Apoptosis In Peripheral Blood Mononuclear Cells Research Articles

Serum long non-coding Ribonucleic Acid H19 serves as a biomarker for systemic lupus erythematosus and participates in the disease progression.

This study aimed to investigate the expression of H19 and its possible molecular mechanism in systemic lupus erythematosus (SLE). The expression of H19 and miR-19b in serum and peripheral blood mononuclear cells (PBMCs) were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Receiver operator characteristic (ROC) curve was constructed to evaluate the diagnostic value of serum H19 in SLE. Pearson correlation coefficient was used to analyze the correlation between serum levels of H19 and miR-19b. Flow cytometry and Cell counting kit-8 (CCK-8) assay were performed to detect cell apoptosis and viability. The levels of pro-inflammatory and anti-inflammatory factors were measured by enzyme-linked immunosorbent assay (ELISA). Luciferase reporter gene assay was conducted to verify the interaction between H19 and miR-19b. The expression of H19 and miR-19b in SLE group were up-regulated and down-regulated, respectively. Serum H19 has certain clinical diagnostic value in SLE. In in vitro studies, overexpression of H19 can significantly inhibit the viability of PBMCs and promote apoptosis and inflammatory response of PBMCs by interacting with miR-19b. The expression of H19 is upregulated in patients with SLE and plays a role in cell function and inflammation by targeting miR-19b in PBMCs, which may be one of the pathological mechanisms of SLE.

Altered apoptosis and proliferation in milk cells and PBMc from BLV-infected bovines with different proviral loads: Possible role of the BCL-2 family proteins, TNF-alpha, and receptors

Bovines infected by bovine leukemia virus (BLV) are characterized by presenting low proviral load (LPL) or high proviral load (HPL). It is reported that animals with HPL in peripheral blood mononuclear cells (PBMCs) present a decrease in apoptosis, an increase in viability and the proliferation rate, while animals that maintain an LPL have an intrinsic ability to control the infection, presenting an increased apoptosis rate of their PBMCs. However, there is little information on the effect of BLV on these mechanisms when the virus infects somatic milk cells (SC). This study investigates the mechanisms underlying apoptosis in milk and blood from BLV-infected animals with HPL and LPL. Relative levels of mRNA of tumor necrosis factor-α (TNF-α), TNF receptor 1 (TNF-RI), TNF receptor 2 (TNF-RII), anti-apoptotic B-cell lymphoma 2 protein (Bcl-2), and pro-apoptotic Bcl-2-like protein 4 (Bax) were measured in SC and PBMCs using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay. A significant decrease in the expression of TNF-α in SC from HPL animals vs non-infected bovines was observed, but the infection in SC with BLV did not show a modulation on the expression of TNF receptors. A significant increase in TNF-RI expression in PBMCs from HPL bovines compared to LPL bovines was observed. No significant differences in PBMCs between HPL and LPL compared to non-infected animals concerning TNF-α, TNF-RI, and TNF-RII expression were found. There was a significant increase of both Bcl-2 and Bax in SC from LPL compared to non-infected bovines, but the Bcl-2/Bax ratio showed an anti-apoptotic profile in LPL and HPL bovines compared to non-infected ones. Reduced mRNA expression levels of Bax were determined in the PBMCs from HPL compared to LPL subjects. In contrast, BLV-infected bovines did not differ significantly in the mRNA expression of Bax compared to non-infected bovines. Our data suggest that the increased mRNA expression of Bax corresponds to the late lactation state of bovine evaluated and the exacerbated increase of mRNA expression of Bcl-2 may be one of the mechanisms for the negative apoptosis regulation in the mammary gland induced by BLV infection. These results provide new insights into the mechanism of mammary cell death in HPL and LPL BLV-infected bovine mammary gland cells during lactation.

Urine-Derived Renal Epithelial Cells (URECs) from Transplanted Kidneys as a Promising Immunomodulatory Cell Population.

Kidney transplantation is a lifesaving procedure for patients with end-stage kidney disease (ESKD). Organs derived from donation after cardiac death (DCD) are constantly increasing; however, DCD often leads to ischaemia-reperfusion (IR) and Acute Kidney Injury (AKI) events. These phenomena increase kidney cell turnover to replace damaged cells, which are voided in urine. Urine-derived renal epithelial cells (URECs) are rarely present in the urine of healthy subjects, and their loss has been associated with several kidney disorders. The present study aimed to characterize the phenotype and potential applications of URECs voided after transplant. The results indicate that URECs are highly proliferating cells, expressing several kidney markers, including markers of kidney epithelial progenitor cells. Since the regulation of the immune response is crucial in organ transplantation and new immunoregulatory strategies are needed, UREC immunomodulatory properties were investigated. Co-culture with peripheral blood mononuclear cells (PBMCs) revealed that URECs reduced PBMC apoptosis, inhibited lymphocyte proliferation, increased T regulatory (Treg) cells and reduced T helper 1 (Th1) cells. URECs from transplanted patients represent a promising cell source for the investigation of regenerative processes occurring in kidneys, and for cell-therapy applications based on the regulation of the immune response.

LOW DOSE OF ESMOLOL ATTENUATES SEPSIS-INDUCED IMMUNOSUPPRESSION VIA MODULATING T-LYMPHOCYTE APOPTOSIS AND DIFFERENTIATION.

Background: Immunosuppression caused by immune cell apoptosis and an imbalance of T helper 2 cells (T H 2) and T helper 1 cells (T H 1), is associated with poor outcomes in septic patients. Esmolol was reported to improve survival by modulating immune responses in septic shock. Whether esmolol could alleviate sepsis-induced immunosuppression and the optimal dose are unclear. Methods: Four hours after cecal ligation and puncture (CLP), Wistar rats were randomized into CLP, CLP + E-5 (esmolol: 5 mg·kg -1 ·h -1 ) and CLP + E-18 (esmolol: 18 mg·kg -1 ·h -1 ) groups. Eight rats were underwent sham operation. Eighteen hours after CLP, hemodynamics and organ histological injuries were evaluated, peripheral blood mononuclear cells apoptosis and T-lymphocyte subsets counts were determined by flow cytometry, and the expression of p-Akt, Bcl-2, cleaved Caspase-3, and p-Erk1/2 in splenic CD4 + T-lymphocytes was determined by western blot and immunohistochemistry. β 1 -Adrenoreceptor expressions were evaluated using real-time polymerase chain reaction and immunohistochemistry. Results: Cecal ligation and puncture induced tachycardia, hypotension, hyperlactatemia, and multiple organ injury. Heart rate was unchanged in the CLP + E-5 group but decreased in the CLP + E-18 group. Hypotension, lactatemia, and multiple organ injuries were improved only in the CLP + E-5 group. T-lymphocyte apoptosis and T H 2/T H 1 ratio was decreased in CLP + E-5 but not in CLP + E-18. p-Akt and Bcl-2 expressions were increased, while cleaved Caspase-3 and p-Erk1/2 expressions were decreased in CLP + E-5. β 1 -Adrenoreceptor expressions were unchanged in both CLP + E-5 and CLP + E-18 groups. Conclusions: Low dose of esmolol reduced T-lymphocyte apoptosis and restored T H 2/T H 1 ratio in septic shock. Esmolol might modulate Akt/Bcl-2/Caspase-3 pathway to relieve T-lymphocyte apoptosis and inhibit Erk1/2 activity to decrease T H 0 differentiation to T H 2. Esmolol may be a potential immunoregulator of septic shock.

Association between oxidative stress, mitochondrial function of peripheral blood mononuclear cells and gastrointestinal cancers

BackgroundThe incidence and mortality rate of gastrointestinal cancers are high worldwide. Increasing studies have illustrated that the occurrence, progression, metastasis and prognosis of cancers are intimately linked to the immune system. Mitochondria, as the main source of cellular energy, play an important role in maintaining the physiological function of immune cells. However, the relationship between mitochondrial function of immune cells and tumorigenesis has not yet been systematically investigated.MethodsA total of 150 cases, including 60 healthy donors and 90 primary gastrointestinal cancer patients without anti-tumor treatments (30 with gastric cancer, 30 with liver cancer and 30 with colorectal cancer) were involved in our study. The oxidant/antioxidant and cytokine levels in plasma, the ROS level, mitochondrial function and apoptosis ratio of peripheral blood mononuclear cells (PBMCs) were evaluated.ResultsThe imbalance between oxidant and antioxidant in plasma was discovered in the primary gastrointestinal cancer patients. The levels of cell reactive oxygen species (ROS) and mitochondrial ROS in PBMCs of primary gastrointestinal cancers were significantly increased compared with that in healthy donors. Meanwhile, the ATP content, the mtDNA copy number and the mitochondrial membrane potential (MMP) in PBMCs of patients with primary gastrointestinal cancers were lower than those in control group. The decreased MMP also occurred in immune cells of gastrointestinal cancers, including T cell, B cell, NK cell and monocyte. Furthermore, the PBMCs apoptosis ratio of primary gastrointestinal cancer patients was significantly higher than that of control group. Importantly, an increase of IL-2 and IL-6 and a decrease of IgG in plasma were found in the patients with primary gastrointestinal cancers. These changes of mitochondrial function in immune cells were consistent among primary gastrointestinal cancers without anti-tumor treatments, such as liver cancer, gastric cancer and colorectal cancer.ConclusionOur study demonstrated that the imbalance of oxidation/antioxidation in primary gastrointestinal cancer patients without anti-tumor treatments results in excessive ROS. The oxidative stress was associated to the mitochondrial dysfunction, the apoptosis of immune cells and eventually the abnormal immune function in primary gastrointestinal cancers. The application of immune cell mitochondrial dysfunction into clinical evaluation is anticipated.

Inhibition of caspase-1-dependent apoptosis suppresses peste des petits ruminants virus replication.

Peste des petits ruminants (PPR), caused by the PPR virus (PPRV), is an acute and fatal contagious disease that mainly infects goats, sheep, and other artiodactyls. Peripheral blood mononuclear cells (PBMCs) are considered the primary innate immune cells. PBMCs derived from goats were infected with PPRV and analyzed to detect the relationship between PPRV replication and apoptosis or the inflammatory response. Quantitative real-time polymerase chain reaction was used to identify PPRV replication and cytokines expression. Flow cytometry was conducted to detect apoptosis and the differentiation of CD4+ and CD8+ T cells after PPRV infection. PPRV stimulated the differentiation of CD4+ and CD8+ T cells. In addition, PPRV induced apoptosis in goat PBMCs. Furthermore, apoptosis and the inflammatory response induced by PPRV could be suppressed by Z-VAD-FMK and Z-YVAD-FMK, respectively. Moreover, the virus titer of PPRV was attenuated by inhibiting caspase-1-dependent apoptosis and inflammation. This study showed that apoptosis and the inflammatory response play an essential role in PPR viral replication in vitro, providing a new mechanism related to the cell host response.

HERC6 is upregulated in peripheral blood mononuclear cells of patients with systemic lupus erythematosus and promotes the disease progression

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease. Peripheral blood mononuclear cells (PBMCs) are any peripheral blood cell with round nuclei, including lymphocytes (T cells, B cells) and monocytes, whose physicochemical properties are randomized by obvious immune changes, and are a potentially effective source of SLE blood test samples and therapeutic targets. This study aimed to explore the upregulation molecules of PBMCs in patients with SLE and to explore their biological role. Homologous to the E6-AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain (RLD) containing E3 ubiquitin protein ligase family member 6 (HERC6) expression was found significantly upregulated in four Gene Expression Omnibus gene sets. Moreover, HERC6 expression was upregulated in PBMCs from SLE patients compared with that in PBMCs from normal donors. HERC6 was significantly associated with SLE clinical phenotypes such as complement C3 content, erythrocyte sedimentation rate, and SLE disease activity index. In vitro, knockdown of HERC6 inhibited PBMC apoptosis, inflammatory response, and janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway, while overexpression of HERC6 led to the opposite results. In addition, AG490, a JAK/STAT pathway inhibitor, reversed the promoting effect of HERC6 overexpression on PBMC apoptosis and inflammation. In conclusion, the level of HERC6 in PBMCs in patients with SLE was upregulated. Overexpression of HERC6 promoted PBMC apoptosis and inflammatory response, which was involved in the JAK/STAT pathway.

Radioprotective Effect of Rosmarinus officinalis L (Rosemary) Essential Oil on Apoptosis, Necrosis and Mitotic Death of Human Peripheral Lymphocytes (PBMCs).

Background: Rosemary plant, with phenolic compounds, is known as an antioxidant herb and able to scavenge free radical agents in the biological environment; therefore, it is expected that the rosemary essential oil (R-EO) shows the radioprotective effect to protect individuals who are physically in contact with ionizing radiation.Objective: This study aimed to assess the radioprotective effect of R-EO on human peripheral blood mononuclear cells (PBMCs).Material and Methods: In this experimental study, the toxicity of the rosemary essential oil on PBMCs was assessed by the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cells were irradiated at 0. 25 and 200 cGy using a 6 MV X-ray linear accelerator. The survival, apoptosis, necrosis, and survival enhancement factors of cells were analyzed by MTT and flow cytometry analyses with a non-toxic concentration of the rosemary essential oil (IC10).Results: Irradiation of cells in the presence of R-EO caused a significant increase in cell survival compared with the control in both 25 and 200 cGy radiation doses. Also, the percentages of apoptosis and necrosis of cells showed a significant decrease compared with the control.Conclusion: Rosemary essential oil as a natural and non-toxic compound could show favorable radioprotective effects in such a way that significantly increases the survival rate and decreases the percentage of apoptosis and necrosis of PBMCs.

MiR-204-5p in vivo inhibition cause diminished CD45RO cells rate in lungs of melanoma B16-bearing mice.

The treatment of melanoma remains a challenge, despite novel approaches recently becoming available for disseminated tumors. RNA targeting is being intensively studied in various types of disease. The aim of the present study was to explore whether the in vivo use of a microRNA (miR)-204-5p inhibitor affected melanoma progression, and whether its metastasis affects target organ remodeling. CD45RO+, CD3+, CD8+, forkhead box P3+, smooth muscle α-actin+ cells in the lungs of B16 melanoma-bearing mice were evaluated using immunohistochemistry following miR-204-5p inhibitor transfection. Next, CD45RO expression in peripheral blood mononuclear cells (PBMCs), as well as the apoptosis of these cells, were measured by flow cytometry. The results revealed that the number of CD45RO+ cells was decreased in the lungs of B16 melanoma-bearing mice and CD45RO+ PBMCs following the use of an miR-204-5p inhibitor, which was associated with increased levels of PBMC apoptosis. In conclusion, the findings of the present study suggested that targeting miR-204-5p in melanoma metastasis target organs could be used to develop novel approaches for the treatment of disseminated forms of the disease.

Regulatory effects of a novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae on mice immune cells

BackgroundThe giant panda (Ailuropoda melanoleuca) is a well-known, rare and endangered species. Baylisascaris schroederi is a pathogenic ascarid. Infection with B. schroederi may cause death in giant pandas. At present, the immune evasion mechanism of B. schroederi is little known. Cysteine protease inhibitors (CPI) play important roles in the regulation of host immune responses against certain nematodes. In this study, we focused on the analysis of the regulation of B. schroederi migratory larvae CPI (rBsCPI-1) on mice immune cells.MethodsFirst, the pattern recognition receptors on the surface of peripheral blood mononuclear cells (PBMCs) and the signal pathways that transduce extracellular signals into the nucleus activated by rBsCPI-1 were identified. Then, the regulatory effects of rBsCPI-1 on PBMCs physiological activities were detected. Finally, the effects of rBsCPI-1 on TLR signaling pathway activation and NF-κB phosphorylation in mice immunized with recombinant protein were analysed.ResultsThe results suggested that rBsCPI-1 secreted by B. schroederi migratory larvae is mainly recognized by TLR2 and TLR4 on PBMCs. Extracellular signals are transduced into the nucleus through the MAPK and NF-κB signaling pathways, enhancing the phagocytosis, migration, and apoptosis of PBMCs; meanwhile, rBsCPI-1 induces high expression of NO. Thus, rBsCPI-1 plays a role in immune regulation. In addition, the high expression of negative regulatory factors also ensured that TLR activation is maintained at the optimal level.ConclusionsrBsCPI-1 can transduce regulatory signals into immune cells by activating the TLR2/4-NF-κB/MAPK signaling pathway, having a certain regulatory effect on the physiological activities. Meanwhile, rBsCPI-1 can maintain the immune response in a balance by limiting the over-activation of the TLRs signaling pathway and thus contributes to B. schroederi immune evasion.Graphical

Effects of Ionizing Radiation on Human Peripheral Blood Mononuclear Cells (PBMCs) in the Presence of Mentha-Pulegium Essential Oil: A Study on the Radioprotective Effect.

Background: The Mentha-Pulegium essential oil (MP-EO) contains different antioxidant compounds and reduces the indirect effects of dispersed ionizing radiation on biological systems.Objective: The current study aimed to assess a possible radio-protective effect of MP-EO on peripheral blood mononuclear cells (PBMCs).Material and Methods: In this experimental study, MP-EO was firstly prepared and PBMCs were then irradiated in various groups with doses of 25 and 200 cGy of X-rays in the presence of IC10 of MP-EO. After incubation times of 48h and 72h, the survival, apoptosis, and necrosis percentages of PBMCs were determined by MTT assay and flow cytometry analyses; the radio-protective effect of MP-EO was examined.Results: In the presence of 80 µg/ml (IC10) MP-EO, the mean survival percentage of irradiated PBMCs by radiation doses of 25 and 200 cGy was significantly increased after 48h of incubation compared with the control. At 72h of incubation, the mean survival percentage of irradiated PBMCs was significantly increased only at 25 cGy. The percentage of apoptosis and necrosis of PBMCs was significantly reduced in the presence of the MP-EO at both incubation times and radiation doses; therefore, the highest reduction was at 200 cGy and 48h incubation compared to the control.Conclusion: MP-EO as a natural, non-toxic, and cost-effective compound can exhibit a favorable in-vitro radio-protective effect by increasing the survival and decreasing the percentage of apoptosis and necrosis of irradiated PBMCs.

Preparation of the sphingolipid fraction from mycelia of Cordyceps sinensis and its immunosuppressive activity

Ethnopharmacological relevanceCordyceps sinensis (CS) is an herbal tonic in traditional Chinese medicine and is used to treat a wide range of disorders, including immune, kidney, respiratory, lung and cardiovascular diseases, in China. Most studies are focused mainly on nucleotides and polysaccharides from CS and consider them to be the main active ingredients, while other ingredients are often disregarded. Hundreds of sphingolipids have been identified from CS and showed inhibitory effects on mouse splenic lymphocytes. Aim of the studyThis study aimed to establish a method for preparing a fraction of sphingolipids from the mycelial powder of CS and evaluate its immunosuppressive activity. Materials and methodsFraction of sphingolipids (Fr-SPLs) were prepared by silica gel chromatography and reversed-phase chromatography. Its components were identified and quantified by Quadrupole-Orbitrap UHPLC–MS/MS. PBMCs were prepared from human blood, and splenic lymphocytes, B cells, and T cells were prepared from mouse spleens. The inhibitory effect of Fr-SPLs on cell viability was evaluated by CCK-8 assay. PBMC apoptosis and the ratio of CD4+ T cells and CD8+ T cells were quantified by flow cytometry analysis. The expression of IL-2, IL-10, and TNF-α in PBMCs was detected by ELISA kits. ResultsA fraction containing 84.83% of sphingolipids (SPLs) was prepared from the mycelia of CS and named Fr-SPLs. 15 SPLs were identified from the Fr-SPLs. Fr-SPLs significantly inhibited the viability of human peripheral blood mononuclear cells (PBMCs) with an IC50 value of 9.82 μg/mL and promoted PBMC apoptosis in a dose-dependent manner. Moreover, Fr-SPLs inhibited the viability of mouse splenocytes, as well as that of B cells and T cells derived from splenocytes. Furthermore, Fr-SPLs reduced the production of IL-2, IL-10, and TNF-α in PBMCs. ConclusionsFr-SPLs show immunosuppressive activity, and this study will be useful for preparing immunosuppressive components from CS and its mycelia for hyperimmune disease.

Transcript Expression Profiles and MicroRNA Regulation Indicate an Upregulation of Processes Linked to Oxidative Stress, DNA Repair, Cell Death, and Inflammation in Type 1 Diabetes Mellitus Patients.

Type 1 diabetes (T1D) arises from autoimmune-mediated destruction of insulin-producing β-cells leading to impaired insulin secretion and hyperglycemia. T1D is accompanied by DNA damage, oxidative stress, and inflammation, although there is still scarce information about the oxidative stress response and DNA repair in T1D pathogenesis. We used the microarray method to assess mRNA expression profiles in peripheral blood mononuclear cells (PBMCs) of 19 T1D patients compared to 11 controls and identify mRNA targets of microRNAs that were previously reported for T1D patients. We found 277 differentially expressed genes (220 upregulated and 57 downregulated) in T1D patients compared to controls. Analysis by gene sets (GSA and GSEA) showed an upregulation of processes linked to ROS generation, oxidative stress, inflammation, cell death, ER stress, and DNA repair in T1D patients. Besides, genes related to oxidative stress responses and DNA repair (PTGS2, ATF3, FOSB, DUSP1, and TNFAIP3) were found to be targets of four microRNAs (hsa-miR-101, hsa-miR148a, hsa-miR-27b, and hsa-miR-424). The expression levels of these mRNAs and microRNAs were confirmed by qRT-PCR. Therefore, the present study on differential expression profiles indicates relevant biological functions related to oxidative stress response, DNA repair, inflammation, and apoptosis in PBMCs of T1D patients relative to controls. We also report new insights regarding microRNA-mRNA interactions, which may play important roles in the T1D pathogenesis.

Effect of fresh soy milk and its compounds on apoptosis in human leukemic cells and peripheral blood mononuclear cells


 
 
 
 Purpose: To determine the effect of fresh soy milk and its compounds (coumestrol, daidzein and genistein) on apoptosis in human leukemic cells and peripheral blood mononuclear cells (PBMC).
 Methods: The apoptotic effect of fresh soy milk and its compounds on human leukemic cells (K562, Jurkat, U937) and PBMC was determined by flow cytometry. The PBMC from healthy donors were isolated by conducting density gradient centrifugation principle. Lymphoprep and cytotoxicity of the compounds was evaluated by 3-(4,5- ‘;1dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) MTT) assay.
 Results: PBMC treatment of daidzein and genistein had a significantly higher half-maximal inhibitory concentration (IC50) value (p < 0.01) and (p < 0.0001), compared to the leukemia cells. In addition, soy milk had a significantly higher IC50 value (p < 0.05) in PBMC than the leukemia cells. Coumestrol induced a significantly increased apoptotic cells in K562, U937 cells (p < 0.01) and Jurkat cells (p < 0.05). However, daidzein only increased apoptosis (p < 0.05) in K562 and U937. Genistein reported a significantly increased apoptotic cell in K562, Jurkat (p < 0.01) and U937 cells (p < 0.001). Nonetheless, in PBMC, significant apoptosis by the compounds did not occur. However, only soy milk induced a significantly increased apoptosis in PBMC (p < 0.05).
 Conclusion: The findings indicate that coumestrol, daidzein, genistein and soy milk inhibits the viability of leukemia cells by apoptosis induction, but they are non-toxic toward normal cells. Therefore, the results suggest that the compound alone, and thus, soy milk might have a therapeutic effect in leukemia.
 
 
 

Dimethylaminoparthenolide (DMAPT) as an alternative approach for treatment of Familial Mediterranean Fever (FMF).

Objective(s): Familial Mediterranean Fever (FMF) is a hereditary auto-inflammatory disorder that is caused by mutations in the Mediterranean fever (MEFV) gene and is associated with an increase in pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and interleukin-18 (IL-18), leading to excess inflammation. Colchicine is a common drug widely used for treatment of FMF attacks, but about 5–15% of the patients show resistance to the regular colchicine treatment. In this study, we used dimethylamino-parthenolide (DMAPT), as a small molecule inhibitor of Nuclear factor-κB (NF-κB), NLR family Pyrin domain containing 3 (NLRP3), and cysteine-aspartic acid protease 1(Caspase-1) on FMF-derived peripheral blood mononuclear cells (PBMCs).Materials and Methods:The effects of DMAPT and colchicine on metabolic activity and apoptosis of FMF-derived PBMCs were evaluated by MTT and Annexin V/PI assays, respectively. Also, the expression levels of NF-κB, NLRP3, MEFV, CASP1, and IL-1β mRNA were investigated using a TaqMan real-time PCR, and the protein levels of IL-1β, IL-18, and IL-37 were assessed via an enzyme-linked immunosorbent assay (ELISA) in LPS/ ATP-stimulated PBMCs.Results:DMAPT decreased the expression levels of NFκB (0.38±0.096, P<0.0001), NLRP3 (0.39±0.12, P<0.001), MEFV (0.384±0.145, P<0.001), CASP1 (0.48±0.13, P=0.0023), and IL-1β (0.09±0.09, P<0.0001) and reduced the secretion levels of IL-1β (8.92±5.3 vs. 149.85±20.92, P<0.0001), IL-18 (135±32.1 vs. 192±22.18, P=0.01), and IL-37 (27.5±6.3 vs. 78.19±14.3, P<0.0001) as compared to untreated cells.Conclusion:Given the obtained results in comparison with previous research, the future clinical development of DMAPT could result in the expansion of new anti-inflammatory therapeutics for FMF disorder.

Immunomodulatory Effects of Canine Adipose Tissue Mesenchymal Stem Cell-Derived Extracellular Vesicles on Stimulated CD4+ T Cells Isolated from Peripheral Blood Mononuclear Cells.

Adipose tissue-derived mesenchymal stem cells (ADSCs) have anti-inflammatory and immunomodulatory characteristics. Many studies have suggested that the immunomodulation of ADSCs is largely mediated by secreted paracrine factors. Various factors are secreted from ADSCs, among which extracellular vesicles are considered to play a major role in the communication between ADSCs and target cells. Several studies have reported the function of canine ADSC-derived extracellular vesicles (cADSC-EVs), but few studies have reported the immunomodulatory effects of cADSC-EVs on immune cells. The purpose of this study was to investigate the effects of cADSC-EVs on in vitro-stimulated CD4+ T cells isolated from peripheral blood mononuclear cells (PBMCs). cADSC-EVs were isolated from cADSCs under naive conditions or primed conditions by tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ). The expression levels of several microRNAs in cADSC-EVs were altered by priming with TNFα and IFNγ. Culturing PBMCs stimulated with concanavalin A in the presence of naive or primed cADSC-EVs inhibited the differentiation of PBMCs and CD4+ T cells and promoted apoptosis of PBMCs. CD4+, CD8+, and CD4+CD8+ T cells were decreased, while CD3+CD4−CD8− T cells were increased. T helper (Th) 1, Th2, Th17, and regulatory T (Treg) cells were analyzed by flow cytometry. cADSC-EVs inhibited the proliferation of Th1 and Th17 cells and enhanced Th2 and Treg cell proliferation. However, CD4+ T cells that had incorporated labeled cADSC-EVs comprised only a few percent of all cells. Therefore, these responses of stimulated CD4+ T cells may be due to not only direct effects of cADSC-EVs but also to indirect effects through interactions between cADSC-EVs and other immune cells. In conclusion, cADSC-EVs exert immunosuppressive effects on stimulated CD4+ T cells in vitro. These findings may be useful for further studies of immune diseases.

The Effect of Global DNA Methylation on PDCD5 Expression in the PBMC of Occupational Chromate Exposed Workers.

To evaluate the alteration of protein of programmed cell death 5 (PDCD5) in peripheral blood mononuclear cells (PBMC) and DNA methylation caused by hexavalent chromium exposure. There were 112 workers and 56 controls in this study. The chromium in RBC and urine, PBMC with PDCD5+, DNA methylation, urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and score of DNA damage were measured. In chromate exposed workers, the percent of PBMC with PDCD5+, urine 8-OHdG, and score of DNA damage were significantly higher, whereas global DNA methylation was significantly lower. The binary logistic regression and generalized linear mixed model analysis showed that the percent of PBMC with PDCD5+ was significantly associated with global DNA hypomethylation. The aberrant DNA hypomethylation plays an important role in PBMC apoptosis of occupational hexavalent chromium exposure.

Apoptotic effect of berberine via Bcl-2, ROR1, and mir-21 in patients with B-chronic lymphocytic leukemia.

Berberine is a natural isoquinoline alkaloid that has been shown to inhibit the proliferation and induce apoptosis in a wide variety of tumor cells. However, the action mechanism of berberine in CLL cells is unknown. The previous study has shown that berberine leads to reduced viability and elevated levels of apoptosis in PBMCs of CLL patients. CLL cells are characterized by remarkable expression of Bcl-2 and ROR1 which leads to activation and survival and increases disease progression in patients. High-level expression of miR-21 in patients with CLL is associated with a higher risk of death. Here we investigated the anticancer effects of berberine upon peripheral blood mononuclear cells (PBMCs) of CLL patients. To evaluate the expression of anti-apoptotic proteins and ROR1 using flow cytometry and western blot, PBMCs were treated with 25 μM of berberine for 24 hr. The expression levels of mir-21 were evaluated by real-time PCR. Examination of treated cells demonstrated that berberine decreased Bcl-2 and ROR1 levels. Although western blot results did not show any change in Bax as a pro-apoptotic protein, an increased Bax/Bcl-2 ratio indicated that mitochondrial pathway is involved in berberine-induced apoptosis of CLL cells. Interestingly, berberine could reduce the expression of miR-21 in comparison to the untreated group. Our findings describe some of the molecular mechanisms of berberine by decreasing Bcl-2, ROR1, and mir-21 which may be considered as a novel apoptosis inducer in CLL cells.

Modulatory functions of recombinant electron transfer flavoprotein α subunit protein from Haemonchus contortus on goat immune cells in vitro

Suppression and modulation of the host immune response to parasitic nematodes have been extensively studied. In the present study, we cloned and produced recombinant electron transfer flavoprotein α subunit (ETFα) protein from Haemonchus contortus (rHCETFα), a parasitic nematode of small ruminants, and studied the effect of this protein on modulating the immune response of goat peripheral blood mononuclear cells (PBMCs) and monocytes. Immunohistochemical tests verified that the HCETFα protein was localized mainly in the intestinal wall and on the body surface of worms. Immunoblot analysis revealed that rHCETFα was recognized by the serum of goats artificially infected with H. contortus. Immunofluorescence analysis indicated that rHCETFα bound to the surface of PBMCs. rHCETFα was co-incubated with goat PBMCs to observe the immunomodulatory effects exerted by HCETFα on proliferation, apoptosis, cytokine secretion and nitric oxide (NO) production. The results showed that rHCETFα suppressed the proliferation of goat PBMCs stimulated by concanavalin A and induced apoptosis in goat PBMCs. After rHCETFα exposure, IL-2, IL-4, IL-17A and TNF-α expression was markedly reduced, whereas secretion of TGF-β1 was significantly elevated, in goat PBMCs. Moreover, rHCETFα up-regulated NO production in a dose-dependent manner. FITC-dextran internalization assays showed that rHCETFα inhibited phagocytosis of goat monocytes. These results elucidate the interaction between parasites and hosts at the molecular level, suggest a possible immunomodulatory target and contribute to the search for innovative proteins that may be candidate targets for drugs and vaccines.

Characterization of a rhodanese homologue from Haemonchus contortus and its immune-modulatory effects on goat immune cells in vitro

BackgroundModulation of the host immune response by nematode parasites has been widely reported. Rhodaneses (thiosulfate: cyanide sulfurtransferases) are present in a wide range of organisms, such as archaea, bacteria, fungi, plants and animals. Previously, it was reported that a rhodanese homologue could be bound by goat peripheral blood mononuclear cells (PBMCs) in vivo.MethodsIn the present study, we cloned and produced a recombinant rhodanese protein originating from Haemonchus contortus (rHCRD), a parasitic nematode of small ruminants. rHCRD was co-incubated with goat PBMCs to assess its immunomodulatory effects on proliferation, apoptosis and cytokine secretion.ResultsWe verified that the natural HCRD protein localized predominantly to the bowel wall and body surface of the parasite. We further demonstrated that serum produced by goats artificially infected with H. contortus successfully recognized rHCRD, which bound to goat PBMCs. rHCRD suppressed proliferation of goat PBMCs stimulated by concanavalin A but did not induce apoptosis in goat PBMCs. The production of TNF-α and IFN-γ decreased significantly, whereas secretion of IL-10 and TGF-β1 increased, in goat PBMCs after exposure to rHCRD. rHCRD also inhibited phagocytosis by goat monocytes. Moreover, rHCRD downregulated the expression of major histocompatibility complex (MHC)-II on goat monocytes in a dose-dependent manner, but did not alter MHC-I expression.ConclusionsThese results propose a possible immunomodulatory target that may help illuminate the interactions between parasites and their hosts at the molecular level and reveal innovative protein species as candidate drug and vaccine targets.