MMP1 and MMP11 Expression in Peripheral Blood Mononuclear Cells upon Their Interaction with Breast Cancer Cells and Fibroblasts

Tumor-infiltrating immune cells phenotype is associated with tumor progression. However, little is known about the phenotype of the peripheral blood mononuclear cells (PBMC) from breast cancer patients. We investigated MMP1 and MMP11 expression in PBMC from breast cancer patients and we analyzed gene expression changes upon their interaction with cancer cells and cancer-associated fibroblasts (CAF). We measured the impact of PBMC on proinflammatory gene expression in breast cancer cells, normal fibroblast (NF), and CAF and the impact on proliferation and invasiveness capacity of breast cancer cells. Gene expression of MMP1 and MMP11 in PBMC from breast cancer patients (n = 54) and control (n = 28); expression of IL1A, IL6, IL17, IFNβ, and NFĸB in breast cancer cell lines (MCF-7 and MDA-MB-231); and, additionally, IL10 and MMP11 in CAF and NF were analyzed by qRT-PCR before and after co-culture. Our results show the existence of a subpopulation of breast cancer patients (25.9%) with very high levels of MMP11 gene expression in PBMC. Also, gene expression of MMP1 and MMP11 increases in PBMC after co-culture with breast cancer cell lines, NF or CAF. PBMC from healthy or breast cancer patients induce an increased proliferation rate on MCF-7 and an increased invasiveness capacity of MDA-MB-231. Finally, we show a differential expression profile of inflammatory genes in NF and CAF when co-cultured with control or breast cancer PBMC. We have observed that MMPs’ expression in PBMC is regulated by the microenvironment, while the expression of inflammatory genes in NF or CAF is differentially regulated by PBMC. These findings confirm the importance of the crosstalk between stromal cells and suggest that PBMC would play a role in promoting aggressive tumor behavior.     

大鼠吸入氡及其子体后的DNA损伤效应

研究氡及其子体对大鼠外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)、肺细胞、支气管肺泡灌洗液(Bronchi alveolar lavage fluid, BALF)细胞以及脾和胸腺细胞DNA的损伤作用。雄性SD大鼠吸入氡及其子体的累积剂量分别为66、111、174工作水平月(Working level month, WLM)后,收集BALF细胞,制备肺、脾脏和胸腺组织的单细胞悬液,采用单细胞凝胶电泳(Single cell gel electrophoresis,SCGE)技术,检测细胞DNA链的断裂改变。结果表明, 大鼠吸入氡及其子体后,各照射组PBMC、BALF和肺细胞DNA链断裂的迁移长度显著增加(p<0.01)。其中, PBMC与肺细胞DNA损伤有相关性(r =0.887, p<0.01),直线拟合(R2=0.787)的回归方程为Y=0.978X-0.12。在本实验的照射剂量下,氡及其子体能引起PBMC、BALF和肺细胞的DNA链断裂的损伤。     

Biocompatibility of polyglycidol with human peripheral blood mononuclear cells

Polyglycidol (PGly), a hyperbranched water‐soluble polyether with numerous terminal hydroxyl groups, has structural similarities with polyethylene glycol. Our laboratory is exploring drug delivery using hyperbranched PGly‐cytokines conjugates. Using human peripheral blood mononuclear cells and a tumor derived human B cell line, HBL‐1; various PGly concentrations were tested for cytotoxicity. Our data indicated that hyperbranched PGly was similar in toxicity to PEG 8000, a commercial polymer. It was concluded that hyperbranched PGly did not exhibit significant cytotoxicity and showed promise for use as a biocompatible polymer. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples

Background: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. Methods: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 10⁶ cells/mL to 1.67 × 10⁶ cells/mL. Results: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 10⁶ cells/mL. Conclusion: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.     

Assessment of in vitro antioxidant, antibacterial and immune activation potentials of aqueous and ethanol extracts of Phyllanthus niruri

BACKGROUND: Recently much attention has been paid to biologically active plants because of their low production cost and fewer adverse effects compared with chemical drugs. In the present investigation the bioactivity of Phyllanthus niruri ethanol and aqueous extracts was evaluated in vitro. RESULTS: The ethanol extract of P. niruri showed a high level of flavonoid content (123.9 ± 0.002 mg g^?1), while the aqueous extract showed the highest 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH; IC₅₀6.85 ± 1.80 µmol L^?1) and 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS; 46.44 ± 0.53 µmol L^?1) free radical scavenging activities with high phenol content (376 ± 0.02 mg g^?1) and elevated levels of ferric reducing antioxidant power (FRAP; 23 883 ± 0.019 mmol g^?1) with excellent antibacterial activity against Staphylococcus aureus (20 mm inhibition zone) and Streptococcus agalactiae (12 mm inhibition zone), respectively, in addition to the best immune activation potential of human peripheral blood mononuclear cells (450.5%). CONCLUSIONS: It is clear from our results that both extracts of P. niruri has excellent bioactivity roles via elevated levels of antibacterial, antioxidant and percentage of peripheral blood mononuclear cell proliferation, which could lead to the development of medications for clinical use. Copyright © 2012 Society of Chemical Industry     

Cytokine Secretion Dynamics of Isolated PBMC after Cladribine Exposure in RRMS Patients

Cladribine (CLD) treats multiple sclerosis (MS) by selectively and transiently depleting B and T cells with a secondary long-term reconstruction of the immune system. This study provides evidence of CLD’s immunomodulatory role in peripheral blood mononuclear cells (PBMCs) harvested from 40 patients with untreated relapsing-remitting MS (RRMS) exposed to CLD. We quantified cytokine secretion from PBMCs isolated by density gradient centrifugation with Ficoll–Paque using xMAP technology on a FlexMap 3D analyzer with a highly sensitive multiplex immunoassay kit. The PBMC secretory profile was evaluated with and without CLD exposure. PBMCs isolated from patients with RRMS for ≤12 months had significantly higher IL-4 but significantly lower IFN-γ and TNF-α secretion after CLD exposure. PBMCs isolated from patients with RRMS for >12 months had altered inflammatory ratios toward an anti-inflammatory profile and increased IL-4 but decreased TNF-α secretion after CLD exposure. CLD induced nonsignificant changes in IL-17 secretion in both RRMS groups. Our findings reaffirm CLD’s immunomodulatory effect that induces an anti-inflammatory phenotype.     

Quantifying the Detrimental Effects of Multiple Freeze/Thaw Cycles on Primary Human Lymphocyte Survival and Function

The use of cryopreserved peripheral blood mononuclear cells is common in biological research. It is widely accepted that primary cells are rendered unusable by several freezing cycles, although this practice might be very helpful when the biological material is valuable and its re-collection is impractical. To determine the extent to which primary cells undergoing repeated freezing cycles are comparable to one another and to fresh samples, we evaluated overall lymphocyte viability, their proliferation and cytokine production capabilities, as well as the levels of 27 cell subtypes in ten human peripheral blood mononuclear cells frozen for five years and repeatedly thawed. As expected, we observed a progressive increase in cell death percentages on three rounds of thawing, but the frequency of the main lymphocyte subsets was stable across the three thawings. Nevertheless, we observed a significant reduction of B cell frequency in frozen samples compared to fresh ones. On repeated thawings and subsequent conventional stimulation, lymphocyte proliferation significantly decreased, and IL-10, IL-6, GM-CSF, IFN-gamma, and IL-8 showed a trend to lower values.     

人肝再生增强因子对人外周血单个核细胞增殖的影响及其机制

目的探讨人肝再生增强因子(hALR)对人外周血单个核细胞(PBMC)增殖的影响及其机制。方法梯度离心分离PBMC,将细胞分为正常对照组、刀豆蛋白A(ConA)(5mg/L)组和ConA(5mg/L)+hALR(30mg/L)组,分别培养10min、30min、1h、2h、4h后,MTT法检测各组各时间点细胞增殖水平,钙荧光指示剂法检测细胞内游离Ca2+浓度的变化,Western blot法检测细胞内胞外信号调节激酶(ERK)的磷酸化程度。结果4h内,各组细胞的增殖水平差异无统计学意义,但ConA组细胞增殖水平已表现出逐渐升高的趋势。正常对照组细胞内Ca2+浓度在加入Fluo-3/AM后2h达高峰,ConA组1h达高峰,ConA+hALR组10min达高峰。正常对照组细胞内磷酸化的ERK随培养时间的延长而逐渐减少;ConA组细胞内磷酸化的ERK在30min达高峰,之后逐渐降低;ConA+hALR组细胞内磷酸化的ERK在2h前明显低于ConA组。结论hALR可能通过影响细胞内Ca2+浓度的变化峰值和磷酸化的ERK,来抑制人PBMC的免疫功能,从而影响其增殖。