scholarly journals Cardiomyocyte Transplantation after Myocardial Infarction Alters the Immune Response in the Heart

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1825
Author(s):  
Praveen Vasudevan ◽  
Markus Wolfien ◽  
Heiko Lemcke ◽  
Cajetan Immanuel Lang ◽  
Anna Skorska ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Immune Response ◽  
Cell Transplantation ◽  
Flow Cytometric Analysis ◽  
Capillary Density ◽  
Flow Cytometric ◽  
Cardiac Pump Function ◽  
Different Response ◽  
First Time ◽  
Positive Effect

We investigated the influence of syngeneic cardiomyocyte transplantation after myocardial infarction (MI) on the immune response and cardiac function. Methods and Results: We show for the first time that the immune response is altered as a result of syngeneic neonatal cardiomyocyte transplantation after MI leading to improved cardiac pump function as observed by magnetic resonance imaging in C57BL/6J mice. Interestingly, there was no improvement in the capillary density as well as infarct area as observed by CD31 and Sirius Red staining, respectively. Flow cytometric analysis revealed a significantly different response of monocyte-derived macrophages and regulatory T cells after cell transplantation. Interestingly, the inhibition of monocyte infiltration accompanied by cardiomyocyte transplantation diminished the positive effect of cell transplantation alone. The number of CD68+ macrophages in the remote area of the heart observed after four weeks was also different between the groups. Transcriptome analysis showed several changes in the gene expression involving circadian regulation, mitochondrial metabolism and immune responses after cardiomyocyte transplantation. Conclusion: Our work shows that cardiomyocyte transplantation alters the immune response after myocardial infarction with the recruited monocytes playing a role in the beneficial effect of cell transplantation. It also paves the way for further optimization of the efficacy of cardiomyocyte transplantation and their successful translation in the clinic.

scholarly journals Optimized protocols for isolation, fixation, and flow cytometric characterization of leukocytes in ischemic hearts

2019 ◽  
Vol 317 (3) ◽  
pp. H658-H666 ◽  
Author(s):  
Roman Covarrubias ◽  
Mohamed Ameen Ismahil ◽  
Gregg Rokosh ◽  
Tariq Hamid ◽  
Federica Accornero ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Immune Cells ◽  
Cell Separation ◽  
Flow Cytometric Analysis ◽  
Left Ventricular ◽  
Coronary Perfusion ◽  
Flow Cytometric ◽  
Mediators Of Inflammation ◽  
Cell Fixation

Immune activation post-myocardial infarction is an orchestrated sequence of cellular responses to effect tissue repair and healing. However, excessive and dysregulated inflammation can result in left ventricular remodeling and pathological alterations in the structural and mechanical attributes of the heart. Identification of key pathways and critical cellular mediators of inflammation is thus essential to design immunomodulatory therapies for myocardial infarction and ischemic heart failure. Despite this, the experimental approaches to isolate mononuclear cells from the heart are diverse, and detailed protocols to enable maximum yield of live cells in the shortest time possible are not readily available. Here, we describe optimized protocols for the isolation, fixation, and flow cytometric characterization of cardiac CD45+ leukocytes. These protocols circumvent time-consuming coronary perfusion and density-mediated cell-separation steps, resulting in high cellular yields from cardiac digests devoid of contaminating intravascular cells. Moreover, in contrast to methanol and acetone, we show that cell fixation using 1% paraformaldehyde is most optimal as it does not affect antibody binding or cellular morphology, thereby providing a considerable advantage to study activation/infiltration-associated changes in cellular granularity and size. These are highly versatile methods that can easily be streamlined for studies requiring simultaneous isolation of immune cells from different tissues or deployment in studies containing a large cohort of samples with time-sensitive constraints. NEW & NOTEWORTHY In this article, we describe optimized protocols for the isolation, fixation, and flow cytometric analysis of immune cells from the ischemic/nonischemic hearts. These protocols are optimized to process several samples/tissues, simultaneously enabling maximal yield of immune cells in the shortest time possible. We show that the low-speed centrifugation can be used as an effective alternative to lengthy coronary perfusion to remove intravascular cells, and sieving through 40-μm filter can replace density-mediated mononuclear cell separation which usually results in 50–70% cell loss in the sedimented pellets. We also show that cell fixation using 1% paraformaldehyde is better than the organic solvents such as methanol and acetone for flow cytometric analysis.

The Studies on the T-Cell Receptor Repertoire Diversity and Flow Cytometric Analysis in a Patient with MAS/HPS after Unrelated Peripheral Blood Stem Cell Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4970-4970
Author(s):  
Xin Du ◽  
Yangqiu Li ◽  
Suxia Geng ◽  
Jianyu Weng ◽  
Zesheng Lu ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Nk Cell ◽  
Flow Cytometric Analysis ◽  
Blood Stem Cell ◽  
Flow Cytometric

Abstract Macrophage activation syndrome (MAS) /Hemophagocytic syndrome (HPS) is characterized by proliferation of activated macrophages under conditions such as infection(C Clin Infect Dis 2004)lymphoma(Aouba A Am J Hematol 2004), autoimmune disease(Kaneko K Clin Rheumatol 2005), solid organ transplantation(Akamatsu N,Transplant Proc 2006;). There have been several reports of MAS /HPS after hematopoietic stem cell transplantation, involving not only allogeneic,but also autologous transplantation(Sreedharan A Bone Marrow Transplantation,2006). Generally, MAS /HPS is a cytokine-related disorder.But at present, its clinical characteristics remain unknown. We firstly study here the T-cell receptor repertoire diversity and flow cytometric analysis in MAS /HPS after unrelated peripheral blood stem cell transplantation. The CDR3 of TCR Vα and Vβ subfamily genes were amplified in peripheral blood mononuclear cells from the patient with MAS/HPS after unrelated peripheral blood stem cell using RT-PCR for detection of the distribution of TCR Vα and Vβ repertoire, the PCR products were further analyzed by genescan technique for the CDR3 size, to evaluating clonality of the detectable TCR Vα and VβT cells. Lymphocyte subsets in the peripheral blood were detected by monoclone antibody and flow cytometry including T lymphocyte subsets and NK cells. Flow-cytometric analysis showed CD56+ CD16+ cell 68.65% and CD3+ cell 11.79% in the lymphocyte population;CD16+CD69+ cell 68.51% and CD25+CD16+ cell 31.59% in NK cell. In the T lymphocytic subsets, CD25 + CD3+ cell 62%; CD69+CD3+ cell 75.81%; CD25CD4+ cell 0.81%,CD25CD8+ cell 3.48%; CD69CD4+ cell 0.31%, CD69+CD8+ cell 16.86%.The results show that the main activated lymphocytes is NK cell in patient at diagnosed with MAS/HPS. Of interest, it was only after the addition of high-dose IVIG 1g/kg/d for two days (Ostronoff et al BMT2006) to the treatment that MAS remitted. There are 23 Vα and 15Vβ subfamily T cells could be identified in this time, and the clonal expansion T cells could be found in TCR Vα5, 13, 20; TCR Vβ4, 11, 15 and 21subfamilies. Billiau et al (Blood 2005)describes the immunohistochemical findings on liver tissues from 5 children with MAS in the context of a different type of hemophagocytic syndrome (HPS) in liver transplantation. This study is the first directly to substantiates the presumed immunopathogenesis of MAS by documenting in situ expression of IFN-γ+ by activated CD8+ lymphocytes, and of IL-6 and TNF-α+ by hemophagocytosing macrophages, on liver tissues of patients with MAS. We found no evidence of potential infectious, autoimmune or malignant triggers of R-HPS in our patient, despite extensive investigations. We conclued that the skew distribution and clonal expansion of TCR Vα and Vβ subfamily T cells underscore the primary role of T cells in the pathogenesis of MAS/HPS.

scholarly journals A preliminary Study on the Effect of Head and Neck Chemoradiotherapy on Systematic Immunity

Dose-Response ◽  
2019 ◽  
Vol 17 (4) ◽  
pp. 155932581988418 ◽  
Author(s):  
Weiqiang Huang ◽  
Yao Fan ◽  
Xiaoya Cheng ◽  
Huazhen Liang ◽  
Hua Pan ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Head And Neck ◽  
Concurrent Chemoradiotherapy ◽  
Flow Cytometric Analysis ◽  
Dual Effect ◽  
Blood Tests ◽  
Flow Cytometric ◽  
Before And After ◽  
Preliminary Study

Background: This study was designed initially to explore the effect of chemoradiotherapy on patients diagnosed with head and neck cancer (HNC) with respect to the alteration of systematic immunity. Methods: We did a retrospective study enrolling patients received concurrent chemoradiotherapy (CCRT), with or without induction chemotherapy (IC). Blood tests were performed before IC, before and after CCRT. Flow cytometric analysis and turbidimetric inhibition immunoassay were used for detection. Results: A total number of 58 patients were included from April 1, 2018, to March 31, 2019. Levels of immunoglobulins (Ig), including IgA, IgG, and IgM, declined after 2 to 3 cycles of IC and CCRT, respectively. Serum level of total hemolytic complement (CH50) increased ( P < .001) after IC, but kept stably post-CCRT. Natural killer (NK) cells decreased ( P < .01) after IC and enhanced ( P < .001) post-CCRT. The number of CD3+CD4+ T cells got increased ( P < .01) after IC and decreased ( P < .001) post-CCRT. Consistently, both IC and CCRT induced the increase in CD3+CD8+ T cells significantly ( P < .001 vs P < .01). Conclusion: Both radiotherapy (RT) and chemotherapy (CT) induced dual effect of immune response. Concurrent chemoradiotherapy created an active immune response based on the effect induced by IC, suggesting that RT exerted a potential function on mobilizing immune system.

scholarly journals An Immunoregulatory Role for Complement Receptors in Murine Models of Breast Cancer

Antibodies ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 2
Author(s):  
Fazrena Nadia Md Akhir ◽  
Mohd Hezmee Mohd Noor ◽  
Keith Weng Kit Leong ◽  
Jamileh A. Nabizadeh ◽  
Helga D. Manthey ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Immune Response ◽  
Tumor Growth ◽  
Complement Activation ◽  
Mammary Carcinoma ◽  
Flow Cytometric Analysis ◽  
Receptor Expression ◽  
Complement Receptors ◽  
Flow Cytometric ◽  
Cancer Models

The complement system has demonstrated roles in regulating tumor growth, although these may differ between tumor types. The current study used two murine breast cancer models (EMT6 and 4T1) to investigate whether pharmacological targeting of receptors for complement proteins C3a (C3aR) and C5a (C5aR1) is protective in murine breast cancer models. In contrast to prior studies in other tumor models, treatment with the selective C5aR1 antagonist PMX53 had no effect on tumor growth. However, treatment of mice with a dual C3aR/C5aR1 agonist (YSFKPMPLaR) significantly slowed mammary tumor development and progression. Examination of receptor expression by quantitative polymerase chain reaction (qPCR) analysis showed very low levels of mRNA expression for either C3aR or C5aR1 by EMT6 or 4T1 mammary carcinoma cell lines compared with the J774 macrophage line or bone marrow-derived macrophages. Moreover, flow cytometric analysis found no evidence of C3aR or C5aR1 protein expression by either EMT6 or 4T1 cells, leading us to hypothesize that the tumor inhibitory effects of the dual agonist are indirect, possibly via regulation of the anti-tumor immune response. This hypothesis was supported by flow cytometric analysis of tumor infiltrating leukocyte populations, which demonstrated a significant increase in T lymphocytes in mice treated with the C3aR/C5aR1 agonist. These results support an immunoregulatory role for complement receptors in primary murine mammary carcinoma models. They also suggest that complement activation peptides can influence the anti-tumor response in different ways depending on the cancer type, the host immune response to the tumor and levels of endogenous complement activation within the tumor microenvironment.

scholarly journals Ploidy Levels and Genome Sizes of Berberis L. and Mahonia Nutt. Species, Hybrids, and Cultivars

HortScience ◽  
2010 ◽  
Vol 45 (7) ◽  
pp. 1029-1033 ◽  
Author(s):  
Todd J. Rounsaville ◽  
Thomas G. Ranney
Keyword(s):  
Pisum Sativum ◽  
Ploidy Level ◽  
Flow Cytometric Analysis ◽  
Internal Standard ◽  
Berberis Thunbergii ◽  
Ploidy Levels ◽  
Flow Cytometric ◽  
Representative Species ◽  
Species Hybrids ◽  
First Time

An extensive survey of genome sizes and ploidy levels was conducted for a diverse collection of Berberis and Mahonia taxa (Berberidaceae). Propidium iodide flow cytometric analysis was conducted using Pisum sativum L. ‘Ctirad’ (2C DNA = 8.76 pg) as an internal standard to determine genome sizes. Mean 1CX genome sizes varied between the two Mahonia subgenera (Occidentales = 1.17 ± 0.02, Orientales = 1.27 ± 0.01), whereas those of Berberis subgenera were similar (Australes = 1.45 ± 0.03, Septentrionales = 1.47 ± 0.02) and each significantly larger than those of Mahonia. Traditional cytology was performed on representative species to calibrate genome sizes with ploidy levels. Polyploidy among both wild and cultivated taxa was found to be rare. Although the majority of species were determined to be diploid with 2n = 2x = 28, artificially induced autopolyploid Berberis thunbergii seedlings were confirmed to be tetraploid and an accession of Mahonia nervosa was confirmed to be hexaploid. Genome size and ploidy level reports for the majority of taxa sampled are presented for the first time and are intended to be of use to plant breeders, ecologists, and systematists.

scholarly journals Flow cytometric characterisation of the complex polyploid genome of Saccharum officinarum and modern sugarcane cultivars

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Cushla J. Metcalfe ◽  
Jingchuan Li ◽  
Debora Giorgi ◽  
Jaroslav Doležel ◽  
Nathalie Piperidis ◽  
...  
Keyword(s):  
Genome Structure ◽  
Flow Cytometric Analysis ◽  
Saccharum Officinarum ◽  
Flow Karyotype ◽  
Flow Cytometric ◽  
Structure Flow ◽  
Flow Cytometric Sorting ◽  
Polyploid Genome ◽  
First Time ◽  
Simple Sequence

AbstractSugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genotype, except R570, suggesting that the peaks may represent ancestral Saccharum sub genomes. The ability to flow sort Saccharum chromosomes will allow us to isolate and analyse chromosomes of interest and further examine the structure and evolution of the sugarcane genome.

DNA content and proliferative activity of myoepitheliomas

1989 ◽  
Vol 103 (12) ◽  
pp. 1192-1197 ◽  
Author(s):  
A. El-Naggar ◽  
J. G. Batsakis ◽  
M. A. Luna ◽  
H. Goepfert ◽  
M. E. Tortoledo
Keyword(s):  
Dna Content ◽  
Proliferative Activity ◽  
Clinical Evidence ◽  
Flow Cytometric Analysis ◽  
S Phase ◽  
Phase Fraction ◽  
Proliferative Capacity ◽  
Biological Behaviour ◽  
Flow Cytometric ◽  
First Time

AbstractThis report adds 16 myoepitheliomas of salivary glands to the 47 already recorded in the literature. It includes, for the first time, a flow cytometric analysis of their ploidy (DNA content) and proliferative capacity (S-phase fraction). Thirteen myoepitheliomas were diploid; three were aneuploid in their DNA content. A high proliferative capacity was always associated with an abnormal DNA content. Only one diploid myoepithelioma had a high S-phase fraction. Both flow-cytometric parameters are good predictors of an aggressive biological behaviour. Recurrences, however, were all the outcome of incomplete primary removal of the myoepitheliomas. Four of the twelve (33 per cent) diploid myoepitheliomas recurred and one, with high S-phase fraction, led to the death of the patient. Two of the three (67 per cent) aneuploid myoepitheliomas recurred. Extensive loco-regional invasion by one killed the patient. The other has clinical evidence of distant metastasis.

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