Untreated Peripheral Blood Lymphocytes Research Articles

Pinus roxburghii constituents augment human lymphocytes mediated cytotoxicity towards cancer cells.

Background: Pinus roxburghii has been used in the Himalayan region as folkloric remedy while nothing is yet known about its immunomodulatory potential. Methods: The crude extracts of green and fallen pine needles were subjected to sequential fractionation to get partially purified fractions. Human peripheral blood lymphocytes (PBL) were used to analyze immunoenhancing potential of these fractions. Microcytotoxicity assay was employed to investigate improved cancer cells killing capabilities of PBL against various cancer cell lines. GC-MS was carried out to identify the major compounds in the bioactive fractions. Results: The lymphocyte proliferation assay depicted the immunoenhancing potential of extracts and fractions of fallen and green needles of P. roxburghii. The ethyl acetate fractions of both fallen and green needles displayed highest mitogenic activity on human PBL. Both fractions heightened the expression of cell surface markers (CD3, CD8, and CD56) and significantly increased the production of cytokines (IL-2, IFN-γ and TNF-α). The enhanced intracellular granulysin (immunomarker for activated CTLs and NK cells) expression also confirmed immune stimulatory potential of these fractions on human lymphocytes. The ethyl acetate fractions of pine needles enhanced the cytotoxicity of PBL towards various cancer cells (HCT-116, HeLa, PC-3 and A549) as compared to untreated PBL. GC-MS analysis showed presence of major compounds like 3-α-mannobiose, octakis (trimethylsilyl) ether, methyloxime in ethyl acetate fraction of the green needles and cyclodecasiloxane, eicosamethyl in ethyl acetate fraction of the fallen needles. Conclusion: The bioactive fractions of the fallen and green needles of P. roxburghii stimulate cancer cells killing potential of human lymphocytes.

Pectin-guar gum-zinc oxide nanocomposite enhances human lymphocytes cytotoxicity towards lung and breast carcinomas

Pectin-guar gum-zinc oxide (PEC-GG-ZnO) nanocomposite was prepared by precipitation technique. The composite was characterized by using FT-IR, XRD, HRTEM, SAED, EDS, and SEM. TEM images showed the hexagonal shape of nanocomposite with the size range of 50–70 nm. Further, PEC-GG-ZnO was used as an immunomodulator for the first time to improve the cancer cells killing capabilities of human peripheral-blood lymphocytes (PBL). The lymphocyte proliferation assay proved the immunostimulatory property of the PEC-GG-ZnO which increased with the increase in concentration (25 μg/ml to 200 μg/ml). ELISA detection confirmed a significant increase in the release of IFN-γ, IL-2 and TNF-α cytokines and flow cytometry analysis revealed enhanced expression of CD3, CD8, and CD56 after treating PBL with PEC-GG-ZnO as compared to PEC and GG treatment. Moreover, we also found that nanocomposite pretreated human PBL displayed enhanced cytotoxicity towards lung (A549) and breast carcinoma (MCF-7) cells as compared to untreated PBL. The microcytotoxicity assay also demonstrated that with increase in effector: target ratios from 2.5:1 to 20:1, there was an increase in the cancer cell death. Taken together, the current data corroborates the immunostimulatory activities of PEC-GG-ZnO, a novel nanocomposite, hence it can serve as a promising cancer therapeutic agent.

Evaluation of in vitro antioxidant, antimicrobial, genotoxic and anticancer activities of lichen Cetraria islandica

In this study, the antioxidant, antimicrobial, genotoxic and anticancer activities of Cetraria islandica methanol extract were determined by using free radical and superoxide anion scavenging activity, reducing power, determination of total phenolic compounds and flavonoid contents, broth microdilution minimal inhibitory concentration against five bacterial and five fungal species, cytokinesis block micronucleus (MN) assay on peripheral blood lymphocytes (PBLs) and the microculture tetrazolium test on FemX (human melanoma) and LS174 (human colon carcinoma) cell lines. As a result of the study, we found that C. islandica methanol extract exhibited moderate free-radical-scavenging activity with IC50 values 678.38μg/ml. Moreover, the tested extract had effective reducing power and superoxide anion radical scavenging. The minimal inhibitory concentration values against the tested microorganisms ranged from 0.312 to 5mg/ml. The extract increased MN frequency in a dose dependent manner, but it was significant in higher tested concentrations (50, 100 and 200μg/ml). No significant differences were observed between NDI values in all treatments and untreated PBLs. In addition, the tested extract had strong anticancer activity towards both cell lines with IC50 values of 22.68 and 33.74μg/ml. It can be concluded that the tested extract exhibited a certain level of in vitro antioxidant, antimicrobial, genotoxic and anticancer activities.

Investigation of constitutive and cytokine-induced expression of pSTAT 1, 3, and 5 in PBL of breast cancer patients

3042 Background: Although cytokines mediate their effects by latent transcription factors, STATs, their dysregulation has not been much investigated in peripheral blood lymphocytes (PBL) of breast cancer patients. As alterations in the JAK-STAT pathway contribute to immunosuppression in cancer patients in this study, aside from baseline-level, we investigated the level and cytokine-mediated activation of STAT1, 3, and 5 in healthy controls and breast cancer patients. Methods: PBL (5x106/ml) of breast cancer patients in all four clinical stages, as well as healthy controls, were treated in vitro with culture medium (CM) alone, IFNα (250 IU/ml CM) and IL-2 (500 IU/ml) for 24h at 37°C. Induction of phosphorylated STAT proteins was assessed in cellular lysates by Western blotting using monoclonal anti-STAT1, 3, and 5 antibodies. Treated PBL were also analyzed for IFNγ using an intracellular flow cytometry assay. Results: Our results indicate that untreated PBL of breast cancer patients, compared to healthy controls, express a significantly lower baseline level of pSTAT1, 3, and 5. The induction of pSTAT1 expression by IFNα, similar to the induction of pSTAT3 and pSTAT5 by IL-2, is significant for healthy controls and breast cancer patients, although the effect in patients is significantly below that for healthy controls. Moreover, the induction of pSTAT5 by IL-2 is of a higher magnitude than pSTAT3. PBL of breast cancer patients, compared to healthy controls, have a lower level of IFNγ. The production of IFNγ in PBL is enhanced by IFNα, as well as IL-2, although in breast cancer patients to a much lower level than in healthy controls. Conclusions: These results provide evidence that evaluation of STAT expression in PBL of breast cancer patients could be of diagnostic and therapeutic significance for cytokine-based therapy. No significant financial relationships to disclose.

The frequency of sister chromatid exchange and micronuclei in evaluation of cytogenetic activity of Kombucha on human peripheral blood lymphocytes

Kombucha is a refreshing beverage obtained by the fermentation of sweetened black tea with a ?tea fungus? (symbiotic culture of acetic acid bacteria and yeasts). It is consumed due to its potential beneficial effects on human health. The aim of this study was to investigate activity of Kombucha on human peripheral blood lymphocytes in vitro. We analyzed Kombucha made from different substrates: Camellia sinensis and Satureja montana, and effects of substrates alone. The frequencies of sister chromatid exchange (SCE) and micronuclei (MN) were scored as genetic endpoints and mitomycin C was used as model mutagen. Kombucha from Camellia sinensis and Camellia sinensis substrate increased frequency of MN and SCE on mitomycin C-treated and -untreated peripheral blood lymphocytes. However, Kombucha from Satureja montana reduced incidence of MN on mitomycin C-treated and -untreated peripheral blood lymphocytes, while SCE frequency was higher than control value. In our pilot study we showed for the first time that Kombucha from different substrates induced different effects on mitomycin C-treated and -untreated peripheral blood lymphocytes.

In vitro cytopathic effects of mycotoxin T-2 on human peripheral blood T lymphocytes

The trichothecene mycotoxin T-2 is reported to exhibit immunotoxic activity. The potential presence of T-2 in foods renders it as public health hazard and its toxicity needs to be better understood. We investigated the in vitro effects of T-2 at sub-toxic (0.1 ng/ml) and toxic (10 ng/ml) levels on freshly isolated human peripheral blood lymphocytes (PBLs). We observed no direct influence on untreated PBLs. The toxic dose of T-2, however, totally inhibited phytohemagglutinin-induced T lymphocyte proliferation and caused early apoptosis that peaked after 8 h of exposure. Both major T lymphocyte subsets (CD4 + and CD8 +) were affected as they appeared to show a positive response to T-2 at 8 h followed by their sharp reduction after 96 h. Further investigation on the naïve (CD45RA +) and memory (CD45RO +) subpopulations confirmed these observations and indicated that T-2 affected equally all the subpopulations studied, although PHA preferentially stimulated CD45RO + T lymphocytes. Sub-toxic T-2 appeared to exhibit co stimulatory properties to PHA-stimulated cells. These results support the hypothesis that T-2 affects the activation-induced cell death mechanism of T lymphocytes.

Dopaminergic modulation of oxidative stress and apoptosis in human peripheral blood lymphocytes: evidence for a D1-like receptor-dependent protective effect

Dopamine (DA) is a neurotransmitter in the central and peripheral nervous system, which can be either cytotoxic or cytoprotective under selected conditions. Such effects involve oxidative mechanisms and are likely to play a role in neurodegenerative disorders. Because increasing evidence points to peripheral blood lymphocytes (PBL) as a feasible model for studying DA-related mechanisms of cell death and survival, we have explored in these cells the effects of DA on oxidative metabolism and apoptosis. Our results show that, whereas DA 100–500 μM resulted in increased intracellular reactive oxygen species (ROS) levels and apoptotic cell death through oxidative stress, DA 0.1–5 μM decreased ROS levels and apoptosis. DA (both 1 and 500 μM) partially counteracted the decrease in Cu/Zn superoxide dismutase levels observed in untreated PBL. However, whereas the effect of the low dose lasted for the whole incubation period (24 h), the effect of DA 500 μM was transient. DA-dependent reduction of ROS levels and apoptosis was prevented by D1-like (but not D2-like) receptor antagonism. The present findings add knowledge about the sensitivity of PBL to DA and strengthen the rationale for exploiting these cells as an easily accessible peripheral model for the ex vivo investigation of oxidative stress-related dopaminergic mechanisms underlying human neurodegenerative diseases.

DUTPase in human neoplastic cells as a potential target for therapeutic intervention.

With the exception of brain, most human tissues analysed contain dUTPase protein detectable by immunohistochemistry. Non-dividing tissues like untreated peripheral blood lymphocytes (PBLs) contain basal levels of cytoplasmic dUTPase and express additional, nuclear dUTPase upon mitogenic stimulation. Normal, proliferating tissues like intestinal mucosa or germinal centres within tonsils contain cytoplasmic as well as nuclear dUTPase in accordance with a proposed role for dUTPase during cell division. Notably, no dUTPase is detectable during mitosis. The failure to stain dUTPase in normal brain tissue by immunohistochemistry while mRNA is readily detectable by Northern blotting cannot be explained at this moment. Epithelial tumours, such as adenocarcinoma of the lung, breast, colon, vulva or nasopharynx contain cells which are either positive for both or only one of the subcellular forms of the dUTPase and show variable numbers of dUTPase-positive cells. High levels of dUTPase correlate with a poor p rognosis regarding the progression of colorectal carcinoma. Of the intracranial tumours tested, neuroepithelial tumours show almost exclusively nuclear expression whereas meningiomas of higher grades of malignancy (WHO grade II and III) also contain cells with additional cytoplasmic dUTPase. The dUTPase is detectable in other malignancies including tumours derived from lymphatic tissues like Burkitts lymphoma or non-Hodgkins-lymphoma. The downregulation of dUTPase protein during apoptosis or the inhibition of dUTPase during nerve cell development in Drosophila melanogaster suggests a possible role of the enzyme during apoptosis. In line with these observations, inhibition of dUTPase by antisense in p53-deficient tumour cells hints at a possible route of treatment of p53-deficient tumours which are otherwise resistant to therapies like irradiation. The expression of the enzyme in normal tissues indicates that sublethal levels of dUTPase inhibitors may also exert an unwanted mutagenic effect. Keywords: peripheral blood lymphocytes, dUTPase, cerebral B-cell non-Hodgkin lymphoma(pCNSL), ANTISENSE EXPRESSION, APOPTOSIS

The effect of vitamin C or vitamin E supplementation on basal and H2O2-induced DNA damage in human lymphocytes.

There is a wealth of epidemiological information on antioxidants and their possible prevention of disease progression but very little of the research on antioxidants has involved intervention studies. In this study, the potential protective effect of vitamin C or E supplementation in vivo against endogenous and H2O2-induced DNA damage levels in lymphocytes was assessed. The supplementation involved fourteen healthy male and female non-smokers mean age 25-53 (SD 1.82) years, who were asked to supplement an otherwise unchanged diet with 1000 mg vitamin C daily for 42 d or 800 mg vitamin E daily for 42 d. DNA damage in H2O2-treated peripheral blood lymphocytes (PBL) and untreated PBL before and after supplementation, and during a 6-week washout period was assessed using an ELISA. At each sampling time-point, the red cell concentrate activities of superoxide dismutase, catalase and glutathione peroxidase were also determined. Supplementation with vitamin C or vitamin E decreased significantly H2O2-induced DNA damage in PBL, but had no effect on endogenous levels of DNA damage. The activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase were suppressed during the supplementation period. These supplementation regimens may be used to limit the possible adverse effects of reactive oxygen species (including those produced during the course of an immune response) on lymphocytes in vivo, and so help to maintain their functional capacity.

Interferon-β Increases the Sensitivity of Endometrial Cancer Cells to Cell-Mediated Cytotoxicity

Established monolayer cell lines derived from human endometrial carcinoma (Ishikawa and HEC-50) were sensitive to the cytotoxic activity of peripheral blood lymphocytes (PBLs). The percentages of hormone-responsive Ishikawa cells lysed by the cytotoxic activity of lymphocytes were 45.7 ± 4.3 (mean ± SE), 31.5 plusmn; 4.1, and 20.1 ± 1.6, at effector/target (E/T) ratios of 50:1, 25:1, and 12:1, respectively. Values of 44.7 ± 5.4%, 29.4 ± 4.6%, and 20 ± 4.9% were obtained when non-hormone-responsive HEC-50 cells were used as targets at the same E/T ratio. The percentages of epithelial and stromal cells, isolated from human endometrial cancer, lysed by the cytotoxic activity of lymphocytes were 40 ± 5.4 and 23.2 ± 3.8, respectively, at an E/T ratio of 25:1. The addition of interferon-β (IFN-β) to the culture increased tumor target cell sensitivity to the lytic activity of untreated PBL. The increase produced by 10 [U/ml of IFN-β ranged between 0.60- and 0.89-fold (P < 0.01 Student's t test, two-tailed, unpaired) in Ishikawa cells and between 0.37- and 0.72-fold P < 0.05) in HEC-50 cells. Higher concentrations of IFN-β (100 and 1000 IU/ml) were less effective in increasing the sensitivity of the target cells. There was no significant increase in the cytotoxic activity of lymphocytes treated with IFN-β whereas cytotoxic activity toward untreated tumor target cells increased when lymphocytes were treated with IFN-α. The effects of IFN-β were also evaluated using epithelial and stromal cells derived from human endometrial cancer. It was found that IFN-β at low concentrations (10 IU/ml) significantly increased the sensitivity of both epithelial and stromal cells, by 48 and 73%, respectively. Our data indicate that Ishikawa, HEC-50, epithelial, and stromal cells may provide a useful experimental model for studying the effects of immunomodulant agents such as IFN-β in hormone-related tumors. IFN-β increases endometrial target cell sensitivity, rather than the lytic activity of lymphocytes.

The in vitro effect of human polymorphonuclear leukocyte azurophil granule components on natural killer cell cytotoxicity

We previously demonstrated that human polymorphonuclear leukocyte (PMN) secretions are capable of activating and inhibiting natural killer cell (NK) cytotoxicity depending on the eliciting PMN stimulus. Serum-opsonized zymosan induced PMN to secrete substances that enhanced NK activity in vitro. Serum-opsonized zymosan stimulates the release of PMN azurophil granules, which contain both human neutrophil peptides (HNPs) and neutral serine proteases (NSPs). When HNPs and NSPs were tested for their ability to activate NK cells in peripheral blood lymphocytes, all but cathepsin G consistently enhanced cytotoxicity above control values. HNP-induced cytotoxicity was significantly enhanced within 12 h, peaking at approximately 24 h. Of the HNPs, HNP-1 was the most potent activator, enhancing NK activity at 1.25 micrograms/ml. Interleukin-2 and interferon-gamma were not involved in this activational process, as antibodies to interleukin-2 and interferon-gamma did not block activation by HNPs and NSPs, and interleukin-2 receptor expression was unaltered after 24 h of incubation. Enzymatically inactivated elastase and cathepsin G produced equivalent activational effects to their active counterparts. Antisera to elastase and cathepsin G decreased but did not eliminate NK activation over untreated peripheral blood lymphocytes. These data suggest that certain PMN azurophil granule components, including HNPs and NSPs directly increase the cytotoxic activity of NK cells.

The streptococcal preparation OK-432 specifically augments the susceptibility of human urinary bladder tumor cells to autologous peripheral blood lymphocytes.

The streptococcal preparation OK-432 was tested for its ability to enhance the susceptibility of fresh urinary bladder tumor (UBT) cells to autologous peripheral blood lymphocytes (PBL) in patients with UBT. PBL treated with OK-432 at 0.1 Klinishe Einheit (KE)/ml for 18 hours killed the human T24-lined UBT cells and freshly separated autologous UBT cells more efficiently than untreated PBL. Treatment of K562 erythroleukemia cells with OK-432 at 0.1 KE/ml for 18 hours had no effect on their susceptibility to lysis by fresh PBL. In contrast, treatment of T24 and fresh autologous UBT cells with OK-432 resulted in an enhancement of their susceptibility to PBL. The susceptibility of autologous UBT cells to both large granular lymphocytes (LGL) and T-lymphocytes was also enhanced by treatment of tumor cells with OK-432. Binding of PBL to T24 and fresh autologous UBT cells was also augmented by treatment of the tumor cells with OK-432. The frequency of binding of OK-432 to fresh UBT cells was positively correlated with the increased target sensitivity to autologous PBL. The inhibition of RNA synthesis in fresh UBT cells by OK-432 was also associated with the elevated susceptibility to autologous PBL. These results indicate that OK-432 activates the autologous tumor killing system through stimulation of effector cells and elevation of target susceptibility to effector cells in patients with UBT, and suggest that the OK-432-augmented target sensitivity to PBL may be oriented specifically to UBT cells and local immunotherapy with OK-432 may be remarkably beneficial in the treatment of UBT.

In vitro activation of lymphocytes by interleukin 2 in patients with renal cell carcinoma

Interleukin-2-stimulated lymphocytes or lymphokine-activated killer cells (LAK) have been shown to have anti-tumor activity which is not present in the fresh untreated peripheral blood lymphocytes. This activity has been evaluated in our laboratory using renal cell carcinoma as a target tumor. Two of 8 patients demonstrated significant lysis of autologous tumor while an additional 3 patients lysed the target cells but to a less significant degree. It is not clear why the LAK phenomenon against autologous tumors occurs in vitro in some patients and not others. By identifying the response of patients in vitro, one might be able to select an appropriate population for meaningful clinical trials.

Mitogenic capacity of oxidized human B cell lines.

A combination of neuraminidase and galactose oxidase alters the membrane glycoproteins to generate highly reactive aldehyde groups on galactose moieties. Human B cell lines oxidized in this way were able to induce strong proliferative responses from normal untreated peripheral blood lymphocytes. Oxidized cell lines that stimulate well were also correlated to some extent with their ability to bind responder lymphocytes. The present investigation demonstrates the usefulness of oxidized B cell lines as a novel mitogen for use in the study of lymphocyte activation.

Interferon acts directly on human B lymphocytes to modulate immunoglobulin synthesis.

At different times of exposure, interferon (IFN) enhanced and suppressed pokeweed mitogen- (PWM) induced IgG synthesis by human peripheral blood lymphocytes (PBL). Pretreatment of PBL and IFN frequently increased antibody production by more than 100% when compared with that by untreated PBL. Results of experiments in which PBL were separated into T and B subpopulations indicated that IFN preparations acted directly on B cells. Thus, mixtures of IFN-treated B cells and untreated T cells from 5 of 7 persons tested produced 81% to 500% more IgG than untreated, matched control cells. However, IFN-treated monocytes mixed with untreated B and T cells or IFN-treated T cells mixed with untreated B cells failed to enhance IgG production significantly in similar assays. In contrast to the pretreatment protocol, when IFN was present in the incubation mixture throughout the PWM assay, IgG production decreased. Sephadex chromatography of the IFN and tests of the resulting fractions indicated that the IgG production-enhancing activity was located in the fraction carrying the antiviral activity.

Studies of two subpopulations of human lymphocytes differing in responsiveness to concanavalin A.

We have identified two populations of human lymphocytes differing in responsiveness to the plant mitogen concanavalin A (Con-A). When peripheral blood lymphocytes are passed through a nylon column a population of lymphocytes highly responsive to Con-A adheres to the fibers while a second population of cells relatively unresponsive to Con-A emerges from the column. The untreated peripheral blood lymphocytes are termed "unfiltered" cells while the lymphocytes which pass through the column are termed "filtered" cells. Under standard assay conditions the Con-A-stimulated DNA synthesis is 6.5-fold greater, and the percentage blast formation is four-to fivefold greater in the unfiltered than in the filtered population. Mixing unfiltered with filtered cells fails to induce responsiveness in the latter indicating that a "helper" cell is not involved. The failure of filtered cells to respond to Con-A is specific for that mitogen since both populations respond nearly equally to erythroagglutinating phytohemagglutinin (E-PHA) and the poke weed mitogen (PWM). Binding studies with Con-A-(131)I demonstrate that the unfiltered population possesses approximately three times as many Con-A receptor sites per cell as the filtered cells, although both cell populations bind the mitogen with the same affinity (apparent association constant [K] of 1.67 x 10(6)m(-1)). The relationship between Con-A binding and lymphocyte activation was determined by measuring the effect on DNA synthesis of incubating the two lymphocyte populations with increasing amounts of Con-A. The concentration of Con-A required for half-maximal stimulation of DNA synthesis was 5-14 times greater for the filtered cells. However in the presence of very high Con-A concentrations the filtered cells achieved a maximal rate of DNA synthesis approaching that of the unfiltered population. These data implicate the decreased number of Con-A receptor sites on the filtered cells in their failure to respond to low concentrations of Con-A. A crucial event in the activation of lymphocytes by plant mitogens may be the binding of a critical number of the mitogen molecules to the cell surface.