Antioxidant Activity of Resveratrol Diastereomeric Forms Assayed in Fluorescent-Engineered Human Keratinocytes.

Resveratrol is a powerful antioxidant molecule. In the human diet, its most important source is in Vitis vinifera grape peel and leaves. Resveratrol exists in two isoforms, cis- and trans. The diastereomeric forms of many drugs have been reported as affecting their activity. The aim of this study was to set up a cellular model to investigate how far resveratrol could counteract cytotoxicity in an oxidant agent. For this purpose, a keratinocyte cell line, which was genetically engineered with jelly fish green fluorescent protein, was treated with the free radical promoter Cumene hydroperoxide. The antioxidant activity of the trans-resveratrol and its diastereomeric mixture was evaluated indirectly in these treated fluorescent-engineered keratinocytes by analyzing the cell number and cell proliferation index. Our results demonstrate that cells, which were pre-incubated with resveratrol, reverted the oxidative damage progression induced by this free radical agent. In conclusion, fluorescent-engineered human keratinocytes represent a rapid and low-cost cellular model to determine cell numbers by studying emitted fluorescence. Comparative studies carried out with fluorescent keratinocytes indicate that trans-resveratrol is more efficient than diastereomeric mixtures in protecting cells from the oxidative stress.

Extended lifespan of normal human B lymphocytes experimentally infected by SV40 or transfected by SV40 large T antigen expression vector.

SV40 footprints were detected in different lymphoproliferative disorders and in blood specimens of healthy donors. However, little is known on the ability of SV40 to infect/transform normal human B-lymphocytes. In this in vitro study, experimental SV40 infection and SV40 Tag transfection of normal human B-lymphocytes from healthy blood donors were carried out. In SV40 infected/transfected purified B-cells, during the time course analyses, viral DNA sequences were detected by PCR, while Tag mRNA and protein were revealed by RT-PCR and immunocytochemistry, respectively. Trypan blue and Alamar blue assays showed an increase in number of cells and cell viability of infected/transfected B-cells up to day 50, then a drastic and constant cell number reduction was observed in cultures. Approximately 50% of both infected and transfected B-cells appeared morphologically transformed. SV40 viral progeny and its titer from infected B-cells was determined by plaque assay in permissive CV-1 cells. Our data indicate that human B-cells can be efficiently infected by SV40, release a viral progeny, while at the same time are transformed. SV40 infected/Tag transfected B-cells may represent an experimental model of study for investigating new biomarkers and targets for innovative therapeutic approaches in human B-cell malignancies.

Association between the JC polyomavirus infection and male infertility.

In recent years the incidence of male infertility has increased. Many risk factors have been taken into consideration, including viral infections. Investigations into viral agents and male infertility have mainly been focused on human papillomaviruses, while no reports have been published on polyomaviruses and male infertility. The aim of this study was to verify whether JC virus and BK virus are associated with male infertility. Matched semen and urine samples from 106 infertile males and 100 fertile males, as controls, were analyzed. Specific PCR analyses were carried out to detect and quantify large T (Tag) coding sequences of JCV and BKV. DNA sequencing, carried out in Tag JCV-positive samples, was addressed to viral protein 1 (VP1) coding sequences. The prevalence of JCV Tag sequences in semen and urine samples from infertile males was 34% (72/212), whereas the BKV prevalence was 0.94% (2/212). Specifically, JCV Tag sequences were detected in 24.5% (26/106) of semen and 43.4% (46/106) of urine samples from infertile men. In semen and urine samples from controls the prevalence was 11% and 28%, respectively. A statistically significant difference (p<0.05) in JCV prevalence was disclosed in semen and urine samples of cases vs. controls. A higher JC viral DNA load was detected in samples from infertile males than in controls. In samples from infertile males the JC virus type 2 strain, subtype 2b, was more prevalent than ubiquitous type 1. JCV type 2 strain infection has been found to be associated with male infertility. These data suggest that the JC virus should be taken into consideration as an infectious agent which is responsible for male infertility.

Simian virus 40 efficiently infects human T lymphocytes and extends their lifespan.

The relevance of viral infections to the onset and progression of human hematologic malignancies and other blood diseases is still a matter of active investigation. Purified human T lymphocytes isolated from the peripheral blood mononuclear cells of healthy blood donors were experimentally infected with simian virus 40 (SV40), a small DNA tumor virus. SV40-positive T lymphocytes extended their lifespan up to day 80 postinfection (PI). Expression of viral antigens, such as the large T antigen and the viral capsid protein VP1 from the early and late regions, respectively, was detected up to day 40 PI. SV40 viral progeny were continuously produced from day 10 to 40 PI. SV40 DNA sequences were detected in infected T cells for up to 80 days. Our data indicate that human T lymphocytes can be efficiently infected with SV40. Although T cells infected by SV40 were not immortalized, 30% of these lymphocytes appeared to be morphologically transformed with an enlarged T-cell shape. Our investigation provides a simple model for studying the interactions of human T lymphocytes with this small DNA tumor virus and it might represent an experimental tool for investigating new biomarkers and targets for innovative therapeutic approaches.

Merkel cell polyomavirus DNA sequences in the buffy coats of healthy blood donors.

Merkel cell polyomavirus (MCPyV), a DNA tumor virus, has been found to be associated with Merkel cell carcinoma and chronic lymphocytic leukemia. MCPyV sequences have also been detected in various normal tissues in tumor-affected patients. Immunologic studies have detected MCPyV antibodies in as many as 80% of healthy blood donors. This high seroprevalence suggests that MCPyV infection is widespread in humans. In our study, buffy coats, which were examined for MCPyV DNA Tag sequences, showed a prevalence of 22%. Viral DNA load was revealed in blood samples from 10 to 100 molecules/100 000 cells. DNA sequencing confirmed that polymerase chain reaction amplicons belong to the MCPyV strain, MKL-1. To interpret the putative role of MCPyV in chronic lymphocytic leukemia, we may infer that, during a long period of viral persistence in blood cells, this DNA tumor virus may generate mutants, which are able to participate as cofactors in the multistep process of cell transformation.

Simian virus 40 sequences in blood specimens from healthy individuals of Casale Monferrato, an industrial town with a history of asbestos pollution.

OBJECTIVE: Asbestos is considered the main agent in causing the onset of the malignant pleural mesothelioma (MM), a fatal cancer of increasing incidence worldwide. Other factors may contribute to the onset/progression of MM, such as genetic predisposition and infection by oncogenic viruses, like simian virus 40 (SV40). SV40 was administered to human populations mainly with SV40-contaminated anti-polio vaccines. SV40 footprints have been detected in specific human tumours, including MM, and in healthy blood donors. The aim of this study was to verify the presence of SV40 sequences in buffy coats of healthy blood donors, inhabitants of Casale Monferrato, where MM is 10 times more prevalent compared to other areas. METHODS: DNA from 148 buffy coats of healthy blood donors were qualitatively and quantitatively PCR analyzed for SV40 sequences. RESULTS: SV40 sequences were detected in 24 out of 148 (16%) samples. Quantitative real time PCR analyses carried out in SV40-positive samples indicated a viral copy number in the range of 10-10,000 per 100,000 cells. CONCLUSIONS: SV40 sequences are present in blood samples of healthy donors from Casale Monferrato with a prevalence similar to that reported in previous investigations of healthy donors from asbestos-free areas. Altogether these data suggest that SV40 is circulating in the human population.

Simian virus 40 in humans.

Simian virus 40 (SV40) is a monkey virus that was administered to human populations by contaminated vaccines which were produced in SV40 naturally infected monkey cells. Recent molecular biology and epidemiological studies suggest that SV40 may be contagiously transmitted in humans by horizontal infection, independently from the earlier administration of SV40-contaminated vaccines.SV40 footprints in humans have been found associated at high prevalence with specific tumor types such as brain and bone tumors, mesotheliomas and lymphomas and with kidney diseases, and at lower prevalence in blood samples from healthy donors. Contrasting reports appeared in the literature on the circulation of SV40 in humans by contagious transmission and its association, as a possible etiologic cofactor, with specific human tumors. As a consequence of the conflicting results, a considerable debate has developed in the scientific community. In the present review we consider the main results obtained by different groups investigating SV40 sequences in human tumors and in blood specimens, the putative role of SV40 in the onset/progression of specific human tumors, and comment on the hypotheses arising from these data.

Expression, pharmacological profile, and functional coupling of A2B receptors in a recombinant system and in peripheral blood cells using a novel selective antagonist radioligand, [3H]MRE 2029-F20.

In this study, we compared the pharmacological and biochemical characteristics of A(2B) adenosine receptors in recombinant (hA(2B)HEK293 cells) and native cells (neutrophils, lymphocytes) by using a new potent 8-pyrazole xanthine derivative, [(3)H]N-benzo[1,3]dioxol-5-yl-2-[5-(1,3-dipropyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yl-oxy]-acetamide] ([(3)H]MRE 2029-F20), that has high affinity and selectivity for hA(2B) versus hA(1),hA(2A), and hA(3) subtypes. [(3)H]MRE 2029-F20 bound specifically to the hA(2B) receptor stably transfected in human embryonic kidney (HEK) 293 cells with K(D) of 2.8 +/- 0.2 nM and B(max) of 450 +/- 42 fmol/mg of protein. Saturation experiments of [(3)H]MRE 2029-F20 binding in human neutrophils and lymphocytes detected a single high-affinity binding site with K(D) values of 2.4 +/- 0.5 and 2.7 +/- 0.7 nM, respectively, and B(max) values of 79 +/- 10 and 54 +/- 8 fmol/mg of protein, respectively, in agreement with real-time reverse transcription polymerase chain reaction studies showing the presence of A(2B) mRNA. The rank order of potency of typical adenosine ligands with recombinant hA(2B) receptors was consistent with that typically found for interactions with the A(2B) subtype and was also similar in peripheral blood cells. 5'-N-Ethyl-carboxamidoadenosine stimulated cAMP accumulation in both hA(2B)HEK293 and native cells, whereas phospholipase C activation was observed in recombinant receptors and endogenous subtypes expressed in neutrophils but not in lymphocytes. MRE 2029-F20 was revealed to be a potent antagonist in counteracting the agonist effect in both signal transduction pathways. In conclusion, [(3)H]MRE 2029-F20 is a selective and high-affinity radioligand for the hA(2B) adenosine subtype and may be used to quantify A(2B) endogenous receptors. In this work, we demonstrated their presence and functional coupling in neutrophils and lymphocytes that play a role in inflammatory processes in which A(2B) receptors may be involved.

Synthesis, radiolabeling, and preliminary biological evaluation of [3H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine, a potent antagonist radioligand for the P2X7 receptor.

The design, synthesis, and preliminary biological evaluation of the first potent radioligand antagonist for the P2X(7) receptor, named [(3)H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine (compound 13), are reported. This compound bound to human P2X(7) receptors expressed in HEK transfected cells with K(D) and B(max) value of 3.46+/-0.1 nM and 727+/-73 fmol/mg of protein, respectively. The high affinity and facile labeling makes it a promising radioligand for a further characterization of P2X(7) receptor subtype.

Alteration of A(3) adenosine receptors in human neutrophils and low frequency electromagnetic fields.

The present study was designed to evaluate the binding and functional characterization of A(3) adenosine receptors in human neutrophils exposed to low frequency, low energy, pulsing electromagnetic fields (PEMFs). Great interest has grown concerning the use of PEMF in the clinical practice for therapeutic purposes strictly correlated with inflammatory conditions. Saturation experiments performed using the high affinity and selective A(3) adenosine antagonist 5N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([3H]-MRE 3008F20) revealed a single class of binding sites with similar affinity in control and in PEMF treated human neutrophils (K(D)=2.36+/-0.16 and 2.45+/-0.15 nM, respectively). PEMFs treatment revealed that the receptor density was statistically increased (P<0.01) (B(max)=451+/-18 and 736+/-25fmolmg(-1) protein, respectively). Thermodynamic data indicated that [3H]-MRE 3008F20 binding in control and in PEMF-treated human neutrophils was entropy and enthalpy driven. Competition of radioligand binding by the high affinity A(3) receptor agonists, N(6)-(3-iodo-benzyl)-2-chloro-adenosine-5'-N-methyluronamide (Cl-IB-MECA) and N(6)-(3-iodo-benzyl)adenosine-5'-N-methyl-uronamide (IB-MECA), in the absence of PEMFs revealed high and low affinity values similar to those found in the presence of PEMFs. In both experimental conditions, the addition of GTP 100 microM shifted the competition binding curves of the agonists from a biphasic to a monophasic shape. In functional assays Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and their potencies were statistically increased after exposure to PEMFs. These results indicate in human neutrophils treated with PEMFs the presence of significant alterations in the A(3) adenosine receptor density and functionality.

Effects of doxazosin and propranolol on A2A adenosine receptors in essential hypertension.

A2A adenosine receptors inhibit neutrophil adhesion and superoxide anion generation. The aim of the present study was to evaluate the effect of antihypertensive treatment with doxazosin or propranolol on the binding and functional parameters of A2A adenosine receptors of lymphocytes and neutrophils in essential hypertensive patients. Two groups of previously untreated, essential hypertensive patients were studied. The mean affinity (K(d)) and density (B(max)) of adenosine receptors, by the A2A selective radioligand [3H]-ZM-241385 binding assays, and EC50, by cAMP assays, were obtained first on no medication and a second time after treatment for up to 13 weeks with doxazosin (13 patients) or propranolol (8 patients). A third group of 15 healthy normotensive volunteers matched by age, sex, and body mass index was used as a control. Binding and functional parameters of the A2A adenosine receptors were significantly higher in the 2 hypertensive groups than in controls (P always <0.0001), both in lymphocyte and neutrophil membranes. After treatment with propranolol, the binding parameters did not change significantly, whereas after treatment with doxazosin, K(d), B(max), and EC50 values returned to control levels. In never-treated essential hypertensive patients, lower affinity, higher density, and impaired function of A2A adenosine receptors are present. The binding and functional parameters of A2A adenosine receptors appear to be normalized after treatment with doxazosin but not with propranolol.