Hair analysis in order to evaluate drug abuse in driver's license regranting procedures.

In Italy, driving under the influence of drugs determines the suspension of the offender's driver's license. To regain the license the person must be drug free during an observation period. People whose license has been revoked or suspended can obtain, or re-obtain their driver's license subject to the judgment of a medical commission. The exclusion of illicit drug use is determined by means of toxicological analysis, mainly on urine or hair matrices. We reported the results of several years of experience of the forensic toxicology laboratory of the University of Macerata in the use of hair analysis for the assessment of past exposure to drugs in people suspected of driving under the influence of drugs. From 2004 to 2013, 8612 hair samples, were analyzed for opiates, cocaine and delta-9-tetrahydrocannabinol (Δ(9)-THC) using gas chromatography/mass spectrometry (GC/MS) method. We used a cutoff (SoHT or national guidelines) to determine the positive data, regardless of the hair sample concentrations. 1213 samples resulted positive, 71.7% were positive for cocaine and metabolites, 19.8% for morphine and metabolites, 8.5% for Δ(9)-THC. We also studied the timeframe of the abuse, as well as gender and age distribution of positive subjects. Moreover, we analyzed the possible deterrent effect of the hair analysis on driving under the influence of psychoactive substances.

relA over-expression reduces tumorigenicity and activates apoptosis in human cancer cells.

We previously demonstrated that bcl-2 over-expression increases the malignant behaviour of the MCF7 ADR human breast cancer cell line and enhances nuclear factor-kappa B (NF-kappa B) transcriptional activity. Here, we investigated the direct effect of increased NF-kB activity on the tumorigenicity of MCF7 ADR cells by over-expressing the NF-kappa B subunit relA/p65. Surprisingly, our results demonstrated that over-expression of relA determines a considerable reduction of the tumorigenic ability in nude mice as indicated by the tumour take and the median time of tumour appearance. In vitro studies also evidenced a reduced cell proliferation and the activation of the apoptotic programme after relA over-expression. Apoptosis was associated with the production of reactive oxygen species, and the cleavage of the specific substrate Poly-ADP-ribose-polymerase. Our data indicate that there is no general role for NF-kappa B in the regulation of apoptosis and tumorigenicity. In fact, even though inhibiting NF-kappa B activity has been reported to be lethal to tumour cells, our findings clearly suggest that an over-induction of nuclear NF-kappa B activity may produce the same effect.

bcl-2 over-expression enhances NF-kappaB activity and induces mmp-9 transcription in human MCF7(ADR) breast-cancer cells.

bcl-2 expression is often associated with poor prognosis in several types of tumors; however, the role of this molecule in breast cancer is still controversial. We found earlier that over-expression of bcl-2 in a human breast-cancer cell line (MCF7(ADR)) enhances its tumorigenicity and metastatic potential by inducing metastasis-associated properties such as increased secretion of the matrix metalloproteinase-9 (mmp-9). In the present study, we investigated the effect of bcl-2 over-expression on the activity of the transcription factor NF-kappaB, an important regulator of genes involved in tumor progression and invasion. Transient transfection experiments indicate that over-expression of bcl-2 in the MCF7(ADR) cell line, enhances NF-kappaB-dependent transcriptional activity. Mobility-shift analysis revealed an increase of NF-kappaB DNA-binding in bcl-2-over-expressing clones that correlated with lower levels of the NF-kappaB cytoplasmic inhibitor IkappaBalpha. Moreover, point mutations of 2 highly conserved residues within the BH1 and BH2 domains that abrogate the interaction of bcl-2 with bax, or deletion of the N-terminal BH4 domain, completely eliminate the ability of this molecule to up-regulate NF-kappaB-dependent transactivation. Since mmp-9 is a NF-kappaB-regulated gene, we also investigated whether bcl-2 over-expression up-regulated mmp-9 transcription. We found that induction of mmp-9 mRNA correlates with the activation of an mmp-9-promoter-reporter-gene construct in transient transfection assay, and a mutation of the (-600)mmp-9-NF-kappaB binding element abolishes this effect. The overall data indicate that bcl-2-mediated regulation of NF-kappaB-transcription-factor activity may represent an important mechanism for the promotion of malignant behavior in MCF-7(ADR) cells.

Vitamin D3: a transcriptional modulator of the interferon-gamma gene.

1Alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] exerts several effects on the immune system, by regulating lymphocyte proliferation, differentiation of monocytes and secretion of cytokines as IL-2, granulocyte-macrophage colony-stimulating factor and IFN-gamma in T cells. Here, we analyze the effect of 1,25-(OH)2D3 on IFN-gamma gene transcription. Transient transfection assays in Jurkat T cells indicate that activation of the IFN-gamma promoter is down-regulated by 1,25-(OH)2D3. This effect is enhanced by retinoid X receptor (RXR), and a functional vitamin D3 receptor (VDR) DNA-binding domain in necessary for repression. We delineated two important promoter regions mainly involved in this modulation. The first of these is situated at the level of a promoter-silencer previously characterized and binds the heterodimer VDR-RXR in electrophoretic mobility shift assay. Residual negative regulation was also detected at the level of the promoter fragment -108 to +64 bp from the transcription start site and, surprisingly, the activity of the IFN-gamma enhancer from -108 to -36 bp in the context of a heterologous promoter was not affected by 1,25-(OH)2D3. Moreover, binding activity for VDR-RXR has been detected in the IFN-gamma minimal promoter, suggesting a possible mechanism of interference with transcription initiation/progression. The overall data indicate that direct modulation of the IFN-gamma promoter activity is one of the possible mechanisms involved in the repressive effect of 1,25-(OH)2D3 on IFN-gamma gene expression.

Integrin-mediated ras-extracellular regulated kinase (ERK) signaling regulates interferon gamma production in human natural killer cells.

Recent evidence indicates that integrin engagement results in the activation of biochemical signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that beta1 integrin ligation on human natural killer (NK) cells results in the activation of Ras/mitogen-activated protein kinase pathways. Formation of Shc-growth factor receptor-bound protein 2 (Grb2) and Shc-proline-rich tyrosine kinase 2-Grb2 complexes are the receptor-proximal events accompanying the beta1 integrin-mediated Ras activation. In addition, we demonstrate that ligation of beta1 integrins results in the stimulation of interferon gamma (IFN-gamma) production, which is under the control of extracellular signal-regulated kinase 2 activation. Overall, our data indicate that beta1 integrins, by delivering signals capable of triggering IFN-gamma production, may function as NK-activating receptors.

Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-kappaB downregulation in transcriptional repression of the p40 gene.

Interleukin 12 (IL-12), produced by myelomonocytic cells, plays a pivotal role in the development of T helper 1 (Th1) cells, which are involved in the pathogenesis of chronic inflammatory autoimmune disorders. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] inhibits IL-12 production by activated macrophages and dendritic cells, thus providing a novel interpretation to its immunosuppressive properties. 1,25(OH)2D3 significantly inhibits mRNA expression for both IL-12 p35 and p40 subunits acting at the transcriptional level. The effect of 1,25(OH)2D3 on p40 promoter activation was analyzed by cotransfecting monocytic RAW264.7 cells with p40 promoter/reporter constructs and expression vectors for vitamin D3 receptor (VDR) and/or retinoid X receptor (RXRalpha). We observed transcriptional repression of the p40 gene by 1,25(OH)2D3, which required coexpression of VDR with RXR and an intact VDR DNA-binding domain. The repressive effect maps to a region in the p40 promoter containing a binding site for NF-kappaB (p40-kappaB). Deletion of the p40-kappaB site abrogates part of the inhibitory effect on the p40 promoter, confirming the functional relevance of this site. Activation of monocytic THP-1 cells in the presence of 1,25(OH)2D3 results in reduced binding to the p40-kappaB site. Thus, 1,25(OH)2D3 may negatively regulate IL-12 production by downregulation of NF-kappaB activation and binding to the p40-kappaB sequence.

Interaction of NF-kappaB and NFAT with the interferon-gamma promoter.

Interferon-gamma (IFN-gamma) is a pleiotropic lymphokine whose production is restricted to activated T cells and NK cells. Along with other cytokines, IFN-gamma gene expression is inhibited by the immunosuppressant cyclosporin A. We have previously identified an intronic enhancer region (C3) of the IFN-gamma gene that binds the NF-kappaB protein c-Rel and that shows partial DNA sequence homology with the cyclosporin A-sensitive NFAT binding site and the 3'-half of the NF-kappaB consensus site. Sequence analysis of the IFN-gamma promoter revealed the presence of two additional C3-related elements (C3-1P and C3-3P). In addition, an NF-kappaB site (IFN-gamma kappaB) was identified within the promoter region. Based on this observation, we have analyzed the potential role of NF-kappaB and NFAT family members in regulating IFN-gamma transcription. Electrophoretic mobility shift assay analysis demonstrated that after T cell activation, the p50 and p65 NF-kappaB subunits bind specifically to the newly identified IFN-gamma kappaB and C3-related sites. In addition, we identified the NFAT proteins as a component of the inducible complexes that bind to the C3-3P site. Site-directed mutagenesis and transfection studies demonstrate that calcineurin-inducible transcriptional factors enhance the transcriptional activity of the IFN-gamma promoter through the cyclosporin-sensitive C3-3P site, whereas NF-kappaB proteins functionally interact with the C3-related sites. In addition, when located downstream to the beta-galactosidase gene driven by the IFN-gamma promoter, the intronic C3 site worked in concert with both the IFN-gamma kappaB and the C3-3P site to enhance gene transcription. These results demonstrate that the coordinate activities of NFAT and NF-kappaB proteins are involved in the molecular mechanisms controlling IFN-gamma gene transcription.

Identification of a Stat-6-responsive element in the promoter of the human interleukin-4 gene.

Interleukin (IL)-4 is an immunomodulatory cytokine produced by a number of cell types including T cells, basophils, and mast cells. This pleiotropic cytokine has a number of immunoregulatory functions; however, the molecular mechanisms controlling the transcription of this gene are not yet completely understood. Several studies have implicated a possible autoregulatory mechanism for its own expression. Here, we have identified a Stat-6-responsive element (Stat-6RE) in the promoter of the human IL-4 gene. Utilizing electrophoretic mobility shift analysis, we have demonstrated the presence of two specific IL-4-responsive DNA-protein complexes in nuclear extracts of both human Th1 and Th2 clones. Phytohemagglutinin-blasted peripheral blood T cells also generated an inducible complex in response to stimulation with IL-4 and the IL-4-like cytokine IL-13. Transient transfection of the murine pre-B cell line BA/F3 stably transfected with the full-length human IL-4 receptor alpha chain demonstrated the ability of multicopy Stat-6RE to initiate transcription from a heterologous promoter upon IL-4 or IL-13 stimulation. These results indicate a possible autocrine mechanism for the regulation of IL-4 gene transcription through the Stat-6RE as well as a possible mechanism for IL-13 regulation of the human IL-4 promoter.

Retinoic acid-induced transcriptional modulation of the human interferon-gamma promoter.

Disregulation of vitamin A metabolism is able to generate different immunological effects, including altered response to infection, reduced IgG production, and differential regulation of cytokine gene expression (including interleukin-2 and -4 and interferon-gamma (IFN-gamma)). In particular, IFN-gamma gene expression is significantly affected by vitamin A and/or its derivatives (e.g. retinoic acid (RA)). Here, we analyze the effect of retinoic acid on IFN-gamma transcription. Transient transfection assays in the human T lymphoblastoid cell line Jurkat demonstrated that the activation of the IFN-gamma promoter was significantly down-regulated in the presence of RA. Surprisingly, two different AP-1/CREB-ATF-binding elements situated in the initial 108 base pairs of the IFN-gamma promoter and previously shown to be critical for transcriptional activity were unaffected by RA. Utilizing promoter deletions and electrophoretic mobility shift analysis, we identified a USF/EGR-1-binding element cooperating in the modulation of IFN-gamma promoter activity by RA. This element was found to be situated in a position of the IFN-gamma promoter close to a silencer element previously identified in our laboratory. These results suggest that direct modulation of IFN-gamma promoter activity is one of the possible mechanisms involved in the inhibitory effect of retinoids on IFN-gamma gene expression.

The nuclear factor YY1 suppresses the human gamma interferon promoter through two mechanisms: inhibition of AP1 binding and activation of a silencer element.

Our group has previously reported that the nuclear factor Yin-Yang 1 (YY1), a ubiquitous DNA-binding protein, is able to interact with a silencer element (BE) in the gamma interferon (IFN-gamma) promoter region. In this study, we demonstrated that YY1 can directly inhibit the activity of the IFN-gamma promoter by interacting with multiple sites in the promoter. In cotransfection assays, a YY1 expression vector significantly inhibited IFN-gamma promoter activity. Mutation of the YY1 binding site in the native IFN-gamma promoter was associated with an increase in the IFN-gamma promoter activity. Analysis of the DNA sequences of the IFN-gamma promoter revealed a second functional YY1 binding site (BED) that overlaps with an AP1 binding site. In this element, AP1 enhancer activity was suppressed by YY1. Since the nuclear level of YY1 does not change upon cell activation, our data support a model that the nuclear factor YY1 acts to suppress basal IFN-gamma transcription by interacting with the promoter at multiple DNA binding sites. This repression can occur through two mechanisms: (i) cooperation with an as-yet-unidentified AP2-like repressor protein and (ii) competition for DNA binding with the transactivating factor AP1.

Activation of nuclear factor of activated T cells in a cyclosporin A-resistant pathway.

The mechanism of action of the immunosuppressive drug cyclosporin A (CsA) is the inactivation of the Ca2+/calmodulin-dependent serine-threonine phosphatase calcineurin by the drug-immunophilin complex. Inactive calcineurin is unable to activate the nuclear factor of activated T cells (NFAT), a transcription factor required for expression of the interleukin 2 (IL-2) gene. IL-2 production by CsA-treated cells is therefore dramatically reduced. We demonstrate here, however, that NFAT can be activated, and significant levels of IL-2 can be produced by the CsA-resistant CD28-signaling pathway. In transient transfection assays, both multicopy NFAT- and IL-2 promoter-beta-galactosidase reporter gene constructs could be activated by phorbol 12-myristate 13-acetate (PMA)/alpha-CD28 stimulation, and this activation was resistant to CsA. Electrophoretic mobility shift assay showed the induction of a CsA-resistant NFAT complex in the nuclear extracts of peripheral blood T cells stimulated with PMA plus alphaCD28. Peripheral blood T cells stimulated with PMA/alphaCD28 produced IL-2 in the presence of CsA. Collectively, these data suggest that NFAT can be activated and IL-2 can be produced in a calcineurin independent manner.

Gradual loss of T-helper 1 populations in spleen of mice during progressive tumor growth.

BACKGROUND: The carefully orchestrated events that result in a protective immune response are coordinated to a large extent by cytokines produced by T helper 1 (Th1) and T helper 2 (Th2) T-cell subsets, which are two arms of the immune system. Th1 cells preferentially produce interleukin 2 (IL-2), interferon gamma (IFN gamma), and tumor necrosis factor (TNF), resulting in a cellular response that helps to eliminate infected cells. In contrast, Th2 cells produce IL-4, IL-5, IL-6, and IL-10 and stimulate an antibody response that helps to prevent the cells from becoming infected. The clinical progression of several infectious diseases, including human immunodeficiency virus, some types of parasitoses, and tuberculosis, is thought to be associated with the predominance of a Th2-type T-cell response. Recent reports have demonstrated the presence of T cells producing Th2 lymphokines (IL-4, IL-6, and IL-10) in tumor-infiltrating lymphocytes of renal cell carcinoma. PURPOSE: The purpose of this study was to investigate at the molecular level whether there was any change in the splenic T cells of mice with progressively growing tumors from a Th1 to a Th2 DNA-binding pattern or phenotype. METHODS: Splenic T cells from mice bearing renal cell carcinoma or MCA-38 colon carcinoma were tested for cytokine production after in vitro activation. Nuclear extracts of splenic T cells were used for the DNA-binding assay using IFN-gamma core promoter region, the kappa B (kappa B) site from immunoglobulin gene, and the nuclear factor of activated T-cell (NFAT) site from IL-2 gene. RESULTS: Splenic T cells from mice bearing renal cell carcinoma or MCA-38 colon carcinoma preferentially produced Th2 cytokines (i.e., IL-4) upon activation and showed a marked decrease in Th1 cytokine (particularly IFN gamma) production compared with the production observed in normal splenic T cells. The DNA-binding assay with the IFN-gamma core promoter region confirmed the gradual decline in the nuclear transcription factors associated with the Th1 phenotype during tumor progression in both tumor models. Renal cell carcinoma-bearing mice, successfully treated with flavone-8-acetic acid and recombinant human IL-2, showed a reversion to a Th1-like pattern. In addition, nuclear extracts of T cells from tumor-bearing animals showed a Th2-type kappa B-binding pattern. Moreover, the NFAT complex present in the normal splenic T cells was lost at the later stages of tumor progression; instead, a new complex was present in mice bearing long-term tumors. CONCLUSION: T cells from tumor-bearing mice lose the Th1 phenotype with progressive tumor growth.

Negative transcriptional regulation of the interferon-gamma promoter by glucocorticoids and dominant negative mutants of c-Jun.

Interferon-gamma (IFN-gamma) is an immunoregulatory cytokine expressed in large granular lymphocytes and T cells. However, the molecular mechanisms underlying IFN-gamma gene transcription have not been fully defined. Here, we analyze the mechanisms responsible for the inhibition of IFN-gamma promoter activity by the glucocorticoid hormone dexamethasone. Cotransfection assays performed in Jurkat T cells demonstrated that the activity of the initial 108 base pairs of the IFN-gamma promoter was down-regulated in the presence of dexamethasone. Furthermore, utilizing electrophoretic mobility shift analysis, we identified activator protein 1 AP-1-cAMP response element binding protein-activating transcription factor (CREB-ATF) binding elements situated in positions of the IFN-gamma promoter previously identified as essential for promoter activity. Moreover, dominant negative mutants of the c-Jun proto-oncogene were able to mimic the same down-regulatory effect exerted by dexamethasone, and mutations that abolished the binding of the AP-1 CREB-ATF factors were able to block the glucocorticoid effect. These results suggest a model involving the inhibition of IFN-gamma AP-1 CREB-ATF DNA binding complexes as one of the mechanisms involved in the negative regulatory action of glucocorticoids on IFN-gamma gene expression and support the relevance of AP-1 CREB-ATF binding factors during the transcriptional activation of the IFN-gamma promoter in T cells.

Characterization of a silencer regulatory element in the human interferon-gamma promoter.

Previous analysis of the human interferon-gamma (IFN-gamma promoter indicated that the region of DNA from -251 to -215 (designated here as BE (binding element)) possessed silencer activity, as deletion of this region caused an increase in promoter activity. Based on this finding, we have conducted a series of experiments to characterize BE function and analyze the binding proteins which interact with this region. Transient transfection assays in the Jurkat T cell line revealed that the BE region possesses silencer activity, which is orientation-dependent when reinserted 5' to the IFN-gamma core promoter. However, when the BE region was inserted in front of a heterologous promoter (thymidine kinase (TK)), a mild enhancer activity was observed. Utilizing the electrophoretic mobility shift assay, we have identified two major DNA-protein complexes (designated as S and E complexes) which interact with this region. Mutational analysis indicated that the silencer activity observed with the IFN-gamma promoter correlated with the S complex and the enhancer activity correlated with the E complex. Preliminary characterization of these two DNA-protein complexes has demonstrated the presence of multiple proteins in each complex. We have found that the S protein complex has a recognition sequence similar to the nuclear factor AP2, and we have identified the nuclear factor Yin-Yang 1 (YY1) as one of the proteins in the E complex.