Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

BACKGROUND: Increasing evidence suggests a double-faceted role of alpha-synuclein (α-syn) following infection by a variety of viruses, including SARS-CoV-2. Although α-syn accumulation is known to contribute to cell toxicity and the development and/or exacerbation of neuropathological manifestations, it is also a key to sustaining anti-viral innate immunity. Consistently with α-syn aggregation as a hallmark of Parkinson's disease, most studies investigating the biological function of α-syn focused on neural cells, while reports on the role of α-syn in periphery are limited, especially in SARS-CoV-2 infection. RESULTS: Results herein obtained by real time qPCR, immunofluorescence and western blot indicate that α-syn upregulation in peripheral cells occurs as a Type-I Interferon (IFN)-related response against SARS-CoV-2 infection. Noteworthy, this effect mostly involves α-syn multimers, and the dynamic α-syn multimer:monomer ratio. Administration of excess α-syn monomers promoted SARS-CoV-2 replication along with downregulation of IFN-Stimulated Genes (ISGs) in epithelial lung cells, which was associated with reduced α-syn multimers and α-syn multimer:monomer ratio. These effects were prevented by combined administration of IFN-ß, which hindered virus replication and upregulated ISGs, meanwhile increasing both α-syn multimers and α-syn multimer:monomer ratio in the absence of cell toxicity. Finally, in endothelial cells displaying abortive SARS-CoV-2 replication, α-syn multimers, and multimer:monomer ratio were not reduced following exposure to the virus and exogenous α-syn, suggesting that only productive viral infection impairs α-syn multimerization and multimer:monomer equilibrium. CONCLUSIONS: Our study provides novel insights into the biology of α-syn, showing that its dynamic conformations are implicated in the innate immune response against SARS-CoV-2 infection in peripheral cells. In particular, our results suggest that promotion of non-toxic α-syn multimers likely occurs as a Type-I IFN-related biological response which partakes in the suppression of viral replication. Further studies are needed to replicate our findings in neuronal cells as well as animal models, and to ascertain the nature of such α-syn conformations.

Preliminary study on HUVEC permeability changes in FNAIT induced by CD36 antibody / 中国输血杂志

【Objective】 To explore the pathogenesis of fetal edema caused by CD36 antibody in fetal/neonatal alloimmune thrombocytopenia (FNAIT), and to provide reference for clinical prevention and treatment. 【Methods】 The established CD36 monoclonal antibody was incubated with human peripheral blood mononuclear cells (PBMC), and the concentrations of cytokines (TNF-α and IL-1β) in the supernatant of cell culture were detected by ELISA. The permeability of endothelial cells were investigated by detecting the fluorescence intensity of FITC-albumin by incubating cytokine-rich cell supernatant with human umbilical vein endothelial cells (HUVEC). 【Results】 Flow cytometry showed that CD36 monoclonal antibody could bind to human monocytes. Compared with isotype IgG control, increased cytokine TNF-α (pg/mL) (407.73±20.40 vs 29.38 ±4.72, P<0.05) and IL-1β (pg/mL) (247.14±83.59 vs 53.68±26.96, P<0.05) were detected in the supernatant of cell culture after incubation of CD36 monoclonal antibody with human PBMC. Detection of fluorescence intensity of FITC-albumin in transwell cultured HUVEC showed that cytokine-rich cell supernatant derived from CD36 monoclonal antibody incubated with human PBMC can increase the permeability of endothelial cells significantly (CD36 antibody vs isotype IgG, MFI value: 492±16 vs 320±11, P<0.05). 【Conclusion】 The effect of CD36 monoclonal antibody on PBMC can increase HUVEC permeability, which may be one of the pathogenesis of fetal edema with FNAIT.

Assessment of cytotoxicity, genotoxicity, and mutagenicity of the dexchlorpheniramine reference standard and pharmaceutical formula in human peripheral blood mononuclear cells

Abstract Dexchlorpheniramine is a first-generation classical antihistamine, clinically used to treat allergies. The main objective of our study was to evaluate the effects of the dexchlorpheniramine reference standard (DCPA Ref. St) and a pharmaceutical formula on DNA in human peripheral blood mononuclear cells (PBMCs). We exposed PBMCs to five different concentrations (0.5, 2.5, 5, 10, and 50 ng/mL) of DCPA Ref. St DCPA Ref. St and pharmaceutical formula in order to evaluate their cytotoxic, genotoxic, and mutagenic potential. The results showed that both dexchlorpheniramine formulations did not affect PBMC viability and CD3+, CD4+, or CD8+ lymphocyte subpopulations. The DCPA Ref. St and pharmaceutical formula neither induced genotoxic or mutagenic effects nor numerical or structural chromosomal alterations in PBMCs after 24 hours of exposure.

The expression and clinical significance of promyelocytic leukemia zinc finger in human peripheral blood with asthma / 中华急诊医学杂志

Objective:To investigate the expression of promyelocytic leukemia zinc finger (PLZF) in human peripheral blood with asthma and its clinical significance.Methods:Forty patients with stable asthma from May 2021 to October 2021 in the Department of Respiratory Medicine of the Shanghai Fifth People's Hospital were enrolled, and forty healthy controls were recruited in the study. The levels of cytokines in serum were measured by enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR (qPCR) was used to detect the expression of PLZF mRNA in plasma. The level and distribution of PLZF+ cells in PBMCs were detected by flow cytometry after isolating peripheral blood mononuclear cells (PBMCs). Independent sample t test, Mann-Whitney U test, χ 2 test, ROC curve and Logistic regression were used to analyze the results with SPSS 26.0 and Graphpad Prism 7.0. A P<0.05 was considered statistically significant. Results:The levels of cytokines IFN-γ, IL-2, IL-4, TNF-α and IL-17 in human peripheral blood from the asthma group were obviously higher than those in the control group ( P<0.05), whereas there was no significant difference in the level of cytokine IL-10 between the two groups. The level of PLZF mRNA in PBMCs from the asthma group was significantly up-regulated compared to that in the control group [(3.40%±2.52%) vs. (1.23%±0.78%), P<0.05]. CD8+PLZF+ and Vβ11+PLZF+T cells in the asthma group were significantly outnumbered than those in the control group ( P<0.05). Logistic regression and ROC curve analysis showed that PLZF expression in PBMC was a risk factor for the development of asthma ( OR =3.67, AUC=0.87, P<0.05). Conclusions:The high expression of PLZF in peripheral blood may play an important role in the development of asthma, which needs to be further confirmed by large sample studies.

Detection of Serum Antibodies and Peripheral Blood Mononuclear Cells Response to Escherichia coli Antigens in Humans

Abstract Among the microorganisms that make up the intestinal microbiota, stands out Escherichia coli, which has as main ecological niche, the large human intestine. Its importance stands out in being part of the pioneer's commensal microorganisms on the colonization of the intestinal mucosa and its pathogenic role causing extra and intra intestinal diseases. The aim of the study was to evaluate the antibody production and proliferative response of Peripheral Blood Mononuclear Cells (PBMC) to E. coli antigens. The bacteria were grown on Brain Heart Infusion broth medium at 35 ºC for 72 hours. Pellet bacteria were lysed for one hour at room temperature with an 8M sodium guanidine solution. After spin and dialysis, the protein antigens were measured in the supernatant by protein assay. The antigens were characterized by polyacrylamide gel electrophoresis and the antigenic profile by western blotting. The presence of specific IgG and IgA antibodies were evaluated using thirty normal human sera by an indirect ELISA. The response of PBMC to E. coli antigens was assessed by MTT metabolization. The results demonstrated that the antigens were composed of proteins of different sizes and they were recognized by antibodies present in normal human serum. Human sera presented high titers of IgG and IgA antibodies to E. coli antigens when compared to the results of lipopolysaccharide. We also showed that total E. coli antigens induced PBMC proliferation at different antigen concentrations. Taken together the results suggest that the antigens from E. coli can induce local and systemic immune responses.

Multivariate analysis of peripheral blood mononuclear cells from children donors / 中国输血杂志

【Objective】 To analyze the characteristics of peripheral blood mononuclear cells (PBMC) collection in healthy children, and explore the factors affecting collection efficiency (CE). 【Methods】 The PBMC data, involving 70 episodes of apheresis from 42 children during January 2017 and June 2020 were retrospectively analyzed All children were collected in Zhujiang Hospital of Southern Medical University. 【Results】 Multiple linear regression analysis showed that mononuclear cells (MNC) in donor collections from healthy children were positively correlated with anticoagulant dosage, lymphocyte count and monocyte count (P<0.05), meanwhile, negatively correlated with age and platelet count. The PBMC CE was negatively correlated with age, platelet count, and processed whole blood volume (P<0.05). CD34+ cells (×107 /kg)was negatively correlated with age, meanwhile, positively correlated with numbers of collection and processed whole blood volume(r=-0.79). No statistical differences in red blood cell count, platelet count, lymphocyte count, monocyte count of healthy child donors were notable before versus after apheresis. 【Conclusion】 MNC can be collected effectively in children of different ages. The PBMC collection efficiency was related to age. Meanwhile, the higher the lymphocytes and monocytes were before apheresis, the more MNC were collected. The efficiency of MNC collection would decrease when the apheresis volume of the children exceeded their total blood volume twice. However, the absolute value of CD34+ cells in the final yields would increase.

Key Genes of Peripheral Blood Mononuclear Cells in Hepatocellular Carcinoma and Effective Chinese Herbs： A Bioinformatics Analysis and Systematic Review / 中国实验方剂学杂志

Objective:To explore the key gens of peripheral blood mononuclear cells in hepatocellular carcinoma（HCC-PBMC） and potentially effective Chinese herbs based on bioinformatics， and to verify the clinical efficacy of these Chinese herbs via a systematic review. Method:The chips GSE58208 and GSE36076 of HCC-PBMC were downloaded from the Gene Expression Omnibus （GEO）， followed by the identification of differentially expressed genes （DEGs） using RStudio. After protein-protein interaction （PPI） analysis by STRING， the DAVID was employed for gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis. The DEGs of HCC-PBMC were visualized by Cytoscape. The key genes of HCC-PBMC were calculated by CytoHubba plug-in and mapped with those in Coremine Medical for screening out the potential Chinese herbs for the treatment of HCC， which were then included for subsequent systematic review. Result:A total of 203 DEGs were obtained （194 up-regulated and nine down-regulated）. As revealed by DAVID analysis， the DEGs were mainly enriched in such biological processes and signaling pathways as transcriptional regulation of DNA template， hydrolysis of ribonucleic acid phosphodiester bond， positive regulation of intranuclear mitosis and division， skeletal muscle fiber development， activation of mitogen-activated protein kinase（MAPK）activity， Fanconi anemia pathway， and metabolic pathway. The key genes of HCC-PBMC were calculated by Cytoscape to be<italic> </italic>GTPase IMAP family member 1 （GIMAP1）， GTPase IMAP family member 4 （GIMAP4）， GTPase IMAP family member 6 （GIMAP6）， GTPase IMAP family member 7 （GIMAP7）， GTPase IMAP family member 8 （GIMAP8）， interleukin-1<italic>β </italic>（IL-1<italic>β）</italic>， CX3C chemokine receptor 1 （CX3CR1）， C-C chemokine receptor type 2 （CCR2）， Toll-like receptor 7（TLR7）， and epidermal growth factor（EGF）. Through Coremine Medical analysis， it was concluded that Ginseng Radix et Rhizoma， Curcumae Longae Rhizoma， Centellae Herba， and Hedyotidis Herba were closely related to the key genes. Ginseng Radix et Rhizoma has the effects of tonifying and benefiting lung and spleen and enhancing strength， suitable for the liver depression and spleen deficiency syndrome or Qi-Yin deficiency syndrome of HCC. Hence， Si Junzitang with Ginseng Radix et Rhizoma as the sovereign medicinal was included for systematic review. It has been confirmed that Ginseng Radix Et Rhizoma was superior to western medicine alone in improving the overall clinical efficacy， alleviating TCM syndrome， elevating serum CD4⁺and CD4⁺/CD8⁺levels， and reducing the serum CD8⁺and TBIL levels （<italic>P</italic><0.01）， with high safety. Conclusion:This study conducted at the gene level has provided new ideas for clinical diagnosis and treatment of HCC. The systematic review of Ginseng Radix Et Rhizoma against HCC provides a basis for the clinical prevention and treatment of HCC with TCM.

In vitro Antioxidant and Anti-inflammatory Activities of the Flower Extracts of Hibiscus vitifolius L

Objective: To determine the antioxidant and anti-inflammatory activities of the solid powder extracted from the ethyl acetate fraction of the flower Hibiscus vitifolius L. Methods: The flower extract assessed for antioxidant activity using the 1,1–diphenyl-2-picryl-hydrazile (DPPH) radical scavenging assay and the reduced power assay was performed using the Ferric Reducing Capacity (FRC) assay. In vitro anti-inflammatory activity was assessed using human peripheral blood mononuclear cells (PBMC) induced by lipopolysaccharide (LPS) to test the production method of nitric oxide (NO). Results: The solid powder extracted from the ethyl acetate fraction of the flower Hibiscus vitifolius L showed good antioxidant activity in the scavenging DPPH radicals and the FRC assay compared to the standard sample. This powder sample also showed good anti-inflammatory activity in cell viability (LPS induced PBMC) assay and nitric oxide (NO) assay. Conclusion: These results suggest that the powder sample extracted from the ethyl acetate fraction of the flower Hibiscus vitifolius L has substantial antioxidant and anti-inflammatory activity.

Inulin Induces IL-10 Secretion and Increased FOXP3 Gene Expression in Human Peripheral Blood Mononuclear Cells

ABSTRACT Inulin is an effective prebiotic and its potential in modulating systemic immunity have been proposed. A subpopulation of T cells, named T regulatory cells (Tregs), expressing the Forkhead boxP3 transcription factor are key mediators of peripheral tolerance and suppress undesirable immune responses. These Tregs can be induced by cytokine transforming growth factor beta (TGF-β) and interleukin 10 (IL-10). This work aimed to evaluate inulin effects on human peripheral blood mononuclear cells (PBMC) in vitro. PBMC were incubated with inulin, and the expression of TGF-(1, FOXP3 and IL-10 was analyzed. Increased supernatant IL-10 levels were observed in PBMC of inulin-treated group (p=0.03). Moreover, FOXP3 gene expression was 7.6 fold higher in inulin-treated PBMC, whereas a trend in TGF-β1 expression was detected (p=0.055). These data suggest that inulin induces an immunosuppressive environment in cultured PBMC by promoting FOXP3 gene expression and IL-10 secretion. These studies offer prospects for further fundamental research in this field.

Effects of IL-7 and IL-21 modified NK-92MI cells on themselves and T cells from normal human peripheral blood / 中国肿瘤生物治疗杂志

@#[Abstract] Objective：To investigate whether the proliferation and cytotoxicity of NK-92MI cells can be improved by IL-7 and IL-21 genes modification, and determine the effects of this genetically modified NK-92MI cells on T cells from normal human peripheral blood. Methods：IL-7 and IL-21 gene fragments were constructed into electroporation vector by genetic engineering method, and NK92MI/IL-21 and NK-92MI/IL-7&21 cells were constructed by electroporation transfection. The in vitro proliferation and cytotoxicity of NK-92MI, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells were measured by cell count and flow cytometry assays. Then, normal human PBMCs were co-cultured with NK-92MI, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells in vitro respectively, and the phenotype change of T cells was measured by flow cytometry. In addition, the cytotoxicity between the activated T cells and three NK-92MI cell lines (NK-92MI, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells) as well as the cytotoxicity of the three NK-92MI cells on tumor cells after co-incubation with activated T cells were detected. Results: NK-92MI/IL-21 cell line (highly expressing IL-21) and NK-92MI/IL-7&21 cell line (highly expressing both IL-7 and IL-21) were successfully constructed. The toxicity of NK-92MI, NK-92MI/IL-21 and NK92MI/IL-7&21 cells on Jurkat and K562 cells showed no difference, while the proliferation of NK-92MI/IL-21 and NK-92MI/IL-7&21 cells was increased compared with NK-92MI cells. Furthermore, NK-92MI/IL-21 and NK-92MI/IL-7&21 cells promoted the activation of T cells to a certain degree, and the activated T cells showed merely no cytotoxicity on NK-92MI, NK-92MI/IL-21 and NK-92MI/IL7&21 cells; Meanwhile, the activated T cells did not affect the cytotoxicity of the three NK cells (NK-92MI, NK-92MI/IL-21, and NK92MI/IL-7&21 cells) on K562 cells under their co-existence. Conclusion: The in vitro proliferation of NK-92MI/IL-21 and NK-92MI/ IL-7&21 cells were enhanced after gene modification, which could also stimulate and promote the activation of T cells from peripheral blood. The cytotoxicity assay showed that the activated T cells had no cytotoxicity on NK-92MI, NK-92MI/IL-21, and NK-92MI/IL-7& 21 cells. Meanwhile, the presence of the activated T cells did not affect the cytotoxicity of NK-92MI cells.

Effect and mechanism of mesenchymal stem cell derived from different sources on inhibiting follicular helper T cells / 器官移植

Objective To investigate the inhibitory effect and underlying mechanism of mesenchymal stem cell (MSC) derived from different sources on follicular helper T cell (Tfh cell). Methods Umbilical cord-derived MSC (UC MSC), bone marrow-derived MSC (BM MSC) and fat-derived MSC (Fat MSC) were co-cultured with peripheral blood mononuclear cell (PBMC) for 48 h. A control group was established. Flow cytometry was adopted to calculate the proportion of Tfh cells among the lymphocytes in four groups. The content of interleukin (IL)-21 in the supernatant was detected by enzyme-linked immune absorbent assay (ELISA) in four groups. BM MSC was co-cultured with PBMC, and supplemented with indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyl tryptophan (1-MT), IL-10 antibody, human leukocyte antigen (HLA)-G antibody in the 1-MT group, IL-10 inhibition group, HLA-G inhibition group and BM MSC group without addition of other substances. After 48 h culture, flow cytometry was used to detect the percentage of Tfh cells among lymphocytes. Results Flow cytometry demonstrated that compared with the control group, the proportion of Tfh cells in the BM MSC group was significantly decreased (P<0.05). Compared with the BM MSC group, the percentage of Tfh cells in the UC MSC and Fat MSC groups was significantly higher (both P<0.05). ELISA revealed that compared with the control group, the IL-21 content in the BM MSC group was significantly decreased (P<0.05). Compared with the BM MSC group, the IL-21 contents were considerably higher in the UC MSC and Fat MSC groups (both P<0.05). The analysis of underlying mechanism revealed that the proportions of Tfh cells in the 1-MT, IL-10 inhibition and the HLA-G inhibition groups were (1.75±0.07)%, (1.31±0.09)% and (1.50±0.03)%, respectively, which were significantly higher than (1.03±0.43)% in the BM MSC group (all P<0.05). Conclusions BM MSC exerts the highest inhibitory effect upon the differentiation of Tfh cell and IL-21. The mechanism underlying suppressing the differentiation of Tfh cells differentiation is probably correlated to promoting the secretion of IDO.

Expressions and clinical significance of CXC chemokine receptor 1 and 2 and CXCL8 in hepatitis B related hepatocellular carcinoma / 中国免疫学杂志

Objective:To investigate the expression levels of CXCR1,CXCR2 and their common ligand CXCL8 in peripheral blood mononuclear cells (PBMCs) and liver biopsy from the patients with hepatitis B related hepatocellular carcinoma and their clinical significances.Methods:Quantitative real time polymerase chain reaction (qRT-PCR) was used to detect the mRNA levels of CXCR1,CXCR2,CXCL8 in the peripheral blood mononuclear cells of thirty-six hepatitis B related hepatocellular carcinoma and the protein levels of CXCR1 and CXCR2 and CXCL8 in liver biopsy were detected by SP immunohistochemical method.The level of C-reactive protein in serum was determined by chemiluminescence immunoassay respectively.Then,the correlations between CRP and the mRNA of CXCR1,CXCR2 and CXCL8 were analyzed.Results:The mRNA levels of CXCR1 (0.952 7±0.197 2),CXCR2 (0.896 9±0.173 0),CXCL8 (1.771 9±0.248 9) in the PBMCs of hepatitis B related hepatocellular carcinoma were significantly higher than those in controls (P＜0.01).And the protein levels of CXCR1,CXCR2 and CXCL8 were also obviously increased in liver biopsy of hepatitis B related hepatocellular carcinoma (P＜0.05).In addition,there was positive correlations between the level of serum C-reactive protein and the mRNA expression of CXCR1 (r =0.54,P＜0.01),CXCR2 (r =0.49,P＜0.01),CXCL8 (r =0.63,P＜0.01).Conclusion:The levels of CXCR1,CXCR2 and CXCL8 significantly increased in hepatitis B related hepatocellular carcinoma patients and positively correlated with serum CRP,suggesting that CXCR1,CXCR2 and their common ligand CXCL8 signal transduction process may play a key role in the pathological process of hepatitis B related hepatocellular carcinoma,which may provide a new direction for the immune intervention therapy of hepatocellular carcinoma.

Function of IgD on lymphocyte activation and effect of hIgD-Fc-Ig fusion protein on human PBMC proliferation / 中国药理学与毒理学杂志

OBJECTIVE This study aimed to investigate the influence of IgD on T/B cell activationand construct hIgD-Fc-Ig fusion protein to competitive inhibition IgD binding with IgDR. METHODS T/B cells were sorted by magnetic cell sorting. The differences of mIgD and IgD-R level between different T/B cell subtypes were detected by FCM. Serum IgD level was detected by ELISA. Human IgD-Fc-IgG1- Fc sequence was amplified by cross- PCR and then subcloned into PET28a(+ ) empty vector. After prokaryotic expression through escherichia coli, we obtained the hIgD-Fc-Ig fusion protein by affinity chromatograph. Western blot was used to identify the hIgD- Fc- Ig fusion protein. Human peripheral blood monouclear cells (PBMC) and fibroblast like synoviocytes (FLS) proliferation were detected using a cell counting kit-8 (CCK-8). RESULTS The percentage of CD3+/CD4+, CD3+/IgD+, CD3+/CD4+/IgD+, CD3+/IgD-R+ and CD3+/CD4+/IgD-R+ cells increased significantly in RA patients comparing to healthy people. IgD can stimulate PBMC proliferation. IgD (1, 3, 10, 30 μg·mL-1) stimulate PBMC proliferation significantly after 24 h. We obtained stable and active hIgD-Fc-Ig fusion protein. The hIgD-Fc-Ig fusion protein showed no effect on PBMC proliferation. But it could downregulate human IgD protein promoting proliferation effects in human PBMC. CONCLUSION This result suggests that IgD and IgDR play an important role on T/B cell activation in RA patients and the hIgD-Fc-Ig fusion protein may competitively inhibit IgD's function and may play an therapeutic role in autoimmune diseases.

Assessment of immunological markers and booster effects of Ag85B peptides, Ag85B, and BCG in blood of BCG vaccinated children: a preliminary report

PURPOSE: In the present study, the protective immunological markers in serum and peripheral blood mononuclear cells (PBMCs) of bacillus Calmette-Guerin (BCG) vaccinated and unvaccinated children were evaluated after vaccination. Further, PBMCs of children with low protective levels were boosted with BCG, Ag85B, and Ag85B peptides to study their booster effects to increase waning BCG induced immunity. MATERIALS AND METHODS: Fifty children from 1 month to 18 years of age were randomized for the study. Blood samples were collected from 27 participants with/without BCG vaccination. Immunological markers (anti-BCG, interferon gamma [IFN-gamma], and adenosine deaminase activity) were assessed in both serum and PBMCs of children. Children with low levels of protective immunological markers were further recruited and their PBMCs were boosted with BCG, Ag85B, and Ag85B peptides. RESULTS: Children in age group of 4-6 years were associated with significantly (p<0.05) higher BCG-specific IgG and IFN-gamma levels compared to those in age group greater than 10 years. Vaccinated children had greater repertoire of immunological memory which on in vitro stimulation with BCG showed increase in BCG-specific response compared to unvaccinated controls. Assessment of booster effects of BCG, Ag85B, and Ag85B peptides in PBMCs of children revealed greater potential of peptides to boost BCG induced immunity compared to BCG and Ag85B. CONCLUSION: To conclude, children within age 4-6 years are associated with high immunological markers which eventually diminish with age thereby suggesting need for booster dose in later years. Mycobacterium tuberculosis peptides along with BCG may be used as attractive candidates to boost such waning BCG induced immunity in children.

Enhancement of viability of radiosensitive (PBMC) and resistant (MDA-MB-231) clones in low-dose-rate cobalt-60 radiation therapy / Aumento de viabilidade de clones radiossensível (PBMC) e resistente (MDA-MB-231) na cobaltoterapia em taxa de dose reduzida

Abstract Objective: In the present study, the authors investigated the in vitro behavior of radio-resistant breast adenocarcinoma (MDA-MB-231) cells line and radiosensitive peripheral blood mononuclear cells (PBMC), as a function of different radiation doses, dose rates and postirradiation time kinetics, with a view to the interest of clinical radiotherapy. Materials and Methods: The cells were irradiated with Co-60, at 2 and 10 Gy and two different exposure rates, 339.56 cGy.min–1 and the other corresponding to one fourth of the standard dose rates, present over a 10-year period of cobalt therapy. Post-irradiation sampling was performed at pre-established kinetics of 24, 48 and 72 hours. The optical density response in viability assay was evaluated and a morphological analysis was performed. Results: Radiosensitive PBMC showed decrease in viability at 2 Gy, and a more significant decrease at 10 Gy for both dose rates. MDAMB- 231 cells presented viability decrease only at higher dose and dose rate. The results showed MDA-MB-231 clone expansion at low dose rate after 48–72 hours post-radiation. Conclusion: Low dose rate shows a possible potential clinical impact involving decrease in management of radio-resistant and radiosensitive tumor cell lines in cobalt therapy for breast cancer. .

Resumo Objetivo: Neste estudo foi investigado o comportamento in vitro de linhagem de adenocarcinoma mamário (MDA-MB-231), radiorresistente, e de células mononucleadas do sangue periférico (PBMC) frente a diferentes doses de radiação, taxas de dose e cinética de tempo pós-irradiação, considerando o interesse para a radioterapia clínica. Materiais e Métodos: As linhagens foram irradiadas com Co-60, com 2 e 10 Gy, em duas taxas de exposição distintas, 339,56 cGy.min–1 e um quarto da primeira, presentes em um período de 10 anos da cobaltoterapia, respectivamente. Coletas pós-irradiação foram realizadas em cinética pré-estabelecida de 24, 48 e 72 horas. Avaliou-se a resposta da densidade óptica em ensaio de viabilidade e foi feita uma análise morfológica. Resultados: Células PBMC, sensíveis à radiação, apresentaram diminuição da viabilidade a 2 Gy e fortemente a 10 Gy em ambas as taxas de dose. Células MDA-MB-231 apresentaram diminuição da viabilidade apenas com dose e taxa de doses elevadas. Os resultados também demonstraram expansão clonal de MDA-MB-231 em baixa dose e taxa de dose pós 48–72 horas pós-irradiação. Conclusão: Baixa taxa de dose apresenta possível impacto clínico de redução do controle tumoral de linhagens radiorresistentes em câncer de mama em cobaltoterapia. .

Expression of interleukins 7 & 8 in peripheral blood mononuclear cells from patients with metabolic syndrome: A preliminary study.

Background& objectives: Metabolic syndrome (MS) is a common but serious public health problem in developed countries. Chronic inflammation plays a key role in MS. Interleukins (IL)-7 and 8 are considered to have proinflammatory effects and may be involved in the pathogenesis of MS. Therefore, the aim of this study was to determine gene expression level of IL-7 and IL-8 in peripheral blood mononuclear cells (PBMCs) of patients with MS compared to healthy control subjects. Methods: Using real-time RT-PCR, the relative amounts of IL-7 and IL-8 mRNA were determined in PBMCs from 20 female patients with MS and compared with those of 20 healthy control subjects. Biochemical and anthropometric parameters of MS were also assessed. Results: Total cholesterol, triglyceride, and fasting blood sugar were significantly higher in MS patients compared to healthy subjects. There were no significant differences in HDLc and LDLc between the two groups. IL-8 expression in PBMC was significantly decreased in MS versus control subjects (fold of change was 0.395 ± 0.1824), while no difference in the IL-7 expression was detected between them. IL-8 expression had negative correlation with MS components especially with triglyceride and total cholesterol (r=0.5, P<0.001). Interpretation & conclusions: In this preliminary study, no detectable differences were found in IL-7 expression and decreased expression of IL-8 in PBMCs of MS patients as compared to those of control subjects. Study on a larger population and investigating the mechanisms involved can reveal more details.

Dipyrone & 2,5-dimethylcelecoxib suppress Th2-related chemokine production in monocyte.

Background & objectives: Selective cyclooxygenase-2 (COX-2) inhibitor is a form of non steroidal anti-inflammatory drug (NSAID) and is commonly used in autoimmune and rheumatic diseases to control inflammation and alleviate pain. Tumour necrosis factor-alpha (TNF-α) production and an imbalance of T helper 1 (Th1)/Th2 contribute to the pathogenesis of autoimmune and also anti-tumour activity. Dipyrone is a NSAID used to treat pain worldwide. The celecoxib analogue, 2,5-dimethylcelecoxib (DMC), lacks COX-2 inhibitory activity but exhibits anti-tumour properties. However, the effects and the mechanisms of dipyrone and 2,5-dimethylcelecoxib on tumour necrosis factor (TNF)-α and Th1- and Th2-related chemokines in monocytes remain poorly defined. This study was carried out to investigate the effects of dipyrone and 2,5-dimethylcelecoxib on the expression of Th1 (IP-10) and Th2 (I-309 and MDC) and TNF-α in human monocytes and the associated intracellular mechanism. methods: THP-1 cells and peripheral blood mononuclear cells (PBMCs) were pre-treated with dipyrone (10-9 – 10-4 M) and 2,5-dimethylcelecoxib (10-9 – 10-5 M) 2 h before lipopolysaccharide (LPS) stimulation. Cell supernatant was collected 24 h after LPS stimulation. TNF-α, I-309, MDC and IP-10 concentrations of cell supernatants were determined using ELISA. Intracellular signaling was evaluated by western blot. results: Dipyrone and 2,5-dimethylcelecoxib downregulated LPS-induced Th2-related chemokine I-309 and macrophage derived chemokine (MDC) production. Only high dose of 2,5-dimethylcelecoxib (10-5 M), but not dipyrone downregulated LPS-induced IP-10. Only very high dose of 2,5-dimethylcelecoxib had effect on LPS-induced TNF-α expression in PBMCs. Dipyrone and 2,5-dimethylcelecoxib suppressed LPS-induced p65 and JNK MAPK (C-Jun N-terminal kinase mitogen activated protein kinase). expression. Interpretation & conclusions: Dipyrone and 2,5-dimethylcelecoxib downregulated LPS-induced Th2-related chemokine I-309 and MDC in THP-1 cells. The suppressive effect on Th2-related

The Expression of HDAC2 In PeripheraI BIood MononucIear CeIIs from Patients with Sudden SensorineuraI Hearing Loss / 听力学及言语疾病杂志

Objective To evaluate expression of HDAC2 in peripheral blood mononuclear cells(PBMCs)from glucocorticoid-resistant versus glucocorticoid-sensitive patients with sudden sensorineural hearing loss and identi-ty the relationship between the level of HDAC2 and glucocorticoid insensitivity.Methods PBMCs were collected from10 patients with deviation of nasal septum (control group)and 20 sudden sensorineural hearing loss patients be-fore and after intratympanic methylprednisolone for 10 days.We divided the SSNHL patients into 2 groups (GC sensitive group and GC insensitive group)according to their hearing recovery.Real time PCR and HDAC2 Assay Kit were used to detect the expression level of HDAC2 mRNA and HDAC2 activity in PBMCs.The data were analyzed with SPSS 17.0 software.ResuIts Before intratympanic methylprednisolone,the level of HDAC2 activity were sig-nificantly depressed in SSNHL patients,while the HDAC2 mRNA expressing much higher than the control group. The expression level of HDAC2 mRNA increased significantly after intratympanic methylprednisolone.The HDAC2 activity in GC sensitive group increased significantly.ConcIusion Knockdown of HDAC2 expression induces corti-costeroid in-sensitivity.Glucocorticoids can increase the expression of HDAC2 mRNA.HDAC2 activity can be down-regulated by post-translational modifications.

"Transplant human PBMC to establish a ""human-mouse""xenogeneic graft-versus-host disease model" / 中国免疫学杂志

Objective:To explore the suitable mouse strains,and establish stable human-miceX-GVHD model.Methods:This study selected the Nude Mice and NOD/SCID, and gave them sublethal dose of γ-ray irradiation whole body , and then intraperitoneal transplanted human peripheral blood mononuclear cells ( PBMC) to establish xenogeneic acute graft-versus-host disease model.By detection of human T cells in the mice′s tail venous blood,tissues,organs of the infiltration and other indicators (By flow cy-tometry and immunohistochemistry ) ,we compared the human immune cell infiltration rates in the two mouse model and recorded the survival time.Finally,we determined the appropriate strains of mice to establish X-GVHD.Optimization means were transfer ways and the appropriate amount of human PBMC ,and the best time to observe the changes of T cell phenotype and function.Results:The NOD/SCID mouse was more suitable for inducing human-mouseX-GVHD model,and there were no significant differences between intrap-eritoneal injection and intravenous injection.Transfer human PBMC more than 5 ×107 can establish human-mouseX-GVHD model.Using the optimized experimental conditions to establish the human-mouseX-GVHD model,we found the 7-11 days was the best time to observe the changes of T cell phenotype and function ,and the average survival time was(14.16±1.77)days.Conclusion:Human-miceX-GVHD model can be successfully established by intraperitoneal injection of 5×107 human PBMC into NOD/SCID, and the best time to observe the changes of T cell phenotype and function is between 7-11 days.

Expansion of new type cytokine induced killer cells in vitro with peripheral blood mononuclearcells in ad-vanced breast cancer in PBMC / 实用肿瘤学杂志

Objective To investigate new type cytokine induced killer cells expansion using advanced breast cancer′s peripheral blood .Methods peripheral blood mononuclear cells were isolated from 8 advanced breast cancer volunteers and co -cultured with Cytokine induced killer cells .These cells were placed in plastic flasks containing CIK-MediumTM supplemented with 10% auto-plasma in the presence of IL -2 ( 1 000 IU/mL) .The cultures were fed with CIK-MediumTM supplemented with IL -2 following the proliferation capacity . Cell proliferation was measured by cell counting during the cultivation .Fourteen days after cultivation ,cell mark-ers CD3/CD16/CD56 were examined by flow cytometry .51Cr and MTT assays were employed in cytotoxicity as-says.Cytokines were assayed by ELISA method .Results CD16+,CD16+CD56+,CD56+CIK cells were 5.8~11.6%in 2 ×107 fresh PBMCs and 95.2~97.6%in co-cultured cells after 18 days cultivation .The in vitro ex-pansion rate of new type cytokine induced killer cells was up to more than 8.2 ×108 in total,the cytotoxicity are ef-fective killing cells against MCF 7 and BT20 breast cancer cell lines .New type cytokine induced killer cells expand-ed from all PBMCs and secreted cytokines IFN -and TNF-.Conclusion The present culture could be useful to clarify the mechanisms of CIK cells expansion in vitro and feasible for breast cancer immmuno cell therapy .