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Purpose: The identification of predictive biomarkers for neoadjuvant chemoradiation therapy (CRT) is a current clinical need. The heterodimer Ku70/80 plays a critical role in DNA repair and cell death induction after damage. The aberrant expression and localization of these proteins fail to control DNA repair and apoptosis. sClusterin is the Ku70 partner that sterically inhibits Bax-dependent cell death after damage in some pathologic conditions. This study sought to evaluate the molecular relevance of Ku70-Ku80-Clu as a molecular cluster predicting the response to neoadjuvant CRT in patients with locally advanced rectal cancer (LARC)., Methods and Materials: Patients enrolled in this study underwent preoperative CRT followed by surgical excision. A retrospective study based on individual response, evaluated by computed tomography and diffusion-weighted magnetic resonance imaging, identified responder (56%) and no-responder patients (44%). Ku70/80 and Clu expression were observed in biopsy specimens obtained before and after treatment with neoadjuvant CRT from the same LARC patients. In vitro studies before and after irradiation were also performed on radioresistant (SW480) and radiosensitive (SW620) colorectal cancer cell lines, mimicking sensitive or resistant tumor behavior., Results: We found a conventional nuclear localization of Ku70/80 in pretherapeutic tumor biopsies of responder patients, in agreement with their role in DNA repair and regulating apoptosis. By contrast, in the no-responder population we observed an unconventional overexpression of Ku70 in the cytoplasm (P<.001). In this context we also overexpression of sClu in the cytoplasm, which accorded with its role in stabilizing of Bax-Ku70 complex, inhibiting Bax-dependent apoptosis. Strikingly, Ku80 in these tumor tissues was lost (P<.005). In vitro testing of colon cancer cells finally confirmed the results observed in tumor biopsy specimens, proving that Ku70/80-Clu deregulation is extensively involved in the resistance mechanism., Conclusion: These results strongly suggest a potential role of these proteins as a new prognostic tool to predict the response to chemoradiation in LARC., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Alternative splicing (AS) is a process in which precursor messenger RNA (pre-mRNA) splicing sites are differentially selected to diversify the protein isoform population. Changes in AS patterns have an essential role in normal development, differentiation and response to physiological stimuli. It is documented that AS can generate both "risk" and "protective" splice variants that can contribute to the pathogenesis of several diseases including atherosclerosis. The main endothelial receptor for oxidized low-density lipoprotein (ox-LDLs) is LOX-1 receptor protein encoded by the OLR1 gene. When OLR1 undergoes AS events, it generates three variants: OLR1 , OLR1D4 and LOXIN. The latter lacks exon 5 and two-thirds of the functional domain. Literature data demonstrate a protective role of LOXIN in pathologies correlated with LOX-1 overexpression such as atherosclerosis and tumors. In this review, we summarize recent developments in understanding of OLR1 AS while also highlighting data warranting further investigation of this process as a novel therapeutic target.

A 60-year-old man with left flank pain, fever, and nausea underwent an abdominal ultrasound and showed left hydroureteronephrosis without urinary calculi. Computed tomography urography showed moderate left hydroureteronephrosis and a hypodense paravesical mass of 1.7 cm with mild contrast enhancement just below the ipsilateral ureterovesical junction. Contrast-enhanced magnetic resonance imaging showed a 48 cc prostate and confirmed a roundish mass, protruding into the bladder, hyperintense on T2-weighted images, hypointense on T1-weighted images, and with mild inhomogeneous contrast enhancement. Cystoscopy with cold cup biopsy was carried out. Histologic analysis revealed the presence of ectopic prostatic tissue with no evidence of malignancy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Transcriptional mechanisms epigenetically-regulated in tumoral tissues point out new targets for anti-cancer therapies. Carnitine palmitoyl transferase I (CPT1) is the rate-limiting enzyme in the transport of long-chain fatty acids for β-oxidation. Here we identified the tumor specific nuclear CPT1A as a product of the transcript variant 2, that doesn't retain the classical transferase activity and is strongly involved in the epigenetic regulation of cancer pro-survival, cell death escaping and tumor invasion pathways. The knockdown of CPT1A variant 2 by small interfering RNAs (siRNAs), was sufficient to induce apoptosis in MCF-7, SK-BR3 and MDA-MB-231 breast cancer cells. The cell death triggered by CPT1A silencing correlated with reduction of HDAC activity and histone hyperacetylation. Docking experiments and molecular dynamics simulations confirmed an high binding affinity of the variant 2 for HDAC1. The CPT1A silenced cells showed an up-regulated transcription of pro-apoptotic genes (BAD, CASP9, COL18A1) and down-modulation of invasion and metastasis related-genes (TIMP-1, PDGF-A, SERPINB2). These findings provide evidence of the CPT1 variant 2 involvement in breast cancer survival, cell death escape and invasion. Thus, we propose nuclear CPT1A as a striking tumor specific target for anticancer therapeutics, more selective and effective as compared with the well-known HDAC inhibitors.

The identification of new biomarkers and targets for tailored therapy in human colorectal cancer (CRC) onset and progression is an interesting challenge. CRC tissue produces an excess of ox-LDL, suggesting a close correlation between lipid dysfunction and malignant transformation. Lectin-like oxidized LDL receptor-1 (LOX-1) is involved in several mechanisms closely linked to tumorigenesis. Here we report a tumor specific LOX-1 overexpression in human colon cancers: LOX-1 results strongly increased in the 72% of carcinomas (P<0.001), and strongly overexpressed in 90% of highly aggressive and metastatic tumours (P<0.001), as compared to normal mucosa. Moreover LOX-1 results modulated since the early stage of the disease (adenomas vs normal mucosa; P<0.001) suggesting an involvement in tumor insurgence and progression. The in vitro knockdown of LOX-1 in DLD-1 and HCT-8 colon cancer cells by siRNA and anti-LOX-1 antibody triggers to an impaired proliferation rate and affects the maintenance of cell growth and tumorigenicity. The wound-healing assay reveals an evident impairment in closing the scratch. Lastly knockdown of LOX-1 delineates a specific pattern of volatile compounds characterized by the presence of a butyrate derivative, suggesting a potential role of LOX-1 in tumor-specific epigenetic regulation in neoplastic cells. The role of LOX-1 as a novel biomarker and molecular target represents a concrete opportunity to improve current therapeutic strategies for CRC. In addition, the innovative application of a technology focused to the identification of LOX-1 driven volatiles specific to colorectal cancer provides a promising diagnostic tool for CRC screening and for monitoring the response to therapy.

The up-regulation of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, plays a fundamental role in the pathogenesis of atherosclerosis. Moreover, OLR1 polymorphisms were associated with increased susceptibility to acute myocardial infarction (AMI) and coronary artery diseases (CAD). In these pathologies, the identification of therapeutic approaches that can inhibit or reduce LOX-1 overexpression is crucial. Predictive analysis showed a putative hsa-miR-24 binding site in the 3'UTR of OLR1, 'naturally' mutated by the presence of the rs1050286 single nucleotide polymorphism (SNP). Luciferase assays revealed that miR-24 targets OLR1 3'UTR-G, but not 3'UTR-A (P < 0.0005). The functional relevance of miR-24 in regulating the expression of OLR1 was established by overexpressing miR-24 in human cell lines heterozygous (A/G, HeLa) and homozygous (A/A, HepG2) for rs1050286 SNP. Accordingly, HeLa (A/G), but not HepG2 (A/A), showed a significant down-regulation of OLR1 both at RNA and protein level. Our results indicate that rs1050286 SNP significantly affects miR-24 binding affinity to the 3'UTR of OLR1, causing a more efficient post-transcriptional gene repression in the presence of the G allele. On this basis, we considered that OLR1 rs1050286 SNP may contribute to modify OLR1 susceptibility to AMI and CAD, so ORL1 SNPs screening could help to stratify patients risk., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The anterior nutcracker syndrome is defined by the compression of the left renal vein between the aorta and superior mesenteric artery, usually related to the occurrence of hematuria. We report the case of an uncommon complication of the nutcracker syndrome. A 75-year-old woman was referred to our institution for left flank pain without hematuria. Multiphasic computer tomography urography showed a condition of left renal vein entrapment between the aorta and superior mesenteric artery with the development of left gonadal vein varicosities at the level of the renal hilum; a pyeloureteral junction compression with dilation of the pyelocalyceal system coexisted. To our knowledge, this is the first report of the association between nutcracker syndrome and pyeloureteral junction obstruction., (© 2015 S. Karger AG, Basel.)

Nutcracker syndrome (NCS) refers to the compression of the left renal vein (LRV) between the aorta and the superior mesenteric artery. It can cause both microscopic and gross haematuria, with or without flank pain. The diagnosis is often delayed in a majority of symptomatic patients. On the other hand, the use of CT in routine abdominal explorations has increased the detection of the compression of the LRV in healthy and asymptomatic patients, but its diagnostic value remains uncertain. In this paper, we report 3 cases of the NCS associated with an increased blood flow in the LRV, due to different conditions, which we believe could produce the appearance of clinical symptoms., (© 2015 S. Karger AG, Basel.)

Background: In the few studies available, the risk of developing systemic reactions (SR) to hymenoptera stings in patients with previous large local reactions (LLRs) to stings ranges from 0 to 7 %. We evaluated both retrospectively and prospectively the risk of SRs in patients with LLRs to stings., Methods: An overall number of 477 patients, 396 with an SR as the first manifestation of allergy and 81 with a history of only LLRs after hymenoptera stings, were included in the study. All patients had clinical history and allergy testing (skin tests and/or specific IgE) indicative of allergy to venom of only one kind of Hymenoptera. Of the 81 patient with LLRs, 53 were followed-up for 3 years by annual control visits, while the 396 patients with SR were evaluated retrospectively., Results: Among the 396 patients with an SR, only 17 (4.2 %) had had a previous LLR as debut of allergy, after an history of normal local reactions to Hymenoptera stings. All the 81 patients with a history of only LLRs had previously had at least two LLRs, with an overall number of 238 stings and no SR. Among the 53 patients who were prospectively evaluated we found that 31 of them (58.3 %) were restung by the same type of insect, with an overall number of 59 stings, presenting only LLRs and no SR., Conclusions: Our findings confirm that patients with repeated LLRs to stings had no risk of SR, while a single LLR does not exclude such risk. This has to be considered in the management of patients with LLRs.

Introduction: Hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE type I) or dysfunction (C1-INH-HAE type II) is a rare disease characterized by recurrent episodes of edema with an estimated frequency of 1:50,000 in the global population without racial or gender differences. In this study we present the results of a nationwide survey of C1-INH-HAE patients referring to 17 Italian centers, the Italian network for C1-INH-HAE, ITACA., Methods: Italian patients diagnosed with C1-INH-HAE from 1973 to 2013 were included in the study. Diagnosis of C1-INH-HAE was based on family and/or personal history of recurrent angioedema without urticaria and on antigenic and/or functional C1-INH deficiency., Results: 983 patients (53% female) from 376 unrelated families were included in this survey. Since 1973, 63 (6%) patients diagnosed with C1-INH-HAE died and data from 3 patients were missing when analysis was performed. Accordingly, the minimum prevalence of HAE in Italy in 2013 is 920:59,394,000 inhabitants, equivalent to 1:64,935. Compared to the general population, patients are less represented in the early and late decades of life: men start reducing after the 5(th) decade and women after the 6(th). Median age of patients is 45 (IQ 28-57), median age at diagnosis is 26 years (IQ 13-41). C1-INH-HAE type 1 are 87%, with median age at diagnosis of 25 (13-40); type 2 are 13% with median age at diagnosis of 31 (IQ 16-49). Functional C1INH is ≤50% in 99% of patients. Antigen C1INH is ≤50% in 99% of type 1. C4 is ≤50% in 96% of patients. The chance of having C1-INH-HAE with C4 plasma levels >50% is < 0.05., Conclusion: This nationwide survey of C1-INH-HAE provides for Italy a prevalence of 1:64,935. C1-INH-HAE patients listed in our database have a shorter life expectancy than the general population. An increased awareness of the disease is needed to reduce this discrepancy. Measurement of C4 antigen can exclude diagnosis of C1-INH-HAE with an accuracy > 95%. This parameter should be therefore considered for initial screening in differential diagnosis of angioedema.

Background: Acute myocardial infarction (AMI), is related to a diffuse active inflammation of the coronary tree associated with rupture of one of the multiple vulnerable plaques. The presence of soluble mediators of inflammation with their synergic or antagonistic actions coordinates the physiological response determining the plaque fate and the fatal event. The present study focus on the cytokines network operating in human coronary plaques of patients died from AMI and controls, pointing out that coronaries of AMI patients produce PTX3 protein twice as that of controls and express high level of PTX3 mRNA., Results: The presence of CX3CR1 polymorphisms is significantly correlated with the incidence and the outcome of acute myocardial infarction inducing in the whole coronary tree a strong recruitment of Th1 polarized inflammation that is directly correlated to PTX3 expression., Conclusions: Moreover we found a positive correlation between the expression of PTX3 in the plaque and the content of macrophage cells showing a M2 polarization indicating the possible role of this chemokine as mediator of immune response that would orchestrate plaque evolution and inflammatory cell type activation., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Starting from a orexin 1 receptor selective antagonist 4,4-disubstituted piperidine series a novel potent 5-azaspiro[2.4]heptane dual orexin 1 and orexin 2 receptor antagonist class has been discovered. SAR and Pharmacokinetic optimization of this series is herein disclosed. Lead compound 15 exhibits potent activity against orexin 1 and orexin 2 receptors along with low cytochrome P450 inhibition potential, good brain penetration and oral bioavailability in rats., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: In prostate cancer the secreted form of clusterin (sCLU) has been described as an anti-apoptotic protein whose expression is increased after therapeutic intervention, whereas, the nuclear protein form nCLU was reported to have pro-apoptotic properties., Methodology: In order to provide new therapeutic approaches targeting CLU, we developed a strategy based on exon skipping by using a lentiviral construct to preferentially induce the nuclear spliced form of the protein. The molecular construct was transduced in LNCaP cells for testing the modulation of sensitivity of the transduced cells to pro-apoptotic stress., Results and Conclusions: We showed an increase of nCLU/sCLU expression ratio in the prostate cancer cell line "LNCaP" after lentiviral vector-U7 nCLU transduction. Moreover, we showed a significant inhibition of cell proliferation in nCLU-U7 LNCaP cells after treatment with cisplatin and after exposure to ionizing radiation compared to control cells. Finally, we showed that nCLU-U7 LNCaP cells exposure to UV-C significantly reduced an increase of cell death compared to control. Finally, we showed that modulating nCLU expression had profound impact on Ku70/Bax interaction as well as Rad17 expression which could be a key mechanism in sensitizing cells to cell death. In conclusion, this is the first report showing that increasing of nCLU/sCLU expression ratio by using an "on demand alternative splicing" strategy successfully increased sensitivity to radiotherapy and chemotherapy of prostate cancer cells.

Fractalkine is a proinflammatory chemokine that participates in atherosclerotic process mediating the interactions of vascular cells and leukocytes and selective recruitment of Th1 lymphocytes, through interaction with CX3CR1 receptor. The polymorphism of the fractalkine receptor 280M-containing haplotype, which codifies for a receptor with minor expression and with a reduced binding capability, represents a novel protective factor of atherosclerotic disease. We investigated the association among CX3CR1 genotype, the inflammatory infiltrate subpopulations recruited in the plaque, and the in situ expression of fractalkine and its receptor, in patients who died of myocardial infarction (AMI) compared with subjects who died of noncardiac causes. Patients with nonlethal AMI (AMI survivors) were also investigated to correlate the CX3CR1 polymorphisms and the incidence of lethal AMI. A strong T cells infiltrate was found in infarct related artery (IRA) plaques of AMI patients presenting the V249 T280 haplotype (84%). Conversely, a decreased T cell recruitment was associated with I249T280 haplotype in the controls (64%). The significant higher presence of the variant allele I249 in homo- and heterozygosis, found in controls (91%) and in AMI survivors (94%), with respect to the patients who died of AMI (48%), showed the relevance of this polymorphism both in the onset and outcome of acute myocardial infarction. The presence of CX3CR1 polymorphisms could influence the incidence and the outcome of acute myocardial infarction, altering the inflammation of the whole coronary tree by the impaired recruitment of Th1 polarized subpopulation in the coronary plaque.

Introduction: Sodium-N-chlorine-p-toluene sulfonamide, commonly known as chloramine-T, is a derivative of chlorine which is widely used as a disinfectant. For many years, chloramine-T has been described as a cause of immediate-type hypersensitivity, especially with regard to asthma and rhinitis, and as a cause of occupational dermatoses in cleaning personnel in hospitals, although no anaphylactic reaction has yet been reported. Hence, to the best of our knowledge we present the first case of anaphylaxis to chloramine-T with evidence of specific immunoglobulin E antibodies., Case Presentation: We describe the case of a 25-year-old Caucasian woman who was in good health and with a negative history for atopy, including no respiratory symptoms of rhinitis or asthma, and with no professional exposure to chloramine-T. She, while showering, applied a chloramine-T solution to a skin area with folliculitis on her leg, and within a few minutes developed generalized urticaria and angioedema, followed by vomiting and collapse with loss of consciousness. A skin prick test with a chloramine-T solution at 10mg/mL concentration was positive, and specific immunoglobulin E to chloramine-T was quantified at a value of 2.9 optical density as measured by the enzyme allergosorbent test technique., Conclusion: The strict cause-effect relationship and the results of the skin test and the in vitro test make certain the causative role of chloramine-T in this case of anaphylaxis. This suggests that chloramine-T, based on its wide use as a disinfectant, should be considered a possible cause in anaphylaxis of unknown origin.

The construction of a EP(4) antagonists pharmacophore model and the discovery of a highly potent oxepinic series of EP(4) antagonists is discussed. Compound 1a exhibits an excellent selectivity profile toward EP(2) receptor subtype and low cytochrome P450 inhibition potential., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The influence of the microenvironment through the various steps of cancer progression is signed by different cytokines and growth factors, that could directly affect cell proliferation and survival, either in cancer and stromal cells. In colon cancer progression, the cooperation between hypoxia, IL-6 and VEGF-A165 could regulate the DNA repair capacity of the cell, whose impairment is the first step of colon cancer development. This cooperation redirects the activity of proteins involved in the metabolic shift and cell death, affecting the cell fate. The pathways triggered by micro environmental factors could modulate cancer-related gene transcription, affecting also small non coding mRNA, microRNAs. MicroRNAs have emerged as key post-transcriptional regulators of gene expression, directly involved in human cancers. The present review will focus first on the intertwined connection between cancer microenvironment and aberrant expression of microRNAs which contribute to carcinogenesis. In particular, the epigenetic mechanisms triggered by tissue microenvironment will be discussed, in view of the recent identification of miRNAs able to directly or indirectly modulate the epigenetic machinery (epi-miRNAs) and that are involved in the epithelial to mesenchimal transition and metastases development.

Diagnosis and treatment of hypersensitivity to Hymenoptera venom took a landmark step forward in the late 1970s with the introduction of venom as an adequate material instead of whole body extracts. Since then, venom immunotherapy (VIT) has provided allergic subjects with a complete protection from fatal anaphylaxis and prevented about 90% of all reactions to stings. The cross-reactivity among some venom components, particularly important in the case of cross-reacting carbohydrate determinants, has often made it difficult to recognize the true causative venom to be used in VIT. Recently, the introduction of purified and recombinant allergens, such as Api m 1 from honeybee venom, Ves v 5 from yellow jacket venom, and Pol d 5 from wasp venom, have allowed a more precise diagnosis with identification of the causative venom component. This paves the way for a patient-tailored VIT in the near future. Another issue which needs to be addressed is the improvement in the safety of VIT with honeybee venom, which is significantly less favourable in comparison to vespid venom. A number of molecular approaches are under investigation in order to achieve this objective. Alternative routes of administration, such as the sublingual and the intralymphatic, have also been proposed, but there are not yet sufficient data available to demonstrate their feasibility. This review also presents patents on new trends in therapies for the management of hypersensitivity to hymenoptera venom.

Objectives: The identification of useful markers for early diagnosis of human colon cancer is a major goal still in progress. Clusterin is a pleiotropic protein with a broad range of functions. It has recently drawn much attention because of its association with cancer promotion and metastasis. It is involved in prosurvival and apoptosis processes that are carried out by two different isoforms. Secreted clusterin isoform (sCLU) is cytoprotective and its prosurvival function is the basis of the current phase I/II clinical trials against prostate, lung, and breast cancers. We have already shown that in colorectal cancer (CRC) there is an increased expression of sCLU. In this report, we investigated whether sCLU is released in the blood and stool of colon cancer patients in order to study sCLU as a potential diagnostic molecular marker for colon cancer screening., Methods: The quantitative expression of sCLU was determined by dot blot immunodosage in the serum and stool of CRC patients (n=63) and age-matched controls without clinical history of neoplasia, CRC, or systemic or bowel inflammatory disease (n=50). Unpaired t-tests and Mann-Whitney U-tests were used for continuous variables. The diagnostic performance of clusterin was appraised by means of receiver operating characteristic (ROC) curves., Results: We found a significant increase of sCLU in the serum and stool of CRC patients (P=0.0002 and P<0.000, respectively) as compared with controls. ROC curves provided cutoff points showing a trade-off between sensitivity and specificity. With a cutoff point of 88.5 microg/ml, sCLU in blood showed a 55.6% sensitivity and 100% specificity, and with a cutoff point of 34.6 microg/g, the stool test reached 66.7% sensitivity and 84% specificity in discriminating between nonneoplastic and colorectal neoplastic lesions. Human cancer xenografts in nude mice indicated a positive correlation between increasing serum clusterin level and tumor size., Conclusions: This study highlights the potential of clusterin detection in stool to be a valuable tool to improve the effectiveness and efficiency of large-scale clinical cancer screening.

Activation of pro-survival pathways and apoptotic cell death escape are considered hallmarks of oncogenic cell transformation. Tissue microenvironment strongly influences tumorigenesis, redirecting some pathways versus a persisting pro-survival state. Here, we report evidence on the role of interleukin 6 (IL-6) in affecting pro-survival pathways in colon cancer progression, modulating the expression and the molecular interactions among the pro-apoptotic factor Bax, the DNA repair proteins Ku70/86 and Clusterin isoforms. In human colorectal carcinomas (n = 50) at different stages of disease, we found an increased IL-6 production, the loss of Ku86 and Clusterin 50-55 kDa pro-apoptotic isoform. Conversely, we observed the overexpression of Bax and the 40 kDa prosurvival sClusterin (sCLU) isoform. Bax co-localized with Ku70 that was found atypically expressed in the cytoplasm of advanced stage colon cancers (Dukes'C-D; n = 22). IL-6 treatment of a colon cancer cell line, Caco-2, modulated the expression of genes involved in tumor invasion and apoptosis, as observed by microarrays. In particular, IL-6 downmodulated Bax expression at mRNA level. Concomitantly, IL-6 exposure influenced Bax also at protein level acting on the Bax-Ku70-sCLU physical interactions in the cytoplasm, by affecting the Ku70 acetylation and phosphorylation state, thus leading to the inhibition of Bax pro-apoptotic activity. In addition, we found that IL-6 treatment induced a significant downregulation of Ku86 and a strong increase of sCLU, confirming tumor biopsies data. In contrast Somatostatin treatment of Caco-2 cells was able to restore apoptosis, demonstrating that Ku70-Bax-CLU interactions could be dynamically modulated. Hence, IL-6 could favor tumor expansion, promoting cell survival and apoptosis escape throughout the different stages of tumor evolution. Uncovering the molecular mechanisms of action of these factors may offer strategies for selectively manipulate the cancer cells sensitivity to therapy.

The transition from normal to malignant phenotype implies the activation of some pathways that underlie the aberrant clone expansion. In some way, the conventional function of proteins involved in DNA repair, cell death/growth induction, vascularization, and metabolism is inhibited or shifted toward other pathways by soluble mediators that orchestrate such change depending on the microenvironment conditions. The adenoma-carcinoma sequence of the colon represents one of the most well studied and characterized models of human tumor progression. In this section, we focus our attention on defined pathways that underlie the initiation, promotion, and progression of colon cancer, conferring aggressiveness to the neoplastic cells. Clusterin (CLU) is a pleiotropic protein with a broad range of functions. It has recently drawn much attention because of its association with cancer promotion and metastasis. It is involved in prosurvival and apoptosis processes that are carried out by two different forms. sCLU is cytoprotective and its prosurvival function is the basis of the current Phase I/II clinical trials. In colorectal cancer an increase of sCLU expression occurs, whereas the nuclear proapoptotic form is downregulated. Several controversial data have been published on colon cancer discussing its role as tumor suppressor or prosurvival factor in colon cancer. Here, we report the dynamic interaction of the different forms of CLU with their partners DNA-repair protein Ku70 and proapoptotic factor Bax during colon cancer progression, which seems to be a crucial point for the neoplastic cell fate. We also highlight that the appearance and the progressive increase of the sCLU in colorectal tumors correlate to a significant increase of CLU in serum and stool of patients. On the basis of results obtained by CLU immuno-dosage in blood and stool of colon cancer patients, we report that sCLU could represent a diagnostic molecular marker for colon cancer screening.

In no other field has the function of clusterin (CLU) been more controversial than in cancer genetics. After more than 20 years of research, there is still uncertainty with regard to the role of CLU in human cancers. Some investigators believe CLU to be an oncogene, others-an inhibitor of tumorigenesis. However, owing to the recent efforts of several laboratories, the role of CLU in important cellular processes like proliferation, apoptosis, differentiation, and transformation is beginning to emerge. The "enigmatic" CLU is becoming less so. In this chapter, we will review the work of research teams interested in understanding how CLU is regulated by oncogenic signaling. We will discuss how and under what circumstances oncogenes and epigenetic factors modify CLU expression, with important consequences for mammalian tumorigenesis.

Cell transformation is strictly linked to important metabolic changes which are instrumental for initial survival of cancer cells and subsequent spreading of disease. Early (i.e., anerobic glycolysis) and late metabolic changes (i.e., fatty acid metabolism) are required for progression and clinical emergence of cancer. Besides well-known tumor suppressors and oncogenes, several metabolic genes have been found implicated in this multistep process, among which are fatty acid synthase (FASN) and carnitine palmitoyl transferase I (CPT I). An intriguing link between these metabolic shifts and a change in the balance between nuclear and secreted forms of CLU (nCLU/sCLU) has been suggested. The shifting balance between CLU forms during tumor progression, by affecting the fate of the cell, seems to be strongly influenced by the metabolic shift occurring in the different steps of tumor progression.

Cancer cells need to interact synergistically with their surrounding microenvironment to form a neoplasm and to progress further to colonize distant organs. The microenvironment can exert profound epigenetic effects on cells through cell-derived interactions between cells, or through cell-derived factors deposited into the microenvironment. Tumor progression implies immune-escaping and triggers several processes that synergistically induce a cooperation among transformed and stromal cells, that compete for space and resources such as oxygen and nutrients. Therefore, the extra cellular milieu and tissue microenvironment heterotypic interactions cooperate to promote tumor growth, angiogenesis, and cancer cell motility, through elevated secretion of pleiotropic cytokines and soluble factors. Clusterin (CLU), widely viewed as an enigmatic protein represents one of the numerous cellular factors sharing the intracellular information with the microenvironment and it has also a systemic diffusion, tightly joining the "In and the Out" of the cell with a still debated variety of antagonistic functions. The multiplicity of names for CLU is an indication of the complexity of the problem and could reflect, on one hand its multifunctionality, or alternatively could mask a commonality of function. The posited role for CLU, further supported as a cytoprotective prosurvival chaperone-like molecule, seems compelling, in contrast its tumor suppressor function, as a guide of the guardians of the genome (DNA-repair proteins Ku70/80, Bax cell death inducer), could really reflect the balanced expression of its different forms, most certainly depending on the intra- and extracellular microenvironment cross talk. The complicated balance of cytokines network and the regulation of CLU forms production in cancer and stromal cells undoubtedly represent a potential link among adaptative responses, genomic stability, and bystander effect after oxidative stresses and damage. This review focuses on the tumor-microenvironment interactions strictly involved in controlling local cancer growth, invasion, and distant metastases that play a decisive role in the regulation of CLU different forms expression and release. In addition, we focus on the pleiotropic action of the extracellular form of this protein, sCLU, that may play a crucial role in redirecting stromal changes, altering intercellular communications binding cell surface receptors and contributing to influence the secretion of chemokines in paracrine and autocrine fashion. Further elucidation of CLU functions inside and outside ("in and out") of cancer cell are warranted for a deeper understanding of the interplay between tumor and stroma, suggesting new therapeutic cotargeting strategies.

Allergic disorders are viewed generally as organ diseases and thus referred to organ specialists, such as the ear, nose and throat specialist for rhinitis, the pulmonologist for asthma, the dermatologist for dermatitis, and so on. Indeed, the systemic nature of allergy is made evident by the fact that the same individual may develop during the life different manifestations to a given allergen. This is true for example in sensitisation to house dust mites, which may start in childhood as atopic dermatitis and later express as asthma or rhinitis. The major player in driving the immune response is the T lymphocyte, and the T helper subpopulations--Th1 and Th2--as well as the T regulatory cells, are involved in orienting tolerance or reactivity to allergens. Interesting observations on the systemic or organ-specific actions of T cells were obtained by transplantations from allergic donors to non-allergic recipients. Bone marrow is able to transfer all allergic manifestations, while lung transplantation transfers only asthma. A number of factors are involved in the expression of allergy as a systemic or organ disease and deserve deeper investigations. They include the antigen presenting cells, the homing of T cells, the cytokine and chemokine pattern, and the adhesion molecules.

Growth factors and their respective receptors are key regulators in development and homeostasis of the nervous system, and changes in the function, expression, or downstream signaling of growth factors are involved in many neuropathological disorders. Recently, research has yielded a rich harvest of information about molecules and gene, and currently the assumption "a gene-a protein", where each gene encodes the structure of a single protein, is becoming a paradox. In the past years, the discovery of synergic or antagonistic proteins deriving from the same gene is a novelty upsetting. In some way, the conventional function of proteins involved in DNA repair, cell death/growth induction, vascularization, and metabolism is inhibited or shifted toward other pathways by soluble mediators that orchestrate such change depending on the microenvironment conditions. In this chapter, we focus on the antithetic properties that proteins could exert, depending on the microenvironment that orchestrates the complex networks among proteins and their respective partners.

Given the increasing incidence of colorectal cancer (CRC), performing new and cost-effective stool tests is of particular importance for early diagnosis and treatment. In the present review, we describe the main characteristics, and the performance of the most recently developed stool tests in the screening setting of colorectal tumoral diseases. Most of the studies reported high sensitivity both for adenomas and CRC diagnosis; less than half studies reported also high specificity with respect to stage and localization of the tumor. However, the performance of every single test was extremely variable, reaching >95% specificity for most of DNA-based markers, although lacking sensitivity even in case of invasive CRC. A new potential stool marker of colon cancer is clusterin, a protein of particular interest for its high sensitivity and positive predictive value in patients with highly aggressive CRC.

Carnitine palmitoyl transferase I (CPT1) catalyzes the transport of long-chain fatty acids into mitochondria for beta-oxidation. A link between CPT1 and apoptosis has been suggested on the basis of several experimental data. Nevertheless, results are contradictory about the effective role of CPT1 in cell survival control and cancer development. Conversely, Fatty acid synthase (FAS) enzyme, required for the synthesis of fatty acids, is found over-expressed in tumors and inhibition of FAS triggers apoptosis in human cancer cells. We have studied the tumor-specific modulation of CPT1 and FAS in human colorectal cancer (n = 11) and breast carcinomas (n = 24). CPT1 was significantly decreased in the cytoplasm of tumoral samples (p < or = 0.04), whereas FAS was increased (p < or = 0.04). A striking CPT1 nuclear localization was evident in the tumors (p < or = 0.04). In the nuclear environment the protein would modulate the levels of acetyl/acyl-CoA implicated in the regulation of gene transcription. At this purpose, we performed in vitro experiments using epithelial neoplastic (MCF-7, Caco-2, HepG2 cells) and non neoplastic cell lines (MCF-12F) confirming a nuclear localization of CPT1 protein exclusively in neoplastic cells. Moreover histone deacetylase (HDAC) activity showed significantly higher levels in nuclear extracts from neoplastic than from control cells. HDAC1 and CPT1 proteins coimmunoprecipitated in nuclear extracts from MCF-7 cells. The treatment with HDAC inhibitors such as trichostatin A and butyrate significantly decreased nuclear expression of CPT1 and its bond to HDAC1. We also identified the existence of CPT1A mRNA transcript variant 2 in MCF-7, beside to the classic isoform 1. The peculiar localization of CPT1 in the nuclei of human carcinomas and the disclosed functional link between nuclear CPT1 and HDAC1 propose a new role of CPT1 in the histonic acetylation level of tumors.

Purpose: In order to characterize the biological effects and molecular mechanism underlying indocyanine-green (ICG)-mediated photo-oxidative cell damage, human cultured retinal pigmented epithelium (RPE) cells preloaded with ICG were exposed to 810-nm laser irradiation. Cell viability and death induction were examined, as well as the modulation of proteins involved in cell death and DNA repair., Methods: ARPE-19 cells preloaded with 100 microM ICG were irradiated using continuous and micropulsed 810-nm laser for the dye photoactivation, and cell viability and apoptosis were evaluated. The expression and subcellular localization of Bax, Ku70, Ku80 and clusterin/ApoJ were analyzed by immunocytochemistry and Western blot., Results: ICG photoactivation induced apoptosis in RPE cells. The micropulsed laser irradiation induced a higher percentage of cell killing as compared to continuous wave. Cell killing was inhibited by sodium azide, suggesting the involvement of reactive oxygen species in the laser-induced cell damage. Bax was strongly induced after 4 and up to 24 h of treatment. The nuclear proapoptotic isoform of clusterin/ApoJ was selectively upregulated after 24 h of treatment. The DNA repair machinery was upregulated after 4 and up to 24 h., Conclusion: These data elucidate some molecular mechanisms involved in cell death induced by ICG photosensitization. The increase and relocalization of Bax into the mitochondria and the upregulation and translocation of the proapoptotic isoform of clusterin/ApoJ in the nucleus demonstrated the involvement of these proteins in the photo-oxidative cell death pathway. These data point out new molecular targets and suggest potential applications in the therapy of the retinal diseases that could benefit by selective RPE treatment.

The current burden of allergic diseases, estimated by both direct and indirect costs, is very relevant. In fact the cost estimation for rhinitis amount globally to 4-10 billion dollars/year in the U.S. and to an average annual cost of 1089 euros per child/adolescent and 1543 euros per adult in Europe. The estimated annual costs in Northern America for asthma amounted to 14 billion dollars. Consequently, preventive strategies aimed at reducing the clinical severity of allergy are potentially able to reduce its costs. Among them, specific immunotherapy (SIT) joins to the preventive capacity the carryover effect once treatment is discontinued. A number of studies, mainly conducted in the US and Germany demonstrated a favourable cost-benefit balance. In the nineties, most surveys on patients with allergic rhinitis and asthma reported significant reductions of the direct and indirect costs in subjects treated with SIT compared to those treated with symptomatic drugs. This is fully confirmed in recent studies conducted in European countries: in Denmark the direct cost per patient/year of the standard care was more than halved following SIT; in Italy a study on Parietaria allergic patients demonstrated a significant difference in favor of SIT plus drug treatment for three years versus drug treatment alone, with a cost reduction starting from the 2nd year and increasing to 48% at the 3rd year, with a highly statistical significance which was maintained up to the 6th year, i.e. 3 years after stopping immunotherapy, corresponding to a net saving for each patient at the final evaluation of 623 euros per year; in France a cost/efficacy analysis comparing SIT and current symptomatic treatment in adults and children with dust mite and pollen allergy showed remarkable savings with SIT for both allergies in adults and children.

Acute myocardial infarction (AMI) associated with unfavorable prognosis is likely to be the consequence of a diffuse active chronic inflammatory process that destabilizes the whole coronary tree and myocardium, suggesting a possible common causal agent underlying both conditions. The main objective of this study was to investigate whether Chlamydia pneumoniae (CP) infection occurred beyond the coronary plaques, namely in the myocardium of individuals who died of AMI. The presence of CP cell wall antigen (OMP-2) and CP-HSP60 was investigated in the myocardium and coronary plaques of 10 AMI and 10 age-matched control patients by immunohistochemistry, electron microscopy, and molecular biology. OMP-2 antigens were found in the unaffected myocardium of 9 of 10 AMI patients. Conversely, only 1 of 10 control patients exhibited a positive staining for CP. Moreover, OMP-2 and CP-HSP60 were detected in the whole coronary tree. CP presence was strongly associated with a T-cell inflammatory infiltrate. Our results suggest that CP may underlie both coronary and myocardial vulnerabilities in patients who died of AMI and corroborate the notion that CP may act by reducing cardiac reserves, thus worsening the ischemic burden of myocardium.

Allergic rhinitis and asthma constitute a global health problem because of their very high prevalence and the consequent burden of disease, concerning medical and economical issues. Among the treatments of allergy, specific immunotherapy has the capacity to favourably alter the natural history of the disease both during and after its performance and thus to reduce the direct and indirect costs of allergic rhinitis and asthma. A number of studies reported such cost reduction for traditional, subcutaneous immunotherapy and recent data demonstrate that also sublingual immunotherapy (SLIT) is associated to economic advantages and/or monetary savings, specifically in terms of reduction of disease economic burden. Only few formal economic assessments of SLIT have been carried out so far, this article will present and discuss the published studies addressed to this issue. The data obtained, although the number of studies is still limited, provide preliminary evidence supporting a SLIT effect on sparing costs for respiratory allergy.

Background: In sensitized subjects Hymenoptera stings may provoke the awakening of mediated systemic reactions of I type IgE, which can sometimes be serious. Considering the type of work performed activity and the high frequency of reported hymenoptera sting episodes, a sample of 206 Forestry Department agents was surveyed who worked outside urban areas in the Marche Region., Objectives: The aim of the study was to analyse the prevalence of stings and their possible systemic reactions, as well as to evaluate the type of occupational risks involved., Methods: A total of 206 agents were examined and questioned about the number of stings suffered during work and about the kind of subsequent skin and systemic reactions; they were then classified according to the method proposed by H.L. Mueller., Results: 179 agents reported having suffered from hymenoptera stings and, of these, 53 subjects (29,6%) remembered that one episode at least occurred during work. Among 175 operators (98%), 4 had a regular reaction, with appearance of a generalized urticaria and uneasiness. In the remaining 4 agents (2%) there was a local extensive reaction, which was not associated with systemic reactions and they were all referred to allergological examination. 19 agents (10,6%) suffered more than 5 stings altogether, but none developed a systemic reaction. 87% of the subjects practised self-medication, 7% reported to the casualty department of the local hospital or to their own doctor, and 6% undertook no cure at all., Conclusions: Epidemiological studies agree in recognizing that, in the general population, the percentage of systemic reactions after one or more hymenoptera stings varies from 0,15% to 3,3%. In categories of workers occupationally at risk, the prevalence of systemic reactions varies from 4,5% to 26%. The prevalence of systemic reactions in Forestry Department agents was 2%, which is similar to the prevalence in the general population. Therefore, rather than occupational risk, there appeared to be a generic risk made more serious by working conditions for Forestry Department agents due to their possible exposure to hymenoptera stings. The occupational health physician needs to monitor these events, due to the fact that frequent exposure to stings, above all occurring within a short period of time (less than two months) favours an increase in the tendency to develop systemic reactions, with a more serious prognosis, especially when working in isolated conditions.

The efficacy and safety of sublingual immunotherapy (SLIT) are currently supported by clinical trials, meta-analysis and post-marketing surveys. Practice parameters for clinical use of SLIT are proposed here by a panel of Italian specialists, with reference to evidence based criteria. Indications to SLIT include allergic rhinoconjunctivitis, asthma, and isolated conjunctivitis (strength of recommendation: grade A). As to severity of the disease, SLIT is indicated in moderate/severe intermittent rhinitis, persistent rhinitis and mild to moderate asthma (grade D). SLIT may be safely prescribed also in children aged three to five years (grade B), and its use in subjects aged more than 60 years is not prevented when the indications and contraindication are ascertained (grade D). The choice of the allergen to be employed for SLIT should be made in accordance with the combination of clinical history and results of skin prick tests (grade D). Polysensitisation, i.e. the occurrence of multiple positive response does not exclude SLIT, which may be done with the clinically most important allergens (grade D). As to practical administration, co-seasonal, pre co-seasonal, and continuous schedules are available, being the latter recommended for perennial allergens or for pollens with particularly prolonged pollination, such as Parietaria (grade D). For pollens with relatively short pollination, such as grasses and trees (cypress, birch, alder, hazelnut, olive) the pre co-seasonal and perennial schedules are preferred (grade C). The build-up phases suggested by manufacturers can be safely used (grade A), but they can be modified according to the patient's tolerance (grade C). A duration of SLIT of 3-5 years is recommended to ensure a long-lasting clinical effect after the treatment has been terminated (grade C).

The content and relative variations of individual carotenoids during the leaflet development stages (I, II, III, A and P) of six species of Ceratozamia (Cycads) were investigated. There is an unusual, transitory and marked presence of six red stroma keto-carotenoids in the first development stages, while the thylakoidal carotenoids showed a low concentration during the same period. As no official A1cm1% coefficients were available, it was necessary to calculate these for the following stroma carotenoids: semi-beta-carotenone (major component), triphasiaxanthin, ceratoxanthin, ceratozamiaxanthin, kuesteriaxanthin and ceratoxanthone. The stroma keto-carotenoids, which reached the highest content in the first development stage (average of 78% of total carotenoids), were always present in the five species: C. fuscoviridis, C. robusta, C. spinosa, C. kuesteriana and C. hildae, but never in C. mexicana. From stage II, the stroma keto-carotenoids decreased and finally disappeared in the green adult leaflets. The thylakoidal carotenoids showed a minimum at stage III, and then increased to a maximum in the perennial leaflets. Among these, beta-carotene showed an anomalous and characteristic behaviour, being a minor component during leaflet development (from stage I to A). In stage P it was markedly exceeded not only by lutein but also by alpha-carotene, neoxanthin and violaxanthin, and in C. robusta also by lutein-5,6-epoxide. Additionally, the alpha/beta ratio in these species is unusual: it increased from 0.3-0.5 to 1.5-3.0 during leaflet development. Moreover, antheraxanthin amounts are very small, while zeaxanthin was present only in the evergreen leaflets of C. mexicana in small quantities. Lutein-5,6-epoxide represented more than 5% of thylakoidal carotenoids in the leaflets of all the species. A revision of the taxonomic rank of C. fuscoviridis is recommended.

Objective: Clusterin is a heterodimeric glycoprotein which is implicated in several biological processes. The nuclear (n-CLU) and cytoplasmic secreted (s-CLU) isoforms have recently been described, but their role is still unclear. The aim of this study is to investigate the expression of clusterin and its isoforms during proliferative arrest and apoptosis of vascular smooth muscle cells (SMCs)., Methods and Results: Clusterin expression was evaluated by immunohistochemistry and Western blotting in human arteries and rat aortas. In human diffuse myointimal thickening, clusterin was detected in cell cytoplasm and extracellular space, whereas it was practically absent in the media. In rat aortas 15 days after ballooning, intimal cells (IT cells) overexpressed s-CLU and n-CLU, the latter mainly in the inner neointima; clusterin expression decreased at 60 days. In vitro, IT cells maintained high clusterin expression and its antisense markedly reduced proliferation and increased apoptosis. Western blotting showed that all-trans retinoic acid-induced proliferative arrest and increased alpha-smooth muscle actin expression did associate to s-CLU and B-myb reduction, whereas bax-related apoptosis was associated to a shift from the s-CLU to n-CLU isoform., Conclusions: Clusterin overexpression characterized neointimal SMCs; s-CLU expression decreased in IT cells during all-trans retinoic acid-induced proliferative arrest and redifferentiation, whereas n-CLU overexpression was characteristic of apoptosis. Clusterin was detected in human arterial myointimal thickening and absent in the underlying media. Rat neointimal cells overexpressed clusterin and clusterin antisense oligonucleotide reduced proliferation and increased apoptosis. All-trans retinoic acid-induced proliferative arrest showed association with s-CLU reduction and n-CLU overexpression with apoptosis, supporting a different biological role of these isoforms.

Somatostatin is a peptide hormone that exerts antisecretory and antiproliferative activities on some human tumors. The Ku70/86 heterodimer acts as regulatory subunit of the DNA dependent protein kinase and its DNA binding activity mediates DNA double strands breaks repair that is crucial to maintain the genetic integrity of the genome. The activation of the heterodimer regulates cell cycle progression and the activity of nuclear transcription factors involved in DNA replication and cell proliferation. Moreover Ku86 behaves as a receptor for the growth inhibitory tetradecapeptide, somatostatin. Herein we report that somatostatin treatment to a colon carcinoma cell line (Caco-2) inhibits cell growth and, at same time, strongly modulates the activation of Ku70/86 heterodimer and the levels of Ku86 in the nucleus by increasing its specific mRNA level. Our findings are consistent with the hypothesis that somatostatin controls cell cycle progression and DNA repair through a new signalling pathway that involves the regulation of Ku86 level and modulates the Ku70/86 activity in the nucleus.

Clusterin is a ubiquitous secretory heterodimeric disulfide-linked glycoprotein, which is implicated in several physiological processes, including immune regulation, cell adhesion and morphological transformation, lipid transportation, tissue remodelling, membrane recycling and cell-cell interactions. A large number of studies have focused their interest on clusterin gene products as mediators of cell cycle progression and cell death induction, although data on the different isoforms and their role in the different cell processes are still obscure. Recently, an increased clusterin expression in breast cancer has been reported. In order to elucidate the role of clusterin in tumor progression and whether one of its isoforms is preferentially expressed in tumorigenesis, we examined its presence throughout the different steps of human colon carcinoma, one of the best-characterized models of human tumor progression. The immunohistochemical observation of 30 bioptic and surgical colon specimens demonstrated a cell compartment clusterin translocation from the nucleus to the cytoplasm directly related to tumor progression. In fact, a nuclear localization found in healthy colonic mucosa is consistent with the involvement of the proapoptotic nuclear form in the regulation of cell cycle progression and in cell death induction. The progression towards high-grade and metastatic carcinoma leads to cytoplasmic clusterin distribution. Protein extracts from freshly isolated cells of the same patients confirm in high-grade carcinomas with metastatic nodes the complete loss of the proapoptotic nuclear form and a cytoplasmic overexpression of the highly glycosylated form. Data obtained from in vitro experiments confirm that this form is released in the extracellular space and corresponded to the fully glycosylated one. These data suggest that the controversial data on clusterin function in tumors may be related to the pattern shift of its isoform production. As the secreted form of clusterin is correlated to cell matrix formation, cell membrane remodeling and cell-cell adhesion, the overexpression of this form in highly aggressive tumors and metastatic nodes could be a potential new prognostic and predictive marker for colon carcinoma aggressiveness.

The Ubiquitination Factor E4A gene (UBE4A) encodes for a U-box-type ubiquitin ligase, originally described as an E4 ubiquitination factor. UBE4A is a mammalian homolog of Saccharomyces cerevisiae Ufd2. The UBE4A gene has been mapped on the human chromosome region 11q23.3, a critical region involved in some specific cancers such as neuroblastoma. Northern blots analysis on foetal and adult human tissues revealed a single band of approximately 7.5 kb transcript most abundant in the heart, skeletal muscle and kidney. We generated a polyclonal antibody to UBE4A and performed immunoblot and immunohistochemical analysis. The UBE4A protein appeared as a single band of approximately 125 kDa. UBE4A was present in the skeletal muscle, kidney and liver; a faint band was visible in peripheral blood leukocytes and spleen. We did not reveal expression of UBE4A in whole brain, colon, lung and heart. At the cellular level, UBE4A results predominantly expressed in the nucleus and the cytoplasm of cortical neurons and liver and in the nucleus of tubular kidney cells. In the liver, the nucleus of similar cells appeared to be unstained or stained at different levels suggesting that UBE4A may have a cell cycle dependent expression or a role of in cell cycle control. In conclusion, our results show that UBE4A is expressed in different tissues in a pattern that seems to be dependent from cell type and cell cycle and that UBE4A might have a specific role in different biochemical processes other than ubiquitination, including growth or differentiation.

Psoriasis is a chronic inflammatory skin disorder showing multifactorial inheritance. Linkage studies have mapped disease susceptibility loci to several genomic regions, including the chromosome 16 interval that contains the CARD15/NOD2 gene. CARD15 has been involved in Crohn's Disease (CD) susceptibility and it has been hypothesised that it may also contribute to the pathogenesis of psoriasis. To test this hypothesis we studied the distribution of 3 CARD15 SNPs in an Italian case-control data set. We failed to observe any significant difference between patients and controls, thereby excluding the presence of a strong genetic association between CARD15 gene polymorphisms and psoriasis, in the Italian population.

Background: Multinodular goitre (MNG) is a common disorder characterised by an enlargement of the thyroid, occurring as a compensatory response to hormonogenesis impairment. The incidence of MNG is dependent on sex (female:male ratio 5:1) and several reports have documented a genetic basis for the disease. Last year we mapped a MNG locus to chromosome Xp22 in a region containing the peroxiredoxin IV (Prx-IV) gene. Since Prx-IV is involved in the removal of H2O2 in thyroid cells, we hypothesize that mutations in Prx-IV gene are involved in pathogenesis of MNG., Methods: Four individuals (2 affected, 2 unrelated unaffected) were sequenced using automated methods. All individuals were originated from the original three-generation Italian family described in previous studies. A Southern blot analysis using a Prx-IV full-length cDNA as a probe was performed in order to exclude genomic rearrangements and/or intronic mutations. In addition a RT-PCR of PRX-IV was performed in order to investigate expression alterations., Results: No causative mutations were found. Two adjacent nucleotide substitutions were detected within introns 1 and 4. These changes were also detected in unaffected individuals, suggesting that they were innocuous polymorphisms. No gross genomic rearrangements and/or restriction fragment alterations were observed on Southern analysis. Finally, using RT-PCR from tissue-specific RNA, no differences of PRX-IV expression-levels were detected between affected and unaffected samples., Conclusions: Based on sequence and genomic analysis, Prx-IV is very unlikely to be the MNG2 gene.

The Ku70/80 heterodimer is the regulatory subunit of the DNA-dependent protein kinase (DNA-PK) and its DNA-binding activity mediates DNA double-strand breaks repair. Although Ku80 was recently proposed as a caretaker gene involved in the control of genome integrity, no data are available on Ku70/80 DNA-binding activity in human tumors. Heterodimer DNA-binding activity and protein expression were assayed by electrophoretic-mobility-shift-assay (EMSA) and Western blot analysis, in nuclear and cytoplasmic extracts from eight breast, seven bladder primary tumors and three metastatic nodes from breast cancers. Corresponding normal tissues of the same patients were used as controls. Ten out of 15 tumors showed nuclear Ku-binding activity 3-10 times higher than in the normal tissues, irrespective of bladder or breast origin. Conversely, in 5/15 primary tumors and in all the metastatic nodes analysed, nuclear Ku-activity was 1.5-4.5-fold lower than in the corresponding normal tissues. Cytoplasmic heterodimer activity significantly differed between tumor and normal tissues, displaying a 2-10-fold increase in neoplastic tissues. Three different patterns combining both Ku expression and activity with tumor characteristics were identified. In low aggressive breast tumors p70/p80 proteins were expressed in tumor but not in normal tissues. The heterodimer binding-activity matched the protein levels. In non-invasive bladder carcinomas no significant differences in protein expression between tumor and the corresponding normal tissues were found, however heterodimer binding-activity was increased in tumor samples. In breast and bladder tumors, at the advanced stage and in node metastases, the binding activity was strongly reduced in tumor biopsies, however no differences were demonstrated between normal and tumor protein levels. Our results suggest a different modulation of Ku70/80 DNA-binding activity in human neoplastic tissues, possibly related to tumor progression. Findings provide further data on tissue-specific protein expression and post-translational regulation of heterodimer activity.

A new method for the determination of benzene at trace levels in air is presented. The method consists of the collection of air samples on adsorbent cartridges with simultaneous adsorption of pre-established amounts of D6-labeled internal standard. Desorption from the cartridge is performed by solid-phase microextraction (SPME) with analysis by gas chromatography/mass spectrometry (GC/MS) using an ion trap mass spectrometer. The influence of several parameters (type of SPME fiber, temperature, time, for example) was investigated, and good linearity in the range 10-400 ng of C6D6, with a coefficient of variance (CV) around 3-5%, was obtained. The method was tested by sampling air in a town center in Italy, and a benzene concentration of approximately 50 microg/m(3) was determined. The maximum limit recommended by the European Community is 10 microg/m(3)., (Copyright 2001 John Wiley & Sons, Ltd.)

The monitoring of air pollution requires simple, rapid and sensitive sampling and analytical techniques with minimal sample manipulation that are usable for routine analyses. In our laboratories we have developed a method for the analysis of air samples collected by adsorbent cartridges based on solid phase microextraction (SPME) coupled to gas chromatography/mass spectrometry. We investigated the influence of some variables (time and temperature) using toluene as analyte (one of the most common air pollutants) and toluene-d(8) on the SPME extraction, and the sensitivity (LOQ) of the method. We then tested the method on an air sample collected in an industrial area and carried out characterisation of the volatile organic compounds present., (Copyright 1999 John Wiley & Sons, Ltd.)

The Fenton reaction for the degradation of surfactants has been investigated and partial degradation products have been identified and characterized by mass spectrometry for the case of lauryl sulphate. The polar soluble products were investigated by liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization (ESI), and the volatile products leaving the mixture during the reaction were trapped by means of solid-phase microextraction (SPME) and investigated by gas chromatography/mass spectrometry (GC/MS) with electron and chemical ionization. The oxidation leads to the formation of products with hydroxyl and epoxide groups due to insertion of oxygen atoms or to aldehydes derived from the loss of the hydrophilic sulphate group. The extent of mineralization is dependent on peroxide and iron concentrations. Copyright 1999 John Wiley & Sons, Ltd.

CD4+ cells from young (3 months) and old (19 months) mice were stimulated by plate-bound anti-CD3 monoclonal antibody (mAb) alone or also by soluble anti-CD28 mAb. Supernatants were analysed by enzyme-linked immunosorbent assay (ELISA) to determine cytokine concentrations. Total RNA was extracted from cells, reverse transcribed and the cDNA amplified by polymerase chain reaction (PCR) to evaluate the amount of specific mRNA. The results indicate that anti-CD3 alone is not sufficient to induce interleukin-2 (IL-2) production in CD4+ cells from both young and old mice. However, anti-CD28, together with anti-CD3 mAb, induces a much higher production of IL-2 in CD4+ cells from young as compared with old mice. Conversely, interferon-gamma (IFN-gamma) production is also induced by anti-CD3 alone and is higher in CD4+ cells from old as compared with young mice. Upon addition of anti-CD28 mAb, IFN-gamma production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL-4 and IL-10 is induced by anti-CD3 mAb but it is increased by the addition of anti-CD28 mAb. CD4+ cells from old mice produce more IL-4 and IL-10 as compared with cells from young mice. The amounts of cytokine specific mRNA in CD4+ cells from young and old mice parallel the cytokine levels in culture supernatants. Results on the mRNA turnover indicate that when CD4+ cells are stimulated by anti-CD3 or costimulated also by anti-CD28 mAb, the IFN-gamma, IL-4 and IL-10 specific mRNAs are more stable in old than in young mice, suggesting that mRNA stability has a relevant role in the different patterns of cytokine production.