scholarly journals OP0334 GGTASE DEFICIENT MACROPHAGES ALTER INTEGRIN EXPRESSION ON LYMPHOCYTES AND FACILITATE DEVELOPMENT OF ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 205-206
Author(s):  
K. M. Andersson ◽  
E. Malmhäll-Bah ◽  
M. C. Erlandsson ◽  
M. Brisslert ◽  
M. Akula ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Type I ◽  
Lymphocyte Function ◽  
Cd4 Cells ◽  
Rho Proteins ◽  
Integrin Receptors ◽  
Hox Proteins ◽  
Mixed Genetic Background ◽  
Leukocyte Influx ◽  
And Control

Background:Geranylgeranyltransferase type I (GGTaseI) is the enzyme responsible for the prenylation/ lipidation of the RhoA family proteins, which keeps them attached to the cell membrane. We reported that GGTaseI-deficient (GLC) mice develop a spontaneous and age-dependent arthritis, reproducing the pathology of RA1. Targeting GGTaseI activates RhoA proteins.Objectives:To study which of the activated Rho proteins is responsible for development of arthritis, we deleted individual RhoA, Rac1 or Cdc42 genes in GLC mice. We study consequences of GGTaseI deficiency for lymphocyte function.Methods:Double deficient mice that lack Rac1 (GLC Rac1fl/fl), RhoA (GLC RhoAfl/fl) and Cdc42 (GLC Cdc42fl/fl) were developed by Cre-technology using the LysM-promotor, and were on a mixed genetic background (129Ola/Hsd-C57BL/6)2. Joints of the hind paws were assessed for signs of arthritis histologically and by micro CT at age of 16 weeks. Phenotype of spleen CD4 and CD8 T cells was analysis by flow cytometry. Proliferation and cytokine production was assessed in spleen cultures by ELISA. Gene expression profile was analyzed by RT-PCR.Results:Deletion of Rho proteins had divergent effect on development of arthritis in GLC mice. We observed a reduction of the arthritis index in GLC Rac1fl/fl (n=19, p=0.027) and GLC RhoAfl/fl (n=4, p=0.007) mice compared to GLC (n=16), while GLC Cdc42fl/fl (n=4) had no change in arthritis development. GLC RhoAfl/fl mice increased the bone mass compared to GLC (p=0.029).Flow cytometry analysis showed that RA-prone GLC and GLC Cdc42fl/fl mice had lower number of CD4 cells in spleen. CD4 cells of RA-prone GLC and GLC Cdc42 mice had significantly higher subsets of the regulatory FoxP3+ and FOXp3+CD25+ cells (p=0.016-0.029 and p=0.016-0.029 respectively) compared to control and GLC RhoAfl/fl mice. Additionally, RA-prone mice had higher expression of receptors to extracellular matrix proteins collagen (α2β2) and fibronectin (α5β1) compared to control mice (p=0.016 and p=0.011 resp) and to RA-protected mice (GLC Rac1fl/fl and GLC RhoAfl/fl, p=0.0004 and p=0.011, resp). In total, both the number of FoxP3+ CD4 cells and the expression of α5β1 receptors on CD4 cells correlated strongly with the synovitis score (r=0.72, p=0.0017 and r=0.59, p=0.012, respectively).GGTaseI gene lays under the control of HOX proteins essential for cell homing. Importantly, HOX regulate the expression of integrins. Studying the expression of HoxA genes in spleen, we found that RA prone GLC and GLC Cdc42 mice tended to have lower expression of HoxA2 and higher expression of HoxA9 compared to RA-protected GLC Rac1 and GLC RhoA and to control mice. The Hoxa9/Hoxa2 ratio was significantly higher in RA prone mice compared to RA-protected mice (p=0.0085) and control mice (p=0.019). This ratio correlated with α5β1 receptors (r=0.55, p=0.0084), FOXP3+ CD4 cells (r=0.50, p=0.017), and the arthritis index (r=0.50, p=0.033).Conclusion:Taken together this study shows that Rho proteins play divergent role in development of arthritis. Activation of Rac1 and RhoA by GGTaseI deletion changes the pattern of HOXA proteins and increases expression of integrin receptors, which facilitates leukocyte influx in the paw joints. Deletion of Rac1 and RhoA has RA-protective effect in GLC mice.References:[1]Khan, O.M., et al.J Clin Invest121, 628 (2011).[2]Akula, M.K., et al.Nat Commun10, 3975 (2019).Disclosure of Interests:None declared

scholarly journals Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1320
Author(s):  
Kristýna Pekárková ◽  
Jakub Soukup ◽  
Marie Kostelanská ◽  
Jan Širc ◽  
Zbyněk Straňák ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cord Blood ◽  
Extracellular Vesicles ◽  
Correlation Coefficients ◽  
Flow Cytometer ◽  
Liquid Biopsies ◽  
Physiological Conditions ◽  
Flow Cytometric ◽  
Term Births ◽  
And Control

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods—one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.

scholarly journals OP0083 INFECTIOUS AND AUTOINFLAMMATORY MODIC TYPE 1 CHANGES HAVE DIFFERENT PATHOMECHANISMS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 45.2-45
Author(s):  
I. Heggli ◽  
R. Schüpbach ◽  
N. Herger ◽  
T. A. Schweizer ◽  
A. Juengel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cell ◽  
Cell Populations ◽  
Gene Set ◽  
Go Enrichment ◽  
Modic Type ◽  
And Control ◽  
Infectious Etiology

Background:Modic type 1 changes (MC1) are vertebral bone marrow (BM) edema that associate with non-specific low back pain (LBP). Two etiologies have been described. In the infectious etiology the anaerobic aerotolerant Cutibacterium acnes (C. acnes) invades damaged intervertebral discs (IVDs) resulting in disc infection and endplate damage, which leads to the evocation of an immune response. In the autoinflammatory etiology disc and endplate damage lead to the exposure of immune privileged disc cells and matrix to leukocytes, thereby evoking an immune response in the BM. Different etiologies require different treatment strategies. However, it is unknown if etiology-specific pathological mechanisms exist.Objectives:The aim of this study was to identify etiology-specific dysregulated pathways of MC1 and to perform in-depth analysis of immune cell populations of the autoinflammatory etiology.Methods:BM aspirates and biopsies were obtained from LBP patients with MC1 undergoing spinal fusion. Aspirates/biopsies were taken prior screw insertion through the pedicle screw trajectory. From each patient, a MC1 and an intra-patient control aspiration/biopsy from the adjacent vertebral level was taken. If C. acnes in IVDs adjacent to MC1 were detected by anaerobic bacterial culture, patients were assigned to the infectious, otherwise to the autoinflammatory etiology.Total RNA was isolated from aspirates and sequenced (Novaseq) (infectious n=3 + 3, autoinflammatory n=5 + 5). Genes were considered as differentially expressed (DEG) if p-value < 0.01 and log2fc > ± 0.5. Gene ontology (GO) enrichment was performed in R (GOseq), gene set enrichment analysis (GSEA) with GSEA software.Changes in cell populations of the autoinflammatory etiology were analyzed with single cell RNA sequencing (scRNAseq): Control and MC1 biopsies (n=1 + 1) were digested, CD45+CD66b- mononuclear cells isolated with fluorescence activated cell sorting (FACS), and 10000 cells were sequenced (10x Genomics). Seurat R toolkit was used for quality-control, clustering, and differential expression analysis.Transcriptomic changes (n=5 + 5) of CD45+CD66b+ neutrophils isolated with flow cytometry from aspirates were analyzed as for total bulk RNAseq. Neutrophil activation (n=3 + 3) was measured as CD66b+ expression with flow cytometry. CD66bhigh and CD66blow fractions in MC1 and control neutrophils were compared with paired t-test.Results:Comparing MC1 to control in total bulk RNAseq, 204 DEG in the autoinflammatory and 444 DEG in the infectious etiology were identified with only 67 shared genes (Fig. 1a). GO enrichment revealed “T-cell activation” (p = 2.50E-03) in the autoinflammatory and “complement activation, classical pathway” (p=1.1E-25) in the infectious etiology as top enriched upregulated biological processes (BP) (Fig 1b). ScRNAseq of autoinflammatory MC1 showed an overrepresentation of T-cells (p= 1.00E-34, OR=1.54) and myelocytes (neutrophil progenitor cells) (p=4.00E-05, OR=2.27) indicating an increased demand of these cells (Fig. 1c). Bulk RNAseq analysis of neutrophils from the autoinflammatory etiology revealed an activated, pro-inflammatory phenotype (Fig 1d), which was confirmed with more CD66bhigh neutrophils in MC1 (+11.13 ± 2.71%, p=0.02) (Fig. 1e).Figure 1.(a) Venn diagram of DEG from total bulk RNAseq (b) Top enriched upregulated BP of autoinflammatory (left) and infectious (right) etiology (c) Cell clustering of autoinflammatory MC1 BM (d) Enrichment of “inflammatory response” gene set in autoinflammatory MC1 neutrophils (e) Representative histogram of CD66b+ expression in MC1 and control neutrophils.Conclusion:Autoinflammatory and infectious etiologies of MC1 have different pathological mechanisms. T-cell and neutrophil activation seem to be important in the autoinflammatory etiology. This has clinical implication as it could be explored for diagnostic approaches to distinguish the two MC1 etiologies and supports developing targeted treatments for both etiologies.Disclosure of Interests:None declared

scholarly journals A genetically stable Zika virus vaccine candidate protects mice against virus infection and vertical transmission

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Awadalkareem Adam ◽  
Camila R. Fontes-Garfias ◽  
Vanessa V. Sarathy ◽  
Yang Liu ◽  
Huanle Luo ◽  
...  
Keyword(s):  
T Cell ◽  
Vertical Transmission ◽  
Zika Virus ◽  
Type I ◽  
Post Vaccination ◽  
Vaccine Trials ◽  
Specific Memory ◽  
Cell Immunity ◽  
Cell Antibody ◽  
And Control

AbstractAlthough live attenuated vaccines (LAVs) have been effective in the control of flavivirus infections, to date they have been excluded from Zika virus (ZIKV) vaccine trials due to safety concerns. We have previously reported two ZIKV mutants, each of which has a single substitution in either envelope (E) glycosylation or nonstructural (NS) 4B P36 and displays a modest reduction in mouse neurovirulence and neuroinvasiveness, respectively. Here, we generated a ZIKV mutant, ZE4B-36, which combines mutations in both E glycosylation and NS4B P36. The ZE4B-36 mutant is stable and attenuated in viral replication. Next-generation sequence analysis showed that the attenuating mutations in the E and NS4B proteins are retained during serial cell culture passages. The mutant exhibits a significant reduction in neuroinvasiveness and neurovirulence and low infectivity in mosquitoes. It induces robust ZIKV-specific memory B cell, antibody, and T cell-mediated immune responses in type I interferon receptor (IFNR) deficient mice. ZIKV-specific T cell immunity remains strong months post-vaccination in wild-type C57BL/6 (B6) mice. Vaccination with ZE4B-36 protects mice from ZIKV-induced diseases and vertical transmission. Our results suggest that combination mutations in E glycosylation and NS4B P36 contribute to a candidate LAV with significantly increased safety but retain strong immunogenicity for prevention and control of ZIKV infection.

Abstract 2155: Plasma Adiponectin Levels in Patients with Atrial Fibrillation

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Masayuki Shimano ◽  
Rei Shibata ◽  
Yukiomi Tsuji ◽  
Noriyuki Ouchi ◽  
Yasuya Inden ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Collagen Type ◽  
Collagen Type I ◽  
High Plasma ◽  
P Wave ◽  
Atrial Remodeling ◽  
Type I ◽  
Plasma Adiponectin ◽  
Type Iii ◽  
And Control

The occurrence and development of atrial fibrillation (AF) are associated with changes in electrical properties and cardiac structure, known as electrical and structural atrial remodeling. AF characterized by atrial remodeling also occurs with obesity-related conditions. Adiponectin, an adipose tissue-derived hormone, exerts beneficial effects on the heart in various pathological conditions. These observations led us to speculate that adiponectin levels affect the development and prevalence of AF. Here, we investigated a potential association between circulating adiponectin levels and atrial remodeling in patients with AF. We measured plasma adiponectin levels, serum carboxy-terminal telopeptide of collagen type I (CITP) levels, as a collagen type I degradation marker, and serum type III procollagen-N-peptide (PIIINP) levels, as a collagen type III synthesis marker in consecutive 414 patients; 225 paroxysmal AF, 81 persistent AF and 108 paroxysmal supra-ventricular tachycardia without AF history (control) patients, who admitted for scheduled radiofrequency catheter ablation. Plasma adiponectin levels were significantly higher in patients with persistent AF compared to paroxysmal AF and control patients (p<0.05). Serum CITP levels, but not serum PIIINP levels, were also higher in patients with persistent AF compared to paroxysmal AF and control patients (p<0.05). In addition, a positive correlation was observed between adiponectin levels and CITP levels (r=0.39, p<0.005) or the P wave duration (r=−0.31, p<0.05) in patients with persistent AF. High plasma adiponectin levels are associated with the presence of persistent AF, which is accompanied by increased CITP levels. Hyperadiponectinemia might also attenuate atrial conduction disturbance. Thus, measurement of plasma adiponectin could be useful for assessment of AF.

scholarly journals Leukocytes in bovine virgin mammary gland: flow cytometry imaging during development and resolution of induced influx

2001 ◽  
Vol 46 (No. 7–8) ◽  
pp. 190-198
Author(s):  
Z. Sládek ◽  
D. Ryšánek ◽  
M. Faldyna
Keyword(s):  
Flow Cytometry ◽  
Mammary Gland ◽  
Blood Cells ◽  
Cell Types ◽  
Mammary Glands ◽  
Mononuclear Phagocytes ◽  
Peripheral Blood Cells ◽  
Leukocyte Influx ◽  
Separate Region ◽  
Blood Leukocyte

Distribution of leukocyte types present in virgin bovine mammary glands was analysed in dot plots obtained by flow cytometry (FACS) of samples collected from 10 non-pregnant heifers after induction of leukocyte influx. Changes of percentage of leukocyte types during development and resolution of induced influx in comparison with blood leukocyte pattern allow identification of these cell types on FACS dot plot. The positions of mammary gland granulocyte and lymphocyte regions were identical with those of the corresponding peripheral blood cells. Two basic morphologically distinct types occupying separate regions in dot plots were observed in the population of mononuclear phagocytes (MoP): non-vacuolised monocyte-like macrophages (MoMAC) and vacuolised macrophages (MAC). Influx resolution was characterised by a marked shift of the MoMAC region towards that of MAC recognisable in dot plots by a separate region of intermediate MoP forms. The study provides a pattern of dynamics of percentages of mammary gland leukocyte types during influx development and resolution as imaged by FACS.

Role of Lymphocyte Subsets in Pathogenesis of Chronic Rheumatic Heart Disease

1998 ◽  
Vol 6 (2) ◽  
pp. 104-107
Author(s):  
Rajendar K Suri ◽  
Neerod K Jha ◽  
Harpreet Vohra ◽  
Ratna S Manjari ◽  
Rajam Venkateshwaran ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Heart Disease ◽  
Rheumatic Heart Disease ◽  
Left Atrial ◽  
Left Atrial Appendage ◽  
Lymphocyte Subsets ◽  
Cd4 Cells ◽  
Activated T Cells ◽  
Rheumatic Heart

Analyses of lymphocyte subsets using flow cytometry were conducted to determine the significance of these cells in the pathogenesis of chronic rheumatic heart disease. Lymphocytes (B cells, T cells, CD4 cells, CD8 suppressor or cytotoxic T cells, activated T cells, and natural killer cells) were measured in blood and left atrial appendage samples of 30 patients with rheumatic heart disease and 10 patients with acyanotic congenital heart disease. Monoclonal fluorescent-labeled antibodies were used to identify various cells by flow cytometry. There was a significant increase in CD4 cells and activated T cells with a significant decrease in B cells in the left atrial appendage tissue of patients with rheumatic heart disease compared to those in the control group. There was no significant difference between the two groups in the distribution pattern of T lymphocytes in peripheral blood. These changes in rheumatic heart disease reflect an abnormal immunoregulatory mechanism with an ongoing enhanced immunological process continuing into the chronic phase of the disease. In our opinion, this persistent T cell response may lead to fresh damage to the myocardium and deformation of the heart valves.

Anova Tests for Homogeneity of Variance: Nonnormality and Unequal Samples

1977 ◽  
Vol 2 (3) ◽  
pp. 187-206 ◽  
Author(s):  
Charles G. Martin ◽  
Paul A. Games
Keyword(s):  
Error Rate ◽  
Type I Error ◽  
Type I ◽  
Empirical Comparison ◽  
Jackknife Test ◽  
Type I Error Rate ◽  
Power And Control ◽  
Homogeneity Of Variance ◽  
Test Use ◽  
And Control

This paper presents an exposition and an empirical comparison of two potentially useful tests for homogeneity of variance. Control of Type I error rate, P(EI), and power are investigated for three forms of the Box test and for two forms of the jackknife test with equal and unequal n's under conditions of normality and nonnormality. The Box test is shown to be robust to violations of the assumption of normality. The jackknife test is shown not to be robust. When n's are unequal, the problem of heterogeneous within-cell variances of the transformed values and unequal n's affects the jackknife and Box tests. Previously reported suggestions for selecting subsample sizes for the Box test are shown to be inappropriate, producing an inflated P(EI). Two procedures which alleviate this problem are presented for the Box test. Use of the jack-knife test with a reduced alpha is shown to provide power and control of P(EI) at approximately the same level as the Box test. Recommendations for the use of these techniques and computational examples of each are provided.

Sign in / Sign up

Export Citation Format

Share Document