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Unusual and discrepant ABO phenotypes are often due to genetic variants that lead to altered levels or activity of ABO transferases and consequently to altered expression of ABO antigens. This report describes eight genetic alterations found in 15 cases with reduced or undetectable expression of ABO antigens. Forward and reverse ABO grouping was performed by standard gel or tube methods. Adsorption-heat elution and saliva testing for H and A substances followed the AABB technical manual procedures. Genomic DNA extracted from whole blood was PCR-amplified to cover the entire ABO coding sequence, splice junctions, proximal promoter, and intron 1 enhancer. Amplification products were sequenced by next-generation or Sanger dideoxy methods, either directly or after cloning into a bacterial plasmid vector. Eight unreported alleles were found in the 15 cases analyzed. Alleles ABO*A(28+1C) and ABO*A(29-5G) harbor variants that alter the consensus sequence at the intron 1 donor and acceptor splice sites, respectively. The other alleles harbor variants that alter the consensus sequence at transcription factor-binding sites in the intron 1 enhancer: specifically, ABO*A(28+5792T), ABO*A(28+5859A) , and ABO*A(28+5860G) at GATA-1 sites; ABO*B(28+5877T) and ABO*B(28+5878G) at a RUNX1 site; and ABO*A(28+5843A) at or near a C/EBP site. Molecular and serologic characterization of ABO alleles can help in their future identification and in the resolution of discrepancies., Unusual and discrepant ABO phenotypes are often due to genetic variants that lead to altered levels or activity of ABO transferases and consequently to altered expression of ABO antigens. This report describes eight genetic alterations found in 15 cases with reduced or undetectable expression of ABO antigens. Forward and reverse ABO grouping was performed by standard gel or tube methods. Adsorption-heat elution and saliva testing for H and A substances followed the AABB technical manual procedures. Genomic DNA extracted from whole blood was PCR-amplified to cover the entire ABO coding sequence, splice junctions, proximal promoter, and intron 1 enhancer. Amplification products were sequenced by next-generation or Sanger dideoxy methods, either directly or after cloning into a bacterial plasmid vector. Eight unreported alleles were found in the 15 cases analyzed. Alleles ABO*A(28+1C) and ABO*A(29–5G) harbor variants that alter the consensus sequence at the intron 1 donor and acceptor splice sites, respectively. The other alleles harbor variants that alter the consensus sequence at transcription factor–binding sites in the intron 1 enhancer: specifically, ABO*A(28+5792T), ABO*A(28+5859A) , and ABO*A(28+5860G) at GATA-1 sites; ABO*B(28+5877T) and ABO*B(28+5878G) at a RUNX1 site; and ABO*A(28+5843A) at or near a C/EBP site. Molecular and serologic characterization of ABO alleles can help in their future identification and in the resolution of discrepancies.

Background: Alterations involving the RET kinase are implicated in the pathogenesis of lung, thyroid and other cancers. However, the clinical activity of multikinase inhibitors (MKIs) with anti-RET activity in RET-altered patients appears limited, calling into question the therapeutic potential of targeting RET. LOXO-292 is a selective RET inhibitor designed to inhibit diverse RET fusions, activating mutations and acquired resistance mutations., Patients and Methods: Potent anti-RET activity, high selectivity, and central nervous system coverage were confirmed preclinically using a variety of in vitro and in vivo RET-dependent tumor models. Due to clinical urgency, two patients with RET-altered, MKI-resistant cancers were treated with LOXO-292, utilizing rapid dose-titration guided by real-time pharmacokinetic assessments to achieve meaningful clinical exposures safely and rapidly., Results: LOXO-292 demonstrated potent and selective anti-RET activity preclinically against human cancer cell lines harboring endogenous RET gene alterations; cells engineered to express a KIF5B-RET fusion protein -/+ the RET V804M gatekeeper resistance mutation or the common RET activating mutation M918T; and RET-altered human cancer cell line and patient-derived xenografts, including a patient-derived RET fusion-positive xenograft injected orthotopically into the brain. A patient with RET M918T-mutant medullary thyroid cancer metastatic to the liver and an acquired RET V804M gatekeeper resistance mutation, previously treated with six MKI regimens, experienced rapid reductions in tumor calcitonin, CEA and cell-free DNA, resolution of painful hepatomegaly and tumor-related diarrhea and a confirmed tumor response. A second patient with KIF5B-RET fusion-positive lung cancer, acquired resistance to alectinib and symptomatic brain metastases experienced a dramatic response in the brain, and her symptoms resolved., Conclusions: These results provide proof-of-concept of the clinical actionability of RET alterations, and identify selective RET inhibition by LOXO-292 as a promising treatment in heavily pretreated, multikinase inhibitor-experienced patients with diverse RET-altered tumors.

In the present study, the phenotype of melanoma cells resistant to dabrafenib (a B-RAF inhibitor) was investigated, to shed more light on melanoma resistance to B-RAF inhibition. Melanoma cells resistant to dabrafenib were generated using 3 different cell lines, A375, 397 and 624.38, all carrying B-RAFV600E, and they were characterized by cytofluorometric analysis, Ion Torrent technology, immunofluorescence and biochemistry. All dabrafenib-resistant cells showed, in addition to a re-activation of MAPK signaling, morphological changes compared to their sensitive counterparts, accompanied by an increase in CD90 (mesenchymal marker) expression and a decrease in E-cadherin (epithelial marker) expression, suggesting an epithelial-to-mesenchymal-like phenotypic transition. However, melanoma cells with TGF-β1-induced epithelial-to-mesenchymal transition (EMT) were more sensitive to dabrafenib treatment compared to the sensitivity noted in the non-TGF‑β1‑induced EMT melanoma cells, suggesting that TGF-β1-induced EMT was not associated with dabrafenib resistance. Although dabrafenib-resistant cells exhibited increased cell motility and E-cadherin/vimentin reorganization, as expected in EMT, all of them showed unvaried E-cadherin mRNA and unchanged Snail protein levels, while Twist1 protein expression was decreased with the exception of A375 dabrafenib-resistant melanoma cells, where it was unaffected. These findings suggest a distinct active EMT-like process adopted by melanoma cells under drug exposure. Furthermore, dabrafenib-resistant cells exhibited stem cell-like features, with Oct4 translocation from the cytoplasm to peri-nuclear sites and nuclei, and increased CD20 expression. In conclusion, our data, in addition to confirming that resistance to dabrafenib is dependent on re-activation of MAPK signaling, suggest that this resistance is linked to a distinct active EMT-like process as well as stem-cell features adopted by melanoma cells.

Epidemiologic data for carcinogenicity in those exposed to lead (Pb) suggests relations with cancers although the totality of the evidence is inconsistent. Alterations in the expression of ERBB receptors have been studied during the development and malignant transformation of different kinds of human tumors where they induce proliferation, angiogenesis and metastasis generation. Relevant clinical data demonstrate the role of ERBB2 receptors in the development and malignancy of human cancers. Therefore, the objective of the present investigation is to give more information on the link between plasma mRNA expression in ERBB2 gene and lead blood levels in a healthy population. Blood samples, socio‑demographic, exposure and health data were obtained from 48 healthy men. Real‑time polymerase chain reaction assays were performed to detect ERBB2 gene transcripts, ΔΔCt method was used to quantify gene expression. Pb blood level was assayed using high‑resolution sector field inductively coupled mass spectrometry and is expressed in µg/dl. Plasma mRNA expression in ERBB2 gene was 6.44±3.07 ΔΔCt; Pb blood levels was 16.07±6.74 µg/dl. Regression analysis revealed a significant association (r2=0.5345; p<0.0001) between Pb levels and mRNA expression in ERBB2. So far, it has still not been established if the expression of ERBB2 receptors is influenced by Pb exposure. On the base of the above reported data, we believe an in vitro study might be useful, to understand the molecular mechanisms implicated.

Tumor spreading is associated with the degradation of extracellular matrix proteins, mediated by the overexpression of matrix metalloproteinase 9 (MMP-9). Although, such overexpression was linked to epigenetic promoter methylation, the role of intragenic methylation was not clarified yet. Melanoma was used as tumor model to investigate the relationship between the DNA intragenic methylation ofMMP9 gene and MMP-9 overexpression at transcriptional and protein levels. Computational analysis revealed DNA hypermethylation within the intragenic CpG-2 region of MMP9 gene in melanoma samples with high MMP-9 transcript levels. In vitro validation showed that CpG-2 hotspot region was hypermethylated in the A375 melanoma cell line with highest mRNA and protein levels of MMP-9, while low methylation levels were observed in the MEWO cell line where MMP-9 was undetectable. Concordant results were demonstrated in both A2058 and M14 cell lines. This correlation may give further insights on the role of MMP-9 upregulation in melanoma.

We performed a comparative study between two human metastatic melanoma cell lines (A375 and 526), and melanocytes (FOM78) by gene expression profiling and pathway analysis, using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) software. Genes involved in Ran signaling were significantly over-represented (p ≤ 0.001) and up-regulated in melanoma cells. A melanoma-associated molecular pathway was identified, where Ran, Aurora Kinase A (AurkA) and TERT were up-regulated, while c-myc and PTEN were down-regulated. A consistent high Ran and AurkA gene expression was detected in about 48% and 53%, respectively, of 113 tissue samples from metastatic melanoma patients. AurkA down-regulation was observed in melanoma cells, by Ran knockdown, suggesting AurkA protein is a Ran downstream target. Furthermore, AurkA inhibition, by exposure of melanoma cells to MLN8054, a specific AurKA inhibitor, induced apoptosis in both melanoma cell lines and molecular alterations in the IPA-identified molecular pathway. These alterations differed between cell lines, with an up-regulation of c-myc protein level observed in 526 cells and a slight reduction seen in A375 cells. Moreover, Ran silencing did not affect the A375 invasive capability, while it was enhanced in 526 cells, suggesting that Ran knockdown, by AurkA down-regulation, resulted in a Ran-independent enhanced melanoma cell invasion. Finally, AurK A inhibition induced a PTEN up-regulation and its action was independent of B-RAF mutational status. These findings provide insights relevant for the development of novel therapeutic strategies as well as for a better understanding of mechanisms underlying therapy resistance in melanoma., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Understanding molecular mechanisms involved in melanoma resistance to drugs is a big challenge. Experimental evidences suggested a correlation between mutational status in B-RAF and melanoma cell susceptibility to drugs, such as paclitaxel, doxorubicin and temozolomide, which generate an accumulation of hydrogen peroxide (H2O2) in the cells. We investigated the survival phenotype and the protein level of c-myc, a B-RAF target molecule, in melanoma cells, carrying a different mutational status in B-RAF, upon paclitaxel, doxorubicin and H2O2 treatment. For the first time, we reported c-myc modulation is critical for melanoma drug response. It appeared drug-specific and post-transcriptionally driven through PP2A; in correlation, cell pre-treatment with okadaic acid (OA), a specific PP2A inhibitor, as well as PP2A silencing of melanoma cells, was able to increase melanoma cell drug-sensitivity and c-myc protein level. This is relevant for designing efficacious therapeutic strategies in melanoma.

Importance: Exposure to trauma increases the risk for developing threat (ie, fear) symptoms, such as reexperiencing and hyperarousal symptoms, and loss (ie, dysphoria) symptoms, such as emotional numbing and depressive symptoms. While preclinical data have implicated the activated dynorphin/κ-opioid receptor (KOR) system in relation to these symptoms, the role of the KOR system in mediating these phenotypes in humans is unknown. Elucidation of molecular targets implicated in threat and loss symptoms is important because it can help inform the development of novel, mechanism-based treatments for trauma-related psychopathology., Objective: To use the newly developed [11C]LY2795050 radiotracer and high-resolution positron emission tomography to evaluate the relation between in vivo KOR availability in an amygdala-anterior cingulate cortex-ventral striatal neural circuit and the severity of threat and loss symptoms. We additionally evaluated the role of 24-hour urinary cortisol levels in mediating this association., Design, Setting, and Participants: This cross-sectional positron emission tomography study under resting conditions was conducted at an academic medical center. Thirty-five individuals representing a broad transdiagnostic and dimensional spectrum of trauma-related psychopathology, ranging from nontrauma-exposed psychiatrically healthy adults to trauma-exposed adults with severe trauma-related psychopathology (ie, posttraumatic stress disorder, major depressive disorder, and/or generalized anxiety disorder)., Main Outcomes and Measures: [11C]LY2795050 volume of distribution values in amygdala-anterior cingulate cortex-ventral striatal neural circuit; composite measures of threat (ie, reexperiencing, avoidance, and hyperarousal symptoms) and loss (ie, emotional numbing, major depressive disorder, and generalized anxiety disorder symptoms) symptoms as assessed using the Clinician-Administered PTSD Scale, Hamilton Depression Rating Scale, and Hamilton Rating Scale for Anxiety; and 24-hour urinary cortisol levels., Results: [11C]LY2795050 volume of distribution values in an amygdala-anterior cingulate cortex-ventral striatal neural circuit were negatively associated with severity of loss (r = -0.39; 95% CI, -0.08 to -0.66), but not threat (r = -0.03; 95% CI, -0.30 to 0.27), symptoms; this association was most pronounced for dysphoria symptoms (r = -0.45; 95% CI, -0.10 to -0.70). Path analysis revealed that lower [11C]LY2795050 volume of distribution values in this circuit was directly associated with greater severity of loss symptoms and indirectly mediated by 24-hour urinary cortisol levels., Conclusions and Relevance: Results of this study suggest that KOR availability in an amygdala-anterior cingulate cortex-ventral striatal neural circuit mediates the phenotypic expression of trauma-related loss (ie, dysphoria) symptoms. They further suggest that an activated corticotropin-releasing factor/hypothalamic-pituitary-adrenal axis system, as assessed by 24-hour urinary cortisol levels, may indirectly mediate this association. These results may help inform the development of more targeted, mechanism-based transdiagnostic treatments for loss (ie, dysphoric) symptoms.

Attentional bias to threat is a key endophenotype that contributes to the chronicity of trauma-related psychopathology. However, little is known about the neurobiology of this endophenotype and no known in vivo molecular imaging study has been conducted to evaluate candidate receptor systems that may be implicated in this endophenotype or the phenotypic expression of trauma-related psychopathology that comprises threat (ie, re-experiencing, avoidance, and hyperarousal) and loss (ie, emotional numbing, depression/dysphoria, generalized anxiety) symptomatology. Using the radioligand [(11)C]OMAR and positron emission tomography (PET), we evaluated the relationship between in vivo cannabinoid receptor type 1 (CB1) receptor availability in the amygdala, and performance on a dot-probe measure of attentional bias to threat, and clinician interview-based measures of trauma-related psychopathology. The sample comprised adults presenting with a broad spectrum of trauma-related psychopathology, ranging from nontrauma-exposed, psychiatrically healthy adults to trauma-exposed adults with severe trauma-related psychopathology. Results revealed that increased CB1 receptor availability in the amygdala was associated with increased attentional bias to threat, as well as increased severity of threat, but not loss, symptomatology; greater peripheral anandamide levels were associated with decreased attentional bias to threat. A mediation analysis further suggested that attentional bias to threat mediated the relationship between CB1 receptor availability in the amygdala and severity of threat symptomatology. These data substantiate a key role for compromised endocannabinoid function in mediating both the endophenotypic and phenotypic expression of threat symptomatology in humans. They further suggest that novel pharmacotherapies that target the CB1 system may provide a more focused, mechanism-based approach to mitigating this core aspect of trauma-related psychopathology.

Cutaneous melanoma is an aggressive cancer with a poor prognosis for patients with advanced disease. The identification of several key molecular pathways implicated in the pathogenesis of melanoma has led to the development of novel therapies for this devastating disease. In melanoma, both the Ras/Raf/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways are constitutively activated through multiple mechanisms. Targeting various effectors of these pathways with pharmacologic inhibitors may inhibit melanoma cell growth and angiogenesis. Ongoing clinical trials provide hope to improve progression-free survival of patients with advanced melanoma. This review summarizes the most relevant studies focused on the specific action of these new molecular targeted agents. Mechanisms of resistance to therapy are also discussed.

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited syndrome, caused by germline mutations in the adenomatous polyposis coli (APC) suppressor gene. Patients with colorectal polyps are more likely to develop a malignant condition with poor prognosis. Typical FAP is characterized by hundreds to thousands of colorectal adenomatous polyps and by several extra-colonic manifestations; an attenuated form of polyposis (AFAP), presenting less than 100 adenomas and later onset, has been reported. In this study we have examined five Sicilian families affected by FAP syndrome, in order to provide predictive genetic testing for the affected families, as well as to contribute to mutation catalog enrichment. We have detected different APC mutations in these five pedigrees, confirming the remarkable heterogeneity of the mutational spectrum in FAP., (Copyright © 2014 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.)

Introduction: Recent confirmatory factor analytic studies of the dimensional structure of posttraumatic stress disorder (PTSD) suggest that this disorder may be best characterized by five symptom dimensions-re-experiencing, avoidance, numbing, dysphoric arousal, and anxious arousal. Hypothalamic-pituitary-adrenal (HPA) axis dysregulation in PTSD and has been attributed to enhanced glucocorticoid responsiveness. However, little is known about how altered HPA-axis function is related to this contemporary phenotypic model of PTSD., Methods: We compared morning plasma cortisol levels of drug-free civilian adults with PTSD (N = 29) to trauma-exposed (TC; N = 12) and non-trauma-exposed healthy controls (HC; N = 23). We then examined the relation between cortisol levels and a contemporary 5-factor 'dysphoric arousal' model of PTSD symptoms among individuals with PTSD., Results: After adjustment for white race/ethnicity, education, lifetime alcohol use disorder, and current smoking status, the PTSD (Cohen's d = 1.1) and TC (Cohen's d = 1.3) groups had significantly lower cortisol levels than the HC group; cortisol levels did not differ between the TC and PTSD groups. Except for age (r = -.46), none of the other demographic, trauma-related, or clinical variables, including lifetime mood/anxiety disorder and severity of current depressive and anxiety symptoms, were associated with cortisol levels. In a stepwise linear regression analysis, age (β = -.44) and severity of emotional numbing symptoms (β = -.35) were independently associated with cortisol levels in the PTSD group; none of the other PTSD symptom clusters or depression symptoms were significant. Post hoc analyses revealed that severity of the emotional numbing symptom of restricted range of affect (i.e., unable to have loving feelings) was independently related to cortisol levels (β = -.35)., Conclusion: These results suggest that trauma-exposed civilian adults with and without PTSD have significantly lower cortisol levels compared to healthy, non-trauma-exposed adults. They further suggest that low cortisol levels among adults with PTSD may be specifically linked to emotional numbing symptomatology that is unique to the PTSD phenotype and unrelated to depressive symptoms., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

HNPCC is an autosomal inherited cancer syndrome characterized by germinal and somatic mutations of DNA mismatch repair (MMR) genes. The inherited mutation in one allele together with an acquired defect in the other allele of an MMR gene leads to accelerate tumor progression. In this study we analyzed a cohort of 11 subjects belonging to four Sicilian families with HNPCC suspected by molecular analysis of coding regions of hMSH2 (NC&#95;000002) and hMLH1 (NC&#95;000003) genes. Molecular analysis has detected the presence of two mutations in gene MSH2 and one mutation in MHL1 gene. In addition, we found a novel mutation consisting in a G deletion at 914 codon of the exon 16 in the MSH2 gene. This deletion leads to a stop codon due to a frame-shift, resulting in a truncated protein. We extended genetic analysis to the other family members and the same mutation was detected in three sisters and in one of the two healthy daughters. This mutation is correlated with clinical findings revealed in genealogic tree and it represents a novel mutation responsible of HNPCC., (Copyright © 2014 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.)

Posttraumatic Stress Disorder (PTSD) is a prevalent, chronic, and disabling anxiety disorder that may develop following exposure to a traumatic event. The majority of individuals with PTSD often have comorbid psychiatric conditions such as major depression, generalized anxiety disorder, and substance use disorders, and are at increased risk for suicide. Despite the public health significance of PTSD, relatively little is known about the etiology or pathophysiology of this disorder, and pharmacotherapy development to date has been largely opportunistic instead of mechanism-based. One promising target for modulation is Tropomyosin Receptor Kinase B (TrkB), the receptor for Brain-Derived Neurotrophic Factor (BDNF), a signaling pathway important for neuronal plasticity, survival, and growth. The following discusses how genetic and environmental alterations to this signaling pathway may contribute to anatomical and functional changes in the hippocampus, amygdala, anterior cingulate cortex, ventromedial prefrontal cortex, and the nucleus accumbens. Changes in these brain regions may in turn contribute to the predisposition to or maintenance of some of the clinical manifestations of PTSD, including intrusive memories, hyperarousal, increased fear, and emotional numbing.

Endocannabinoids and their attending cannabinoid type 1 (CB1) receptor have been implicated in animal models of post-traumatic stress disorder (PTSD). However, their specific role has not been studied in people with PTSD. Herein, we present an in vivo imaging study using positron emission tomography (PET) and the CB1-selective radioligand [(11)C]OMAR in individuals with PTSD, and healthy controls with lifetime histories of trauma (trauma-exposed controls (TC)) and those without such histories (healthy controls (HC)). Untreated individuals with PTSD (N=25) with non-combat trauma histories, and TC (N=12) and HC (N=23) participated in a magnetic resonance imaging scan and a resting PET scan with the CB1 receptor antagonist radiotracer [(11)C]OMAR, which measures the volume of distribution (VT) linearly related to CB1 receptor availability. Peripheral levels of anandamide, 2-arachidonoylglycerol, oleoylethanolamide, palmitoylethanolamide and cortisol were also assessed. In the PTSD group, relative to the HC and TC groups, we found elevated brain-wide [(11)C]OMAR VT values (F(2,53)=7.96, P=0.001; 19.5% and 14.5% higher, respectively), which were most pronounced in women (F(1,53)=5.52, P=0.023). Anandamide concentrations were reduced in the PTSD relative to the TC (53.1% lower) and HC (58.2% lower) groups. Cortisol levels were lower in the PTSD and TC groups relative to the HC group. Three biomarkers examined collectively--OMAR VT, anandamide and cortisol--correctly classified nearly 85% of PTSD cases. These results suggest that abnormal CB1 receptor-mediated anandamide signaling is implicated in the etiology of PTSD, and provide a promising neurobiological model to develop novel, evidence-based pharmacotherapies for this disorder.

Heparin-binding EGF-like growth factor (HB-EGF), a member of the family of epidermal growth factors (EGFs), is involved in several biological processes and tumor formation. Several lines of evidence show that HB-EGF plays a key role in the acquisition of malignant phenotype. Studies show that HB-EGF expression is essential in oncogenesis of cancer-derived cell lines. HB-EGF is a promising target for cancer therapy. The aim of this study was to find new insights on the biological features of the soft tissue sarcomas, in order to consider the possibility to use HB-EGF as an immuno-target in histotype characterization and to facilitate therapeutic intervention. In our study we did HB-EGF-immunostaining on tissue samples collected from 43 human soft tissue sarcomas. We analyzed HB-EGF immunoexpression in some types of tumors such as clear cell sarcomas, leiomyosarcomas, phyllodes sarcomas, chondrosarcomas and liposarcomas. In relation to the different histotypes, we detected different immunostaining localization. From our results it was evident that pleomorphic cells, a signal of tumor progression, were HB-EGF immunostained, and this was accompanied by an extracellular matrix immunostaining. Moreover statistical analysis showed a correlation between HB-EGF immunostaining and the different types of analyzed soft tissue sarcomas. In conclusion, in some types of soft tissue sarcoma HB-EGF could be considered a useful diagnostic marker for their characterization., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Background: Transfusion therapy remains the main treatment for patients with severe haemoglobinopathies, but can cause adverse reactions which may be classified as immediate or delayed. The use of targeted prevention with drugs and treatments of blood components in selected patients can contribute to reducing the development of some reactions.The aim of our study was to develop an algorithm capable of guiding behaviours to adopt in order to reduce the incidence of immediate transfusion reactions., Materials and Methods: Immediate transfusion reactions occurring over a 7-year period in 81 patients with transfusion-dependent haemoglobinopathies were recorded. The patients received transfusions with red cell concentrates that had been filtered prestorage. Various measures were undertaken to prevent transfusion reactions: leucoreduction, washing the red blood cells, prophylactic administration of an antihistamine (loratidine 10 mg tablet) or an antipyretic (paracetamol 500 mg tablet)., Results: Over the study period 20,668 red cell concentrates were transfused and 64 adverse transfusion reactions were recorded in 36 patients. The mean incidence of reactions in the 7 years of observation was 3.1‰. Over the years the incidence gradually decreased from 6.8‰ in 2004 to 0.9‰ in 2010., Discussion: Preventive measures are not required for patients who have an occasional reaction, because the probability that such a type of reaction recurs is very low. In contrast, the targeted use of drugs such as loratidine or paracetamol, sometimes combined with washing and/or double filtration of red blood cells, can reduce the rate of recurrent (allergic) reactions to about 0.9‰. The system for detecting adverse reactions and training staff involved in transfusion therapy are critical points for reliable collection of data and standardisation of the detection system is recommended for those wanting to monitor the incidence of all adverse reactions, including minor ones.

Aim: Cells involved in atherogenesis produce growth factors crucial for the progression of the atherosclerotic lesions. One of them is the heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, synthesized as a transmembrane precursor (proHB-EGF). This anchored insoluble juxtacrine growth factor can be converted into a soluble molecule with paracrine activity and mature HB-EGF is released in the extracellular matrix from the cell surface. HB-EGF is a potent stimulator of cell proliferation, migration and cell motility and several studies show that HB-EGF is associated with pathologies of hyperplasia of smooth muscle cells including atherosclerosis., Methods: We localized HB-EGF by immunohistochemistry within the atherosclerotic lesions collected from right or left internal carotid artery of 20 patients with evident clinical symptoms., Results: In the 20 samples we tested, the proportion of positive samples was significant. Considering the only positive samples the proportion difference related to the gender of patients was highly significant., Conclusion: The aim of our investigation was to better understand if this growth factor exerts its role through a juxtacrine or paracrine mechanism, or both in the process of atherogenesis. According to the results, the paracrine role of HB-EGF was clear.

We have examined the influence of the nitric oxide (NO)-modified anti-inflammatory drug (S,R)-3-phenyl-4,5-dihydro-5-isoxasole acetic acid (VGX-1027) named GIT-27NO or the NO-modified antiviral drug saquinavir (Saq) named Saq-NO on two colon cancer cell lines, mouse CT26CL25 and human HCT116. The effects of the drugs on cell viability, apoptosis, proliferation, and metastatic potential were analyzed. The release of NO and oxygen and nitrogen species was also determined. The efficacy of the drugs was evaluated in vivo in BALB/c mice injected with CT26CL25 cells. Both agents suppressed the growth of colon cancer cells in vitro and reduced tumor volume in syngeneic BALB/c mice. However, their mechanisms of action were different because GIT-27NO released larger amounts of nitrite than Saq-NO in cell cultures and its antitumor action depended on the intracellular NO release inside the cells. On the contrary, Saq-NO released barely detectable amounts of NO and its antitumor action was NO-independent. In fact, cotreatment with an NO-peroxynitrite scavenger revealed that GIT-27NO but not Saq-NO acts through peroxynitrite-mediated cell destruction. At the cellular level, GIT-27NO prevalently induced proapoptotic signals followed by caspase-dependent apoptosis. In contrast, Saq-NO blocked cell proliferation, changed the adhesive, migratory, and invasive properties of the cells, and decreased metastatic potential in vivo. In conclusion, differences in NO release and oxidative stress generation between GIT-27NO and Saq-NO resulted in different mechanisms that caused cell death.

Breast cancer is the worldwide leading cause of cancer incidence among women. Night shift work exposure has been recently considered one of the significant breast cancer risk factors in industrialized countries. The mechanisms by which this work exposure may be responsible for cancer development is still discussed. In the last 15 years, many authors have paid attention to the relationship between night shift work and breast cancer risk. In the current study, eight case-control studies and four prospective epidemiological studies describing such relationship are discussed. A positive correlation between night shift work and breast cancer risk was described in 8 out of 12 studies. However, different reasons suggest that some of these studies have an Achilles heel according to the International Agency of Cancer (IARC) indications. Both the circadian system alteration and the melatonin output reduction, related to the exposure to light-at-night during night shift work, remain the most valid hypotheses on the causal relation of shift work and breast cancer. Overall, the results of the present study suggest that there is an association between night shift work and breast cancer development in western countries. However, further studies are needed to confirm such association and to understand which biomolecular mechanisms may be involved in the pathogenesis of cancer diagnosed in patients with night shift work exposure.

The tumor microenvironment has been largely studied as a dynamic system orchestrated by inflammatory cells, including cancer cells, stroma as well as the extracellular matrix. It is useful to describe and predict the phenotypic characteristics of cancer. Furthermore, a better understanding of its interplay with the various aspects of the tumor cells may be utilized for the discovery of novel molecular targets. Liver cancer is considered a model of the relation occurring between the tumor micro-environment and tumor development. The chronic inflammatory status of the liver, sustained by the infection of hepatitis viruses, as well as the production of cytokines and growth factors within the parenchyma, lead to an intricate microenvironment. The identification of novel molecular therapeutic targets may improve the outcome of patients with liver cancer as it remains the third leading cause of cancer death worldwide. In the present study, the tumor microenvironment in hepatocellular carcinoma (HCC) was explored by a review of the literature. Studies on hepatitis virus infections and the consequent chronic inflammatory status were examined. In this context, immune-mediated and/or virus-related molecular mechanisms have been hypothesized as being responsible for liver cancer development. The interlink among HCC microenvironment components, comprising cellular elements, cytokines, growth factors and several proteins is also described together with the role of matrix metalloproteinases in HCC development. Finally, the rationale for targeting tumor-stromal interface is summarized in the context of new therapeutic opportunities.

The most common therapeutic approach for many cancers is chemotherapy. However, many patients relapse after treatment due to the development of chemoresistance. Recently, targeted therapies represent novel approaches to destroy cancer cells. The PI3K/PTEN/AKT pathway is a key signaling pathway involved in the regulation of cell growth. Dysregulated signaling of this pathway may be associated with activating mutations of PI3K-related genes. Analyses of these mutations reveal that they increase the PI3K signal, stimulate downstream Akt signaling, promote growth factor-independent growth and increase cell invasion and metastasis. In this review, we summarize the PI3K/PTEN/AKT pathway genetic alterations in cancer and their potential clinical applications.

We have analyzed the proteomes of two human melanoma cell lines (A375 and 526), and of the human melanocytes, (FOM 78), by two-dimensional electrophoresis (2D-PAGE) and liquid chromatography - tandem mass spectrometry (LC-MS/MS). Our comparative proteomic analysis revealed that six proteins were over-expressed in both melanoma cell lines as compared to melanocytes: galectin-1, inosine-5'-monophosphate dehydrogenase 2, serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform, protein DJ-1, cyclophilin A and cofilin-1. We show, for the first time, that only specific isoforms of these molecules are over-expressed in melanoma. Different protein profiles were also found between each individual melanoma cell line and the melanocytes. s-Methyl-5-thioadenosine phosphorylase, ubiquitin and ribosomal protein S27 a precursor, the basic form of protein DJ-1, annexin a1, proliferation associated protein 2g4, isoform alfa-enolase of alfa-enolase, protein disulfide-isomerase precursor, and elongation factor 2 were more strongly expressed in A375 cells compared to melanocytes. In 526 cells, 60s acidic ribosomal protein p1 and calreticulin precursor were more highly expressed than in melanocytes. These molecular differences may help in better understanding melanoma development and its different responsiveness to therapies. The identified proteins could be exploited as biomarkers or therapeutic targets for melanoma., (© 2011 Landes Bioscience)

Background: ATRX is a severe X-linked disorder characterized by mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassemia. The disease is caused by mutations in ATRX gene, which encodes a protein belonging to the SWI/SNF DNA helicase family, a group of proteins involved in the regulation of gene transcription at the chromatin level. In order to identify specific genes involved in the pathogenesis of the disease, we compared, by cDNA microarray, the expression levels of approximately 8500 transcripts between ATRX and normal males of comparable age., Methods: cDNA microarray was performed using total RNA from peripheral blood mononuclear cells of ATRX and normal males. Microarray results were validated by quantitative real-time polymerase chain reaction., Results: cDNA microarray analysis showed that 35 genes had a lower expression (30-35% of controls) while 25 transcripts had a two-fold higher expression in comparison to controls. In the microarray results the probe for oligophrenin-1, a gene known for its involvement in mental retardation, showed a decreased hybridization signal. However, such gene was poorly expressed in blood mononuclear cells and its decrease was not confirmed in the quantitative real-time RT-PCR assay. On the other hand, the expression of an homologous gene, the GTPase regulator associated with the focal adhesion kinase 1/Oligophrenin-1-like (GRAF1/OPHN-1-L), was relatively high in blood mononuclear cells and significantly decreased in ATRX patients. The analysis of the expression pattern of the GRAF1/OPHN-1-L gene in human tissues and organs revealed the predominant brain expression of a novel splicing isoform, called variant-3., Conclusions: Our data support the hypothesis of a primary role for altered gene expression in ATRX syndrome and suggest that the GRAF1/OPHN-1-L gene might be involved in the pathogenesis of the mental retardation. Moreover a novel alternative splicing transcript of such gene, predominantly expressed in brain tissues, was identified.

Objectives: Host factors, including genetic polymorphisms, may explain some of the individual differences in cervical cancer occurrence, and susceptibility information may be useful to address effective and specific preventive strategies for different countries. The purpose of the present study was to investigate the role of p53 codon 72, glutathione S-transferase class mu (GSTM1), glutathione S-transferase class theta (GSTT1), and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms on the risk for infection and/or of cervical intraepithelial lesions in women attending a colposcopy service in Catania, Sicily, with an already reported high prevalence of human papillomavirus., Methods: To identify the association among individual genetic polymorphisms, human papillomavirus infection, and histological findings, a case-control study was designed. Furthermore, to assess the combined effects of these polymorphisms on cervical cancer risk, combined genotype frequencies were compared among case patients and controls., Results: Women homozygous for the p53 codon 72 Arg genotype were at a 5.6-fold higher risk for developing cervical intraepithelial neoplasia (CIN) 2 or 3 compared with those showing homozygosity for the Pro genotype or heterozygosity for the Pro/Arg genotype. The GSTM1 and GSTT1 null genotypes were overrepresented in infected patients and in women with CIN 2 or 3, although without any significant associations. A decreased risk for CIN of individuals homozygous for the MTHFR T allele was shown., Conclusions: After multiple logistic analyses, the presence of the allele 677T of the MTHFR gene was the best explaining protective factor against cervical carcinogenesis, and the allelic distribution in the control group followed the Hardy-Weinberg equilibrium expectations. However, the findings of our study still remain to be confirmed by additional and larger population-based surveys.

Introduction: Human papillomavirus (HPV) infection has been strongly and consistently associated with cervical carcinoma and its cytologic precursors, such as squamous intraepithelial lesions. A cross-sectional survey was conducted with the aim of estimating the prevalence of cervical HPV infection in women attending a service of colposcopy in Catania, Eastern Sicily, Italy., Methods: The prevalence of type-specific HPV was examined in women with negative colposcopic results and cervical intraepithelial neoplasia grades 1, 2, or 3, with the aim of providing some cross-sectional figures on the local epidemiology of HPV infection., Results: Human papillomavirus DNA was found in 62.1% of women with negative colposcopic results and in 73.2% with positive colposcopic results. Among high-risk types, a predominance of HPV-16 (51.5% of infected women) was shown followed by HPV-56 (29.7%). An age-related pattern was described with a peak in HPV prevalence among women younger than 25 years, followed by the expected decline in prevalence and a second characteristic peak in the perimenopausal or postmenopausal years, useful to design future control strategies., Conclusions: The age-related pattern of HPV prevalence and the presence of uncommon high-risk genotypes and their role in the pathogenesis of cervical cancer need to be addressed by specific epidemiologic studies to design large-scale screening programs and multivalent vaccine strategies.

Aims: Heparin-binding epidermal growth factor is a member of the EGF family, it is a potent mitogen for smooth muscle cells and has been implicated in atherosclerosis, angiogenesis. In athererogenesis, HB-EGF has been detected in medial smooth cells and foamy macrofages. In this work, we have investigate about immunohistocemical localization of HB-EGF in atherosclerotic plaques., Materials and Methods: Three cases of man affected by atherosclerosis have been examined. We have collected and examined atherosclerotic plaques by immunohistochemical procedure in optical microscopy. Samples have been incubated with primary Ac (anti-human HB-EGF- goat IgG)., Results: In the three examined cases, results are partly overlap-ping, but with some difference in relation to location of positivity to HB-EGF. Only in one case, HB-EGF staining is rather weak and located just below endothelium where is a thickened area of tissue rich in fibres and few cells, In another case, positivity to HB-EGF is present in an area of connective tissue of the intima. In the last case, positivity to HB-EGF is evident in the context of a presumed elastic tissue with fusiform cells following fibres orientation, and that could be fibroblasts or smooth muscle cells., Conclusions: These results indicate that HB-EGF is involved in the development of atherosclerotic plaques and that HB-EGF is a possible target for atherosclerosis therapy.

Background and Aim: Gallbladder wall thickening and impaired contractility are currently reported in cirrhotic patients and often related to portal hypertension and hepatic failure. The purpose of this work was to evaluate, by ultrasonographic method, gallbladder wall thickness and gallbladder emptying after a standard meal in normal subjects and in patients with compensated liver cirrhosis without gallstones., Methods: Twenty-three patients with Child-Pugh class A liver cirrhosis and twenty healthy controls were studied. Gallbladder wall thickness (GWT), gallbladder fasting volume (FV), residual volume (RV), and maximum percentage of emptying (%E) were calculated. Measurements of mean portal velocity, portal vein flow, and serum albumin were performed too. Statistical analysis was assessed by Student's "t test" for unpaired data., Results: GWT was 0.60 +/- 0.22 cm in cirrhotic patients and 0.21 +/- 0.06 cm in controls (P < .0001). FV and RV were, respectively, 37.8 +/- 3.7 cm(3) and 21.8 +/- 3 cm(3) in cirrhotic patients, 21.9 +/- 4.2 cm(3) and 4.6 +/- 2.2 cm(3) in healthy volunteers (P < .0001). %E was smaller in cirrhotics (42.6 +/- 7.8) as compared to controls (80.3 +/- 7.2; P < .0001)., Conclusions: In patients with compensated liver cirrhosis without gallstones gallbladder wall thickness is increased whereas its contractility is reduced. These early structural and functional alterations could play a role in gallstone formation in more advanced stages of the disease.

Purpose: Prostate cancer remains the second most frequent cause of tumor-related deaths in the Western world. Additional markers for the identification of prostate cancer development and progression are needed. Osteopontin (OPN), which activates matrix metalloproteinases (MMP), is considered a prognostic biomarker in several cancers. "In silico" and experimental approaches were used to determine whether OPN-mediated MMP activation may be a signal of prostate cancer progression., Experimental Design: Pearson correlation coefficients were computed for each OPN/MMP pair across seven publicly available prostate cancer gene expression data sets. Using Gene Set Enrichment Analysis, 101 cancer-related gene sets were analyzed for association with OPN and MMP-9 expression. OPN, MMP-9, MMP-2 tissue inhibitor of metalloproteinase-1 plasma levels, and MMP gelatinase activity were measured by ELISA and zymography in 96 and 92 patients with prostate cancer and benign prostatic hyperplasia, respectively, and 125 age-matched healthy men., Results: Computational analyses identified a significant correlation only between MMP-9 and OPN, and showed significant enrichment scores in "cell proliferation", "genes constituting the phosphoinositide-3-kinase predictor", "proliferation signature", and "tumor metastasis" gene sets in association with both OPN and MMP-9. Plasma analyses revealed a significant increase in OPN and MMP-9 levels and activity in patients with prostate cancer in association with clinical variables (prostate-specific antigen > 4 ng/mL and Gleason score > 7). Significant correlation between OPN and MMP-9 levels were also observed. Mean plasma levels of OPN and MMP-9 decreased in patients with prostate cancer within 6 months after prostatectomy., Conclusions: The concordant computational and experimental data indicate that the extent of OPN pathway activation correlates with prostate cancer progression.

The treatment of thalassemia is still essentially based on continuous transfusion supporting using red cell concentrates (RCC) prepared in different ways. For patients with sickle-cell disorders, either urgent or chronic red blood cell transfusion therapy, is widely used in the management of sickle cell disease (SCD) because it reduces HbS level and generally prevents recurrent vaso-occlusive disease (VOD). Recently, the introduction of pre-storage filtration to remove leukocytes and the use of techniques for multicomponent donation have increased the types of blood components available for transfusion purposes. The clinical effects of different types of blood components in thalassaemic and sickle-cell patients have not been extensively studied so far. We evaluated the impact of the various different blood components currently available on transfusion needs, transfusion intervals and adverse reactions in order to determine which is the most advantageous for transfusion-dependent thalassaemic and sickle-cell patients followed in our centre. We believe that the optimal characteristics of the RCC are aged less than 10 days from time of collection; Hb content greater than 56 g per unit; Hct: 55-60%; volume (including additive) 300 mL+/-20%; leucodepleted to less than 200,000 leukocytes per unit; low cytokine content (achievable by pre-storage filtration carried out between two and 24 hours after the collection); lack of microaggregates (achievable by pre-storage filtration or filtration in the laboratory) and protein content less than 0.5 g per unit for patients allergic to plasma proteins (achievable with manual or automated washing). It is still recommended that the blood transfused should be as fresh as possible, compatible with the centre's product availability and the centre's organisation should be continuously adapted to this aim. We always transfuse blood within 10 days of its collection, respecting Rh and Kell system phenotypes. Pre-storage filtration is strongly recommended, both in order to prevent adverse reactions through the marked leucodepletion (less than 200,000 leukocytes per unit) and for a better standardisation of the final product, including the certainty that the product does not contain clots, an assurance that bed-side filtration cannot give. The RCC should be produced using a method causing as little as possible stress to the red cell membrane. The use of RCC with a high content of Hb (less than 56 g per unit) is strongly recommended, because our study clearly shows that this reduces the number of exposures to donors and the number of accesses to hospital, thus improving the patient's quality of life.

Introduction: Fulminant hepatic failure is the end result of many different acute damage to the liver. In the present study we compared the clinical to the experimental experience and we postulated the usage of Vascular Endothelial Growth Factor in the clinical arena as a potential treatment in alternative to liver transplantation., Materials and Methods: Twelve patient diagnosed with fulminat hepatic failure have been enclosed in the present study. Each patient underwent trans-jugular liver biopsy in order to assess the degree of liver necrosis as well as the following biochemical investigation: AST ALT, Total Bilirubin, &#95;gt, alkaline phosphatase, prothrombin time., Results: Twenty-five percent of those patients required support in the Intensive Care Unit without need for transplantation. Forty-one percent of those patients underwent liver transplantation, and 36% of them died before the liver become available. These results were compared with an experiment, previously performed by our group, where 260 rats were poisoned with CCl4 and subsequently treated with Vascula Endothelial Growth Factor (VEGF)., Conclusion: The rate of the hepatic regeneration has been found to be critical in the prognosis of patients diagnosed with fulminant hepatic failure.

Background and Aims: Few studies in literature have investigated the gastric emptying of solids in elderly subjects. We assessed the differences between young and elderly subjects in the gastric emptying rate of solids by a radioisotopic method., Methods: Two groups of 15 elderly male subjects (mean age 68.20 years and 77.26 years, respectively) and a group of young male subjects (mean age 30.23 years) underwent a radioisotopic study of gastric emptying after eating a radiolabeled solid meal. Half-time of gastric emptying (T1/2) and emptying index (EI), i.e. rate of gastric emptying at 120 min, were measured with two opposing detectors connected to a computerized rate-meter. Results are expressed as means +/- SD., Results: Significantly different values were obtained in the two groups both at T1/2 (183+/-88 and 195+/-75, respectively) and EI (0.40+/-0.3 and 0.36+/-0.4), compared with young subjects (T1/2=53+/-23; EI=1.10+/-0.3) (p<0.0001)., Conclusions: Gastric emptying of solids is significantly delayed in elderly men; this variable must be taken into account when studies on gastric emptying rates are performed.

In this study, by using different techniques (i.e. Northern blot hybridization, RT-PCR and Southern blot hybridization) on various normal rat tissues, we were able to identify liver, kidney, heart, small intestine, brain, spleen, stomach and prostate as tissues in which the ApoH gene is transcribed. Moreover, for some of these tissues, by in situ hybridization, we found a specific localization of apoH transcripts. For instance epithelial cells of the bile ducts in liver and of the proximal tubules in kidney are the major sites of apoH synthesis. Our data suggest that some of the different physiological roles proposed for apoH could correlate with its direct expression, while others could correlate with its absorption from bloodstream or adjacent cells.

Introduction: Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen. The objective of this study was to verify the proregenerative effects of VEGF in an experimental model of acute liver failure., Materials and Methods: Sixty four rats that underwent intraperitoneal injection of carbon tetrachloride (CCl(4)) were randomly divided into two groups: group B animals received intravenous injection of VEGF(164) 1 hour following CCl(4) poisoning. Group A hosts were untreated. To obtain daily liver function tests (LFTs) and histological samples, on each day up to 8 days we sacrificed four rats in each group., Results: The laboratory examinations showed notable alteration of LFTs in group A, while group B revealed only slight changes. The histological examination showed greater liver damage in group A compared with group B., Conclusion: Our results suggest that administration of exogenous VEGF protects the liver from CCl(4)-induced acute hepatic failure. Further studies are underway to assess whether exogenous VEGF is effective in other liver injuries.

Basal cell carcinoma (BCC) is the most common skin cancer, and its incidence is increasing. It was proposed that the RAS oncogene significantly contributes to skin cancer development. Numerous BRAF mutations have been detected in melanoma biopsy specimens and cell lines. For the first time, in the present study, tumor biopsy specimens from 78 patients with BCC were screened for BRAF mutation within exons 11 and 15. Our results indicate that the BRAF gene does not appear to be frequently mutated in nonmelanoma skin tumors such as BCC. These data suggest that other gene alterations may cause tumor development.

Partial deletions of the DAZ gene cluster are thought to cause spermatogenesis impairment. The presence of homologous copies of this gene in the Y chromosome does not allow PCR to be used for the identification of this abnormality. Hence, sequence family variants (SFV), following amplification of sY581, sY587 and sY586 and subsequent enzymatic digestion with Sau3A, DraI and TaqI, respectively, and the dual fiber fluorescence in situ hybridization (FISH) have been used to this aim. However, SFV is not always able to identify single DAZ gene copy deletions. We report a quantitative real-time PCR application to evaluate partial deletions of the DAZ gene cluster. To accomplish this, we designed a probe on exon 6 of the DAZ gene which is repeated 3 times in DAZ1, once in DAZ2 and DAZ3 and twice in DAZ4. Five normozoospermic healthy men (C1-C5) having 4 DAZ gene copies by SFV were selected. Fiber-FISH confirmed this outcome in C1-C4, but not in C5 who had an incomplete DAZ gene cluster. The men underwent then quantitative real-time PCR and C1 was arbitrarily selected as calibrator for the calculation of the DAZ gene signals because of the lowest variation in the threshold cycles. Real-time PCR identified 7.2+/-0.05 signals in C2-C4 and 5.4+/-0.05 signals in C5. The overall coefficient of variation was 1.4+/-0.2%. The loss of two signals in this subject may relate to a deletion of both DAZ2 and DAZ3 or of DAZ4 gene. Since SFV showed clearly the presence of DAZ2, it may be hypothesized that C5 lacks DAZ4. In conclusion, these data suggested that quantitative real-time PCR seems to be an effective and reproducible technique that can be used to study the DAZ gene cluster. In addition, the probe chosen for this approach may give indication on the DAZ gene copy deleted.

In this study, we evaluated the expression of genes probably involved in spermatogenesis in the mouse. We examined cytosolic chaperonin theta subunit (CCTtheta), Ngg1 interacting factor 3 like 1 binding protein 1 (NIF3L1 BP1) and apolipoprotein H (ApoH) expression during mouse onto-geny using RT-PCR. Testicular tissue was obtained from mice 3, 6, 8, 10, 12, 14, 18, 20 and 40 (adult) days after birth. For each mouse, one testis was used for histological examination, whereas RNA was extracted from the controlateral testis for expression analysis. RT-PCR analysis showed that CCTtheta gene expression was low until day 10, but increased drastically afterwards. At this age, spermatocytes started to be present in the mouse testis. Therefore, CCT protein could be involved in chromatin packaging and remodeling during spermiogenesis, as also suggested by other studies. NIF3L1 BP1 expression increased steadily during ontogenesis reaching maximum levels in the adult mouse when all germ cell stages are present. This finding suggests that NIF3L1 BP1 is a gene not expressed by a specific germ cell type. ApoH expression was very low or absent during prepuberal stages, whereas it was detectable in the adult testis when spermatogenesis was completed. This suggests that ApoH may be involved in clearing apoptotic bodies during spermatogenesis since apoptotic events increase during spermatogenesis. This study contributes to understanding the role played by genes important for spermatogenesis.

The epidemiological impact of Acinetobacter baumannii nosocomial infections in a Sicilian intensive care unit (ICU) was investigated to determine the Acinetobacter-specific infection rates, to estimate the preventable proportion of Acinetobacter infections, i.e., those resulting from cross-transmission, and to investigate the molecular epidemiology of antimicrobial resistance in Acinetobacter. The impact of Acinetobacter nosocomial infection in the ICU was determined to be 3.0 new cases per 100 admissions. Site-specific rates confirmed that ICU-acquired pneumonia was the most important infection type. The incidence rate, adjusted by the number of patient-days, was 3.3 infections/1000 patient-days. The estimated preventable proportion of A. baumannii nosocomial infections in the ICU was 66.7%. A class 1 integron, characterised by its gene cassette content, was present in all A. baumannii isolates of four different pulsed-field gel electrophoresis types, and was associated significantly with clones implicated in cross-transmission episodes. Furthermore, the same integron was detected in two genetically distinct isolates responsible for recurrent infection in the same patient, suggesting the occurrence of horizontal gene transfer in vivo. Even in an endemic setting with low infection rates, spread of A. baumannii was caused mainly by infection control shortcomings that require appropriate surveillance and control policies.

Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by mutations in the gene coding for the low density lipoprotein receptor (LDL-R). It is characterized by a high concentration of low density lipoprotein (LDL), which frequently gives rise to premature coronary artery disease. We studied the probands of five FH Sicilian families with 'definite' FH and one proband of Paraguayan descent with homozygous FH who has been treated with an effective living-donor liver transplantation. In order to seek the molecular defect in these six families, we used direct sequencing to define the molecular defects of the LDL-R gene responsible for the disease. We described three novel missense mutations (C100Y, C183Y and G440C), two frameshift mutations (g.1162delC in exon 8 and g.2051delC in exon 14) and one mutation (g.2390-1Gright curved arrow A) at splicing acceptor consensus sequences located in intron 16 of the LDL-R gene; the analysis of cDNA of this splicing mutation showed the activation of a cryptic splice site in intron 16 and the binding studies showed a reduction in internalisation of LDL-DIL in the proband's cultured fibroblasts. Moreover, a g.2051delC in exon 14 was identified in the proband of Paraguayan ancestry with clinical features of homozygous FH. The mutation identified in the South American patient represents the first description of a variant in South American patients other than Brazilian FH patients. The 5 mutations identified in the Sicilian patients confirm the heterogeneity of LDL-R gene mutations in Sicily.

Numerous BRAF mutations have been detected in melanoma biopsy specimens and cell lines. In contrast, several studies report lack of BRAF mutations in uveal melanoma including primary and metastatic choroidal and ciliary body melanomas. To our knowledge, for the first time, here we report a case of choroidal melanoma harboring the BRAF mutation (V600E). The activation of RAF/MEK/ERK pathway, although independent of BRAF mutation, was reported in uveal melanoma. The presence of V600E mutation indicates that the RAF/MEK/ERK pathway, in addition to cutaneous melanoma progression, may play a role in the choroidal melanoma development.

Mutation of BRAF has been proposed to contribute to melanoma development. However, it remains unclear whether or not BRAF mutation is associated with any particular stage of melanoma progression. Tumor biopsy specimens from patients with melanoma were analyzed to determine whether the frequency of BRAF mutation in metastatic melanoma differed from primary melanoma. BRAF mutation was present in 15 of 23 (61%) patients with primary melanoma and in 7 of 12 (58%) patients with metastatic melanoma. These results suggest that BRAF mutation in melanoma is most likely to occur prior to the development of metastatic disease.

Bovine seminal-ribonuclease (BS-RNase) is a member of the 'ribonucleases with special biological actions' family since it possesses specific anti-tumour, anti-spermatogenic and embryotoxic activities and exerts an immunosuppressive effect on T lymphocytes. In previous studies it was demonstrated that BS-RNase induced apoptosis in proliferating, malignant and normal cells and that telomerase activity loss also caused apoptotic death in neoplastic cells. Since an obvious relationship between cell proliferation and telomerase activity exists, the aim of this work was to study if the pro-apoptotic cytotoxic action exerted by BS-RNase on proliferating malignant cells (HT29) and proliferating normal cells (PHA-stimulated lymphocytes) could be linked to a possible BS-RNase effect on telomerase activity. In BS-RNase-treated HT29 cells (Na-butyrate-differentiated or not) and human lymphocytes (proliferating or not), we investigated cell vitality (MTT method) and morphology (SEM), BS-RNase localization (immunofluorescence), telomerase activity (TRAP-ELISA method), hTR mRNA expression (RT-PCR), and hTERT levels (western blot). While no BS-RNase effect was detectable on not proliferating cells, a clear relationship was noticed between the diminished number of vital elements of both proliferating cell populations after treatment (48 h and 72 h for HT29 and PHA-stimulated lymphocytes, respectively) with 50 microg/ml BS-RNase and the decrease of their telomerase activity. At the same time, we found that hTR levels, the RNA subunit of telomerase, in proliferation-inhibited BS-RNase-treated cells were diminished. Moreover, by immunofluorescence technique, we detected BS-RNase in the HT29 cell nucleolus after 3-h treatment. Therefore, as hTR has been recently proven to co-fractionate with nucleoli, we hypothesize that a BS-RNase direct action on the telomerase hTR subunit could be a possible mechanism of action by which BS-RNase exerts its pro-apoptotic effects only on proliferating cells.

Aim: Hepatitis C virus (HCV) is one of the most common blood-borne pathogens transmitted from patients to health care workers (HCWs). The Centers for Disease Control and Prevention (CDC) have developed a set of universal precautions to help prevent transmission of blood-borne pathogens between patients and HCWs in health care settings. HCV infection status among HCWs and proportion of HCWs experiencing occupational blood exposure accidents were monitored to assess the risk of HCV infection among HCWs at a hospital in Catania, Italy., Methods: The number of HCWs reporting occupational blood exposure accidents during 1999 and 2004 were compared to examine whether there was any change in the incidence of these accidents among 900 HCWs. HCV infection status of these HCWs was also analyzed in 1999 and 2004 to determine how many were infected with HCV during this time period., Results: HCV infection was detected in 21 out of 900 subjects in 1999. The remaining 879 HCWs remained HCV-negative until they were last tested in 2004. There was a statistically significant decrease in the number of HCWs that experienced occupational blood exposure accidents from 306 in 1999 to 240 in 2004 (P = 0.001)., Conclusions: The finding that all 871 HCV-negative HCWs remained HCV-negative from 1999 until 2004 supports the view that the set of universal precautions recommended by the CDC are helpful for preventing HCV transmission from patients to HCWs. HCWs must continue following these precautions to prevent transmission of HCV and other blood-borne pathogens between patients and HCWs in the future.

Aim: It has been previously suggested that t(14;18) translocation of bcl-2 to the immuno-globulin heavy chain (IgH) locus may contribute to pathogenesis of lymphoproliferative disorders related to hepatitis C virus (HCV) infection, including type II mixed cryoglobulinemia (MC)., Methods: In this study, the presence or absence of t(14;18) translocation was determined in tumor biopsy specimens and peripheral blood mononuclear cells (PBMCs) for 48 NHL patients with chronic HCV infection., Results: In tumor biopsy specimens from 32 HCV-positive NHL patients, bcl-2/IgH translocation was detected in 1 of 13 patients with MC syndrome (7.7%) and 3 of 19 patients without MC syndrome (15.8%). In PBMCs from 23 HCV-positive NHL patients, this translocation was observed in 3 of 6 patients with MC syndrome (50%) and 4 of 17 patients without MC syndrome (23.5%). Interestingly, bcl-2/IgH translocation was found in 2 extranodal marginal zone B-cell lymphoma tissues from HCV-infected patients., Conclusions: However, additional studies are required to better clarify the relationship between this translocation and extranodal marginal zone B-cell lymphoma development. Although the frequency of bcl-2/IgH translocation in PBMCs from patients with chronic HCV infection is higher than that of other NHL patients, this increased translocation rate remains to be elucidated.

We tracked the results of 394 manual or automatic red blood cell exchanges done with a cell separator in 20 sickle cell patients at high risk for recurrent complications. Over an average of 6 years, none of the patients developed complications related to the procedure or to the increased blood use. It was safe and effective in preventing complications of sickle cell disease, and if done automatically, reduced iron overload. Ferritin levels also decreased in patients treated with automatic red blood cell exchange. Furthermore, using Single Donor Red Blood Cell units (SDRC) we reduced the potential exposure to transfusion transmitted infectious diseases (TTI).

Interleukin (IL)-1beta-treated rat islets of Langerhans were exposed in vitro either to the imidazoline compound, Efaroxan, or to the selective inducible nitric oxide synthase (iNOS) inhibitor, 1400W, in a medium containing a high concentration of glucose (16.7 mmol/L). Our data have evidenced the following: (i) addition of Efaroxan to islet cultures inhibited IL-1beta activation of ICE (cysteine protease IL-1beta converting enzyme) while addition of 1400W did not; (ii) Efaroxan completely inhibited IL-1beta-induced suppression of insulin secretion and induction of iNOS mRNA transcripts, and, in addition, counteracted islet beta-cell protein profile alterations, Bax-cytochrome c translocation, caspase activation, and apoptosis; (iii) 1400W inhibited IL-1beta induction of iNOS, but failed to completely counteract the other cytotoxic effects; (iv) the two compounds, moreover, exerted different effects on manganese superoxide dismutase (MnSOD), in fact, while Efaroxan inhibited the early stimulatory effect of IL-1beta on MnSOD, 1400W did not. Thus, Efaroxan completely protected islet beta cells from damage caused by IL-1beta-induced toxicity, while compound 1400W only inhibited NO radical production without altering the cytokine's cytotoxicity. Our observations have evidenced that suppression of ICE activation is required to counteract IL-1beta-mediated islet beta cell toxicity, and that IL-1beta-induced apoptosis is NO-independent and involves the cytochrome c-mitochondrial pathway., (2004 Wiley-Liss, Inc.)

Previous studies have detected high levels of matrix metalloproteinases (MMPs) in metastatic prostate cancer. In this study, we recruited 40 patients with prostate cancer (PCa): 20 presented organ-confined carcinoma and 20 had metastatic cancer. We also recruited 40 subjects for control groups, 20 with benign prostate hyperplasia (BPH) and 20 healthy males with similar characteristics. All of the patients were monitored at the beginning (time 0) and after 90 days. We analyzed the plasma concentrations of MMP-2, MMP-9, MMP-13, TIMP-1 and the enzyme activity of MMP-2 and MMP-9,using specific ELISA tests. The plasma concentrations of MMP-2, MMP-9 and MMP-13 were higher in PCa patients with metastasis than in the other groups, and in these patients decreased markedly after therapy began. For MMP-2 and MMP-9, greater differences were observed in enzyme activity than in plasma concentrations. TIMP-1 was reduced in PCa patients with metastasis, even if the intergroup differences were not statistically significant. Our results suggest that the plasma concentration and activity of MMPs, in association with PSA determination, could play a role in diagnosis, monitoring therapy and evaluating malignant progression in PCa.