Human Peripheral Leukocytes Research Articles

Preclinical development of small-molecule CRM1 inhibitors as novel therapy for the treatment of colorectal cancer (CRC).

430 Background: CRM1 (XPO1) is a key nuclear export protein which controls the location of multiple tumor suppressor (TSP) and growth regulatory (GRP) proteins including p53, PI3K/AKT, Wnt/ß-catenin and NF-kB. Forced nuclear expression of TSP and GRP by CRM1 inhibition can lead to apoptosis in cancer cells while sparing normal cells. Methods: Novel small-moleculeCRM1 inhibitors were synthesized and nuclear distribution studies were performed in cells transfected with HIV-rev GFP proteins. Cell proliferation studies were performed in 16 CRC cell lines: LS-123, SW-626, Colo-201, Colo-205, Colo-320DM, Colo-320HSR, Lovo, DLD-1, HCT-15, WiDi, LS-174T, LS-180, SW-620, C2BBe1, HCT-8, HCT-116, and in human peripheral leukocytes (PBMC). Cellular distribution and apoptosis assays were performed on HCT-116. Antitumor activity is assessed in human HCT-116 xenografts in scid-mice. Results: The lead CRM1 inhibitor, KPT-0127, blocks CRM1 mediated nuclear export of HIV-Rev-GFP, FOXO, and p53 with an IC50 of ∼300 nM. KPT-0127 is cytotoxic to various CRC cell lines with EC50s of 0.07-1.1 μM; in 9 CRC lines EC50s were < 0.3 mM. In contrast, normal cell lines and PBMCs had EC50 > 5-20 μM. In HCT- 116 cells, KPT-0127 induces cell cycle arrest at both G1/S and G2/M checkpoints and dose dependently increases nuclear p53, followed by an increase in caspase 3. KTP-0127 10μM shows no significant effect on 37 proteins including several cysteine proteases. In mice, KPT-0127 given by SC injection of 30-100 mg/kg leads to serum levels exceeding the effective CRM1 inhibitory concentration for at least 4 hours and is well tolerated. KPT-0127 given SC to mice bearing HCT-116 colon xenografts results in dose-dependent antitumor activity. Conclusions: The novel small- molecule CRM1 inhibitor KTP-0127 kills CRC lines with multiple TSP, GRP, and oncogenic abnormalities, including p53 mutations/deletions and PTEN deficiency/AKT activation, while sparing normal cells. This likely reflects the ability of CRM1 inhibition to affect multiple critical and non-redundant regulatory pathways. These results support the development of CRM1 inhibitors for the treatment of CRC. IND-enabling CMC and toxicology work are in preparation. [Table: see text]

Human Spinal Cord Injury Causes Specific Increases in Surface Expression of Beta Integrins on Leukocytes

Spinal cord injury (SCI) activates circulating leukocytes that migrate into the injured cord and bystander organs using adhesion molecule-mediated mechanisms. These cells cause oxidative damage, resulting in secondary injury to the spinal cord, as well as injury to bystander organs. This study was designed to examine, over a 6-h to 2-week period, changes in adhesion molecule surface expression on human peripheral leukocytes after SCI (9 subjects), using as controls 10 uninjured subjects and 6 general trauma patients (trauma controls, TC). Both the percentage of cells expressing a given adhesion molecule and the average level of its expression was quantified for both circulating neutrophils and monocytes. The percentage of neutrophils and monocytes expressing the selectin CD62L was unchanged in TC and SCI patients after injury compared to uninjured subjects. Concurrently, the amount of surface CD62L on neutrophils was decreased in SCI and TC subjects, and on monocytes after SCI. The percentage of neutrophils expressing α4 decreased in TC, but not in SCI, subjects. Likewise, the percentage of neutrophils and monocytes expressing CD11d decreased markedly in TC subjects, but not after SCI. In contrast, the mean surface expression of α4 and CD11d by neutrophils and monocytes increased after SCI compared with uninjured and TC subjects. The percentage of cells and surface expression of CD11b were similar in neutrophils of all three groups, whereas CD11b surface expression increased after SCI in monocytes. In summary, unlike changes found after general trauma, the proinflammatory stimulation induced by SCI increases the surface expression of adhesion molecules on circulating neutrophils and monocytes before they infiltrate the injured spinal cord and multiple organs of patients. Integrins may be excellent targets for anti-inflammatory treatment after human SCI.

Increased monocyte chemoattractant protein-1 levels indicating early vascular damage in lean young PCOS patients

Serum levels of monocyte chemoattractant protein-1 and C-reactive protein levels, as early signs of vascular damage and cardiovascular disease, were increased in young, lean patients with polycystic ovary syndrome. Serum monocyte chemoattractant protein-1 levels were positively correlated with serum LH, T, and C-reactive protein levels.

Identification of Early Response Genes in Human Peripheral Leukocytes Infected with Orientia tsutsugamushi: The Emergent of a Unique Gene Expression Profile for Diagnosis of O. tsutsugamush Infection~!2010-03-24~!2010-06-08~!2010-08-02~!

Abstract : Scrub typhus, caused by infection with Orientia tsutsugamushi, is one of the most common rickettsial diseases in the Asia-Pacific region. The disease can cause up to 35% mortality if left untreated. In order to get a better understanding of the host responses to O. tsutsugamushi infection, freshly isolated peripheral blood mononuclear cells (PBMC) were infected with O. tsutsugamushi. The infected cells were collected at 1 h, 4 h, 8 h or 18 h post infection. The gene expression profiles were monitored by cDNA microarray. Among the 7,489 genes, 658 genes were up or down regulated by 2-fold upon infection. ANOVA t-test revealed 432 genes with statistically significant fold change (p < 0.05). Semi-quantitative PCR using specific primers that flank the mRNA splicing sites of these 658 genes was carried out to verify the microarray results. More than 9000 semi-quantitative PCR reactions were performed using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene. Of these reactions, 22 genes were confirmed to exhibit up or down regulation. Quantitative PCR using 18S rRNA as the reference further confirmed two genes (NM_001547 and NM_006187) that exhibited significant increase of expression. Analysis of these 22 genes and the specific increase of both NM_001547 and NM_006187 suggest that the 22 genes are unique to O. tsutsugamushi infection and have the potential use for differentiating infections caused by virus, bacteria, parasites and other pathogens.

Prevention by l-carnitine of DNA damage induced by propionic and l-methylmalonic acids in human peripheral leukocytes in vitro

Propionic acidemia (PAemia) and methylmalonic acidemia (MMAemia) are inborn errors of propionate metabolism characterized by the accumulation of, respectively, propionic and l-methylmalonic acids (and their metabolites) in the blood and tissues of affected patients. The conditions lead to severe metabolic complications in the neonatal period and to long-term neurological manifestations. Treatment for these disorders consists of a protein-restricted diet, supplemented with synthetic formulas of amino acids, but excluding isoleucine, threonine, valine and methionine; and l-carnitine, to promote detoxication. In vitro and in vivo studies have demonstrated that lipid and protein oxidative damage may be involved in the pathophysiology of these diseases, but DNA damage has not been fully investigated. In this work, we evaluated in vitro the effects of PA and MMA, in the presence or absence of l-carnitine, on DNA damage in peripheral leukocytes, as determined by the alkaline comet assay, using silver staining and visual scoring. PA and MMA induced a DNA damage index (DI) significantly higher than that of the control group. l-Carnitine significantly reduced PA- and MMA-induced DNA damage, in a concentration-dependent manner. Our findings indicate that PA and MMA induce DNA damage and l-carnitine is able to prevent this damage.

AML1 enhances the expression of leukotriene B 4 type‐1 receptor in leukocytes

Leukotriene B(4) type-1 receptor (BLT1), which plays a role in various inflammatory diseases, is exclusively expressed in peripheral leukocytes, which suggests that its expression is stringently regulated. However, the precise mechanism of BLT1 expression is not fully understood. Here we report that acute myeloid leukemia 1 (AML1/Runx1) is involved in the enhancement of BLT1 expression in leukocytes. In retinoic acid (RA)-stimulated human promyelocytic leukemia (HL-60) cells, the transcription of the BLT1 gene was found to be significantly activated. RA did not directly modulate the BLT1 promoter, suggesting enhancers in other loci. DNase I-hypersensitivity analyses revealed an activated region, termed AE-BLex, at the intron-I:exon-II boundary. AE-BLex acts as an enhancer for the BLT1 promoter and possesses 2 AML1 recognition sites. The importance of AML1 was determined using electrophoretic mobility shift assays, reporter assays, and knockdown experiments. We demonstrated that the enhancement of BLT1 expression during the RA-induced differentiation of HL-60 cells is due to a loosening of the chromatin structure around AE-BLex, which leads to the incremental binding of AML1. The AML1/AE-BLex complex was confirmed in other BLT1- expressing leukemia cell lines and human peripheral leukocytes. Thus, AML1 enhances BLT1 expression by binding to AE-BLex, which is accessible in leukocytes.

Evaluation of DNA damage after tourniquet-induced ischaemia/reperfusion injury during lower extremity surgery

Ischemia/reperfusion (I/R) injury represents a source of substantial morbidity and mortality in various statuses that is, coronary bypass, myocardial infarction, and so on. Oxygen free radicals, formed during I/R, have been proposed as one of the main causes of tissue injury and play important role in I/R injury. Leucocytes have been shown to play an important role in the development of tissue injury after I/R. Accordingly, numerous studies have shown that even short-time I/R-induced DNA damage can be investigated in human peripheral leucocytes using the alkaline single-cell gel-electrophoresis assay (comet assay). After ischaemia, the genotoxic damage detected in human peripheral leucocytes locally in the reperfused tissue results in numerical, morphologic and biochemical alterations of all circulating white blood cells in the human organism. It leads particularly to the release of substantial amounts of oxygen radicals and other reactive agents. Simultaneously, local ischaemia in the reperfused tissue is extended to the whole body systemically through these activated inflammatory cells and, possibly, results in secondary detectable tissue damage in endothelial cells of the systemic circulation inducing prolonged DNA damage even in the early reperfusion period. Thus, we aimed to investigate whether the I/R during the routinely practised operation causes DNA damage, since other published studies were on animal and in vitro models and did not exactly reflect the operation procedure in the clinic. We measured DNA damage (single-strand breaks, oxidised purines and pyrimidines) by modified alkaline comet assay using two bacterial enzymes (formamidopyrimidine glycosylase (Fpg), endonuclease III (EndoIII)), which recognise oxidised purines and pyrimidine bases, in patients who had lower extremity surgery. There was no statistically significant difference in DNA damage between time periods (before surgery, after I/R; T1, T2 and T3). According to our findings, we indicate that at the molecular level DNA is damaged due to the I/R during the routinely practised operation; however, this effect could not be determined phenotypically. Thus, we may suggest that the surgeons and anaesthetist/operation room personnel could be informed about that fact and encouraged to use antioxidants and/or apply prophylaxis. Our findings showed that I/R injury did not induce DNA damage. Further studies of this approach are needed.

The expression of leukotriene B4 type‐1 receptor, BLT1, is facilitated by AML1 in leukocytes

Leukotriene B4 (LTB4) is a lipid mediator that activates leukocytes through a specific G protein‐coupled receptor, BLT1. BLT1 plays important roles in host defense and pathogenesis of inflammatory and immune diseases. BLT1 is exclusively expressed in peripheral leukocytes, suggesting that its expression is stringently regulated. However, the precise mechanism of BLT1 expression is not fully understood. Here, we show that acute myeloid leukemia 1 (AML1/Runx1) is involved in the enhancement of BLT1 expression in leukocytes. In retinoic acid (RA)‐stimulated HL‐60 cells, the transcription of the BLT1 gene is significantly activated. RA did not directly modulate the BLT1 promoter, suggesting an enhancer(s) in other loci. DNase I‐hypersensitivity analyses revealed an activated region, termed AE‐BLex, at the intron‐I:exon‐II boundary. AE‐BLex acts as an enhancer for the BLT1 promoter and possesses two AML1 recognition sites. We demonstrated that the enhancement of BLT1 expression during the RA‐induced differentiation of HL‐60 cells is due to a loosening of the chromatin structure around AE‐BLex, which leads to the incremental binding of AML1. The AML1/AE‐BLex complex was confirmed in other BLT1‐expressing leukemia cell lines and human peripheral leukocytes. Thus, AML1 enhances BLT1 expression by binding to AE‐BLex, which is accessible in leukocytes.

Differential expression of immunomodulatory galectin-1 in peripheral leukocytes and adult tissues and its cytosolic organization in striated muscle

Galectin-1 (Gal-1) is important in immune function and muscle regeneration, but its expression and localization in adult tissues and primary leukocytes remain unclear. To address this, we generated a specific monoclonal antibody against Gal-1, termed alphahGal-1, and defined a sequential peptide epitope that it recognizes, which is preserved in human and porcine Gal-1, but not in murine Gal-1. Using alphahGal-1, we found that Gal-1 is expressed in a wide range of porcine tissues, including striated muscle, liver, lung, brain, kidney, spleen, and intestine. In most types of cells, Gal-1 exhibits diffuse cytosolic expression, but in cells within the splenic red pulp, Gal-1 showed both cytosolic and nuclear localization. Gal-1 was also expressed in arterial walls and exhibited prominent cytosolic and nuclear staining in cultured human endothelial cells. However, human peripheral leukocytes and promyelocytic HL60 cells lack detectable Gal-1 and also showed very low levels of Gal-1 mRNA. In striking contrast, Gal-1 exhibited an organized cytosolic staining pattern within striated muscle tissue of cardiac and skeletal muscle and colocalized with sarcomeric actin on I bands. These results provide insights into previously defined roles for Gal-1 in inflammation, immune regulation and muscle biology.

Immunomodulatory Effects of Radiofrequency Ablation in a Breast Cancer Model

Radiofrequency ablation (RFA), a minimally invasive surgical procedure has an increasing application in the surgical treatment of tumors. Data indicate that RFA might stimulate anti-cancer immunity possibly through the induction of necrosis and heat shock proteins (HSP) expression. This study tests a hypothesis that RFA leads to bidirectional immunoregulation. Experimental rat breast tumors were treated with RFA, surgical excision or sham operation. RFA resulted in the highest NK cells infiltration, increased HSP70 expression and activation of caspase-3 enzyme in the tumor margins. A significant reduction of the circulatory regulatory T (Treg) cells was found in both RFA- and excision-treated rats, although less pronounced in the RFA-group. The splenocyte proliferation to tumor cell lysate was stronger in the RFA-treated rats in comparison with untreated tumor-bearing rats. The potential role of self-HSP for immunomodulation was examined using in vitro proliferation assay to tetanus toxoid using human peripheral leukocytes. The response to the tetanus toxoid was significantly suppressed by HSP90 plus auto-antibodies versus HSP90 or auto-antibodies alone. In conclusion, RFA reduced the circulatory Tregs although not as efficient as tumor excision. HSPs plus natural antibodies suppress the anti-tumor response probably by stimulating Tregs. Therefore, RFA may play a role in anti-cancer therapy if combined with Tregs suppression.

Effect of Recombinant Heat Shock Protein 70 of Mycobacterial Origin on Cytotoxic Activity and Immunophenotype of Human Peripheral Blood Mononuclear Leukocytes

Recombinant heat shock protein of mycobacterial origin with a molecular weight 70 kDa in concentration 0.1 microg/ml in vitro activates cytotoxic activity of mononuclear lymphocytes from peripheral blood of healthy donors against K-562 human erythroblastic leukemia cells sensitive to natural killers. In other concentrations (1.0 microg/ml and 10 microg/ml) this heat shock protein did not significantly affect functional activity of mononuclear leukocytes. Expression of CD4, CD25, CD16 and CD56 on membrane of mononuclear leukocytes was also studied.

Identification of the 11 β-hydroxysteroid Dehydrogenase Type 1 mRNA and Protein in Human Mononuclear Leukocytes

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion between inactive 11-ketoglucocorticoids and their active 11beta-hydroxy derivatives, such as cortisol and corticosterone. We have investigated the expression of 11beta-HSD1 in freshly isolated human peripheral mononuclear leukocytes (MNL). The presence of 11beta-HSD1 mRNA was demonstrated in total RNA by RT-PCR using specific primers designed on the 4th and 5th exons of the human 11beta-HSD1 gene. Fragments of the expected size were consistently detected on agarose gels, and sequencing showed complete identity with the corresponding sequence deposited in GenBank. The occurrence of 11beta-HSD1 protein was established by Western immunoblot analysis with a specific polyclonal antibody. Enzyme oxo-reductase activity was investigated by incubating 12 samples of MNL isolated from from 8 subjects with [3H]cortisone and formation of cortisol was established only in 4 subjects (yield range: 0.15-1.3%) after acetylation and TLC, blank subtraction and correction for losses. 18beta-Glycyrrhetinic acid, an inhibitor of 11 beta-HSD1, reduced cortisol production below detection limit. Dehydrogenase activity could not be demonstrated. It is suggested that, although enzyme activity of 11beta-HSD1 in circulating MNL is low, it is apparently ready for enhancement after MNL migration to sites of inflammation.

Genotoxicity assays analysis for carbon nanotubes: friends or foes? Preliminary results on human peripheral leukocytes

The literature is full of information on the biological effects, both in vitro and in vivo, of Carbon Nanotubes (CNTs) and almost all the data published are in agreement on the toxicity related to physicochemical properties. The gap in the knowledge of the effects of CNTs on biological systems is instead evident when approaching the damage induced to DNA by CNTs. In fact, genotoxicity data is still missing. Is this due to a lack of interest in this field of research or it is triggered by the fact that standard procedures for genotoxicity assays are still to come for CNTs? Actually, the second question seems to be the more appropriate, but also for science, 'in media stat virtus' remains valid. This brief review, including some preliminary results on human peripheral leukocytes, will try to highlight this problem as seen from the genotoxicological point of view.

Assessment of genetic damage in peripheral blood of human volunteers exposed (whole-body) to a 200 μT, 60 Hz magnetic field

Aim: To investigate the extent of damage in nucleated cells in peripheral blood of healthy human volunteers exposed to a whole-body 60 Hz, 200 μT magnetic field.Materials and methods: In this study, 10 male and 10 female healthy human volunteers received a 4 h whole-body exposure to a 200 μT, 60 Hz magnetic field. In addition, five males and five females were treated in a similar fashion, but were exposed to sham conditions. For each subject, a blood sample was obtained prior to the exposure period and aliquots were used as negative- (pre-exposure) and positive- [1.5 Gray (Gy) 60Cobalt (60Co) γ-irradiation] controls. At the end of the 4 h exposure period, a second blood sample was obtained. The extent of DNA damage was assessed in peripheral human blood leukocytes from all samples using the alkaline comet assay. To detect possible clastogenic effects, the incidence of micronuclei was assessed in phytohemagglutinin (PHA)-stimulated lymphocytes using the cytokinesis-block micronucleus assay.Results: There was no evidence of either increased DNA damage, as indicated by the alkaline comet assay, or increased incidence of micronuclei (MN) in the magnetic field exposed group. However, an in vitro exposure of 1.5 Gy γ-irradiation caused a significant increase in both DNA damage and MN induction.Conclusions: This study found no evidence that an acute, whole-body exposure to a 200 μT, 60 Hz magnetic field for 4 hours could cause DNA damage in human blood.

Expression of enzymatically active L-DOPA decarboxylase in human peripheral leukocytes

L-DOPA decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyses the decarboxylation of L-DOPA to dopamine. Dopamine (DA) has been found to be a regulating factor of the proliferation and differentiation of different leukocyte subtypes. In the present study, we report the expression of the gene that codes for the L-DOPA decarboxylase in human peripheral leukocytes and in T-lymphocytes, as well as the simultaneous detection of both neural and non-neural type DDC mRNA in the cellular components of this specialized connective tissue type. Furthermore, we have detected the neural type DDC transcript which lacks exon 3 and the alternative 37 kD alt-DDC protein isoform which lacks exons 10–15 but includes an alternative exon 10 in human peripheral leukocytes. Treatment of white blood cells with Triton X-114 resulted in the recovery of DDC in the detergent enriched and highly hydrophobic phases, suggesting association of DDC molecules with membranes in the studied cells. Enzymatic activity experiments revealed that DDC is active towards the decarboxylation of L-DOPA. The expression of enzymatically active DDC in human leukocytes could indicate a cross-talk between the nervous and the immune systems and raises new questions about the regulatory role of DDC in immune responses.

Effects of Physiological Concentrations of Heavy Metals Both Individually and in Mixtures on the Viability and Function of Peripheral Blood Human Leukocytes In Vitro

Among environmental contaminants recognized for their toxicity and global distribution, heavy metals are elements known to exert serious ecological consequences. Published experiments on the immunotoxic effects of metals such as methylmercury (MeHg), cadmium (Cd), and lead (Pb) were often conducted at concentrations higher than those present in the environment or those in human blood. In the present study the in vitro effects on human blood of environmentally relevant concentrations of MeHg (33–200 μg/L), Cd (3.1–16 μg/L), and Pb (75–207 μg/L) were assessed individually and in mixtures on the viability and immune competence of peripheral blood leukocytes (PBLs). At MeHg concentrations of 120 and 200 μg/L both lymphocyte proliferation, as measured by [3H]thymidine incorporation, and natural killer (NK) cytotoxity activity, as determined by dioctadecyloacarbocyanine, were suppressed. Our results showed an increase of intracellular thiols in lymphocytes and in monocytes at all the concentrations of metals tested. A decrease in the level of metallothionein (MT) was seen in monocytes in presence of Hg at concentration of 120 μg/L and higher. For lymphocytes, a significant increase of MT in groups containing the lower concentrations of Cd, and Hg was noted. In summary, it appears that Hg represents the most toxic metal at environmentally relevant concentrations on human peripheral mononuclear cells. The effects of Hg exposure were greater on lymphocytes and NK cells than on monocytes.

Antifungal activity of human polymorphonuclear leucocytes against yeast cells of Paracoccidioides brasiliensis

Human peripheral blood polymorphonuclear leucocytes (PMN) were examined in vitro for antifungal activity against yeast cells of Paracoccidioides brasiliensis. The yeast cell of this fungus was resistant to killing by PMN. However, PMN exhibited a fungistatic effect on the fungal isolates employed in a long-term (∼72 h) assay. Lysates of PMN did not show a fungistatic or fungicidal effect, indicating that live PMN are necessary for the antifungal effect. Interferon-Γ (IFN-Γ) enhanced the antifungal activity of PMN. IFN-Γ-treated PMN killed isolate Bt-4 within 2 h of coculture, and after 24 h a still greater killing effect was observed. By contrast, IFN-Γ-treated PMN did not show a significant killing effect on isolate Tatu, but did exhibit an enhanced fungistatic effect on this isolate. In contrast, tumor necrosis factor-α and interleukin 8 had no effect on the antifungal activity of PMN. Scanning electron microscopy revealed that the surface structure of the fungal cell was apparently damaged by PMN within 24 h of cocultures. Based on these novel findings, we speculate that human PMN might play a role in host resistance in early infection with this fungus due to their antifungal activity.

Effects ofHimatanthus lancifoliuson Human Leukocyte Chemotaxis and their Adhesion to Integrins

The aim of this work was to investigate the anti-inflammatory activities of the uleine-rich fraction extracted from the barks of Himatanthus lancifolius (Muell. Arg.) Woodson (Apocynaceae). To achieve this, we focused on its in vitro effects on some steps of the inflammatory response using peripheral human leukocytes. The results presented herein show that the uleine-rich fraction significantly inhibits the migration of casein-induced granulocytes and their adhesion to fibronectin and vitronectin, along with mononuclear cells, by down-regulating the expression of alpha 4beta1 and alpha5beta1 integrins. The data suggest that H. LANCIFOLIUS has the potential of interferring with leukocyte trafficking through its uleine-rich fraction, emphasizing its usefulness in inflammatory conditions. DEXA:dexamethasone disodium phosphate FN:fibronectin PMN:polymorphonuclear URF:uleine-rich fraction VN:vitronectin.

Comparative genotoxicity of cobalt nanoparticles and ions on human peripheral leukocytes in vitro

Owing to the increasing development of nanotechnology, there is a need to assess how engineered nanomaterials can interact with living cells. The main purpose of the present study was to assess whether metal cobalt nanoparticles (CoNP 100-500 nm) are genotoxic compared to cobalt ions (Co(2+)). Uptake experiments were carried out by incubating peripheral blood leukocytes (PBLs) with (57)Co(2+) (added to stable Co(2+) 10(-2) M to obtain concentrations in the range of 10(-5) to 10(-4) M) or with (60)CoNP for 24 and 48 h. Whereas intracellular Co(2+) showed slight or no variations over the baseline levels, CoNP were taken up efficiently leading to intracellular CoNP concentrations of 485 +/- 106.1 and 970 +/- 99 fg per cell after 24 and 48 h, respectively. The genotoxicity end points considered in this study were the frequency of binucleated micronucleated (BNMN) cells and the percentage of tail DNA (% Tail DNA) fragmentation by means of the comet assay. Genotoxic effects were evaluated by incubating PBLs of three healthy donors with subtoxic concentrations (10(-5) to 8 x 10(-5)M) of Co(2+) in the form of cobalt chloride, CoNP and 'washed' CoNP, the latter to exclude any interference by Co(2+). On a group basis, Co(2+) induced a clear trend in the increase of the BNMN frequency, whereas CoNP showed only minor changes. Moreover, we observed a high variability among donors in the induction of micronuclei. The comet assay showed a statistically significant dose-related increase in % Tail DNA for CoNP (P < 0,001), whereas Co(2+) did not induce significant changes over control values. These findings suggest that nanosized Co can be internalized by human leukocytes and can interact with DNA leading to the observed genotoxic effects, which are, however, modulated both by donor's characteristics and/or by Co(2+) release.

Normal human primary CD4+ T lymphocytes synthesize and release functional osteoprotegerin in vitro

Osteoprotegerin (OPG) acts as a decoy receptor for receptor activator of nuclear factor-κB ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL). OPG regulates bone remodeling and the immune response. The primary objective was to decipher, among human peripheral blood mononuclear leukocytes (PBML) that produce OPG, the subset(s) responsible for this synthesis and its regulation. To this end, normal human PBML and CD4-, 8-, 19-, 14-enriched subpopulations were studied in vitro for OPG synthesis. PBML were subjected to adherence and immunomagnetic separation, and OPG expression was analyzed by PCR, northern and western blotting, and ELISA. The antiapoptotic effects of OPG were studied on TRAIL-stimulated RPMI 8226 myeloma cells. OPG was time-dependently produced by primary CD4+ T lymphocytes exclusively. OPG secretion was upregulated by anti-CD3 antibody stimulation or incubation with interleukin (IL)-4, IL-1β, TNF-α, GM-CSF, and vitamin D3. In contrast, IL-10 inhibited the basal and IL-4-induced production of OPG by T cells. Conditioned media from activated T lymphocytes decreased TRAIL-induced apoptosis of RPMI 8226 cells. This effect was reversed by addition of RANKL to the T-cell conditioned media. As human immunodeficiency virus-1 (HIV-1) targets CD4+ T cells, we evaluated the effects of recombinant HIV-1 gp120 proteins on OPG synthesis. The gp120 from three different HIV-1 strains significantly reduced the basal output of OPG from T cells. Furthermore, all four protease inhibitors (PIs) used in highly active antiretroviral therapy decreased OPG synthesis by human blood T cells, nelfinavir being the most efficient PI. The simultaneous presence of an HIV-1 gp120 and a PI abrogated the basal output of OPG. In conclusion, these results highlight a new role for T lymphocytes involved in pathologies. Activated CD4+ T cells could, through OPG release, have a paracrine effect on adjacent cells and contribute to reduce the local process of bone remodeling and cellular apoptosis.