Cultures Of Human Peripheral Blood Research Articles

Comparison of the cytotoxic effects of bulk and nanosized CeO2 on lymphocyte cells

Cerium(IV) oxide (CeO2) is widely used in industrial fields such as biomedical, glass, electronics, automotive, and pharmacology. In this study, the cytotoxic effects on human peripheral blood cultures of two forms of cerium(IV) oxide with different particle sizes (Bulk-sized Cerium(IV) oxide: BC and Nano-sized Cerium(IV) oxide: NC) in concentrations range of 0.001-200 ppm were investigated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were used to determine the cytotoxicity of these forms. According to the test results, it was determined that both forms caused severe cytotoxicity at all concentrations studied. It was observed that cytotoxicity increased with increasing concentration. NCs are more toxic at all concentrations except 100 and 200 ppm concentrations.

Genoprotective role of purslane methanol extract against somatic mutations induced by bifenthrin, a third generation prethyroid insecticide

In this study, in vitro and in vivo genotoxic effects of Bifenthrin (BIF), an important insecticide used in agricultural production, storage and processing, were investigated. The genoprotective properties of purslane (Portulaca oleracea L.) against the genotoxic effects of BIF were also determined by using the methanol (POmet) extract of this plant. In vivo experiments were performed with somatic mutation and recombination test (SMART) in Drosophila melanogaster. In in vitro studies, human peripheral blood cultures were prepared and different concentrations of BIF were applied to lymphocyte cells in accordance with the procedure of both the micronucleus (MN) and sister chromatid exchange (SCE) assay. The results obtained from all applied tests showed that BIF is genotoxic and induces chromosomal mutations. Later, another experiment was conducted and it was determined that the genotoxic effects of BIF were reduced with POmet (1:1 v/v). This result, which was observed in all in vivo and in vitro tests, shows that purslane plant is a potent radical scavenger. Due to the healing properties of POmet, gas chromatography-mass spectrometry (GS-MS) method was used to determine the components in its content. Some of the components found in the highest ratio in this extract are γ-sitosterol (21.86%), 13-docosenamide, (13.30%), palmitic acid (12.85%), stigmasteol (6.64%), campesterol (5.69%), linoleicacid (5.46%) and 2-methyl-1-hexadecanol (3.88%).

Investigation of the Genotoxicity of Aluminum Oxide, β-Tricalcium Phosphate, and Zinc Oxide Nanoparticles In Vitro.

The aim of this study was to investigate the genotoxicity of aluminum oxide (Al2O3), β-tricalcium phosphate (β-TCP) (Ca3(PO4)2), and zinc oxide (ZnO) nanoparticles (NPs) that were 4.175, 9.058, and 19.8 nm sized, respectively, on human peripheral blood lymphocytes using micronucleus (MN) and chromosome aberration (CA) techniques. Aluminum oxide and β-TCP NPs did not show genotoxic effects on human peripheral blood cultures in vitro, even at the highest concentrations; therefore, these materials may be suitable for use as biocompatible materials. It was observed that, even at a very low dose (≥12.5 ppm), ZnO NPs had led to genotoxicity. In addition, at high concentrations (500 ppm and above), ZnO NPs caused mortality of lymphocytes. For these reasons, it was concluded that ZnO NPs are not appropriate for using as a biocompatible biomaterial.

The impact of concentration and administration time on the radiomodulating properties of undecylprodigiosin in vitro.

Undecylprodigiosin pigment (UPP) is reported to display cytotoxic activity towards various types of tumours. Nevertheless, its efficacy in modifying the cellular response to ionising radiation is still unknown. In this study, the radiomodulating effects of UPP were investigated. The effects of UPP were assessed in vitro by treating cultures of human peripheral blood with UPP and ionising radiation using two treatment regimens, the UPP pre-irradiation treatment and UPP post-irradiation treatment. The activity of UPP was investigated evaluating its effects on the radiation-induced micronuclei formation, cell proliferation, and induction of apoptosis. The redox modulating effects of UPP were examined measuring the catalase activity and the level of malondialdehyde, as a measure of oxidative stress. The results showed that UPP effects on cellular response to ionising radiation depend on its concentration and the timing of its administration. At low concentration, the UPP displayed radioprotective effects in γ-irradiated human lymphocytes while at higher concentrations, it acted as a radiosensitiser enhancing either mitotic catastrophe or apoptosis depending on the treatment regimen. The UPP modified redox processes in cells, particularly when it was employed prior to γ-irradiation. Our data highlight the importance of further research of the potential of UPP to sensitize tumour cells to radiation therapy by inhibiting pathways that lead to treatment resistance.

The effect of core and lanthanide ion dopants in sodium fluoride-based nanocrystals on phagocytic activity of human blood leukocytes

Sodium fluoride-based β-NaLnF4 nanoparticles (NPs) doped with lanthanide ions are promising materials for application as luminescent markers in bio-imaging. In this work, the effect of NPs doped with yttrium (Y), gadolinium (Gd), europium (Eu), thulium (Tm), ytterbium (Yb) and terbium (Tb) ions on phagocytic activity of monocytes and granulocytes and the respiratory burst was examined. The surface functionalization of <10-nm NPs was performed according to our variation of patent pending ligand exchange method that resulted in meso-2,3-dimercaptosuccinic acid (DMSA) molecules on their surface. Y-core-based NCs were doped with Eu ions, which enabled them to be excited with UV light wavelengths. Cultures of human peripheral blood (n = 8) were in vitro treated with five different concentrations of eight NPs for 24 h. In summary, neither type of nanoparticles is found toxic with respect to conducted test; however, some cause toxic effects (they have statistically significant deviations compared to reference) in some selected doses tested. Both core types of NPs (Y-core and Gd-core) impaired the phagocytic activity of monocytes the strongest, having minimal or none whatsoever influence on granulocytes and respiratory burst of phagocytic cells. The lowest toxicity was observed in Gd-core, Yb, Tm dopants and near-infrared nanoparticles. Clear dose-dependent effect of NPs on phagocytic activity of leukocytes and respiratory burst of cells was observed for limited number of samples.

Titanium dioxide nanoparticles: an in vitro study of DNA binding, chromosome aberration assay, and comet assay.

Engineered titanium dioxide nanoparticles (TiO2 NPs) are extensively used in cosmetic, pharmaceutical and other industries globally due to their unique properties, which has raised concern for biosafety. Genotoxicity assessment is an important part of biosafety evaluation; we report in vitro cytogenetic assays for NPs considering their unique physicochemical characteristics to fill the gap of laboratory data regarding biological safety along with mechanistic study for mode of interaction of NP with genetic material. Comet and chromosome aberration assay (CA assay) using short-term human peripheral blood cultures following exposure to TiO2 NPs; along with physicochemical parameters for stability of nano form in cultures; and DNA binding activity were carried out. The dynamic light scattering and zeta potential measurements revealed mono dispersion in media. The fluorescence spectroscopy for binding affinity of TiO2 NPs and human genomic DNA showed binding constant (Kb), 4.158×106M-1 indicating strong binding affinity and negative ΔG0 value suggesting spontaneous DNA binding supporting its genotoxic potential. Following in vitro exposure to TiO2 NPs for 24h, the cultures were analyzed for comet and CA assays, which showed significant results (p<0.05) for % DNA intensity in tail, Olive Tail Moment and frequency of Chromosomal aberrations (CA) at 75 and 125μM but not at 25μM.

Highly angiogenic CXCR4+CD31+ monocyte subset derived from 3D culture of human peripheral blood

Ex vivo expansion of human circulating angiogenic cells is a major challenge in autologous cell therapy for ischemic diseases. Here, we demonstrate that hematosphere-derived CXCR4+CD31+ myeloid cells using peripheral blood possess robust proangiogenic capacity such as formation of vessel-like structures and tip cell-like morphology in Matrigel. We also found that CD31 positive myeloid cells are principal cellular component of hematospheres by magnetic cell sorting. Flow cytometry analysis showed that fresh peripheral blood contained 40.3 ± 15.2% of CXCR4+CD31+ myeloid cells, but at day 5 of hematosphere culture, most of myeloid cells were CXCR4+CD31+ by 86.9 ± 5.4%. Hematosphere culture significantly increased the production of angiogenic niche-supporting cytokines. Moreover, CD31-homophilic interaction and VEGF–VEGF receptor loop signaling were essential for sphere formation and acquisition of angiogenic capacity in hematospheres. Matrigel plug and ischemic hindlimb model provide in vivo evidence that hematosphere-derived myeloid cells have highly vasculogenic capacities, participate in new and mature vessel formation, and exert therapeutic effects on ischemic hindlimb. In conclusion, our strategy for ex vivo expansion of human CXCR4+CD31+ angiogenic cells using hematospheres provides an autologous therapeutic cell source for ischemic diseases and a new model for investigating the microenvironment of angiogenesis.

Bystander effect in human lymphocytes incubated with irradiated mitochondrial DNA deficient yeast cells

The comparison of strength of bystander effect induction in human lymphocytes by rho 0 (complete loss of mitochondrial DNA) and rho + (with fully functional mtDNA) strains of Saccharomyces cerevisiae was carried out. The cultures of human peripheral blood were experimentally contaminated with nonirradiated or X-ray irradiated yeast cells (haploid rho 0 and rho + strains of S. cerevisiae ). The doses of irradiation were 1 and 10 Gy. Well spread human metaphases were scored for chromosomal aberrations (CA). It was found that nonirradiated yeast cells (no matter rho 0 or rho + ) had no effect on lymphocytes chromosomal stability. Statistically significant increased level of CA was observed in lymphocytes incubated with irradiated rho + yeast cells. The increasing of the CA was dose independent. However, after cocultivation with irradiated rho 0 cells the bystander effect was revealed for dose 10 Gy only and his manifestation was mild in comparison with rho + data. Thus, our findings suggest that strength of bystander effect depends on existence of mitochondrial DNA and normally functioning mitochondria.

Evaluation of in vitro anti-genotoxic potential of melatonin against arsenic and fluoride in human blood cultures

Melatonin has been known for its anti-oxidant potential but not much studied for the anti-genotoxic potential. The current study elucidate the role of melatonin against in vitro genototxicity by arsenic (As) and fluoirde (F). Human peripheral blood cultures were exposed to As (1.4μM) and F (34μM), alone and in combination, with and without melatonin (0.2mM). Ethyle methane sulphonate (EMS; 1.9mM) was selected as the positive control to analyze the genotoxic parameters like sister chrmatid exchanges (SCEs), cell cycle proliferative index (CCPI) and primary DNA damage. The frequency of SCE/cell, SCE/chromosome and primary DNA damage reduced significantly (p<0.001) accompanied with a marked increase in CCPI upon addtion of melatonin. Similarly marked recovery was attained by melatonin from As and F induced primary DNA damage as studied using comet assay. The results clearly indicate towards the protection of lymphocytes from toxic effects af As and F by melatonin in vitro.

PHELLODENDRON AMURENSE BARK EXTRACT ENHANCES RADIOSENSITIVITY BY INHIBITION OF NF-KAPPA B IN TRANSGENIC ADENOCARCINOMA OF MOUSE PROSTATE MODEL AND HUMAN PROSTATE CANCER CELLS

RESULTS: IL-18Bpa was expressed and secreted by the prostate cancer cell lines DU145 and PC3, but not by LNCaP and CWR22, upon interferon(IFN) stimulation. IFN-induced secretion of IL-18Bpa was enhanced by added TNF, IFNand IFN. The IL-18Bpa secreted from DU145 and PC3 functionally inhibited IL-18. Conditioned medium from IL-18Bpa-overexpressed PC3 cells suppressed CD8+ IFN+ cells and CD4+ cells and appeared to decrease TH1 cells in human peripheral blood cultures. Immunohistochemical analyses showed positive IL18Bpa staining in prostate cancer cells as well as in macrophages in radical prostatectomy specimens. Significant differences in post-DRE urinary IL-18Bpa levels (normalized by total protein) were found between cases with and without cancer on biopsy (P = 0.02) and serum IL-18Bpa levels correlated with Gleason score (P = 0.03). CONCLUSIONS: Our finding of elevated IL-18Bpa secretion from prostate cancer cells suggests an attempt by cancer to escape immune surveillance. IL-18Bpa merits further study as a marker of aggressive prostate cancer and as a therapeutic target.

Effects of interleukin-2 and interleukin-2-activated cells on in vitro myelopoiesis

Lymphokine-activated killer (LAK) cells from human peripheral blood mononuclear cells cultured with recombinant interleukin-2 (IL-2) have been used clinically in adoptive immunotherapy for cancer patients. To study the influence of LAK cells and IL-2 on haematopoiesis, an in vitro assay system for colony formation of granulocyte-macrophage progenitor cells (GM-CFC) was used. LAK cells from cultures of either human peripheral blood (PB) or human bone marrow (BM) mononuclear cells were both inhibitory to allogeneic BM-derived GM-CFC. Inhibitory activity could be transferred with supernatants from co-cultures of LAK cells and BM targets, but also from the IL-2 activated PB- or BM-derived cells alone. The inhibitory activity from the initially non-cytotoxic/non-inhibitory BM population was rapidly induced by IL-2 activation, and preceded the generation of cytotoxic LAK cells in the culture. These experiments show that inhibition of haematopoietic progenitor cells by IL-2 is not dependent on generation of cytotoxic LAK cells, but rather the result of IL-2-induced cytokine production. We conclude that the synergistic action of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) may contribute to inhibition, but that also other cytokines are responsible for the observed inhibition of BM-derived GM-CFC.

Lactoferrin Acts as an Alarmin to Promote the Recruitment and Activation of APCs and Antigen-Specific Immune Responses

Lactoferrin is an 80-kDa iron-binding protein present at high concentrations in milk and in the granules of neutrophils. It possesses multiple activities, including antibacterial, antiviral, antifungal, and even antitumor effects. Most of its antimicrobial effects are due to direct interaction with pathogens, but a few reports show that it has direct interactions with cells of the immune system. In this study, we show the ability of recombinant human lactoferrin (talactoferrin alfa (TLF)) to chemoattract monocytes. What is more, addition of TLF to human peripheral blood or monocyte-derived dendritic cell cultures resulted in cell maturation, as evidenced by up-regulated expression of CD80, CD83, and CD86, production of proinflammatory cytokines, and increased capacity to stimulate the proliferation of allogeneic lymphocytes. When injected into the mouse peritoneal cavity, lactoferrin also caused a marked recruitment of neutrophils and macrophages. Immunization of mice with OVA in the presence of TLF promoted Th1-polarized Ag-specific immune responses. These results suggest that lactoferrin contributes to the activation of both the innate and adaptive immune responses by promoting the recruitment of leukocytes and activation of dendritic cells.

Genotoxic effects of white fluorescent light on human lymphocytes in vitro

Sources of light beams such as white fluorescent light, are present in our daily life to meet the needs of life in the modern world. This study was conducted with the objective of determining the possible genotoxic, cytotoxic and aneugenic effects caused by this agent in different stages of the cell cycle (G0/early G1, S, and late G2), using different cytogenetic parameters (sister chromatid exchanges––SCE, chromosome aberrations––CA, and detection of aneugenic effects) in lymphocytes from temporary cultures of human peripheral blood. WFL showed a genotoxic effect in vitro, expressed by an increase in the frequency of SCE's, regardless of the cell cycle stage. However, no increase in the frequency of CAs was observed. In addition, disturbances in cell cycle kinetics and chromosomal segregation were also observed. Taken together, such data may contribute to a better understanding and a different management in the use of phototherapy for some pathological conditions.

In situ hybridization by scanning electron microscopy for painting, centromeric, and YAC localization

The hybridization site of a DNA probe was detected using a scanning electron microscope (SEM), modifying the standard in situ hybridization (ISH) method. The experiments were performed on human metaphases obtained from lymphocyte cultures of human peripheral blood. The libraries and probes used were: 1-chromosome library for the painting of chromosome 1 (wcp 1), an alphoid centromere-specific probe of chromosome 8 (pZ8.4), and the yeast artificial chromosome (YAC) 964-C10 mapped at band p13 on chromosome 12. These probes were labeled by nick translation with biotin and displayed with a gold-conjugated anti biotin goat antibody. The gold signal was amplified by silver enhancement. The chromatides appeared as packages of thin filaments 120 nm high; some of them collapsed, probably due to ISH procedures. All the probes were clearly detected as small gold particles grouped on the surface of the target chromosomes and chromosome sites. Thus, this procedure is useful to clarify the positional relationship between the chromatin filaments and the probe.

Induction of Chromosomal Aberrations and Sister Chromatid Exchanges by Cyproterone Acetate in Human Lymphocytes

The genotoxicity study of cyproterone acetate used as antiandrogen was carried out on human lymphocyte chromosomes using chromosomal aberrations and sister chromatid exchanges as parameters. The study was carried out in the presence as well as absence of metabolic activation (S9 mix). The effect of cyproterone acetate was studied as 5, 10, 20, 30 µM and was found to be genotoxic at 20 and 30 µM both in the presence as well as absence of metabolic activation system. The results suggest a genotoxic and cytotoxic effect of cyproterone acetate in human peripheral blood cultures in vitro.

Cytogenetic evaluation of two nitroimidazole derivatives

5-Nitroimidazoles are a well-established group of antiprotozoal and antibacterial agents. Thanks to their antimicrobial activity these chemotherapeutic agents inhibit the growth of both anaerobic bacteria and certain anaerobic protozoa such as Trichomonas vaginalis, Entamoeba histolytica and Giardia lamblia. The aim of the present study is to achieve a precise characterization of the genotoxic activity of these compounds and to establish the value of cytogenetic assays in order to determine the effect of these drugs, at therapeutic doses, to settle an improved risk assessment. Two nitroimidazole were studied, metronidazole and ornidazole, at four different concentrations (0.1, 1, 10 and 50 μg/ml of peripheral blood lymphocyte culture). Endpoints analyzed included: mitotic index (MI), replication index (RI), sister chromatid exchange (SCE) and chromosomal aberrations (CA). An analysis of variance test (ANOVA) was performed to evaluate the results. A significant decrease ( P<0.0001) in MI as well as an increase in SCE ( P<0.0001) and CA (0.0001) frequencies for both drugs was observed. No modifications in RI were found. The results suggest a genotoxic and cytotoxic effect of MTZ and ONZ in human peripheral blood cultures in vitro.

Enhanced Cytokine Synthesis of Leukocytes by a β‐Glucan Preparation, SCG, Extracted from a Medicinal Mushroom, Sparassis crispa

Sparassis crispa is edible mushroom recently cultivable in Japan. It contains significantly high content (∼40%) of 6‐branched 1,3‐β‐D‐glucan showing antitumor activity in mice. We recently purified a β‐glucan preparation designated as “SCG.” It was considered worth while to test SCG in vitro with whole blood collected from human volunteers. The present study is focusing on the cytokine productivity of SCG in an in vitro human system. The following results were observed: (i) SCG dose dependently enhanced IL‐8 synthesis of whole blood cell culture of human peripheral blood. (ii) IL‐8 synthesis was enhanced in both PBMC and PMN cultures. (iii) IL‐8 synthesis was induced in the culture with autologous plasma, but significantly reduced after 56°C treatment. (iv) The activity was also weak in heat inactivated fetal calf serum (FCS). (v) A complement fragment, C5a, was released by SCG dependently upon dose and kinetics. (vi) Anti‐SCG natural antibody was detected in human plasma. From these facts, SCG was observed to have the capacity to activate human leukocytes and related immune system.

Cytogenetic damage in human lymphocytes following GMSK phase modulated microwave exposure.

The present study investigated, using in vitro experiments on human lymphocytes, whether exposure to a microwave frequency used for mobile communication, either unmodulated or in presence of phase only modulation, can cause modification of cell proliferation kinetics and/or genotoxic effects, by evaluating the cytokinesis block proliferation index and the micronucleus frequency. In the GSM 1800 mobile communication systems the field is both phase (Gaussian minimum shift keying, GMSK) and amplitude (time domain multiple access, TDMA) modulated. The present study investigated only the effects of phase modulation, and no amplitude modulation was applied. Human peripheral blood cultures were exposed to 1.748 GHz, either continuous wave (CW) or phase only modulated wave (GMSK), for 15 min. The maximum specific absorption rate (approximately 5 W/kg) was higher than that occurring in the head of mobile phone users; however, no changes were found in cell proliferation kinetics after exposure to either CW or GMSK fields. As far as genotoxicity is concerned, the micronucleus frequency result was not affected by CW exposure; however, a statistically significant micronucleus effect was found following exposure to phase modulated field. These results would suggest a genotoxic power of the phase modulation per se.

Evaluation of genetic damage induced by a nitroimidazole derivative in human lymphocytes: Tinidazole (TNZ)

One of the useful drugs in the treatment against infestations caused by Trichomonas vaginalis, Entamoeba histolytica and Giardia lamblia is Tinidazole (TNZ) 1-[2-(ethylsulfonyl) ethyl]-2-methyl-5-nitroimidazole) (Gilman R.H., Marquis G.S., Miranda E., Vestegui M., Martinez H., 1988. Rapid reinfection by Giardia lamblia after treatment in a hyperendemic third world community. Lancet i, 343–345). We decided to evaluate the potential genetic damage induced by TNZ using different biological biomarkers such as the mitotic index (MI), sister chromatid exchange (SCE) and cell proliferation kinetics (CPK). We observed a significant decrease ( P<0.0005) in the MI as well as an increase ( P<0.0005) in SCE frequency and no modifications in the replication index (RI). The results obtained suggest a potential genotoxic and cytotoxic effect of TNZ in human peripheral blood cultures in vitro.

Cytological effects of 60 Hz magnetic fields on human lymphocytes in vitro: sister-chromatid exchanges, cell kinetics and mitotic rate.

Incubation for 72 h of human peripheral blood cultures in the presence of 60 Hz sinusoidal magnetic fields (MF) at magnetic flux densities of 1.0, 1.5, and 2.0 mT led to stimulation of lymphocyte proliferation but had no influence on the frequency of sister-chromatid exchanges (SCE). The cytotoxic potential of MF combined with the mutagen Mitomycin-C also was analyzed. An opposite effect between MF exposure and Mitomycin-C treatment in terms of cell kinetics and mitotic rate was found, whereas no variation in SCE frequency was observed for this coexposure condition.