bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Assessment of Clinically Relevant Extracellular Matrix Markers in a Bleomycin‐Induced Mouse Model of Lung Fibrosis
Authors: Young Alan , Nath Puneeta , Leeming Diana , Karsdal Morten , Brockbank Sarah , Rider David , Cruwys Simon 
Published: November 2023

The bleomycin‐induced lung fibrosis model is routinely used to investigate efficacy of potential therapies for treating idiopathic pulmonary fibrosis (IPF). The aim here was to characterize a bleomycin model over an extended time course and correlate inflammation, lung function changes, collagen deposition and pathology with systemic markers reflecting extracellular matrix (ECM) metabolism assessed by MMP derived collagen fragments (C1M, C3M); markers associated with the diagnosis and prognosis of human idiopathic pulmonary fibrosis (IPF) (Jenkins et al, Lancet Respir Med 2015).MethodsC57BL/6J mice were anaesthetized and dosed with bleomycin (BLM) intratracheally (0.03 units/mouse). Control animals received saline. Animals were observed daily and bodyweight measured from D3. Lung function was assessed on days −1, 3, 7, 14, 21, 28, 35, 42 and 56. Animals were sacrificed on days 3, 7, 14, 21, 28, 35, 42, and 56 and serum samples taken. Markers of the formation and degradation of collagen type I (C1M) and collagen type III (C3M) were measured. Lungs from separate cohorts were lavaged for mediator and inflammatory cell analysis, then perfused and snap‐frozen for determination of hydroxyproline and protein levels. Another cohort had their lungs inflated with phosphate buffered formalin and used for histopathological analysis (including Modified Ashcroft score) and subsequent image analysis.ResultsBLM elicited a decrease in bodyweight, peaking on D7. BLM induced lung inflammation within the BALF with increases in neutrophils (peaking at D7, +135% p<0.05), lymphocytes (peaking at D14, +5240% p<0.0001) and macrophages (peaking at D21, +134%, p<0.05). Lung wet weight also increased following BLM‐administration, peaking on D14 (+44%, p<0.0001). There were observable BLM‐induced changes in lung function (peaking at D7), which were fully resolved by D21. Levels of hydroxyproline were elevated from D21 onwards (+63%, p<0.05). Pathology also increased over this time period, with a clear inflammatory response being seen at D7 and D14 and fibrotic lesions increasing in severity and prevalence from D21. The ECM degradation biomarker C1M was elevated in serum on D7 and 14 (+32%, p<0.01), with C3M increasing on D7,14 and 21 (+15–25%, all p< 0.05). These biomarkers were not observed in other models of inflammation (e.g., LPS‐induced lung inflammation).These data suggest that in this BLM‐induced lung fibrosis model, there is an early inflammatory phase, followed by a generation of fibrosis and collagen deposition. Clinically relevant biomarkers associated with diagnosis and progression of IPF can be measured in this model and may improve the predictability and translatability of such models when considering assessment of novel mechanisms and compoundsSupport or Funding InformationCharles River, Nordic Biosciences and Grunenthal
Influenza A infection decrease the population of tissue resident alveolar macrophages
Authors: Gonzalez‐Gonzalez Francisco , McQuattie‐Pimentel Alexandra , Soberanes Saul , Casalino‐Matsuda Marina , Chi Monica , Piseaux Raul , Sporn Peter , Budinger Scott 
Published: November 2023

RationaleInfluenza A pneumonia is the most common cause of death from an infectious agent and can become an important contributor to overall mortality during pandemics. Severe influenza A pneumonia results in the development of the acute respiratory distress syndrome, which is the proximal cause of death in most of these patients. It is now recognized that alveolar macrophages represent a heterogeneous population of cells. Tissue resident alveolar macrophages are long lived, self‐renewing cells with developmental origins outside of the bone marrow. During infection, monocytes are recruited to the lung where they differentiate into alveolar macrophages. The role and fate of these different macrophage populations during the course of influenza A pneumonia is not known.HypothesisInfluenza A infection results in the replacement of tissue resident alveolar macrophages with monocyte derived cells that persist after injury.MethodsWe infected ten to twelve week old C57Bl/6 mice intratracheally with lethal and sublethal doses of influenza A/WSN/33. We analyzed inflammatory cell populations in lung digests at different time points after the influenza A infection using multi‐color flow cytometry. We developed a shielded bone marrow chimera system to unambiguously identify tissue resident and alveolar macrophage populations during the development and resolution of influenza A infection.ResultsIn the early stages of influenza A infection, tissue‐resident alveolar macrophages could be readily distinguished from monocyte‐derived cells using flow cytometry. Tissue‐resident alveolar macrophages were rapidly depleted during influenza A infection accompanied by an influx of monocyte derived cells into the lung. Using bone marrow chimeric mice generated with lung protection followed by myeloablation, we observed that monocyte derived cells persisted in the lung after the resolution of infection for as long as 10 months but could no longer be distinguished from tissue resident cells on the basis of flow cytometry.ConclusionsInfluenza A infection results in the dose‐dependent depletion of tissue resident alveolar macrophages in the lung. Whether this results from direct infection by the influenza A virus or secondary effects related to lung tissue destruction and inflammation is not clear. Monocytes recruited to the lung during influenza A infection differentiate into alveolar macrophages in the local microenvironment of the lung and persist long after the resolution of infection. Our results suggest that severe influenza infection results in permanent changes in tissue resident alveolar macrophage ontogeny.
Developing an In Vivo Model of Reinke's Edema
Authors: Durkes Abigail , Sivasankar Preeti 
Published: November 2023

Approximately 7.5 million Americans suffer from difficulty using their voice. Phonatory difficulties are often multifactorial with Reinke's edema being a common underlying pathology. The most common cause of Reinke's edema is smoking. Reflux of stomach acids into the larynx and excessive voice use are also implicated in causing edema. There is currently no reproducible animal model of this common human laryngeal disease. We set out to determine if primary cigarette exposure could be 1) accomplished in an inhalation chamber designed for pigs; and 2) if cigarette smoke exposure could differentially affect the vocal folds of exposed pigs similarly to the human disorder of Reinke's edema. The pig was chosen as the animal model because the pig larynx offers the greatest structural, cellular, immunologic, and neuroanatomical similarity to the human larynx. Twelve Sinclair miniature pigs were randomly assigned to 2 treatment groups (baseline and high cigarette exposure). Six pigs assigned to the baseline cigarette smoke were exposed to 3 cigarettes per day for 20 days. Six pigs assigned to the high cigarette smoke were exposed to 15 cigarettes per day for 20 days. A week prior to treatment, each group of pigs was habituated to the chamber and human contact to reduce stress and ease treatment administration. Behavior modification was accomplished using positive reinforcement. Vocal fold, nasal mucosa, and trachea were examined histologically. Vocal fold ultrastructural alterations, epithelial intercellular space diameter, and microridge height were examined via transmission electron microscopy (TEM). Real‐time polymerase chain reaction was used to investigate genes associated with Reinke's edema in human beings. All animals tolerated the chamber without any signs of distress. Animals on average coughed more in the high exposure group compared with the low exposure (P<0.05). Histology and ultrastructure of the vocal fold were preserved even in the high exposure group of animals. Gene expression data is on‐going. The data obtained from this study offer a potential novel experimental methodology to simulate the development of Reinke's edema in healthy pigs where there is currently no reproducible animal model. The success of this methodology could impact future research on prevention, improving early diagnosis, and therapeutic options for Reinke's edema.Support or Funding InformationNIH R01DC011759
ATF3 protects LPS‐induced acute pancreatic inflammation via modulating NFκB‐mediated iNOS production in mice
Authors: Chen Yu‐Min , Tseng Ya‐Ping , Liu Chin‐Hung , Lin Heng , Cheng Ching‐Feng , Lai Pei‐Fang 
Published: November 2023

A state of excessive endotoxin (lipopolysaccharide, LPS) in the circulation is called endotoxemia. An overproduction of proinflammatory mediators in acute pancreatic inflammation, which may lead to insulin resistance, pancreas dysfunction and even death. Activating transcription factor (ATF)‐3, a subunit belongs to ATF/cAMP responsive element‐binding protein (CREB) family of transcription factor protects against stress‐induced acute kidney injury. The previous results suggest that ATF3 contributes to the negative regulation of inflammatory cytokine gene expression by recruiting histone deacetylase (HDAC)‐1 to ATF/NFκB‐binding sites. Biological activities and pathological effects of ATF3 in sepsis is unclear. The aim of this study is to assess the possible protective effects of ATF3 in endotoxemia induced by LPS in vivo studies. ATF3‐knockout (KO) and wild‐type mice (WT) were injected with LPS (10 mg/kg, ip) to induce endotoxemia. Survival rate in mouse model of endotoxemia, circulating white blood cells (WBC) level, serum NO expression, glucose level, histology, and the mRNA expression of inflammatory factors were investigated. The decreased circulating WBC levels, increased serum NO and glucose levels were significantly obtained in KO mice after LPS induction. Furthermore, inflammatory factor expressions, macrophage infiltration and β cell damage in KO mice after LPS induction were significantly reversed by injected adeno‐associated virus (AAV) to restore ATF3 gene expression. These results demonstrated that ATF3 plays a protective role to prevent LPS‐induced pancreas inflammation in experimental sepsis.Support or Funding Informationsupported by MOST and Tzu Chi Medical Center
Model Systems to Study the Pathogenesis of Zika Virus‐Mediated Eye Disease
Authors: Martinez Laura Elizabeth , Contreras Deisy , Jones Melissa Kaye , Gangalapudi Vineela , Tang Jie , Wang Shaomei , Arumugaswami Vaithilingaraja 
Published: November 2023

Zika virus (ZIKV) is a teratogenic vector‐borne viral pathogen that causes detrimental congenital eye disease, and other disorders such as microcephaly, intrauterine growth restriction, and sensorineural hearing loss. Eye malformations in newborns to mothers infected with ZIKV during pregnancy, such as macular pigment mottling, loss of foveal reflex, intra‐retinal hemorrhages, chorioretinal atrophy, and blindness have been reported. The disease manifestations of congenital ZIKV eye infection are not well understood. Our overall goal is to understand the mechanisms of ZIKV‐mediated ocular injury. We utilized both cell‐based in vitro and animal model systems to address this goal. We first evaluated the permissiveness of fetal retinal pigment epithelium (RPE) cells to ZIKV PRVABC59 (Asian genotype) infection. The RPE is an epithelial cell monolayer present in between photoreceptor cells and the highly vascularized choroid plexus. We observed that RPEs are highly susceptible to ZIKV infection that lead to reduced cell viability and apoptosis. Transcriptomic analysis of infected cells revealed the activation of inflammatory and VEGF signaling pathways, and dysregulation of factors involved in cell survival (PI3K/Akt signaling). Induced pluripotent stem cell‐derived retinal stem cells (iRSCs) supported ZIKV replication at a lower level compared to that of RPE cells. We then investigated the ZIKV disease pathogenesis using a mouse model. The type‐I interferon receptor knockout (ifnar1−/− or A129) mice were infected with ZIKV through subcutaneous route. Infected animals showed high viremia at 3 days post infection (dpi). Affected mice showed weight loss and signs of posterior paraplegia at 7 dpi, and reached 100% mortality. ZIKV did not establish efficient infection in wild‐type SJL mice and the infected mice did not show changes in body weight or mortality. Brain tissues from infected ifnar1−/− mice had the highest viral load, followed by spleen, thymus, eyes, kidney, and heart. Histopathological analysis showed neuroinflammation characterized by extensive perivascular cuffing with neutrophils and mononuclear cells. Infected eyes showed infiltration of cells in the vitreous space and ganglion cell layer. Immunohistochemistry revealed infiltration of macrophages (CD11) and T‐cells (CD4) in the infected retina. We are currently investigating the molecular mechanisms involved in ZIKV infection of the developing eye using a mouse pregnancy model. In conclusion, we provide evidence that fetal retinal epithelial cells and retinal stem cells are susceptible to ZIKV infection, and the described animal model is useful for studying the Zika virulence factors contributing to congenital eye injury and disease pathogenesis.Support or Funding InformationCedars‐Sinai Medical Center
Method Development for the Micromolar Analysis of Pyocyanin (PYO) in Blood and Urine via Mass Spectrometry (MS)
Authors: Mawhinney Thomas Patrick , Chance Deborah L. , Waters James K. , Mossine Valeri V. 
Published: November 2023

The virulence factor, pyocyanin (PYO), is one of several toxins that are produced by the opportunistic Gram negative microbe Pseudomonas aeruginosa (PA) and is capable of affecting a broad range of cellular constituents and pathways. It is a soluble, redox‐active blue phenazine pigment that is zwitterionic and, at the pH of blood, freely moves through the cytoplasmic membrane and has been shown to be important in PA virulence. Intracellularly, PYO increases reactive oxygen species (ROS) which can directly and/or indirectly cause cell damage. Its cytotoxicity had been demonstrated on numerous cell lines which can result in morphological changes that include PYO‐induced cell vacuolation, along with a drop in the cellular viability, metabolic mitochondrial activity, and decreased transcriptional and translational activity. A major difficulty in research moving forward is the measurement of PA‐derived PYO in blood and urine from patients with bacteremia, urinary tract infections, cystic fibrosis, and others. In this study, we address the need for a more sensitive method of analysis by synthesizing internal standards (IS) of PYO with stable isotopes to be used for MS analysis.Initial experiments demonstrated that by reacting phenazine methosulfate (PMS) with H2O18 (98% purity), PYO‐O18 (m/z, 213 M+H+) was produced in near quantitative yields. In a parallel fashion, by reacting phenazine with dimethyl sulfate‐D6, crystalline PMS(D3) was produced. When this product was individually reacted with H2O16 and H2O18, PYO‐D3 (m/z, 214 M+H+), and PYO‐D3O18 (m/z, 216 M+H+) were formed, respectively. With this library of isotopically labeled PYO analogues, differing in atomic mass units (amu) of 2, 3 and 5 (i.e., PYO‐O18, PYO‐D3 and PYO‐D3O18, respectively) from PYO (m/z, 211 M+H+), an analytical scheme was devised. Bovine serum, fortified with PYO, and spiked with PYO‐D3 as the IS, was first used to assist in the development of a PYO isolation scheme amenable to MS analysis (Orbitrap) via direct infusion, and by LC‐MS analysis. Preliminary results indicated that PYO, with a level of detection of ≤40 ng/mL, is achievable. Current research demonstrates that by utilizing both PYO‐D3 and PYO‐D3O18 the unambiguous identification and accurate measurement of bacterial exotoxin PYO in both human serum and urine is possible in the low ng/mL range.Support or Funding InformationSupported by the Leda J. Sears Trust Fund, the Cystic Fibrosis Association of Missouri, and the University of Missouri Agricultural Experiment Station Laboratories.
Modeling the ischemic neurovascular unit in a dish using patient‐specific induced pluripotent stem cells
Authors: Page Shyanne 
Published: November 2023

The blood‐brain barrier (BBB) is a component of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs), astrocytes and neurons. It plays an important function in the maintenance of brain homeostasis by tightly regulating the entrance of water and solutes into the brain and by blocking the entrance of potentially neurotoxic molecules. Dysfunction of the BBB is a key element observed in various neurological disorders and, in particular, following stroke injury.Stroke is the fifth cause of death in the US and the third leading cause of death in women. It is also a major cause of permanent disability. A key element of ischemic stroke (the most common type of stroke) pathophysiology is the opening of the BBB leading to cerebral edema onset and neural cell death by excitotoxicity. Notably, there is a growing literature highlighting the presence of a sexual dimorphism in stroke. Pre‐menopausal women have lesser stroke events than age‐matched men, however stroke outcome is worsened in post‐menopausal women. Interestingly, the nature such neuroprotection in pre‐menopausal women remains yet to be determined, as current in vitro models of the BBB cannot differentiate the effect of sex from the effects of sexual hormones.In this project, we designed a sex‐specific in vitro model of the BBB using patient‐derived induced pluripotent stem cells (iPSCs), using one male (CTR66M) and one female cell line (IMR90). We were capable of generating three different cell types from these iPSCs: astrocytes, BMECs and neurons. We did not observe a sexual dimorphism in the differentiation of these cell types in normal conditions, suggesting that biological sex may have not an impact on the differentiation and maturation of the BBB.Next, we challenged our cells to oxygen‐glucose deprivation (OGD) stress to model ischemic stroke injury in vitro. Cells were given DMEM –glucose and placed in a normobaric, hypoxic chamber for six hours. Following OGD stress, cells were given DMEM +glucose and re‐oxygenated for 24 hours to determine the effect of re‐oxygenation on barrier recovery. In order to assess barrier integrity, transendothelial electrical resistance (TEER) and permeability to sodium fluorescein was used. To determine any changes in cellular metabolism, an MTS or LDH assay was used.Preliminary data suggests the ability of the different cell types to respond to OGD stress in similar fashion as in vivo studies, as we noted a reversible BBB opening, astrocyte activation and pruning of neurites. In conclusion, our study demonstrates the possibility to obtain a sex‐specific in vitro model of the neurovascular unit, as well as its ability to respond to OGD stress. We are currently investigating the role of the HIF1‐alpha pathway in the response to ischemic stroke in astrocytes, neurons and BMECs.
Regulator of G‐protein signaling 6 (RGS6) expression in human substantia nigra pars compacta (SNc) and loss in Parkinson's disease (PD)
Authors: Luo Zili , Ahlers Katelin E , Yang Jianqi , Chakravarti Bandana , Stevens Hanna E , Narayanan Nandakumar S , Fisher Rory A 
Published: November 2023

Parkinson's disease (PD) is a devastating neurodegenerative disorder characterized by the progressive loss of dopamine (DA) neurons in the SNc. The reason for the greater sensitivity of these neurons to degeneration and their loss with aging in PD is unknown. Although there are treatments targeting symptoms of this disease there are no interventions that protect DA neurons from degeneration. We and our collaborators have recently discovered that RGS6 is restrictively expressed in and required for the adult survival of SNc DA neurons that undergo degeneration in PD. Mice lacking RGS6 exhibit a late onset age‐dependent loss of SNc DA neurons accompanied by reduced striatal and SNc DA content and motor deficits as well as increased sensitivity to 6‐OHDA‐induced neurodegeneration and behavioral deficits. These findings suggest a critical pathogenic link between RGS6 loss and DA neuron degeneration in PD. Here we tested this hypothesis by a quantitative analysis of RGS6 expressing neurons in the SNc of human control and PD patients (n = 5 each). Immunohistochemical analysis of RGS6 and tyrosine hydroxylase (TH) expression and unbiased sterology was used to quantify the numbers of RGS6+ and TH+ SNc neurons. We found that RGS6 is exclusively expressed in DA neurons in the SNc of humans and there is a dramatic (73%) and selective loss of these RGS6+ TH+ neurons in PD patients. Consistent with our hypothesis, all surviving SNc TH+ neurons in normal and PD patients express RGS6. Given our evidence that RGS6 is required for survival of SNc DA neurons in mice, these findings suggest that RGS6 loss in human SNc DA neurons renders them susceptible to neurodegeneration characteristic of PD. These findings provide new evidence suggesting that RGS6 may have a critical neuroprotective role in PD in both mice and humans. Together our findings identify RGS6 as a potential novel therapeutic target for PD treatment.Support or Funding InformationNIH CA161882, MJ Fox 11551
Investigating the Underlying Mechanisms of Chemosensory Dysfunctions in Alzheimer's Disease Using Caenorhabditis elegans with Mutations in Presenilin 1
Authors: Parvand Mahraz , Bozorgmehr Tahereh , Rankin Catharine 
Published: November 2023

BackgroundMany cases of familial Alzheimer's disease (FAD) are linked to mutations of the presenilin (PS) genes. These genes are orthologous with sel‐12 genes in Caenorhabditis elegans, a transparent roundworm. C.elegans have a small nervous system consisting of 302 neurons, making it an ideal candidate for system‐level genetic analysis of neural circuits and behavior. Olfactory dysfunction is an early symptom of AD. However, the underlying mechanisms of these dementia‐linked deficits are unknown.MethodsChemotaxis experiments were conducted on worms with a mutation in sel‐12 to gain a better understanding of the relationship between presenilin 1 (PS1) mutations in AD and olfactory deficits. To understand this phenomenon further, we examined various stages of the worm's life cycle to determine when this deficit appeared. This provided preliminary insight into the dynamics of PS1 mutations and its subsequent effect on cellular pathways involved in chemosensation. Since C. elegans are innately repulsed by the odorant octanol and attracted by the odorant diacetyl, we utilized these two odorants in chemotaxis assays to assess the olfactory function of sel‐12 mutants.ResultsAdult sel‐12 mutants had a significantly decreased sensitivity to octanol and diacetyl compared to wild‐type worms. Introducing human wild‐type PS1 into the nervous system of C. elegans rescued olfactory defects, while a PS1 mutant from Alzheimer's patients did not. Moreover, C. elegans sel‐12 mutants presented olfactory deficits from hatching, and this deficit increased as worms aged, similar to the neurodegenerative progression of AD.ConclusionsThis study demonstrates that mutations in C. elegans orthologue of PS1 are associated with altered chemosensation, and that these deficits were rescued by the wild‐type human PS gene in the nervous system.Future DirectionsIn order to localize the region(s) where wild‐type sel‐12 function is required for normal olfaction, sel‐12 and PS1 rescues will be conducted in specific neurons that are responsible for the detection of octanol and diacetyl. We will also investigate the morphology of these neurons and compare them to those of wild‐type worms.