Irritable bowel syndrome: new findings in pathophysiological and therapeutic field.

Irritable bowel syndrome (IBS) is a high prevalence disease, whose symptoms are reported by a large number of young adults with significant effects on quality of life and social costs. Traditionally, IBS has been treated with dietary and lifestyle modification, fiber supplementation, psychological and pharmacological therapy. Since its complex and multifactorial etiopathogenesis is only partially known, therapeutic choices may be difficult and not always effective. New research efforts focused on the role of relationship between central nervous system and gut disorders (brain-gut axis), altered composition of gut microbiota (e.g. an eight times increased risk for IBS after Salmonella infection), immune activation with an increased number of T lymphocytes and mast cells associated with mucosa as well as an increased level of pro-inflammatory cytokines (IL-10 and IL-12, suggesting Th1 polarization), visceral hypersensitivity causing perception of pain even for minimal abdominal distension. Based on these findings, new possibilities of treatment are emerging with encouraging outcomes. Attention is directed to drugs that showed good tolerability profile and poor systemic absorption, which may make them suitable for repeated or long term treatments, as frequently required in patients with IBS. They have been successfully used drugs such as tachykinin receptors antagonists, tryptophan hydroxylase inhibitors, bile acid sequestrants, µ agonist and δ antagonist opioid receptors. Recent studies are discussed in this review, focusing both on new therapeutic approaches and innovative adaptation of previously available treatments.

Control of Aurora-A stability through interaction with TPX2.

The Aurora-A kinase has well-established roles in spindle assembly and function and is frequently overexpressed in tumours. Its abundance is cell cycle regulated, with a peak in G2 and M phases, followed by regulated proteolysis at the end of mitosis. The microtubule-binding protein TPX2 plays a major role in regulating the activity and localisation of Aurora-A in mitotic cells. Here, we report a novel regulatory role of TPX2 and show that it protects Aurora-A from degradation both in interphase and in mitosis in human cells. Specifically, Aurora-A levels decrease in G2 and prometaphase cells silenced for TPX2, whereas degradation of Aurora-A is impaired in telophase cells overexpressing the Aurora-A-binding region of TPX2. The decrease in Aurora-A in TPX2-silenced prometaphases requires proteasome activity and the Cdh1 activator of the APC/C ubiquitin ligase. Reintroducing either full-length TPX2, or the Aurora-A-binding region of TPX2, but not a truncated TPX2 mutant lacking the Aurora-A-interaction domain, restores Aurora-A levels in TPX2-silenced prometaphases. The control by TPX2 of Aurora-A stability is independent of its ability to activate Aurora-A and to localise it to the spindle. These results highlight a novel regulatory level impinging on Aurora-A and provide further evidence for the central role of TPX2 in regulation of Aurora-A.

Liver fibrosis: evaluation with diffusion-weighted magnetic resonance imaging in patients with chronic liver disease.

AIM: Liver fibrosis is often a possible evolution of chronic liver disease (CLD), with a risk of progression to cirrhosis. This study was designed to determine if the measure of apparent diffusion coefficient (ADC) is clinically accurate in the staging of fibrosis. METHODS: The study was conducted in the period 2008-2012. We recruited 84 patients with CLD. The control group included 67 patients whose laboratory, ultrasound and magnetic resonance imaging exams demonstrated liver's normal conditions. For ethical reasons, these patients did not undergo liver biopsy. Patients were examined using diffusion-weighted magnetic resonance imaging with a 1.5 Tesla magnet and with single shot echo-planar technique. Patients did undergo liver biopsy and the samples were evaluated with the Metavir score (F0-F4), Ishak score (0-6) and Brunt score (0-6). Patients were divided into three groups according to the different degree of fibrosis and the ADC was compared with U-test of Mann-Whitney. Moreover, it was used the analysis Receiver Operating Characteristic (ROC). RESULTS: A significant difference between group 1 (F0-F1) and group 3 (F3-F4) was found, with P=0.0024 and between group 2 (F2) and group 3, with P=0.027, but there was no significant difference of the ADC values in group 1 and group 2. CONCLUSION: The study showed a correlation between reduction of ADC and increasing of liver fibrosis degree. The ADC seems to be useful in staging liver fibrosis in patients with CLD, in particular to distinguish the later stages of fibrosis from early and intermediate stages.

ALFI: Cell cycle phenotype annotations of label-free time-lapse imaging data from cultured human cells.

Detecting and tracking multiple moving objects in a video is a challenging task. For living cells, the task becomes even more arduous as cells change their morphology over time, can partially overlap, and mitosis leads to new cells. Differently from fluorescence microscopy, label-free techniques can be easily applied to almost all cell lines, reducing sample preparation complexity and phototoxicity. In this study, we present ALFI, a dataset of images and annotations for label-free microscopy, made publicly available to the scientific community, that notably extends the current panorama of expertly labeled data for detection and tracking of cultured living nontransformed and cancer human cells. It consists of 29 time-lapse image sequences from HeLa, U2OS, and hTERT RPE-1 cells under different experimental conditions, acquired by differential interference contrast microscopy, for a total of 237.9 hours. It contains various annotations (pixel-wise segmentation masks, object-wise bounding boxes, tracking information). The dataset is useful for testing and comparing methods for identifying interphase and mitotic events and reconstructing their lineage, and for discriminating different cellular phenotypes.

[Pancreatic hyperenzymemia: new advances in the field of clinical-diagnostic approach, with particular attention about Gullo's syndrome]. / Iperenzimemia pancreatica: recenti acquisizioni in tema di approccio clinico-diagnostico, con particolare attenzione alla sindrome di Gullo.

An increase in serum levels of pancreatic enzymes is a well-known manifestation of pancreatic disease, especially of inflammatory or neoplastic nature, even if several extrapancreatic diseases can equally cause that increase. In addition to this pathological type of hyperenzymemia, different "non-pathological" forms have also been identified, including macroamylasemia, salivary, and mixed salivary and pancreatic hyperamylasemia, in all of which only amylase elevations are seen. Nevertheless, in 1996 a new syndrome characterized by an abnormal, chronic, benign increase in levels of serum amylase, pancreatic isoamylase, lipase and trypsin, asymptomatic and usually discovered incidentally, was described for the first time by Lucio Gullo et al. Hyperamylasemia/hyperlipasemia's observation is nowadays on the increase because general practitioners tend to include more frequently amylase and lipase in routine blood tests and, moreover, because the constant evaluation of this biochemical alteration in the Emergency Unit: for this reason, this syndrome was clearly identified only recently. Therefore, it's characterized by serum elevation of all pancreatic enzymes in the absence of underlying diseases; it occurs in either sporadic or familial form and it persists over time with considerable fluctuation in serum enzyme concentrations, including frequent normalizations. Proper diagnosis of this form of hyperenzymemia is important because it reassures the subjects having this anomaly that the syndrome is benign, and because it can prevent multiple and expensive diagnostic tests or useless hospitalizations or therapies.

Aurora-A inactivation causes mitotic spindle pole fragmentation by unbalancing microtubule-generated forces.

BACKGROUND: Aurora-A is an oncogenic kinase playing well-documented roles in mitotic spindle organisation. We previously found that Aurora-A inactivation yields the formation of spindles with fragmented poles that can drive chromosome mis-segregation. Here we have addressed the mechanism through which Aurora-A activity regulates the structure and cohesion of spindle poles. RESULTS: We inactivated Aurora-A in human U2OS osteosarcoma cells either by RNA-interference-mediated silencing or treating cultures with the specific inhibitor MLN8237. We show that mitotic spindle pole fragmentation induced by Aurora-A inactivation is associated with microtubule hyperstabilisation. Silencing of the microtubule-stabilising factor ch-TOG prevents spindle pole fragmentation caused by inactivation of Aurora-A alone and concomitantly reduces the hyperstabilisation of microtubules. Furthermore, decreasing pole-directed spindle forces by inhibition of the Eg5 kinesin, or by destabilisation of microtubule-kinetochore attachments, also prevents pole fragmentation in Aurora-A-inactivated mitoses. CONCLUSIONS: Our findings indicate that microtubule-generated forces are imbalanced in Aurora-A-defective cells and exert abnormal pressure at the level of spindle poles, ultimately causing their fragmentation. This study therefore highlights a novel role of the Aurora-A kinase in regulating the balance between microtubule forces during bipolar spindle assembly.

The Aurora-A/TPX2 Axis Directs Spindle Orientation in Adherent Human Cells by Regulating NuMA and Microtubule Stability.

Mitotic spindle orientation is a crucial process that defines the axis of cell division, contributing to daughter cell positioning and fate, and hence to tissue morphogenesis and homeostasis.1,2 The trimeric NuMA/LGN/Gαi complex, the major determinant of spindle orientation, exerts pulling forces on the spindle poles by anchoring astral microtubules (MTs) and dynein motors to the cell cortex.3,4 Mitotic kinases contribute to correct spindle orientation by regulating nuclear mitotic apparatus protein (NuMA) localization,5-7 among which the Aurora-A centrosomal kinase regulates NuMA targeting to the cell cortex in metaphase.8,9 Aurora-A and its activator targeting protein for Xklp2 (TPX2) are frequently overexpressed in cancer,10-12 raising the question as to whether spindle orientation is among the processes downstream the Aurora-A/TPX2 signaling axis altered under pathological conditions. Here, we investigated the role of TPX2 in the Aurora-A- and NuMA-dependent spindle orientation. We show that, in cultured adherent human cells, the interaction with TPX2 is required for Aurora-A to exert this function. We also show that Aurora-A, TPX2, and NuMA are part of a complex at spindle MTs, where TPX2 acts as a platform for Aurora-A regulation of NuMA. Interestingly, excess TPX2 does not influence NuMA localization but induces a "super-alignment" of the spindle axis with respect to the substrate, although an excess of Aurora-A induces spindle misorientation. These opposite effects are both linked to altered MT stability. Overall, our results highlight the importance of TPX2 for spindle orientation and suggest that spindle orientation is differentially sensitive to unbalanced levels of Aurora-A, TPX2, or the Aurora-A/TPX2 complex.

Excess TPX2 Interferes with Microtubule Disassembly and Nuclei Reformation at Mitotic Exit.

The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent-or independent-on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition.

The gut microbiota and its correlations with the central nervous system disorders.

A mutual impact of gastrointestinal tract (GIT) and central nervous system (CNS) functions has been recognized since the mid-twentieth century. It is accepted that the so-called gut-brain axis provides a two-way homeostatic communication, through immunological, hormonal and neuronal signals. A dysfunction of this axis has been associated with the pathogenesis of some diseases both within and outside the GIT, that have shown an increase in incidence over the last decades. Studies comparing germ-free animals and animals exposed to pathogenic bacterial infections, probiotics or antibiotics suggest the participation of the microbiota in this communication and a role in host defense, regulation of immunity and autoimmune disease appearance. The GIT could represent a vulnerable area through which pathogens influence all aspects of physiology and even induce CNS neuro-inflammation. All those concepts may suggest the modulation of the gut microbiota as an achievable strategy for innovative therapies in complex disorders. Moving from this background, the present review discusses the relationship between intestinal microbiota and CNS and the effects in health and disease. We particularly look at how the commensal gut microbiota influences systemic immune response in some neurological disorders, highlighting its impact on pain and cognition in multiple sclerosis, Guillain-Barrè Syndrome, neurodevelopmental and behavioral disorders and Alzheimer's disease. In this review we discuss recent studies showing that the potential microbiota-gut-brain dialogue is implicated in neurodegenerative diseases. Gaining a better understanding of the relationship between microbiota and CNS could provide an insight on the pathogenesis and therapeutic strategies of these disorders.

Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory profile and suitable for topical pulmonary administration.

A novel glucocorticoids series of (GCs), 6α,9α-di-Fluoro 3-substituted C-16,17-isoxazolines was designed, synthesised and their structure-activity relationship was evaluated with glucocorticoid receptor (GR) binding studies together with GR nuclear translocation cell-based assays. This strategy, coupled with in silico modelling analysis, allowed for the identification of Cpd #15, an isoxazoline showing a sub-nanomolar inhibitory potency (IC50=0.84 nM) against TNFα-evoked IL-8 release in primary human airways smooth muscle cells. In Raw264.7 mouse macrophages, Cpd #15 inhibited LPS-induced NO release with a potency (IC50=6 nM)>10-fold higher with respect to Dexamethasone. Upon intratracheal (i.t.) administration, Cpd #15, at 0.1 µmol/kg significantly inhibited and at 1 µmol/kg fully counteracted eosinophilic infiltration in a model of allergen-induced pulmonary inflammation in rats. Moreover, Cpd #15 proved to be suitable for pulmonary topical administration given its sustained lung retention (t1/2=6.5h) and high pulmonary levels (>100-fold higher than plasma levels) upon intratracheal administration in rats. In summary, Cpd #15 displays a pharmacokinetic and pharmacodynamic profile suitable for topical treatment of conditions associated with pulmonary inflammation such as asthma and COPD.

Thrombin activatable fibrinolysis inhibitor (TAFI) does not inhibit in vitro thrombolysis by pharmacological concentrations of t-PA.

TAFI (thrombin activatable fibrinolysis inhibitor) is a plasma procarboxypeptidase that upon activation inhibits the fibrinolytic process by removing the C-terminal lysines from partially degraded fibrin. The generation of activated TAFI (TAFIa) has been suggested to represent a mechanism of thrombus resistance to thrombolytic therapy. However, the ability of TAFI to inhibit fibrinolysis by pharmacological concentrations of t-PA has not been properly investigated. We used an in vitro model consisting of 125I-fibrin blood clots submerged in autologous defibrinated plasma. Upon addition of t-PA (125-5,000 ng/ml) and CaCl2 (25 mM), samples were incubated at 37 degrees C, and clot lysis was measured at intervals from the radioactivity released into solution. The role of TAFI was assessed either by neutralizing the generated TAFIa with the specific inhibitor PTI (50 microg/ml) or by enhancing TAFI activation through the addition of recombinant soluble thrombomodulin (solulin, 1 microg/ml). In our clot lysis model, activation of TAFI amounted to about 20% of inducible carboxypeptidase activity. Addition of PTI, however, produced a significant increase in the extent of lysis only at concentrations of t-PA equal to or lower than 250 ng/ml. When solulin was added to the plasma surrounding the clot, about 70% of TAFI was activated within 15 min. Under these conditions, inhibition of clot lysis was very marked in samples containing 125 or 250 ng/ml of t-PA, but negligible in those containing pharmacological concentrations of the activator (1,000 and 5,000 ng/ml). Additional experiments suggest that loss of fibrin-dependence by elevated concentrations of t-PA may be one of the mechanisms explaining the lack of effect of TAFIa. Our data indicate that, under our experimental conditions, clot lysis by pharmacological concentrations of t-PA is not influenced by TAFIa even after maximal activation of this procarboxy-peptidase.

Fibrin down-regulates LPS- and PMA-induced tissue factor expression by blood mononuclear cells.

Several studies indicate that fibrin may play a functional role in inflammation by modulating a variety of cellular functions. We investigated the effect of fibrin on tissue factor (TF) production by blood mononuclear cells (MNC). Citrated human blood was recalcified and incubated at 37 degrees C for 1-4 h. The resulting clot was lysed by the addition of tissue plasminogen activator (t-PA) and MNC were isolated by density gradient centrifugation. A control blood sample was processed in the same way but omitting calcium addition and clot formation. Clot- and blood-derived MNC did not express detectable TF activity and antigen whatever the incubation time. Clot-derived MNC, however, generated on average 5 fold less TF (activity and antigen) than control cells, when stimulated with lipopolysaccharide (LPS, I microg/ml) for 3 h at 37 degrees C. A reduced TF response of clot-derived cells was also observed at mRNA level as indicated by RT-PCR and in situ hybridization. The effect was dependent on the incubation time within the clot, could not be reversed by enhancing LPS concentration or by adding serum, and was maintained if LPS was replaced by the tumor promoter PMA. A reduced TF response was also found when washed MNC were incorporated for 1 h at 37 degrees C within purified fibrin but not when the cells were incubated with fibrinogen, thrombin or fibrin split products alone. indicating that contact with fibrin was responsible for the inhibition of TF production. Fibrin-induced down-regulation of TF response to LPS and PMA by MNC may represent a negative feed-back aimed at limiting excessive blood clotting activation in immunoinflammatory diseases.

Identification of the major degradation products of 4-methoxy-2-(3-phenyl-2-propynyl)phenol formed by exposure to air and light.

Exposure of 4-methoxy-2-(3-phenyl-2-propynyl)phenol (CO/1828) to air and light (accelerated by temperature) yields 1-(2-hydroxy-5- methoxyphenyl)-3-phenylpropynone as the major degradation product. With extraction, this product rapidly degrades to 5-methoxyaurone and 6-methoxyflavone. In addition, a mixture of dimeric and heterodimeric compounds that are not fully identified was observed. These results indicate the formation of a reactive ortho-quinone methide as an unstable intermediate. This hypothesis is supported by evidence that the aurone slowly isomerizes into the flavone in control samples. Identification of compounds was accomplished with mass spectrometry, nuclear magnetic resonance spectrometry, UV-high-performance liquid chromatography, and comparison with authentic samples.

Sobrerol enantiomers and racemates: solid-state spectroscopy, thermal behavior, and phase diagrams.

The characterization of the solid state of sobrerol enantiomers and racemates has been accomplished by a number of techniques on solid phase such as thermal analysis (DSC) and spectroscopy (IR, 13C NMR, and X-ray diffraction both on powders and on single crystal). Experimental and theoretical binary phase diagrams of cis- and trans-sobrerol enantiomers and their mixtures have been drawn and are discussed. Thermal analysis allowed, moreover, the detection of cis racemate polymorphism. Finally, the quantitative analysis of the cis racemate as an impurity of the trans racemate by means of microcalorimetric determinations is reported.

Metabolism of (-)-6(S)-hydroxy-4(R)-(1-hydroxy-1-methylethyl)-1- cyclohexene-1-ethanol in rat and dog.

1. The metabolism of a new mucoactive drug, chemically (-)-6(S)-hydroxy-4(R)- (1-hydroxy-1-methylethyl)-1-cyclohexene-1-ethanol (CO/1408), has been studied in rat and dog after a single oral dose; eight metabolites were identified. 2. Oxidation of the primary and secondary alcohol groups, hydroxylation in allylic positions and conjugation with glucuronic acid occurred in both species. Products of oxidation on the double bond have not been identified. 3. Using reversed-phase h.p.l.c. and beta-cyclodextrin in the eluent it was found that the glucuronide metabolites varied with species and with the biological fluid examined.