Increased levels of circulating soluble CD226 in multiple sclerosis.

BACKGROUND: The glycoprotein CD226 plays a key role in regulating immune cell function. Soluble CD226 (sCD226) is increased in sera of patients with several chronic inflammatory diseases but its levels in neuroinflammatory diseases such as multiple sclerosis (MS) are unknown. OBJECTIVE: To investigate the presence and functional implications of sCD226 in persons with multiple sclerosis (pwMS) and other neurological diseases. METHODS: The mechanisms of sCD226 production were first investigated by analyzing CD226 surface expression levels and supernatants of CD3/CD226-coactivated T cells. The role of sCD226 on dendritic cell maturation was evaluated. The concentration of sCD226 in the sera from healthy donors (HD), pwMS, neuromyelitis optica (NMO), and Alzheimer's disease (AD) was measured. RESULTS: CD3/CD226-costimulation induced CD226 shedding. Addition of sCD226 to dendritic cells during their maturation led to an increased production of the pro-inflammatory cytokine interleukin (IL)-23. We observed a significant increase in sCD226 in sera from pwMS and NMO compared to HD and AD. In MS, levels were increased in both relapsing-remitting multiple sclerosis (RRMS) and secondary-progressive multiple sclerosis (SPMS) compared to clinically isolated syndrome (CIS). CONCLUSION: Our data suggest that T-cell activation leads to release of sCD226 that could promote inflammation and raises the possibility of using sCD226 as a biomarker for neuroinflammation.

Vitamin D/CD46 Crosstalk in Human T Cells in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), in which T-cell migration into the CNS is key for pathogenesis. Patients with MS exhibit impaired regulatory T cell populations, and both Foxp3+ Tregs and type I regulatory T cells (Tr1) are dysfunctional. MS is a multifactorial disease and vitamin D deficiency is associated with disease. Herein, we examined the impact of 1,25(OH)2D3 on CD4+ T cells coactivated by either CD28 to induce polyclonal activation or by the complement regulator CD46 to promote Tr1 differentiation. Addition of 1,25(OH)2D3 led to a differential expression of adhesion molecules on CD28- and CD46-costimulated T cells isolated from both healthy donors or from patients with MS. 1,25(OH)2D3 favored Tr1 motility though a Vitamin D-CD46 crosstalk highlighted by increased VDR expression as well as increased CYP24A1 and miR-9 in CD46-costimulated T cells. Furthermore, analysis of CD46 expression on T cells from a cohort of patients with MS supplemented by vitamin D showed a negative correlation with the levels of circulating vitamin D. Moreover, t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis allowed the visualization and identification of clusters increased by vitamin D supplementation, but not by placebo, that exhibited similar adhesion phenotype to what was observed in vitro. Overall, our data show a crosstalk between vitamin D and CD46 that allows a preferential effect of Vitamin D on Tr1 cells, providing novel key insights into the role of an important modifiable environmental factor in MS.

The Secretive Liaison of Particulate Matter and SARS-CoV-2. A Hypothesis and Theory Investigation.

As the novel coronavirus disease sweeps across the world, there is growing speculation on the role that atmospheric factors may have played on the different distribution of SARS-CoV-2, and on the epidemiological characteristics of COVID-19. Knowing the role that environmental factors play in influenza virus outbreaks, environmental pollution and, in particular, atmospheric airborne (particulate matter, PM) has been considered as a potential key factor in the spread and mortality of COVID-19. A possible role of the PM as the virus carrier has also been debated. The role of PM in exacerbating respiratory and cardiovascular disease has been well recognized. Accumulating evidence support the hypothesis that PM can trigger inflammatory response at molecular, cellular and organ levels. On this basis, we developed the hypothesis that PM may play a role as a booster of COVID-19 rather than as a carrier of SARS-CoV-2. To support our hypothesis, we analyzed the molecular signatures detected in cells exposed to PM samples collected in one of the most affected areas by the COVID-19 outbreak, in Italy. T47D human breast adenocarcinoma cells were chosen to explore the global gene expression changes induced by the treatment with organic extracts of PM 2.5. The analysis of the KEGG's pathways showed modulation of several gene networks related to the leucocyte transendothelial migration, cytoskeleton and adhesion system. Three major biological process were identified, including coagulation, growth control and immune response. The analysis of the modulated genes gave evidence for the involvement of PM in the endothelial disease, coagulation disorders, diabetes and reproductive toxicity, supporting the hypothesis that PM, directly or through molecular interplay, affects the same molecular targets as so far known for SARS-COV-2, contributing to the cytokines storm and to the aggravation of the symptoms triggered by COVID-19. We provide evidence for a plausible cooperation of receptors and transmembrane proteins, targeted by PM and involved in COVID-19, together with new insights into the molecular interplay of chemicals and pathogens that could be of importance for sustaining public health policies and developing new therapeutic approaches.

Eomes-Dependent Loss of the Co-activating Receptor CD226 Restrains CD8+ T Cell Anti-tumor Functions and Limits the Efficacy of Cancer Immunotherapy.

CD8+ T cells within the tumor microenvironment (TME) are exposed to various signals that ultimately determine functional outcomes. Here, we examined the role of the co-activating receptor CD226 (DNAM-1) in CD8+ T cell function. The absence of CD226 expression identified a subset of dysfunctional CD8+ T cells present in peripheral blood of healthy individuals. These cells exhibited reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program upon stimulation. CD226neg CD8+ T cells accumulated in human and mouse tumors of diverse origin through an antigen-specific mechanism involving the transcriptional regulator Eomesodermin (Eomes). Despite similar expression of co-inhibitory receptors, CD8+ tumor-infiltrating lymphocyte failed to respond to anti-PD-1 in the absence of CD226. Immune checkpoint blockade efficacy was hampered in Cd226-/- mice. Anti-CD137 (4-1BB) agonists also stimulated Eomes-dependent CD226 loss that limited the anti-tumor efficacy of this treatment. Thus, CD226 loss restrains CD8+ T cell function and limits the efficacy of cancer immunotherapy.

Increased IL-2 and Reduced TGF-ß Upon T-Cell Stimulation are Associated with GM-CSF Upregulation in Multiple Immune Cell Types in Multiple Sclerosis.

Granulocyte macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory cytokine produced by immune cells. Recent evidence suggests that GM-CSF plays an important role in multiple sclerosis (MS) pathogenesis. We investigated the expression and regulation of GM-CSF in different immune cells in MS. We also investigated the differentiation and frequency of GM-CSF-producing Th cells that do not co-express interferon (IFN)-γ or interleukin-17 (IL-17) (Th-GM cells) in MS. We found a significant increase in the percentage of GM-CSF-expressing Th cells, Th1 cells, Th-GM cells, cytotoxic T (Tc) cells, monocytes, natural killer (NK) cells, and B cells in PBMC from MS patients stimulated with T cell stimuli. Stimulated PBMC culture supernatants from MS patients contained significantly higher levels of IL-2, IL-12, IL-1ß, and GM-CSF and significantly lower levels of transforming growth factor (TGF-)ß. Blocking IL-2 reduced the frequency of Th-GM cells in PBMC from MS patients. The frequency of Th-GM cells differentiated in vitro from naïve CD4+ T cells was significantly higher in MS patients and was further increased in MS with IL-2 stimulation. These findings suggest that all main immune cell subsets produce more GM-CSF in MS after in vitro stimulation, which is associated with defective TGF-ß and increased IL-2 and IL-12 production. Th-GM cells are increased in MS. GM-CSF may be a potential therapeutic target in MS.

Differential Expression of HERV-W in Peripheral Blood in Multiple Sclerosis and Healthy Patients in Two Different Ethnic Groups.

Overexpression of the Human endogenous retrovirus W (HERV-W) group of inherited retroviruses has been consistently linked with Multiple Sclerosis (MS). However most of the studies on this link have focused on European genetic groups with a very high risk of MS and it is not clear that this relationship holds for all ethnic groups. This study examined via qPCR the RNA expression in peripheral blood of HERV-W (the multiple sclerosis associated retrovirus variant MSRV) of MS patients and healthy controls from two ethnic groups with very different risk rates of MS. Population one was derived from the UK with a Northern European genetic background and an MS risk rate of 108/100,000, population two was derived from the republic of Tatarstan, Russian Federation, with a mixed Russian (Eastern European) and Tartar (Turkic or Volga/Urals) population with an MS risk rate of 21-31/100,000. The Russian population displayed a significantly higher basal level of expression of MSRV in both healthy and MS individuals when compared to the British control population with a trend in the Russian population towards higher expression levels in MS patients than healthy patients.

The Soluble Form of Toll-Like Receptor 2 Is Elevated in Serum of Multiple Sclerosis Patients: A Novel Potential Disease Biomarker.

Multiple sclerosis (MS) is an immune-mediated inflammatory demyelinating disease of the central nervous system. It was previously shown that toll-like receptor (TLR)-2 signaling plays a key role in the murine experimental autoimmune encephalomyelitis (EAE) model of MS, and that TLR2-stimulation of regulatory T cells (Tregs) promotes their conversion to T helper 17 (Th17) cells. Here, we sought potential sources of TLR2 stimulation and evidence of TLR2 activity in MS patient clinical samples. Soluble TLR2 (sTLR2) was found to be significantly elevated in sera of MS patients (n = 21), in both relapse and remission, compared to healthy controls (HC) (n = 24). This was not associated with the acute phase reaction (APR) as measured by serum C-reactive protein (CRP) level, which was similarly increased in MS patients compared to controls. An independent validation cohort from a different ethnic background showed a similar upward trend in mean sTLR2 values in relapsing-remitting MS (RRMS) patients, and significant differences in sTLR2 values between patients and HC were preserved when the data from the two cohorts were pooled together (n = 41 RRMS and 44 HC, P = 0.0006). TLR2-stimulants, measured using a human embryonic kidney (HEK)-293 cells transfectant reporter assay, were significantly higher in urine of MS patients than HC. A screen of several common urinary tract infections (UTI)-related organisms showed strong induction of TLR2-signaling in the same assay. Taken together, these results indicate that two different markers of TLR2-activity-urinary TLR2-stimulants and serum sTLR2 levels-are significantly elevated in MS patients compared to HC.

Do Antiretroviral Drugs Protect From Multiple Sclerosis by Inhibiting Expression of MS-Associated Retrovirus?

The expression of human endogenous retroviruses (HERVs) has been associated with Multiple Sclerosis (MS). The MS-related retrovirus (MSRV/HERV-W) has the potential to activate inflammatory immunity, which could promote both susceptibility and progression toward MS. A connection between HERVs and MS is also supported by the observation that people infected with the human immunodeficiency virus (HIV) may have a lower risk of developing MS than the HIV non-infected, healthy population. This may be due to suppression of HERV expression by antiretroviral therapies (ART) used to treat HIV infection. In this pilot study, we compared RNA expression of the envelope gene of MSRV/HERV-W, as well as Toll-like receptors (TLR) 2 and 4, in a small cohort of HIV+ patients with MS patients and healthy controls (HC). An increased expression of MSRV/HERV-Wenv and TLR2 RNA was detected in blood of MS patients compared with HIV patients and HC, while TLR4 was increased in both MS and HIV patients. There was, however, no difference in MSRV/HERV-Wenv, TLR2 and TLR4 expression between ART-treated and -untreated HIV patients. The viral protein Env was expressed mainly by B cells and monocytes, but not by T cells and EBV infection could induce the expression of MSRV/HERV-Wenv in Lymphoblastoid cell lines (LCLs). LCLs were therefore used as an in vitro system to test the efficacy of ART in inhibiting the expression of MSRV/HERV-Wenv. Efavirenz (a non-nucleoside reverse transcriptase inhibitor) alone or different combined drugs could reduce MSRV/HERV-Wenv expression in vitro. Further, experiments are needed to clarify the potential role of ART in protection from MS.

EBV Infection Empowers Human B Cells for Autoimmunity: Role of Autophagy and Relevance to Multiple Sclerosis.

The efficacy of B cell depletion therapy in multiple sclerosis indicates their central pathogenic role in disease pathogenesis. The B lymphotropic EBV is a major risk factor in multiple sclerosis, via as yet unclear mechanisms. We reported in a nonhuman primate experimental autoimmune encephalomyelitis model that an EBV-related lymphocryptovirus enables B cells to protect a proteolysis-sensitive immunodominant myelin oligodendrocyte glycoprotein (MOG) epitope (residues 40-48) against destructive processing. This facilitates its cross-presentation to autoaggressive cytotoxic MHC-E-restricted CD8+CD56+ T cells. The present study extends these observations to intact human B cells and identifies a key role of autophagy. EBV infection upregulated APC-related markers on B cells and activated the cross-presentation machinery. Although human MOG protein was degraded less in EBV-infected than in uninfected B cells, induction of cathepsin G activity by EBV led to total degradation of the immunodominant peptides MOG35-55 and MOG1-20 Inhibition of cathepsin G or citrullination of the arginine residue within an LC3-interacting region motif of immunodominant MOG peptides abrogated their degradation. Internalized MOG colocalized with autophagosomes, which can protect from destructive processing. In conclusion, EBV infection switches MOG processing in B cells from destructive to productive and facilitates cross-presentation of disease-relevant epitopes to CD8+ T cells.

Human endogenous retroviruses and multiple sclerosis: Causation, association, or after-effect?

From the early days of MS discovery, infections have been proposed as a possible cause of the disease. In the last three decades, an association between human endogenous retrovirus expression and MS has been further investigated and confirmed. Nevertheless, the role of such retroviruses in the disease needs clarification. In this review, we introduce MSRV/HERV-W and describe its association with MS. We then summarize the evidence for the involvement of MSRV/HERV-W in the aetiology and progression of MS and its possible role as biomarker and drug target. Biological mechanisms for HERV effects in MS may involve the activation of innate immune pathways by the envelope protein of MSRV (MSRVEnv). In addition to in vitro and experimental studies, further insight on how HERVs may influence immune-mediated pathology in MS may also come from the use of antiretroviral treatments in patients.

The association between human endogenous retroviruses and multiple sclerosis: A systematic review and meta-analysis.

BACKGROUND: The interaction between genetic and environmental factors is crucial to multiple sclerosis (MS) pathogenesis. Human Endogenous Retroviruses (HERVs) are endogenous viral elements of the human genome whose expression is associated with MS. OBJECTIVE: To perform a systematic review and meta-analysis and to assess qualitative and quantitative evidence on the expression of HERV families in MS patients. METHODS: Medline, Embase and the Cochrane Library were searched for published studies on the association of HERVs and MS. Meta-analysis was performed on the HERV-W family. Odds Ratio (OR) and 95% confidence interval (CI) were calculated for association. RESULTS: 43 reports were extracted (25 related to HERV-W, 13 to HERV-H, 9 to HERV-K, 5 to HRES-1 and 1 to HER-15 family). The analysis showed an association between expression of all HERV families and MS. For HERV-W, adequate data was available for meta-analysis. Results from meta-analyses of HERV-W were OR = 22.66 (95%CI 6.32 to 81.20) from 4 studies investigating MSRV/HERV-W (MS-associated retrovirus) envelope mRNA in peripheral blood mononuclear cells, OR = 44.11 (95%CI 12.95 to 150.30) from 6 studies of MSRV/HERV-W polymerase mRNA in serum/plasma and OR = 6.00 (95%CI 3.35 to 10.74) from 4 studies of MSRV/HERV-W polymerase mRNA in CSF. CONCLUSIONS: This systematic review and meta-analysis shows an association between expression of HERVs, and in particular the HERV-W family, and MS.

EBV Infection and Multiple Sclerosis: Lessons from a Marmoset Model.

Multiple sclerosis (MS) is thought to be initiated by the interaction of genetic and environmental factors, eliciting an autoimmune attack on the central nervous system. Epstein-Barr virus (EBV) is the strongest infectious risk factor, but an explanation for the paradox between high infection prevalence and low MS incidence remains elusive. We discuss new data using marmosets with experimental autoimmune encephalomyelitis (EAE) - a valid primate model of MS. The findings may help to explain how a common infection can contribute to the pathogenesis of MS. We propose that EBV infection induces citrullination of peptides in conjunction with autophagy during antigen processing, endowing B cells with the capacity to cross-present autoantigen to CD8+CD56+ T cells, thereby leading to MS progression.

Lymphocryptovirus Infection of Nonhuman Primate B Cells Converts Destructive into Productive Processing of the Pathogenic CD8 T Cell Epitope in Myelin Oligodendrocyte Glycoprotein.

EBV is the major infectious environmental risk factor for multiple sclerosis (MS), but the underlying mechanisms remain obscure. Patient studies do not allow manipulation in vivo. We used the experimental autoimmune encephalomyelitis (EAE) models in the common marmoset and rhesus monkey to model the association of EBV and MS. We report that B cells infected with EBV-related lymphocryptovirus (LCV) are requisite APCs for MHC-E-restricted autoaggressive effector memory CTLs specific for the immunodominant epitope 40-48 of myelin oligodendrocyte glycoprotein (MOG). These T cells drive the EAE pathogenesis to irreversible neurologic deficit. The aim of this study was to determine why LCV infection is important for this pathogenic role of B cells. Transcriptome comparison of LCV-infected B cells and CD20(+) spleen cells from rhesus monkeys shows increased expression of genes encoding elements of the Ag cross-presentation machinery (i.e., of proteasome maturation protein and immunoproteasome subunits) and enhanced expression of MHC-E and of costimulatory molecules (CD70 and CD80, but not CD86). It was also shown that altered expression of endolysosomal proteases (cathepsins) mitigates the fast endolysosomal degradation of the MOG40-48 core epitope. Finally, LCV infection also induced expression of LC3-II(+) cytosolic structures resembling autophagosomes, which seem to form an intracellular compartment where the MOG40-48 epitope is protected against proteolytic degradation by the endolysosomal serine protease cathepsin G. In conclusion, LCV infection induces a variety of changes in B cells that underlies the conversion of destructive processing of the immunodominant MOG40-48 epitope into productive processing and cross-presentation to strongly autoaggressive CTLs.

TLR2 stimulation regulates the balance between regulatory T cell and Th17 function: a novel mechanism of reduced regulatory T cell function in multiple sclerosis.

CD4(+)CD25(hi) FOXP3(+) regulatory T cells (Tregs) maintain tolerance to self-Ags. Their defective function is involved in the pathogenesis of multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. However, the mechanisms of such defective function are poorly understood. Recently, we reported that stimulation of TLR2, which is preferentially expressed by human Tregs, reduces their suppressive function and skews them into a Th17-like phenotype. In this study, we tested the hypothesis that TLR2 activation is involved in reduced Treg function in MS. We found that Tregs from MS patients expressed higher levels of TLR2 compared with healthy controls, and stimulation with the synthetic lipopeptide Pam3Cys, an agonist of TLR1/2, reduced Treg function and induced Th17 skewing in MS patient samples more than in healthy controls. These data provide a novel mechanism underlying diminished Treg function in MS. Infections that activate TLR2 in vivo (specifically through TLR1/2 heterodimers) could shift the Treg/Th17 balance toward a proinflammatory state in MS, thereby promoting disease activity and progression.

Identification of pathway-based toxicity in the BALB/c 3T3 cell model.

The particulate matter represents one of the most complex environmental mixtures, whose effects on human health and environment vary according to particles characteristics and source of emissions. The present study describes an integrated approach, including in vitro tests and toxicogenomics, to highlight the effects of air particulate matter on toxicological relevant endpoints. Air samples (PM2.5) were collected in summer and winter at different sites, representative of different levels of air pollution. Samples organic extracts were tested in the BALB/c 3T3 CTA at a dose range 1-12m(3). The effect of the exposure to the samples at a dose of 8m(3) on the whole-genome transcriptomic profile was also assessed. All the collected samples induced dose-related toxic effects in the exposed cells. The modulated gene pathways confirmed that toxicity was related to sampling season and sampling site. The analysis of the KEGG's pathways showed modulation of several gene networks related to oxidative stress and inflammation. Even if the samples did not induce cell transformation in the treated cells, gene pathways related to the onset of cancer were modulated as a consequence of the exposure. This integrated approach could provide valuable information for predicting toxic risks in humans exposed to air pollution.

Multiple Sclerosis between Genetics and Infections: Human Endogenous Retroviruses in Monocytes and Macrophages.

The etiology of multiple sclerosis (MS) is still unknown, but there is strong evidence that genetic predisposition associated with environmental factors can trigger the disease. An estimated 30 million years ago, exogenous retroviruses are thought to have integrated themselves into human germ line cells, becoming part of human DNA and being transmitted over generations. Usually such human endogenous retroviruses (HERVs) are silenced or expressed at low levels, but in some pathological conditions, such as MS, their expression is higher than that in the healthy population. Three HERV families have been associated with MS: HERV-H, HERV-K, and HERV-W. The envelope protein of MS-associated retrovirus (MSRV) from the HERV-W family currently has the strongest evidence as a potential trigger for MS. In addition to expression in peripheral immune cells, MSRV is expressed in monocytes and microglia in central nervous system lesions of people with MS and, through the activation of toll-like receptor 4, it has been shown to drive the production of proinflammatory cytokines, reduction of myelin protein expression, and death of oligodendrocyte precursors. In conclusion, the association between HERVs and MS is well documented and a pathological role for MSRV in MS is plausible. Further studies are required to determine whether the presence of these HERVs is a cause or an effect of immune dysregulation in MS.

Cancer-related genes transcriptionally induced by the fungicide penconazole.

Penconazole is a systemic triazole fungicide mainly used on grapes. The UE Maximum Residue Level (MRL) for penconazole is set at 0.2ppm in wine and grapes. In the aim of identifying potential biomarkers of exposure to penconazole and possibly highlighting its endocrine disrupting mode of action, we used a transcriptomics-based approach to detect genes, that are transcriptionally modulated by penconazole, by using an appropriate in vitro model. T-47D cells were treated with commercial penconazole or penconazole contaminated grape extracts for 4h at doses close to the MRL. The whole-genome transcriptomic profile was assessed by using genome 44K oligo-microarray slides. The list of common genes generated by the two treatments could be representative of potential markers of exposure. In order to understand the role of these genes in key events related to adversity, a pathway analysis was performed on a list of genes with the same modulation trend (up or down). The analysis returned a set of genes involved in Thyroid Cancer Pathway, thus confirming a role of penconazole in endocrine disrupting mediated effects and strongly suggesting a possible mode of action in thyroid carcinogenesis.

Different sensitivity of BALB/c 3T3 cell clones in the response to carcinogens.

Cell transformation assays (CTAs) are currently regarded as the only possible in vitro alternative to animal testing for carcinogenesis studies. CTAs have been proposed as screening tests for the carcinogenic potential of compounds that have no evidence of genotoxicity but present structural alerts for carcinogenicity. We have extensively used the BALB/c 3T3 model based on the A31 cell clone to test single chemicals, complex mixtures and environmental pollutants. In the prevalidation study carried out by ECVAM, the improved protocol is based on BALB/c 3T3 A31-1-1 cells, a clone derived by A31 cells, that is very sensitive to PAH-induced transformation. The present study was performed in the aim to compare the results obtained with the two different clones exposed to different classes of carcinogens. Cells were treated with PAHs (3-methylcholanthrene, benzo(a)pyrene), alkylating agents (melphalan) and aloethanes (1,2-dibromoethane). The induction of cytotoxicity and the onset of chemically transformed foci were evaluated by two experimental protocols, differing for cell seeding density and chemical treatment duration. The A31-1-1 cells showed higher inherent transformation rate after PAHs treatment, but they were insensitive to 1,2-dibromoethane at concentrations that usually induced transformation in A31 cells. As 1,2-dibromoethane is bioactivated to reactive forms able to bind DNA mainly through the conjugation with intracellular glutathione, these results suggested a reduced activity of phase-2 enzymes involved in glutathione conjugation in A31-1-1 cells. Our results give evidence that inherent metabolic capacity of cells may play a critical role in in vitro cell transformation, cautioning against possible misclassification of chemicals.

BALB/c 3T3 cell transformation assay for the prediction of carcinogenic potential of chemicals and environmental mixtures.

The prediction of the carcinogenic risk for humans is mostly based on animal experiments. For the last 20 years, however, the scientific community has paid great attention to alternative strategies in compliance with common moral and ethical values. The new European chemical regulation REACH (Reg. EC 1907/2006) requires the performance of new studies in vertebrates only as a last resort. REACH asks for the development of validated in vitro protocols that can replace, in the medium to the long term, animal bioassays. An in vitro cell transformation assay (CTA) is proposed as an alternative to in vivo carcinogenicity testing. This assay is reported in the list of accepted methods for REACH (Reg. EC 440/2008). The BALB/c 3T3 model represents one of the most well-known CTAs and is regarded as a useful tool to screen single chemicals or complex mixtures for carcinogenicity prediction. In this study we used a modified protocol to highlight the transforming potential of three single compounds, ethinylestradiol (EE), azathioprine (AZA-T), melphalan, and two polychlorinated biphenyls (PCBs) mixtures, which are known or suspected to be human carcinogens. We also evaluated the activity of the antioxidant alpha-lipoic acid (ALA), a promising tumor chemopreventive. A significant increase in transformation frequency was observed when the BALB/c 3T3 cells were exposed to EE, AZA-T or melphalan as well as after PCBs treatment. On the contrary, ALA did not induce any increase of foci occurrence. Our results confirm the suitability of the improved protocol to discriminate carcinogenic compounds and support the use of BALB/c 3T3 cell transformation assay as a possible alternative to predict carcinogenic risk to humans.

Gene expression changes in medical workers exposed to radiation.

The use of nuclear resources for medical purposes causes considerable concern about occupational exposure. Nevertheless, little information is available regarding the effects of low-dose irradiations protracted over time. We used oligomicroarrays to identify the genes that are transcriptionally regulated by persistent exposure to extremely low doses of ionizing radiation in 28 exposed professionals (mean cumulative effective dose +/- SD, 19 +/- 38 mSv) compared with a matched sample of nonexposed subjects. We identified 256 modulated genes from peripheral blood mononuclear cells profiles, and the main biological processes we found were DNA packaging and mitochondrial electron transport NADH to ubiquinone. Next we investigated whether a different pattern existed when only 22 exposed subjects with accumulated doses >2.5 mSv, a threshold corresponding to the natural background radiation in Italy per year, and mean equal to 25 +/- 41 mSv were used. In addition to DNA packaging and NADH dehydrogenase function, the analysis of the higher-exposed subgroup revealed a significant modulation of ion homeostasis and programmed cell death as well. The changes in gene expression that we found suggest different mechanisms from those involved in high-dose studies that may help to define new biomarkers of radiation exposure for accumulated doses below 25 mSv.