Evaluating the effects of polices on nursing care as promoted by the Veneto region: the research protocol and its implications for public health.

Study design: Multi-centre mixed-method study design organised into several phases. Background: The Veneto region has recently defined a set of policies on nursing care by determining the needed amount of daily care in minutes and by initiating a systematic measurement of nursing outcomes; also, with a more recent policy, missed nursing care (MNC) has been established as a process measure of interest. To measure the effect of these policies, a research protocol - aimed at evaluating several end points - has been designed, involving a large target population and hospital units. The aim of this manuscript is to briefly present the research protocol and to discuss the public health implications of its expected end-points. Methods: The endpoints of the protocol are: (a) to describe the frequency of MNC as perceived by nurses; (b) to identify contributing factors; (c) to identify practices adopted in low-occurrence MNC units and to assess the effectiveness of implementing them in units with higher levels of MNC; (d) to explore the relationship between the amount of nursing care provided, MNC, and patient outcomes; and (e) to validate a tool that measures MNC as perceived by patients/caregivers. A total of 3,460 nurses, 5,000 patient/day and 160 nursing coordinators of the medical and surgical units of public hospitals in the Veneto Region will be included. Conclusions: Measuring the association between the amount of nursing care and patient outcomes, as well as evaluating the role of MNC as perceived by nurses and patients in hindering or increasing the risk of some patient outcomes can provide a body of evidence capable of further informing policies in the field, both at the national and at the international level. Moreover, emerging good practices capable of preventing or minimising MNC, sharing and implementing them in other units where high levels of missed care are reported and evaluating their effectiveness, can also inform public health policies.

NOTCH1-mutated chronic lymphocytic leukemia cells are characterized by a MYC-related overexpression of nucleophosmin 1 and ribosome-associated components.

In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.

CD49d prevails over the novel recurrent mutations as independent prognosticator of overall survival in chronic lymphocytic leukemia.

CD49d, the alpha-chain of the integrin heterodimer α4ß1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53. In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53, NOTCH1, SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P<0.0001). Consistently, high CD49d expression identified CLL subsets with inferior OS in the context of each category of a previously reported hierarchical risk stratification model. Moreover, by evaluating the relative importance of biological prognosticators by random survival forests, CD49d was selected among the top-ranked OS predictor (variable importance =0.0410), along with IGHV mutational status and TP53 abnormalities. These results confirmed CD49d as an independent negative OS prognosticator in CLL also in comprehensive models comprising the novel recurrent mutations. In this context, TP53 disruption and NOTCH1 mutations retained prognostic relevance, in keeping with their roles in CLL cell immuno-chemoresistance.

NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation.

In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by γ-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541-7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC-mediated epigenetic repression of CD20 expression.

Effects of six months of vitamin D supplementation in patients with heart failure: a randomized double-blind controlled trial.

BACKGROUND AND AIM: Low plasma vitamin D levels have been associated with heart failure (HF). This research attempts to explain the role of vitamin D supplementation on myocardial function in elderly patients with HF. METHODS AND RESULTS: Twenty-three chronic HF patients were randomized in a small parallel group, double-blind, placebo-controlled trial. All patients, with a mean age of 74 years and vitamin D levels <30 ng/mL, received 800,000 IU (4000 IU/daily) of cholecalciferol or placebo for 6 months. The outcomes measured at baseline and after 6 months were ejection fraction (EF) and other echocardiography parameters, carboxyterminal propeptide of procollagen type I (PIP), natriuretic peptides, lipid profile, renin, parathyroid hormone, blood pressure, and body mass index (BMI). In 13 patients under active treatment for 6 months, mean plasma 25-hydroxy vitamin D concentrations (15.51 vs. -1.40 ng/mL, p < 0.001) and plasma calcium (from 9.3 to 9.6 mmol/L, p < 0.05) increased significantly. However, other biomarkers of bone metabolism did not differ between the treatment and placebo groups. EF increased significantly in the intervention group (6.71 vs. -4.3%; p < 0.001), and the serum concentration of PIP increased only in the placebo group after 6 months (1140.98 vs. -145 mcg/L; p < 0.05). Systolic blood pressure was lower after 6 months of cholecalciferol treatment (from 129.6 to 122.7 mm Hg, p < 0.05). No significant variations were observed for other parameters. CONCLUSIONS: Six months of vitamin D supplementation significantly improves EF in elderly patients with HF and vitamin D deficiency.

Anti-angiogenic effects of two cystine-knot miniproteins from tomato fruit.

BACKGROUND AND PURPOSE: Cystine-knot miniproteins are characterized by a similar molecular structure. Some cystine-knot miniproteins display therapeutically useful biological activities, as antithrombotic agents or tumour growth inhibitors. A critical event in the progression of tumours is the formation of new blood vessels. The aim of this work was to test two tomato cystine-knot miniproteins for their effects on endothelial cell proliferation and angiogenesis in vitro. EXPERIMENTAL APPROACH: Two tomato cystine-knot miniproteins (TCMPs) were expressed and purified either as recombinant or as native proteins from tomato fruits. The Matrigel assay was used to investigate the effects of TCMPs on in vitro angiogenesis. Viability and proliferation of endothelial cells were tested. Extracellular signal-regulated kinase (ERK)1/2 phosphorylation was assayed in either HUVEC or A431 epidermal growth factor receptor (EGFR)-overexpressing cells treated with TCMPs. EGFR phosphorylation was tested in A431 cells. KEY RESULTS: Both recombinant and native TCMPs inhibited in vitro angiogenesis of HUVEC cells at concentrations of 15-100 nM. The anti-angiogenic effect of TCMPs was associated with the inhibition of ERK phosphorylation. The two miniproteins did not alter the viability and proliferation of the endothelial cells. CONCLUSIONS AND IMPLICATIONS: The anti-angiogenetic properties of TCMPs are of potential pharmacological interest because they are common and natural components of the human diet, they possess low toxicity, they are active at submicromolar concentrations, they share a common molecular structure that can be used as a molecular platform for the design of molecules with enhanced biological activity.

Preclinical ex vivo expansion of peripheral blood CD34+ selected cells from cancer patients mobilized with combination chemotherapy and granulocyte colony-stimulating factor.

BACKGROUND AND OBJECTIVES: Ex vivo peripheral blood progenitor cell (PBPC) expansion has been proposed as a strategy to increase the number of haematopoietic progenitors available for cell transplantation. We have expanded CD34+ cells from PBPCs obtained from four patients with haematological malignancies and one patient with an Ewing's sarcoma. MATERIALS AND METHODS: Cells were expanded in the Dideco 'Pluricell system'. After 12 days in culture, we evaluated cell phenotype, total nucleated cells, CD34+ fold increase, cell apoptosis and colony assay of expanded cells. Cell engraftment has been evaluated by transplanting two groups of irradiated non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice with expanded and non-expanded cell populations. RESULTS: Total nucleated cells and CD34+ cells increased 59.5 and 4.0 times, respectively. The expanded cells were mainly constituted of myeloid and megakaryocytic cells. A significant increase in the number of colony-forming unit-granulocyte macrophage (CFU-GM) was observed in the CFU assay. Ten mice transplanted with expanded cells showed a best overall survival (80%) compared to 10 mice transplanted with non-expanded cells (20%). Human CD45+ cells were detected by flow cytometry and polymerase chain reaction in bone marrow and spleen of transplanted animals. The relative low engraftment level obtained with the expanded cells suggests a loss of SCID repopulating cells maybe due to cell differentiation during expansion. CONCLUSIONS: We have demonstrated the feasibility of the ex vivo expansion of mobilized PBPCs from cancer patients, evidencing a clonal expansion of CFUs and the ability of the expanded cells to engraft the bone marrow and spleen of immunosuppressed mice. The differentiation of the CD34+ stem cell compartment could be further minimized by ameliorating the expansion conditions.

Telomerase expression in B-cell chronic lymphocytic leukemia predicts survival and delineates subgroups of patients with the same igVH mutation status and different outcome.

Activation of telomerase reverse transcriptase (hTERT) is essential for unlimited cell growth and plays a critical role in tumorigenesis. We investigated hTERT gene expression in 134 B-cell chronic lymphocytic leukemia (B-CLL) cases and evaluated its prognostic value with other prognostic markers (IgVH mutation status, CD38 and ZAP-70 expression). Real-time PCR assays to quantify either all hTERT transcripts (AT) or only the full length (FL) transcript encoding the functional protein were developed. hTERT-AT levels strongly correlated with hTERT-FT levels (r=0.743, P<0.0001); both inversely correlated with the percentage of IgVH mutation (P<0.005) and were significantly higher in unmutated than in mutated cases (P=0.004 and P=0.001, respectively). The hTERT values which best discriminated between the unmutated and mutated IgVH cases were 150 and 40 copies for hTERT-AT and hTERT-FL, respectively. Using these cut-off values, there was a significant difference in the survival of patients with high or low hTERT levels (P<0.0001). Unmutated cases with low hTERT levels had an overall survival close to mutated cases with high hTERT levels. Thus, this work identifies hTERT-RNA level as a new prognostic marker in B-CLL, and may be used to identify previously unrecognized patient groups with the same IgVH mutation status and different disease outcomes.

Insulin and glucose mediate opposite intracellular ionized magnesium variations in human lymphocytes.

Insulin is capable of increasing intracellular magnesium, although very little is known about the effect of insulin on the biologically active fraction of magnesium, i.e. the ionized quota (Mg(i)(2+)), its interactions with glucose, and the cellular mechanisms involved in these processes. We studied the interactions of the effects of insulin and glucose on intracellular ionized magnesium in human lymphocytes. Mg(i)(2+) was measured using a fluorimetric method and the Mg(2+)-sensitive dye, furaptra. We found that insulin significantly increases the Mg(i)(2+)(without insulin 227 +/- 14 microM, with 10 microU/ml, insulin 301 +/- 30 microM, P<0.0001, n = 12) in a dose-dependent manner in all three glucose concentrations tested (5, 7 and 15 mmol/l). The half-maximal effect of insulin was approximately 0.8 microU/ml. Glucose and insulin showed opposite effects in their ability to modify Mg(i)(2+) in lymphocytes. Inhibitors of the membrane Na(+)- Mg(2+) transport system and of phosphatidylinositol (PI) 3-kinase abolish the insulin-mediated increase of Mg(i)(2+), thus suggesting that insulin is capable of increasing Mg(i)(2+) by modulating the activity of this transport system, possibly through the mediation of PI 3-kinase activation. Taking into account the relationship between insulin and glucose plasma levels and their opposing effects on Mg(i)(2+), this mechanism may represent the two limbs of a biphasic regulatory system of Mg(i)(2+) in both physiological and pathological conditions.

Evaluation of platelet turnover by flow cytometry.

The number of circulating newly produced platelets depends on the thrombopoietic capacity of bone marrow as well as platelet removal from the bloodstream. Flow cytometric analysis with thiazole orange (TO), a fluorescent dye that crosses platelet membranes and binds intracellular RNA, has been used to measure circulating reticulated platelets (RPs) with high RNA content as an index of platelet turnover. We first assessed the specificity of TO flow cytometry and then applied this method in the diagnosis of thrombocytopenia caused by impaired platelet production or increased destruction. We also explored the utility of TO flow cytometry to predict thrombocytopoiesis after chemotherapy-induced bone marrow aplasia. Venous blood, anticoagulated with K(2)EDTA, was incubated with 0.6 microg/ml TO plus an anti-GPIIIa monoclonal antibody. The mean percentage of RPs in control subjects (n = 23) was 6.13 +/- 3.09%. RPs were 10.41 +/- 9.02% in patients (n = 10) with hematological malignancies during aplasia induced by chemotherapy and a significant increase in RPs (35.45 +/- 6.11%) was seen in the recovery phase. In 10 patients with idiopathic thrombocytopenic purpura, the percentage of TO positive platelets was 67.81 +/- 18.79 (P < 0.001 vs. controls). In patients with thrombocytopenia associated with hepatic cirrhosis (n = 21; 21.04 +/- 16.21%, P < 0.001 vs. controls) or systemic lupus erythematosus (n = 6, 29.08 +/- 15.57%; P < 0.001 vs. controls) increases in TO-stained platelets were also observed. Measurement of TO positive platelets may be a reliable tool for the laboratory identification of platelet disorders, with a higher sensitivity than measurement of platelet volume. Measurement of RPs may also prove useful to recognize the underlying pathogenetic mechanisms in thrombocytopenia.

Levels of F2-isoprostanes in systemic sclerosis: correlation with clinical features.

OBJECTIVE: Oxidative stress may be one of the important complex pathogenetic mechanisms that lead to damage in scleroderma; free radicals may provoke endothelial injury, fibroblast proliferation and fragmentation of autoantigens favouring induction of autoantibodies. The present study investigates the oxidant status in scleroderma patients by measuring the urinary concentration of 8-isoprostaglandin-F2alpha, an F2-isoprostane, and a product of free radical-mediated peroxidation of arachidonic acid. METHODS: Forty-three scleroderma patients (42 women and 1 man, mean age 54.1 yr, mean disease duration 9.0 yr) underwent clinical evaluation and instrumental investigations in order to assess skin, vascular, lung and heart involvement. Von Willebrand factor was evaluated as marker of vascular dysfunction in 36 out of the 43 cases. The urinary level of 8-isoprostaglandin-F2alpha was measured in all scleroderma patients and in the 43 age- and sex-matched healthy controls. RESULTS: Urinary levels of 8-isoprostaglandin-F2alpha were higher in scleroderma patients than in the healthy control group (341.7 vs 147.6 pg/mg creatinine; P < 0.001). Values of 8-isoprostaglandin-F2alpha were strongly correlated with the nailfold videocapillaroscopy pattern and lung involvement (P = 0.002 and 0.003, respectively), showing increasing levels with the progression of pulmonary severity. Correlation between 8-isoprostaglandin-F2alpha level and von Willebrand factor narrowly failed to reach statistical significance (P = 0.05). There was no correlation between 8-isoprostaglandin-F2alpha concentration and disease activity, vascular, skin and heart involvement, disease pattern or autoantibody profile. CONCLUSIONS: Our study further supports the role of oxidant stress in the pathogenesis of scleroderma, showing a strong correlation between a marker of free radical damage with both the severity of lung involvement and the videocapillaroscopic patterns.

Catecholamine-induced regulation in vitro and ex vivo of intralymphocyte ionized magnesium.

Despite the importance of the adrenergic activity and of the metabolism of magnesium in some important cardiovascular pathologies, very little is known about how intracellular ionized magnesium (Mgi2+) is regulated by catecholamines. We made an in-vitro study of the variations in the concentration of ionized magnesium in human lymphocytes using the fluorescent probe furaptra in response to different catecholamines. We also made an ex-vivo study of the changes in intracellular ionized magnesium in lymphocytes in 20 subjects with essential arterial hypertension, 10 treated with 120 mg/d of propranolol and 10 with placebo. Norepinephrine and isoproterenol significantly decrease Mgi2+ and this effect is blocked by beta-blockers but not by alpha-blockers. The EC50 of the effect of norepinephrine is within the range of concentrations physiologically present in plasma. The substitution of extracellular sodium with choline blocks the decrease in intracellular ionized magnesium induced by norepinephrine, which leads us to suppose that the magnesium-reducing effect of catecholamines is a result of the activation of a Na+-Mg2+ exchanger. We were not able to demonstrate any change in intracellular ionized magnesium after 1 and 17 days of active treatment in essential hypertensives. The impossibility of demonstrating ex vivo the mechanism of catecholamine-mediated regulation that is evident in vitro is perhaps due to our experimental conditions or to substances which in vivo inhibit the action of the catecholamines on magnesium, such as insulin and/or glucose.

[Italian translation and validation of the ICNP Beta (International Classification for Nursing Practice]. / Traduzione e validazione italiana della Classificazione Internazionale per la Pratica infermieristica (ICNP Beta).

The visibility of nursing care can be measured through the results on the patient and studying the existing data on patient care. An important area is tied up for a common language and terminology because it is important to have a universal understanding. For this reason the International Council of Nurses (ICN) has elaborated an international terminology for Nursing Practice that can be useful to pick up and to catalogue the problems of nursing nature in diagnosis also finding a system of classification of the activities The Consociazione Association, as representative of the ICN in Italy, through the School of Advanced Nursing of the University "La Sapienza" of Rome, has set up a Working group for the translation of the document ICNP version Beta. The present article explains the difficulties related to the Italian translation, the working group, the procedure, the validation and the met problems along with a vision for the future development and utilization.

[The utilization of the ICNP Beta version taxonomy system for the nursing protocols' construction]. / Un'esperienza applicativa del sistema tassonomico ICNP nella costruzione di protocolli infermieristici.

The ICNP Beta version system has been used for the revision of hospital protocols on the nursing management of the central venous catheters. Despite the taxonomy defects of clarity and functionality, our experience has shown an increase of understanding and accord between builders professionals and protocol's users. Besides all. It has also been important to sensitize the participants to the experimentation of an encoded international language.

[Pilot training experience in lip-reading skills for nurses in intensive care units]. / Un'esperienza pilota di addestramento alla lettura labiale per infermieri delle unità di terapia intensiva.

UNLABELLED: In the intensive care unit (ICU) of Mestre hospital (Italy) a research was carried out to analyse the possibility to improve nurses' lip-reading skills. METHODS: A specialized speech therapist organized a 20 hour training course for 34 health workers in ICU. RESULTS: The participants had a lip-reading test at the beginning and at the end of the course and six months later. The final test revealed that participants could recognize a greater number of typical words in ICU in comparison to the initial test. Yet, after 6 months the skill decreased to the level shown at the beginning of the course. On the other hand, the trend shows that some participants' skill did not decrease. Neither the starting level nor intermediate stage are conditioned by sex, age, profession, and experience. In order to measure clinic efficacy, the participants had another test to understand if improved lip-reading skill could influence the following variables: word comprehension, nurse attitude to communication toward non-speaking people, patient emotional status and nurse emotional status. The training course seems to have had good effects on nurses' attitudes towards their patients and on their communication. Yet, study results do not show if increased lip-reading skills have real positive effects on ICU communication and whether this method is better than usual communication methods (e.g. mimic or alphabetic board).

CD30L up-regulates CD30 and IL-4 expression by T cells.

CD30L is frequently expressed on acute myeloid leukemia (AML) blasts. Its presence is associated with the co-expression of interleukin-4 (IL-4) receptor and with the expansion of specific T-helper 2 (Th2) cell subsets producing IL-4 and expressing CD30. Recombinant CD30L-bearing cells up-regulated the expression of surface CD30 and increased the production of IL-4 and soluble (s) CD30 by co-cultured T cells. These findings were confirmed with AML blasts expressing surface CD30L, where blocking anti-CD30 antibodies completely abolished the release of sCD30 and reduced the production of IL-4. Our data indicates a direct role of CD30L(+) neoplastic cells in driving the immune response toward a Th2-polarized non-protective state.

NCX4016 (NO-Aspirin) has multiple inhibitory effects in LPS-stimulated human monocytes.

NCX4016 (2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester, NicOx S.A., France) is an anti-thrombotic agent, chemically related to acetylsalicylic acid (ASA) and able to release NO. We tested the effects of NCX4016 and ASA on the release of the thromboxane (TX) A(2) metabolite TXB(2), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), expression and activity of tissue factor (TF) in stimulated, adherent human monocytes. Both ASA and NCX4016 1 - 1000 micromol l(-1) dose-dependently reduced TXB(2) concentration, measured by RIA in the supernatant of 10 microg ml(-1) LPS-stimulated cells. NCX4016 activity was comparable to that of equimolar ASA when incubation lasted 6 h (NCX4016 30 micromol l(-1): -86.0+/-10.1%, NCX4016 300 micromol l(-1): -92.2+/-9.0%, ASA 30 micromol l(-1): -92.3+/-7.5%, ASA 300 micromol l(-1): -97.3+/-1.0%, n=6, M+/-s.d.). Most of the activity of NCX4016 up to 100 micromol l(-1) was prevented by 10 micromol l(-1) ODQ, inhibitor of cyclic GMP. NCX4016 100 - 300 micromol l(-1) reduced TNF-alpha (NCX4016 300 micromol l(-1)=-77.2+/-19.9%, n=6) and IL-6 (NCX4016 300 micromol l(-1): -61.9+/-15.2%, n=6) in LPS stimulated monocytes while ASA had no significant effects. TF activity (NCX4016 300 micromol l(-1): 53.7+/-39.9%, n=4) and immunoreactive TF (NCX4016 300 micromol l(-1): -93.9+/-7.9%, n=7), measured in the supernatant of stimulated cells, were also dose-dependently inhibited by NCX4016 but not by ASA. The present results indicate that NCX4016 inhibits TXA(2) generation as well as cytokine release and TF in human monocytes partly via NO-dependent mechanisms. NCX4016 may have a favourable profile of activities in the clinical setting of athero-thrombosis.