[Atrial fibrillation: use of healthcare resources and expenditures in the Lazio region (Italy)]. / Fibrillazione atriale: attività assistenziale e assorbimento di risorse nella Regione Lazio.

Atrial fibrillation (AF) is the most common chronic cardiac arrhythmia and is an important risk factor for mortality and morbidity related mainly to an increased risk of cerebrovascular events and heart failure.An observational cross-sectional study was performed to evaluate the use of healthcare resources (including hospital and outpatient care) by patients with AF in the Lazio region (central Italy), from 1 January 2006 to 31 December 2008.Atrial fibrillation is an important source of healthcare resource utilization because of repeated emergency room visits, hospital admissions, outpatient consultations and procedures and extensive use of laboratory tests and pharmacological treatments.Results show that 55% of costs are attributable to hospital admissions and Emergency Room visits, 37% to pharmacological treatment and the remaining 8% to outpatient care. These results are consistent with the international literature.The impact of AF in terms of cost is not negligible and it is therefore desirable to implement an organizational scheme that safeguards the appropriateness of care, taking charge of the patient as early as possible. The aims of early diagnosis of AF are to improve the appropriateness of care and optimize the use of specialized tests, thereby reducing hospital admissions for complications or recurrences of AF.

The folding and aggregation properties of a single KH-domain protein: Ribosome binding factor A (RbfA) from Pseudomonas aeruginosa.

BACKGROUND: Ribosome-binding factor A from the pathogenic bacterium Pseudomonas aeruginosa (PaRbfA) is a small ribosome assembly factor, composed by a single KH domain, involved in the maturation of the 30S subunit. These domains are characterized by the ability to bind RNA or ssDNA and are often located in proteins involved in a variety of cellular functions. However, although the ability of proteins to fold properly, to misfold or to aggregate is of paramount importance for their cellular functions, limited information is available on these dynamic properties in the case of KH domains. METHODS: PaRbfA thermodynamic stability and folding mechanism: Far-UV CD and fluorescence spectroscopy, stopped-flow kinetics and chevron plot analysis, site-directed mutagenesis. Fibrils characterization: FT-IR spectroscopy, Thioflavin T fluorescence, Transmission Electron Microscopy (TEM) and X-ray fibrils diffraction. RESULTS: Quantitative analysis of the (un)folding kinetics of PaRbfA show that, in vitro, the protein folds via a 3-states mechanism involving a transiently populated folding intermediate. We also provide experimental evidences that PaRbfA can form ordered fibrils endowed with cross-ß structure even in mild conditions. CONCLUSION: These results lead to the hypothesis that the folding intermediate of PaRbfA may expose (some of) the predicted amyloidogenic regions, which could act as aggregation nuclei in the fibrillogenesis. GENERAL SIGNIFICANCE: The methodological approach presented herein could be readily adapted to verify the ability of other KH domain proteins to form cross-ß structured fibrils and to transiently populate a folding intermediate.

UVB radiation induced effects on cells studied by FTIR spectroscopy.

We have made a preliminary analysis of the results about the effects on tumoral cell line (lymphoid T cell line Jurkat) induced by UVB radiation (dose of 310 mJ/cm(2)) with and without a vegetable mixture. In the present study, we have used two techniques: Fourier transform infrared spectroscopy (FTIR) and flow cytometry. FTIR spectroscopy has the potential to provide the identification of the vibrational modes of some of the major compounds (lipid, proteins and nucleic acids) without being invasive in the biomaterials. The second technique has allowed us to perform measurements of cytotoxicity and to assess the percentage of apoptosis. We already studied the induction of apoptotic process in the same cell line by UVB radiation; in particular, we looked for correspondences and correlations between FTIR spectroscopy and flow cytometry data finding three highly probable spectroscopic markers of apoptosis (Pozzi et al. in Radiat Res 168:698-705, 2007). In the present work, the results have shown significant changes in the absorbance and spectral pattern in the wavenumber protein and nucleic acids regions after the treatments.

Ultrasound delivery of Surface Enhanced InfraRed Absorption active gold-nanoprobes into fibroblast cells: a biological study via Synchrotron-based InfraRed microanalysis at single cell level.

Ultrasound (US) induced transient membrane permeabilisation has emerged as a hugely promising tool for the delivery of exogenous vectors through the cytoplasmic membrane, paving the way to the design of novel anticancer strategies by targeting functional nanomaterials to specific biological sites. An essential step towards this end is the detailed recognition of suitably marked nanoparticles in sonoporated cells and the investigation of the potential related biological effects. By taking advantage of Synchrotron Radiation Fourier Transform Infrared micro-spectroscopy (SR-microFTIR) in providing highly sensitive analysis at the single cell level, we studied the internalisation of a nanoprobe within fibroblasts (NIH-3T3) promoted by low-intensity US. To this aim we employed 20 nm gold nanoparticles conjugated with the IR marker 4-aminothiophenol. The significant Surface Enhanced Infrared Absorption provided by the nanoprobes, with an absorbance increase up to two orders of magnitude, allowed us to efficiently recognise their inclusion within cells. Notably, the selective and stable SR-microFTIR detection from single cells that have internalised the nanoprobe exhibited clear changes in both shape and intensity of the spectral profile, highlighting the occurrence of biological effects. Flow cytometry, immunofluorescence and murine cytokinesis-block micronucleus assays confirmed the presence of slight but significant cytotoxic and genotoxic events associated with the US-nanoprobe combined treatments. Our results can provide novel hints towards US and nanomedicine combined strategies for cell spectral imaging as well as drug delivery-based therapies.

Differential effects on membrane permeability and viability of human keratinocyte cells undergoing very low intensity megasonic fields.

Among different therapeutic applications of Ultrasound (US), transient membrane sonoporation (SP) - a temporary, non-lethal porosity, mechanically induced in cell membranes through US exposure - represents a compelling opportunity towards an efficient and safe drug delivery. Nevertheless, progresses in this field have been limited by an insufficient understanding of the potential cytotoxic effects of US related to the failure of the cellular repair and to the possible activation of inflammatory pathway. In this framework we studied the in vitro effects of very low-intensity US on a human keratinocyte cell line, which represents an ideal model system of skin protective barrier cells which are the first to be involved during medical US treatments. Bioeffects linked to US application at 1 MHz varying the exposure parameters were investigated by fluorescence microscopy and fluorescence activated cell sorting. Our results indicate that keratinocytes undergoing low US doses can uptake drug model molecules with size and efficiency which depend on exposure parameters. According to sub-cavitation SP models, we have identified the range of doses triggering transient membrane SP, actually with negligible biological damage. By increasing US doses we observed a reduced cells viability and an inflammatory gene overexpression enlightening novel healthy relevant strategies.

Exploring mental status in Friedreich's ataxia: a combined neuropsychological, behavioral and neuroimaging study.

Despite much evidence of cognitive and affective disorders in Friedreich's ataxia (FRDA), the nature of mental status in FRDA has received little systematic attention. It has been proposed that the cerebellum may interfere indirectly with cognition through the cerebello-cortical loops, whereas the role of pathological changes in different areas of the central nervous system is still undetermined. In the present study, 13 patients with molecularly determined FRDA and a group of matched controls were evaluated by a comprehensive battery of neuropsychological tests and the Minnesota Multiphasic Personality Inventory. A repetitive task of simple visual-reaction times was used to investigate implicit learning in all subjects. Pathological changes in cortical areas were explored comparing cerebral activations of patients and controls during finger movements (functional MRI). The intelligence profile of FRDA patients is characterized by concrete thinking, poor capacity in concept formation and visuospatial reasoning. FRDA patients show reduced speed of information processing. The learning effect seen in controls was notably absent in patients with FRDA. The patients' personality is characterized by some pathological aspects and reduced defensiveness. Patterns of cortical activation during finger movements are heterogeneous in patients compared to controls. Cognitive impairment, mood disorders and motor deficits in FRDA patients may be the result of the cumulative damage caused by frataxin deficiency not only in the cerebellum and spinal cord but also in other brain areas.

Folate-based single cell screening using surface enhanced Raman microimaging.

Recent progress in nanotechnology and its application to biomedical settings have generated great advantages in dealing with early cancer diagnosis. The identification of the specific properties of cancer cells, such as the expression of particular plasma membrane molecular receptors, has become crucial in revealing the presence and in assessing the stage of development of the disease. Here we report a single cell screening approach based on Surface Enhanced Raman Scattering (SERS) microimaging. We fabricated a SERS-labelled nanovector based on the biofunctionalization of gold nanoparticles with folic acid. After treating the cells with the nanovector, we were able to distinguish three different cell populations from different cell lines (cancer HeLa and PC-3, and normal HaCaT lines), suitably chosen for their different expressions of folate binding proteins. The nanovector, indeed, binds much more efficiently on cancer cell lines than on normal ones, resulting in a higher SERS signal measured on cancer cells. These results pave the way for applications in single cell diagnostics and, potentially, in theranostics.

Detection of epidermal growth factor receptor mRNA in peripheral blood: a new marker of circulating neoplastic cells in bladder cancer patients.

Despite the large number of studies performed in solid tumors, few attempts at molecular detection of urothelial cells in blood have been made. Specifically, only uroplakin II (UP-II) and cytokeratin 20 (CK-20) have been suggested as tumor markers in the blood of bladder cancer patients. Epidermal growth factor receptor (EGFR) mRNA expression was found in the blood of patients with some types of carcinoma; nevertheless, its expression has been never investigated in the blood of patients with urothelial tumors. We used a EGFR-based reverse transcription-PCR assay for the detection of tumoral cells in the blood of 27 patients with bladder cancer, in 30 healthy donors, and in 9 patients with cystitis. EGFR expression was compared with that of known markers of circulating epithelial cells, CK-19 and CK-20, and to a urothelial-specific marker, UP-II. Analysis by reverse transcription-PCR and Southern blot hybridization showed no evidence of EGFR and UP-II mRNA expression in any of the samples used as controls. Analysis of healthy donors showed mRNA expression for CK-19 and CK-20 in 6 of 30 and in 4 of 30 samples, respectively. All patients with cystitis resulted negative for EGFR expression, whereas 3 of 9, 2 of 9, and 3 of 9 were found expressing CK-19, CK-20, and UP-II, respectively. Among blood samples from tumoral patients, 74% had EGFR mRNA and 41% had positive signals for CK-19, whereas positivity for CK-20 and UP-II was found in 15% and 37% of patients, respectively. These results seem to indicate that EGFR mRNA in the blood may be a useful tumor marker in bladder cancer patients, as well as in other patients with epithelial tumors.

[Endothelial progenitor cells in systemic sclerosis: their possible role in angiogenesis]. / Progenitori delle cellule endoteliali di origine midollare in corso di sclerosi sistemica: possibile ruolo nell'angiogenesi.

BACKGROUND: Recently, several studies have demonstrated the presence of circulating endothelial progenitors (CEPs) responsible for angiogenesis. Notably, these cells are able to migrate to ischemic tissues and differentiate in situ in mature endothelial cells. Aim of this study was to assess the presence of CEPs in the peripheral blood of patients with Sistemic Sclerosis (SSc) and evaluate their significance as an attempt of re-vascularization MATERIAL AND METHODS: Samples of peripheral blood from 40 healthy subjects and 56 patients with SSc were studied. Five-parameter, 3-color flow cytometry was performed with a FACScan. CEPs were defined as CD45 negative, CD34 and CD133 positive. In addition, plasma levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were detected by commercial ELISA (R&D Systems). RESULTS: Levels of CEPs (CD133+/CD34+/CD45-) were significantly higher in patients with SSc in comparison to HC (P = 0.01). No correlation was found between CEPs and any clinical parameter of disease neither activity score. CEPs were significantly higher in the group of patients with early disease, while their number decreased in the late phases of disease. Plasma levels of VEGF, but not bFGF, were significantly higher in SSc in comparison to HC (P<0.001) but no correlation was found between VEGF concentrations and CEP number. CONCLUSIONS: The presence of CEPs in patients with SSc suggest that sclerodermic hypoxic tissues could induce the mobilization of bone-marrow derived cells in an attempt to provided new vessels, in the early phase of the disease, at least.

A comparison of two GM-CSF schedules to counteract the granulo-monocytopenia of carboplatin-etoposide chemotherapy.

In order to obtain the beneficial effects from granulocyte-macrophage colony-stimulating factor (GM-CSF) on granulo-monocyte recovery with the minimum dose and toxicity, we compared the effect of two different GM-CSF schedules (5 micrograms/kg/day subcutaneously, days 5 to > 18 versus days 12 to > 18 on the cytopenias which follow cytostatic treatment with carboplatin (400 mg/m2 intravenous (i.v.) day 1) and etoposide (100 mg/m2 i.v. days 1 to > 3). 13 patients entered the study for a total of 36 evaluable cycles. The cytostatic treatment produced a neutropenia that persisted for up to day 22 (absolute neutrophil count (ANC) < 1000/microliters in 25% and ANC < 2000 in 50% of control cycles). Early GM-CSF administration markedly increased the leucocyte nadir and produced two waves of leucocytosis: an early one, linked to marrow reserve release and presumably of no value to the patients; and a delayed one, due to marrow precursor and progenitor cell proliferation, in which the granulomonocytosis was associated with a marked eosinophilia. The delayed GM-CSF administration markedly increased the leucocyte nadir and accelerated granulo-monocyte recovery (with an only modest eosinophilia), so that chemotherapy could be repeated every 21 days in all the patients.

High levels of transforming growth factor-alpha (TGF-alpha) mRNA may predict local relapses in early stage urinary bladder cancer.

Elevated expression of transforming growth factor-alpha (TGF-alpha) gene has been previously reported in some types of human neoplasms, but its role in the pathogenesis of bladder cancer has still not been investigated. In the present study, we analysed 28 samples of early stage bladder tumours for the presence of TGF-alpha mRNA using reverse transcription-polymerase chain reaction (RT-PCR). We detected TGF-alpha mRNA in 71% (20/28) of these samples. When we related the expression levels of TGF-alpha with local relapses of patients during a follow-up of 2 years, we found that a high TGF-alpha expression level in bladder cancer was significantly associated with local relapses in patients with early stage tumours. The appearance of early relapses in tumours with high TGF-alpha expression levels may suggest the existence of an additional marker in the prediction of local relapses in patients with superficial disease.

C3 synthesis and CRs expression during differentiation of a murine stem cell line.

C3 production, release and CRs expression during the neutrophilic differentiation of a murine non tumorigenic cell line is investigated. The murine non tumorigenic cell line 32DCl3(G) which undergoes terminal differentiation into polymorphonuclear granulocytes when cultured in presence of G-CSF was selected as a suitable in vitro model for this study. The results show that as the cells progress into the differentiation program, levels of C3 mRNA increase, accompanied by increased C3 production. As differentiation progresses the cells gradually express CRs on their surface; these are undetectable on the surface of undifferentiated cells. As a consequence of CRs appearance, cells become able to bind C3 through receptorial binding. Differences were found in the modality of C3 secretion: differentiated cells tend to store C3 in their intracellular compartments rather than secrete it continuously into the medium and they respond to membrane stimulation with increased secretion of C3. Treatment of 32DCl3(G) with TNF-alpha increased C3 production in a time- and dose-dependent fashion. Cell response to this stimulus progressively increases during the differentiation process suggesting that they acquire functionality in the signal transduction mechanisms.

Detection of basic fibroblast growth factor mRNA in urinary bladder cancer: correlation with local relapses.

Natural history of bladder cancer is characterized by high risk of disease progression even for patients with a clinical diagnosis of superficial disease; in these tumors, the occurrence of local relapse is known to be dependent on the angiogenesis rate. Basic fibroblast growth factor (bFGF), has been described to be elevated in urine and serum of patients with bladder cancer. We investigated the expression of bFGF at mRNA level in a panel of 32 transitional cell tumors of the urinary bladder and in normal bladder tissues used as controls. Expression of bFGF was found elevated in most tumors of high stage, where its presence was found correlated with the occurrence of early local relapses. Furthermore, bFGF was found highly expressed in the majority of tumors showing a high bcl-2 expression rate. Our data suggest that bFGF expression could contribute to the progression of disease; it may provide a prognostic indicator in the identification of patients with high risk for occurrence of local relapses.

Involvement of bcl-2 and bax gene expression in apoptosis and differentiation of the non-tumorigenic murine hematopoietic cell line, 32DC13(G).

32DCl3(G) is an interleukin-3 (IL-3) dependent, non-tumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocyte-colony stimulating factor (G-CSF). This line therefore offers a convenient system to study the expression of genes involved in apoptosis and differentiation. In our experiments we have acquired evidence that during the differentiation pathway, likewise in apoptosis induced by IL-3 deprivation, detectable levels of bax mRNA appear, while bcl-2 expression decreases. These events are under the control of the p53 tumor-suppressor gene. In these cells, an overexpression of exogenous wild-type p53 leads to a decrease in bcl-2 mRNA and to the appearance of box mRNA, which instead is absent in the parental cells growing in IL-3 conditioned medium. Furthermore, results from experiments on p53 transfected cells demonstrate that excess wild-type p53 activity, on its own, fails to elicit apoptosis as long as IL-3 is present and does not induce differentiation if G-CSF is not added to the culture medium. We conclude that in apoptosis and differentiation of 32DCl3(G) the alterate ratio of bcl-2 and box gene expression, modulated by p53, is an early event dependent on IL-3 withdrawal and that the appearance of bax and the decrease of bcl-2 expression are necessary, but not sufficient for the acquisition of a completely mature granulocytic phenotype.

Early blastic transformation of a myeloproliferative disorder with t(8;21) and progressive aberrations of chromosome 8.

We describe a case of a patient affected by a chronic myeloproliferative disorder with t(8;21)(q22;q22) and trisomy 8 at diagnosis. At the time of blastic metamorphosis, 2 months later, trisomy 8 metaphases were significantly reduced, while a predominance of t(8;21) was present. Finally, in the phase of leukemic regrowth following chemotherapy administration, monosomy 8 associated with der(21)t(8;21) was the predominant cytogenetic abnormality.

Variable levels of bcl-2, bcl-x and bax mRNA in bladder cancer progression.

We investigated the expression of the anti-apoptotic genes bcl-2 and bcl-X and the pro-apoptotic gene bax in bladder tumors and normal samples from urinary bladder, using RT-PCR analysis. Bcl-2 mRNA was not detected in any of the normal samples, while it was found expressed in 66% of the low stage tumors and in 100% of the high stage tumors. Bax expression had an inverse progress, being present in 62% of the normal tissues examined, in 16% of the low stage tumors and in 14% of the high stage. Bcl-X gene expression was quite variable among all samples (37% in normal tissues, 50% in the low stage tumors and 14% in the high stage). bcl-X mRNA was only found in the isoform bcl-XL, with anti-apoptotic functions, whereas no sample expressed the isoform bcl-XS, which is known to suppress bcl-2 functions. Most samples expressing bcl-2 did not express bcl-X, and vice versa. These results, besides confirming the potential role of these genes in the pathogenesis of low stage bladder cancer strengthen the hypothesis concerning their possible interaction in the progression of disease.

[The outpatient care in the Latium region (Italy) in 2001]. / L'assistenza specialistica ambulatoriale nella regione Lazio nell'anno 2001.

The Region of Latium has been operating an Outpatient Care Information System (SIAS) since 1997 to monitor the supply of outpatient care in a territory with a population of over five million. The present work has the aim of describing the outpatient care in the region, in terms of number of facilities involved by category (public and private, operating in the regional public health system) and volume of procedures rendered to residents in 2001. Of the 971 outpatient facilities operating in hospitals and elsewhere--37% state managed and 67% private--distributed in a non-uniform manner throughout the region, the majority is concentrated in the city of Rome, which by itself accounts for 49% of its total amount of facilities, and in a lesser measure in the other provincial capitals (Viterbo, Rieti, Frosinone, Latina). In 2001, 71 million procedures were performed, comprising 17 million prescriptions, for an economic value of over 400 million Euros. The three specialties of greatest use were Lab Analysis, Physical Therapy and Rehabilitation, and Radiology, making up 88% of the total outpatient procedures performed within the precinct of the regional health service, in respective measures of 57%, 27%, and 4%. It is noted that the public facilities are prevalently polyspecialistic while a great number of private facilities are monospecialistic and perform procedures almost exclusively (96%) in the three specialties of greatest use. The other specialties which receive notable use are Cardiology, Eye Care, Orthopedics and Neurology. In general, the greater the number of facilities there are in either the public or private sector, the greater the level of activity in terms of procedures performed, with the exception of the area of Physical Therapy and Rehabilitation where the correlation is inversely proportioned; in fact, for this specialty the public facilities, which are represented in a much greater number throughout the region, supply only 7% of the volume of activity.

Sialidase NEU4 is involved in glioblastoma stem cell survival.

The human sialidase, NEU4, has emerged as a possible regulator of neuronal differentiation and its overexpression has been demonstrated to promote the acquisition of a stem cell-like phenotype in neuroblastoma cells. In this paper, we demonstrated that glioblastoma stem cells (GSCs) isolated from glioblastoma multiforme (GBM) cell lines and patients' specimens as neurospheres are specifically marked by the upregulation of NEU4; in contrast, the expression of NEU4 is very low in non-neurosphere-differentiated GBM cells. We showed that NEU4 silencing by miRNA or a chemical inhibitor of its catalytic activity triggered key events in GSCs, including (a) the activation of the glycogen synthase kinase 3ß, with the consequent inhibition of Sonic Hedgehog and Wnt/ß-catenin signalling pathways; (b) the decrease of the stem cell-like gene expression and marker signatures, evidenced by the reduction of NANOG, OCT-4, SOX-2, CD133 expression, ganglioside GD3 synthesis, and an altered protein glycosylation profile; and (c) a significant decrease in GSCs survival. Consistent with this finding, increased NEU4 activity and expression induced in the more differentiated GBM cells by the NEU4 agonist thymoquinone increased the expression of OCT-4 and GLI-1. Thus, NEU4 expression and activity appeared to help to determine the molecular signature of GSCs and to be closely connected with their survival properties. Given the pivotal role played by GSCs in GBM lethality, our results strongly suggest that NEU4 inhibition could significantly improve current therapies against this tumour.

A study of fine specificity of monoclonal antibodies to yeast iso-1-cytochrome c.

Seven monoclonal antibodies, prepared to yeast holo- or apo-iso-1-cytochrome c by the method of Köhler and Milstein (Goding, J. W. (1983) Monoclonal Antibodies: Principles and Practice, Academic Press, Orlando, FL) were characterized by cross-reaction with a panel of evolutionarily related cytochromes c, apocytochromes c, fragments and homologous and hybrid fragment complexes, inhibition, competitive inhibition, and complementation and fluorescence titration. The results have permitted us to assign the specifically recognized amino acids as follows. IgG1 monoclonals: 4-74-6, Leu-63 and/or Asn-67 and/or Asn-68; 4-128-6, Glu-93; 4-145-10, Thr-74; 2-96-12, Asp-65; 2-34-19, Lys-59; and 10-28-86, trimethyl-lysine 77. IgM monoclonal 39-14, Pro-30 and His-31. With mAb 4-128-6 substitution of glutamic acid 93 with alanine, as it occurs in Candida cytochrome c, has resulted in a decrease in affinity by a factor of 10(4). A calculation appears to show that this value is too large to be accounted for solely by the sum of energy losses due to disruption of charge neutralization and changes of hydrophobic interaction including van der Waals interaction. This and similar results with mAb 10-28-86 have led us to the idea that some new extra interatomic interaction sensitive to differences in configuration of atomic groups may be present and perturbed in the substitution. Furthermore, the assumption of the presence of such interaction can explain the striking similarity between the antigen-antibody interaction (e.g. Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3998-4002) and a model system of horse cytochrome c three-fragment complex (Juillerat, M. A., and Taniuchi, H. (1986) J. Biol. Chem. 261, 2697-2711) with respect to the high specificity of interacting residues in the interface. Thus, by analogy to the hypothesis developed in the model system (Fisher, A., and Taniuchi, H. (1988) FASEB J. 2, A1338), we hypothesize that a closed interaction loop would be formed on the basis of contacting groups including glutamic acid 93 across or within the interface between the antigen and mAb 4-128-6 and mediate delocalized interaction to generate extra binding energy.