scholarly journals Profiling of Microrna Expression within the Cells of Peripheral Blood Mononuclear after an Infection with Serotype-2 of Dengue Virus: Preliminary Study

2018 ◽  
Vol 11 (2) ◽  
pp. 923-927 ◽  
Author(s):  
Sri Masyeni ◽  
Usman Hadi ◽  
K Kuntaman ◽  
Yorapermata Dewi
Keyword(s):  
Dengue Virus ◽  
Mononuclear Cells ◽  
Gradient Centrifugation ◽  
Dengue Infection ◽  
Virus Titer ◽  
Specific Protein ◽  
Peripheral Blood Mononuclear ◽  
Non Coding Rna ◽  
Ficoll Density Gradient Centrifugation ◽  
Time Of Infection

The role of microRiboNucleic Acids (miRNA), a small-non coding RNA has been associated with immune regulation in various viral infectionincluding dengue infection. The microRNA will bind a specific protein target in order to encourage an explosive expression of various cytokines, known as cytokines storm in Dengue infection.The objective of this study aimed to determine and evaluate themicroRNAs profile expression withinperipheral blood mononuclear cells having been infected with one of the dengue virus serotype.To obtained the PBMCs from a healthy donor, Ficoll density gradient centrifugation was used to isolate the PBMCs and then followed infecting it with a DENV-2 clinical isolate. Prior to PBMCs isolation, the virus has been propagated and having titration to get an optimal virus titer. We conducted the infection at the multiplication of infections 4 PFU/106 cells.MiRCURYLNATMExiqon was utilized on purpose to extract the RNA. Quantitative Real-Time PCR was applied in order for the miRNAs relative expression to be measured. The preliminary result reveals that miR-150, miR-146a, hsa-let-7e expression were increased 1.74 folds, 2 folds, and 1.49 foldsrespectively at 12 hours post-infection on PBMCs upon DENV-2 infection.The expression of microRNAswas discovered to behigher inPBMCsat the time of infection withDENV-2.ThemiRNAs expression in the uninfected PMBCs was lower than that of the miRNA. This high expression of miRNAsin dengue infection may proceedto dengue infection pathogenesis.

scholarly journals Expression of Four Cytokine/Chemokine Genes in Peripheral Blood Mononuclear Cells infected with Dengue Virus

2019 ◽  
Vol 7 (4) ◽  
pp. 75
Author(s):  
Dewa Ayu Putri Sri Masyeni ◽  
Usman Hadi ◽  
Kuntaman Kuntaman ◽  
Benediktus Yohan ◽  
Nur Ita Margyaningsih ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Dengue Infection ◽  
Peripheral Blood Mononuclear ◽  
Study Objective ◽  
Genes Expression ◽  
Blood Mononuclear Cells

Overproduction of numerous pro-inflammatory cytokines, during dengue virus (DENV) infection, has been related to plasma leakage in the vascular endothelium and studied elsewhere with conflicting results. The current study objective is to evaluate the expression of four cytokine/chemokine genes following DENV-2 infection within peripheral blood mononuclear cells (PBMC) isolated from a healthy donor. Venous blood was drawn,  and PBMCs were isolated using Ficoll density gradient centrifugation. Cells were maintained in culture medium and infected with Indonesian isolate of DENV-2. Cells were harvested and followed by total RNA extraction and reverse-transcription into cDNA using oligo d(T) primers and Reverse Transcriptase enzyme system. The SYBR Green-based quantitative qRT-PCR was used to calculate the relative expression of IL-6, IL-8, IP-10 and MIP-1β- encoding genes during infection time points, compared to uninfected cell controls. The observation of the cytokine was on the 6 and 18 hours post-infection. The different expression profiles of cytokines/chemokines were observed. The up-regulation of gene expression was observed for IL-8 and IP-10. In contrast, the down-regulatory of IL-6 and MIP-1β genes expression was documented during the infection period. The cytokine IL-8 and IP-10 are potent chemoattractants  in the recruitment  of neutrophil, basophil, and lymphocytes in response to an infection. The highlight of this study is on the up-regulation of IL-8 and IP-10 genes expression which may confirm the roles of these chemokines in the pathogenesis of dengue infection.

scholarly journals Correlation of miR-150, hsa-let-7e, and miR-146a and gene expression of IL-6, IL-8, IP-10, and MIP-1β during dengue virus infection

Narra J ◽  
2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Sri Masyeni ◽  
Kuntaman Kuntaman ◽  
Aryati Aryati ◽  
Muchlis AU Sofro ◽  
Usman Hadi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dengue Virus ◽  
Mononuclear Cells ◽  
Dengue Infection ◽  
Denv Infection ◽  
Vital Role ◽  
Gene Expressions ◽  
Peripheral Blood Mononuclear ◽  
Infected Pbmcs ◽  
Causality Tests

Growing evidence suggests that microRNAs (miRNAs) play a pivotal role in viral infection. The objective of this study was to assess the association between the expression of miR-150, hsa-let-7e, and miR-146a on cytokine expression during dengue infection. Dengue virus (DENV) strain SJN-006, a serotype 2 DENV strain of the Cosmopolitan genotype, isolated in Bali, Indonesia, was used to infect peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. The relative gene expressions of miR-150, hsa-let-7e, and miR-146a as well as the gene expression of cytokines (IL-6, IL-8, IP-10, and MIP-1β) were determined using quantitative real time - polymerase chain reaction (qRT-PCR) at 6, 12 and 24 hours post infection (hpi). Correlations between the microRNAs and cytokines were analyzed by means of causality tests. Our data suggests that miR-150 and hsa-let-7e were significantly higher in infected-PBMCs after 12 hpi compared to the uninfected-PBMCs (p<0.05). The causality tests demonstrated that miR-150 and hsa-let-7e were negatively correlated with IL-8 expression, meanwhile miR-146a was the contrast. DENV infection was negatively and positively correlated with miR-150 and hsa-let-7e, respectively, after 24 hpi. In conclusion, our data demonstrates the vital role of miR-150, hsa-let-7e, and miR-146a in regulating IL-8 expression with possible different pathways.

scholarly journals Induksi Ekspresi Gen Sitokin/Kemokin pada Sel Makrofag Manusia yang Dipapar Virus Dengue Isolat Indonesia

2014 ◽  
Vol 1 (3) ◽  
pp. 146-157
Author(s):  
Siti Warnasih
Keyword(s):  
Dengue Virus ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Gradient Centrifugation ◽  
Gene Expression Profiles ◽  
Host Gene ◽  
Peripheral Blood Mononuclear ◽  
Expression Levels ◽  
Dengue Disease ◽  
Encoding Genes

Dengue is one of the world's most important arbovirus disease. Dengue pathogenesis has not been yet fully understood. It has been reported that there is involvement of the host immune factors and viral factors. Several studies have shown that concentrations of cytokines/chemokines on blood are significantly increased during infection and viral factors are also involved in disease severity. Therefore, characterization of host gene expression profiles in response to dengue virus infection of different serotypes could provide input for understanding the pathogenesis of dengue. The purpose of this research was to determine expression profiles of the genes (mRNA) of cytokines/chemokines as immune response that are released by monocyte derived macrophages (MDM) cells (host gene) exposed dengue viruses. Four dengue serotypes of Indonesia isolate were used in this study. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from blood cells of healthy donors by Ficoll gradient centrifugation techniques and then differentiated into MDM cells. Quantitative real time RT-PCR was used to quantify expression levels of cytokine/chemokine-encoding genes from MDM cells infected dengue. Four cytokine/chemokine-encoding genes i.e IP-10, MCP-1, IL-10, and MIP-1β known involved in dengue pathogenesis. Measurement of the expression levels of cytokines/ chemokines showed that the dengue virus of serotypes DENV-1 and DENV-3 caused an increase in the expression of genes encoding cytokine IL-10 and chemokine IP-10 is higher than other serotypes. Further research is needed to better determine the pathogenesis of dengue disease.

scholarly journals Impact of SARS-CoV-2 infection on the recovery of peripheral blood mononuclear cells by density gradient

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria D. I. Manunta ◽  
Giuseppe Lamorte ◽  
Francesca Ferrari ◽  
Elena Trombetta ◽  
Mario Tirone ◽  
...  
Keyword(s):  
Density Gradient ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Early Phase ◽  
Mononuclear Cells ◽  
Early Stage ◽  
Gradient Centrifugation ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Circulating Lymphocytes

AbstractSARS-CoV-2 virus infection is responsible for coronavirus disease (COVID-19), which is characterised by a hyperinflammatory response that plays a major role in determining the respiratory and immune-mediated complications of this condition. While isolating peripheral blood mononuclear cells (PBMCs) from whole blood of COVID-19 patients by density gradient centrifugation, we noticed some changes in the floating properties and in the sedimentation of the cells on density medium. Investigating this further, we found that in early phase COVID-19 patients, characterised by reduced circulating lymphocytes and monocytes, the PBMC fraction contained surprisingly high levels of neutrophils. Furthermore, the neutrophil population exhibited alterations in the cell size and in the internal complexity, consistent with the presence of low density neutrophils (LDNs) and immature forms, which may explain the shift seen in the floating abilities and that may be predictive of the severity of the disease. The percentage of this subset of neutrophils found in the PBMC band was rather spread (35.4 ± 27.2%, with a median 28.8% and IQR 11.6–56.1, Welch’s t-test early phase COVID-19 versus blood donor healthy controls P < 0.0001). Results confirm the presence of an increased number of LDNs in patients with early stage COVID-19, which correlates with disease severity and may be recovered by centrifugation on a density gradient together with PBMCs.

Abstract 20: Resolution Phenotype of Monocytes and Macrophages is Altered in Peripheral Arterial Disease

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Melinda S Schaller ◽  
Laura Menke ◽  
Mian Chen ◽  
Warren J Gasper ◽  
S. Marlene Grenon ◽  
...  
Keyword(s):  
Peripheral Arterial Disease ◽  
Cell Surface ◽  
Phagocytic Activity ◽  
Mononuclear Cells ◽  
Gradient Centrifugation ◽  
Arterial Disease ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Peripheral Blood Mononuclear ◽  
Peripheral Arterial

Introduction: Peripheral arterial disease (PAD) is a chronic disease characterized by systemic inflammation. Monocytes (Mo) and macrophages play a central role in vascular inflammation and its resolution. We hypothesize that impaired resolution in PAD results in poor clinical outcomes. Methods: Resolution phenotype was assessed by phagocytic activity of leukocytes, Mo cell surface markers, and cytokine profiling of Mo-derived macrophages (MDM). Phagocytosis and cell-surface markers were determined by flow cytometry. MDMs were generated from peripheral blood mononuclear cells via density gradient centrifugation. Cytokines were measured by ELISA following MDM differentiation and subsequent stimulation with LPS. Results: Circulating Mo and neutrophils (PMN) isolated from PAD patients (n=9) demonstrated significantly lower phagocytic activity (Mo: >30%, p<.001; PMN: >25%, p<.01, Fig. 1) as compared to healthy subjects (HS) (n=14). Cell-surface marker analysis demonstrated a higher proportion of the pro-inflammatory intermediate Mo subset (CD14 ++ 16 + , 1.8-fold, p=.04) in PAD compared to HS. MDM from PAD subjects retain their intrinsic inflammatory program by producing more IL-6 (PAD 3138±2676 ng/mL, HS 731±854 ng/mL p=.03) and IL-1β (PAD 244±236 ng/mL, HS 24.1±23.8 ng/mL p=.04) than those from HS. Upon stimulation with LPS, MDM from PAD subjects secrete more IL-6 (PAD 23353±22483 ng/mL, HS 5097±5836 ng/mL p=.05) than those from HS. Conclusions: Circulating Mo and PMN in patients with PAD have substantially lower phagocytic activity as well as a greater proportion of the pro-inflammatory intermediate Mo subset compared to HS. MDM preserve their elevated inflammatory state throughout culture and retain a heightened response upon latter stimulatory cues. Collectively these data demonstrate a heightened inflammatory and impaired resolution phenotype in PAD that has potential implications for disease progression and response to interventions.

scholarly journals Predominance of HLA-Restricted Cytotoxic T-Lymphocyte Responses to Serotype-Cross-Reactive Epitopes on Nonstructural Proteins following Natural Secondary Dengue Virus Infection

1998 ◽  
Vol 72 (5) ◽  
pp. 3999-4004 ◽  
Author(s):  
Anuja Mathew ◽  
Ichiro Kurane ◽  
Sharone Green ◽  
Henry A. F. Stephens ◽  
David W. Vaughn ◽  
...  
Keyword(s):  
Virus Infection ◽  
Dengue Virus ◽  
T Lymphocyte ◽  
Mononuclear Cells ◽  
Cytotoxic T Lymphocyte ◽  
Nonstructural Proteins ◽  
Dengue Virus Infection ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Lymphocyte Responses

ABSTRACT We examined the memory cytotoxic T-lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue virus infection. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue virus proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for NS1.2a (NS1 and NS2a collectively) and NS3 proteins, respectively. All CTL lines derived from both patients were cross-reactive with other serotypes of dengue virus. The CD8+ NS1.2a-specific lines from patient KPP94-037 were HLA B57 restricted, and the CD8+ NS3-specific lines from patient KPP94-024 were HLA B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA DR7 restricted. A majority of the CD8+ CTLs isolated from patient KPP94-024 were found to recognize amino acids 221 to 232 on NS3. These results demonstrate that in Thai patients after symptomatic secondary natural dengue infections, CTLs are mainly directed against nonstructural proteins and are broadly cross-reactive.

scholarly journals Studies on Immunomodulatory Effect of Casein Phospho Peptide Isolated from Cultured Dairy Product

2020 ◽  
pp. 28-40
Author(s):  
Indumathi Mullaiselvan ◽  
Vijayarani Kanagaraj ◽  
Saranya Sekar ◽  
Baskaran Dharmar ◽  
Rathnapraba Sambandan
Keyword(s):  
Amino Acid ◽  
Bioactive Peptides ◽  
Mononuclear Cells ◽  
Dairy Product ◽  
Fermented Milk ◽  
Specific Protein ◽  
Immunomodulatory Effect ◽  
Peripheral Blood Mononuclear ◽  
Protein Fragments ◽  
Ft Ir

Bioactive peptides have been defined as specific protein fragments that have an impact on body functions or conditions and may ultimately influence health. Fermented milk is a dairy product which has abundance of bioactive peptides. In this study, Casein Phospho peptide (CPP) was isolated by enzymatic hydrolysis of fermented milk using trypsin. The molecular weight of the Casein Phospho peptide was 3.5 KDa. The anti-bacterial activity of Casein Phospho peptide was determined using four pathogens such as, Escherichia coli, Bacillus cereus, Staphylococcus aureus and Salmonella enterica. Casein phospho peptide formed a zone of inhibition against the pathogens. The bioactive peptides were characterized using Fourier Transform Infra-Red Spectroscopy (FT-IR), Casein Phospho peptide had aliphatic amine, acetyl amino I and acetyl amino II functional groups. The HPLC analysis of Casein Phospho peptide revealed that the major amino acid present was L- Glutamic acid and the amino acid present in lesser concentration was Leucine. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from human blood and the cells were treated with Casein Phospho peptide to assess the immunomodulatory effect. Casein Phospho peptide was able to produce a higher concentration of IL-10 anti-inflammatory cytokines when treated with PBMCs.

scholarly journals The Increased RNase Activity of IRE1α in PBMCs from Patients with Rheumatoid Arthritis

2019 ◽  
Vol 9 (3) ◽  
pp. 505-509 ◽  
Author(s):  
Mahdieh Ahmadiany ◽  
Mahshid Alavi-Samani ◽  
Zahra Hashemi ◽  
Mohammad Amin Moosavi ◽  
Marveh Rahmati
Keyword(s):  
Rheumatoid Arthritis ◽  
Er Stress ◽  
Mononuclear Cells ◽  
Gradient Centrifugation ◽  
Rnase Activity ◽  
Healthy Controls ◽  
Peripheral Blood Mononuclear ◽  
Micro Rnas ◽  
Density Gradient Centrifugation Method ◽  
Gene Expression Levels

Purpose: Despite recent advances in the diagnosis and treatment of rheumatoid arthritis (RA), this inflammatory disease remains a challenge to patients and physicians. Recent evidence highlights the contribution of endoplasmic reticulum (ER) stress in the pathogenesis and treatment of RA. Herein, we study the expression of the ER stress sensor inositol-requiring enzyme 1α (IRE1α), as well as XBP1 splicing and the regulated IRE1-dependent decay (RIDD), in peripheral blood mononuclear cells (PBMCs) from patients with RA compared with healthy controls. Methods: The PBMCs from blood samples of RA patients and healthy volunteers were isolated by a density gradient centrifugation method using Ficoll. The gene expression levels of GRP78/ Bip, IRE1, XBP1s, micro-RNAs (miRNAs) were evaluated by real-time PCR. Results: The expression of GRP78, IRE1, and XBP1s were increased in PBMCs of RA patients compared with healthy controls. We further show that the RIDD targets (miRNA-17, -34a, -96, and -125b) were downregulated in RA samples. Conclusion: This study can expand our knowledge on the importance of RNase activity of IRE1α in RA and may offer new potentials for developing novel diagnostic and/or therapeutic biomarkers.

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