Label-free electrochemical biosensor for direct detection of Oncostatin M (OSM) inflammatory bowel diseases (IBD) biomarker in human serum.

Oncostatin M (OSM) is an interleukin-6 (IL-6) member family cytokine implicated in the pathogenesis of chronic diseases including inflammatory bowel disease (IBD). OSM is a novel diagnostic biomarker over-expressed in the serum of IBD patients. This paper reports on the first electrochemical OSM immunosensor, developed using a multistep fabrication process aimed at covalently immobilizing OSM antibodies on a mixed self-assembled monolayer coated gold working electrode. Cyclic voltammetry, atomic force microscopy (AFM), IR spectroscopy and optical characterizations were used to validate the sensor functionalization protocol. Electrochemical impedance spectroscopy (EIS) measurements were performed to assess the reliability of the immunosensor preparation and to verify the antibody-antigen complexes formation. The label-free immunosensor showed high sensitivity identifying OSM at clinically relevant concentrations (37-1000 pg mL-1) with low detection limit of 2.86 pg mL-1. Both sensitivity and selectivity of the proposed immunosensor were also demonstrated in human serum in the presence of interfering biomarkers, making it an innovative potential platform for the OSM biomarker detection in IBD patients' serum.

Anthracycline-related cardiotoxicity in patients with breast cancer harboring mutational signature of homologous recombination deficiency (HRD).

BACKGROUND: The BRCA proteins play a key role in the homologous recombination (HR) pathway. Beyond BRCA1/2, other genes are involved in the HR repair (HRR). Due to the prominent role in the cellular repair process, pathogenic or likely pathogenic variants (PV/LPVs) in HRR genes may cause inadequate DNA damage repair in cardiomyocytes. PATIENTS AND METHODS: This was a multicenter, hospital-based, retrospective cohort study to investigate the heart toxicity from anthracycline-containing regimens (ACRs) in the adjuvant setting of breast cancer (BC) patients carrying germline BRCA PV/LPVs and no-BRCA HRR pathway genes. The left ventricular ejection fraction (LVEF) was assessed using cardiac ultrasound before starting ACR therapy and at subsequent time points according to clinical indications. RESULTS: Five hundred and three BC patients were included in the study. We predefined three groups: (i) BRCA cohort; (ii) no-BRCA cohort; (iii) variant of uncertain significance (VUS)/wild-type (WT) cohort. When baseline (T0) and post-ACR (T1) LVEFs between the three cohorts were compared, pre-treatment LVEF values were not different (BRCA1/2 versus HRR-no-BRCA versus VUS/WT cohort). Notably, during monitoring (T1, median 3.4 months), patients carrying BRCA or HRR no-BRCA germline pathogenic or likely pathogenic variants showed a statistically significant reduction of LVEF compared to baseline (T0). To assess the relevance of HRR on the results, we included the analysis of the subgroup of 20 BC patients carrying PV/LPVs in other genes not involved in HRR, such as mismatch repair genes (MUTYH, PMS2, MSH6). Unlike HRR genes, no significant differences in T0-T1 were found in this subgroup of patients. CONCLUSION: Our data suggest that deleterious variants in HRR genes, leading to impaired HR, could increase the sensitivity of cardiomyocytes to ACR in early BC patients. In this subgroup of patients, other measurements, such as the global longitudinal strain, and a more in-depth assessment of risk factors may be proposed in the future to optimize cardiovascular risk management and improve long-term survival.

Nutritional management of the patient with pancreatic cancer: from the diagnostic and therapeutic pathway to an integrated hospital-territory approach.

Abstract: Pancreatic cancer is associated to a high risk of malnutrition and neoplastic cachexia even at first diagnosis. Malnutrition is a negative prognostic factor for the outcome of surgery or medical oncology treatments. Despite the good awareness of the problem and the knowledge of the guidelines, the early recognition of malnutrition and its management are still uneven, mainly due to the lack of implementation of standardized and shared protocols and the shortage of dedicated clinical nutritionists and dieticians. An early and appropriate nutritional intervention is mandatory to improve the outcome of patients with pancreatic cancer at any stage of disease. The Mini Nutritional Assessment is useful tool to screen patients malnourished or at risk of malnutrition. The need for the establishment and implementation of an integrated hospital - territorial assistance as well as a home-delivered nutrition service is discussed.

Nivolumab in metastatic melanoma: good efficacy and tolerability in elderly patients.

Background: Nivolumab is an anti-PD-1 antibody that restores the antitumour immune function of T cells, blocking the binding of PD-1 with its ligand PD-L1. PD-1 is expressed on T cells and interacts with PD-L1 on tumour cells. The PD-1-PD-L1 link inhibits T cell activation. In metastatic melanoma, PD-1-PD-L1 binding plays a critical role, and the advent of the immune checkpoint inhibitor nivolumab has delivered new and effective treatment options with proven clinical benefit. In the present study, we evaluated the efficacy of nivolumab in elderly patients with metastatic melanoma. Methods: The study enrolled 55 elderly patients (75 years of age and older) with a diagnosis of metastatic melanoma. Primary endpoints of the study were progression-free survival (pfs) and the objective response rate; secondary endpoints were overall survival, reduction in serum lactate dehydrogenase (ldh) from before to after treatment, and tolerability. Results: Nivolumab was well tolerated and resulted in good disease control, with a manageable toxicity profile and significant clinical benefit. The duration of pfs was 5.1 months (95% confidence interval: 3.5 months to 6.8 months). A significant correlation was observed between reduction in serum ldh and pfs: 0.60 (95% confidence interval: 0.28 to 0.86; p = 0.002). Conclusions: Nivolumab is an immunotherapy treatment that has proved to be an effective and well-tolerated therapeutic option in elderly patients with metastatic melanoma.

Efficacy and safety of Everolimus and Exemestane in hormone-receptor positive (HR+) human-epidermal-growth-factor negative (HER2-) advanced breast cancer patients: New insights beyond clinical trials. The EVA study.

BACKGROUND: The BOLERO-2 trial reported efficacy and safety of Everolimus (EVE) and Exemestane (EXE) combination in HR+ advanced breast cancer (ABC) patients. The BALLET trial further evaluated the safety of EVE-EXE in HR+ ABC patients, without reporting efficacy data. Aim of the EVA real-life study was to collect data of efficacy and safety of EVE-EXE combination in the clinical setting, as well as exploring efficacy according to EVE Dose-Intensity (DI) and to previous treatment with Fulvestrant. PATIENTS AND METHODS: This study aimed to describe the outcome of ABC pts treated with EVE-EXE combination in terms of median duration of EVE treatment and ORR in a real-life setting. RESULTS: From July 2013 to December 2015, the EVA study enrolled 404 pts. Median age was 61 years (33-83). Main metastatic sites were: bone (69.1%), soft tissue (34.7%) and viscera (33.2%). Median number of previous treatments was 2 (1-7). 43.3% of the pts had received Fulvestrant. Median exposure to EVE was 31.0 weeks (15.4-58.3) in the whole population. No difference was observed in terms of EVE exposure duration according to DI (p for trend = 0.27) or type of previous treatments (p = 0.33). ORR and Disease Control Rate (DCR) were observed in 31.6% and 60.7% of the patients, respectively, with the lowest ORRs confined in CHT pre-treated patients or in those who received the lowest DI of EVE. Grade 3-4 adverse events (AEs) were reported in 37.9% of the patients. Main AEs were: stomatitis (11.2%), non-infectious pneumonitis - NIP (3.8%), anaemia (3.8%) and fatigue (3.2%). CONCLUSIONS: The EVA study provided new insights in the use of EVE-EVE combination in HR+ ABC pts many years after the publication of the pivotal trial. The combination is safe and the best response could be obtained in patients receiving the full dose of EVE and/or after hormone-therapy as Fulvestrant in ABC.

Fibrous scaffolds fabricated by emulsion electrospinning: from hosting capacity to in vivo biocompatibility.

Electrospinning is a versatile method for preparing functional three-dimensional scaffolds. Synthetic and natural polymers have been used to produce micro- and nanofibers that mimic extracellular matrices. Here, we describe the use of emulsion electrospinning to prepare blended fibers capable of hosting aqueous species and releasing them in solution. The existence of an aqueous and a non-aqueous phase allows water-soluble molecules to be introduced without altering the structure and the degradation of the fibers, and means that their release properties under physiological conditions can be controlled. To demonstrate the loading capability and flexibility of the blend, various species were introduced, from magnetic nanoparticles and quantum rods to biological molecules. Cellular studies showed the spontaneous adhesion and alignment of cells along the fibers. Finally, in vivo experiments demonstrated the high biocompatibility and safety of the scaffolds up to 21 days post-implantation.

[Long-term survival metastatic ovarian cancer elderly patient]. / Paziente anziana con carcinoma ovarico metastatico lungo-sopravvivente.

Ovarian cancer is the sixth diagnosed cancer among women worldwide, it has a high mortality and in most cases it's diagnosed in advanced stage (stage III-IV). Combination platinum-paclitaxel chemotherapy administered every 3 weeks is considered the gold standard for first-line treatment of patients with advanced ovarian cancer. Elderly patients with ovarian cancer represents a subgroup with poor prognosis because they are often treated less radically for comorbidities and age. In the present article, we report a case of a 85 year old woman who was diagnosed with stage IV ovarian carcinoma for the presence of peritoneal carcinomatosis ab initio, not radically debulked and then treated with weekly schedule platinum-based and paclitaxel. Despite not being able to complete the chemotherapy, the patient achieved excellent results and represents a case of long survival.

Nanowalled polymer microtubes fabricated by using strained semiconductor templates.

Herein we describe the realization of nanowalled polymeric microtubes through a novel and versatile approach combining the layer-by-layer (LbL) deposition technique, the self-rolling of hybrid polymer/semiconductor microtubes and the subsequent removal of the semiconductor template. The realized channels were characterized in detail using scanning electron and atomic force microscopes. Additionally, we report on the incorporation of a dye molecule within the nanowalls of such microtubes, demonstrating a distribution of the fluorescence signal throughout the whole channel volume. This approach offers the possibility to tailor the properties of micro/nanotubes in terms of size, wall thickness and composition, thus enabling their employment for several applications.

Uptake and distribution of labeled antibodies into pH-sensitive microgels.

We investigated the uptake and release of labeled antibodies from pH-sensitive hydrogel microparticles (i.e. microgels) by means of fluorescence analysis of labeled biological samples. The poly(methacrylic acid) (PMAA) hydrogel is a carbon-based network having carboxylic groups on the surface that dissociate according to their acid-base equilibrium. The ability of the PMAA microgel to encapsulate and release anti-CD4 and anti-CD8 monoclonal antibodies (MAbs), differing for the isotype and labeled with highly photostable fluorophore, was studied in solution by photoluminescence spectroscopy. The experimental results indicated that the uptake and release of the tested antibodies were controlled by pH. Furthermore, confocal microscopy analysis in the solid state revealed that the distribution of the labeled antibodies either on the surface or in the core of the microgel matrix was related to the specific properties of these MAbs.

Study of the surface morphology of a cholesteryl tethering system for lipidic bilayers.

The immobilization of functional molecules embedded in lipidic membranes onto inorganic substrates is of great interest for numerous applications in the fields of biosensors and biomaterials. We report on the preparation and the morphological characterization of a tethering system for lipidic bilayers, which is based on cholesteryl derivatives deposited on hydrophilic surfaces by self-assembling and microcontact printing techniques. The investigation of the structural properties of the realized films by atomic, lateral, and surface potential microscopy allowed us to assess the high quality of the realized cholesteryl layers.

Formation and characterization of glutamate dehydrogenase monolayers on silicon supports.

In this paper we have tested two different procedures (the "three-step" and the "four-step" procedures) for the covalent immobilization of glutamate dehydrogenase (GDH) onto silicon supports. Atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FT-IR), fluorescence spectroscopy and an enzymatic assay were used to probe the structure and activity of the immobilized enzyme. Our results demonstrate that coupling through the "three-step" procedure does not significantly affect either the fold pattern or the activity of the enzyme, suggesting that this method could be ideally suited to the development of high quality monolayers for use in enzyme-based planar biosensors.

Self-assembling of proteins and enzymes at nanoscale for biodevice applications.

Different nanotechnological strategies have been selected to implement biomolecular devices following a bottom-up or top-down approach depending on the biomolecule and on its functionality. Biomolecules have particular functionality and self-assembling capabilities that can be exploited for the implementation of both bioelectronic devices and multipurpose engineered biosurfaces. Surface preparation with supramolecular methods and microcontact printing have been developed and optimised to realise suitable functionalised surfaces. These surfaces can be used to link metalloproteins and enzymes for the implementation of nanobioelectronic devices and planar biosensors or to bind cells in order to promote their growth along predefined tracks and grooves. Some possible applications of these biosurfaces are shown and discussed. Results are presented for the realisation of a biomolecular nanodevice working in air based on the metalloprotein azurin immobilised in the solid state, the formation and characterisation of functional glutamate Dehydrogenase monolayers for nanobiosensing applications, the results of soft lithography processes on azurin for biosensor implementation, and the development of physiological self-assembled patterns of laminin-1 for cell culture applications and hybrid devices.

Continuous apomorphine infusion and neuropsychiatric disorders: a controlled study in patients with advanced Parkinson's disease.

The aim of this study was to assess whether patients with Parkinson's disease (PD) develop cognitive and psychiatric complications more frequently during prolonged therapy with continuous apomorphine infusion compared with standard oral treatment. Thirty consecutive PD patients with severe motor fluctuations were included in the study. Twelve patients accepted the treatment with subcutaneous continuous apomorphine infusion, while the remaining 18 preferred to continue with oral dopaminergic therapy. The two groups were evaluated with neuropsychological, psychiatric, and motor tests at baseline and after 1 year. The off daily duration and the levodopa dosage were significantly reduced in infused patients. The neuropsychiatric assessment did not change in both groups compared with baseline, except for a significant improvement of mood in the apomorphine group.

Optical characterization of glutamate dehydrogenase monolayers chemisorbed on SiO2.

This paper describes the formation of glutamate dehydrogenase monolayers on silicon dioxide, and their characterization by means of physical techniques, i.e., fluorescence spectroscopy and Fourier-transform infrared spectroscopy. Detailed investigations of the intrinsic stability of native proteins in solution were carried out to elucidate the occurrence of conformational changes induced by the immobilization procedure. The enzyme monolayers were deposited on SiO2 after preexposing silicon surfaces to 3-aminopropyltriethoxysilane and reacting the silylated surfaces with glutaric dialdehyde. The optical characterization demonstrates that the immobilization does not interfere with the fold pattern of the native enzyme. In addition, fluorescence spectroscopy, thermal denaturation, and quenching studies performed on the enzyme in solution well describe the folding and unfolding properties of glutamate dehydrogenase. The photophysical studies reported here are relevant for nanobioelectronics applications requiring protein immobilization on a chip.

New approaches to breast cancer: oxaliplatin combined with 5-fluorouracil and folinic acid in pretreated advanced breast cancer patients: preliminary reports.

Oxaliplatin is a platinum compound that inhibits DNA synthesis. This drug has a broad spectrum of antineoplastic activity, and its results in breast cancer are promising. We began a phase II study in pretreated advanced breast cancer patients using oxaliplatin together with 5-fluorouracil and folinic acid, a combination based on the efficacy of both drugs in breast cancer and their different toxicity profiles. Seventeen patients with advanced breast cancer were treated with oxaliplatin, 5-fluorouracil, and folinic acid, and preliminary data were analyzed. The mean number of courses per patient was 2.82 (range 1-8). The main toxicity was gastrointestinal, with nausea and vomiting G2-3 in 53% of the patients. Hematologic toxicity was moderate with neutropenia G2-3 in 13% of the patients. Among 10 evaluable patients we obtained partial response in one and stabilized the disease in two patients. No data on survival were evaluated. The small number of enrolled and evaluable patients does not permit any conclusions to be drawn. The study is ongoing.

The damage to a person caused by venous thromboembolism in the civil responsibility.

The venous thromboembolism can clinically show itself as deep venous thrombosis or as pulmonary embolism. Both serious and potentially fatal, for this high incidence, they assume importance in social economic sphere. The authors take into account the medicolegal diagnostics methodology of the deep venous thrombosis and of the pulmonary embolism, the traumatic and post traumatic etiology, to determine the connection of causality and the estimating parameters of the damage to a person in the sphere of civil responsibility. To attain to a certain diagnosis of thromboembolism, since its difficult cause of paucisymtomaticity or asymtomaticity of the pathology after an attentive evaluation of symptoms, clinic manifestations and factors of risk, it can't be disregarded to utilize scientific diagnostic criteria, and instrumental ascertainments, serial too, helped by conventional means of standardization, such as the new American system of classification CEAP. The following phases of medicolegal ascertainment consist in identifying the causal connection between disease and event and in estimating of the damage to a person, with rigorous and objective methodology and using tabular orientation guides, that have to indicate the percentage incidence of the undergone disablement on the person's validity for indemnity. It is showed the particular delicacy of the medical examiner's evaluation in thromboembolic disease, in the sphere of civil responsibility, both for the difficulties of the diagnostic identification of the deep venous thrombosis, and of the pulmonary embolism, and for the determination of the connection of causality with traumatic events and with following operation of orthopedics-traumatology and neurosurgery (sector on which the most difficult problems of professional responsibility can connect) and finally for the real evaluation of the consequent damage to a persons, in order to its indemnity.

Effect of serum albumin on estrogen metabolism in human cancer cell lines.

The observation that charcoal-treated fetal bovine serum (ctFBS) was able to modify one of main pathways of estrogens in cancer cells in culture, prompted us to initiate the present study. The active component of serum was isolated using native preparative polyacrylamide gel electrophoresis (PAGE). Under analysis with SDS-PAGE, a M(W) of 68 kDa and mobility of authentic bovine serum albumin (BSA) was observed. The addition of BSA to the serum free culture medium of HEC 1A human endometrial cancer cell line, resulted in an alteration of estradiol (E2) metabolism similar to that observed in the presence of ctFBS. BSA in fact, much enhanced 16 alpha-hydroxylation and significantly reduced 2-hydroxylation of E2 in HEC 1A cells. Comparable results were obtained with different endometrial (Ishikawa) and mammary (MCF-7) tumor cell lines having a different metabolic conversion rate of E2. Several albumin preparations from either bovine or human serum had the same effect; besides, BSA activity was unaffected by treatment with dextran-charcoal or heat. In the light of the present results, the inclusion of serum albumin (SA) in the formulation of media for studies evaluating steroid metabolism in cultured cells should be carefully considered.

Different conversion metabolic rates of testosterone are associated to hormone-sensitive status and -response of human prostate cancer cells.

The main goal of the present work was to compare the ability of human prostate cancer (PCa) cells to metabolize testosterone (T) in living conditions. To this end we studied three different human PCa cell lines (LNCaP, DU145 and PC3) having different hormone-sensitive status and capability of response to androgens. We used an original approach which allows the evaluation of conversion metabolic rates in growing cells after administration of labeled steroid precursor (presently T), at physiological concentrations (1-10 nM). Analysis of both precursor degradation and formation of several products was carried out using reverse phase-high performance liquid chromatography (RP-HPLC) and "on line" radioactive detection. Comparison of the three human PCa cells revealed that their metabolic aptitude differed in many respects: (i) rates of precursor degradation, (ii) different products' formation, and (iii) extent of conjugate production. In detail, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione (A-4-ene-Ad); both DU145 and LNCaP cells mostly retained high levels of unconverted T, with a limited production of A-4-ene-Ad and its 17-keto derivatives (if any). Either LNCaP or DU145 cells generated a relatively high amount of dihydrotestosterone (DHT). In contrast, neither DHT nor its main metabolites were detected in PC3 cells at both short and longer incubation times. As expected, T degradation and A-4-ene-Ad production were highly correlated (r = 0.97; P < 0.03); similarly, A-4-ene-Ad and DHT formation showed a negative, significant correlation. Negligible production of conjugates was noted in both PC3 and DU145 cells, whilst it was remarkable in LNCaP cells (ranging from 43 to 57%). Overall, our data indicate that human PCa cells degrade T quite differently, favoring alternatively reductive or oxidative patterns of androgen metabolism.

Androgen receptor assays in specimens of prostatic tissue obtained by transurethral resection and transvesical adenomectomy.

The main goal of this study was to ascertain whether routine transurethral resection (TUR) of prostate may provide useful material for the evaluation of androgen receptor (AR) status. At the same time, either intracellular distribution of binding affinity and capacity of receptor molecules were particularly taken into account. Based on our previous findings in breast and endometrial cancer, we suggest that a "functional" receptor status may correspond to the presence of type I (high affinity, low capacity) AR in both soluble and nuclear fractions. However, the precise significance of type II (lower affinity, higher capacity) binding sites remains to be clarified. Ten samples of large prostatic adenomas, obtained by transvesical adenomectomy (TVA), were compared with ten parallel specimens obtained by an in vitro TUR, whereby a pure cutting current was used. The AR assay was carried out with a standard competition method using tritiated mibolerone as the radioligand and Scatchard analysis for data processing. No significant difference between the TUR and TVA groups emerged concerning type I AR content of soluble, nuclear or soluble together with nuclear fractions; this was also true when the results were expressed either as fmol/ml homogenate or as fmol/mg DNA. Similarly, concentrations of type II AR in TVA and TUR samples did not differ significantly in either cell compartment, although they were widely scattered, especially in the soluble fraction. In the light of our findings, it is suggested that TUR specimens represent suitable material for receptor studies, provided that only cutting current is employed and that the use of coagulation current, to control bleeding from the prostatic bed, is confined to the final step of the TUR procedure.

Steroid patterns of benign breast disease.

We briefly review some biochemical aspects of benign breast disease (BBD), mainly focusing on free and conjugate estrogen content of breast cyst fluid (BCF), also in relation to cyst type. Evidence is reported that high K(+)-type I-cysts clearly associate with low Cl- levels and accumulate significantly higher quantities of dehydroepiandrosterone sulfate (DHAS) and estrone-3-sulfate (E1S). In spite of the limited number of cases, both increasing DHAS and E1S levels correlate with the increment of K+ to Na+ ratio. A positive correlation was also found between DHAS and E1S. Using electrochemical detection (ECD) on-line to high performance liquid chromatography (HPLC) in the reverse phase mode, we also studied the free estrogen profile. We observed that in type I BCF there are significantly increased amounts of free estrone (E1). The E1S to E1 ratio was significantly different in the two cyst subpopulations; again, a positive correlation was found between free and sulfated E1 (r = 0.820, p less than 10(-6). This last, together with other experimental observations, allows us to hypothesize that in BCF a main pathway of steroids should be E1S----E1. Besides, high specific activity of sulfatase, as well as beta-glucuronidase enzymes, has been demonstrated for BBD. Preliminary information is also reported concerning the BCF pattern of free estrogens, including the highly polar ones, i.e., catecholestrogens (CCE) and the parent methoxy (MeO) conjugates, which represent, in BCF, a predominant portion of all free estrogens. Both CCE levels and ratios appear unevenly distributed in the two different cyst types. In addition, some BCFs show very high concentrations of 16 alpha-OH-E1. Further studies are needed to answer the main question: whether estrogen patterns could represent additive parameters to further categorize breast cystic disease (BCD) or whether they are of minor interest to determine patients' risk of developing breast cancer.