Immunological evidence of an early seroconversion to oncogenic Merkel cell polyomavirus in healthy children and young adults

Oncogenic Merkel cell polyomavirus (MCPyV) provokes a widespread and asymptomatic infection in humans. Herein, sera from healthy children and young adults (HC, n = 344) aged 0–20 years old were evaluated for anti-MCPyV immunoglobulin G (IgG) and IgM antibodies employing a recently developed immunoassay. Serum MCPyV IgG data from healthy subjects (HS, n = 510) and elderlies (ES, n = 226), aged 21–65/66–100 years old, from our previous studies, were included. The anti-MCPyV IgG and IgM rates in HC sera were 40.7% and 29.7%, respectively. A lower prevalence of anti-MCPyV IgGs was found in HC aged 0–5 years old (13%) compared to 6–10 (52.3%), 11–15 (60.5%) and 16–20 years old (61.6%) cohorts. Age-stratified HCs exhibited similar anti-MCPyV IgM rates (27.9%–32.9%). Serological profiles indicated that anti-MCPyV IgGs and IgMs had low optical densities (ODs) during the first years of life, while IgM ODs appeared to decrease throughout young adulthood. A lower anti-MCPyV IgGs rate was found in HC (40.7%) than HS (61.8%) and ES (63.7%). Upon the 5-years range age-stratification, a lower anti-MCPyV IgGs rate was found in the younger HC cohort aged 0–5 years old compared to the remaining older HC/HS/ES cohorts (52.3%–72%). The younger HC cohort exhibited the lowest anti-MCPyV IgG ODs than the older cohorts. Low anti-MCPyV IgMs rates and ODs were found in the 21–25 (17.5%) and 26–30 (7.7%) years old cohorts. Our data indicate that, upon an early-in-life seroconversion, the seropositivity for oncogenic MCPyV peaks in late childhood/young adulthood and remains at high prevalence and relatively stable throughout life.     

Reduction of urinary 8-epi-prostaglandin F2α during cyclo-oxygenase inhibition in rats but not in man

1. 8-epi-prostaglandin (PG) F_2α, a major F₂ isoprostane, is produced in vivo by free radical-dependent peroxidation of lipid-esterified arachidonic acid. Both cyclo-oxygenase isoforms (COX-1 and COX-2) may also form free 8-epi-PGF_2α as a minor product. It has been recently seen in human volunteers that the overall basal formation of 8-epi-PGF_2α in vivo is mostly COX-independent and urinary 8-epi-PGF_2α is therefore an accurate marker of ‘basal'' oxidative stress in vivo.
2. To test the validity of this marker in the rat, we evaluated in vivo the effect of COX inhibition on the formation of 8-epi-PGF_2α vs prostanoids. Two structurally unrelated COX inhibitors (naproxen: 30 mg kg⁻¹ day⁻¹; indomethacin: 4 mg kg⁻¹ day⁻¹) were given i.p. to rats kept in metabolic cages. In vivo formation of 8-epi-PGF_2α was assessed by measuring its urinary excretion. Prostanoid biosynthesis was assessed by measuring urinary excretion of major metabolites of thromboxane (TX) and prostacyclin (2,3-dinor-TXB₁ and 2,3-dinor-6-keto-PGF_1α). All compounds were selectively measured by immunopurification/gas chromatography-mass spectrometry.
3. Naproxen reduced urinary excretion of 2,3-dinor-TXB₁ and 2,3-dinor-6-keto-PGF_1α but, unexpectedly, also that of 8-epi-PGF_2α (82, 49 and 52% inhibition, respectively). Indomethacin had a similar effect (77, 69 and 55% inhibition). Esterified 8-epi-PGF_2α in liver and plasma remained unchanged after indomethacin.
4. These findings prompted us to re-assess the contribution of COX activity to the systemic production of 8-epi-PGF_2α in man. We gave naproxen (1 g day⁻¹) to healthy subjects (four nonsmokers and four smokers). Urinary 8-epi-PGF_2α remained unchanged in the two groups (9.63±0.99 before vs 10.24±1.01 after and 20.14±3.00 vs 19.03±2.45 ng h⁻¹ 1.73 m⁻²), whereas there was a marked reduction of major urinary metabolites of thromboxane and prostacyclin (about 90% for both 11-dehydro-TXB₂ and 2,3-dinor-TXB₂; >50% for 2,3-dinor-6-keto-PGF_1α).
5. To investigate whether rat COX-1 produces 8-epi-PGF_2α more efficiently than human COX-1, we measured the ex vivo formation of 8-epi-PGF_2α and TXB₂ simultaneously in whole clotting blood. Serum levels of 8-epi-PGF_2α and TXB₂ were similar in rats and man.
6. We conclude that a significant amount of COX-dependent 8-epi-PGF_2α is present in rat but not in human urine under normal conditions. This implies that urinary 8-epi-PGF_2α cannot be used as an index of near-basal oxidant stress in rats. On the other hand, our data further confirm the validity of this marker in man.

     

Characterization of the Ca2+ responses evoked by ATP and other nucleotides in mammalian brain astrocytes

1. This study was aimed at characterizing ATP-induced rises in cytosolic free calcium ion, [Ca²⁺]_i, in a population of rat striatal astrocytes loaded with the fluorescent Ca²⁺ probe Fura2, by means of fluorescence spectrometry.
2. ATP triggered a fast and transient elevation of [Ca²⁺]_i in a concentration-dependent manner. The responses of the purine analogues 2-methylthio-ATP (2-meSATP), adenosine-5′-O-(2-thiodiphosphate) (ADPβS), as well as uridine-5′-triphosphate (UTP) resembled that of ATP, while α,β-methylene-ATP (α,β-meATP) and β,γ-methylene-ATP (β,γ-meATP) were totally ineffective.
3. Suramin (50 μM) had only a minor effect on the ATP response, whereas pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (5 μM) significantly depressed the maximum response.
4. Extracellular Ca²⁺ did not contribute to the observed [Ca²⁺]_i rise: removing calcium from the extracellular medium (with 1 mM EGTA) or blocking its influx by means of either Ni²⁺ (1 mM) or Mn²⁺ (1 mM) did not modify the nucleotide responses.
5. Furthermore, after preincubation with 10 μM thapsigargin, the nucleotide-evoked [Ca²⁺]_i increments were completely abolished. In contrast, 10 mM caffeine did not affect the responses, suggesting that thapsigargin-, but not caffeine/ryanodine-sensitive stores are involved.
6. Both application of the G-protein blocker guanosine-5′-O-(2-thiodiphosphate) (GDPβS) (1 mM) and preincubation with pertussis toxin (PTx) (350 ng ml⁻¹) partially inhibited the nucleotide-mediated responses. Moreover, the phospholipase C (PLC) inhibitor U-73122, but not its inactive stereoisomer U-73343 (5 μM), significantly reduced the ATP-evoked [Ca²⁺]_i rise.
7. In conclusion, our results suggest that, in rat striatal astrocytes, ATP-elicited elevation of [Ca²⁺]_i is due solely to release from intracellular stores and is mediated by a G-protein-linked P2Y receptor, partially sensitive to PTx and coupled to PLC.

     

Potential of Gene Therapy in Bone Marrow Transplantation

Gene therapy, initiated as a treatment for inherited disorders such as adenosine deaminase deficiency, is now a promising therapeutic strategy for malignancies and other acquired diseases. In particular, in the field of bone marrow transplantation (BMT) for haematological malignancies, the gene transfer of the suicide gene HSV-TK into donor lymphocytes allows control of the severe complication graft-versus-host disease (GvHD). The transfer of the HSV-TK suicide gene confers selective sensitivity to the drug ganciclovir, allowing in vivo elimination of the donor T-cells if severe GvHD occurs. In Italy, the first pilot study on delayed infusion of genetically engineered donor lymphocytes after T-depleted allogeneic BMT documented efficacy of engineered donor lymphocytes in terms of anti-tumour activity and efficiency of the suicide system. GvHD developed in 3 out of 8 patients and was successfully treated by ganciclovir administration.     

Modulation of feeding-induced activation of mesolimbic dopamine transmission by appetitive stimuli and its relation to motivational state

We have previously shown in non-deprived rats that feeding of an unfamiliar palatable food (Fonzies(R)) phasically stimulates in vivo dopamine (DA) transmission in the medial nucleus accumbens (NAc) and this effect undergoes habituation after a previous (24 h) Fonzies meal (Bassareo & Di Chiara 1997, J. Neurosci., 17, 851-861). The present study shows that an unfamiliar food (Kinder(R)) with a taste and composition (milk chocolate) different from that of Fonzies, also induces a release of DA in the NAc subjected to one-trial habituation. Habituation was taste specific as no cross-habituation was observed between Fonzies and Kinder. In undeprived rats, a 40-min exposure to an intrinsic appetitive stimulus (food smell arising from a Fonzies-filled plastic box) also prevented the increase in dialysate DA associated with Fonzies feeding, and this effect was partially reversed by food deprivation. Food deprivation also prevented habituation of Fonzies-induced increase of dialysate DA in the NAc. Predictive association of an empty plastic box to Fonzies feeding resulted in the acquisition of appetitive properties by the box and in facilitation (rather than inhibition) of the phasic responsiveness of DA transmission to Fonzies feeding. A 10-min pre-exposure to appetitive olfactory stimuli intrinsic to Fonzies still prevented, like a 40-min pre-exposure, the NAc DA response to Fonzies feeding; however, a 5-min pre-exposure to these appetitive stimuli did not prevent the DA response in the NAc. These results show that the phasic responsiveness of NAc DA transmission to an unfamiliar palatable food is under strong modulatory control by primary (consummatory) and secondary (appetitive) stimuli, and that the sign and extent of this control depends on the nature of the appetitive stimulus, delay of reward and motivational state (deprivation).     

Mianserin markedly and selectively increases extracellular dopamine in the prefrontal cortex as compared to the nucleus accumbens of the rat

The atypical antidepressant mianserin, administered at doses of 1, 5 and 10 mg/kg SC, dose-dependently increased up to about 6 times extracellular dopamine in the medial prefrontal cortex of the rat, as estimated by vertical concentric microdialysis probes. Mianserin failed to modify extracellular dopamine in the nucleus accumbens. Mianserin also dose-dependently increased extracellular noradrenaline in the prefrontal cortex. Yohimbine, an ₂ antagonist, increased extracellular dopamine in the prefrontal cortex but the maximal increase was lower than that elicited by mianserin. Yohimbine also increased extracellular noradrenaline in the prefrontal cortex, but to a lesser extent than dopamine. Clonidine, an ₂ agonist, decreased extracellular dopamine and noradrenaline in the prefrontal cortex but failed to affect extracellular dopamine in the nucleus accumbens. Ritanserin, a 5HT₂ antagonist, at doses of 1.0 mg/kg, failed to increase extracellular dopamine in the prefrontal cortex, but significantly potentiated the increase in extracellular noradrenaline due to yohimbine. Ritanserin failed to potentiate the increase in extracellular noradrenaline elicited by yohimbine in the prefrontal cortex. The results are interpreted to indicate that mianserin increases extracellular DA as a result of the concurrent blockade of ₂ and 5HT₂ receptors. Failure to affect extracellular dopamine in the nucleus accumbens is explained as due to the lack of a significant effect of ₂ and 5HT₂ tone on DA release in the nucleus accumbens as compared to the prefrontal cortex. The results are consistent with the postulated relationship between antidepressant drug action and the ability to increase extracellular dopamine in the prefrontal cortex.     

Differential scanning calorimetry differences in micronized and unmicronized nimesulide uptake processes in biomembrane models.

Nimesulide release from micronized and unmicronized drug particles was tested at pH 7.4 by measuring the transfer to dimyristoylphosphatidylcholine liposomes (multilamellar and unilamellar vesicles), chosen as a biomembrane model. The perturbing effect of increasing molar fractions of pure nimesulide on the thermotropic behaviour of dimyristoylphosphatidylcholine liposomes was investigated by differential scanning calorimetry. In order to study the drug dissolution process by its uptake into void liposomes, measurements were carried out on suspensions of blank liposomes added to weighed amounts of free powdered nimesulide (micronized and unmicronized). The amount of drug transferred was quantified by comparing the effect caused by the dissolved and released drug to that caused by the free drug that had been previously molecularly dissolved in the liposomes. The calorimetric results show that the dissolution rate depends on the nimesulide form (micronized or unmicronized), and that the transfer to the void liposomes is quicker when the drug is in a micronized form. The uptake was faster when unilamellar vesicles were used instead of multilamellar vesicles because of the greater lipid surface. The calorimetric technique could represent an alternative 'in vitro' method that can be applied to the study of the dissolution kinetics directly at the site of drug uptake, mimicking a biological system.     

Prognostic value of CD40 in adult soft tissue sarcomas.

PURPOSE: The purpose is to evaluate the expression of CD40, a membrane protein predominantly expressed on B cells, dendritic cells, and macrophages, in a series of adult soft tissue sarcomas and to test its possible prognostic value. EXPERIMENTAL DESIGN: CD40 expression was studied by immunohistochemistry. Correlations with other baseline characteristics of patients and tumors were analyzed with chi(2) test. The prognostic value was studied with univariable and multivariable analysis adjusted by age, sex, tumor size, grade, location, and distant metastases. RESULTS: Eighty-two patients, between January 1994 and May 2001, were analyzed. Membrane or cytoplasmic staining for CD40 protein was absent in 30% of the tumors but present in <10% of cells in 22 (27%), in 10% to 50% in 23 (28%), and in >50% of cells in 12 (15%) tumors. There was no correlation between CD40 expression and age, sex, size, grade, and location of the primary tumor and distant metastases. With 61 patients (74.4%) progressed and 31 (37.8%) dead, CD40 expression was a significant prognostic factor for disease-free and overall survival at univariable and multivariable analysis. Patients with tumors expressing CD40 in >50% of cells had a dramatically unfavorable prognosis with median disease-free and overall survival of 7 and 17 months, respectively, and hazard ratios of relapse and death as compared with patients with CD40-negative tumors of 2.89 (95% confidence interval: 1.26-6.60) and 6.92 (95% confidence interval: 2.18-22.0), respectively. CONCLUSIONS: These data suggest that expression of CD40 protein in >50% of cells might indicate an unfavorable prognosis in adult soft tissue sarcomas.