The use of an amphiphilic aryleneethynylene fluorophore as a plasma membrane marker in fixed and living mammalian cells and liposome model systems is demonstrated. We show here that the optical properties of the novel dye are almost independent on pH, in the range 5.0–8.0. Spectroscopic characterization performed on unilamellar liposomes ascertained that the fluorescence intensity of the aryleneethynylene fluorophore greatly increases after incorporation in lipidic membranes. Experiments performed on different mammalian cells demonstrated that the novel membrane marker exhibits fast staining and a good photostability that make it a suitable tool for live cell imaging. Importantly, the aryleneethynylene fluorophore was also shown to be a fast and reliable blue membrane marker in classical multicolor immunofluorescence experiments. This study adds new important findings to the recent exploitation of the wide class of aryleneethynylene molecules as luminescent markers for biological investigations.

Anisakis simplex is a parasite that, if present in uncooked and contaminated saltwater fish, can invade the human gut. Two different clinical situations are recognized: the first, known as a gastrointestinal disease, varying from an asymptomatic episode to vomiting and diarrhea, and the second, classified as an adverse reaction to food, characterized by a wide spectrum of allergic reactions like rhinitis, conjunctivitis, or even anaphylaxis causing hypotension and/or shock. The intestinal epithelium, the major defense system against external molecules, represents an open gate for toxins and allergens if its protective function is compromised. Previous data have demonstrated a strict relationship between an altered intestinal permeability (I.P.) and worsening of the clinical manifestations in patients with adverse reactions to the food. In this article we evaluated the sensitization to A. simplex among patients who referred clinical symptoms of allergy. All subjects underwent commonly used alimentary skin prick test for food allergens, to which Ani s1, an A. simplex allergen, was added. In addition, in A. simplex-sensitized subjects, I.P. was determined upon their enrolment to the study (time 0) and after 6 months of consuming a raw fish-free diet (time 6). Five hundred and forty subjects were screened, and 170 had a positive skin prick test, 87 (51.2%) of whom were positive to Ani s1. Increased I.P. was evidenced in A. simplex-sensitized subjects with worse clinical symptoms, which receded after 6 months' elimination of raw seafood. With our data we demonstrated that the alimentary habit to eat raw fish represents a high risk for the integrity of the intestinal mucosa, and we suggest that this pathological situation may constitute an ideal, under-estimated, open gate for molecules that predispose to other, more important pathologies.

Background: Fatigue, pain and depression are common problems among long-term cancer survivors (BCS) which in some patients may persist for many years after healing and the completion of treatment. Several studies have reported that increased serum levels of chemokines and growth factors are particularly significantly correlated with the coexistence of these disorders in cancer survivors. The aim of this study was to assess whether the altered imbalance of pro-inflammatory cytokines, growth factors and chemokine serum levels are associated to presence of fatigue and pain in long-term breast cancer survivors . Methods: Ninety-three BCS were enrolled in this study and blood samples taken from each. Serum levels of 25 analytes including cytokines, growth factors and chemokines were tested by enzyme immunoassay using the flexible Bio-Plex System. Participants also completed a questionnaire measuring demographic, clinical and behavioral variables. Results: Non-parametric discriminant analysis showed that fatigued BCS had significantly higher serum levels of FGF and lower IL-4 and IL-8 compared to the non-fatigued group, while BCS with pain had an increase in eotaxin serum levels and lower IL-4 and Il-7 compared to the group without pain. Univariate analysis showed a statistically significant difference in both mental and physical qol, with levels lower in the subgroup who presented pain than in those without: p = 0.0003 and p < 0.0001 respectively. A lower value of Rantes (p = 0.0131) in breast cancer survivors with pain compared to the group without pain, and a higher median value of TNF-α (p = 0.054) in the pain group than in those without pain was observed. The level of depression was higher than the score of 50 on the Zung scale in fatigued survivors compared to non-fatigued survivors (p = 0.0006). Conclusions: Our results suggest that an altered balance of chemokines, cytokines and growth factors serum levels may be associated to presence of symptoms such as fatigue and pain in breast cancer survivors at an average of 5 years after diagnosis.

Background. Bone metastases are a common event in breast cancer and other tumours, such as prostate and lung cancer. The occurrence of SRE is frequent in presence of bone metastases. The impact of SRE on the quality of life is well established in all tumour types. The use of denosumab, a molecular target drug thatinhibits osteoclast activation, has changed the management of bone metastases in advanced cancer. Methods. Data of 81 pts with bone metastases from breast, prostate and lung cancer, treated in the last year with denosumab, were collected from three Institutions. The occurrence of SRE and safety of denosumab were evaluated in patients that cross from bisphosphonate to denosumab and in pts treated with denosumab upfront. Results. Breast cancer was 70% (N = 57), prostate 16% (N = 13), lung 14% (N = 11). Median age was 69 (34-86), median time from diagnosis to metastasis was 30 months (0-231), bone metastatic sites <3 in 43% pts, = 3 in 57% pts, 51% had extrabone disease. Previous treatment with bisphosphonate was in 38% pts (N = 31), 94% breast (N = 29) and 6% prostate (N = 2), respectively (“cross-over population”). Median time of bisphosphonate treatment was overall 31 months (2-114), 33 mos in breast (2-114), 6 mos in prostate (5.8-6.2). SRE before treatment with denosumab occurred in 54% pts: 80% breast, 7% prostate, 13% lung, respectively. SRE during treatment with denosumab was seen in 5% pts; 50% breast, 25% prostate, 25% lung, respectively. Hypocalcemia was seen in 10% overall: 63% breast, 25% prostate, 12% lung; median time of hypocalcemia was 24 days for all types of tumours. All patients had oral supplementation with calcium as prevention of hypocalcemia. ONJ was seen in 1% of the population. Bone disease progression was seen in 11% pts (56% breast, 22% prostate, 22% lung). Conclusion. Use of denosumab results safe in a population of patients with bone metastases and previous use of bisphosphonate, independently of tumour type; the occurrence of hypocalcemia is easily manageable with a fast recovery; nevertheless, supplementation with calcium is strongly recommended.

Effects of novel sintetic peroxisome proliferator-activation receptor  (PPARs) agonist on neuronal differentiation in the human neuroblastoma SH-SY5Y cell line MALLAMACI R1, LAGHEZZA A3,LOIODICE F3, VITIELLO F2, BUTTIGLIONE M2 1Dip.Pharmaceutical Biology, Faculty of Pharmacy, University of Bari 2Dip.Biomedical Sciences and Human Oncology, Faculty of Medicine, University of Bari 3Dip. Pharmaceutical Chemistry, Faculty of Pharmacy, University of Bari BACKGROUND: PPARs are a subfamily of the nuclear hormone receptor that heterodimerizes with the retinoid X receptor to act as a transcriptional regulator. Recently it  has been shown that the activation of PPARγ isoform promotes neuronal differentiation. AIM: In this study we have investigated the capability of synthetic compounds endowed with different activity profile on PPARα/γ subtypes to induce neuronal differentiation and neurite outgrowth. The experiments were carried out on cells of  the human neuroblastoma SH-SY5Y line. Retinoic acid (RA) was used as a  positive control for neurite outgrowth. METHODS: SH-SY5Y cultures were maintained in DMEM supplemented with 10% FCS. 10µM RA,  0,5 to 25µM PPAR agonist was added to the culture media in the experimental cultures. Cell viability was tested using the MTT assay. Changes in the expression and re-organization of specific markers involved in neuronal differentiation were investigated using immunofluorescence. RESULTS: Synthetic PPAR agonists promote cell differentiation and the outgrowth of cell processes in a concentration-dependent manner. The maximal effect was obtained at a concentration of 25µM. At this concentration we obtained a significant increase of the expression of neurofilament-200 (NF-H) and Gap-43; this agrees with a hypothesis of cell differentiation induced by these molecules.  CONCLUSION: Our results suggest that sintetic PPAR agonist promotes neuronal differentiation and neurite outgrowth in  SH-SY5Y human neuroblastoma cells. Further work on this subject is underway in our laboratories.

FISH testing of HER2 IHC 1+ early breast cancer with unfavorable prognostic factors Background HER2-positive tumors are associated with a poor prognosis and a shortened disease-free and overall survival as well as with other unfavorable prognostic tumor characteristics (high histological grade, high proliferative index, negative or low estrogen receptor expression, etc.). HER2-positive tumors are also responsive to treatment with trastuzumab in reducing the risk of recurrence and improving survival. The aim of this study is to assess the incidence of HER2 gene amplification in selected tumors with adverse prognostic features which scored 1+ by immunohistochemistry (IHC). Methods 75 women with infiltrating ductal carcinoma (IDC) and infiltrating lobular carcinoma (ILC) scoring 1+ by IHC were included. Forty-eight invasive breast carcinoma samples were selected according to unfavorable prognostic tumor characteristics and tested by FISH. HER2 amplification was evaluated using Vysis HER2/Cep17 probe (Path Vysion HER2 DNA Probe Kit®, Abbott Molecular, IL); ratio–based amplification was considered present when the HER2/Cep17 ratio was 2 or more and copy number-based amplification was considered present when the mean HER2 copy number was more than 6, in agreement with the ASCO/CAP/SIAPEC guidelines. Results In 2013, 331 patients with invasive breast tumors were tested by IHC; 75 cases (23%) were scored 1+ of which 62 cases (19%) of IDC and 13 cases (4%) of ILC. Forty-eight invasive breast carcinoma samples (64%) were selected according to one or more unfavorable prognostic tumor characteristics; 22 out of 48 tumors (46%) showed high histological grade (G3); 27 cases (56%) had high proliferative index (Ki-67≥30%); 32 tumor samples (67%) were node-positive; and 29 cases (60%) showed vascular invasion. FISH was performed on 31 of the 1+ patients with adverse tumor characteristics and 7 IDC out of 48 (14.6%) showed HER2 amplification. Conclusions Our preliminary retrospective data suggest that 7 patients out of 48 (14.6%) scoring 1+ by IHC show HER2 amplification, in agreement with the most recently published literature data. In order to not deny the benefit deriving from trastuzumab administration, in breast cancer patients showing IHC 1+, it is advisable to test HER2 gene amplification by FISH. Bibliografia Goldhirsch A, Gelber RD, Piccart-Gebhart MJ, de Azambuja E, Procter M, Suter TM, Jackisch C, Cameron D, Weber HA, Heinzmann D, Dal Lago L, McFadden E, Dowsett M, Untch M, Gianni L, Bell R, Köhne CH, Vindevoghel A, Andersson M, Brunt AM, Otero-Reyes D, Song S, Smith I, Leyland-Jones B, Baselga J; Herceptin Adjuvant (HERA) Trial Study Team. 2 years versus 1 year of adjuvant trastuzumab for HER2-positive breast cancer (HERA): an open-label, randomised controlled trial. Lancet. 2013 Sep 21;382(9897):1021-8. Iorfida M, Dellapasqua S, Bagnardi V, Cardillo A, Rotmensz N, Mastropasqua MG, Bottiglieri L, Goldhirsch A, Viale G, Colleoni M. HER2-negative (1+) breast cancer with unfavorable prognostic features: to FISH or not to FISH? Ann Oncol. 2012 May;23(5):1371-2. Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE Jr, Davidson NE, Tan-Chiu E, Martino S, Paik S, Kaufman PA, Swain SM, Pisansky TM, Fehrenbacher L, Kutteh LA, Vogel VG, Visscher DW, Yothers G, Jenkins RB, Brown AM, Dakhil SR, Mamounas EP, Lingle WL, Klein PM, Ingle JN, Wolmark N. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 2005 Oct 20;353(16):1673-84.

Background HER2-positive tumors are associated with a poor prognosis and a shortened disease-free and overall survival as well as with other unfavorable prognostic tumor characteristics (high histological grade, high proliferative index, negative or low estrogen receptor expression, etc.). HER2-positive tumors are also responsive to treatment with trastuzumab in reducing the risk of recurrence and improving survival. The aim of this study is to assess the incidence of HER2 gene amplification in selected tumors with adverse prognostic features which scored 1+ by immunohistochemistry (IHC). Methods 75 women with infiltrating ductal carcinoma (IDC) and infiltrating lobular carcinoma (ILC) scoring 1+ by IHC were included. Forty-eight invasive breast carcinoma samples were selected according to unfavorable prognostic tumor characteristics and tested by FISH. HER2 amplification was evaluated using Vysis HER2/Cep17 probe (Path Vysion HER2 DNA Probe Kit®, Abbott Molecular, IL); ratio–based amplification was considered present when the HER2/Cep17 ratio was 2 or more and copy number-based amplification was considered present when the mean HER2 copy number was more than 6, in agreement with the ASCO/CAP/SIAPEC guidelines. Results In 2013, 331 patients with invasive breast tumors were tested by IHC; 75 cases (23%) were scored 1+ of which 62 cases (19%) of IDC and 13 cases (4%) of ILC. Forty-eight invasive breast carcinoma samples (64%) were selected according to one or more unfavorable prognostic tumor characteristics; 22 out of 48 tumors (46%) showed high histological grade (G3); 27 cases (56%) had high proliferative index (Ki-67≥30%); 32 tumor samples (67%) were node-positive; and 29 cases (60%) showed vascular invasion. FISH was performed on 28 of the 1+ patients with adverse tumor characteristics and 6 IDC out of 48 (12.5%) showed HER2 amplification. Conclusions Our preliminary retrospective data suggest that 6 patients out of 48 (12.5%) scoring 1+ by IHC show HER2 amplification, in agreement with the most recently published literature data. In order to not deny the benefit deriving from trastuzumab administration, in breast cancer patients showing IHC 1+, it is advisable to test HER2 gene amplification by FISH.

We explored the hypothesis that an altered microenvironment (intestinal adenomatous polyp) could modify the differentiation program of bone marrow-derived stem cells (BMSCs), involving them in colon carcinogenesis. Sublethally irradiated 8-week-old female Apc(Min/+) mice were transplanted with bone marrow (BM) cells obtained from either male age-matched Apc(Min/+) (Apc-Tx-Apc) or wild type (WT) (WT-Tx-Apc) mice. At 4 and 7 weeks after transplantation, BM-derived colonocytes were recognized by colocalization of Y-chromosome and Cdx2 protein (specific colonocyte marker). Polyp number, volume, and grade of dysplasia were not influenced by irradiation/transplantation procedures since they were similar in both untreated female Apc(Min/+) and Apc-Tx-Apc mice. At 4 and 7 weeks after transplantation, a progressive significant reduction of polyp number and volume was observed in WT-Tx-Apc mice. Moreover, the number of WT-Tx-Apc mice with a high-grade dysplastic polyps significantly decreased as compared to Apc-Tx-Apc mice. Finally, at 4 and 7 weeks after transplantation, WT-Tx-Apc mice showed a progressive significant increase of Y+/Cdx2+ cells in "normal" mucosa, whereas, in the adenomatous tissue, Y+/Cdx2+ cells remained substantially unvaried. Our findings demonstrate that WT BMSCs do not participate in polyp development but rather inhibit their growth. The substitution of genotypically altered colonocytes with Y+/Cdx2+ cells probably contributes to this process.

Background: The cancer disease is the clinical condition most often associated with the concept of protein-calorie malnutrition that affects 8% to 84% of cancer patients and can worsen until determining cachexia also called “Wasting Disease”. Cancer-Related Anorexia-Cachexia Syndrome (CACS), is a complex metabolic syndrome characterized by a negative protein balance and energy caused both by a reduced caloric intake from both metabolic abnormalities associated with loss of muscle mass with or without loss of mass fat and considerable weight loss; it is estimated that about 20% of all cancer deaths occur for malnutrition. It is necessary to complement clinical examination of the patient also with review the nutritional status in order to evaluate the therapeutic process in these patients. Objective: The aim of this study was to assess the prevalence of malnutrition or the risk of malnutrition in patients undergoing to surgery for colorectal carcinoma. Methods: A total of 53 patients, 26 male and 27 female media age: 70,5 (42-86 yrs) undergoing to surgery for colorectal carcinoma in our Institute between November 2014 and April 2015, were evaluated to determine individual nutritional status, using the Mini Nutritional Assessment (MNA®) that includes 18 items grouped in four rubrics: anthropometric assessment, general assessment and lifestyle, short dietary assessment and subjective perception of health and nutrition. Results: The overall mean score for the MNA, was 26.2 ± 13.2 (range 6.0-27.0);15/53 patients (24.5%) presented a normal nutritional status (mean 24.8; range 24.0 to 27.5),25/53 (47%) reported a risk of malnutrition while 13/53 (24.5%) reported a severemalnutrition that was found to be more common in men than in women. Besides, allmalnourished patients had in the previous six months from the date of diagnosis, asignificant weight loss (> 10 kg), muscle mass loss and severe reduction in the intake of food due loss of appetite and altered taste perception. Instead, patients at risk for malnutrition reported a weight loss of 3 to 5 kg and a moderate reduction in food intake. Conclusions: More than 50% of patients had moderate or severe malnutrition and the majority of them needed nutritional intervention before and during chemotherapy. Our preliminary data show the importance of nutritional assessment in cancer patients in order to set the appropriate treatment plan, improve its quality of life and increase overall survival in cancer patient.