Human Peripheral Blood Mononucleated Cells Research Articles

Immunophenotype and antitumor activity of cytokine-induced killer cells from patients with hepatocellular carcinoma.

Cytokine-induced killer (CIK) cells are heterogeneous lymphocytes from human peripheral blood mononucleated cells (PBMCs) co-cultured with several cytokines. The main purpose of this study is to evaluate the functional characteristics and anticancer ability of CIK cells from hepatocarcinoma (HCC) patients. CIK cells were activated ex-vivo and expanded from PBMCs from HCC patients. The immunophenotype and the ex-vivo killing ability of CIK cells were evaluated. Human CIK cells were intravenously injected into NOD/SCID mice to evaluate the in vivo anticancer ability. More than 70% of CIK cells were CD3+CD8+, and 15%-30% were CD3+CD56+. These cells expressed an increased number of activated natural killer (NK) receptors, such as DNAM1 and NKG2D, and expressed low-immune checkpoint molecules, including PD-1, CTLA-4, and LAG-3. Among the chemokine receptors expressed by CIKs, CXCR3 and CD62L were elevated in CD8+ T cells, representing the trafficking ability to inflamed tumor sites. CIK cells possess the ex-vivo anticancer activity to different cell lines. To demonstrate in vivo antitumor ability, human CIK cells could significantly suppress the tumor of J7 bearing NOD/SCID mice. Furthermore, human immune cells could be detected in the peripheral blood and on the tumors after CIK injection. This study revealed that CIK cells from HCC patients possess cytotoxic properties, and express increased levels of effector NK receptors and chemokine molecules and lower levels of suppressive checkpoint receptors. CIK cells can suppress human HCC ex-vivo and in vivo. Future clinical trials of human CIK cell therapy for HCC are warranted.

Effects of Medical Diagnostic X-rays Delivered at 0.01 or 0.05 mGy on Human Blood Cells

Background: Low-dose X-rays are commonly used in medical imaging to help in the diagnosis ofdiseases. However, the deleterious effects of exposure to medical diagnostic low-dose X-rays remaina highly debated topic. The objective was to study the effects of medical diagnostic X-rays on humanblood cells.
 Materials and Methods: We studied the effects of medical diagnostic low-dose X-rays (80kVp), i.e.,0.01 or 0.05 mGy, after the in vitro exposure of human red blood cells (RBCs) and peripheralblood mononucleated cells (PBMCs).Cells with no irradiation served as the control group. The biologicalendpoints that were used to determine the effects of medical diagnostic low-dose X-rays were hemolysisfor RBCs and mitochondrial membrane potential, lysosomes, and the cell cycle for PBMCs.
 Results: Ourresults showed no changes in the hemolysis of RBCs and mitochondrial membrane potential, lysosome, orcell cycle in cells exposed to these low doses of X-rays when compared to the corresponding nonirradiatedcells at all harvest timepoints.
 Conclusion: These results suggested that there were no deleterious effectsof diagnostic low-dose X-rays when human RBCs and PBMCs were exposed in vitro.
 Bangladesh Journal of Medical Science Vol.20(1) 2021 p.136-144

Establishing cryopreservation protocol for human Peripheral Blood Mononucleated Cells (PBMC) for generation of NK cells for immunotherapy

Background & Aim The human peripheral blood mononucleated cells (PBMC) which largely consist of lymphocytes have been identified as one of the potential sources for cell-based immunotherapy due to its ease of isolation and expansion. These valuable mononucleated cells can be stored under cryopreservation state before the mononucleated cells are thawed for therapeutic purposes or expansion. This study was performed to determine the feasibility to isolate and cryopreserved PBMC with no significant effect on NK cells phenotype and proliferation ability. Methods, Results & Conclusion Method PBMC were isolated from whole blood samples collected from consenting six healthy donors using density gradient centrifugation. Cells viability and total nucleated count after isolation was determined before dividing the PBMC population into two groups. The mononucleated cells in one group were were analyzed as freshly isolated PBMC (Group A) while the mononucleated cells in the other group were cryopreserved for 1 month (Group B) under liquid nitrogen before analysis. Both groups were analysed for NK cells phenotype and ability to proliferate. Results The mean post isolated PBMC was 0.98 × 106 cells/ml whole blood. The cell viability was high at 99.2±0.75% for Group A and 92.5±2.5% for Group B. We compared the observed expression of NK cells phenotype (CD56+CD3−) for both groups and found no significant difference in expression prior to culture. NK cells in both groups also exhibit the ability to proliferate under in-vitro culture for 21 days. Upon harvest, the expanded cells consisting mainly of NK cells with a mean of 75.1±16.6% (Group A) and 69.6±30.6% (Group B) and were on average (Group A: 61.8%, Group B: 54.6%) higher than in NK cells proportion prior to culture. The mean fold expansion on NK cells were 96.1±68.2 fold (Group A) and 122.2±30.9 fold (Group B). Conclusion We conclude that it is feasible to cryopreserve PBMC for 1 month for generation of NK cells without any significant effect on its phenotype and proliferation ability. However, it is recommended to repeat this study with PBMC which have been cryopreserved for a longer period. From this study, we can conclude that it is feasible to cryopreserve PBMC for generation of NK cells for immunotherapy.

P2X7 Receptor Stimulation Is Not Required for Oxalate Crystal-Induced Kidney Injury

Oxalate crystal-induced renal inflammation is associated with progressive kidney failure due to activation of the NLRP3/CASP-1 inflammasome. It has been suggested previously that purinergic P2X7 receptor signaling is critical for crystal-induced inflammasome activation and renal injury. Therefore, we investigated the role of the P2X7 receptor in response to crystal-induced cytokine release, inflammation, and kidney failure using in vitro and in vivo models. Dendritic cells and macrophages derived from murine bone marrow and human peripheral blood mononucleated cells stimulated with calcium-oxalate crystals, monosodium urate crystals, or ATP lead to the robust release of interleukin-1beta (IL-1ß). Treatment with the P2X7 inhibitor A740003 or the depletion of ATP by apyrase selectively abrogated ATP-induced, but not oxalate and urate crystal-induced IL-1ß release. In line with this finding, dendritic cells derived from bone marrow (BMDCs) from P2X7−/− mice released reduced amounts of IL-1ß following stimulation with ATP, while oxalate and urate crystal-induced IL-1ß release was unaffected. In sharp contrast, BMDCs from Casp1−/− mice exhibited reduced IL-1ß release following either of the three stimulants. In addition, P2X7−/− mice demonstrated similar degrees of crystal deposition, tubular damage and inflammation when compared with WT mice. In line with these findings, increases in plasma creatinine were no different between WT and P2X7−/− mice. In contrast to previous reports, our results indicate that P2X7 receptor is not required for crystal-induced CKD and it is unlikely to be a suitable therapeutic target for crystal-induced progressive kidney disease.

Albumin binding and anticancer effect of magnesium oxide nanoparticles.

BackgroundRecently, nanomaterials have moved into biological and medicinal implementations like cancer therapy. Therefore, before clinical trials, their binding to plasma proteins like human serum albumin (HSA) and their cytotoxic effects against normal and cancer cell lines should be addressed.MethodsHerein, the interaction of magnesium oxide nanoparticles (MgO NPs) with HSA was studied by means of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and docking studies. Afterwards, the cytotoxic impacts of MgO NPs on human leukemia cell line (K562) and peripheral blood mononucleated cells (PBMCs) were evaluated by MTT and flow cytometry assays to quantify reactive oxygen species (ROS) generation and apoptosis.ResultsIt was demonstrated that MgO NPs spontaneously form a static complex with HSA molecules through hydrophobic interactions. Docking study based on the size of NPs demonstrated that different linkages can be established between MgO NPs and HSA. The CD investigation explored that MgO NPs did not alter the secondary structure of HSA. Cellular studies revealed that MgO NPs induced cytotoxicity against K562 cell lines, whereas no adverse effects were detected on PBMCs up to optimum applied concentration of MgO NPs. It was exhibited that ROS production mediated by IC50 concentrations of MgO NPs caused apoptosis-associated cell death. The pre-incubation of K562 with ROS scavenger (curcumin) inhibited the impact of MgO NPs -based apoptosis on cell fate, revealing the upstream effect of ROS in our system.ConclusionIn summary, MgO NPs may exhibit strong plasma distribution and mediate apoptosis by ROS induction in the cancer cell lines. These data demonstrate a safe aspect of MgO NPs on the proteins and normal cells and their application as a distinctive therapeutic approach in the cancer treatment.

Cytogenetic effects of Jacareubin from Calophyllum brasiliense on human peripheral blood mononucleated cells in vitro and on mouse polychromatic erythrocytes in vivo

Jacareubin is a xanthone isolated from the heartwood of Calophyllum brasiliense with antibacterial and gastroprotective properties and the intention for clinical use as an anti-cancer treatment (due to the similar chemical structure to other anti-neoplastic drugs) requires an investigation of whether this compound can generate adverse effects on non-transformed cells. Jacareubin (0.5–1000μM in DMSO) was more cytotoxic on phytohemagglutinin (PHA)-stimulated normal human peripheral blood mononuclear cells (PBMCs; IC50 at 72h by MTT: 85.9μM) than on G0 phase-PBMCs (IC50 315.6μM) using trypan blue exclusion and formazan metabolism assays. Jacareubin had lower toxicity on PBMCs than Taxol (1μM). Jacareubin presented cytostatic activity because it inhibited PHA-stimulated PBMCs proliferation (from 2.5μM; CFSE dilution and replication index). Jacareubin induced PBMCs arrest in G0/G1 phase of the cell cycle (from 5μM) as evaluated by DNA content. Moreover, Jacareubin generated genotoxicity by breaking DNA strands selectively in PHA-stimulated PBMCs (from 5μM) rather than on resting PBMCs using the single-cell gel electrophoresis assay and increasing the frequency of micronucleated (MN) PBMCs in vitro (from 5μM) and frequency of hypodiploid cells (from 10μM). When 100mg/kg Jacareubin was injected i.p. into mice (a fifth of the LD50; 0.548g/kg. Approximately to 300μM in vitro), we observe no increase in the MN level in bone marrow cells. Jacareubin can be consider for further anti-tumoural activity due to its preferential genotoxic, cytotoxic and cytostatic actions on proliferating cells rather than on resting cells and the lack of in vivo genotoxicity.

Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells.

Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non–transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours.

Mesenchymal STEM CELLS Favor Tumor Growth By Generating Granulocyte-like Myeloid Derived Suppressor CELLS in CML Patients

INTRODUCTION. The complex interplay between cancer cells and immune system allows neoplastic cells to evade immune surveillance and expand. Recently, our and another group have demonstrated that a subpopulation of myeloid cells, defined as "granulocytic myeloid-derived suppressor cells" (G-MDSC), plays an important role for immune escape in chronic myeloid leukemia (CML) patients by reducing T cell activation. The aim of this study was to evaluate the influence of Mesenchymal stem cells (MSC) on generation of MDSCs by comparing CML MSCs (n=10) with healthy donors (HD) MSC (n=8).METHODS. G-MDSC (CD11b+CD33+CD14-HLADR- cells) were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis by cytofluorimetric analysis. Immuno-suppressive activity was tested through incubation of G-MDSC with autologous CFSE-labeled T cells and stimulation with phytohaemagglutinin (PHA). Controls included a positive T cell proliferation control (T cells plus PHA) and a negative one (T cells only). After three days, T cell proliferation was analyzed by flow cytometry. For G-MDSC generation, human peripheral blood mononucleated cells (PBMC) from HD were cultured alone and with MSC of CML (n=10) or HD (n=8) (1:100 ratio). After one week, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype was confirmed by cytofluorimetric analysis. Expression of ARG1, NOS2, PTGS2, TNFα, TGFβ, IL6, IL10, IL1β was also evaluated using real time PCR.RESULTS. Percentage of cells with a G-MDSC phenotype was greater in PB obtained from CML patients than HD (82.5±9.6% vs 56,2±5.4%, p<0.0001). G-MDSC were able to inhibit T cell proliferation compared to positive control (25±5% vs 48±7.6%, p=0.0057). To investigate if CML MSC may be involved in G-MDSC generation, we incubated HD PBMC with CML or HD MSC for one week. After magnetic isolation, we found that only CML MSC-educated G-MDSC acquired immune-suppressive ability, inhibiting T cell proliferation compared to G-MDSCs control (isolated from PBMC cultured in medium alone) (32±12% vs 63±5.9%, p=0.003). On the contrary, HD MSC-educated G-MDSC did not show any suppressive effect. We also found that CML MSC-educated G-MDSC expressed higher level of the following immune modulatory factors: TNFα (20.8±19.3, p=0.006), IL1β (47.3±25.2, p=0.001), PTGS2 (20.7±10.9, p=0.002) and IL6 (33.8±13.9, p=0.004) compared to HD MSC-educated G-MDSCs (arbitrarily 2-ΔΔCt value: 1). MSC WE also observed ane an up-regulation of PTGS2 (19±4.4, p=0.04), TGFβ (6±3, p=0.01) and IL6 (5±2.8, p=0.04) in CML MSCs at time 0 with a great variability among the patients (calculated value of 2-ΔΔCt in HD MSC was 1). After 48 h of co-culture with PBMC, CML MSC showed statistically significant up-regulation of ARG1 (23.5±11.9, p=0.02), TGFβ (4.8±3, p=0.04), IL10 (5.6±2.8, p=0.03) and IL6 (54.3±23, p=0.02) expression, suggesting that multiple mechanisms are involved in MDSC induction by CML MSC.CONCLUSION. Our work demonstrates that CML MSCs are able to activate MDSCs favoring cancer immune evasion in CML patients. DisclosuresPalumbo:Novartis: Honoraria, Other: Advisory Board.

Abstract LB-B12: Using MiXeno Mouse Models as Robust Tool to Evaluate T Cell Dependent Immunotherapies

Abstract Backgrounds: Fighting cancer by taking advantage of a patient's own immune system is showing considerable success in oncology research. Using therapeutic antibodies against tumor-associated antigens or modulating the host immune system, has been shown to prolong survival of patients with certain solid tumors or leukemia, particularly when combined with conventional treatments. Recent fast-track designations and regulatory approvals (including the first FDA approvals of anti-PD-1 agents in advanced melanoma) have signified the coming of age of cancer immunotherapy as a treatment paradigm, creating both potential patient benefits and commercial success for the pharmaceutical industry. A lack of experimental immunotherapy models is a major obstacle toward answering these questions and developing better treatments, and there is a high unmet need for new preclinical models to help drive forward immune therapy research and to recapitulate clinical treatments. Results: Crown Bioscience has created transient human immunity in mice (MiXeno) for testing human immunotherapeutics, particularly T cell engaged immunotherapies by mixing human peripheral blood mononucleated cells (PBMC) with xenograft models. Our data showed that anti-human PD-1 or PD-L1 antibodies demonstrated significant anti-tumor activities in several Mixeno models, including A375 melanoma model and KARPAS-299 lymphoma model. The emergence of the efficacy coincided with the T cell reconstitution in mouse blood. T cells are also recruited to the tumor microenvironment. The number of T cells in the tumor microenvironment varies significant and is largely dependent on tumor cells itself. Conclusion: The MiXeno model provides an alternative to the full stem cell reconstitution approach, and may allow T cell engaged immunotherapies (monotherapies or combinational therapies) to be evaluated in xenograft models. Citation Format: Juan Zhang, Meng Qiao, Qian Shi. Using MiXeno Mouse Models as Robust Tool to Evaluate T Cell Dependent Immunotherapies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B12.

Up-regulation of Bcl-2 expression in cultured human lymphocytes after exposure to low doses of gamma radiation.

Lymphocytes have demonstrated complex molecular responses to induced stress by ionizing radiation. Many of these reactions are mediated through modifications in gene expressions, including the genes involved in apoptosis. The primary aim of this study was to assess the effects of low doses of ionizing radiation on the apoptotic genes, expression levels. The secondary goal was to estimate the time-effect on the modified gene expression caused by low doses of ionizing radiation. Mononuclear cells in culture were exposed to various dose values ranged from 20 to 100 mGy by gamma rays from a Cobalt-60 source. Samples were taken for gene expression analysis at hours 4, 24, 48, 72, and 168 following to exposure. Expression level of two apoptotic genes; BAX (pro-apoptotic) and Bcl-2 (anti-apoptotic) were examined by relative quantitative real-time polymerase chain reaction (PCR), at different time intervals. Radio-sensitivity of peripheral blood mononucleated cells (PBMCs) was measured by the Bcl-2/BAX ratio (as a predictive marker for radio-sensitivity). The non-parametric two independent samples Mann–Whitney U-test were performed to compare means of gene expression. The results of this study revealed that low doses of gamma radiation can induce early down-regulation of the BAX gene of freshly isolated human PBMCs; however, these changes were restored to near normal levels after 168 hours. In most cases, expression of the Bcl-2 anti-apoptotic gene was up-regulated. Four hours following to exposure to low doses of gamma radiation, apoptotic gene expression is modified, this is manifested as adaptive response. Modification of these gene expressions seems to be a principle pathway in the early radioresistance response. In our study, we found that these changes were temporary and faded completely within a week.

Genotoxic and cytostatic effects of 6-pentadecyl salicylic anacardic acid in transformed cell lines and peripheral blood mononuclear cells

In Mexico, as in many other countries, traditional medicine is used for the treatment of several diseases. In particular, Amphipterygium adstringens infusion is used for gastritis, gastric ulcers, and gastric cancer. Extracts from this tree have microbicidal effects against Helicobacter pylori, an important risk factor for gastric cancer development. Anacardic acids are constituents of A. adstringens, and 6-pentadecyl salicylic acid (6-PSA) is the most abundant. However, there is a lack of information regarding the effects of 6-PSA on cancer cells. Therefore, we investigated whether 6-PSA has differential effects on the induction of genotoxicity, cytostaticity, and apoptosis in normal human peripheral blood mononucleated cells (PBMCs), bone marrow polychromatic erythrocytes of Balb/c mice, and human transformed cell lines derived from both gastric cancer (AGS cells) and leukaemia (K562 cells).Treatment with 6-PSA (30–150μM) reduced the viability of AGS and K562 cells together with a moderate, but significant, increase in the frequency of micronucleated cells and the induction of DNA breakage (Comet Assay). Moreover, 6-PSA increased the apoptosis rate in both the AGS and K562 cell lines in a caspase 8-dependent manner. In contrast, neither cytotoxicity nor genotoxicity were observed in PBMCs or bone marrow polychromatic erythrocytes of Balb/c mice after treatment with low doses of 6-PSA (0.2–2.0mg/Kg). Instead, 6-PSA treatment resulted in the inhibition of PBMC proliferation, which was reversible after the compound was removed. Additionally, 6-PSA treatments (2–20mg/Kg) increased the frequency of mature polychromatic erythrocytes in the bone marrow, suggesting a possible effect on the differentiation process of immune cells.The present results indicate that 6-PSA induces cytotoxicity and moderate genotoxicity, together with an increase in the apoptosis rate, in a caspase 8-dependent manner in gastric cancer cells. In contrast, a low toxicity was observed when PBMCs were exposed to 6-PSA.

Synthesis and evaluation of the cytotoxic activity of 1,2-furanonaphthoquinones tethered to 1,2,3-1H-triazoles in myeloid and lymphoid leukemia cell lines

Leukemia is the most common blood cancer, and its development starts at diverse points, leading to distinct subtypes that respond differently to therapy. This heterogeneity is rarely taken into account in therapies, so it is still essential to look for new specific drugs for leukemia subtypes or even for therapy-resistant cases. Naphthoquinones (NQ) are considered privileged structures in medicinal chemistry due to their plethora of biological activities, including antimicrobial and anticancer effects. Nitrogen-containing heterocycles such as 1,2,3-1H-triazoles have been identified as general scaffolds for generating glycosidase inhibitors. In the present study, the NQ and 1,2,3-1H-triazole cores have been combined to chemically synthesize 18 new 1,2-furanonaphthoquinones tethered to 1,2,3-1H-triazoles (1,2-FNQT). Their cytotoxicities were evaluated against four different leukemia cell lines, including MOLT-4 and CEM (lymphoid cell lines) and K562 and KG1 (myeloid cell lines), as well as normal human peripheral blood mononucleated cells (PBMCs). The new 1,2-FNQT series showed high cytotoxic potential against all leukemia cell lines tested, and some compounds (12o and 12p) showed even better results than the classical therapeutic compounds such as doxorubicin or cisplatin. Others compounds, such as 12b, are promising because of their high selectivity against lymphoblastic leukemia and their low activity against normal hematopoietic cells. The cells of lymphoid origin (MOLT and CEM) were generally more sensitive than the myeloid cell lines to this series of compounds, and most of the compounds that showed the highest cytotoxicity were similarly active against both cell lines.

Genome-wide age-related changes in DNA methylation and gene expression in human PBMCs.

Aging is a progressive process that results in the accumulation of intra- and extracellular alterations that in turn contribute to a reduction in health. Age-related changes in DNA methylation have been reported before and may be responsible for aging-induced changes in gene expression, although a causal relationship has yet to be shown. Using genome-wide assays, we analyzed age-induced changes in DNA methylation and their effect on gene expression with and without transient induction with the synthetic transcription modulating agent WY14,643. To demonstrate feasibility of the approach, we isolated peripheral blood mononucleated cells (PBMCs) from five young and five old healthy male volunteers and cultured them with or without WY14,643. Infinium 450K BeadChip and Affymetrix Human Gene 1.1 ST expression array analysis revealed significant differential methylation of at least 5 % (ΔYO > 5 %) at 10,625 CpG sites between young and old subjects, but only a subset of the associated genes were also differentially expressed. Age-related differential methylation of previously reported epigenetic biomarkers of aging including ELOVL2, FHL2, PENK, and KLF14 was confirmed in our study, but these genes did not display an age-related change in gene expression in PBMCs. Bioinformatic analysis revealed that differentially methylated genes that lack an age-related expression change predominantly represent genes involved in carcinogenesis and developmental processes, and expression of most of these genes were silenced in PBMCs. No changes in DNA methylation were found in genes displaying transiently induced changes in gene expression. In conclusion, aging-induced differential methylation often targets developmental genes and occurs mostly without change in gene expression.Electronic supplementary materialThe online version of this article (doi:10.1007/s11357-014-9648-x) contains supplementary material, which is available to authorized users.

Hard-metal (WC–Co) particles trigger a signaling cascade involving p38 MAPK, HIF-1α, HMOX1, and p53 activation in human PBMC

Hard-metals are made of tungsten carbide (WC) and metallic cobalt (Co) particles and are important industrial materials produced for their extreme hardness and high wear resistance properties. While occupational exposure to metallic Co alone is apparently not associated with an increased risk of cancer, the WC-Co particle mixture was shown to increase the risk of lung cancer in exposed workers. We have previously shown that WC-Co specifically induces a burst of reactive oxygen species (ROS) and in vitro mutagenic/apoptogenic effects in human peripheral blood mononucleated cells (PBMC) used as a validated experimental model. In the present study, PBMCs were treated during a short period (15 min) to focus on the very rapid ROS burst induced by WC-Co. We investigated by microarray the response to WC-Co versus Co(2+) ions (CoCl(2)) after 15 min exposure and found that the oxidative stress response HMOX1 gene was highly expressed in WC-Co-treated samples. This result was confirmed by qRT-PCR, and western blotting was carried out to analyze translational and post-translational regulation of genes belonging to the HMOX1 pathway. We show here that WC-Co, and metallic Co particles although with slower kinetics, but not CoCl(2) or WC alone, induced a temporally ordered cascade of events. This cascade implies p38/MAP kinase activation, HIF-1α stabilization, HMOX1 transcriptional activation, and ATM-independent p53 stabilization. These events, and in particular HIF-1α stabilization, could contribute to the carcinogenic activity of WC-Co dusts.

Molecular characterisation of human peripheral blood stem cells

Peripheral blood mononucleated cells consist of haematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). To date there is no well-defined isolation or characterisation protocol of stem cells from human adult peripheral blood mononucleated cells. Our aim in this study was to isolate and characterise mononucleated cells from human peripheral blood. Peripheral blood mononucleated cells were isolated using the Ficoll-Paque density gradient separation method and cultured in complete medium. After 4 days of culture, adherent and suspension mononucleated cells were separated and cultured for 14 days in an in-vitro culture selection. Stem cells in the isolated mononucleated cells were characterised using a multidisciplinary approach which was based on the expression of stem cell markers, morphology and the capacity to self-renew or proliferate and differentiate into specialised cells. Reverse transcription polymerase chain reaction was used to identify the expression of an HSC marker (signalling lymphocytic activation molecule family member 1, SLAMF1) and a MSC marker (CD105). Results revealed that adherent mononucleated cells were positive for MSC markers, whereas mononucleated cells in suspension were positive for HSC markers. The isolated adherent and suspension mononucleated cells were able to maintain their stem cell properties during in-vitro culture by retaining their capacity to proliferate and differentiate into osteoclast and osteoblast cells, respectively, when exposed to the appropriate induction medium. The isolated mononucleated cells consisted of suspension HSCs and adherent MSCs, both of which have the capability to proliferate and differentiate into mature cells. We have shown that suspension HSCs and adherent MSCs can be obtained from an in-vitro culture of peripheral blood mononucleated cells.

The significance of the host inflammatory response on the therapeutic efficacy of cell therapies utilising human adult stem cells

Controlling the fate of implanted hMSCs is one of the major drawbacks to be overcome to realize tissue engineering strategies. In particular, the effect of the inflammatory environment on hMSCs behaviour is poorly understood. Studying and mimicking the inflammatory process in vitro is a very complex and challenging task that involves multiple variables. This research addressed the questions using in vitro co-cultures of primary derived hMSCs together with human peripheral blood mononucleated cells (PBMCs); the latter are key agents in the inflammatory process. This work explored the in vitro phenotypic changes of hMSCs in co-culture direct contact with monocytes and lymphocytes isolated from blood using both basal and osteogenic medium. Our findings indicated that hMSCs maintained their undifferentiated phenotype and pluripotency despite the contact with PBMCs. Moreover, hMSCs demonstrated increased proliferation and were able to differentiate specifically down the osteogenic lineage pathway. Providing significant crucial evidence to support the hypothesis that inflammation and host defence mechanisms could be utilised rather than avoided and combated to provide for the successful therapeutic application of stem cell therapies.

English

Stem cells are known to have the ability to renew themselves and differentiate into a diverse range of specialized cell types. Currently, the differentiation potential of human peripheral blood mononucleated cells in suspension as stem cells is not well-understood. The aim of this study was to investigate the differentiation potential of suspension mononucleated cells from human peripheral blood to differentiate. The osteoblast and osteoclast differentiation potential of suspension peripheral blood mononucleated cells were examined by molecular, biochemical and cell morphology analyses. The expression of osteoblast marker (osteonectin, SPARC) and osteoclast marker (tartrate resistant acid phosphatase, TRAP) as well as high alkaline phosphate (ALP) and TRAP enzyme activity were observed at days 14 and 10 of osteoblast and osteoclast differentiation, respectively. Morphology analyses showed that mononucleated cells successfully differentiated into osteoblasts and osteoclasts. The existence of stem cells in mononucleated cells was evaluated by the expression of a stem cell factor (KIT) and a haematopoietic stem cell marker (signalling lymphocytic activation molecule family member 1, SLAMF1). This study has shown that these suspension mononucleated cells possess differentiation potential through in vitro study. Human peripheral blood suspension mononucleated cells that have multi-lineage differentiation potential may provide a new source of stem cells that may be used for bone regeneration and tissue engineering applications.

The Critical Role of IL-34 in Osteoclastogenesis

It has been widely believed that the cytokines required for osteoclast formation are M-CSF (also known as CSF-1) and RANKL. Recently, a novel cytokine, designated IL-34, has been identified as another ligand of CSF1R. This study was to explore the biological function, specifically osteoclastogenesis and bone metabolism, of the new cytokine. We produced recombinant mouse IL-34 and found that together with RANKL it induces the formation of osteoclasts both from splenocytes as well as dose-dependently from bone marrow cells in mouse and these cells also revealed bone resorption activity. It also promotes osteoclast differentiation from human peripheral blood mononucleated cells. Finally, we show that systemic administration of IL-34 to mice increases the proportion of CD11b+ cells and reduces trabecular bone mass. Our data indicate that IL-34 is another important player in osteoclastogenesis and thus may have a role in bone diseases. Strategies of targeting CSF1/CSF1R have been developed and some of them are already in preclinical and clinical studies for treatment of inflammatory diseases. Our results strongly suggest the need to revisit these strategies as they may provide a new potential pharmaceutical target for the regulation of bone metabolism in addition to their role in the treatment of inflammatory diseases.

Probing of Actinobacillus pleuropneumoniae ApxIIIA toxin-dependent cytotoxicity towards mammalian peripheral blood mononucleated cells

BackgroundActinobacillus pleuropneumoniae, the causative bacterial agent of porcine pleuropneumonia, produces Apx toxins which belong to RTX toxin family and are recognized as the major virulence factors. So far, their target receptor(s) has not been identified and the disease cytopathogenesis remains poorly understood. Production of an active Apx toxin and characterization of its toxic activity constitute the premises necessary to the description of its interaction with a potential receptor. From this point of view, we produced an active recombinant ApxIIIA toxin in order to characterize its toxicity on peripheral blood mononucleated cells (PBMCs) isolated from several species.FindingsToxin preparation exercises a strong cytotoxic action on porcine PBMCs which is directly related to recombinant ApxIIIA since preincubation with polymyxin B does not modify the cytotoxicity rate while preincubation with a monospecific polyclonal antiserum directed against ApxIIIA does. The cell death process triggered by ApxIIIA is extremely fast, the maximum rate of toxicity being already reached after 20 minutes of incubation. Moreover, ApxIIIA cytotoxicity is species-specific because llama, human, dog, rat and mouse PBMCs are resistant. Interestingly, bovine and caprine PBMCs are slightly sensitive to ApxIIIA toxin too. Finally, ApxIIIA cytotoxicity is cell type-specific as porcine epithelial cells are resistant.ConclusionWe have produced an active recombinant ApxIIIA toxin and characterized its specific cytotoxicity on porcine PBMCs which will allow us to get new insights on porcine pleuropneumonia pathogenesis in the future.

In vitro expression of hard metal dust (WC–Co) — responsive genes in human peripheral blood mononucleated cells

Hard metals consist of tungsten carbide (WC) and metallic cobalt (Co) particles and are important industrial materials produced for their extreme hardness and high wear resistance properties. While occupational exposure to metallic Co alone is apparently not associated with an increased risk of cancer, the WC–Co particle mixture was shown to be carcinogenic in exposed workers. The in vitro mutagenic/apoptogenic potential of WC–Co in human peripheral blood mononucleated cells was previously demonstrated by us. This study aimed at obtaining a broader view of the pathways responsible for WC–Co induced carcinogenicity, and in particular genotoxicity and apoptosis. We analyzed the profile of gene expression induced in vitro by WC–Co versus control (24 h treatment) in human PBMC and monocytes using microarrays. The most significantly up-regulated pathways for WC–Co treated PBMC were apoptosis and stress/defense response; the most down-regulated was immune response. For WC–Co treated monocytes the most significantly up- and down-regulated pathways were nucleosome/chromatin assembly and immune response respectively. Quantitative RT-PCR data for a selection of the most strongly modulated genes ( HMOX1, HSPA1A, HSPA1L, BNIP3, BNIP3L, ADORA2B, MT3, PLA2G7, TNFAIP6), and some additionally chosen apoptosis related genes ( BCL2, BAX, FAS, FASL, TNFα), confirmed the microarray data after WC–Co exposure and demonstrated limited differences between the Co-containing compounds. Overall, this study provides the first analysis of gene expression induced by the WC–Co mixture showing a large profile of gene modulation and giving a preliminary indication for a hypoxia mimicking environment induced by WC–Co exposure.