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The spectral fingerprinting of the excitation emission matrix (EEM) of fluorescent substances is demonstrated using polychromatic light sources and tri-chromatic image detectors. A model of the measured fingerprints explaining their features and classification performance, based on the polychromatic excitation of the indicators is proposed. Substantial amount of spectral information is retained in the fingerprints as corroborated by multivariate analysis and experimental conditions that favor such situation are identified. In average, for five different substances, the model shows a fitting goodness measured by the Pearson's r coefficient and the root mean square deviation of 0.8541 and 0.0247 respectively, while principal component classification patterns satisfactorily compare with the EEM spectroscopy classification and respectively explain 96% and 93% of the information in the fist two principal components. The measurements can be performed using regular computer screens as illumination and web cameras as detectors, which constitute ubiquitous and affordable platforms compatible with distributed evaluations, in contrast to regular instrumentation for EEM measurements.

Objective: To investigate potential associations between toll-like receptor 4 (TLR4) gene polymorphisms and susceptibility to, clinical features, and severity of Behçet's disease (BD)., Methods: A total of 189 Italian patients who satisfied the International Study Group criteria for BD and 210 healthy age- and sex-matched blood donors were genotyped for two coding single nucleotide polymorphisms of TLR4 (Asp299Gly and Thr399Ile) by molecular methods. The patients were subgrouped according to the presence or absence of clinical manifestations. Severity score was calculated., Results: The distribution of allele and genotype frequencies did not differ significantly between the BD patients and the healthy controls. No significant associations were found when BD patients with and those without clinical manifestations were compared. No association between TLR4 polymorphisms and severity score was observed., Conclusion: Our data suggest that the TLR4 gene polymorphisms are not associated with susceptibility to, clinical expression of, and severity of BD in Italian patients.

Objective: To investigate potential associations between A-13G and G79A polymorphisms of the protein Z gene and venous thrombosis and other clinical manifestations in Italian patients with Behçet's disease (BD)., Methods: 176 Italian patients who satisfied the International Study Group criteria for BD and 134 healthy age- and sex- matched blood donors were genotyped for A-13G and G79A polymorphisms of the protein Z gene by molecular methods. 113 and 112 of the 176 BD patients were also genotyped for factor V Leiden and prothrombin gene G20210A polymorphisms. Serological HLA class B51 typing was performed by a standard microlymphocytotoxicity technique. The patients were subgrouped according to the presence or absence of clinical manifestations., Results: The distribution of allele and genotype frequencies of A-13G and G79A polymorphisms did not differ significantly between BD patients and healthy controls.The frequencies of carriage rates of protein Z G79A and A-13G polymorphisms in BD patients with and without DVT were similar. Similarly, no associations between thrombotic events and the protein Z gene polymorphisms studied were observed in BD patients carrying factor V Leiden or prothrombin gene G20210A mutations. No significant associations were observed between protein Z polymorphisms and the occurrence of specific clinical findings., Conclusion: No association between DVT and A-13G or G79A polymorphisms of the protein Z gene was found in Italian BD patients. Furthermore, these protein Z polymorphisms in BD do not seem to increase the risk of DVT due to factor V Leiden or prothrombin gene G20210A mutations.

Nanostructured molecular assemblies may provide additional sensing properties not found in other arrangements of the same basic constituents. Among three-dimensional structures, nanotubes are particularly appealing for applications as chemical sensors, because of the potential inclusion of different guests inside the cavity or the induced modification of the skeletal interaction after analyte binding. Porphyrins are a class of compounds characterized by brilliant sensing properties, appearing also in non-ordered solid-state aggregates. In recent years, it was reported that aggregation of oppositely charged porphyrins led to the formation of self-assembled nanotubes and in this paper their sensing properties, both in solution and in the solid state, have been investigated. The interactions of porphyrin nanotubes with guest molecules have been monitored by following the changes in their UV-vis spectra. The results obtained have been exploited to build up a sensing platform based on a computer screen as a light source and a digital camera as detector. Porphyrin nanostructures exhibited an enhanced sensitivity to different compounds with respect to those shown by single porphyrin subunits. The reason for the increased sensitivity may be likely found in an additional sensing mechanism related to the modulation of the strength of the forces that keep the supramolecular ensemble together.

The computer screen photo-assisted technique (CSPT) utilizes consumer electronic devices such as computer screens and web cameras for distributed chemical sensing. Key to this method is the development of small and disposable sensing assays able to aid the CSPT optical detection, and the evaluation of multiple indicators. Here we demonstrate CSPT identification of fluorescent indicators from individual 100 microm SU-8 (10) pillars covering an area of 4 mm x 4 mm with a density of 47 elements mm(-2). The extraction of distinctive spectral fingerprints is aided by the exploitation of a three-band Moiré interference that enables the partial Bayer decoding of the CSPT images.

Angle and spectra resolved surface plasmon resonance (SPR) images of gold and silver thin films with protein deposits is demonstrated using a regular computer screen as light source and a web camera as detector. The screen provides multiple-angle illumination, p-polarized light and controlled spectral radiances to excite surface plasmons in a Kretchmann configuration. A model of the SPR reflectances incorporating the particularities of the source and detector explain the observed signals and the generation of distinctive SPR landscapes is demonstrated. The sensitivity and resolution of the method, determined in air and solution, are 0.145 nm pixel(-1), 0.523 nm, 5.13x10(-3) RIU degree(-1) and 6.014x10(-4) RIU, respectively, encouraging results at this proof of concept stage and considering the ubiquity of the instrumentation.

Artificial olfactory systems have been studied for the last two decades mainly from the point of view of the features of olfactory neuron receptor fields. Other fundamental olfaction properties have only been episodically considered in artificial systems. As a result, current artificial olfactory systems are mostly intended as instruments and are of poor benefit for biologists who may need tools to model and test olfactory models. Herewith, we show how a simple experimental approach can be used to account for several phenomena observed in olfaction. An artificial epithelium is formed as a disordered distributed layer of broadly selective color indicators dispersed in a transparent polymer layer. The whole epithelium is probed with colored light, imaged with a digital camera and the olfactory response upon exposure to an odor is the change of the multispectral image. The pixels are treated as olfactory receptor neurons, whose optical properties are used to build a convergence classifier into a number of mathematically defined artificial glomeruli. A non-homogenous exposure of the test structure to the odours gives rise to a time and spatial dependence of the response of the different glomeruli strikingly similar to patterns observed in the olfactory bulb. The model seems to mimic both the formation of glomeruli, the zonal nature of olfactory epithelium, and the spatio-temporal signal patterns at the glomeruli level. This platform is able to provide a readily available test vehicle for chemists developing optical indicators for chemical sensing purposes and for biologists to test models of olfactory system organization.

Xenopus laevis melanophores can be used in high-throughput screens for guanine nucleotide binding protein coupled receptor ligands and have potential as biosensors. Inherent in this immortal cell line is a substantial variability, which macroscopic evaluations disregard. Here we demonstrate a systematic way to incorporate this natural variability in the evaluations. Clusters of similar cells from a sparsely seeded cell culture are examined by imaging changes in cell appearance, pigment motility, and cumulative displacements. The time evolution of the image intensity distributions of clusters upon a pigment-dispersing stimulus is used as a signature of the cell clusters, and their behaviors are classified by multivariate analysis. Conventional image subtraction procedures are used to highlight cumulative and transitory changes in the pigment dynamics, enabling characterization of multiple aspects of the cell response from a single experiment. Additionally, a simple way to accomplish standard optical density changes at the single-cell group level is shown. The present results also provide evidence that natural cell variability arising from a cell culture can enrich the diversity of responses from pigment-containing cells assays and underscore that in conventional macroscopic evaluations these aspects are overlooked and can lead to spurious results.

The combination of computer monitors and webcams has been recently demonstrated to behave as a sort of spectrophotometer able to classify and recognize substances according to their light absorption and emission properties. This measurement technique is known as computer screen photoassisted technique (CSPT). In this paper, it is demonstrated for the first time and in the case of a complex sample such as red wine, that also quantification of integral parameters (colour indicators) and specific compounds (total anthocyanins and polyphenols) is possible through a multivariate analysis of CSPT fingerprints. Most of the properties of the method are due to the combination of light emission and absorption properties that are captured by the CSPT platform. Thanks to this combination, a CSPT fingerprint may contain a comparable amount of information with respect to standard spectrophotometers. Wine measurements were performed on intact samples without the intervention of chemical mediators. The regression models, built by Partial Least Squares, obtained errors of estimation of colour parameters, total polyphenols and anthocyanines that are comparable with those typical of the standard methods in use. Since computation functionalities, video capture and display are embedded in a steadily growing number of ubiquitously distributed equipment (from portable computers to cellular phones), these results indicate a viable methodology for low-cost and largely diffused analytical capabilities.

In the last years a large number of different measurement methodologies were applied to measure the freshness of fishes. Among them the connection between freshness and headspace composition has been considered by gas chromatographic analysis and from the last two decades by a number of sensors and biosensors aimed at measuring some characteristic indicators (usually amines). More recently also the so-called artificial olfaction systems gathering together many non-specific sensors have shown a certain capability to transduce the global composition of the fish headspace capturing the differences between fresh and spoiled products. One of the main objectives related to the introduction of sensor systems with respect to the analytical methods is the claimed possibility to distribute the freshness control since sensors are expected to be "portable" and "simple". In spite of these objectives, until now sensor systems did not result in any tool that may be broadly distributed. In this paper, we present a chemical sensor array where the optical features of layers of chemicals, sensitive to volatile compounds typical of spoilage processes in fish, are interrogated by a very simple platform based on a computer screen and a web cam. An array of metalloporphyrins is here used to classify fillets of thawed fishes according to their storage days and to monitor the spoilage in filleted anchovies for a time of 8 h. Results indicate a complete identification of the storage days of thawed fillets and a determination of the storage time of anchovies held at room temperature with a root mean square error of validation of about 30 min. The optical system produces a sort of spectral fingerprint containing information about both the absorbance and the emission of the sensitive layer. The system here illustrated, based on computer peripherals, can be easily scaled to any device endowed with a programmable screen and a camera such as cellular phones offering for the first time the possibility to fulfil the sensor expectation of diffused and efficient analytical capabilities.

Epithelial-to-mesenchymal transition (EMT) is a coordinated process, occurring both during morphogenesis and tumor progression, that allows epithelial cells to dissociate from initial contacts and migrate to secondary sites. The transcriptional repressors of the Snail family induce EMT in different epithelial cell lines and their expression is strictly correlated with EMT during the development and progression of carcinomas. We have previously shown that EMT in hepatocytes correlates with the downregulation of hepatic differentiation key factors HNFs (hepatocyte nuclear factors), and in particular of HNF4alpha. Here, we demonstrate that Snail overexpression is sufficient (i) to induce EMT in hepatocytes with conversion of morphology, downregulation of several epithelial adhesion molecules, reduction of proliferation and induction of matrix metalloproteinase 2 expression and, (ii) most relevantly, to repress the transcription of the HNF4alpha gene through a direct binding to its promoter. These finding demonstrate that Snail is at the crossroads of the regulation of EMT in hepatocytes by a dual control of epithelial morphogenesis and differentiation., (Copyright 2006 Wiley-Liss, Inc.)

A measuring strategy for the evaluation of a seven parameters colorimetric test using a computer screen photo-assisted technique (CSPT) is demonstrated. CSPT is a versatile approach aimed at point of care or home tests that uses regular computer sets and web cameras as the whole instrument. Issues such as the stability and the equivalency on different platforms of the determinations have been addressed in the present work. The method uses an embedded local reference simultaneously measured with the tests and solves the evaluation as a classification problem. The achieved performance tested along 580 classifications covering all the ranges of the assay, using synthetic samples, yielded 97.2% correct determinations compared with 89.7% for the case of colorimetric determinations. The errors were concentrated in only two parameters that show a significant correlation with a set of quality indices used to assess the performance of the classification.

Distinctive generation of biochemical response patterns of eight different substances, using an assay based on pigment containing cells, was demonstrated. Xenopus laevis melanophores, transfected with human beta(2)-adrenergic receptor, were seeded in a 96 well microplate and used to generate individual biochemical images through a two transient measuring protocol that contributes to highlight the response signatures of the agents. Adequate signal processing creates distinctive patterns in a time-concentration response space suitable for substance classification. The concept of biochemical images is introduced here. The assays were evaluated both with a standard microplate reader and with a computer screen photo-assisted technique (CSPT) yielding similar results. Since CSPT platforms only demand standard computer sets and web cameras as measuring setup, applications for these kind of assays outside main-laboratories were discussed.

The computer screen photo-assisted technique (CSPT), a method for substance classification based on spectral fingerprinting, which involves just a computer screen and a web camera as measuring platform is used here for the evaluation of a prospective enzyme-linked immunosorbent assay (ELISA). A anti-neutrophil cytoplasm antibodies (ANCA-ELISA) test, typically used for diagnosing patients suffering from chronic inflammatory disorders in the skin, joints, blood vessels and other tissues is comparatively tested with a standard microplate reader and CSPT, yielding equivalent results at a fraction of the instrumental costs. The CSPT approach is discussed as a distributed measuring platform allowing decentralized measurements in routine applications, whereas keeping centralized information management due to its natural network embedded operation.

Melanophores are pigmented cells in lower vertebrates capable of quick color changes and thereby suitable as whole cell biosensors. In the frog dermis skin layer, the large and dark pigmented melanophore surrounds a core of other pigmented cells. Upon hormonal stimulation the black-brown pigment organelles will redistribute within the melanophore, and thereby cover or uncover the core, making complex color changes possible in the dermis. Previously, melanophores have only been cultured on flat surfaces. Here we mimic the three dimensional biological geometry in the frog dermis by culturing melanophores on fluorescent plastic microbeads. To demonstrate biosensing we use the hormones melatonin and alpha-melanocyte stimulating hormone (alpha-MSH) as lightening or darkening stimuli, respectively. Cellular responses were successfully demonstrated on single cell level by fluorescence microscopy, and in cell suspension by a fluorescence microplate reader and a previously demonstrated computer screen photo-assisted technique. The demonstrated principle is the first step towards "single well/multiple read-out" biosensor arrays based on suspensions of different selective-responding melanophores, each cultured on microbeads with distinctive spectral characteristics. By applying small amount of a clinical sample, or a candidate substance in early drug screening, to a single well containing combinations of melanophores on beads, multiple parameter read-outs will be possible.

We analyzed the clinical features and prognostic factors for transformation of immunoglobulin Mrelated disorders (IgM-RDs) to malignant lymphoproliferative disease (MLD) in 83 patients with IgM-RDs. We studied 19 patients with type I cryoglobulinemias, 56 patients with type II cryoglobulinemias, 5 patients with peripheral neuropathies (PNs), and 3 patients with idiopathic thrombocytopenic purpuras. Fourteen patients with cryoglobulinemias presented with mild to moderate hepatomegaly with or without splenomegaly. Fourteen patients with type II cryoglobulinemias had arthralgias and/or vascular purpura (12 receiving corticosteroids), and 7 presented with PN. These latter patients and those with PNs without cryoglobulinemia were treated with steroids, cyclophosphamide, or polychemotherapy with/without plasma-exchange. Cumulative probability of evolution to MLD at 5 years was 15% (95% CI; 5%-25%). At a median of 62 months (12-195 months), 8 cases of IgM-RDs (8.4%) evolved to overt Waldenstrom's macroglobulinemia (n = 6), 1 case to non-Hodgkin's lymphoma, and 1 case to B-cell chronic lymphocytic leukemia. At univariate analysis, male sex (P = 0.02), IgM level > or = 3 g/dL (P < 0.0001), detectable Bence Jones proteinuria (P = 0.0005), lymphocytosis (P = 0.049), and high erythrocyte sedimentation rate (P = 0.003) significantly correlated with the evolution risk. Age, blood cell counts, b2-microglobulin level, degree of marrow lymphoplasmacytic infiltration, type of cryoglobulinemia, and hepatitis C virus positivity did not correlate with transformation. Although IgM-RDs represent a distinct clinical entity frequently requiring treatment in view of the IgM-related symptoms, their evolution probability and prognostic factors for malignant transformation seem to widely overlap those described for asymptomatic IgM monoclonal gammopathies.

Objective: To evaluate potential associations of vascular endothelial growth factor (VEGF) gene polymorphisms with Behçet's disease (BD) and disease expression., Methods: Case patients were 122 consecutive Italian patients with BD followed at the Rheumatology, Ophthalmology, and Neurology Units in Bologna, Ferrara, Milano, Palermo, Potenza, Prato, Reggio Emilia, and Trento over a 3-year period (1997-99) and who satisfied the International Study Group criteria for BD. Also selected as a control group were 200 healthy age and sex matched blood donors. All patients with BD and controls were genotyped by polymerase chain reaction and allele-specific oligonucleotide techniques for +936 C/T (rs3025039) and -634 C/G (rs2010963) mutations and for an 18 base pair (bp) insertion/deletion (I/D) polymorphism at -2549 of the the VEGF promoter region. In vitro release of VEGF by peripheral blood mononuclear cells (PBMC) was investigated by ELISA in healthy controls homozygous for the polymorphisms studied., Results: The carriage rates of the alleles I and -634C were significantly more frequent in patients with BD than in healthy controls [p corr = 0.036, OR 1.8 (95% CI 1.1-2.9) and p corr = 0.05, OR 1.8 (95% CI 1.1-3.0), respectively]. While the distribution of allele +936T was similar in patients with BD and healthy controls, its frequency was significantly higher in BD patients with posterior uveitis/retinal vasculitis than in those without (p = 0.022, OR 2.4, 95% CI 1.1-5.0). Lipopolysaccharide-stimulated VEGF production from PBMC of healthy subjects was higher in II homozygous than in DD homozygous., Conclusion: Our data indicate that carriers of -634C and I alleles are associated with susceptibility to developing BD.

Objective: To evaluate the frequency and type of vascular lesions and to study the association of factor V gene G1691A (Leiden) and prothrombin gene G20210A polymorphisms with venous thrombosis in Italian patients with Behçet's disease (BD)., Methods: Included were 118 consecutive Italian BD patients followed over a 3-year period (1997-1999) who satisfied the International Study Group criteria for BD. The control group consisted of 132 healthy Italian blood donors. All BD patients and controls were genotyped by polymerase chain reaction and allele-specific restriction enzyme techniques for factor V Leiden and prothrombin gene G20210A polymorphisms., Results: Vascular lesions were observed in 37 (31.4%) patients. The 2 most common lesions were subcutaneous thrombophlebitis (10.2%) and deep vein thrombosis (DVT) of the legs (22.8%). No significant demographic and clinical differences between patients with and without DVT were present. The distribution of allele and genotype frequencies of prothrombin gene G20210A and factor V Leiden polymorphisms did not differ significantly between BD patients and healthy controls. The frequencies of carriage rates of prothrombin gene G20210A and factor V Leiden polymorphisms in BD patients with and without DVT were similar. However, the frequency of 20210A allele was significantly higher in BD patients with ocular disease than in those without, particularly in the patients with posterior uveitis/retinal vasculitis., Conclusions: The frequency and types of vascular lesions in Italian BD patients were similar to those reported in studies from other countries. No association between factor V Leiden mutation and G20210A mutation in the 3'-untranslated region of the prothrombin gene with DVT was found. However, a prothrombin gene G20210A mutation may influence the development and severity of ocular involvement in BD.

Background: Primary hepatocytes, one of the most widely used cell types for toxicological studies, have a very limited life span and must be freshly derived from mice or even humans. Attempts to use stable cell lines maintaining the enzymatic pattern of liver cells have been so far unsatisfactory. Stress proteins (heat shock proteins, HSPs) have been proposed as general markers of cellular injury and their use for environmental monitoring has been suggested. The aim of this work is to develop a bi-transgenic hepatocyte cell line in order to evaluate the ability of various organic and inorganic chemicals to induce the expression of the HSP70 driven reporter gene. We previously described transgenic mice (Hsp70/hGH) secreting high levels of human Growth Hormone (hGH) following exposure to toxic compounds in vivo and in vitro in primary cultures derived from different organs. In addition, we also reported another transgenic model (AT/cytoMet) allowing the reproducible immortalization of untransformed hepatocytes retaining in vitro complex liver functions., Results: The transgenic mouse line Hsp70/hGH was crossed with the AT/cytoMet transgenic strain permitting the reproducible immortalization of untransformed hepatocytes. From double transgenic animals we derived several stable hepatic cell lines (MMH-GH) which showed a highly-differentiated phenotype as judged from the retention of epithelial cell polarity and the profile of gene expression, including hepatocyte-enriched transcription factors and detoxifying enzymes. In these cell lines, stresses induced by exposure to inorganic [Sodium Arsenite (NaAsO2) and Cadmium Chloride (CdCl2)], and organic [Benzo(a)Pyrene (BaP), PentaChloroPhenol (PCP), TetraChloroHydroQuinone (TCHQ), 1-Chloro-2,4-DiNitro-Benzene (CDNB)] compounds, specifically induced hGH release in the culture medium., Conclusions: MMH-GH, an innovative model to evaluate the toxic potential of chemical and physical xenobiotics, provides a simple biological system that may reduce the need for animal experimentation and/or continuously deriving fresh hepatocytes.

Objective: To determine the type and frequency of clinical features of Behçet's disease in a population of Italian patients., Methods: We retrospectively studied 137 Italian patients (76 males and 61 females, age at onset 29.6 +/- 12.2 [mean +/- SD] years) seen consecutively in nine different referral centers. The duration of follow-up at study entry was 10.9 +/- 8.2 years. Virtually all patients fulfilled the classification criteria developed by the International Study Group for Behçet's disease. The clinical manifestations of the patients were recorded by the attending physicians using specifically designed forms., Results: The most frequent manifestations at disease onset were oral (78.3%) and genital aphthae (29.2%) followed by inflammatory ocular involvement (20%) and arthritis (14.2%). The commonest (>50% of cases) manifestations observed throughout the disease course were oral aphthae (99.3%), genital aphthae (62.8%), various cutaneous lesions including erythema nodosum (81.8%), and inflammatory ocular disease (60.6%). Panuveitis and posterior uveitis/retinitis occurred more frequently in males compared with females (28.9% versus 11.5% and 57.9% versus 36.1%, respectively; p < 0.05). 61.6% of our patients were HLA-B51 positive., Conclusion: Behçet's disease in Italian patients is characterized by a variety of clinical manifestations in agreement with the medical literature. Panuveitis and posterior uveitis/retinitis occur more frequently in male patients.

Melanophores, dark pigment cells from the frog Xenopus laevis, have the ability to change light absorbance upon stimulation by different biological agents. Hormone exposure (e.g. melatonin or alpha-melanocyte stimulating hormone) has been used here as a reversible stimulus to test a new compact microplate reading platform. As an application, the detection of the asthma drug formoterol in blood plasma samples is demonstrated. The present system utilizes a computer screen as a (programmable) large area light source, and a standard web camera as recording media enabling even kinetic microplate reading with a versatile and broadly available platform, which suffices to evaluate numerous bioassays. Especially in the context of point of care testing or self testing applications these possibilities become advantageous compared with highly dedicated comparatively expensive commercial systems.

Factors predicting evolution to symptomatic disease were investigated in 27 patients with smouldering Waldenstrom's macroglobulinemia (SWM) among 172 patients with Waldenstom's macroglobulinemia (WM), selected on the basis of the following criteria: (1) IgM paraprotein > 3 g/dL, and/or (2) bone marrow (BM) lymphoplasmacytoid (LPC) infiltration >or= 30%, and/or (3) diffuse infiltration pattern on BM biopsy, and (4) no treatment requirement for at least 12 months. Cumulative probability of survival was calculated by means of Kaplan-Meier. The Mantel and Haenszel test and multivariate Cox model were used to identify possible predictors for evolution. At a median follow-up of 79 months (range, 14 to 204), 11 patients (40.7%) showed progression to symptomatic disease, with the median interval from diagnosis being 46 months (range, 12 to 154). Event-free survival (EFS) at 5 and 10 years was 65% (95% confidence interval [CI], 45% to 85%) and 53% (95% CI, 31% to 75%), respectively. At multivariate analysis, paraprotein > 3 g/dL (hazard ratio [HR], 15.1; 95% CI, 3.01 to 75.64; P <.0009) and hemoglobin 3 g/dL and hemoglobin levels

Visible absorption features suitable for color recognition and micro-plate reading of a standard bioassay are performed by the combination of a computer screen used as a programmable light source and a web camera as detector. The method provides in this way a highly available platform for 'home tests' or 'self-tests', where the requirement is to monitor well defined assays and the use of economical instrumentation is advantageous.

Nop56p was initially identified in yeast as the third common component of the ribonucleoprotein particles (snoRNPs) assembled on box C/D small nucleolar RNAs (snoRNAs). Thereafter, the characterization of Nop56p homologs in Archaea and in several eukaryotes pointed to the highly conserved structure of this factor. Studies in yeast indicate that Nop56 is not required for the stability of box C/D snoRNAs. Through the isolation of a Xenopus laevis Nop56 cDNA clone, we have been able to characterize the X. laevis Nop56 protein (XNop56p). We showed that it is a common component of X. laevis box C/D snoRNPs and that it displays the same electrophoretic mobility of p62 protein that we previously characterized as a box C/D snoRNP component, not essential for snoRNA stability in X. laevis. Mapping the 5' end of X. laevis Nop56 transcript indicates that it starts with a pyrimidine tract and the analysis of genomic clones revealed a snoRNA encoded in one of NOP56 introns. Although these two characteristics could suggest that XNOP56 is a TOP gene, it is not translationally controlled in a growth-dependent manner.

Introduction: Few well-documented cases of central nervous system involvement in patients with mixed cryoglobulinemia and/or HCV infection have been reported. We can distinguish between acute or subacute diffuse and focal lesions (transient ischemic attack-like syndromes and cerebrovascular accidents)., Methods: A search of two electronic databases (Medline and EMBASE) was conducted from the year of their inception (1966 for Medline and 1988 for EMBASE) to September 2000. The search strategy employed entailed combining these terms: Cryoglobulinemia, Central Nervous System, Hepatitis C, Chronic Hepatitis. Cryoglobulinemia and Central Nervous System were also used as free test words. We analysed articles with case reports and the most frequent articles on the references list., Pathogenesis: The main pathophysiologic mechanism of cerebral involvement is ischemia (or rarely hemorrhage) due to diffuse or segmental vasculitis of the small cerebral vessels. In these cases a brain MRI usually shows single or multiple increased T2 signals. Furthermore an occasional occlusive vasculopathy without vasculitis was documented histologically. In these patients ischemia could be started or enhanced by the engorgement of the microvasculature by clumps of red cells and by aggregates of cryoglobulins. In the same patients vasculitis and hemorheological abnormalities can affect the clinical picture of the cerebral involvement in mixed cryoglobulinemia. Finally, the detection of HCV in the lesions induces a hypothesis that, in some cases, CNS involvement could be directly related to chronic HCV infection, even in the absence of cryoglobulin production., Case Report: We describe a 63 year-old woman with acute severe encephalopathy. Laboratory evaluation revealed a high positive test result for rheumatoid factor (3390 U/ml) and hypocomplementemia (C4 less than 1.67 mg/dl). Protein immunofixation electrophoresis demonstrated 5% monoclonal proteins (IgM/k and IgG/k), 3% cryoglobulins were present, HCV antibody and HCV-RNA (type 2a-2c) were positive. Cryoglobulins were never typed, because they disappeared after plasma exchanges. Liver enzymes, renal function and findings on cerebrospinal fluid were normal. Cerebral CT and MRI were also normal. Antinuclear antibodies, anti nDNA antibodies, antiphospholipid antibodies, lupus anticoagulant, ANCA, Lyme disease serology, complete tests for thrombophilia were negative. Bone aspiration was normal. The patient, in coma, was treated with two plasma exchanges. During the first treatment she recovered consciousness. Prednisone (1 mg/Kg/day) and cyclophosphamide (400 mg iv for three days) were added. After a week two plasma exchanges were performed again. Liver enzymes and rheumatoid factor were analysed monthly for six months and than every two months for another six month period up to the present. Liver enzymes were always normal, rheumatoid factor was always at a lower level than the first evaluation (now it's 311 U/ml). At present she is taking Prednisone 5 mg once a day, neurologic symptoms are absent and neurologic examination is normal., Discussion: We can conclude that: central neurologic involvement may be the clinical presentation of HCV infection and mixed cryoglobulinemia. HCV serologic tests and cryoglobulins should be considered in patient with encephalopathy of non-obvious cause; plasma exchange is the treatment of choice in acute severe forms; in some patients HCV could involve directly CNS, even in the absence of cryoglobulin production.

Objective: To analyze potential associations of Glu-Asp298 polymorphism in exon 7 and 4 a/b polymorphism in intron 4 of the endothelial nitric oxide synthase (eNOS) gene with susceptibility for Behçet's disease (BD)., Methods: Seventy-three consecutive Italian patients who satisfied the International Study Group criteria for BD and 135 healthy blood donor controls from the same geographic area were genotyped by polymerase chain reaction and allele-specific oligonucleotide techniques for eNOS polymorphisms in exon 7 and in intron 4., Results: The distribution of the Glu-Asp298 genotype differed significantly between BD patients and controls (p(corr) = 0.00009). Allele Asp298 was significantly more frequent in BD patients than in controls (p(corr) = 0.0006, OR 2.1, 95% CI 1.5-3.3). The distribution of 4 a/b genotype was similar in patients and controls., Conclusion: Our findings show that Glu-Asp298 polymorphism of eNOS gene is associated with BD susceptibility.

The Xenopus laevis Nop56 gene (XNOP56), coding for a snoRNP-specific factor, belongs to the 5'-TOP gene family. XNOP56, as many 5'-TOP genes, contains an intron-encoded snoRNA. This previously unidentified RNA, named U86, was found as a highly conserved species in yeast and human. While in human it is also encoded in an intron of the hNop56 gene, in yeast it has an unprecedented gene organization: it is encoded inside an open-reading frame. Both in X. laevis and yeast, the synthesis of U86 snoRNA appears to be alternative to that of the cotranscribed mRNA. Despite the overall homology, the three U86 snoRNAs do not show strong conservation of the sequence upstream from the box D and none of them displays significant sequence complementarity to rRNA or snRNA sequences, suggesting a role different from that of methylation., (Copyright 2001 Academic Press.)

Objective: Intercellular adhesion molecule 1 (ICAM-1) is strongly expressed in vascular endothelial cells and perivascular inflammatory infiltrates in immunopathologic studies of Behçet's disease (BD) lesions. ICAM-1 genes may contribute to the inflammatory events responsible for the vessel damage in BD. We examined potential associations of ICAM-1 gene polymorphisms with BD susceptibility., Methods: Case patients were 74 consecutive Italian patients with BD who were followed at the Bologna, Ferrara, Milano, Potenza, Prato, Reggio Emilia, and Trento rheumatology, ophthalmology, and neurology units over a 3 year period (1997-99) who satisfied the International Study Group criteria for BD; 228 healthy Italian blood donors from the same geographic areas were selected as control groups. All BD patients and controls were genotyped by polymerase chain reaction and allele-specific oligonucleotide techniques for ICAM-1 polymorphisms at codon 241 (exon 4) and codon 469 (exon 6)., Results: The frequency of R241 was significantly higher in BD patients than in controls (20.3% vs 5.7%; p = 0.001, pcorr = 0.002, OR 4.2, 95% CI 1.9-9.3). The distribution of E/K 469 genotype was similar in patients and controls. Comparing patients with different clinical features, we found only a trend to higher frequency of R241 in patients with articular manifestations (21.4% vs 12.5%; p = 0.08)., Conclusion: Our findings show that G/R 241 polymorphism of ICAM-1 is associated with BD susceptibility.

U16 belongs to the family of box C/D small nucleolar RNAs (snoRNAs) whose members participate in ribosome biogenesis, mainly acting as guides for site-specific methylation of the pre-rRNA. Like all the other members of the family, U16 is associated with a set of protein factors forming a ribonucleoprotein particle, localized in the nucleolus. So far, only a few box C/D-specific proteins are known: in Xenopus laevis, fibrillarin and p68 have been identified by UV crosslinking and shown to require the conserved boxes C and D for snoRNA interaction. In this study, we have identified an additional protein factor (p62), common to box C/D snoRNPs, that crosslinks to the internal stem region, distinct from the conserved box C/D "core motif," of U16 snoRNA. We show here that, although the absence of the core motif and, as a consequence, of fibrillarin and p68 binding prevents processing and accumulation of the snoRNA, the lack of the internal stem does not interfere with the efficient release of U16 from its host intron and only slightly affects snoRNA stability. Because this region is likely to be the binding site for p62, we propose that this protein plays an accessory role in the formation of a mature and stable U16 snoRNP particle.

The aim of the study was to evaluate the frequency of extra-articular manifestations (EAMs) of rheumatoid arthritis (RA) in a series of patients from nine Italian rheumatology clinics. A total of 587 patients underwent direct questioning, complete physical evaluation, and review of medical records and laboratory data. The relationships between EAMs and the eosinophilic count, IgM rheumatoid factor (RF), and antinuclear antibodies (ANA) were studied. EAMs were present in 240/587 (40.9%) patients. The most common features were sicca syndrome (17.5%) and rheumatoid nodules (16.7%). EAMs were significantly more frequent in male patients (OR = 1.68), patients with ANA positivity (OR = 2.82), high anatomical class (OR = 2.3), and rheumatoid factor seropositivity (OR = 2.22). EAMs were more common in patients from southern Italy than in those from northern Italy (P < 0.001). EAMs seem to be rarer in Italy than in the Anglo-Saxon populations of northern Europe and the USA. Differences in prevalence of EAMs can exist even within the same country.