Clinical evaluation of the perception of post-trauma paresthesia in the mandible, using a biomimetic material: A preliminary study in humans.

There is a great effort from numerous research groups in the development of materials and therapeutic strategies for the functional recovery of patients who have suffered peripheral nerve injuries (PNI). In an article in vivo, the formation of a nerve bridge was observed, reconnecting the distal and proximal stumps, in the sciatic nerve of rats, indicating the effective participation of the biomaterial in the recovery of peripheral nerve injuries. For the current pilot study, 15 cases of multiple fractures of the mandible, with involvement of the inferior alveolar nerve (IAN) were selected and studied: JC (control cases) n = 6 with conventional treatment, and JT (treated cases) n = 9, with the use of biomimetic biomaterial. The evaluation of the return to sensitivity was measured through a self-assessment, where the patients assigned scores from 0 to 10, where zero (0) represented the complete absence of sensitivity and ten (10) the normality of the perception of local sensitivity. Patients were evaluated from the preoperative period to the 360th day. The statistical results obtained by the t-Student, Shapiro-Wilk normality and non-parametric One-Way ANOVA tests indicated statistically significant differences (p < 0.005; 0.005 e 0.5 respectively), between the two treatments, which were reflected in the clinical results observed, we also calculate the size of the effect represented by ϵ2, calculated by Cohen's d. The results indicate a great difference between the treatments performed,ϵ2 = 1.00. In the 6 cases followed up in the JC group, four remained with a significant deficit until the end of the evaluations and two indicated the remission of the lack of sensitivity in this period. In the JT group, in 28 days, all cases indicated complete remission of the lack of sensitivity with healing concentration. In one of the cases where there was a complete rupture of the mental nerve, the (score-10) was observed in 60 days. The observed results indicate the existence of a statistical significance between the groups and an important relationship when using the biomimetic biomaterial during the recovery of the perception of sensitivity in polytraumatized patients, compatible with the results observed in laboratory animals, which may indicate its clinical feasibility in the reduction of sequelae in PNI.

Phase I trial of the oral smoothened inhibitor sonidegib in combination with paclitaxel in patients with advanced solid tumors.

Purpose To establish a recommended phase II dose (RP2D) for the oral smoothened inhibitor sonidegib in combination with paclitaxel; secondary objectives include evaluation of safety, tolerability, markers of Hedgehog (Hh) signaling and preliminary antitumor activity. Methods Patients with advanced solid tumors were enrolled in cohorts of escalating sonidegib dose levels (400mg, 600mg and 800mg orally, once daily on days 1-28) in combination with paclitaxel 80 mg/m2 on days 1, 8 and 15 in 4-weekly cycles. Dose-limiting toxicities (DLTs) were assessed using CTCAE v4. Once the RP2D was defined, patients with advanced ovarian carcinoma were treated at this dose level in an expansion phase. Biomarkers of Hh signaling were assessed by immunohistochemistry in archival tissue and antitumor activity evaluated using RECIST 1.1. Results 18 patients were treated: 3 at 400 mg, 3 at 600 mg and 12 at 800 mg sonidegib. Only one patient treated at 800 mg presented a DLT (prolonged neutropenia resulting in failure to receive 75% of the planned sonidegib dose). However, 4 of 12 patients treated at 800 mg had their sonidegib dose reduced for toxicity after cycle 1. Hh biomarker (SHH, Patched, SMO and GLI1) staining did not correlate with clinical activity. Best response was partial response in 3 patients (2 ovarian, 1 breast cancer) and stable disease >4 cycles in 3 patients (2 ovarian, 1 anal cancer). Conclusions The combination of sonidegib and paclitaxel is tolerable and evidence of antitumor activity was identified. The RP2D of sonidegib was 800 mg in combination with paclitaxel 80mg/m2.

Cell proliferation and drug sensitivity of human glioblastoma cells are altered by the stable modulation of cytosolic 5'-nucleotidase II.

Cytosolic 5'-nucleotidase II (cN-II) has been reported to be involved in cell survival, nucleotide metabolism and in the cellular response to anticancer drugs. With the aim to further evaluate the role of this enzyme in cell biology, we stably modulated its expression the human glioblastoma cell ADF in which the transient inhibition of cN-II has been shown to induce cell death. Stable cell lines were obtained both with inhibition, obtained with plasmids coding cN-II-targeting short hairpin RNA, and stimulation, obtained with plasmids coding Green Fluorescence Protein (GFP)-fused wild type cN-II or a GFP-fused hyperactive mutant (GFP-cN-II-R367Q), of cN-II expression. Silenced cells displayed a decreased proliferation rate while the over expressing cell lines displayed an increased proliferation rate as evidenced by impedance measurement using the xCELLigence device. The expression of nucleotide metabolism relevant genes was only slightly different between cell lines, suggesting a compensatory mechanism in transfected cells. Cells with decreased cN-II expression were resistant to the nucleoside analog fludarabine confirming the involvement of cN-II in the metabolism of this drug. Finally, we observed sensitivity to cisplatin in cN-II silenced cells and resistance to this same drug in cN-II over-expressing cells indicating an involvement of cN-II in the mechanism of action of platinum derivatives, and most probably in DNA repair. In summary, our findings confirm some previous data on the role of cN-II in the sensitivity of cancer cells to cancer drugs, and suggest its involvement in other cellular phenomenon such as cell proliferation.

The purine analog fludarabine acts as a cytosolic 5'-nucleotidase II inhibitor.

For several years the IMP/GMP-preferring cytosolic 5'-nucleotidase II (cN-II) has been considered as a therapeutic target in oncology. Indeed, various reports have indicated associations between cN-II expression level and resistance to anticancer agents in several cancer cell lines and in patients affected with neoplasia, mainly by hematologic malignancies. In this paper we present evidence showing that, among the commonly used cytotoxic nucleoside analogs, fludarabine can act as a cN-II inhibitor. In vitro studies using the wild type recombinant cN-II demonstrated that fludarabine inhibited enzymatic activity in a mixed manner (Ki 0.5 mM and Ki' 9 mM), whereas no inhibition was observed with clofarabine and cladribine. Additional experiments with mutant recombinant proteins and an in silico molecular docking indicated that this inhibition is due to an interaction with a regulatory site of cN-II known to interact with adenylic compounds. Moreover, synergy experiments between fludarabine and 6-mercaptopurine in human follicular lymphoma (RL) and human acute promyelocytic leukemia (HL-60) cells transfected with control or cN-II-targeting shRNA-encoding plasmids, showed synergy in control cells and antagonism in cells with decreased cN-II expression. This is in line with the hypothesis that fludarabine acts as a cN-II inhibitor and supports the idea of using cN-II inhibitors in association with other drugs to increase their therapeutic effect and decrease their resistance.

On the physiological role of cytosolic 5'-nucleotidase II (cN-II): pathological and therapeutical implications.

Among the members of the 5'-nucleotidase family, there is only one membrane-bound ectosolic isoenzyme. This esterase prefers AMP as substrate but can hydrolyze a number of purine and pyrimidine phosphorylated compounds, indicating that no evolutive pressure to develop a more restricted specificity was exerted on this enzyme. On the contrary, five cytosolic isoforms have been evolved, probably by convergent evolution, showing different and restricted substrate specificity. The different isoforms have different level of expression and distribution in organs of vertebrates. The cytosolic nucleotidase specific for IMP and GMP (cN-II), is an enzyme allosterically regulated, structurally strongly conserved and expressed at a low but constant level in all organs and tissues in vertebrates. As far as we know, alteration of cN-II expression is limited to pathological conditions. In this review, we report the results of the modulation of cN-II specific activity exerted by silencing or hyperexpression in different cell types, in the attempt to better understand its role and implications in pathology and therapy.

Mutation analysis of the EGFR gene and downstream signalling pathway in histologic samples of malignant pleural mesothelioma.

BACKGROUND: As epidermal growth factor receptor (EGFR) is involved in the pathogenesis of malignant pleural mesotheliomas (MPMs), the anti-EGFR drugs may be effective in treating MPM patients. Mutations of the EGFR gene or its downstream effectors may cause constitutive activation leading to cell proliferation, and the inhibition of apoptosis and metastases. Consequently, molecular profiling is essential for select patients with MPM who may respond to anti-EGFR therapies. METHODS: After manual macrodissection, genomic DNA was extracted from 77 histological samples of MPM: 59 epithelioid, 10 biphasic, and 8 sarcomatoid. Epidermal growth factor receptor gene mutations were sought by means of real-time polymerase chain reaction (PCR) and direct sequencing, KRAS gene mutations by mutant-enriched PCR, and PIK3CA and BRAF gene mutations by direct sequencing. RESULTS: Gene mutations were identified in nine cases (12%): five KRAS, three BRAF, and one PI3KCA mutation; no EGFR gene mutations were detected. There was no difference in disease-specific survival between the patients with or without gene mutations (P=0.552). CONCLUSIONS: Mutations in EGFR downstream pathways are not rare in MPM. Although none of those found in this study seemed to be prognostically significant, they may support a more specific selection of patients for future trials.

Japanese encephalitis virus RNA detected in Culex pipiens mosquitoes in Italy.

Mosquitoes collected in northern Italy were screened for flavivirus RNA. Positive amplicons were sequenced and found most similar to insect flavivirus (ISF), Usutu virus (USUV) and surprisingly also to Japanese encephalitis virus (JEV). The sequence (167 bp), obtained from one pool of Culex pipiens, was found identical to JEV strains from bats in China. Unfortunately additional sequence data or virus isolations were not obtained in this study. Confirmation of potential introduction of JEV to Italy and other European countries is urgently needed.

Comparative animal study on hard tissue integration and bone formation of different Nobel Biocare implants.

Dental implantation aims at optimal and long-term hard tissue integration. Beside primary stability, loading time and other factors, e.g. the surface of the endosteal part of the implant, is a matter of special importance. In this animal trial, hard tissue integration of two different implant types was studied using radiological, histological and histomorphometric analysis. Two different implants with an oxidized surface (TiUnite; Nobel Biocare AB, Goteborg, Sweden, NobelReplace Tapered Groovy 4.3 x 10 mm and Replace Select Tapered 4.3 x 10 mm) were inserted into the right and left mandibles of 10 German domestic pigs between canine and premolar and immediately provided with a ceramic crown. The primary implant stability was determined using resonance frequency analysis. After 70 days, the test animals were killed and specimens were collected for histological and histomorphometric examination. All implants showed good primary stability after surgery. Histological and histomorphometrical analysis revealed no significant differences in the bone apposition. The immediate loading of the different implant types don't have any negative effects on the bone apposition in the period of 70 days. The long-term effects of immediate loading of these types of implant requires further studies.

Soft tissue integration in the neck area of titanium implants--an animal trial.

Dental implant materials are required to enable good apposition of bone and soft tissues. They must show sufficient resistance to chemical, physical and biological stress in the oral cavity to achieve good long-term outcomes. A critical issue is the apposition of the soft tissues, as they have provided a quasi-physiological closure of oral cavity. The present experiment was performed to study the peri-implant tissue response to non-submerged (1-stage) implant installation procedures. Two different implants types (NobelBiocare, NobelReplace Tapered Groovy 4.3 x 10 mm and Replace Select Tapered TiU RP 4.3 x 10mm) were inserted into the right and left sides of 8 domestic pigs (Sus scrofa domestica) mandibles, between canines and premolars and immediately provided with a ceramic crown. Primary implant stability was determined using ressonance frequency analysis. Soft tissue parameters were assessed: sulcus depth (SDI) and junctional epithelium (JE). Following 70 days of healing, jaw sections were processed for histology and histomorphometric examination. Undecalcified histological sections demonstrated osseointegration with direct bone contact. The soft tissue parameters revealed no significant differences between the two implant types. The peri-implant soft tissues appear to behave similarly in both implant types.

Requirements and infection prophylaxis for internally cooled implant drills.

Implant site preparation is crucially important to long-term success. Heat generation during drilling is unfavourable, since bone is relatively susceptible to heat, depending on its vascularisation and microstructure. Numerous factors such as drilling pressure, number of revolutions, drill design, wear and material, drilling depth and cooling influence heat generation. Internally cooled drills are, therefore, increasingly used, even though the improved cooling effect compared to conventional externally cooled drills is controversial. Internally cooled drills may have the disadvantage of a germ reservoir developing in the cooling channel. This study aimed to examine the effects of disinfection and sterilisation of internally cooled drills. After contamination of the cooling channel with suitable bioindicators (Enterococcus faecium, ATCC 6057 and spores of Bacillus stearothermophilus, ATCC 7953), the drills were disinfected (disinfection solution ID 220, Dürr Dental) and autoclaved (Webeco, E5S90, 134 degrees C, 2.6 bar, 5 min). Disinfection was not completely effective except after pre-cleaning. By means of sterilisation all spores of Bacillus stearothermophilus were completely killed. Internally cooled drills can be successfully disinfected by means of this hygienic procedure routinely used in dental practice and no source of infection is created.

[Retrospective survey of the management of patients treated for acute myocardial infarction in Provence-Alpes-Côte d'Azur]. / Evaluation de la prise en charge de l'infarctus du myocarde dans la région Provence-Alpes-Côte d'Azur.

OBJECTIVE: The aim of the study is to assess the characteristics, outcomes and factors associated with delay of reperfusion therapy in patients with myocardial infarction in the Provence Alpes Côte d'Azur (PACA). METHODS: This retrospective study included all patients hospitalized with myocardial infarction in the PACA between January and June 2000. Myocardial infarction patients were identified using the Programme de Médicalisation des Systèmes d'Information. Univariate and multivariate analysis were performed. RESULTS: A total of 2049 patients were admitted in 74 hospitals. Mean patient age was 68.9. Treatment consisted of coronary angioplasty alone in 53.3% of cases, thrombolysis alone in 4.5% and angioplasty after thrombolysis in 7.7%. The complication rate was 27.4% and mortality was 9.5%. The median time from first awareness of symptoms to the first action taken by the patient (1) was 1 hour 30 minutes. The time from the first action taken by the patient to revascularisation (2) was 3 hours 30 minutes. The time between the first awareness and revascularisation (3) was 8 hours 45 minutes. Multivariate analysis showed that the following factors were associated with increased delay: (1). no prior myocardial infarction, occurrence of symptoms at home. (2). age > 65 years, absence of chest pain, consultation with a physician before hospitalization, non-medical transport, transfer from one hospital to another, treatment by angioplasty alone. (3). age > 65 years, absence of chest pain, consultation with a physician before hospitalization, non-medical transport, treatment by angioplasty alone. CONCLUSIONS: Alerting emergency services more quickly would shorten treatment delay not only by ensuring quicker transport to a properly equipped hospital but also by allowing prompt pre-hospital thrombolytic therapy.

Plasma hormones and metabolites in Piedmontese cows during late pregnancy: relationships with calf birth weight.

Relationships among plasma hormonal and metabolic variables in the last trimester of gestation in 59 Piedmontese dams (n = 15 heifers, n = 44 cows) and the calf birth weight (BWT) class of their offspring were investigated in seven herds. The BWT data were categorized as follows: > 50 kg (BWT-A), 46 to 50 kg (BWT-B), 41 to 45 kg (BWT-C), and < 41 kg (BWT-D). Blood samples were collected at 33, 36, and 39 wk of gestation. Packed cell volume (PCV) and plasma concentrations of insulin, estrone sulfate (E1SO4), NEFA, and creatinine were determined and correlated to BWT class. Creatinine: E1SO4 ratio also was calculated. Duration of gestation was greater for dams producing a BWT-A calf than for the other BWT classes, and calf BWT was heavier (P < 0.001) for calves in the BWT-A vs. BWT-D class. The heaviest calf in BWT-A was associated with the highest calving difficulty score. Insulin and PCV values were not affected by week of gestation, whereas plasma E1SO4, NEFA, and creatinine content increased (P < 0.001) and creatinine:E1SO4 decreased (P < 0.001) during late gestation. Calf BWT class did not affect PCV value. Plasma E1SO4 concentrations were lower (P < 0.01) in BWT-D dams than the other dams, showing the greatest difference at 39 wk of gestation. At 36 and 39 wk of gestation, dams bearing BWT-C and BWT-D calves had a higher (P < 0.01) plasma insulin concentration than those bearing BWT-A and BWT-B calves. Plasma NEFA concentrations at 39 wk of gestation were higher (P < 0.05) in dams of calf BWT-A than in the other dams. We conclude that plasma E1SO4 level is a variable that can be used to monitor problems related to a small size calf. Conversely, the forthcoming birth of a calf with a heavy BW seems to be preceded by a pronounced increase in plasma NEFA level in the dam just a few days before calving.

5'-aminoimidazole-4-carboxamide riboside induces apoptosis in human neuroblastoma cells.

5'-Aminoimidazole-4-carboxamide riboside (AICA riboside) has been previously shown to be toxic to two neuronal cell models [Neuroreport 11 (2000) 1827]. In this paper we demonstrate that AICA riboside promotes apoptosis in undifferentiated human neuroblastoma cells (SH-SY5Y), inducing a raise in caspase-3 activity. In order to exert its effect on viability, AICA riboside must enter the cells and be phosphorylated to the ribotide, since both a nucleoside transport inhibitor, and an inhibitor of adenosine kinase produce an enhancement of the viability of AICA riboside-treated cells. Short-term incubations (2 h) with AICA riboside result in five-fold increase in the activity of AMP-dependent protein kinase (AMPK). However, the activity of AMPK is not significantly affected at prolonged incubations (48 h), when the apoptotic effect of AICA riboside is evident. The results demonstrate that when the cell line is induced to differentiate both toward a cholinergic phenotype (with retinoic acid) or a noradrenergic phenotype (with phorbol esters), the toxic effect is significantly reduced, and in the case of the noradrenergic phenotype differentiation, the riboside is completely ineffective in promoting apoptosis. This reduction of effect correlates with an overexpression of Bcl-2 during differentiation. AICA riboside, derived from the hydrolysis of the ribotide, an intermediate of purine de novo synthesis, is absent in normal healthy cells; however it may accumulate in those individuals in which an inborn error of purine metabolism causes an increase in the rate of de novo synthesis and/or an overexpression of cytosolic 5'-nucleotidase, that appears to be the enzyme responsible for AICA ribotide hydrolysis. In fact, 5'-nucleotidase activity has been shown to increase in patients affected by Lesch-Nyhan syndrome in which both acceleration of de novo synthesis and accumulation of AICA ribotide has been described, and also in other neurological disorders of unknown etiology. Our results raise the intriguing clue that the neurotoxic effect of AICA riboside on the developing brain might contribute to the neurological manifestations of syndromes related to purine dismetabolisms.

Catabolism of exogenous deoxyinosine in cultured epithelial amniotic cells.

Uptake and catabolism of purine nucleosides have been commonly considered as means to salvage the purine ring for nucleic acid synthesis, usually neglecting the destiny of the pentose moiety. With the aim to ascertain if deoxyribose derived from exogenous DNA can be utilised as a carbon and energy source, we studied the catabolism of exogenous deoxyinosine in a cell line derived from human amnion epithelium (WISH). Intact WISH cells catabolise deoxyinosine by conversion into hypoxanthine. The nucleoside enters the cell through a nitrobenzylthioinosine-insensitive equilibrative transport. Deoxyinosine undergoes a phosphorolytic cleavage inside the cell. The purine base diffuses back to the external medium, while the phosphorylated pentose moiety can be further catabolised to glycolysis and citric acid cycle intermediates. Our results indicate that the catabolism of the deoxynucleoside can be considered mainly as a means to meet the carbon and energy requirements of growing cells.

Bovine cytosolic 5'-nucleotidase acts through the formation of an aspartate 52-phosphoenzyme intermediate.

Cytosolic 5'-nucleotidase/phosphotransferase (cN-II), specific for purine monophosphates and their deoxyderivatives, acts through the formation of a phosphoenzyme intermediate. Phosphate may either be released leading to 5'-mononucleotide hydrolysis or be transferred to an appropriate nucleoside acceptor, giving rise to a mononucleotide interconversion. Chemical reagents specifically modifying aspartate and glutamate residues inhibit the enzyme, and this inhibition is partially prevented by cN-II substrates and physiological inhibitors. Peptide mapping experiments with the phosphoenzyme previously treated with tritiated borohydride allowed isolation of a radiolabeled peptide. Sequence analysis demonstrated that radioactivity was associated with a hydroxymethyl derivative that resulted from reduction of the Asp-52-phosphate intermediate. Site-directed mutagenesis experiments confirmed the essential role of Asp-52 in the catalytic machinery of the enzyme and suggested also that Asp-54 assists in the formation of the acyl phosphate species. From sequence alignments we conclude that cytosolic 5'-nucleotidase, along with other nucleotidases, belong to a large superfamily of hydrolases with different substrate specificities and functional roles.

Cytosolic 5'-nucleotidase hyperactivity in erythrocytes of Lesch-Nyhan syndrome patients.

Lesch-Nyhan syndrome is a metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Metabolic consequences of HGPRT deficiency have been clarified, but the connection with the neurological manifestations is still unknown. Much effort has been directed to finding other alterations in purine nucleotides in different cells of Lesch-Nyhan patients. A peculiar finding was the measure of appreciable amount of Z-nucleotides in red cells. We found significantly higher IMP-GMP-specific 5'-nucleotidase activity in the erythrocytes of seven patients with Lesch-Nyhan syndrome than in healthy controls. The same alteration was found in one individual with partial HGPRT deficiency displaying a severe neurological syndrome, and in two slightly hyperuricemic patients with a psychomotor delay. Since ZMP was a good substrate of 5'-nucleotidase producing Z-riboside, we incubated murine and human cultured neuronal cells with this nucleoside and found that it is toxic for our models, promoting apoptosis. This finding suggests an involvement of the toxicity of the Z-riboside in the pathogenesis of neurological disorders in Lesch-Nyhan syndrome and possibly in other pediatric neurological syndromes of uncertain origin.

Identification, separation and characterisation of two forms of cytosolic 5'-nucleotidase/nucleoside phosphotransferase in calf thymus.

Cytosolic 5'-nucleotidase, acting preferentially on IMP, GMP and their deoxyderivatives, endowed with phosphotransferase activity, is a widespread enzyme responsible for the regulation of intracellular IMP and GMP concentrations and the phosphorylation of purine nucleoside pro-drugs. The enzyme activity is stimulated by ATP, ADP and 2,3-bisphosphoglycerate (BPG), and is inhibited by phosphate. Calf thymus possesses two active proteins with a different electrophoretic mobility. In this report we show that the two forms can be separated by ADP-agarose affinity chromatography. Whereas form A binds weakly to the column, form B is tightly bound and is released by the addition of ADP into the elution buffer. The two enzyme forms differ in terms of electrophoretic, chromatographic behaviour and regulatory characteristics. Form B, as already described for the enzyme purified from the same source (Pesi et al., 1996, Biochim Biophys Acta 294, 191-194), exhibits three different sites for the three activators with a synergistic effect between ADP and BPG. Form A has a high affinity regulatory site for BPG, while ADP and ATP appear to share the same low affinity site and no synergistic effect is observed.

Bovine cytosolic IMP/GMP-specific 5'-nucleotidase: cloning and expression of active enzyme in Escherichia coli.

A cDNA coding for bovine cytosolic IMP/GMP-specific 5'-nucleotidase endowed with phosphotransferase activity was cloned from calf thymus RNA, by 5' and 3' rapid amplification of cDNA ends protocols (5' and 3' RACE). Two products were isolated: a 5' RACE 1.6 kb fragment and a 3' RACE 2.0 kb fragment, with an overlapping region of 505 bp, leading to a total length of approx. 2951 bp. The similarity in the coding region to that of the human 5'-nucleotidase cDNA sequence [Oka, Matsumoto, Hosokawa and Inoue (1994) Biochem. Biophys. Res. Commun. 205, 917-922], indirectly identified as a 5'-nucleotidase, was 94% and the deduced amino acid sequences were 99.5% identical. The bovine cDNA sequence included the sequences codifying for six peptides obtained from 5'-nucleotidase/phosphotransferase purified from calf thymus. Northern blots of human mRNA species from different tissues showed a 3.6 kb mRNA expressed at equal levels in most tissues. The cDNA was cloned into a pET-28c expression vector and the protein obtained after induction had a molecular mass of 61 kDa under SDS/PAGE. It exhibited both 5'-nucleotidase and phosphotransferase activity, as well as immunological and kinetic properties similar to those of the enzyme purified from calf thymus. This is the first time that a fully active recombinant 5'nucleotidase has been described.