Differences In Cell Populations Research Articles

The Identification of Potential Anti-Depression/Anxiety Drug Targets by Stress-Induced Rat Brain Regional Proteome and Network Analyses.

Depression and anxiety disorders are prevalent stress-related neuropsychiatric disorders and involve multiple molecular changes and dysfunctions across various brain regions. However, the specific and shared pathophysiological mechanisms occurring in these regions remain unclear. Previous research used a rat model of chronic mild stress (CMS) to segregate and identify depression-susceptible, anxiety-susceptible, and insusceptible groups; then the proteomes of six distinct brain regions (the hippocampus, prefrontal cortex, hypothalamus, pituitary, olfactory bulb, and striatum) were separately and quantitatively analyzed. To gain a comprehensive and systematic understanding of the molecular abnormalities, this study aimed to investigate and compare differential proteomics data from the six regions. Differentially expressed proteins (DEPs) were identified in between specific regions and across all regions and subjected to a series of bioinformatics analyses. Regional comparisons showed that stress-induced proteomic changes and corresponding gene ontology and pathway enrichments were largely distinct, attributable to differences in cell populations, protein compositions, and brain functions of these areas. Additionally, a notable degree of overlap in the significantly enriched terms was identified, potentially suggesting strong connections in the enrichment across different regions. Furthermore, intra-regional and inter-regional protein-protein interaction networks and drug-target-DEP networks were constructed. Integrated analysis of the three association networks in the six regions, along with the DisGeNET database, identified ten DEPs as potential targets for anti-depression/anxiety drugs. Collectively, these findings revealed commonalities and differences across different brain regions at the protein level induced by CMS, and identified several novel protein targets for the development of new therapeutics for depression and anxiety.

Chorioamnionitis accelerates granule cell and oligodendrocyte maturation in the cerebellum of preterm nonhuman primates

BackgroundPreterm birth is often associated with chorioamnionitis and leads to increased risk of neurodevelopmental disorders, such as autism. Preterm birth can lead to cerebellar underdevelopment, but the mechanisms of disrupted cerebellar development in preterm infants are not well understood. The cerebellum is consistently affected in people with autism spectrum disorders, showing reduction of Purkinje cells, decreased cerebellar grey matter, and altered connectivity.MethodsPreterm rhesus macaque fetuses were exposed to intra-amniotic LPS (1 mg, E. coli O55:B5) at 127 days (80%) gestation and delivered by c-section 5 days after injections. Maternal and fetal plasma were sampled for cytokine measurements. Chorio-decidua was analyzed for immune cell populations by flow cytometry. Fetal cerebellum was sampled for histology and molecular analysis by single-nuclei RNA-sequencing (snRNA-seq) on a 10× chromium platform. snRNA-seq data were analyzed for differences in cell populations, cell-type specific gene expression, and inferred cellular communications.Results We leveraged snRNA-seq of the cerebellum in a clinically relevant rhesus macaque model of chorioamnionitis and preterm birth, to show that chorioamnionitis leads to Purkinje cell loss and disrupted maturation of granule cells and oligodendrocytes in the fetal cerebellum at late gestation. Purkinje cell loss is accompanied by decreased sonic hedgehog signaling from Purkinje cells to granule cells, which show an accelerated maturation, and to oligodendrocytes, which show accelerated maturation from pre-oligodendrocytes into myelinating oligodendrocytes.ConclusionThese findings suggest a role of chorioamnionitis on disrupted cerebellar maturation associated with preterm birth and on the pathogenesis of neurodevelopmental disorders among preterm infants.

Abstract 16780: Single Cell Profiles of Human Heart Failure and Myocardial Diseases

Background: Heart failure represents the end-stage of a range of diseases which adversely impact the heart muscle. The extent by which cellular and molecular mechanisms differ between myocardial diseases in heart failure remains incompletely understood. Methods: We performed isolation and droplet-based RNA-sequencing of 394 247 single nuclei from 103 explanted hearts from transplant and ventricular assist device recipients and 7 unused donor hearts without heart disease. After sample and nuclei level quality control, doublet removal and sample integration; principal component analysis and shared nearest neighbor clustering were used to identify distinct cell populations. Cell type composition and transcriptomic profiles were compared across donor hearts and 10 myocardial diseases (hypertrophic, dilated, ischemic, restrictive, arrythmogenic and non-compaction cardiomyopathy; rheumatic and congenital heart disease; cardiac amyloidosis and graft rejection; n=1-45/group). Results: We identified and manually annotated 14 distinct cell populations that were present across controls and myocardial diseases, including transcriptionally diverse subgroups of cardiomyocytes, endothelial cells and pericytes. Compared to controls, we observed disease-specific differences in cell populations with regards to composition, heterogeneity, and specific transcriptional profiles. Conclusions: Our results reveal differences in single cell profiles of individual heart muscle diseases compared to controls without heart disease.

C/EBPα Acts As an RNA Binding Protein Essential for, and Promotes End-Stage Macrophage Differentiation

The transcription factor C/EBPα is a well characterized DNA binding protein with essential functions in controlling and regulating myeloid differentiation and lipid metabolism. Herein, we describe C/EBPα's separate and distinct RNA binding characteristics. Using Chromatin RNA Immunoprecipitation, we identified that C/EBPα interacts primarily with RNA introns in both HL-60 and THP-1 cells, with a preference for a palindromic GC-rich binding motif. Structural prediction in conjunction with RNA electrophoretic mobility shift assays show that C/EBPα interacts with RNA through two previously undescribed domains located towards the proteins N terminus. These domains are distinct from C/EBPα's DNA binding b-ZIP domain which is instead located on the proteins C terminus. Mouse bone marrow transplantation and in vitro cytokine assays reveal that C/EBPα RNA binding appears to be essential for macrophage maturation but not neutrophil differentiation. To better understand these phenotypic differences, we expanded our observations into the transcriptomic space by utilizing single-cell CITE-Seq. In line with biochemical data, notable mature cell populations were absent when the C/EBPα's RNA binding domains are excluded from the protein. Intriguingly, differential gene expression revealed strong, selective upregulation of Lipoprotein Lipase (Lpl) in monocyte, but not neutrophil populations (Panel 1). It has been previously reported that dysregulation of Lpl affects bone marrow monocyte progenitor differentiation and results in dysregulated cellular mobilization from the bone marrow, a phenotype that is commonly seen in cancers such as AML. The increased abundance of monocyte and neutrophil populations in the C/EBPα RNA deletion samples is therefore suggestive that C/EBPα RNA binding is critical in regulating terminal differentiation and mobilization circuits which may become dysregulated or perturbed in diseases such as AML. On-going analyses are investigating the RNA velocity trajectory of captured cell populations in an attempt to better understand at which stage C/EBPα RNA binding becomes essential for terminal maturation and differentiation of select cell populations. Taken together, we demonstrate that C/EBPα is also a bona fida RNA binding protein with unique functions distinct from its DNA binding activity that are essential for monocyte maturation, differentiation and mobilization. Panel 1 - Identified Cell Populations Following scCITE-Seq with C/EBPα Full-Length (WT) and C/EBPα RNA Binding Deletion (DD). Each circle represents a single cell presented in UMAP space. The left panel shows populations identified with the full-length C/EBPα construct. The right panel shows the cell populations identified with the RNA domain deletion construct. Notable differences in cell populations and numbers are evident. Overlayed onto the cells is the expression of Lipoprotein Lipase (Lpl). Expression is shown as a gradient from black up to green, representing a log2(4) expression. Lpl expression is shown to be expressed uniquely in the monocyte population of cells almost exclusively in the RNA deletion sample.

Generation of Human Regulatory Dendritic Cells from Cryopreserved Healthy Donor Cells and Hematopoietic Stem Cell Transplant Recipients.

Acute graft versus host disease (GVHD) remains a significant complication following hematopoietic stem cell transplant (HSCT), despite improved human leukocyte antigen (HLA) matching and advances in prophylactic treatment regimens. Previous studies have shown promising results for future regulatory dendritic cell (DCreg) therapies in the amelioration of GVHD. This study evaluates the effects of cryopreservation on the generation of DCreg, the generation of young and older DCreg in serum-free media, and the feasibility of generating DCreg from young and older HSCT patient monocytes. DCregs were generated in X-vivo 15 serum-free media from donor or patient monocytes. This study includes the use of monocytes from young and older healthy, donor, and HSCT patients with varying hematological diseases. Phenotypic differences in cell populations were assessed via flow cytometry while pro-inflammatory and anti-inflammatory cytokine production was evaluated in culture medium. The number of DCreg generated from cryopreserved monocytes of healthy donors was not significantly different from freshly isolated monocytes. DCreg generated from cryopreserved monocytes had comparable levels of co-stimulatory molecule expression, inhibitory molecule expression, and cytokine production as freshly isolated monocytes. Young and older healthy donor monocytes generated similar numbers of DCreg with similar cytokine production and phenotype. Although monocytes from older HSCT patients generated significantly fewer DCreg, DCreg from young and older HSCT patients had comparable phenotypes and cytokine production. Monocytes from young and older myelodysplastic syndrome (MDS) patients generated reduced numbers of DCreg compared to non-MDS-derived DCreg. We demonstrate that the cryopreservation of monocytes from HSCT patients of varying hematological diseases allows for the cost-effective generation of DCreg on an as-needed basis. Although the generation of DCreg from MDS patients requires further assessment, these data support the possibility of in vitro-generated DCreg as a therapy to reduce GVHD-associated morbidity and mortality in young and older HSCT recipients.

Single-Cell Network-Based Drug Repositioning for Discovery of Therapies against Anti-Tumour Necrosis Factor-Resistant Crohn's Disease.

Primary and secondary non-response affects approximately 50% of patients with Crohn's disease treated with anti-tumour necrosis factor (TNF) monoclonal antibodies. To date, very little single cell research exists regarding drug repurposing in Crohn's disease. We aimed to elucidate the cellular phenomena underlying resistance to anti-TNF therapy in patients with Crohn's disease and to identify potential drug candidates for these patients. Single-cell transcriptome analyses were performed using data (GSE134809) from the Gene Expression Omnibus and Library of Integrated Network-Based Cellular Signatures L1000 Project. Data aligned to the Genome Reference Consortium Human Build 38 reference genome using the Cell Ranger software were processed using the Seurat package. To capture significant functional terms, gene ontology functional enrichment analysis was performed on the marker genes. For biological analysis, 93,893 cells were retained (median 20,163 genes). Through marker genes, seven major cell lineages were identified: B-cells, T-cells, natural killer cells, monocytes, endothelial cells, epithelial cells, and tissue stem cells. In the anti-TNF-resistant samples, the top 10 differentially expressed genes were HLA-DQB-1, IGHG1, RPS23, RPL7A, ARID5B, LTB, STAT1, NAMPT, COTL1, ISG20, IGHA1, IGKC, and JCHAIN, which were robustly distributed in all cell lineages, mainly in B-cells. Through molecular function analyses, we found that the biological functions of both monocyte and T-cell groups mainly involved immune-mediated functions. According to multi-cluster drug repurposing prediction, vorinostat is the top drug candidate for patients with anti-TNF-refractory Crohn's disease. Differences in cell populations and immune-related activity within tissues may influence the responsiveness of Crohn's disease to anti-TNF agents. Vorinostat may serve as a promising novel therapy for anti-TNF-resistant Crohn's disease.

Single-cell analysis of Sézary syndrome reveals novel markers and shifting gene profiles associated with treatment

AbstractCutaneous T-cell lymphomas (CTCLs) are a spectrum of diseases with varied clinical courses caused by malignant clonal proliferation of skin-tropic T cells. Most patients have an indolent disease course managed with skin-directed therapies. In contrast, others, especially in advanced stages of disease or with specific forms, have aggressive progression and poor median survival. Sézary syndrome (SS), a leukemic variant of CTCL, lacks highly consistent phenotypic and genetic markers that may be leveraged to prevent the delay in diagnosis experienced by most patients with CTCL and could be useful for optimal treatment selection. Using single-cell mRNA and T-cell receptor sequencing of peripheral blood immune cells in SS, we extensively mapped the transcriptomic variations of nearly 50 000 T cells of both malignant and nonmalignant origins. We identified potential diverging SS cell populations, including quiescent and proliferative populations shared across multiple patients. In particular, the expression of AIRE was the most highly upregulated gene in our analysis, and AIRE protein expression could be observed over a variety of CTCLs. Furthermore, within a single patient, we were able to characterize differences in cell populations by comparing malignant T cells over the course of treatment with histone deacetylase inhibition and photopheresis. New cellular clusters after progression of the therapy notably exhibited increased expression of the transcriptional factor FOXP3, a master regulator of regulatory T-cell function, raising the potential implication of an evolving mechanism of immune evasion.

070 Comprehensive single-cell analysis of Sézary syndrome reveals novel expression and therapeutic biomarkers

Cutaneous T cell lymphomas (CTCL) are a group of malignancies thought to derive from a clonal proliferation of skin-tropic T cells. Diagnosis is often delayed several years due to variations in presentation and lack of definitive biomarkers, which result in missed early intervention opportunities that may prevent disease progression. In the setting of more aggressive forms of CTCL, most treatments can initially decrease the tumor burden, but often ultimately fail to prevent the selective outgrowth of a resistant population. The lack of effective therapies is due in part to an incomplete understanding of tumor biology. Using single-cell mRNA and adaptive immune receptor sequencing of peripheral blood immune cells in SS, transcriptomic variations were quantified of almost 50,000 malignant and nonmalignant T cells across six SS patients. Within the population of single cells, we identified both quiescent and proliferative populations across multiple patients, and were able to newly identify a large number of genes associated with malignancy. Furthermore, in a single patient, we characterized differences in cell populations comparing malignant T cells after disease progression in the setting of treatment with histone deacetylase inhibition (HDACi) and photopheresis. HDACi therapy led to new transcriptional profiles in a subset of SS cells, characterized by the increased expression of the transcriptional factor FOXP3. These data are critical in defining a compendium of gene expression profiles and intratumoral heterogeneity in SS and CTCL, which are pivotal in our understanding of CTCL biology and in the development of targeted treatments.

Unraveling the Big Sleep: Molecular Aspects of Stem Cell Dormancy and Hibernation.

Tissue-resident stem cells may enter a dormant state, also known as quiescence, which allows them to withstand metabolic stress and unfavorable conditions. Similarly, hibernating mammals can also enter a state of dormancy used to evade hostile circumstances, such as food shortage and low ambient temperatures. In hibernation, the dormant state of the individual and its cells is commonly known as torpor, and is characterized by metabolic suppression in individual cells. Given that both conditions represent cell survival strategies, we here compare the molecular aspects of cellular quiescence, particularly of well-studied hematopoietic stem cells, and torpor at the cellular level. Critical processes of dormancy are reviewed, including the suppression of the cell cycle, changes in metabolic characteristics, and cellular mechanisms of dealing with damage. Key factors shared by hematopoietic stem cell quiescence and torpor include a reversible activation of factors inhibiting the cell cycle, a shift in metabolism from glucose to fatty acid oxidation, downregulation of mitochondrial activity, key changes in hypoxia-inducible factor one alpha (HIF-1α), mTOR, reversible protein phosphorylation and autophagy, and increased radiation resistance. This similarity is remarkable in view of the difference in cell populations, as stem cell quiescence regards proliferating cells, while torpor mainly involves terminally differentiated cells. A future perspective is provided how to advance our understanding of the crucial pathways that allow stem cells and hibernating animals to engage in their ‘great slumbers.’

DOP48 Gene expression landscape of epithelial monolayer in inflammatory bowel disease

Abstract Background Gene expression analyses on IBD tissue are dominated by analyses of biopsy homogenates that are highly heterogeneous due to a difference in cell populations when contrasting inflamed vs. healthy mucosa. We wished to characterise the inflammatory activity of the isolated epithelial monolayer (EM), avoiding the infiltrating inflammatory cells, thereby form a comprehensive understanding of the epithelium’s role in IBD pathobiology. Methods Total RNA was isolated from laser capture microdissected (LCM) colon EM, and sequencing libraries were prepared using TruSeq RNA access kit (Illumina, CA, USA). Gene cluster analysis (WGCNA) was used to stratify the expression data in genetic modules. Following this, we characterised the modules correlated to IBD status, using Metacore pathway analysis, in order to suggest what underlying biological processes contribute to the observed correlation of expression data. Finally, we used immunohistochemistry to verify the protein expression pattern of key players in the suggested pathways, thereby confirming EM involvement in these processes during inflammation. Results Biopsies from active (a) IBD (7 UCa, 5 CDa) and from un-inflamed (u) IBD and healthy control (7 UCu, 5 CDu and 6 HC) were included in the analysis. 6386 genes were differentially expressed (adjusted p-value (pval.) <0.05) in EM from active inflamed colon vs. un-inflamed (Figure 1). Six gene modules showed a significant correlation between inflammatory status and gene expression (Figure 2). Of note was a broad, significant up-regulation of MHC class II presentation machinery, e.g. STAT1 (log2 fold change (lfc) 0.93, p < 0.001), CIITA (lfc 2.57, p < 0.001) and HLA-DRB1 (lfc 3.69, p < 0.001), and a down-regulation unique to epithelial cells of the retinoic acid receptors RARA (lfc −0.79, p < 0.001) and RXRA (log fc −0.56, p < 0.001). The most significantly up-regulated pathway was ‘Immune Response’, induction of antigen presentation machinery by IFN-γ (FDR 2.5 × 10–18), while ‘RXR-dependent regulation of lipid metabolism’ (FDR 2.6 × 10–8) was the most significantly down-regulated pathway. Conclusion Our analyses suggest that the involvement of the EM in IBD inflammation goes far beyond acting as a barrier. The largest genetic modules are dominated by genes important for inflammatory regulation, suggesting that the EM is much more involved in these processes than what is commonly understood. This study lends support to further analyses of interplay between epithelial cells and immune cells, particularly in a pro-inflammatory environment.

Chondrocyte colonisation of a tissue-engineered cartilage substitute under a mechanical stimulus

Cell-free collagen scaffolds as cartilage substitute for small focal defects show promising results in first clinical studies. However, chondrocyte migration between scaffolds and the colonisation process of a cell-free implant is yet to be fully understood. We here focus on mechanobiological interdependencies between cell migration and mechanical stimulus in a 3D environment. We develop an in vitro model composed of a human chondrocyte-seeded collagen base and adjacent cell-free collagen type I scaffolds of varying collagen concentrations. Constructs are either cultured statically or dynamically under the influence of a physiological compression (0.5Hz, 0.5% initial strain). After 20 days we identify vital chondrocytes inside all collagen implants, proving that chondrocytes migrated from the underlying scaffold into the implants. Chondrocytes have not colonised the entire sample and are predominantly found in the bottom of the implant. In static culture conditions, a nearly equal cell number is found inside of all collagen scaffolds. In dynamic culture, the total amount of cells is increased by 30% to 320%, with the highest population in a commercial implant. Differences in cell population between the materials in dynamic culturing can be referred to differences in mechanical properties of the scaffolds, such as strain-rate insensitivity fostering the colonisation process.

Histologic comparison of inflammatory cells in the penile and preputial epithelium of young vs mature beef bulls

There is evidence supporting clearance of the Tritrichomonas foetus in young bulls. The reasons for mature bulls becoming chronic carriers and resolution of venereal infections in young bulls have not been illuminated. It has been hypothesized that there is variation in the inflammatory response in the reproductive tract as the bull ages. Hence, the objectives of this study were to evaluate the immune cell population in the normal bovine penile and preputial epithelium and to examine any differences in cell populations between young and mature age groups. Our hypothesis was that there are significant differences in immune cell types within the penile and preputial epithelium and dermis of young versus mature bulls.

Abstract 1469: Integrated analysis of microRNA, mRNA, and protein expression utilizing MultiOmyxTM and NanoStringTM from formalin-fixed paraffin-embedded, lung, head and neck, breast, and melanoma tumors

Abstract Cancer is characterized as a loss of normal cellular regulation, due to accumulation of mutations and disruption of complex biological pathways. MicroRNAs (miRNAs) regulation of co-stimulatory and immune checkpoint pathways have been implicated as one of the potential mechanisms for cancer evasion in immuno-oncology. It is estimated that 30% of all mRNA expression may be regulated by miRNAs, and some are either oncogenic or tumor suppressive. Complexity of miRNA regulation highlights the need for integrated assays, providing direct correlation between miRNA and mRNA, and protein expression. From a single 4 µm FFPE section, MultiOmyxTM hyperplexed immunofluorescent assay (demonstrated to stain up to 60 protein biomarkers) is utilized to measure CD3, CD4, CD8, CD16, CD56, Granzyme B, FoxP3, ICOS, OX40, OX40L, PD1, PDL1, HLA-DR, and Ki67 protein expression. From an adjacent 10 µm section, NanoStringTM nCounter PanCancer Immune Profiling Panel and Human v3 miRNA expression panel were utilized to comprehensively profile the expression of 770 mRNA and 800 miRNA. Integrating MultiOmyx and NanoString technologies, the current study measured miRNA, mRNA, and protein expression in lung, head and neck, breast, and melanoma samples. For each indication, three samples were selected from a larger sample set, based on high protein expression of lymphocytes and macrophage markers (CD3, CD4, CD8, CD56, CD16), co-stimulator markers (ICOS, OX40), and immune checkpoint markers (PD1, PD-L1). Protein expression results indicate positive correlation between expression of ICOS and OX40 with higher infiltration of Thelper (CD3+CD4+), Tcytotoxic (CD3+CD8+), and effector T cells (CD3+CD8+Granzyme B). NanoString normalized mRNA counts for the protein biomarkers profiled indicate that all markers except for HLA-DR belong to low expressers group with counts ranging from 20-700. Comparison of protein expression to mRNA counts revealed inconsistencies in modulated markers (PD1, PD-L1, ICOS, OX40) which are attributed to differences in population of cells between the two sections. Direct assessment of up regulation of miRNA and down regulation of target mRNA could not be made for miRNAs reported in literature as negative regulator of PD-L1 (miR-34a, 34b, 34c), PD1 (miR-28, miR-107), FoxP3 (miR-210, miR-24, miR-31), and ICOS (miR-101). Analysis of multiple miRNAs (combinatorial targeting) mapped in context to mRNA, and their respective protein expression will be presented from lung, head and neck, breast, and melanoma cancer samples. Citation Format: Qingyan Au, RaghavKrishna Padmanabhan, Nam Tran, Lakshmi Chandramohan, Nicholas Hoe. Integrated analysis of microRNA, mRNA, and protein expression utilizing MultiOmyxTM and NanoStringTM from formalin-fixed paraffin-embedded, lung, head and neck, breast, and melanoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1469. doi:10.1158/1538-7445.AM2017-1469

Osteoclasts in Periprosthetic Osteolysis: The Charnley Arthroplasty Revisited

BackgroundPeriprosthetic osteolysis by polyethylene wear debris–triggered osteoclasts is viewed as the main pathophysiological pathway in aseptic loosening in total hip arthroplasty. The present aim was to study osteoclast occurrence in osteolytic lesions in early and late revisions of the Charnley low-friction torque arthroplasty (CLFA). MethodsBiopsies of the soft interface membrane and the adjacent bone were taken from osteolytic lesions during revision of 16 loose CLFA, early (2-6 years) or late (>10 years) after primary surgery. By light microscopy (LM), cell-dense regions with signs of osteoclast-mediated bone resorption were selected for transmission electron microscopy. Three additional patients were studied in LM for osteoclast markers (tartrate-resistant acid phosphatase and Cathepsin K). ResultsLM disclosed a low-grade chronic inflammation and birefringent particles in most sections. Multiple conglomerates of tartrate-resistant acid phosphatase positive and Cathepsin K positive mononuclear and multinucleated cells were found deep in the fibrous interface membrane. Transmission electron microscopy showed traces of polyethylene-like particles in 67%-100% of the cells. Osteoclast-like cells exhibiting resorptive activity were few (mean, 0.7%; standard deviation, 0.2%), and multinucleated cells, possibly osteoclast precursor cells, located immediately on the bone were also scarce (mean, 2.7%; standard deviation, 5.3%). Multinucleated (odds ratio, 3.0; 95% confidence interval, 1.7-5.5) and macrophage-like cells (odds ratio, 3.6; 95% confidence interval, 2.2-5.6) were typically located deeper in the inflammatory interface membrane with a pathologic appearance with distension and abundance of phagocytic vacuoles. There were no systematic differences in cell populations between early or late revisions. ConclusionDespite probable ongoing osteoclastogenesis in the osteolytic lesions, there were few sites of osteoclast-mediated bone resorption. These findings attach a contributing biological explanation to the longevity of the CLFA.

Loss of CCR5 signaling results in early NK cell and neutrophil recruitment into the liver and increased hepatic inflammation in a mouse model of acute hepatitis B

Abstract Human genetic studies demonstrate that a non-functional CCR5 receptor sequence variant is associated with enhanced control of acute hepatitis B virus (HBV) infection. To better understand the role of CCR5 in immune responses to HBV, we employed a mouse model of acute HBV in which Ccr5 +/+(WT) or Ccr5 −/−(KO) C57BL/6 mice were intravenously infected with adenovirus containing an overlapping HBV1.3 construct (AdHBV) or vector control (AdShuttle). Mice (N=4–6/group) were euthanized at days 2, 3, 7, 10, and 14 post injection. Increased plasma ALT levels were observed in both Ccr5 WT and KO AdHBV mice at day 7, but levels were significantly higher in the Ccr5 KO compared to WT mice by day 14 (554 vs 274 U/L, p < 0.05). Plasma ALT levels were not significantly different between Ccr5 WT and KO AdShuttle mice. Analysis of intrahepatic leukocytes by flow cytometry revealed significantly increased intrahepatic CD11b+ NK cells (26.3 vs 6.9, % of live leukocytes, p< 0.01) and neutrophils (7.3 vs 1.3, % of live leukocytes, p< 0.05) at day 3 in Ccr5 KO compared to WT AdHBV mice. By day 7, these differences in cell populations were no longer present. At Day 14, significantly higher levels of intrahepatic Ly6c+ macrophages were detected in Ccr5 KO compared to WT AdHBV mice (17.0 vs 6.4, % of live leukocytes, p< 0.01). These differences in intrahepatic leukocytes were not observed in AdShuttle mice. Loss of Ccr5 function did not significantly alter intrahepatic CD4+ and CD8+ populations at the time points investigated. Taken together, these findings suggest that loss of Ccr5 signaling increases intrahepatic innate cells and inflammation during acute HBV infection, providing evidence for a role of CCR5 in regulation of innate immune responses to acute HBV infection.

Modeling Physiological Differences in Cell Populations: Acetone‐Butanol‐Ethanol (ABE) Fermentation in a Cascade of Continuous Stirred‐Tank Reactors

Modeling Physiological Differences in Cell Populations: Acetone‐Butanol‐Ethanol (ABE) Fermentation in a Cascade of Continuous Stirred‐Tank Reactors

Sex Differences in Pain.

Females greatly outnumber males as sufferers of chronic pain. Although social and psychological factors certainly play a role in the differences in prevalence and incidence, biological differences in the functioning of the immune system likely underlie these observed effects. This Review examines the current literature on biological sex differences in the functioning of the innate and adaptive immune systems as they relate to pain experience. With rodent models, we and others have observed that male mice utilize microglia in the spinal cord to mediate pain, whereas females preferentially use T cells in a similar manner. The difference can be traced to differences in cell populations, differences in suppression by hormones, and disparate cellular responses in males and females. These sex differences also translate into human cellular responses and may be the mechanism by which the disproportionate chronic pain experience is based. Recognition of the evidence underlying sex differences in pain will guide development of treatments and provide better options for patients that are tailored to their physiology. © 2016 Wiley Periodicals, Inc.

In vitro and in vivo genotoxicity assessment of selected pharmaceuticals in relation to Escherichia coli and Cyprinus carpio

ABSTRACTGenotoxicity studies (using SOS chromotest and comet assay) of Escherichia coli and carp (Cyprinus carpio) were performed for three pharmaceutically active compounds, ciprofloxacin, 17α-ethinylestradiol and 5-fluorouracil, used in the treatment of humans. The values of genotoxicity induction coefficient (I) in the SOS chromotest clearly showed genotoxicity for ciprofloxacin, both in the presence and in the absence of S9 fraction; 17α-ethinylestradiol demonstrated slight genotoxicity at the highest tested concentration; and 5-fluorouracil did not induce genotoxic effects in Escherichia coli mutants. Statistical analysis of the results of the comet assay revealed significant differences in cell populations derived from carp placed in a solution of 5-fluorouracil in comparison with the negative control. Statistical analysis also showed a significant increase of “% DNA in tail” of comets in cell populations incubated in solutions of 17α-ethinylestradiol at concentrations of 10000, 2000 and 400 µg/L and in solutions of 5-fluorouracil with S9 fraction at concentrations of 50,000 and 2,000 μg/L in comparison with the negative controls.

Online analysis of single cyanobacteria and algae cells under nitrogen-limited conditions using aerosol time-of-flight mass spectrometry.

Metabolomics studies typically perform measurements on populations of whole cells which provide the average representation of a collection of many cells. However, key mechanistic information can be lost using this approach. Investigating chemistry at the single cell level yields a more accurate representation of the diversity of populations within a cell sample; however, this approach has many analytical challenges. In this study, an aerosol time-of-flight mass spectrometer (ATOFMS) was used for rapid analysis of single algae and cyanobacteria cells with diameters ranging from 1 to 8 μm. Cells were aerosolized by nebulization and directly transmitted into the ATOFMS. Whole cells were determined to remain intact inside the instrument through a combination of particle sizing and imaging measurements. Differences in cell populations were observed after perturbing Chlamydomonas reinhardtii cells via nitrogen deprivation. Thousands of single cells were measured over a period of 4 days for nitrogen-replete and nitrogen-limited conditions. A comparison of the single cell mass spectra of the cells sampled under the two conditions revealed an increase in the dipalmitic acid sulfolipid sulfoquinovosyldiacylglycerol (SQDG), a chloroplast membrane lipid, under nitrogen-limited conditions. Single cell peak intensity distributions demonstrate the ability of the ATOFMS to measure metabolic differences of single cells. The ATOFMS provides an unprecedented maximum throughput of 50 Hz, enabling the rapid online measurement of thousands of single cell mass spectra.

The use of noncellularized artificial dermis in the prevention of scar contracture and hypertrophy

Abnormal scarring occurring after wounds and burns remains a major source of functional and cosmetic disorders. The dermal part of the skin is recognized as playing a major role in the contraction process. The skin dermis may be involved in superficial wounds, but when the basal membrane and the sources of keratinocytes, like hair follicles and adnexa, are left intact, little visible scarring is observed. The skin elasticity may be preserved, even when the extracellular matrix is somehow altered. On the opposite extreme, in wounds of extended depth, the loss of dermal component results in the absence of elasticity and fibroblastic cell proliferation, leading to hypertrophy and contracture. These phenomena may be explained either by differences in cell populations, by extracellular matrix reactions to different stimuli, or by chemical control of interactions between cells and matrix.