Metabolic and genetic screening of electromagnetic hypersensitive subjects as a feasible tool for diagnostics and intervention.

Growing numbers of "electromagnetic hypersensitive" (EHS) people worldwide self-report severely disabling, multiorgan, non-specific symptoms when exposed to low-dose electromagnetic radiations, often associated with symptoms of multiple chemical sensitivity (MCS) and/or other environmental "sensitivity-related illnesses" (SRI). This cluster of chronic inflammatory disorders still lacks validated pathogenetic mechanism, diagnostic biomarkers, and management guidelines. We hypothesized that SRI, not being merely psychogenic, may share organic determinants of impaired detoxification of common physic-chemical stressors. Based on our previous MCS studies, we tested a panel of 12 metabolic blood redox-related parameters and of selected drug-metabolizing-enzyme gene polymorphisms, on 153 EHS, 147 MCS, and 132 control Italians, confirming MCS altered (P < 0.05-0.0001) glutathione-(GSH), GSH-peroxidase/S-transferase, and catalase erythrocyte activities. We first described comparable-though milder-metabolic pro-oxidant/proinflammatory alterations in EHS with distinctively increased plasma coenzyme-Q10 oxidation ratio. Severe depletion of erythrocyte membrane polyunsaturated fatty acids with increased ω 6/ ω 3 ratio was confirmed in MCS, but not in EHS. We also identified significantly (P = 0.003) altered distribution-versus-control of the CYP2C19∗1/∗2 SNP variants in EHS, and a 9.7-fold increased risk (OR: 95% C.I. = 1.3-74.5) of developing EHS for the haplotype (null)GSTT1 + (null)GSTM1 variants. Altogether, results on MCS and EHS strengthen our proposal to adopt this blood metabolic/genetic biomarkers' panel as suitable diagnostic tool for SRI.

Xenobiotic sensor- and metabolism-related gene variants in environmental sensitivity-related illnesses: a survey on the Italian population.

In the environmental sensitivity-related illnesses (SRIs), multiple chemical sensitivity (MCS), chronic fatigue syndrome (FCS), and fibromyalgia (FM), the search for genetic polymorphisms of phase I/II xenobiotic-metabolizing enzymes as suitable diagnostic biomarkers produced so far inconclusive results, due to patient heterogeneity, geographic/ethnic differences in genetic backgrounds, and different methodological approaches. Here, we compared the frequency of gene polymorphisms of selected cytochrome P450 (CYP) metabolizing enzymes and, for the first time, the frequency of the xenobiotic sensor Aryl hydrocarbon receptor (AHR) in the three cohorts of 156 diagnosed MCS, 94 suspected MCS, and 80 FM/FCS patients versus 113 healthy controls. We found significantly higher frequency of polymorphisms CYP2C9∗2, CYP2C9∗3, CYP2C19∗2, CYP2D6∗4 and CYP2D6∗41 in patients compared with controls. This confirms that these genetic variants represent a genetic risk factor for SRI. Moreover, the compound heterozygosity for CYP2C9∗2 and ∗3 variants was useful to discriminate between either MCS or FM/CFS versus SMCS, while the PM ∗41/∗41 genotype discriminated between MCS and either SMCS or FM/CFS. The compound heterozygosity for CYP2C9 ∗1/∗3 and CYP2D6 ∗1/∗4 differentiated MCS and SMCS cases from FM/CFS ones. Interestingly, despite the distribution of the AHR Arg554Lys variant did not result significantly different between SRI cases and controls, it resulted useful for the discrimination between MCS and SMCS cases when considered within haplotypes in combination with CYP2C19 ∗1/∗2 and CYP2D6 ∗1/∗4. Results allowed us to propose the genotyping for these specific CYP variants, together with the AHR Arg554Lys variant, as reliable, cost-effective genetic parameters to be included in the still undefined biomarkers' panel for laboratory diagnosis of the main types of environmental-borne SRI.

Diabetes impairs adipose tissue-derived stem cell function and efficiency in promoting wound healing.

Adipose tissue-derived stem cells (ASCs) are gaining increasing consideration in tissue repair therapeutic application. Recent evidence indicates that ASCs enhance skin repair in animal models of impaired wound healing. To assess the therapeutic activity of autologous vs. allogeneic ASCs in the treatment of diabetic ulcers, we functionally characterized diabetic ASCs and investigated their potential to promote wound healing with respect to nondiabetic ones. Adipose tissue-derived cells from streptozotocin-induced type 1 diabetic mice were analyzed either freshly isolated as stromal vascular fraction (SVF), or following a single passage of culture (ASCs). Diabetic ASCs showed decreased proliferative potential and migration. Expression of surface markers was altered in diabetic SVF and cultured ASCs, with a reduction in stem cell marker-positive cells. ASCs from diabetic mice released lower amounts of hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, and insulin-like growth factor-1, growth factors playing important roles in skin repair. Accordingly, the supernatant of diabetic ASCs manifested reduced capability to promote keratinocyte and fibroblast proliferation and migration. Therapeutic potential of diabetic SVF administered to wounds of diabetic mice was blunted as compared with cells isolated from nondiabetic mice. Our data indicate that diabetes alters ASC intrinsic properties and impairs their function, thus affecting therapeutic potential in the autologous treatment for diabetic ulcers.

An Endogenous Electron Spin Resonance (ESR) signal discriminates nevi from melanomas in human specimens: a step forward in its diagnostic application.

Given the specific melanin-associated paramagnetic features, the Electron Spin Resonance (ESR, called also Electron Paramagnetic Resonance, EPR) analysis has been proposed as a potential tool for non-invasive melanoma diagnosis. However, studies comparing human melanoma tissues to the most appropriate physiological counterpart (nevi) have not been performed, and ESR direct correlation with melanoma clinical features has never been investigated. ESR spectrum was obtained from melanoma and non-melanoma cell-cultures as well as mouse melanoma and non-melanoma tissues and an endogenous ESR signal (g = 2.005) was found in human melanoma cells and in primary melanoma tissues explanted from mice, while it was always absent in non-melanoma samples. These characteristics of the measured ESR signal strongly suggested its connection with melanin. Quantitative analyses were then performed on paraffin-embedded human melanoma and nevus sections, and validated on an independent larger validation set, for a total of 112 sections (52 melanomas, 60 nevi). The ESR signal was significantly higher in melanomas (p = 0.0002) and was significantly different between "Low Breslow's and "High Breslow's" depth melanomas (p<0.0001). A direct correlation between ESR signal and Breslow's depth, expressed in millimetres, was found (R = 0.57; p<0.0001). The eu/pheomelanin ratio was found to be significantly different in melanomas "Low Breslow's" vs melanomas "High Breslow's" depth and in nevi vs melanomas "High Breslow's depth". Finally, ROC analysis using ESR data discriminated melanomas sections from nevi sections with up to 90% accuracy and p<0.0002. In the present study we report for the first time that ESR signal in human paraffin-embedded nevi is significantly lower than signal in human melanomas suggesting that spectrum variations may be related to qualitative melanin differences specifically occurring in melanoma cells. We therefore conclude that this ESR signal may represent a reliable marker for melanoma diagnosis in human histological sections.

Increase of plasma IL-9 and decrease of plasma IL-5, IL-7, and IFN-γ in patients with chronic heart failure.

BACKGROUND: Several cytokines are associated with the development and/or progression of chronic heart failure (CHF). Our aim was to look more closely at the cytokine networks involved in CHF, and to assess whether disease etiology affects cytokine expression. The study population was comprised of a) 69 patients with stable CHF, New York Heart Association (NYHA) II/IV classes, secondary to ischaemic (ICM) and non ischaemic dilated (NIDCM) cardiomyopathy and b) 16 control subjects. We analyzed and compared the plasma levels of 27 pro- and anti-inflammatory mediators, in the study population and assessed for any possible correlation with echocardiographic parameters and disease duration. METHODS: 27 cytokines and growth factors were analyzed in the plasma of ICM- (n = 42) and NIDCM (n = 27) NYHA class II-IV patients vs age- and gender-matched controls (n = 16) by a beadbased multiplex immunoassay. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test for multiple comparison. RESULTS: Macrophage inflammatory protein (MIP)-1ß, Vascular endothelial growth factor (VEGF), interleukin (IL)-9, Monocyte chemotactic protein (MCP)-1, and IL-8 plasma levels were increased in both ICM and NIDCM groups vs controls. In contrast, IL-7, IL-5, and Interferon (IFN)-γ were decreased in both ICM and NIDCM groups as compared to controls. Plasma IL-6 and IL-1 ß were increased in ICM and decreased in NIDCM, vs controls, respectively.IL-9 levels inversely correlated, in ICM patients, with left ventricular ejection fraction (LVEF) while IL-5 plasma levels inversely correlated with disease duration, in NYHA III/IV ICM patients.This is the first time that both an increase of plasma IL-9, and a decrease of plasma IL-5, IL-7 and IFN-γ have been reported in ICM as well as in NIDCM groups, vs controls. Interestingly, such cytokines are part of a network of genes whose expression levels change during chronic heart failure. The altered expression levels of MIP-1 ß, VEGF, MCP-1, IL-1 ß, IL-6, and IL-8, found in this study, are in keeping with previous reports. CONCLUSIONS: The increase of plasma IL-9, and the decrease of plasma IL-5, IL-7 and IFN-γ in ICM as well as in NIDCM groups vs controls may contribute to get further insights into the inflammatory pathways involved in CHF.

Biological definition of multiple chemical sensitivity from redox state and cytokine profiling and not from polymorphisms of xenobiotic-metabolizing enzymes.

BACKGROUND: Multiple chemical sensitivity (MCS) is a poorly clinically and biologically defined environment-associated syndrome. Although dysfunctions of phase I/phase II metabolizing enzymes and redox imbalance have been hypothesized, corresponding genetic and metabolic parameters in MCS have not been systematically examined. OBJECTIVES: We sought for genetic, immunological, and metabolic markers in MCS. METHODS: We genotyped patients with diagnosis of MCS, suspected MCS and Italian healthy controls for allelic variants of cytochrome P450 isoforms (CYP2C9, CYP2C19, CYP2D6, and CYP3A5), UDP-glucuronosyl transferase (UGT1A1), and glutathione S-transferases (GSTP1, GSTM1, and GSTT1). Erythrocyte membrane fatty acids, antioxidant (catalase, superoxide dismutase (SOD)) and glutathione metabolizing (GST, glutathione peroxidase (Gpx)) enzymes, whole blood chemiluminescence, total antioxidant capacity, levels of nitrites/nitrates, glutathione, HNE-protein adducts, and a wide spectrum of cytokines in the plasma were determined. RESULTS: Allele and genotype frequencies of CYPs, UGT, GSTM, GSTT, and GSTP were similar in the Italian MCS patients and in the control populations. The activities of erythrocyte catalase and GST were lower, whereas Gpx was higher than normal. Both reduced and oxidised glutathione were decreased, whereas nitrites/nitrates were increased in the MCS groups. The MCS fatty acid profile was shifted to saturated compartment and IFNgamma, IL-8, IL-10, MCP-1, PDGFbb, and VEGF were increased. CONCLUSIONS: Altered redox and cytokine patterns suggest inhibition of expression/activity of metabolizing and antioxidant enzymes in MCS. Metabolic parameters indicating accelerated lipid oxidation, increased nitric oxide production and glutathione depletion in combination with increased plasma inflammatory cytokines should be considered in biological definition and diagnosis of MCS.

Dysfunction of glutathione S-transferase leads to excess 4-hydroxy-2-nonenal and H(2)O(2) and impaired cytokine pattern in cultured keratinocytes and blood of vitiligo patients.

Oxidative stress due to increased epidermal levels of H(2)O(2) with consequent inhibition of catalase activity is generally accepted as a leading cytotoxic mechanism of melanocyte loss in vitiligo. Keratinocyte-derived cytokines are considered key factors in the maintenance of melanocyte structure and functions. We hypothesized that abnormal redox control may lead to impaired cytokine production by keratinocytes, thus causing noncytotoxic defects in melanocyte proliferation and melanogenesis. We found significantly suppressed mRNA and protein expression of glutathione-S-transferase (GST) M1 isoform, and higher-than-normal levels of both 4-hydroxy-2-nonenal (HNE)-protein adducts and H(2)O(2) in the cultures of keratinocytes derived from unaffected and affected skin of vitiligo patients, and in their co-cultures with allogeneic melanocytes. GST and catalase activities, as well as glutathione levels, were dramatically low in erythrocytes, whilst HNE-protein adducts were high in the plasma of vitiligo patients. The broad spectrum of major cytokines, chemokines, and growth factors was dysregulated in both blood plasma and cultured keratinocytes of vitiligo patients, when compared to normal subjects. Exogenous HNE added to normal keratinocytes induced a vitiligo-like cytokine pattern, and H(2)O(2) overproduction accompanied by adaptive upregulation of catalase and GSTM1 genes, and transient inhibition of Erk1/2 and Akt phosphorylation. Based on these results, we suggest a novel GST-HNE-H(2)O(2)-based mechanism of dysregulation of cytokine-mediated keratinocyte-melanocyte interaction in vitiligo.

Platelet-derived growth factor-receptor alpha strongly inhibits melanoma growth in vitro and in vivo.

Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. The expression of platelet-derived growth factor receptors (PDGF-Rs) is reported to be reduced in metastatic melanoma compared with benign nevi or normal skin; we then hypothesized that PDGF-Ralpha may control growth of melanoma cells. We show here that melanoma cells overexpressing PDGF-Ralpha respond to serum with a significantly lower proliferation compared with that of controls. Apoptosis, cell cycle arrest, pRb dephosphorylation, and DNA synthesis inhibition were also observed in cells overexpressing PDGF-Ralpha. Proliferation was rescued by PDGF-Ralpha inhibitors, allowing to exclude nonspecific toxic effects and indicating that PDGF-Ralpha mediates autocrine antiproliferation signals in melanoma cells. Accordingly, PDGF-Ralpha was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the mitogen-activated protein kinase pathway revealed that melanoma cells overexpressing PDGF-Ralpha show a strong reduction of c-Jun phosphorylated in serine 63 and of protein phosphatase 2A/Balpha and a marked increase of p38gamma, mitogen-activated protein kinase kinase 3, and signal regulatory protein alpha1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector overexpressing PDGF-Ralpha reached a significant 70% inhibition of primary melanoma growth (P < .001) and a similar inhibition of tumor angiogenesis. All together, these data demonstrate that PDGF-Ralpha strongly impairs melanoma growth likely through autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control.

Calorimetric and structural studies of the nitric oxide carrier S-nitrosoglutathione bound to human glutathione transferase P1-1.

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S-nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1-1 (hGSTP1-1) at 1.4 A resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild-type hGSTP1-1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild-type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S-nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix alpha2 of hGST P1-1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild-type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix alpha2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S-nitrosylation of Cys47 by GSNO is a likely physiological scenario.

Nitrosylation of human glutathione transferase P1-1 with dinitrosyl diglutathionyl iron complex in vitro and in vivo.

We have recently shown that dinitrosyl diglutathionyl iron complex, a possible in vivo nitric oxide (NO) donor, binds with extraordinary affinity to one of the active sites of human glutathione transferase (GST) P1-1 and triggers negative cooperativity in the neighboring subunit of the dimer. This strong interaction has also been observed in the human Mu, Alpha, and Theta GST classes, suggesting a common mechanism by which GSTs may act as intracellular NO carriers or scavengers. We present here the crystal structure of GST P1-1 in complex with the dinitrosyl diglutathionyl iron ligand at high resolution. In this complex the active site Tyr-7 coordinates to the iron atom through its phenolate group by displacing one of the GSH ligands. The crucial importance of this catalytic residue in binding the nitric oxide donor is demonstrated by site-directed mutagenesis of this residue with His, Cys, or Phe residues. The relative binding affinity for the complex is strongly reduced in all three mutants by about 3 orders of magnitude with respect to the wild type. Electron paramagnetic resonance spectroscopy studies on intact Escherichia coli cells expressing the recombinant GST P1-1 enzyme indicate that bacterial cells, in response to NO treatment, are able to form the dinitrosyl diglutathionyl iron complex using intracellular iron and GSH. We hypothesize the complex is stabilized in vivo through binding to GST P1-1.