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QX-314 (N-ethyl-lidocaine) is a cationic lidocaine derivative that blocks voltage-dependent sodium channels when applied internally to axons or neuronal cell bodies. Coapplication of external QX-314 with the transient receptor potential vanilloid 1 protein (TRPV1) agonist capsaicin produces long-lasting sodium channel inhibition in TRPV1-expressing neurons, suggestive of QX-314 entry into the neurons. We asked whether QX-314 entry occurs directly through TRPV1 channels or through a different pathway (e.g., pannexin channels) activated downstream of TRPV1 and whether QX-314 entry requires the phenomenon of "pore dilation" previously reported for TRPV1. With external solutions containing 10 or 20 mM QX-314 as the only cation, inward currents were activated by stimulation of both heterologously expressed and native TRPV1 channels in rat dorsal root ganglion neurons. QX-314-mediated inward current did not require pore dilation, as it activated within several seconds and in parallel with Cs-mediated outward current, with a reversal potential consistent with PQX-314/PCs = 0.12. QX-314-mediated current was no different when TRPV1 channels were expressed in C6 glioma cells, which lack expression of pannexin channels. Rapid addition of QX-314 to physiological external solutions produced instant partial inhibition of inward currents carried by sodium ions, suggesting that QX-314 is a permeant blocker. Maintained coapplication of QX-314 with capsaicin produced slowly developing reduction of outward currents carried by internal Cs, consistent with intracellular accumulation of QX-314 to concentrations of 50-100 μM. We conclude that QX-314 is directly permeant in the "standard" pore formed by TRPV1 channels and does not require either pore dilation or activation of additional downstream channels for entry.

Specific somatosensations may be processed by different subsets of primary afferents. C-fibers expressing heat-sensitive TRPV1 channels are proposed, for example, to be heat but not mechanical pain detectors. To phenotype in rats the sensory function of TRPV1(+) afferents, we rapidly and selectively silenced only their activity, by introducing the membrane-impermeant sodium channel blocker QX-314 into these axons via the TRPV1 channel pore. Using tandem mass spectrometry we show that upon activation with capsaicin, QX-314 selectively accumulates in the cytosol only of TRPV1-expressing cells, and not in control cells. Exposure to QX-314 and capsaicin induces in small DRG neurons a robust sodium current block within 30 s. In sciatic nerves, application of extracellular QX-314 with capsaicin persistently reduces C-fiber but not A-fiber compound action potentials and this effect does not occur in TRPV1(-/-) mice. Behavioral phenotyping after selectively silencing TRPV1(+) sciatic nerve axons by perineural injections of QX-314 and capsaicin reveals deficits in heat and mechanical pressure but not pinprick or light touch perception. The response to intraplantar capsaicin is substantially reduced, as expected. During inflammation, silencing TRPV1(+) axons abolishes heat, mechanical, and cold hyperalgesia but tactile and cold allodynia remain following peripheral nerve injury. These results indicate that TRPV1-expressing sensory neurons process particular thermal and mechanical somatosensations, and that the sensory channels activated by mechanical and cold stimuli to produce pain in naive/inflamed rats differ from those in animals after peripheral nerve injury.

Background: The E200K mutation of the prion protein gene (PRNP) is the most frequent amino acid substitution in genetic Creutzfeldt-Jakob disease and is the only one responsible for the appearance of clustered cases in the world. In the Israel and Slovakian clusters, age of disease onset was reduced in successive generations but the absence of a clear molecular basis raised the possibility that this event was an observational bias. The aim of the present study was to investigate possible selection biases or confounding factors related to anticipation in E200K CJD patients belonging to a cluster in Southern Italy., Methods: Clinical and demographical data of 41 parent-offspring pairs from 19 pedigrees of the Italian cluster of E200K patients were collected. Age at death of parents was compared with age at death of E200K CJD offspring. Subgroup analyses were performed for controlling possible selection biases, confounding factors, or both., Results: The mean age at death/last follow-up of the parent generation was 71.4 years while that of CJD offspring was 59.3 years with an estimated anticipation of 12.1 years. When the same analysis was performed including only parents with CJD or carrying the E200K mutation (n = 26), the difference between offspring and parents increased to 14.8 years., Conclusions: These results show that early age at death occurs in offspring of families carrying the E200K PRNP mutation and that this event is not linked to observational biases. Although molecular or environmental bases for this occurrence remain unsettled, this information is important for improving the accuracy of information to give to mutated carriers.

Background: The safety of plasma-derived products is of concern for possible transmission of variant Creutzfeldt-Jakob disease. The absence of validated screening tests requires the use of procedures to remove or inactivate prions during the manufacture of plasma-derived products to minimize the risk of transmission. These procedures need proper validation studies based on spiking human plasma or intermediate fractions of plasma fractionation with prions in a form as close as possible to that present in blood., Study Design and Methods: Human albumin was spiked with low-speed or high-speed supernatants of 263K scrapie-infected hamster brain homogenates. Spiked albumin was then passed through a cascade of filters from 100 nm down to 20 to 15 nm. Residual infectivity was measured by bioassay., Results: The overall removal of infectivity spiked into albumin through serial nanofiltration steps was 4 to 5 logs using low-speed supernatant and 2 to 3 logs with high-speed supernatant., Conclusion: These findings confirm the utility of nanofiltration in removing infectivity from plasma (or other products) spiked with scrapie brain homogenate supernatants. However, efficiency is diminished using supernatants that have been ultracentrifuged to reduce aggregated forms of the infectious agent. Thus, filtration removal data based on experiments using "standard" low-speed centrifugation supernatants might overestimate the amount of prion removal in plasma or urine-derived therapeutic products., (© 2011 American Association of Blood Banks.)

Prion diseases are neurodegenerative conditions associated with a misfolded and infectious protein, scrapie prion protein (PrP(Sc)). PrP(Sc) propagate prion diseases within and between species and thus pose risks to public health. Prion infectivity or PrP(Sc) presence has been demonstrated in urine of experimentally infected animals, but there are no recent studies of urine from patients with Creutzfeldt-Jakob disease (CJD). We performed bioassays in transgenic mice expressing human PrP to assess prion infectivity in urine from patients affected by a common subtype of sporadic CJD, sCJDMM1. We tested raw urine and 100-fold concentrated and dialyzed urine and assessed the sensitivity of the bioassay along with the effect of concentration and dialysis on prion infectivity. Intracerebral inoculation of transgenic mice with urine from 3 sCJDMM1 patients failed to demonstrate prion disease transmission, indicating that prion infectivity in urine from sCJDMM1 patients is either not present or is <0.38 infectious units/mL.

To evaluate whether growth discordance is an independent risk factor in the neonatal outcome of the smaller twin, all medical records of twin pregnancies delivered between 26 and 41 weeks during a 5-year period (January 2004-December 2008) were reviewed. Among the 49 selected twins, weight discordance was 15-20% in 7 infants, 21-30% in 16 infants, 31-40% in 16 infants and > 40% in 10 infants. No significant differences between the four groups were found with regards to obstetric complications and neonatal disease. Occurrence of birthweight below the 10th percentile and rate of admission to the neonatal intensive care unit significantly increased as intra-pair birthweight difference increased (p = .03). The > 40% discordant group had a significantly lower gestational age (p = .03), lower birthweight (p = .007) and a significantly higher mortality rate (4/10 versus 3/39 p = .04) in comparison with the other discordant groups. Multiple logistic regression analysis showed that birthweight was the single independent and consistent factor associated with elevated risks of mortality. For every 250 g increase in birthweight, the risk for mortality decreased by about 84% [RR 0.16(CI 0.00-0.70)]. Gestational age was the most reliable predictor for major neonatal complications. For every 1-week increase in gestational age a significant decreased risk for all outcomes was found. Discordance alone should not be considered as a predictor for adverse neonatal outcome. Neonatal outcome in discordant twins appears to be related to gestational age and birthweight rather than to the degree of discordance.

Background: The occurrence of transfusion transmissions of variant Creutzfeldt-Jakob disease (CJD) cases has reawakened attention to the possible similar risk posed by other forms of CJD., Study Design and Methods: CJD with a definite or probable diagnosis (sporadic CJD, n = 741; genetic CJD, n = 175) and no-CJD patients with definite alternative diagnosis (n = 482) with available blood transfusion history were included in the study. The risk of exposure to blood transfusion occurring more than 10 years before disease onset and for some possible confounding factors was evaluated by calculating crude odds ratios (ORs). Variables with significant ORs in univariate analyses were included in multivariate logistic regression analyses., Results: In the univariate model, blood transfusion occurring more than 10 years before clinical onset is 4.1-fold more frequent in sporadic CJD than in other neurologic disorders. This significance is lost when the 10-year lag time was not considered. Multivariate analyses show that the risk of developing sporadic CJD after transfusion increases (OR, 5.05) after adjusting for possible confounding factors. Analysis conducted on patients with genetic CJD did not reveal any significant risk factor associated with transfusion., Conclusion: This is the first case-control study showing a significant risk of transfusion occurring more than 10 years before clinical onset in sporadic CJD patients. It remains questionable whether the significance of these data is biologically plausible or the consequence of biases in the design of the study, but they counterbalance previous epidemiologic negative reports that might have overestimated the assessment of blood safety in sporadic CJD., (© 2010 American Association of Blood Banks.)

The voltage-activated sodium (Nav) channel Nav1.9 is expressed in dorsal root ganglion (DRG) neurons where it is believed to play an important role in nociception. Progress in revealing the functional properties and pharmacological sensitivities of this non-canonical Nav channel has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here, we use a protein engineering approach to dissect the contributions of the four Nav1.9 voltage sensors to channel function and pharmacology. We define individual S3b-S4 paddle motifs within each voltage sensor, and show that they can sense changes in membrane voltage and drive voltage sensor activation when transplanted into voltage-activated potassium channels. We also find that the paddle motifs in Nav1.9 are targeted by animal toxins, and that these toxins alter Nav1.9-mediated currents in DRG neurons. Our results demonstrate that slowly activating and inactivating Nav1.9 channels have functional and pharmacological properties in common with canonical Nav channels, but also show distinctive pharmacological sensitivities that can potentially be exploited for developing novel treatments for pain.

Rare neurodegenerative diseases are fatal and no therapy is available to cure or slow down the progression of disease. We report possibly weaknesses in the management of clinical studies in these diseases, ranging from poor preclinical studies, difficulties in the recruitment of patients, delay in the onset of treatment because of lack in early disease-specific biomarkers, and suboptimal design of Phase II clinical trials. The adoption of innovative statistical approaches in early Phase II trials might improve the screening of drugs in rare neurodegenerative disorders, but this implicates efforts from clinical researchers, statisticians, and regulatory people to the development of new strategies that should maintain rigorous scientific integrity together with a more ethical approach to human experimentations.

Three cases of cystic struma ovarii in women aged 16, 20 and 40 are described. All patients had an asymptomatic ovarian mass at ultrasound scan. The tumours, all of which were unilateral and confined to the ovary, ranged from 7 to 10 cm in the greatest dimension. Two lesions were unilocular, the third multilocular, and all were filled with green fluid. Microscopic examination showed cysts with fibrous wall lined by non-specific-appearing epithelial cells. In the wall of the cysts, there was a small number of thyroid follicles. In one case, an association with a cystic mature teratoma was seen. The paucity of thyroid follicles and the non-specific appearance of the epithelial cells required a careful sampling and immunohistochemical staining for thyroglobulin to establish an exact diagnosis. The postoperative period was uneventful and thyroid function remained normal. In conclusion, cystic struma is probably often underdiagnosed and should be considered when evaluating cystic ovarian tumours whose features are not obviously those of another tumour type. A careful search for thyroid follicles should be undertaken. In problematic cases immunohistochemical staining for thyroglobulin may be required.

The involvement of muscles in the pathogenesis of transmissible spongiform encephalopathies (TSEs) is irregular and unpredictable. We show that the TSE-specific protein (PrP(TSE)) is present in muscles of mice fed with a mouse-adapted strain of bovine spongiform encephalopathy as early as 100 days post-infection, corresponding to about one-third of the incubation period. The proportion of mice with PrP(TSE)-positive muscles and the number of muscles involved increased as infection progressed, but never attained more than a limited distribution, even at the clinical stage of disease. The appearance of PrP(TSE) in muscles during the preclinical stage of disease was probably due to the haematogenous/lymphatic spread of infectivity from the gastrointestinal tract to lymphatic tissues associated with muscles, whereas in symptomatic animals, the presence of PrP(TSE) in the nervous system, in neuromuscular junctions and in muscle fibres suggests a centrifugal spread from the central nervous system, as already observed in other TSE models.

GABA release by dopaminergic amacrine (DA) cells of the mouse retina was detected by measuring Cl- currents generated by isolated perikarya in response to their own neurotransmitter. The possibility that the Cl- currents were caused by GABA release from synaptic endings that had survived the dissociation of the retina was ruled out by examining confocal Z series of the surface of dissociated tyrosine hydroxylase-positive perikarya after staining with antibodies to pre and postsynaptic markers. GABA release was caused by exocytosis because 1) the current events were transient on the millisecond time scale and thus resembled miniature synaptic currents; 2) they were abolished by treatment with a blocker of the vesicular proton pump, bafilomycin A1; and 3) their frequency was controlled by the intracellular Ca2+ concentration. Because DA cell perikarya do not contain presynaptic active zones, release was by necessity extrasynaptic. A range of depolarizing stimuli caused GABA exocytosis, showing that extrasynaptic release of GABA is controlled by DA cell electrical activity. With all modalities of stimulation, including long-lasting square pulses, segments of pacemaker activity delivered by the action-potential-clamp method and high-frequency trains of ramps, discharge of GABAergic currents exhibited considerable variability in latency and duration, suggesting that coupling between Ca2+ influx and transmitter exocytosis is extremely loose in comparison with the synapse. Paracrine signaling based on extrasynaptic release of GABA by DA cells and other GABAergic amacrines may participate in controlling the excitability of the neuronal processes that interact synaptically in the inner plexiform layer.

Statins are potent inhibitors of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase in the cholesterol-biosynthesis pathway. They are either lipophilic (e.g. simvastatin) or hydrophilic [e.g. pravastatin (PRV)] compounds, considered mainly for long-term treatment of hypercholesterolaemic individuals. Beneficial effects of statins are not related exclusively to their lipid-lowering action; they also possess cholesterol-independent, pleiotropic effects (e.g. anti-inflammatory and antioxidant). Recent studies revealed that simvastatin treatment increased survival significantly in scrapie-infected mice. Although PRV treatment results in measurable drug levels in the mouse brain, the anti-prion effect of this compound has not been investigated. Therefore, we aimed to test the potential therapeutic action of PRV in a murine scrapie model. Our study showed that high-dose and long-term oral PRV treatment prolonged survival times of strain 139A scrapie-infected mice significantly (194 versus 177 days) in the absence of any obvious toxicity, suggesting that protective effects of statins may be independent of absolute solvent or water solubility of the drug.

Background: Nociceptive-selective local anesthesia is produced by entry of the permanently charged lidocaine-derivative QX-314 into nociceptors when coadministered with capsaicin, a transient receptor potential vanilloid 1 (TRPV1) channel agonist. However, the pain evoked by capsaicin before establishment of the QX-314-mediated block would limit clinical utility. Because TRPV1 channels are also activated by lidocaine, the authors tested whether lidocaine can substitute for capsaicin to introduce QX-314 into nociceptors through TRPV1 channels and produce selective analgesia., Methods: Lidocaine (0.5% [17.5 mM], 1% [35 mM], and 2% [70 mM]) alone, QX-314 (0.2% [5.8 mM]) alone, and a combination of the two were injected subcutaneously and adjacent to the sciatic nerve in rats and mice. Mechanical and thermal responsiveness were measured, as was motor block., Results: Coapplication of 0.2% QX-314 with lidocaine prolonged the nociceptive block relative to lidocaine alone, an effect attenuated in TRPV1 knockout mice. The 0.2% QX-314 alone had no effect when injected intraplantary or perineurally, and it produced only weak short-lasting inhibition of the cutaneous trunci muscle reflex. Perisciatic nerve injection of lidocaine with QX-314 produced a differential nociceptive block much longer than the transient motor block, lasting 2 h (for 1% lidocaine) to 9 h (2% lidocaine). Triple application of lidocaine, QX-314, and capsaicin further increased the duration of the differential block., Conclusions: Coapplication of lidocaine and its quaternary derivative QX-314 produces a long-lasting, predominantly nociceptor-selective block, likely by facilitating QX-314 entry through TRPV1 channels. Delivery of QX-314 into nociceptors by using lidocaine instead of capsaicin produces sustained regional analgesia without nocifensive behavior.

In vitro and in vivo studies have shown that phthalocyanine tetrasulfonate (PcTS), a cyclic tetrapyrrole compound, is an efficient antiscrapie drug. To investigate the spectrum of PcTS against prion diseases, we tested the effect of PcTS on two mouse-adapted human strains. We also tested PcTS in rodents infected with two scrapie strains (139A and 263K). PcTS treatment significantly prolonged mean survival times of all infected animals. These results show that PcTS is effective on different prion strains, confirming its potential use for prion therapy.

We assessed the role of the APOE genotype and prion protein polymorphism at codon 129 in predicting the clinical duration of 92 neuropathologically confirmed sporadic Alzheimer's disease patients. Analyses of survival showed that the absence of the APOE epsilon 4 allele in heterozygous codon 129 PRNP carriers is a negative predictor of survival. When this subgroup of patients was stratified by sex, the effect of APOE was observed in women, but not in men., ((c) 2008 S. Karger AG, Basel.)

Objective: To investigate how the mode of delivery affects the level oxidative stress in newborns., Design and Methods: 15-F(2t)-isoprostane, as index of oxidative stress, was measured in umbilical cord plasma samples from 37 infants born after vaginal delivery or caesarian section, using specific immuno-affinity extraction and immunoassay., Results: 15-F(2t)-isoprostane levels were higher in infants born after vaginal delivery (n=18) compared to those delivered by elective caesarian section (n=19)., Conclusions: 15-F(2t)-isoprostane is a sensitive biomarker of fetal oxidative stress during labor.

Purpose: To investigate the effect of excision repair cross-complementation group 1 (ERCC1) on treatment response and survival of patients treated with platinum chemotherapy with or without paclitaxel., Patients and Methods: Tumor samples from epithelial ovarian cancer patients were evaluated for ERCC1 mRNA expression and a single nucleotide polymorphism at codon 118 (C>T). Of 178 patients treated with postoperative platinum-based chemotherapy, 75 were also given paclitaxel. For all of these patients, ERCC1 expression and genotype were analyzed for associations with treatment response and survival., Results: Among the 103 patients treated with platinum without paclitaxel, the C/C genotype, compared with C/T and T/T, was associated with greater risk of disease progression and death (hazard ratio [HR], 1.95, P = .051; HR, 2.01, P = .033, respectively); high levels of ERCC1 mRNA, compared with low levels, were associated with greater risk of disease progression (HR, 2.41; P = .014). Similarly, when the ERCC1 data were combined, patients with the C/C genotype and high ERCC1 expression had greater risk for disease progression (HR, 3.73; P = .003) compared with those with low expression and non-C/C genotype. However, for the 75 patients treated with platinum plus paclitaxel, the C/C genotype and high ERCC1 expression were not associated with poor prognosis, suggesting that paclitaxel may help to alleviate ERCC1-related platinum resistance., Conclusion: Ovarian cancer patients with high ERCC1 expression or the C/C genotype at codon 118 may benefit from the combination of platinum and paclitaxel, while those with low ERCC1 expression or the C/T or T/T genotype may respond well to platinum without paclitaxel.

The aim of this study was to investigate whether attention may be specifically impaired in Alzheimer's disease from the early stages of the disease. Subgroups of patients with different types of mild cognitive impairment were selected according to standard criteria. Patients and controls were given tasks exploring various subcomponents of attention and executive functions. Only subgroups of mild cognitive impairment characterized by memory disorders obtained lower scores than controls on attention and executive tasks. On the basis of the scores obtained on the Clinical Dementia Rating at the 1-year follow-up, patients were redistributed into 2 groups: those who developed and those who did not develop dementia. Patients who presented evolution to dementia already had, at baseline, lower scores than patients who did not evolve on tasks exploring attention and executive functions. The results suggest that not only memory disorders but also attention/executive deficits may characterize dementia at the onset.

The purposes of this study were to analyze MDR1 expression in ovarian tumors prior to chemotherapy, to correlate the expression with p16, IGFs, ERalpha, and BRCA1, and to examine the association of MDR1 expression with ovarian cancer prognosis. A primary ovarian cancer cohort of 206 patients after surgery was followed up. MDR1, IGFs, ERalpha, p16, and BRCA1 expressions were analyzed in ovarian tumor samples using quantitative real-time PCR. MDR1 was detected in 177 of 206 specimens. MDR1 expression was positively correlated with IGFBP3, ERalpha, p16, and BRCA1, but not correlated with IGF-II, age, and other clinicopathological parameters. MDR1 expression significantly elevated the risk for disease progression (p = 0.02), and this association remained statistically significant after controlling for patient age and clinicopathological parameters or other correlated genes. No association was found between MDR1 expression and overall survival. MDR1 expression may be an independent marker for ovarian cancer progression and combination of different agents targeting different molecules may improve the outcome of ovarian cancer treatment and prevent drug resistance.

Objectives: The IGF-II gene has four promoters (P1-P4); each initiates promoter-specific transcription. Studies have shown that IGF-II promoters normally active during fetal growth, but silent in postnatal life, are reactivated in cancer. In a previous study, we found IGF-II transcription evaluated at a common translated region was associated with ovarian cancer progression. This study was conducted to further determine which IGF-II promoters were responsible for the association., Methods: Promoter-specific transcription at each IGF-II promoter was analyzed in 201 ovarian tumor samples using quantitative RT-PCR. Cox regression analysis was performed to determine the association of IGF-II promoter-specific expression with patient survival., Results: P3 and P4 transcripts were detected more frequently and at significantly higher levels than the transcripts of P1 and P2. P3 and P4 transcripts were strongly correlated with the common IGF-II translated region and were significantly higher in patients with late stage disease, large residual tumor, suboptimal debulking or serous histology compared to those with early stage, small residual tumor, optimal debulking or non-serous histology. Survival analysis showed that patients with high P3 or P4 expression had a 2-fold increase in risk for death compared to those with low P3 or P4. These associations remained significant after adjustment for patient age at surgery, disease stage, tumor grade and histology. P1 and P2 transcripts, however, were not associated with disease characteristics or survival., Conclusions: These findings suggest that IGF-II transcription from P3 and P4 promoters is important in ovarian cancer and evaluation of IGF-II promoter-specific transcription may have clinical implications in ovarian cancer prognosis and treatment.

Background: The objective of this study was to describe the diagnostic panorama of human transmissible spongiform encephalopathies across 11 countries., Methods: From data collected for surveillance purposes, we describe annual proportions of deaths due to different human transmissible spongiform encephalopathies in eleven EUROCJD-consortium countries over the period 1993-2002, as well as variations in the use of diagnostic tests. Using logistic models we quantified international differences and changes across time., Results: In general, pre-mortem use of diagnostic investigations increased with time. International differences in pathological confirmation of sporadic Creutzfeldt-Jakob disease, stable over time, were evident. Compared to their counterparts, some countries displayed remarkable patterns, such as: 1) the high proportion, increasing with time, of variant Creutzfeldt-Jakob disease in the United Kingdom, (OR 607.99 95% CI 84.72-4363.40), and France (OR 18.35, 95% CI 2.20-152.83); 2) high, decreasing proportions of iatrogenic Creutzfeldt-Jakob disease in France, (OR 5.81 95% CI 4.09-8.24), and the United Kingdom, (OR 1.54 95% CI 1.03-2.30); and, 3) high and stable ratios of genetic forms in Slovakia (OR 21.82 95% CI 12.42-38.33) and Italy (OR 2.12 95% CI 1.69-2.68)., Conclusion: Considerable international variation in aetiological subtypes of human transmissible spongiform encephalopathies was evident over the observation period. With the exception of variant Creutzfeldt-Jakob disease and iatrogenic Creutzfeldt-Jakob disease in France and the United Kingdom, these differences persisted across time.

Background: Methylation-mediated suppression of detoxification, DNA repair, and tumor suppressor genes has been implicated in cancer development and progression. Studies also have indicated that concordant methylation of multiple genes (methylator phenotypes), rather than a single gene, may predict cancer prognosis. The current study was designed to determine whether a methylator phenotype exists in ovarian cancer, whether methylation frequencies differ between malignant ovarian tumors and ovarian tumors with low malignant potential (LMP or borderline), and whether methylation of multiple genes affects patient survival., Methods: The current study included 234 consecutively diagnosed patients with either LMP (n = 19 patients) or malignant (n = 215 patients) ovarian tumors. DNA samples were extracted from fresh frozen tissues and were analyzed for methylation in the promoter region of 6 genes (p16, breast cancer 1 [BRCA1], insulin-like growth factor-binding protein 3 [IGFBP-3], glutathione S-transferase pi 1 [GSTP1], estrogen receptor-alpha [ER-alpha], and human MutL homologue 1 [hMLH1]) by using methylation-specific polymerase chain reaction analysis., Results: The frequencies of methylation in malignant tumors and LMP tumors were 0% and 0% for GSTP1, respectively; 9% and 0% for hMLH1, respectively; 21% and 5% for BRCA1, respectively; 42% and 21% for p16, respectively; 44% and 26% for IGFBP-3, respectively; and 57% and 42% for ER-alpha, respectively. A methylator phenotype was not detected, but a calculated methylation index (MI) that was based on the total number of genes methylated in each tumor was associated with ovarian cancer risk and progression. A higher MI was associated with malignant tumors (odds ratio, 10.11; 95% confidence interval [95% CI], 1.19-85.75) and disease progression (hazards ratio, 6.53; 95% CI, 1.39-30.65)., Conclusions: Although a methylator phenotype was not identified, the current results suggested that methylation of multiple genes may play an important role in ovarian cancer development and progression and may have clinical implications in prognosis.

Aim: Remifentanil hydrochloride is an ultra-short acting m-opioid receptor agonist. This study compared the use of remifentanil with that of fentanyl during elective supratentorial craniotomy in a target controlled infusion (TCI)-propofol anesthesia regimen and evaluated the quality of recovery from anesthesia., Methods: After written informed consent for this prospective study, 40 adult patients were randomly divided into 2 groups: in group F analgesia was provided with fentanyl 2-3 mg kg(-1) h(-1) and in group R with remifentanil 0.25 mg kg(-1) h(-1). Anesthesia was induced with thiopental and pancuronium bromide, and maintained with propofol-TCI, pancuronium, air and oxygen and fentanyl (group F) or remifentanil (group R), respectively. After tracheal intubation, infusion rate of remifentanil was reduced and then adjusted to maintain stable hemodynamics. Hemodynamics and recovery time were monitored for 60 min after surgery. Analgesic requirements, propofol intraoperative consumption, nausea and vomiting in postoperative period were monitored. Recovery was evaluated according to a modified Aldrete score., Results: Baseline hemodynamics were similar in both groups. Mean arterial pressure differed between the 2 groups (P<0.05) with the greatest decrease in group R during dura opening (P<0.001). Postoperative mean arterial pressure was higher in group R. Patients in group R exhibited a faster recovery. The incidence of nausea and vomiting was similar in the 2 groups. Noteworthy, there was a reduction in the amount of propofol used in group R., Conclusions: Remifentanil appears to be a reasonable alternative to fentanyl during elective surgery of supratentorial lesions.

Background: Oxidative stress is believed to be an early event and a key factor in Alzheimer's disease (AD) pathogenesis and progression. In spite of an intensive search for surrogate markers to monitor changes related to oxidative stress in the brain, there is as yet no consensus about which markers to use in clinical studies. The measurement of peripheral anti-oxidants is an alternative way of evaluating the involvement of oxidative stress in the course of the disease. Given the complexity of peripheral anti-oxidant defence, variations in the levels of individual anti-oxidant species may not fully reflect the overall capacity to fight oxidant conditions. We therefore chose to evaluate the total reductive capacity (herein defined as anti-oxidant capacity, AOC) in serum from control subjects and AD patients in order to study the association between peripheral anti-oxidant defence, cognitive impairment and patient survival., Methods: We measured the levels of AOC in serum samples from 26 cognitively normal controls and 25 AD patients (12 post-mortem confirmed) who completed the Cambridge Cognitive Assessment. Cognitive decline was assessed in a subgroup of 19 patients who underwent a second cognitive assessment 2 years after the initial visit., Results: Serum AOC levels were lower in AD patients than in controls and were correlated with their cognitive test scores, although AOC levels were unrelated to cognitive decline assessed two years later. On the other hand, AOC levels were predictive of the length of patients' survival, with higher levels giving longer survival., Conclusion: This study indicates that peripheral anti-oxidant defences are depleted in AD patients. The results suggest that serum AOC is a good index of the general health status and prognosis of patients but does not necessarily reflect the extent to which vulnerable neuronal populations are protected from oxidant processes. Further studies are required to establish whether peripheral AOC measurements may be useful in identifying asymptomatic individuals or those with early symptoms at high risk of developing significant cognitive impairment or dementia.

Purpose: The insulin-like growth factor (IGF) system plays important roles in cancer; blocking IGF signaling has been shown to have therapeutic effects on tumor growth. Many studies have focused on the effect of IGF-I, but few have addressed IGF-II. To assess the role of IGF-II in cancer, we analyzed IGF-II expression in ovarian cancer and examined its association with disease characteristics and prognosis., Experimental Design: Included in the study were 215 patients with primary epithelial ovarian cancer. Fresh tumor specimens were collected during surgery, and the patients were followed for a median of 31 months. Total RNA was extracted from the tumor and analyzed for IGF-II, IGF binding protein 3 (IGFBP-3), and estrogen receptor-alpha expressions using quantitative reverse transcription PCR. Survival analysis was done to examine the associations of IGF-II with disease progression., Results: IGF-II expression was found to be higher in tumors with poor prognosis; this included tumors with advanced stage, poor differentiation, serous histology, and large residual lesions. Patients with high IGF-II had elevated risk for disease progression and death, although the significance became less evident when the analysis was adjusted for clinical and pathologic variables. IGFBP-3 expression was higher in less aggressive tumors, but was not associated with disease progression. The expression of estrogen receptor-alpha had no effect on survival., Conclusion: This study found evidence that IGF-II expression is associated with disease progression, suggesting that IGF-II and IGF signaling are potential targets for ovarian cancer treatment. The study also indicates that IGF-II and IGFBP-3 have limited value in prognosis because of their strong associations with disease stage and tumor grade.

Background: Although epidemiological, clinical, and experimental evidence indicates that the inducible isoform of cyclo-oxygenase (COX-2) may be involved in the pathogenesis of several neurodegenerative disorders, the mechanisms whereby COX-2 contributes to Alzheimer's disease are largely unknown., Objective: To undertake a longitudinal study of CSF levels of a major product of COX activity, prostaglandin E2 (PGE2), in relation to cognitive decline and survival in patients with Alzheimer's disease., Methods: CSF PGE2 was measured on at least three annual visits in 35 controls and 33 Alzheimer patients (26 necropsy confirmed) who completed the Cambridge cognitive assessment (CAMCOG)., Results: Compared with controls, CSF PGE2 was higher in patients with mild memory impairment, but lower in those with more advanced Alzheimer's disease. The median survival time of patients with higher initial PGE2 levels was five years longer than those with lower levels., Conclusions: COX activity in Alzheimer's disease varies with stage of the disease. PGE2 levels correlate positively with patient survival. These findings suggest that inhibition of COX activity does not represent a major target for the pharmacological treatment of Alzheimer's disease.

Frontal variant-Frontotemporal dementia (fvFTD) and Alzheimer's disease (AD) patients matched for severity of dementia at the Clinical Dementia Rating (CDR) received neuropsychological testing in order to explore if the dysexecutive disorder might characterise fvFTD at early stage, when AD is dominated by the episodic memory defect. We also determined if the behavioural syndrome was more severe in fvFTD than AD, and if specific patterns of behavioural symptoms could differentiate the two types of dementia, using the Neuropsychiatry Inventory (NPI). AD patients performed worse than fvFTD not only in memory but also in executive tasks. Apathy and eating disorders proved to be more severe or frequent in fvFTD even if the two groups did not differ in the total NPI score. CDR score significantly correlated with the NPI score in fvFTD and with the MMSE in AD. Our data confirm that the memory disorders may differentiate the two types of dementia; however, the dysexecutive syndrome is as severe, and even more severe in AD. The severity of the behavioural syndrome is comparable in the two groups but the nature of the behavioural disorders may vary to some extent. We conclude that AD dementia at early stage is a behavioural-cognitive syndrome, while in fvFTD the behavioural disorders appear when the cognitive deficit is still relatively mild.

Ectopic mammary gland tissue in the vulva is an uncommon clinical or pathologic finding. Such ectopic tissue can be the site of the same physiologic and pathologic processes found in the normal breast. However, the occurrence of adenocarcinoma is very rare, the first case being reported by Greene in 1935. We here report the 16th case of primary "breast-like" cancer arising in the vulva, together with a critical review of the literature, in order to highlight the dilemmas of a clinical approach to this neoplasm. Clear guidelines for diagnosis and therapy are still unavailable. The main diagnostic criteria suggested by the authors of previous reports are discussed together with our own findings. The therapeutic approach to this rare malignancy is also critically reviewed. In our opinion, when diagnosis of breast-like vulvar cancer is finally confirmed, treatment and follow-up should be the same as that would be chosen in a case of orthotopic breast neoplasm.

A total of 10-15% of human transmissible spongiform encephalopathies (TSEs) or prion diseases are characterised by disease-specific mutations in the prion protein gene (PRNP). We examined the phenotype, distribution, and frequency of genetic TSEs (gTSEs) in different countries/geographical regions. We collected standardised data on gTSEs between 1993 and 2002 in the framework of the EUROCJD collaborative surveillance project. Our results show that clinicopathological phenotypes include genetic Creutzfeldt-Jakob disease (gCJD), fatal familial insomnia (FFI), and Gerstmann-Sträussler-Scheinker disease (GSS). Genetic TSE patients with insert mutation in the PRNP represent a separate group. Point and insertional mutations in the PRNP gene varies significantly in frequency between countries. The commonest mutation is E200K. Absence of a positive family history is noted in a significant proportion of cases in all mutation types (12-88%). FFI and GSS patients develop disease earlier than gCJD. Base pair insertions associated with the Creutzfeldt-Jakob disease (CJD) phenotype, GSS, and FFI cases have a longer duration of illness compared to cases with point mutations and gCJD. Cerebrospinal fluid 14-3-3 immunoassay, EEG, and MRI brain scan are useful in the diagnosis of CJD with point mutations, but are less sensitive in the other forms. Given the low prevalence of family history, the term "gTSE" is preferable to "familial TSE". Application of genetic screening in clinical practice has the advantage of early diagnosis and may lead to the identification of a risk of a TSE.

We examined the electrophysiological properties of a population of identified dopaminergic periglomerular cells of the main olfactory bulb using transgenic mice in which catecholaminergic neurons expressed human placental alkaline phosphatase (PLAP) on the outer surface of the plasma membrane. After acute dissociation, living dopaminergic periglomerular cells were identified by a fluorescently labeled monoclonal antibody to PLAP. In current-clamp mode, dopaminergic periglomerular cells spontaneously generated action potentials in a rhythmic fashion with an average frequency of 8 Hz. The hyperpolarization-activated cation current (Ih) did not seem important for pacemaking because blocking the current with ZD 7288 or Cs+ had little effect on spontaneous firing. To investigate what ionic currents do drive pacemaking, we performed action-potential-clamp experiments using records of pacemaking as voltage command in voltage-clamp experiments. We found that substantial TTX-sensitive Na+ current flows during the interspike depolarization. In addition, substantial Ca2+ current flowed during the interspike interval, and blocking Ca2+ current hyperpolarized the neurons and stopped spontaneous firing. These results show that dopaminergic periglomerular cells have intrinsic pacemaking activity, supporting the possibility that they can maintain a tonic release of dopamine to modulate the sensitivity of the olfactory system during odor detection. Calcium entry into these neurons provides electrical drive for pacemaking as well as triggering transmitter release.

We analysed the effects of 192 IgG-saporin lesions on pnd 7 upon neonatal behavioural responses. Number of ultrasonic vocalisations (USVs) were recorded on pnds 9, 11 and 13. On pnd 13 rats underwent a homing test to measure olfactory orientation towards nest material. 192 IgG-saporin reduced the number of USVs at all ages considered while increasing number of entrancies into the nest area. These data suggest that early damage to the basal forebrain cholinergic nuclei induces significant changes in the rat behavioural repertoire as early as the second-postnatal week.

Objective: To assess the incidence and mortality rates of genetic transmissible spongiform encephalopathy (TSE) diseases in Italy., Methods: The authors have sequenced the prion protein gene (PRNP) in 643 patients referred to the Italian Registry of Creutzfeldt-Jakob disease (CJD) and related disorders between 1993 and 2002. Crude age- and sex-specific incidence and mortality rates were calculated. Differences in morbidity from genetic TSE diseases in the 20 Italian regions were assessed by the standardized morbidity ratio (SMR)., Results: A total of 130 cases were classified as genetic TSE diseases with a mean yearly incidence rate of 0.28 cases per million people. Genetic TSE diseases represent 17.7% of all TSE diseases, including sporadic, iatrogenic, and variant CJD. The most frequent mutation was the V210I (n = 54), and the second most common the E200K (n = 42). Mortality rates for genetic TSE diseases did not increase in any of the age groups under examination over the 10 years of surveillance. The analysis of regional distribution of genetic cases by place of birth revealed that in Campania and Calabria regions the number of genetic TSE cases was higher than in other regions., Conclusions: In Italy the incidence of genetic transmissible spongiform encephalopathy (TSE) diseases is the second highest among European countries. Genetic analysis is important for a correct classification of patients with TSE.

Background: An international study of the epidemiologic characteristics of Creutzfeldt-Jakob disease (CJD) was established in 1993 and included national registries in France, Germany, Italy, the Netherlands, Slovakia, and the United Kingdom. In 1997, the study was extended to Australia, Austria, Canada, Spain, and Switzerland., Methods: Data were pooled from all participating countries for the years 1993 to 2002 and included deaths from definite or probable CJD of all etiologic subtypes., Results: Four thousand four hundred forty-one cases were available for analysis and included 3,720 cases of sporadic CJD, 455 genetic cases, 138 iatrogenic cases, and 128 variant cases. The overall annual mortality rate between 1999 and 2002 was 1.67 per million for all cases and 1.39 per million for sporadic CJD. Mortality rates were similar in all countries. There was heterogeneity in the distribution of cases by etiologic subtype with an excess of genetic cases in Italy and Slovakia, of iatrogenic cases in France and the UK, and of variant CJD in the UK., Conclusions: This study has established overall epidemiologic characteristics for Creutzfeldt-Jakob disease (CJD) of all types in a multinational population-based study. Intercountry comparisons did not suggest any relative change in the characteristics of sporadic CJD in the United Kingdom, and the evidence in this study does not suggest the occurrence of a novel form of human bovine spongiform encephalopathy infection other than variant CJD. However, this remains a possibility, and countries currently unaffected by variant CJD may yet have cases.

Unlabelled: To evaluate the epidemiology of pulmonary candidiasis (PC) and to identify risk factors in premature infants during the 1st month of life, all infants with a birth weight <1250 g admitted to our neonatal intensive care unit with PC between January 1994 and December 2001 were retrospectively reviewed. Infants with PC ( n =20) were compared with a control group ( n =20), matched for gestational age and birth weight, with regard to possible perinatal and postnatal risk factors. Among 325 infants with a birth weight <1250 g, 20 out of 233 ventilated infants (8.6%) developed PC. Candida albicans ( n =12) and C. parapsilosis ( n =4) were the predominant isolates. Neonates with PC were significantly different from controls with regard to male prevalence ( P =0.002), rates of preterm premature rupture of membranes (PPROM) ( P =0.02), longer duration of antibiotic therapy ( P =0.01) and of ventilation ( P =0.02). The difference between groups did not attain significance with regard to postnatal dexamethasone administration, duration of central vein catheterisation and duration of parenteral nutrition. Multivariate logistic regression analysis indicated as significant predictors of PC, among perinatal data, the male gender (OR =26.3; 95%CI 2.44 to 284) and PPROM (OR =12.3; 95%CI 1.16 to 130) and, among postnatal data, the duration of ventilation (OR =1.54; 95%CI 1.01 to 2.34)., Conclusion: The presence of preterm premature rupture of membranes and the duration of ventilation are significant risk factors for developing pulmonary candidiasis and should be considered in the preventive efforts to reduce this disease in infants with a birth weight <1250 g.

Background and Objectives: Endoscopic procedures are the gold standard for the diagnosis of esophageal varices but these invasive methods are complex to perform in hemophilic patients co-infected with hepatitis C virus/human immunodeficiency virus (HCV/HIV). Real-time ultrasonography has been reported to be an effective, non-invasive procedure able to monitor patients with chronic liver disease and to give useful information for the diagnosis of liver cirrhosis, portal hypertension and the presence of esophageal varices., Design and Methods: Seventy patients with severe hemophilia were evaluated by esophago-gastro-duodenoscopy (EGDS) and ultrasonography; 40 had HCV/HIV co-infection and 30, comparable for age and HCV exposure time, were HCV+/HIV-. Hepatic longitudinal diameter, splenic longitudinal diameter, portal vein diameter and the average speed of portal flow were measured. The congestion index was calculated., Results: Thirteen out of 40 (32.5%) HCV/HIV coinfected patients had esophageal varices. None out of 30 HCV+/HIV- patients had esophageal varices (p< 0.001). Univariate analysis showed that the 13 HCV/HIV coinfected patients with esophageal varices had significantly higher hepatic longitudinal diameter (p=0.006), splenic longitudinal diameter (p=0.0002), portal vein diameter (p=0.0005) and congestion index (p=0.0001) than did the remaining 27 HCV/HIV coinfected patients. The stepwise logistic regression analysis indicated that, of the various ultrasonographic parameters evaluated, splenic longitudinal diameter and portal vein diameter had the greatest diagnostic efficiency in diagnosing a high proportion of patients with esophageal varices. The diagnostic efficiency of the combined criterion expressed by the area under the ROC curve was 0.8803. Interpretation and Conclusions. Real-time ultrasonography, by evaluation of splenic longitudinal diameter and portal vein diameter, is an effective non-invasive technique able to classify correctly a large proportion of HCV/HIV co-infected hemophilic patients with esophageal varices.

Creutzfeldt-Jakob Disease (CJD) is a rare pathology (about 1 case per million) but it has a great importance for Public Health; the Italian National CJD register has been established in the Istituto Superiore di Sanita (ISS) since 1993, and epidemiological studies on CJD have been carried out as well. This paper reports a mortality study carried out comparing and integrating data from the two available sources: the National CJD Register and the Italian Data Base on Mortality, processed by the ISS Statistics Unit, on the data collected by the Italian Census Bureau (ISTAT). The study allowed to estimate: the underreporting of CJD mortality to both sources, the misclassification of ISTAT data and the integrated mortality rates from CJD in Italy: 1.58 per million persons aged 25 or more, average rate during the period 1993-1999.

To evaluate the ability of the Italian Creutzfeldt-Jakob (CJD) register to detect human transmissible spongiform encephalopathy (TSE) cases we compared mortality data from the CJD register with those obtained from death certificates collected by the Italian National Census Bureau (ISTAT) between 1993 and 1999. We used the method of record linkage to compare and integrate data from these two sources. The integrated estimate of TSE deaths was 457: 183 deaths recorded by the CJD register and ISTAT, 210 cases only by the CJD register, and 64 cases only by ISTAT. The average integrated estimated mortality rate was 1.58 deaths per million people per year over the study period and peaked in 1999 at 2.13. This figure is similar to that obtained from data from the CJD register alone in the years 2000-2002. The increase in mortality rates is likely due to an improvement in case ascertainment. The misclassification of cases by ISTAT was above 50% from 1996 onward, suggesting that using only death certificates is not a reliable way to monitor TSE cases in Italy.

The aim of our study was to evaluate whether the replicate PCR testing may provide more accurate estimates of C. pneumoniae DNA prevalence in PBMC of patients undergoing carotid endarterectomy. Clinical sensitivity and reproducibility of ompA nested touchdown PCR was also performed. Clinical sensitivity and reproducibility was examined by testing C. pneumoniae-negative PBMC spiked with serial dilutions of semipurified C. pneumoniae elementary bodies (from 8 to 0.002 IFU/ml). Detection of C. pneumoniae DNA was performed by ompA nested touchdown PCR. Each clinical and spiked PBMC DNA specimen was analyzed in replicates of 1, 3, 5 and 10. PCR results of serial dilutions of C. pneumoniae DNA performed in replicates of 10 were analysed by probit analysis. C. pneumoniae DNA was detected in 14 of the 30 (46.7 %) PBMC clinical specimens examined when 10 replicates were tested. When we analyzed 1, 3 and 5 replicates, 4 (13.3 %), 7(23.3 %), 12(40 %) of the 30 specimens were positive, respectively. The limit of detection of ompA nested PCR touchdown was 0.008 IFU/ml when 10 replicates were tested. The ompA nested PCR had reproducibility scores of 10 for 10 from 8 to 4 IFU/ml concentration, but scores decreased for smaller numbers of IFU/ml. Our results showed that repeat testing of the same specimen increased clinical sensitivity as well as reproducibility of the ompA nested touchdown PCR. In conclusion the replicate PCR testing improves the performance of ompA nested touchdown PCR and provides a more accurate estimates of the prevalence of C. pneumoniae in PBMC of patients with atherosclerotic cardiovascular disease.

Zidovudine (AZT) is the main therapeutic agent against HIV vertical transmission and is routinely administered to seropositive pregnant women and their newborns. Toxicity after chronic administration as well as citogenetic effects following developmental AZT exposure has been reported. Furthermore, recent animal data indicate alterations of several behavioral endpoints during the entire lifespan of mice and rats after developmental AZT exposure. In this study, we investigated specific central nervous system (CNS) effects of AZT administration during pregnancy on the offspring. CD-1 mouse females were administered twice daily from day 10 of pregnancy until delivery with either AZT (160 mg/kg) or saline (0.9% NaCl). On PND, 60 male offsprings received an intraperitoneal injection of the D1 receptor agonist 2,3,4,5-tetra-hydro-7,8-diol-1-phenyl-(1H)-3-benzazepine (SKF 38393) (0, 3, and 10 mg/kg), and spontaneous behavior was assessed in an automated activity chamber for 40 min. At variance from what observed in control mice that displayed excessive grooming when administered the higher dose of the D1 agonist, SKF 38393 failed to increase duration of grooming in AZT-treated mice. These data suggest that the D1 receptorial dopaminergic subsystem might be hyporesponsive in mice prenatally exposed to AZT.

We examined the behavior of three inbred mouse strains (129/SvPasIco, C57BL/6J, and DBA/2J) exposed to an object soaked with the chemical component of the aversive scent (toluquinone odor) emitted by a myriapod species (Ommatoiulus sabulosus) in the presence of a predator. Subjects were exposed to the odor for three consecutive days. Behavioral responses to the toluquinone odor were characterized both by an approach phase of risk assessment and by a repeated series of approach-avoid episodes. Results indicate that toluquinone exposure reduced completely, and in a strain independent fashion, selected behaviors such as crouching, catching and eating object. Other responses were strain-dependent: the DBA (DBA/2J) strain displayed defensive burying at high levels, C57 (C57BL/6J) mice performed high levels of withdrawal while the 129/Sv (129/SvPasIco) strain showed also high levels of stretch attend posture. Compared to other tasks, this test is ethological, simple, cheap and is not affected by strain differences in appetitive-sensory responses, as shown by some strain-independent responses. These features make this task as a good complement to any exploration-anxiety test battery.

A collaborative study of human transmissible spongiform encephalopathies has been carried out from 1993 to 2000 and includes data from 10 national registries, the majority in Western Europe. In this study, we present analyses of predictors of survival in sporadic (n = 2304), iatrogenic (n = 106) and variant Creutzfeldt-Jakob disease (n = 86) and in cases associated with mutations of the prion protein gene (n = 278), including Gerstmann-Sträussler-Scheinker syndrome (n = 24) and fatal familial insomnia (n = 41). Overall survival for each disease type was assessed by the Kaplan-Meier method and the multivariate analyses by the Cox proportional hazards model. In sporadic disease, longer survival was correlated with younger age at onset of illness, female gender, codon 129 heterozygosity, presence of CSF 14-3-3 protein and type 2a prion protein type. The ability to predict survival based on patient covariates is important for diagnosis and counselling, and the characterization of the survival distributions, in the absence of therapy, will be an important starting point for the assessment of potential therapeutic agents in the future.

Oxidative stress leads to lipid peroxidation and may contribute to the pathogenesis of lesions in multiple sclerosis (MS), an autoimmune disease characterised by inflammatory as well as degenerative phenomena. We previously found that cerebrospinal fluid (CSF) levels of isoprostane 8-epi-PGF2alpha, a marker of free radical damage and lipid peroxidation in vivo, were elevated in MS patients. Such levels were correlated with the degree of disability and reduced in subjects under steroid therapy. Here we investigated weather the CSF isoprostane levels correlated with disease inflammatory activity. To this aim, we enrolled 41 relapsing-remitting (RR) MS patients who underwent at the same time full neurological examination, NMR-imaging brain scan and diagnostic CSF test. No evidence of correlation was found between 8-epi-PGF2alpha levels and the presence of gadolinium (Gd)-enhancing NMR lesions or the time elapsed since the last relapse. We suggest that isoprostanes are not useful as surrogate inflammatory markers in MS. However, they may represent a sensitive index of degenerative phenomena, which can persist also in the absence of inflammatory activity.

In ethological and behavioural toxicological studies, elaborate behavioural patterns shown by the animals under well-established experimental paradigms or naturalistic conditions are routinely observed and split into single behavioural items. Subsequently, these items are analysed in terms of their frequencies and/or durations. Behavioural observations are usually videotaped and scored by dedicated softwares, which collect the sequences of behavioural items together with frequencies and durations. So far, the Cox proportional hazards model, a method originally developed for the analysis of time-to-event data, has been employed for the analysis of the time-structure of behaviour, but its usefulness has been limited because of difficulties in including random effects in the model. Recent developments in mixed models for the analysis of time-to-event data may overcome this limitation and improve the analysis of behavioural patterns. Data from social interactions in mice on the effects of exposure to chlorpyrifos, a widely used organophosphorous pesticide, are presented to illustrate the use of these new statistical methods. Our results suggest that the study of behavioural sequences may highlight the role of the investigated conditions (treatments, genetic condition, social status) in setting behavioural organisations. In addition, the refinement of statistical methods by time-structured analysis provides more detailed information from an experimental data set, thus contributing to the reduction of the number of animals used in this field of the life sciences.

In the retina, dopamine plays a central role in neural adaptation to light. Progress in the study of dopaminergic amacrine (DA) cells has been limited because they are very few (450 in each mouse retina, 0.005% of retinal neurons). Here, we applied transgenic technology, single-cell global mRNA amplification, and cDNA microarray screening to identify transcripts present in DA cells. To profile gene expression in single neurons, we developed a method (SMART7) that combines a PCR-based initial step (switching mechanism at the 5' end of the RNA transcript or SMART) with T7 RNA polymerase amplification. Single-cell targets were synthesized from genetically labeled DA cells to screen the RIKEN 19k mouse cDNA microarrays. Seven hundred ninety-five transcripts were identified in DA cells at a high level of confidence, and expression of the most interesting genes was confirmed by immunocytochemistry. Twenty-one previously undescribed proteins were found in DA cells, including a chloride channel, receptors and other membrane glycoproteins, kinases, transcription factors, and secreted neuroactive molecules. Thirty-eight percent of transcripts were ESTs or coding for hypothetical proteins, suggesting that a large portion of the DA cell proteome is still uncharacterized. Because cryptochrome-1 mRNA was found in DA cells, immunocytochemistry was extended to other components of the circadian clock machinery. This analysis showed that DA cells contain the most common clock-related proteins.

We examined the influence of prenatal stress on 3,4-methylenedioxymethamphetamine (MDMA, 5 mg/kg p.o.) pharmacokinetics in adolescent female SD rats (30 days). Our results indicate that the metabolic rate of MDMA was higher in the prenatal stress group than in the control group. Moreover, MDMA-induced motor alterations were increased in prenatally stressed rats. These findings provide evidence that (i) prenatal stress increases sensitivity to MDMA, (ii) these effects are already detectable at the adolescent stage and (iii) early differences in metabolism may play a role in the behavioural changes associated with this drug of abuse.

The identification of new molecular prognostic and predictive factors for ovarian cancer may contribute in deciding individual therapeutic strategies; on the other hand, there has been growing interest in new biologic therapies to correct molecular or genic lesions of neoplastic cells (genic therapy), or to activate the specific immune response (immunological therapy). Chemotherapy collateral toxic effects, as myelotoxicity, should be reduced through transfection of genes that modulate drug resistance in stem cells. The data at present available suggest then the potential role of these new treatments, are more specific and less toxic than current therapies; however, other biological-molecular studies are required to obtain the clinical applications of the results: Aim of this study is to provide a review of the most interesting data in ovarian cancer biologic therapy.

Infant rodents emit ultrasonic vocalizations (USVs) when isolated from the mother and littermates. While USVs reportedly promote maternal behavior, their communicatory role is still controversial. We performed a detailed behavioral characterization in 7-day-old CD-1 Swiss mice to describe (a) behavioral patterns accompanying or immediately preceding or following the USV and (b) how selected USV features are modulated according to the behavioral pattern being performed. The present results represent an ethogram of mouse pup behavior in close temporal proximity to USV, providing further information to evaluate the functional role of USVs in the framework of the mother-offspring relationship. A significant increase of head rising behavior immediately prior to the USV and a behavioral activation displayed by pups in conjunction with it suggest that pups behave in a way that may favor interaction with their mothers., (Copyright 2003 Wiley Periodicals, Inc. Dev Psychobiol 44: 37-44, 2004.)

For ethical reasons, the least number of animals possible should be used in biomedical research, though not so few as to fail to detect biologically important effects or to necessitate the repetition of experiments. We describe biostatistical approaches that can contribute to either reducing the number of animals in single experiments or to increasing the quality of studies so that fewer subsequent studies (and thus animals) will be needed. The described approaches regard different phases of experimentation, specifically: planning the experimental design and calculating the sample size, controlling variability, choosing the response variable, postulating the statistical hypothesis to be tested, choosing the procedure for analysing data, and interpreting and suitably presenting the results.