Inhibition of matrix metalloproteinase-9 secretion by dimethyl sulfoxide and cyclic adenosine monophosphate in human monocytes.

BACKGROUND: Matrix metalloproteinases (MMPs), including MMP-9, are an integral part of the immune response and are upregulated in response to a variety of stimuli. New details continue to emerge concerning the mechanistic and regulatory pathways that mediate MMP-9 secretion. There is significant evidence for regulation of inflammation by dimethyl sulfoxide (DMSO) and 3',5'-cyclic adenosine monophosphate (cAMP), thus investigation of how these two molecules may regulate both MMP-9 and tumor necrosis factor α (TNFα) secretion by human monocytes was of high interest. The hypothesis tested in this study was that DMSO and cAMP regulate MMP-9 and TNFα secretion by distinct mechanisms. AIM: To investigate the regulation of lipopolysaccharide (LPS)-stimulated MMP-9 and tumor necrosis factor α secretion in THP-1 human monocytes by dimethyl sulfoxide and cAMP. METHODS: The paper describes a basic research study using THP-1 human monocyte cells. All experiments were conducted at the University of Missouri-St. Louis in the Department of Chemistry and Biochemistry. Human monocyte cells were grown, cultured, and prepared for experiments in the University of Missouri-St. Louis Cell Culture Facility as per accepted guidelines. Cells were treated with LPS for selected exposure times and the conditioned medium was collected for analysis of MMP-9 and TNFα production. Inhibitors including DMSO, cAMP regulators, and anti-TNFα antibody were added to the cells prior to LPS treatment. MMP-9 secretion was analyzed by gel electrophoresis/western blot and quantitated by ImageJ software. TNFα secretion was analyzed by enzyme-linked immuno sorbent assay. All data is presented as the average and standard error for at least 3 trials. Statistical analysis was done using a two-tailed paired Student t-test. P values less than 0.05 were considered significant and designated as such in the Figures. LPS and cAMP regulators were from Sigma-Aldrich, MMP-9 standard and antibody and TNFα antibodies were from R&D Systems, and amyloid-ß peptide was from rPeptide. RESULTS: In our investigation of MMP-9 secretion from THP-1 human monocytes, we made the following findings. Inclusion of DMSO in the cell treatment inhibited LPS-induced MMP-9, but not TNFα, secretion. Inclusion of DMSO in the cell treatment at different concentrations inhibited LPS-induced MMP-9 secretion in a dose-dependent fashion. A cell-permeable cAMP analog, dibutyryl cAMP, inhibited both LPS-induced MMP-9 and TNFα secretion. Pretreatment of the cells with the adenylyl cyclase activator forskolin inhibited LPS-induced MMP-9 and TNFα secretion. Pretreatment of the cells with the general cAMP phosphodiesterase inhibitor IBMX reduced LPS-induced MMP-9 and TNFα in a dose-dependent fashion. Pre-treatment of monocytes with an anti-TNFα antibody blocked LPS-induced MMP-9 and TNFα secretion. Amyloid-ß peptide induced MMP-9 secretion, which occurred much later than TNFα secretion. The latter two findings strongly suggested an upstream role for TNFα in mediating LPS-stimulate MMP-9 secretion. CONCLUSION: The cumulative data indicated that MMP-9 secretion was a distinct process from TNFα secretion and occurred downstream. First, DMSO inhibited MMP-9, but not TNFα, suggesting that the MMP-9 secretion process was selectively altered. Second, cAMP inhibited both MMP-9 and TNFα with a similar potency, but at different monocyte cell exposure time points. The pattern of cAMP inhibition for these two molecules suggested that MMP-9 secretion lies downstream of TNFα and that TNFα may a key component of the pathway leading to MMP-9 secretion. This temporal relationship fit a model whereby early TNFα secretion directly led to later MMP-9 secretion. Lastly, antibody-blocking of TNFα diminished MMP-9 secretion, suggesting a direct link between TNFα secretion and MMP-9 secretion.

A Snapshot of Radiology Training During the Early COVID-19 Pandemic.

PURPOSE: The COVID-19 pandemic transformed the personal and professional lives of radiology trainees. The purpose of this study was to broadly summarize the impact of COVID-19 on radiology trainees and their training programs via data collected during the early pandemic. MATERIALS AND METHODS: An online survey was distributed to radiology chief residents in residencies throughout North America with responses collected between March 20th, 2020 and May 15th, 2020, which coincided with the development of initial COVID-19 peaks in North America. A subset of COVID-19 pandemic questions included resident wellness, imaging opinions, residency infrastructure change, and opinions regarding the Core Exam delay. RESULTS: One hundred forty chief residents from 86 institutions responded to COVID-19-related questions. Nearly all responding programs (99%; 85/86) reported institutional positive cases of COVID-19. Most residents (94%; 132/140) thought laboratory testing provided more value than imaging. Fifty-seven percent of respondents (80/140) would use COVID-19-related terminology when encountering chest CT findings supportive of viral pneumonia in symptomatic patients. There was little reported change in the number of residents on call (no change reported in >80% of programs). Fifty-nine percent of residents (83/140) reported increased stress related to the COVID-19 pandemic. The majority of programs (93%) had fewer residents on service (80/86 responding programs). CONCLUSIONS: COVID-19 dramatically affected radiology residencies during the early pandemic period. As we enter future phases of the COVID-19 pandemic, careful thought should also be given to rebuilding the radiology resident experience.

A Sall1-NuRD interaction regulates multipotent nephron progenitors and is required for loop of Henle formation.

The formation of the proper number of nephrons requires a tightly regulated balance between renal progenitor cell self-renewal and differentiation. The molecular pathways that regulate the transition from renal progenitor to renal vesicle are not well understood. Here, we show that Sall1interacts with the nucleosome remodeling and deacetylase complex (NuRD) to inhibit premature differentiation of nephron progenitor cells. Disruption of Sall1-NuRD in vivo in knock-in mice (ΔSRM) resulted in accelerated differentiation of nephron progenitors and bilateral renal hypoplasia. Transcriptional profiling of mutant kidneys revealed a striking pattern in which genes of the glomerular and proximal tubule lineages were either unchanged or upregulated, and those in the loop of Henle and distal tubule lineages were downregulated. These global changes in gene expression were accompanied by a significant decrease in THP-, NKCC2- and AQP1-positive loop of Henle nephron segments in mutant ΔSRM kidneys. These findings highlight an important function of Sall1-NuRD interaction in the regulation of Six2-positive multipotent renal progenitor cells and formation of the loop of Henle.

The metabolic footprint of the airway bacterial community in cystic fibrosis.

BACKGROUND: Progressive, chronic bacterial infection of the airways is a leading cause of death in cystic fibrosis (CF). Culture-independent methods based on sequencing of the bacterial 16S rRNA gene describe a distinct microbial community that decreases in richness and diversity with disease progression. Understanding the functional characteristics of the microbial community may aid in identifying potential therapies and may assist in management, but current methods are cumbersome. Here, we demonstrate the use of an oxidative metabolic assay as a complement to sequencing methods to describe the microbiome in the airways of patients with CF. METHODS: Expectorated sputum was collected from 16 CF subjects and 8 control subjects. The Biolog Gen III Microplate was used in a community-level physiological profiling (CLPP)-based assay to examine oxidative metabolic activity. 16S rRNA V4 amplicon sequencing was used to characterize the taxonomy and diversity of the samples. Correlations were then identified among the oxidative activity and taxonomy data. In an additional paired analysis, sputum from seven CF subjects were collected at two separate clinic visits and compared for oxidative activity, taxonomy, and diversity. RESULTS: Significant differences in oxidative metabolic activity, microbial taxonomy, and diversity were found between the CF and control sputum samples. Oxidative activity correlated positively with total genera but not with other measures of diversity or taxonomy, demonstrating that the metabolic assay complements the structural aspects of the microbiome. As expected, Pseudomonas was significantly enriched in CF samples, while Streptococcus and Prevotella were similarly abundant in both CF and control samples. Paired analysis of CF samples at separate clinic visits revealed comparable oxidative activity that correlated with similar stability in taxonomy and diversity. CONCLUSIONS: The CLPP assay used in this study complements existing sequencing methods to delineate the oxidative metabolic footprint of the CF airway bacterial community. This method may be useful to study the CF microbial community over time and with changes in disease state.

Corticosteroid therapy and airflow obstruction influence the bronchial microbiome, which is distinct from that of bronchoalveolar lavage in asthmatic airways.

BACKGROUND: The lung has a diverse microbiome that is modest in biomass. This microbiome differs in asthmatic patients compared with control subjects, but the effects of clinical characteristics on the microbial community composition and structure are not clear. OBJECTIVES: We examined whether the composition and structure of the lower airway microbiome correlated with clinical characteristics of chronic persistent asthma, including airflow obstruction, use of corticosteroid medications, and presence of airway eosinophilia. METHODS: DNA was extracted from endobronchial brushings and bronchoalveolar lavage fluid collected from 39 asthmatic patients and 19 control subjects, along with negative control samples. 16S rRNA V4 amplicon sequencing was used to compare the relative abundance of bacterial genera with clinical characteristics. RESULTS: Differential feature selection analysis revealed significant differences in microbial diversity between brush and lavage samples from asthmatic patients and control subjects. Lactobacillus, Pseudomonas, and Rickettsia species were significantly enriched in samples from asthmatic patients, whereas Prevotella, Streptococcus, and Veillonella species were enriched in brush samples from control subjects. Generalized linear models on brush samples demonstrated oral corticosteroid use as an important factor affecting the relative abundance of the taxa that were significantly enriched in asthmatic patients. In addition, bacterial α-diversity in brush samples from asthmatic patients was correlated with FEV1 and the proportion of lavage eosinophils. CONCLUSION: The diversity and composition of the bronchial airway microbiome of asthmatic patients is distinct from that of nonasthmatic control subjects and influenced by worsening airflow obstruction and corticosteroid use.

Airway Inflammation after Bronchial Thermoplasty for Severe Asthma.

RATIONALE: Bronchial thermoplasty is an alternative treatment for patients with severe, uncontrolled asthma in which the airway smooth muscle is eliminated using radioablation. Although this emerging therapy shows promising outcomes, little is known about its effects on airway inflammation. OBJECTIVES: We examined the presence of bronchoalveolar lavage cytokines and expression of smooth muscle actin in patients with severe asthma before and in the weeks after bronchial thermoplasty. METHODS: Endobronchial biopsies and bronchoalveolar lavage samples from 11 patients with severe asthma were collected from the right lower lobe before and 3 and 6 weeks after initial bronchial thermoplasty. Samples were analyzed for cell proportions and cytokine concentrations in bronchoalveolar lavage and for the presence of α-SMA in endobronchial biopsies. MEASUREMENTS AND MAIN RESULTS: α-SMA expression was decreased in endobronchial biopsies of 7 of 11 subjects by Week 6. In bronchoalveolar lavage fluid, both transforming growth factor-ß1 and regulated upon activation, normal T-cell expressed and secreted (RANTES)/CCL5 were substantially decreased 3 and 6 weeks post bronchial thermoplasty in all patients. The cytokine tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in several cell types, was increased in concentration both 3 and 6 weeks post bronchial thermoplasty. CONCLUSIONS: Clinical improvement and reduction in α-SMA after bronchial thermoplasty in severe, uncontrolled asthma is associated with substantial changes in key mediators of inflammation. These data confirm the substantial elimination of airway smooth muscle post thermoplasty in the human asthmatic airway and represent the first characterization of significant changes in airway inflammation in the first weeks after thermoplasty.

Antifibrotic effects of noscapine through activation of prostaglandin E2 receptors and protein kinase A.

Myofibroblast differentiation is a key process in the pathogenesis of fibrotic disease. We have shown previously that differentiation of myofibroblasts is regulated by microtubule polymerization state. In this work, we examined the potential antifibrotic effects of the antitussive drug, noscapine, recently found to bind microtubules and affect microtubule dynamics. Noscapine inhibited TGF-ß-induced differentiation of cultured human lung fibroblasts (HLFs). Therapeutic noscapine treatment resulted in a significant attenuation of pulmonary fibrosis in the bleomycin model of the disease. Noscapine did not affect gross microtubule content in HLFs, but inhibited TGF-ß-induced stress fiber formation and activation of serum response factor without affecting Smad signaling. Furthermore, noscapine stimulated a rapid and profound activation of protein kinase A (PKA), which mediated the antifibrotic effect of noscapine in HLFs, as assessed with the PKA inhibitor, PKI. In contrast, noscapine did not activate PKA in human bronchial or alveolar epithelial cells. Finally, activation of PKA and the antifibrotic effect of noscapine in HLFs were blocked by the EP2 prostaglandin E2 receptor antagonist, PF-04418948, but not by the antagonists of EP4, prostaglandin D2, or prostacyclin receptors. Together, we demonstrate for the first time the antifibrotic effect of noscapine in vitro and in vivo, and we describe a novel mechanism of noscapine action through EP2 prostaglandin E2 receptor-mediated activation of PKA in pulmonary fibroblasts.

Melanopsin-dependent persistence and photopotentiation of murine pupillary light responses.

PURPOSE: To determine the relative contributions of inner and outer retinal photoreception to the pupillary light response. METHODS: Wild-type, retinal degenerate (rd/rd), and melanopsin mutant (opn4(-/-)) mice were tested for pupillary light responsiveness by video pupillometry before, during, and after exposure to supersaturating light intensities. Similar lighting protocols were used to probe responses of intrinsically photosensitive retinal ganglion cells (ipRGCs) recorded with multielectrode arrays ex vivo. RESULTS: Both outer retinal photoreceptors (rods and cones) and inner retinal photoreceptors (intrinsically photosensitive retinal ganglion cells [ipRGCs]) are sufficient to drive the pupillary light response in mice. After supersaturating light exposure, rather than bleaching or adapting, rd/rd mice showed paradoxical potentiation of responses to subsaturating light exposure. opn4(-/-) mice, in contrast, could not sustain pupillary constriction under continuous bright illumination, and showed desensitization after bright-light exposure. Both the intensity of light necessary to induce potentiation and the spectral sensitivity for sustained and potentiated responses differed from that necessary to trigger pupillary constriction, suggesting that photopotentiation is dependent on a pigment-state distinct from that triggering the pupillary light response itself. Multielectrode array recordings of ipRGCs from rd/rd retinas demonstrated persistent cell firing under continuous light exposure but did not show potentiation. CONCLUSIONS: Unique photoreceptive properties of intrinsically photosensitive RGCs confer resistance to bleaching and/or adaptation under continuous bright illumination to the pupillary light response and suggest the presence of a photopigment with multiple absorption states.