Sensitive and quantitative method to evaluate DNA methylation of the positive regulatory domains (PRDI, PRDII) and cAMP response element (CRE) in human endothelial nitric oxide synthase promoter.

Nitric oxide plays a prominent role in the cardiovascular system and much attention has been devoted in the last years on deciphering the regulation of human endothelial nitric oxide synthase (eNOS) expression. Epigenetic-based mechanisms have a key role in the eNOS expression and their pathologic perturbations may have profound effects on the steady state RNA levels in the endothelium. The human eNOS promoter lacks a canonical TATA box and it does not contain a proximal CpG island. A differentially DNA methylated region (DMR) in the native eNOS proximal promoter is involved in gene expression regulation. Here we describe a quantitative, sensitive and cost-effective method that, relying on a novel normalization strategy, allows the quantification of DNA methylation status of the positive regulatory domains (PRDI, PRDII) and cAMP response element (CRE) in human eNOS promoter. This technique will enable to explore the functional relevance of DNA methylation perturbations of eNOS promoter both under pathological and physiological conditions.

Two siblings affected by Netherton/Comèl syndrome. Diagnostic pathology and description of a new SPINK5 variant.

Netherton syndrome is a severe, autosomal recessive form of ichthyosis associated with mutations in the SPINK5 gene encompassing three main clinical findings: 1) ichthyosiform dermatitis and/or ichthyosis linearis circumflexa, 2) hair shaft defects with peculiar "trichorrhexis invaginata" (bamboo pole hair) findings, 3) atopic dermatitis. We describe two siblings affected by Netherton/Comèl syndrome who were referred to our Center for Genodermatosis. A diagnostic pathway and the description of a new SPINK5 variant has been determined for these two patients. A novel genetic mutation has been found.

Management of treatment-naïve chronic hepatitis C genotype 1 patients: a cost-effectiveness analysis of treatment options.

New and more promising therapies for chronic hepatitis C (CHC) genotype 1 (G1) naive patients have recently been approved in the United States and Europe, and several more regimens are expected to become available within the next several years. While this scenario unfolds, it is necessary to develop a rational method to allocate current treatment in CHC G1 patients. We performed a cost-effectiveness analysis of boceprevir (BOC)- and telaprevir (TVR)-based triple therapy according to different patients' selection strategies. A semi-Markov model of CHC natural history and progression towards end-stage liver disease was built. We considered 3 selection strategies based on METAVIR fibrosis stage: (i) treat all patients with F1-F4 fibrosis, (ii) only F2-F4 and (iii) only F3-F4. For each strategy, TVR interleukin-28B-guided (IL28B-guided) and BOC rapid virologic response-guided (RVR-guided) therapies were applied. The model assessed the costs and outcomes, using a lifetime and 5-year time horizon, and adopting the Italian National Health System perspective. The incremental cost-effectiveness ratio (ICER) for F1-F4 strategy relative to F3-F4 was €5132 per quality-adjusted life years gained, across TVR IL-28B-guided therapy, and €7042 in the BOC RVR-guided therapy. Conversely, in the 5-year scenario, the ICER for F1-F4 strategy relative to F3-F4 was €1 818 679 (TVR IL28B-guided) and €1 866 437 (BOC RVR-guided) per end-stage liver disease or death (ESLD-D) avoided. In view of anticipated improvement in the efficacy of future regimens, selective treatment of only patients with advanced fibrosis and cirrhosis with TVR or BOC could represent the most cost-effective strategy to optimize resource utilization.

Antiangiogenic therapy inhibits the recruitment of vascular accessory cells to the perivascular niche in glioma angiogenesis.

OBJECTIVE: Antiangiogenic therapies could be limited by various escape mechanisms including bone marrow-derived myeloid cell-induced vasculogenesis. The recruitment of vascular accessory cells (VACs) to the tumor neovasculature is as a multistep process. However, the recruitment process of these cells during antiangiogenic treatment remains unknown. The aim of our study was to characterize the recruitment of VACs during antiangiogenic therapy using sunitinib. METHODS: C6 glioma cells were implanted into dorsal skinfold chambers. Animals received antiangiogenic therapy intraperitoneally for 5 days prior to VAC application intra-arterially. Intravital microscopy was performed during VAC injection and 1 and 48 h after injection. Analyses included total (TVD) and functional vessel densities (FVD), the perfusion index (PI), microvascular permeability, blood flow rate (Q), microvascular diameter (D), red blood cell velocity (RBCV), wall shear rate (γ), wall shear stress (τ), first and firm adhesions of VACs, and accumulation in the perivascular niche. RESULTS: Antiangiogenic therapy resulted in a significant reduction in TVD (365 ± 47 cm/cm(2) vs. 183 ± 37 cm/cm(2)) and FVD (227 ± 65 cm/cm(2) vs. 147 ± 25 cm/cm(2)) and an increase in PI, Q, D, and RBCV. γ and τ remained unaltered. Initial adhesion and firm adhesion were unaffected by antiangiogenic therapy; however, the accumulation in the perivascular niche was significantly diminished in treated tumors (53.7 ± 8% vs. 24.0 ± 17%). CONCLUSIONS: Antiangiogenic treatment inhibits the accumulation of VACs in the perivascular niche and therefore interferes with consecutive neovascularization.

Comparative multiplex dosage analysis in spinocerebellar ataxia type 2 patients.

We developed a new application of comparative multiplex dosage analysis (CMDA) for evaluation of the ataxin 2 gene. Expansions of the triplet CAG can cause spinocerebellar ataxia type 2 (SCA2), a neurodegenerative disease with an autosomal-dominant mode of inheritance. Molecular diagnosis of SCA2 is routinely based on the use of conventional PCR to detect the CAG expansion. However, PCR does not amplify an allele with an expansion of many triplets (>80), which is typically found in infantile and juvenile forms of SCA2, thus leading to false negatives. We propose the analysis of the ATXN2 gene by CMDA to complement existing methods currently used for the detection of large expansions of the CAG repeat. Using CMDA, the presence of any longer mutated allele in a heterozygous patient or fetus would be inferred due to dosage variation of the very frequent normal allele #22. CMDA can be completed in 1 day, at very low cost, and would be a useful tool for prenatal diagnosis and for diagnosis of presymptomatic forms of early-onset SCA2.

[Best practice in risk management of Neisseria meningitidis bacterial meningitis at nurseries]. / Best practice nel risk management della meningite batterica da Neisseria meningitidis negli asili.

In many contexts is often underestimated the biological risk and schools can be an example. Proof of this is the exclusion of work in the school from the example of the work activities of biohazard included in Annex XLIV of Legislative Decree 81/08. Our work proposes a protocol for risk management meningitis contagious in the specific environment of the nursery taking a cue from the specific experience gained by the Service of Prevention and Protection Bambino Gesù Children's Hospital following the course accreditation Joint Commision. This is primary prevention measures (training and information) and secondary (vaccination, reporting of suspected cases, chemoprophylaxis of contacts, contact tracing, counseling) to be applied consistently even and especially in the absence of sick people, at which time the shares are aimed exclusively to control its spread to be taken and monitored, with the cooperation of all subject involved in various capacities in the protection of the health of workers, within these specific working environments.

Prolonged in-hospital exposure to an infant with active pulmonary tuberculosis.

Active pulmonary tuberculosis was diagnosed in a 4-month-old infant 16 days after hospitalization; 186 exposed individuals were traced and one conversion detected. Although the risk of tuberculosis transmission in paediatric hospitals is low, paediatricians in low-incidence countries should maintain a high level of alert for timely identification of cases.

An atypical presentation of diffuse midline pontine glioma in a middle age patient: Case report.

INTRODUCTION: Diffuse midline glioma is a newly WHO defined entity (grade IV) (Louis et al., 2016) which includes diffuse intrinsic pontine glioma (DIPG) reported in pediatric population and, occasionally, in young adults. Here, we present a detailed description of an atypical case of diffuse midline glioma in a 53 years old woman. CASE REPORT: A caucasian woman aged 53 from Ukraine, was referred to another neurological department complaining of 3 months history of progressive postural instability and gait impairment with frequent falling. Magnetic resonance demonstrated two brainstem lesions, hyperintense in FLAIR with "patchy" peripheral enhancement, leptomeningeal and cranial nerves enhancement. CSF was normal. Due to positive antinuclear antibodies test (ANA 1:360), intravenous steroid treatment was administered and reported to initially improve the patient condition. However, the following weeks the lady worsened. Imaging features were unchanged. Because quantiferon test resulted positive, MRI-Spectroscopy showed an inflammatory pattern and MRI perfusion study and brain FDG-PET, were normal, tubercolar granulomatous hypothesis was initially favored. Antitubercular therapy with isoniazid, pyrazinamide, ethambutol and rifampicin was started without any clinical improvement. Hence, the biopsy was proposed. The procedure revealed a diffuse midline pontine glioma. Considering the advanced stage of the disease, radiotherapy was not indicated. Patient died after eight months from the onset of neurological disturbances. CONCLUSION: Our case shows that diffuse midline glioma is a CNS tumor not limited to young population but occurring also in middle aged patients with an insidious pattern. We therefore recommend to perform biopsy at very early stages in patients with atypical brainstem lesions.

Spinal ceramide modulates the development of morphine antinociceptive tolerance via peroxynitrite-mediated nitroxidative stress and neuroimmune activation.

The effective treatment of pain is typically limited by a decrease in the pain-relieving action of morphine that follows its chronic administration (tolerance). Therefore, restoring opioid efficacy is of great clinical importance. In a murine model of opioid antinociceptive tolerance, repeated administration of morphine significantly stimulated the enzymatic activities of spinal cord serine palmitoyltransferase, ceramide synthase, and acid sphingomyelinase (enzymes involved in the de novo and sphingomyelinase pathways of ceramide biosynthesis, respectively) and led to peroxynitrite-derive nitroxidative stress and neuroimmune activation [activation of spinal glial cells and increase formation of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6]. Inhibition of ceramide biosynthesis with various pharmacological inhibitors significantly attenuated the increase in spinal ceramide production, nitroxidative stress, and neuroimmune activation. These events culminated in a significant inhibition of the development of morphine antinociceptive tolerance at doses devoid of behavioral side effects. Our findings implicate ceramide as a key upstream signaling molecule in the development of morphine antinociceptive tolerance and provide the rationale for development of inhibitors of ceramide biosynthesis as adjuncts to opiates for the management of chronic pain.

Biventricular pacing concomitant to on-pump heart surgery: a case series.

AIM: The aim of this paper was to report the authors' experience on biventricular epicardial pacing (BEP) as first-choice procedure concomitant to on-pump heart surgery for other definite indications. METHODS: BEP was performed in 13 consecutive patients with stage IV heart failure (HF) undergoing on-pump cardiac surgery for other definite indications. All patients were treated with optimized pharmacologic therapy, and showed complete left bundle branch block and reduced (<30%) left ventricular ejection fraction. RESULTS: In all patients, effective BEP was achieved. All patients were discharged alive; functional, ECG and echocardiographic parameters showed significant improvement, better observed at 4-month interval. However, a high mortality rate was noticed during follow up (about 70% at 6 months) with a significant number of sudden cardiac deaths. The absence of functional improvement in the mid-term period (4-month control) related to a poor prognosis. CONCLUSIONS: Epicardial lead placement during cardiac surgery of severe HF patients is safe and effective. A clear evaluation of the effect of BEP alone is precluded because of the interference of the concomitant indications for cardiac surgery and the absence of randomization. The high rate of sudden death noticed in this study raises the important question of whether implantation of a defibrillator would be warranted in such population.

Weekly paclitaxel for advanced aggressive classic Kaposi sarcoma: experience in 17 cases.

BACKGROUND: Paclitaxel has proved to be highly effective in the treatment of severe AIDS-related Kaposi sarcoma (KS), for which it is now considered as a second-line monotherapy. Taxanes were recently shown to be active also in classic, endemic and post-transplantation KS. OBJECTIVES: To evaluate the clinical efficacy and tolerability of standardized paclitaxel treatment (100 mg weekly, intravenously) in a homogeneous group of 17 patients with advanced aggressive and refractory classic KS (cKS). METHODS: Seventeen patients with aggressive refractory cKS (stage IIIBc-IVBcv) were treated with intravenous paclitaxel 100 mg weekly. The response to the therapy was evaluated after 12 weeks. A maintenance treatment every 2 weeks was introduced for most of the patients and a final evaluation was made. RESULTS: A partial/complete response was achieved in 14 of 17 patients. Two patients had allergic reactions, for which treatment was discontinued. One patient had progression of disease despite initial improvement. Patients received a mean of 16.8 courses. The treatment was generally well tolerated. Mean time to recurrence was 4.5 months from the end of the therapy and 7.35 months from the 12th course. In four of 10 patients who relapsed at follow-up, the recurrence was mild and responsive to local treatment, while the other six relapsing patients repeated paclitaxel with good response in five of them. CONCLUSIONS: This study shows that low-dose paclitaxel proved to be effective and well tolerated in patients with aggressive refractory cKS, controlling the aggressiveness of the disease. The treatment can be repeated with good response.

[Role of screening tests for indirect diagnosis of tuberculosis in health care workers: Mantoux and the new tests on blood ELISA]. / Ruolo dei test di screening nella diagnosi indiretta di tubercolosi negli operatori sanitari: la Mantoux e il nuovo test ELISA su sangue.

The Tuberculosis infection in recent years has become always more a threat. The failure in the attempt to stop it (O.M.S. Millennium Global Plan) brought to the revision of the world control strategy to at least contain this disease (The Global Plan to Stop TB 2006-2015). Due to these severe facts it is even more important now to elaborate more sensitive and specific methods to find out, as fast as possible, the infected cases. As of today, the main TB infection screening test is the Skin PPD test (Mantoux). Recently new tests for the population screening are in use; these tests are based on the evaluation of immunity cell-mediated. They (QFT-G) do not have the typical limits of the Skin Test and they are more suitable as serial tests and therefore more useful, according to us, in the screening programs of the TB infection in low prevalence countries, like Italy.

Clinical and endoscopic presentation of primary gastric lymphoma: a multicentre study.

BACKGROUND: Although the stomach is the most frequent site of intestinal lymphomas, few data are available on both clinical endoscopic presentation of gastric lymphoma and possible differences between low-grade and high-grade lymphomas. METHODS: Clinical, histological and endoscopic records of consecutive patients with primary low-grade or high-grade lymphoma diagnosed were retrieved. Symptoms were categorized as 'alarm' or 'not alarm'. The endoscopic findings were classified as 'normal' or 'abnormal'. RESULTS: Overall, 144 patients with primary gastric lymphoma were detected, including 74 low-grade and 70 high-grade lymphoma. Alarm symptoms, particularly persistent vomiting and weight loss, were more frequently present in patients with high-grade lymphoma than in those with low-grade lymphoma (54% vs. 28%; P = 0.002). Low-grade lymphomas presented as 'normal' appearing mucosa (20% vs. 0%; P = 0.0004) or petechial haemorrhage in the fundus (9% vs. 0%; P = 0.02) more frequently than high-grade lymphomas, being also more often confined to the antrum (47% vs. 27%, P = 0.03) and associated with Helicobacter pylori infection (88% vs. 52%, P < 0.0001). On the contrary, high-grade lymphomas presented more commonly as ulcerative type (70% vs. 52%; P = 0.03), being also more frequently diagnosed in stage >I when compared with low-grade lymphomas (70% vs. 21%, P < 0.0001). CONCLUSIONS: The overall prevalence of alarm symptoms is quite low and may be absent in more than 70% of patients with low-grade lymphoma.

Autografting followed by nonmyeloablative immunosuppressive chemotherapy and allogeneic peripheral-blood hematopoietic stem-cell transplantation as treatment of resistant Hodgkin's disease and non-Hodgkin's lymphoma.

PURPOSE: To investigate the use of a nonmyeloablative fludarabine-based immunosuppressive regimen to allow engraftment of HLA-sibling donors' mobilized stem cells and induction of a graft-versus-lymphoma effect for patients with advanced resistant Hodgkin's disease and non-Hodgkin's lymphoma. PATIENTS AND METHODS: Fifteen patients with Hodgkin's disease (n = 10) and non-Hodgkin's lymphoma (n = 5) were studied. All patients received cyclophosphamide and granulocyte colony-stimulating factor to mobilize autologous hematopoietic stem cells (HSCs). Subsequently, they received high-dose therapy with carmustine, etoposide, cytarabine, and melphalan and reinfusion of HSCs. At a median of 61 days after engraftment, patients were given fludarabine 30 mg/m(2) with cyclophosphamide 300 mg/m(2) daily for 3 days. Donor-mobilized HSC collections were prepared for fresh infusion and were not T-cell depleted. Methotrexate and cyclosporine were used to prevent graft rejection and as graft-versus-host disease (GVHD) prophylaxis. RESULTS: Combined treatment was well tolerated. After mini-allografting, hematologic recovery was prompt. Thirteen patients had 100% donor cell engraftment. Eleven patients achieved complete remission (CR) after the combined procedure. Nine patients, who were in partial remission after autografting, achieved CR after mini-allografting. Seven patients developed >/= grade 2 acute GVHD (aGVHD) and two developed extensive chronic GVHD (cGVHD). Three patients who received the highest number of donor lymphocyte infusions (DLIs) developed grade 3 GVHD (two patients) and extensive cGVHD (one patient). Ten patients are currently alive, and five are in continuous CR. Seven patients received DLI, with five CRs. Five patients died: one of progressive disease, two of progressive disease combined with aGVHD or cGVHD, one of extensive cGVHD, and one of infection. CONCLUSION: Fludarabine/cyclophosphamide was well tolerated and allowed consistent engraftment in lymphoma allografted patients. Response rates were high in this group of refractory and heavily pretreated patients. This dual procedure seems to be most promising in patients with end-stage malignant lymphomas.

Role of potassium channels in the antinociception induced by agonists of alpha2-adrenoceptors.

1. The effect of the administration of pertussis toxin (PTX) as well as modulators of different subtypes of K+ channels on the antinociception induced by clonidine and guanabenz was evaluated in the mouse hot plate test. 2. Pretreatment with pertussis toxin (0.25 microg per mouse i.c.v.) 7 days before the hot-plate test, prevented the antinociception induced by both clonidine (0.08-0.2 mg kg(-1), s.c.) and guanabenz (0.1-0.5 mg kg(-1), s.c.). 3. The administration of the K(ATP) channel openers minoxidil (10 microg per mouse, i.c.v.), pinacidil (25 microg per mouse, i.c.v.) and diazoxide (100 mg kg(-1), p.o.) potentiated the antinociception produced by clonidine and guanabenz whereas the K(ATP) channel blocker gliquidone (6 microg per mouse, i.c.v.) prevented the alpha2 adrenoceptor agonist-induced analgesia. 4. Pretreatment with an antisense oligonucleotide (aODN) to mKv1.1, a voltage-gated K+ channel, at the dose of 2.0 nmol per single i.c.v. injection, prevented the antinociception induced by both clonidine and guanabenz in comparison with degenerate oligonucleotide (dODN)-treated mice. 5. The administration of the Ca2+-gated K+ channel blocker apamin (0.5-2.0 ng per mouse, i.c.v.) never modified clonidine and guanabenz analgesia. 6. At the highest effective doses, none of the drugs used modified animals' gross behaviour nor impaired motor coordination, as revealed by the rota-rod test. 7. The present data demonstrate that both K(ATP) and mKv1.1 K+ channels represent an important step in the transduction mechanism underlying central antinociception induced by activation of alpha2 adrenoceptors.

Comparative effects of L-methionine, S-adenosyl-L-methionine and 5'-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.

Male Wistar rats, initiated with diethylnitrosamine (DENA), were subjected to a selection treatment, according to the "resistant hepatocyte" model, followed or not followed by phenobarbital (PB). Rats received, for 3 weeks after selection, 4 i.m. doses (96 mmol/kg) of L-methionine, S-adenosyl-L-methionine (SAM), or 5'-methylthioadenosine (MTA), a SAM catabolite formed during polyamine synthesis or by spontaneous splitting of SAM at physiologic temperature and pH. They were then killed. In some rats, SAM and MTA treatments were started 20 weeks after initiation. The animals were killed 3 weeks later and persistent (neoplastic) nodules (PN) were collected. Some rat groups received 1/2 and 1/4 of the above SAM and MTA doses, or 1/8 of the above MTA dose. SAM and MTA, but not methionine, caused a dose-dependent decrease in number and surface area of gamma-glutamyltranspeptidase (GGT)-positive foci, and in labeling index (LI) of focal cells, coupled with remodeling. SAM and MTA liver contents, SAM/S-adenosylhomocysteine (SAH) ratio and overall methylation of liver DNA were low during the development of GGT-positive foci. SAM, but not methionine, caused a dose-dependent recovery of SAM content and DNA methylation, and a partial reconstitution of liver MTA pool. Exogenous MTA only induced the reconstitution of MTA pool, without affecting SAM level and DNA methylation. Recovery of SAM and MTA pool and DNA methylation was found in the rats subjected to SAM plus MTA, indicating the absence of inhibition of DNA methyltransferases in vivo by MTA. MTA also inhibited liver reparative growth in partially hepatectomized rats, without modifying SAM content and DNA methylation of regenerating liver (RL). A high activity of ornithine decarboxylase (ODC) was found in the liver, during the development of preneoplastic foci, and in PN. This activity was inhibited by SAM and MTA treatments. Although MTA was more effective than SAM, the decrease in ODC activity was coupled with a larger fall in DNA synthesis in SAM-treated than in MTA-treated rats. Thus the antipromotion effect of SAM could not merely depend on its (spontaneous) transformation into MTA. Although MTA production may play a role in the SAM antipromotion effect, other mechanisms could be involved. A role of DNA methylation in the inhibition of growth by SAM is suggested. MTA is a potential chemopreventive agent for liver carcinogenesis.

Electrophysiological alterations in hepatic encephalopathy detected by brain mapping.

Analogical electroencephalogram has been used to study electric alterations in the brain during liver encephalopathy. We adopted digital technique brain mapping to study the electric background activity in the brain and to evaluate the diagnostic and prognosis usefulness of this technique compared with the methods routinely used in this disease. We studied 18 patients with liver cirrhosis and varying degrees of liver encephalopathy and 7 healthy control subjects to assess correlation between the severity of encephalopathy and abnormal electric activity in the brain, and to detect the main differences between the brain mapping findings obtained in the two groups. The findings revealed an overall reduction in the rhythm, increased amplitude and anomalous distribution of the waveforms in the cirrhotic patients. Although similar results have already been reported, brain mapping furnished prompt and more easily visualised findings. Moreover, brain mapping facilitated detailed analysis of wave amplitude, frequency and topographic location indicating that this technique is a valid tool in diagnosing brain disorders.

[Effect of treatment with biosynthetic GH on thyroid function in patients with an isolated deficiency of GH]. / Effetti del trattamento con GH-biosintetico sulla funzione tiroidea in soggetti con deficit isolato di GH.

The use of GH treatment in subjects with a GH deficiency has led to contrasting results concerning its impact to develop thyroid hyperfunction, whereas many others have underlined the possible onset of hypothyroidism. A number of studies have been carried out over short periods in subjects with multiple tropin deficiencies, in healthy adults or adults with GH deficiencies, in healthy adults or adults with GH deficiencies. The aim of the present study was to assess the effect of prolonged treatment with biosynthetic GH on thyroid function in children with an isolated idiopathic GH deficiency. The study included 8 children (mean age 10.4 +/- 0.8 years) with GH deficiencies treated with biosynthetic GH and 8 children with familial retarded stature of a similar age (mean age 10.3 +/- 0.7 years) who represented the control group. Serum levels of T3, T4, FT3, FT4 and TSH were measured at the start of the study and after one year of continuous GH treatment in subjects with GH deficiency; the same tests were performed in the control group on recruitment and after one year's observation without therapy. T4 and FT4 levels diminished, but not significantly, whereas there was a significant increase in plasma levels of T3 and FT3 (p less than 0.01); TSH values were significantly reduced in the treated group (p = 0.025). No significant variations in thyroid parameters were found in the control group. These data support the hypothesis of an increased peripheral conversion of T4 into T3 due to GH therapy; in conclusion, however, no significant variation in thyroid function was observed following GH replacement therapy, even if prolonged, in subjects with an idiopathic isolated GH deficiency.