR3DL3 have been somewhat elusive, so we searched extensively for KIR3DL3 transcripts, revealing highly enriched expression in γδ and CD8+ T cells rather than NK cells. These KIR3DL3-expressing cells are rare in the blood and thymus but more common in the lungs and digestive tract. High-resolution flow cytometry and single-cell transcriptomics showed that peripheral blood KIR3DL3+ T cells have an activated transitional memory phenotype and are hypofunctional. The T cell receptor (TCR) usage is biased toward genes from early rearranged TCR-α variable segments or Vδ1 chains. In addition, we show that TCR-mediated stimulation can be inhibited through KIR3DL3 ligation. Whereas we detected no impact of KIR3DL3 polymorphism on ligand binding, variants in the proximal promoter and at residue 86 can reduce expression. Together, we demonstrate that KIR3DL3 is up-regulated alongside unconventional T cell stimulation and that individuals may vary in their ability to express KIR3DL3. These results have implications for the personalized targeting of KIR3DL3/HHLA2 checkpoint inhibition.
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Linfócitos T CD8-Positivos , Células Matadoras Naturais , Humanos , Ligantes , Timo , Receptores de Antígenos de Linfócitos T alfa-beta , Imunoglobulinas , Receptores KIRRESUMO
The pathogenesis of the marked pulmonary microvasculature injury, a distinguishing feature of COVID-19 acute respiratory distress syndrome (COVID-ARDS), remains unclear. Implicated in the pathophysiology of diverse diseases characterized by endothelial damage, including ARDS and ischemic cardiovascular disease, ceramide and in particular palmitoyl ceramide (C16:0-ceramide) may be involved in the microvascular injury in COVID-19. Using deidentified plasma and lung samples from COVID-19 patients, ceramide profiling by mass spectrometry was performed. Compared with healthy individuals, a specific 3-fold C16:0-ceramide elevation in COVID-19 patient plasma was identified. Compared with age-matched controls, autopsied lungs of individuals succumbing to COVID-ARDS displayed a massive 9-fold C16:0-ceramide elevation and exhibited a previously unrecognized microvascular ceramide-staining pattern and markedly enhanced apoptosis. In COVID-19 plasma and lungs, the C16-ceramide/C24-ceramide ratios were increased and reversed, respectively, consistent with increased risk of vascular injury. Indeed, exposure of primary human lung microvascular endothelial cell monolayers to C16:0-ceramide-rich plasma lipid extracts from COVID-19, but not healthy, individuals led to a significant decrease in endothelial barrier function. This effect was phenocopied by spiking healthy plasma lipid extracts with synthetic C16:0-ceramide and was inhibited by treatment with ceramide-neutralizing monoclonal antibody or single-chain variable fragment. These results indicate that C16:0-ceramide may be implicated in the vascular injury associated with COVID-19.
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COVID-19 , Síndrome do Desconforto Respiratório , Lesões do Sistema Vascular , Humanos , Ceramidas , Pulmão/irrigação sanguíneaRESUMO
EVALI is an acute inflammatory disease in response to lung cell injury induced by electronic cigarettes and vaping devices (EV) frequently containing Vitamin E Acetate or tetrahydrocannabinol additives, in the context of risk factors such as microbial exposure. EVALI resembles a respiratory viral illness that may progress to acute respiratory failure and acute respiratory distress syndrome (ARDS) but can also affect extra pulmonary organs. Manifestations may be severe, leading to death or long-term morbidity and current treatments are largely supportive. While COVID-19 has demanded public and research attention, EVALI continues to affect young individuals and its better understanding via research remains a priority. Although clinical research led to improved recognition of triggers, clinical and pathological manifestations, and natural course of EVALI, important questions remain that require a better understanding of disease pathogenesis. Preclinical models utilizing laboratory animals and cell or tissue culture platforms provide insight into the physiologic and mechanistic consequences of acute and chronic EV exposure, including the characteristics of the respiratory dysfunction and inflammatory response. However, a key limitation in the field is the absence of an established animal model of EVALI. Important areas of research emphasis include identifying triggers and risk factors to understand why only certain vapers develop EVALI, the role of specific lung immune and structural cells in the pathogenesis of EVALI, and the most important molecular mediators and therapeutic targets in EVALI. © 2023 American Physiological Society. Compr Physiol 13:4617-4630, 2023.
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COVID-19 , Sistemas Eletrônicos de Liberação de Nicotina , Lesão Pulmonar , Vaping , Estados Unidos , Humanos , Lesão Pulmonar/induzido quimicamente , COVID-19/complicações , Dronabinol/efeitos adversos , Vaping/efeitos adversosRESUMO
Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best preventive action, nearly 50% of all lung cancer diagnoses occur in people who have already quit smoking. Research into treatment options for these high-risk patients has been constrained to rodent models of chemical carcinogenesis, which are time-consuming, expensive, and require large numbers of animals. Here we show that embedding precision-cut lung slices within an engineered hydrogel and exposing this tissue to a carcinogen from cigarette smoke creates an in vitro model of lung cancer premalignancy. Hydrogel formulations were selected to promote early lung cancer cellular phenotypes and extend PCLS viability up to six weeks. In this study, hydrogel-embedded lung slices were exposed to the cigarette smoke derived carcinogen vinyl carbamate, which induces adenocarcinoma in mice. At six weeks, analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content revealed that vinyl carbamate induced the formation of premalignant lesions with a mixed adenoma/squamous phenotype. Two putative chemoprevention agents were able to freely diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue were validated with hydrogel-embedded human PCLS and results showed increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the starting point for more sophisticated ex vivo models and a foundation for the study of carcinogenesis and chemoprevention strategies.
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The pathogenesis of chronic obstructive pulmonary disease (COPD), a prevalent disease primarily caused by cigarette smoke exposure, is incompletely elucidated. Studies in humans and mice have suggested that hypoxia-inducible factor-1α (HIF-1α) may play a role. Reduced lung levels of HIF-1α are associated with decreased vascular density, whereas increased leukocyte HIF-1α may be responsible for increased inflammation. To elucidate the specific role of leukocyte HIF-1α in COPD, we exposed transgenic mice with conditional deletion or overexpression of HIF-1α in leukocytes to cigarette smoke for 7 mo. Outcomes included pulmonary physiology, aerated lung volumes via microcomputed tomography, lung morphometry and histology, and cardiopulmonary hemodynamics. On aggregate, cigarette smoke increased the aerated lung volume, quasi-static lung compliance, inspiratory capacity of all strains while reducing the total alveolar septal volume. Independent of smoke exposure, mice with leukocyte-specific HIF-1α overexpression had increased quasi-static compliance, inspiratory capacity, and alveolar septal volume compared with mice with leukocyte-specific HIF-1α deletion. However, the overall development of cigarette smoke-induced lung disease did not vary relative to control mice for either of the conditional strains. This suggests that the development of murine cigarette smoke-induced airspace disease occurs independently of leukocyte HIF-1α signaling.
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Fumar Cigarros , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Animais , Modelos Animais de Doenças , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Leucócitos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/patologia , Nicotiana/efeitos adversos , Microtomografia por Raio-XRESUMO
Supportive mechanical ventilation is a necessary lifesaving treatment for acute respiratory distress syndrome (ARDS). This intervention often leads to injury exacerbation by ventilator-induced lung injury (VILI). Patterns of injury in ARDS and VILI are recognized to be heterogeneous; however, quantification of these injury distributions remains incomplete. Developing a more detailed understanding of injury heterogeneity, particularly how it varies in space and time, can help elucidate the mechanisms of VILI pathogenesis. Ultimately, this knowledge can be used to develop protective ventilation strategies that slow disease progression. To expand existing knowledge of VILI heterogeneity, we document the spatial evolution of cellular injury distribution and leukocyte infiltration, on the micro- and macroscales, during protective and injurious mechanical ventilation. We ventilated naïve mice using either high inspiratory pressure and zero positive end-expiratory pressure ventilation or low tidal volume with positive end-expiratory pressure. Distributions of cellular injury, identified with propidium iodide staining, were microscopically analyzed at three levels of injury severity. Cellular injury initiated in diffuse, quasi-random patterns, and progressed through expansion of high-density regions of injured cells termed "injury clusters." The density profile of the expanding injury regions suggests that stress shielding occurs, protecting the already injured regions from further damage. Spatial distribution of leukocytes did not correlate with that of cellular injury or ventilation-induced changes in lung function. These results suggest that protective ventilation protocols should protect the interface between healthy and injured regions to stymie injury propagation.
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Síndrome do Desconforto Respiratório , Lesão Pulmonar Induzida por Ventilação Mecânica , Animais , Leucócitos , Camundongos , Respiração com Pressão Positiva/métodos , Respiração Artificial/efeitos adversos , Respiração Artificial/métodos , Volume de Ventilação Pulmonar , Lesão Pulmonar Induzida por Ventilação Mecânica/patologiaRESUMO
Pulmonary macrophages are heterogeneous. Distinct populations of resident tissue macrophages exist in the lung airspace and tissue compartments during homeostasis. During inflammation, these are joined by monocyte-derived recruited macrophages. Flow cytometry can be used to identify and purify lung macrophage subsets. Here, we describe methods for identifying and isolating macrophages from bronchoalveolar lavage and digested lung tissues from mouse and human. We also describe basic staining for flow cytometry analysis of different macrophage subsets.
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Pulmão , Macrófagos , Animais , Líquido da Lavagem Broncoalveolar , Citometria de Fluxo/métodos , Humanos , Macrófagos Alveolares , CamundongosRESUMO
Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, commonly used instillation-based fixation methods can displace airspace cells and mucus into terminal airways and can alter tissue morphology. In comparison, vascular perfusion-fixation techniques are superior at preserving the location and morphology of cells within airspaces and the mucosal lining. However, if positive airway pressure is not simultaneously applied, regions of the lungs may collapse and capillaries may bulge into the alveolar spaces, leading to distortion of the lung anatomy. Herein, we describe an inexpensive method for air-inflation during vascular perfusion-fixation to preserve the morphology and location of airway and alveolar cells and interstitium in murine lungs for downstream histologic studies. Constant air pressure is delivered to the lungs via the trachea from a sealed, air-filled chamber that maintains pressure via an adjustable liquid column while fixative is perfused through the right ventricle.
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Vasos Sanguíneos/fisiologia , Pulmão/fisiologia , Perfusão , Pressão , Alvéolos Pulmonares/fisiologia , Animais , Fixadores , CamundongosRESUMO
Rationale: Interstitial macrophages (IMs) and airspace macrophages (AMs) play critical roles in lung homeostasis and host defense, and are central to the pathogenesis of a number of lung diseases. However, the absolute numbers of macrophages and the precise anatomic locations they occupy in the healthy human lung have not been quantified.Objectives: To determine the precise number and anatomic location of human pulmonary macrophages in nondiseased lungs and to quantify how this is altered in chronic cigarette smokers.Methods: Whole right upper lobes from 12 human donors without pulmonary disease (6 smokers and 6 nonsmokers) were evaluated using design-based stereology. CD206 (cluster of differentiation 206)-positive/CD43+ AMs and CD206+/CD43- IMs were counted in five distinct anatomical locations using the optical disector probe.Measurements and Main Results: An average of 2.1 × 109 IMs and 1.4 × 109 AMs were estimated per right upper lobe. Of the AMs, 95% were contained in diffusing airspaces and 5% in airways. Of the IMs, 78% were located within the alveolar septa, 14% around small vessels, and 7% around the airways. The local density of IMs was greater in the alveolar septa than in the connective tissue surrounding the airways or vessels. The total number and density of IMs was 36% to 56% greater in the lungs of cigarette smokers versus nonsmokers.Conclusions: The precise locations occupied by pulmonary macrophages were defined in nondiseased human lungs from smokers and nonsmokers. IM density was greatest in the alveolar septa. Lungs from chronic smokers had increased IM numbers and overall density, supporting a role for IMs in smoking-related disease.
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Fumar Cigarros/patologia , Pulmão/patologia , Macrófagos Alveolares/patologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Contagem de Células , Feminino , Humanos , Imuno-Histoquímica , Lectinas Tipo C/metabolismo , Leucossialina/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Pessoa de Meia-Idade , Dispositivos Ópticos , Receptores de Superfície Celular/metabolismo , Doadores de TecidosRESUMO
Dendritic cells play a key role in determining adaptive immunity, and there is growing interest in characterizing and manipulating the interactions between dendritic cells and biomaterial surfaces. Contact with several common biomaterials can induce the maturation of immature dendritic cells, but substrates that reduce dendritic cell maturation are of particular interest within the field of cell-based therapeutics where the goal is to reduce the immune response to cell-laden material carriers. In this study, we use a materials-based strategy to functionalize poly(ethylene glycol) hydrogels with immobilized immunosuppressive factors (TGF-ß1 and IL-10) to reduce the maturation of immature dendritic cells. TGF-ß1 and IL-10 are commonly employed as soluble factors to program dendritic cells in vitro, and we demonstrate that these proteins retain bioactivity towards dendritic cells when immobilized on hydrogel surfaces. Following stimulation with lipopolysaccharide (LPS) and/or cytokines, a dendritic cell line interacting with the surfaces of immunosuppressive hydrogels expressed reduced markers of maturation, including IL-12 and MHCII. The bioactivity of these immunomodulatory hydrogels was further confirmed with primary bone marrow-derived dendritic cells (BMDCs) isolated from non-obese diabetic (NOD) mice, as quantified by a decrease in activation markers and a significantly reduced capacity to activate T cells. Furthermore, by introducing a second signal to promote BMDC-material interactions combined with the presentation of tolerizing signals, the multifunctional PEG hydrogels were found to further increase signaling towards BMDCs, as evidenced by greater reductions in maturation markers.
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Materiais Biocompatíveis , Células Dendríticas/imunologia , Animais , Materiais Biocompatíveis/química , Diferenciação Celular/imunologia , Técnicas de Cocultura , Células Dendríticas/citologia , Hidrogéis , Imunossupressores , Interleucina-10 , Ativação Linfocitária , Teste de Materiais , Camundongos , Camundongos Endogâmicos NOD , Polietilenoglicóis , Transdução de Sinais , Linfócitos T/imunologia , Fator de Crescimento Transformador beta1RESUMO
A polymerizable superoxide dismutase mimetic (SODm) was incorporated into poly(ethylene glycol) (PEG) hydrogels to protect encapsulated cells from superoxide-mediated damage. Superoxide and other small reactive oxygen species (ROS) can cause oxidative damage to donor tissue encapsulated within size exclusion barrier materials. To enzymatically breakdown ROS within biomaterial cell encapsulation systems, Mn(III) Tetrakis[1-(3-acryloxy-propyl)-4-pyridyl] porphyrin (MnTTPyP-acryl), a polymerizable manganese metalloporphyrin SOD mimetic, was photopolymerized with PEG diacrylate (PEGDA) to create functional gels. In unmodified PEG hydrogels, a significant reduction in metabolic activity was observed when encapsulated Min6 ß-cells were challenged with chemically generated superoxide. Cells encapsulated within MnTPPyP-co-PEG hydrogels, however, demonstrated greatly improved metabolic activity following various superoxide challenges. Further, cells were encapsulated and cultured for 10 days within MnTPPyP-co-PEG hydrogels and challenged with superoxide on days 4, 6, and 8. At the conclusion of this study, cells in blank PEG hydrogels had no observable metabolic activity but when encapsulated in MnTPPyP-functionalized hydrogels, cells retained 60 ± 5% of the metabolic activity compared to untreated controls.
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Materiais Biomiméticos/química , Hidrogéis/química , Metaloporfirinas/química , Polietilenoglicóis/química , Superóxido Dismutase , Superóxidos/química , Animais , Células Cultivadas , Camundongos , Superóxidos/metabolismo , Fatores de TempoRESUMO
Influencing the host immune system via implantable cell-delivery devices has the potential to reduce inflammation at the transplant site and increase the likelihood of tissue acceptance. Towards this goal, an enzymatically-initiated, dip-coating technique is adapted to fabricate conformal hydrogel layers and to create immunoactive polymer coatings on cell-laden poly(ethylene glycol) (PEG) hydrogels. Glucose oxidase (GOx)-initiated dip coatings enable the rapid formation of uniform, PEG-based coatings on the surfaces of PEG hydrogels, with thicknesses up to 500 µm where the thickness is proportional to the reaction time. Biofunctional coatings were fabricated by thiolating biomolecules that were subsequently covalently incorporated into the coating layer via thiol-acrylate copolymerization. The presence of these proteins was verified via fluorescent confocal microscopy and a modified ELISA, which indicated IgG concentrations as high as 13 ± 1 ng/coated cm² were achievable. Anti-Fas antibody, known to induce T cell apoptosis, was incorporated into coatings, with or without the addition of ICAM-1 to promote T cell interaction with the functionalized coating. Jurkat T cells were seeded atop functionalized coatings and the induction of apoptosis was measured as an indicator of coating bioactivity. After 48 h of interaction with the functionalized coatings, 61 ± 9% of all cells were either apoptotic or dead, compared to only 18 ± 5% of T cells on non-functionalized coatings. Finally, the cytocompatibility of the surface-initiated GOx coating process was confirmed by modifying gels with either encapsulated ß-cells or 3T3 fibroblasts within a gel that contained a PEG methacrylate coating.
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Materiais Revestidos Biocompatíveis/química , Hidrogéis/química , Polietilenoglicóis/química , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Materiais Revestidos Biocompatíveis/farmacologia , Citometria de Fluxo , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Humanos , Hidrogéis/farmacologia , Camundongos , Polímeros/química , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Cell encapsulation has long been investigated as a means to achieve transplant immunoprotection as it creates a physical barrier between allograft tissue and host immune cells. Encapsulation with passive barrier materials alone, however, is generally insufficient to protect donor tissue from rejection, because small cytotoxic molecules produced by activated T cells can diffuse readily into the capsule and mediate allograft death. As a means to provide bioactive protection for polymeric encapsulation devices, we investigated a functionalized polymeric coating that mimics a natural T cell regulation pathway. T cells are regulated in vivo via Fas, a well-known 'death receptor,' whereby effector cells express Fas ligand and elicit T cell apoptosis upon binding the Fas receptor on a T cell surface. Anti-Fas antibodies are capable of replicating this effect and induce T cell apoptosis in solution. Here, an iniferter-based living radical polymerization was utilized to fabricate surface-anchored polymer chains containing poly(ethylene glycol) with covalently incorporated pendant anti-Fas antibody. Using this reaction mechanism, we demonstrate fabrication conditions that yield surface densities in excess of 1.5 ng/cm(2) of incorporated therapeutic, as detected by ELISA. Additionally, we show that coatings containing anti-Fas antibody induced significant T cell apoptosis, 21+/-2% of cells, after 24h. Finally, the incorporation of a T cell adhesion ligand, intracellular adhesion molecule-1, along with anti-Fas antibody, yielded even higher levels of apoptosis, 34+/-1% of T cells, compared to either signal alone.
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Apoptose , Fotoquímica , Polímeros/química , Linfócitos T/citologia , Receptor fas/imunologia , Animais , Adesão Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Células Jurkat , Camundongos , Receptor fas/antagonistas & inibidoresRESUMO
BACKGROUND: DNA microarrays have proven powerful for functional genomics studies. Several technologies exist for the generation of whole-genome arrays. It is well documented that 25mer probes directed against different regions of the same gene produce variable signal intensity values. However, the extent to which this is true for probes of greater length (60mers) is not well characterized. Moreover, this information has not previously been reported for whole-genome arrays designed against bacteria, whose genomes may differ substantially in characteristics directly affecting microarray performance. RESULTS: We report here an analysis of alternative 60mer probe designs for an in-situ synthesized oligonucleotide array for the GC rich, beta-proteobacterium Burkholderia cenocepacia. Probes were designed using the ArrayOligoSel3.5 software package and whole-genome microarrays synthesized by Agilent, Inc. using their in-situ, ink-jet technology platform. We first validated the quality of the microarrays as demonstrated by an average signal to noise ratio of >1000. Next, we determined that the variance of replicate probes (1178 total probes examined) of identical sequence was 3.8% whereas the variance of alternative probes (558 total alternative probes examined) designs was 9.5%. We determined that depending upon the definition, about 2.4% of replicate and 7.8% of alternative probes produced outlier conclusions. Finally, we determined none of the probe design subscores (GC content, internal repeat, binding energy and self annealment) produced by ArrayOligoSel3.5 were predictive or probes that produced outlier signals. CONCLUSION: Our analysis demonstrated that the use of multiple probes per target sequence is not essential for in-situ synthesized 60mer oligonucleotide arrays designed against bacteria. Although probes producing outlier signals were identified, the use of ratios results in less than 10% of such outlier conclusions. We also determined that several different measures commonly utilized in probe design were not predictive of outlier probes.