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Studies of pulmonary inflammation require unique considerations due to the complex structure and composition of the lungs. The lungs have multiple compartments and diverse immune cell populations, with inherently high autofluorescence, and are involved in the host response to pulmonary pathogens. We describe a protocol that accounts for these factors through a novel combination of methodologies-in vivo compartmental analysis and spectral flow cytometry with a broad panel of antibodies. In vivo compartmental analysis enables the precise localization of immune cells within the marginated vasculature, lung interstitium, nonlavageable airways, and lavageable airways of the lungs, as well as the pulmonary lymph nodes. Spectral flow cytometry with a broad panel of antibodies supports an unbiased exploratory approach to investigating diverse immune cell populations during pulmonary inflammation. Most importantly, spectral flow uses cellular autofluorescence to aid in the resolution and identification of immune cell populations. This methodology enables the acquisition of high-quality data compatible with informed gating and dimensionality reduction algorithms. In addition, our protocol emphasizes considerations for compartmentalization of the inflammatory response, spectral flow panel design, and autofluorescence spectra analysis. These methodologies are critical for increasing the rigor of pulmonary research. We apply this protocol for the precise characterization and localization of leukocytes in the pulmonary host response to influenza A virus in C57BL/6J mice. In particular, we demonstrate that this protocol improves the quantification and localization of alveolar macrophages within the airways. The methodology is modifiable and expandable to allow for further characterization of leukocyte populations of special interest.NEW & NOTEWORTHY We describe a novel combination of methodologies that incorporates dual in vivo compartmental analysis using intravascular and intratracheal CD45 labeling, a broad panel of antibodies for identifying lymphoid and nonlymphoid cells, and spectral flow cytometry that uses cellular autofluorescence to aid in resolving and identifying immune cell populations. This methodology allows precise localization of immune cells in the lavageable airways, nonlavageable airways, interstitial lung tissue, and marginated in the lung vasculature.
Assuntos
Pulmão , Pneumonia , Camundongos , Animais , Citometria de Fluxo/métodos , Camundongos Endogâmicos C57BL , Leucócitos , Pneumonia/patologia , AnticorposRESUMO
The extracellular matrix (ECM) imparts critical mechanical and biochemical information to cells in the lungs. Proteoglycans are essential constituents of the ECM and play a crucial role in controlling numerous biological processes, including regulating cellular phenotype and function. Versican, a chondroitin sulfate proteoglycan required for embryonic development, is almost absent from mature, healthy lungs and is reexpressed and accumulates in acute and chronic lung disease. Studies using genetically engineered mice show that the versican-enriched matrix can be pro- or anti-inflammatory depending on the cellular source or disease process studied. The mechanisms whereby versican develops a contextual ECM remain largely unknown. The primary goal of this review is to provide an overview of the interaction of versican with its many binding partners, the "versican interactome," and how through these interactions, versican is an integrator of complex extracellular information. Hopefully, the information provided in this review will be used to develop future studies to determine how versican and its binding partners can develop contextual ECMs that control select biological processes. Although this review focuses on versican and the lungs, what is described can be extended to other proteoglycans, tissues, and organs.
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Matriz Extracelular , Versicanas , Animais , Matriz Extracelular/metabolismo , Pulmão/metabolismo , Camundongos , Versicanas/genética , Versicanas/metabolismoRESUMO
Due to a lack of evidence-based standards for cage-change intervals for antelope ground squirrels (AGS, Ammospermophilus leucurus), we evaluated cage ammonia accumulation in our colony of adult, wild-caught AGS and identified factors that influenced ammonia levels. Intracage ammonia was measured daily in singly housed AGS in static caging that contained a running wheel and 1/2, 3/4, 1, or 2 quart (qt) of corncob bedding. Cages were changed when ammonia levels reached greater than 50ppm, our upper acceptable limit for ammonia based on mouse studies of ammonia aversion and toxicity. We also measured average daily water consumption over 2 wk to examine any correlation between water use and ammonia accumulation. We hypothesized that the desert-dwelling AGS would not reach intracage ammonia levels of greater than 50ppm in a 2-wk interval at any bedding volume. Our data showed that intracage ammonia was highly variable among individuals and was significantly associated with water consumption and bedding volumes. Seventeen percent of AGS on 1/2qt of bedding and 18% on 3/4qt of bedding reached greater than 50ppm ammonia before 7 d. All AGS on 1 and 2qt of bedding remained below 50ppm ammonia for 1 wk. Even when maintained on 2qt of bedding, not all AGS remained below 50ppm ammonia for 2 wk. Therefore, we concluded that the most appropriate option was weekly cage change for singly housed AGS on 1qt of bedding in static caging.
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Amônia , Abrigo para Animais , Sciuridae , Animais , Amônia/análise , Sciuridae/fisiologia , Feminino , Masculino , Ingestão de Líquidos , Criação de Animais Domésticos/métodos , Bem-Estar do AnimalRESUMO
Growing evidence supports a role for versican as an important component of the inflammatory response, with both pro- and anti-inflammatory roles depending on the specific context of the system or disease under investigation. Our goal is to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. In previous work, we showed that LPS triggers a signaling cascade involving TLR4, the Trif adaptor, type I interferons, and the type I interferon receptor, leading to increased versican expression by macrophages. In the present study, we used a combination of chromatin immunoprecipitation, siRNA, chemical inhibitors, and mouse model approaches to investigate the regulatory events downstream of the type I interferon receptor to better define the mechanism controlling versican expression. Results indicate that transcriptional regulation by canonical type I interferon signaling via the heterotrimeric transcription factor, ISGF3, controls versican expression in macrophages exposed to LPS. This pathway is not dependent on MAPK signaling, which has been shown to regulate versican expression in other cell types. The stability of versican mRNA may also contribute to prolonged versican expression in macrophages. These findings strongly support a role for macrophage-derived versican as a type I interferon-stimulated gene and further our understanding of versican's role in regulating inflammation.
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The inter-alpha-trypsin inhibitor (IαI) complex is composed of the bikunin core protein with a single chondroitin sulfate (CS) attached and one or two heavy chains (HCs) covalently linked to the CS chain. The HCs from IαI can be transferred to hyaluronan (HA) through a TNFα-stimulated gene-6 (TSG-6) dependent process to form an HCâ¢HA matrix. Previous studies reported increased IαI, HA, and HCâ¢HA complexes in mouse bronchoalveolar lavage fluid (BALF) post-influenza infection. However, the expression and incorporation of HCs into the HA matrix of the lungs during the clinical course of influenza A virus (IAV) infection and the biological significance of the HCâ¢HA matrix are poorly understood. The present study aimed to better understand the composition of HCâ¢HA matrices in mice infected with IAV and how these matrices regulate the host pulmonary immune response. In IAV infected mice bikunin, HC1-3, TSG-6, and HAS1-3 all show increased gene expression at various times during a 12-day clinical course. The increased accumulation of IαI and HA was confirmed in the lungs of infected mice using immunohistochemistry and quantitative digital pathology. Western blots confirmed increases in the IαI components in BALF and lung tissue at 6 days post-infection (dpi). Interestingly, HCs and bikunin recovered from BALF and plasma from mice 6 dpi with IAV, displayed differences in the HC composition by Western blot analysis and differences in bikunin's CS chain sulfation patterns by mass spectrometry analysis. This strongly suggests that the IαI components were synthesized in the lungs rather than translocated from the vascular compartment. HA was significantly increased in BALF at 6 dpi, and the HA recovered in BALF and lung tissues were modified with HCs indicating the presence of an HCâ¢HA matrix. In vitro experiments using polyinosinic-polycytidylic acid (poly(I:C)) treated mouse lung fibroblasts (MLF) showed that modification of HA with HCs increased cell-associated HA, and that this increase was due to the retention of HA in the MLF glycocalyx. In vitro studies of leukocyte adhesion showed differential binding of lymphoid (Hut78), monocyte (U937), and neutrophil (dHL60) cell lines to HA and HCâ¢HA matrices. Hut78 cells adhered to immobilized HA in a size and concentration-dependent manner. In contrast, the binding of dHL60 and U937 cells depended on generating a HCâ¢HA matrix by MLF. Our in vivo findings, using multiple bronchoalveolar lavages, correlated with our in vitro findings in that lymphoid cells bound more tightly to the HA-glycocalyx in the lungs of influenza-infected mice than neutrophils and mononuclear phagocytes (MNPs). The neutrophils and MNPs were associated with a HCâ¢HA matrix and were more readily lavaged from the lungs. In conclusion, this work shows increased IαI and HA accumulation and the formation of a HCâ¢HA matrix in mouse lungs post-IAV infection. The formation of HA and HCâ¢HA matrices could potentially create specific microenvironments in the lungs for immune cell recruitment and activation during IAV infection.
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alfa-Globulinas , Influenza Humana , Orthomyxoviridae , Camundongos , Animais , Humanos , Ácido Hialurônico/metabolismo , Sulfatos de Condroitina/metabolismo , Pulmão/metabolismo , Orthomyxoviridae/metabolismo , Imunidade Inata , Progressão da DoençaRESUMO
BACKGROUND: Lactate dehydrogenase (LDH) and creatine kinase (CK) have differential tissue activity and isoenzyme profiles. LDH and CK exist as 5 and 3 isoenzymes, respectively, in both serum and cerebrospinal fluid (CSF). Studies have demonstrated that measuring LDH, CK, and their isoenzymes in CSF has diagnostic and prognostic values for dogs and people with neurologic disorders. OBJECTIVES: Iatrogenic blood contamination can distort the results of CSF analysis. The purpose of this study was to determine allowable thresholds of blood contamination (RBC/µL) for accurate measurement of LDH, CK, and their isoenzymes in canine CSF. METHODS: Venous blood and CSF were collected from healthy dogs. Total LDH and CK activity were measured spectrophotometrically. Isoenzyme profiles were determined using gel electrophoresis and densitometric scanning. All samples were analyzed within 6 hours of collection. A nonlinear mixed effects regression model was used to estimate the allowable thresholds of blood contamination for accurate measurement of LDH, CK, and their isoenzymes in canine CSF. RESULTS: The threshold of iatrogenic blood contamination for total LDH and total CK in healthy dogs are 6696 RBC/µL (95% CI 3879-11 187) and 5961 RBC/µL (95% CI 2939-12 085), respectively. LDH-1 is the most sensitive isoenzyme to iatrogenic blood contamination, while LDH-4 is the least sensitive. CONCLUSIONS: These results are important for the interpretation of LDH, CK, and their isoenzymes in canine CSF. Additionally, our methodology is translatable for determining thresholds of acceptable iatrogenic blood contamination in CSF for other diagnostic and prognostic biomarkers of neurologic disease.
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Doenças do Cão , Isoenzimas , Cães , Animais , Creatina Quinase , Eletroforese/veterinária , L-Lactato Desidrogenase , Doença Iatrogênica/veterináriaRESUMO
BACKGROUND: Creatine kinase (CK) exists as three isoenzymes (CK-MM, CK-MB, and CK-BB) that are predominantly expressed in specific tissues and can be detected in both the serum and cerebrospinal fluid (CSF). CSF CK has been relatively unstudied in veterinary medicine, although studies in human medicine have demonstrated that changes in total CSF CK activity can indicate neurologic abnormalities. OBJECTIVES: The purpose of this study was to establish reference intervals for CK and its three major isoenzymes in the serum and CSF of clinically healthy dogs. By establishing a definitive reference interval for this enzyme in healthy canines, the diagnostic use and possible significance of CK in clinical disease can be studied. METHODS: Serum and/or CSF were collected from healthy dogs. Total CK activity was measured spectrophotometrically, and isoenzyme distributions were determined using the QuickGel CK Vis Isoenzyme Kit and a densitometric scanner. Total CK and CK isoenzyme activities were determined within 8 h of collection. RESULTS: The median serum total CK in healthy canines was 159.0 U/L (range: 53.0-539.0 U/L), while the median CSF total CK was 3.7 U/L (range: 2.0-84.0 U/L). CK-BB and CK-MM were approximately equal in the serum, while CK-MM was the predominant isoenzyme in the CSF. CONCLUSIONS: Knowledge of the normal distribution and concentration of CK in canine serum and CSF will set the foundation for future studies of canine CK as a potentially clinically useful biomarker.
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Creatina Quinase , Isoenzimas , Cães , Humanos , AnimaisRESUMO
Versican, a chondroitin sulfate proteoglycan, is an essential component of the extracellular matrix (ECM) in inflammatory lung disease. Versican's potential as an immunomodulatory molecule makes it a promising therapeutic target for controlling host immune responses in the lungs. To establish changes to versican expression and accumulation during influenza A viral pneumonia, we document the temporal and spatial changes to versican mRNA and protein in concert with pulmonary inflammatory cell infiltration. These studies were performed in the lungs of wild-type C57BL6/J mice on days 3, 6, 9, and 12 post-infection with influenza A virus using immunohistochemistry, in situ hybridization, and quantitative digital pathology. Using duplex in situ hybridization, we demonstrate that type I interferon signaling contributes significantly to versican expression in lung stromal cells. Our findings show that versican is a type I interferon-stimulated gene in pulmonary fibroblasts and pericytes in the context of viral pneumonia. These data also provide a guide for future studies to determine the role of versican in the pulmonary immune response to influenza infection.
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Influenza Humana/imunologia , Interferon Tipo I/imunologia , Pulmão/imunologia , Células Estromais/imunologia , Versicanas/imunologia , Animais , Humanos , Influenza Humana/patologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologia , Células Estromais/patologiaRESUMO
Advances in reagents, methodologies, analytic platforms, and tools have resulted in a dramatic transformation of the research pathology laboratory. These advances have increased our ability to efficiently generate substantial volumes of data on the expression and accumulation of mRNA, proteins, carbohydrates, signaling pathways, cells, and structures in healthy and diseased tissues that are objective, quantitative, reproducible, and suitable for statistical analysis. The goal of this review is to identify and present how to acquire the critical information required to measure changes in tissues. Included is a brief overview of two morphometric techniques, image analysis and stereology, and the use of artificial intelligence to classify cells and identify hidden patterns and relationships in digital images. In addition, we explore the importance of preanalytical factors in generating high-quality data. This review focuses on techniques we have used to measure proteoglycans, glycosaminoglycans, and immune cells in tissues using immunohistochemistry and in situ hybridization to demonstrate the various morphometric techniques. When performed correctly, quantitative digital pathology is a powerful tool that provides unbiased quantitative data that are difficult to obtain with other methods.
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Inteligência Artificial , Glicosaminoglicanos/análise , Processamento de Imagem Assistida por Computador , Proteoglicanas/análise , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Proteoglicanas/genética , Proteoglicanas/metabolismoRESUMO
Mice are a common animal model for the study of influenza virus A (IAV). IAV infection causes weight loss due to anorexia and dehydration, which can result in early removal of mice from a study when they reach a humane endpoint. To reduce the number of mice prematurely removed from an experiment, we assessed nutritional gel (NG) supplementation as a support strategy for mice infected with mouse-adapted Influenza A/Puerto Rico/8/34 (A/PR/8/34; H1N1) virus. We hypothesized that, compared with the standard of care (SOC), supplementation with NG would reduce weight loss and increase survival in mice infected with IAV without impacting the initial immune response to infection. To assess the effects of NG, male and female C57BL/6J mice were infected with IAV at low, intermediate, or high doses. When compared with SOC, mice given NG showed a significant decrease in the maximal percent weight loss at all viral doses in males and at the intermediate dose for females. Mice supplemented with NG had no deaths for either sex at the intermediate dose and a significant increase in survival in males at the high viral dose. Supplementation with NG did not alter the viral titer or the pulmonary recruitment of immune cells as measured by cell counts and flow cytometry of cells recovered in bronchoalveolar lavage (BAL) fluid in either sex. However, mice given NG had a significant reduction in IL6 and TNFα in BAL fluid and no significant differences in CCL2, IL4, IL10, CXCL1, CXCL2, and VEGF. The results of this study show that as compared with infected SOC mice, infected mice supplemented with NG have reduced weight loss and increased survival, with males showing a greater benefit. These results suggest that NG should be considered as a support strategy and indicate that sex is an important biologic variable in mice infected with IAV.
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Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Animais , Suplementos Nutricionais , Feminino , Humanos , Pulmão , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Lactate dehydrogenase (LD) exists as 5 isoenzymes (LD-1 through LD-5) that are expressed throughout the body and can be detected in both serum and cerebrospinal fluid (CSF). LD and its isoenzymes have been relatively unstudied in veterinary medicine, although studies in human medicine have demonstrated that changes in total LD activity and atypical isoenzyme patterns can indicate disease processes, including neurologic abnormalities. OBJECTIVES: The purpose of this study was to establish RIs for LD and its isoenzymes in the serum and CSF of clinically healthy dogs. By establishing a definitive RI for this enzyme in healthy canines, further study of the clinical and diagnostic usefulness of LD can be undertaken. METHODS: Serum and atlantoaxial CSF were collected from clinically healthy dogs. Total LD activity was measured spectrophotometrically immediately after collection. Isoenzyme distributions were also determined within 8 hours of collection using the QuickGel LD Isoenzyme technique and a densitometric scanner. RESULTS: The median serum total LD in healthy canines was 69.0 U/L (n = 41; range: 21.0-217.0 U/L), while the median CSF total LD was 10.0 U/L (n = 40; range: 6.0-19.3 U/L). LD-5 is the predominant isoenzyme in canine serum (n = 40), contributing over half of the total enzyme activity. Conversely, in canine CSF (n = 42), LD-1 is the predominant isoenzyme, followed by LD-2 and LD-3. CONCLUSIONS: Knowledge of the distribution and concentration of LD in the serum and CSF of healthy dogs will set the foundation for future studies of canine LD as a potentially clinically useful biomarker.