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1.  Long-acting antipsychotic drugs for the treatment of schizophrenia: use in daily practice from naturalistic observations 
BMC Psychiatry  2012;12:122.
Current guidelines suggest specific criteria for oral or long-acting injectable antipsychotic drugs (LAIs). This review aims to describe the demographic and clinical characteristics of the ideal profile of the patient with schizophrenia treated with LAIs, through the analysis of nonrandomized studies.
A systematic review of nonrandomized studies in English was performed attempting to analyze the factors related to the choice and use of LAIs in daily practice. The contents were outlined using the Cochrane methods for nonrandomized studies and the variables included demographic as well as clinical characteristics. The available literature did not allow any statistical analysis that could be used to identify the ideal profile of patients with schizophrenia to be treated with LAIs.
Eighty publications were selected and reviewed. Prevalence of LAI use ranged from 4.8% to 66%. The only demographic characteristics that were consistently assessed through retrieved studies were age (38.5 years in the 1970’s, 35.8 years in the 1980’s, 39.3 years in the 1990’s, to 39.5 years in the 2000’s) and gender (male > female).
Efficacy was assessed through the use of various symptom scales and other indirect measurements; safety was assessed through extrapyramidal symptoms and the use of anticholinergic drugs, but these data were inconsistent and impossible to pool. Efficacy and safety results reported in the different studies yielded a good therapeutic profile with a maximum of 74% decrease in hospital admissions and the prevalence of extrapyramidal symptoms with LAIs consistently increased at 6, 12, 18, and 24 months (35.4%, 37.1%, 36.9%, and 41.3%, respectively).
This analysis of the available literature strongly suggests that further observational studies on patients with schizophrenia treated with LAIs are needed to systematically assess their demographic and clinical characteristics and the relationships between them and patient outcome.
Besides the good efficacy and safety profile of LAIs, health care staff must also take into account the importance of establishing a therapeutic alliance with the patient and his/her relatives when selecting the most appropriate treatment. LAIs seem to be a good choice not only because of their good safety and efficacy profile, but also because they improve compliance, a key factor to improving adherence and to establishing a therapeutic alliance between patients with schizophrenia, their relatives, and their health care providers.
PMCID: PMC3573926  PMID: 22909285
Delayed-action preparations; Antipsychotic agents; Schizophrenia; Patients; Review
2.  Physician-assessed and patient-reported outcome measures in chemotherapy-induced sensory peripheral neurotoxicity: two sides of the same coin 
Alberti, P. | Rossi, E. | Cornblath, D. R. | Merkies, I. S. J. | Postma, T. J. | Frigeni, B. | Bruna, J. | Velasco, R. | Argyriou, A. A. | Kalofonos, H. P. | Psimaras, D. | Ricard, D. | Pace, A. | Galiè, E. | Briani, C. | Dalla Torre, C. | Faber, C. G. | Lalisang, R. I. | Boogerd, W. | Brandsma, D. | Koeppen, S. | Hense, J. | Storey, D. | Kerrigan, S. | Schenone, A. | Fabbri, S. | Valsecchi, M. G. | Cavaletti, G. | Cavaletti, G. | Cornblath, D.R. | Merkies, I.S.J. | Postma, T.J. | Valsecchi, M.G | Galimberti, S. | Rossi, E. | Cavaletti, G. | Frigeni, B. | Lanzani, F. | Mattavelli, L. | Piatti, ML. | Alberti, P. | Binda, D. | Bidoli, P.. | Cazzaniga, M. | Cortinovis, D. | Bruna, J. | Velasco, R. | Argyriou, AA. | Kalofonos, HP. | Psimaras, D. | Ricard, D. | Pace, A. | Galiè, E. | Briani, C. | Lucchetta, M. | Campagnolo, M. | Dalla Torre, C. | Merkies, ISJ. | Faber, CG. | Merkies, ISJ. | Vanhoutte, EK. | Bakkers, M. | Brouwer, B. | Lalisang, RI. | Boogerd, W. | Brandsma, D. | Koeppen, S. | Hense, J. | Grant, R. | Storey, D. | Kerrigan, S. | Schenone, A. | Reni, L. | Piras, B. | Fabbri, S. | Padua, L. | Granata, G. | Leandri, M. | Ghignotti, I. | Plasmati, R.. | Pastorelli, F. | Postma, TJ. | Heimans, JJ. | Eurelings, M. | Meijer, RJ. | Grisold, W. | Lindeck Pozza, E. | Mazzeo, A. | Toscano, A. | Tomasello, C. | Altavilla, G. | Penas Prado, M. | Dominguez Gonzalez, C. | Dorsey, SG. | Brell, JM.
Annals of Oncology  2013;25(1):257-264.
The perception of the severity and relevance of Chemotherapy Induced Peripheral Neurotoxicity (CIPN) is different for physicians and patients. This study provides the basis for a rationale use of different physician assessed scales and of European Organization for Research and Treatment of Cancer CIPN specific self-report questionnaire (EORTC QOL-CIPN20).
The different perception and assessment of chemotherapy-induced peripheral neurotoxicity (CIPN) between healthcare providers and patients has not yet been fully addressed, although these two approaches might eventually lead to inconsistent, possibly conflicting interpretation, especially regarding sensory impairment.
Patients and methods
A cohort of 281 subjects with stable CIPN was evaluated with the National Cancer Institute—Common Toxicity Criteria (NCI-CTC v. 2.0) sensory scale, the clinical Total Neuropathy Score (TNSc©), the modified Inflammatory Neuropathy Cause and Treatment (INCAT) sensory sumscore (mISS) and the European Organization for Research and Treatment of Cancer CIPN specific self-report questionnaire (EORTC QOL-CIPN20).
Patients' probability estimates showed that the EORTC QLQ-CIPN20 sensory score was overall more highly related to the NCI-CTC sensory score. However, the vibration perception item of the TNSc had a higher probability to be scored 0 for EORTC QLQ-CIPN20 scores lower than 35, as vibration score 2 for EORTC QLQ-CIPN20 scores between 35 and 50 and as grade 3 or 4 for EORTC QLQ-CIPN20 scores higher than 50. The linear models showed a significant trend between each mISS item and increasing EORTC QLQ-CIPN20 sensory scores.
None of the clinical items had a perfect relationship with patients' perception, and most of the discrepancies stood in the intermediate levels of CIPN severity. Our data indicate that to achieve a comprehensive knowledge of CIPN including a reliable assessment of both the severity and the quality of CIPN-related sensory impairment, clinical and PRO measures should be always combined.
PMCID: PMC3868322  PMID: 24256846
chemotherapy; neuropathy; assessment; patient-reported outcome measure; neurotoxicity
3.  RNA-sequencing analysis of Trichophyton rubrum transcriptome in response to sublethal doses of acriflavine 
BMC Genomics  2014;15(Suppl 7):S1.
The dermatophyte Trichophyton rubrum is an anthropophilic filamentous fungus that infects keratinized tissues and is the most common etiologic agent isolated in human dermatophytoses. The clinical treatment of these infections is challenging because only few antifungal drugs are commercially available. To understand the mode of action of cytotoxic drugs against fungi, we evaluated the time-dependent effects of acriflavine on T. rubrum transcriptome using high-throughput RNA-sequencing (RNA-seq) technology.
RNA-seq analysis generated approximately 200 million short reads that were mapped to the Broad Institute's Dermatophyte Comparative Database before differential gene expression analysis was performed. By employing a stringent cut-off threshold of −1.5 and 1.5 log2-fold changes in gene expression, a subset of 490 unique genes were found to be modulated in T. rubrum in response to acriflavine exposure. Among the selected genes, 69 genes were modulated at all exposure time points. Functional categorization indicated the putative involvement of these genes in various cellular processes such as oxidation-reduction reaction, transmembrane transport, and metal ion binding. Interestingly, genes putatively involved in the pathogenicity of dermatophytoses were down-regulated suggesting that this drug interferes with the virulence of T. rubrum. Moreover, we identified 159 novel putative transcripts in intergenic regions and two transcripts in intron regions of T. rubrum genome.
The results provide insights into the molecular events underlying the stress responses of T. rubrum to acriflavine, revealing that this drug interfered with important molecular events involved in the establishment and maintenance of fungal infection in the host. In addition, the identification of novel transcripts will further enable the improvement of gene annotation and open reading frame prediction of T. rubrum and other dermatophyte genomes.
PMCID: PMC4243288  PMID: 25573029
4.  Laparoscopic management of left paraduodenal hernia. Case report and review of literature 
Il Giornale di Chirurgia  2014;35(7-8):185-189.
We report a rare case of left paraduodenal hernia in patient with symptoms of abdominal subobstruction treated successful with laparoscopic management in urgent situation that have reduced the length of stay and postoperative pain. Internal hernia is only 1% of the causes of abdominal obstruction and the left paraduodenal hernia about 50% of them; it is a congenital defect that derive from malrotation and abnormal mesenteric adhesion. The modern imaging techniques help for the correct diagnosis despite difficult identification of the pathology for the various clinical presentation. The treatment of choice is the surgical intervention; the laparoscopic approach is rarely described in literature but it can reduce the morbidity, postoperative pain and the length of hospital stay.
PMCID: PMC4321526  PMID: 25174294
Paraduodenal; Hernia; Obstruction; Laparoscopic; Congenital
5.  Time Course of Corticospinal Excitability and Autonomic Function Interplay during and Following Monopolar tDCS 
While polarity-specific after-effects of monopolar transcranial direct current stimulation (tDCS) on corticospinal excitability are well-documented, modulation of vital parameters due to current spread through the brainstem is still a matter of debate, raising potential concerns about its use through the general public, as well as for neurorehabilitation purposes. We monitored online and after-effects of monopolar tDCS (primary motor cortex) in 10 healthy subjects by adopting a neuronavigated transcranial magnetic stimulation (TMS)/tDCS combined protocol. Motor evoked potentials (MEPs) together with vital parameters [e.g., blood pressure, heart-rate variability (HRV), and sympathovagal balance] were recorded and monitored before, during, and after anodal, cathodal, or sham tDCS. Ten MEPs, every 2.5-min time windows, were recorded from the right first dorsal interosseous (FDI), while 5-min epochs were used to record vital parameters. The protocol included 15 min of pre-tDCS and of online tDCS (anodal, cathodal, or sham). After-effects were recorded for 30 min. We showed a polarity-independent stabilization of cortical excitability level, a polarity-specific after-effect for cathodal and anodal stimulation, and an absence of persistent excitability changes during online stimulation. No significant effects on vital parameters emerged both during and after tDCS, while a linear increase in systolic/diastolic blood pressure and HRV was observed during each tDCS condition, as a possible unspecific response to experimental demands. Taken together, current findings provide new insights on the safety of monopolar tDCS, promoting its application both in research and clinical settings.
PMCID: PMC4104833  PMID: 25101009
transcranial direct current stimulation; transcranial magnetic stimulation; safety; neuromodulation; vital parameters
6.  Differential Effects of G- and F-Actin on the Plasma Membrane Calcium Pump Activity 
Cell biochemistry and biophysics  2013;66(1):187-198.
We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641–1644, 2007). Results show evidences for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca2+ extrusion through the membrane. Our results provide further evidence of the activation–inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts.
PMCID: PMC3894748  PMID: 23152090
PMCA; Cytoskeleton; Actin; Regulation
7.  Functional characterisation of human pulmonary monocyte-like cells in lipopolysaccharide-mediated acute lung inflammation 
We have previously reported the presence of novel subpopulations of pulmonary monocyte-like cells (PMLC) in the human lung; resident PMLC (rPMLC, HLA-DR+CD14++CD16+cells) and inducible PMLC (iPMLC, HLA-DR+CD14++CD16- cells). iPMLC are significantly increased in bronchoalveolar lavage (BAL) fluid following inhalation of lipopolysaccharide (LPS). We have carried out the first functional evaluation of PMLC subpopulations in the inflamed lung, following the isolation of these cells, and other lineages, from BAL fluid using novel and complex protocols.
iPMLC, rPMLC, alveolar macrophages (AM), neutrophils, and regulatory T cells were quantified in BAL fluid of healthy subjects at 9 hours post-LPS inhalation (n = 15). Cell surface antigen expression by iPMLC, rPMLC and AM and the ability of each lineage to proliferate and to undergo phagocytosis were investigated using flow cytometry. Basal cytokine production by iPMLC compared to AM following their isolation from BAL fluid and the responsiveness of both cell types following in vitro treatment with the synthetic corticosteroid dexamethasone were assessed.
rPMLC have a significantly increased expression of mature macrophage markers and of the proliferation antigen Ki67, compared to iPMLC. Our cytokine data revealed a pro-inflammatory, corticosteroid-resistant phenotype of iPMLC in this model.
These data emphasise the presence of functionally distinct subpopulations of the monocyte/macrophage lineage in the human lung in experimental acute lung inflammation.
PMCID: PMC4032498  PMID: 24684897
Monocytes; Macrophages; Acute lung inflammation; Lipopolysaccharide; Multiparameter flow cytometry; Corticosteroid
8.  Comparative metabolism of cellulose, sophorose and glucose in Trichoderma reesei using high-throughput genomic and proteomic analyses 
The filamentous fungus Trichoderma reesei is a major producer of lignocellulolytic enzymes utilized by bioethanol industries. However, to achieve low cost second generation bioethanol production on an industrial scale an efficient mix of hydrolytic enzymes is required for the deconstruction of plant biomass. In this study, we investigated the molecular basis for lignocellulose-degrading enzyme production T. reesei during growth in cellulose, sophorose, and glucose.
We examined and compared the transcriptome and differential secretome (2D-DIGE) of T. reesei grown in cellulose, sophorose, or glucose as the sole carbon sources. By applying a stringent cut-off threshold 2,060 genes were identified as being differentially expressed in at least one of the respective carbon source comparisons. Hierarchical clustering of the differentially expressed genes identified three possible regulons, representing 123 genes controlled by cellulose, 154 genes controlled by sophorose and 402 genes controlled by glucose. Gene regulatory network analyses of the 692 genes differentially expressed between cellulose and sophorose, identified only 75 and 107 genes as being specific to growth in sophorose and cellulose, respectively. 2D-DIGE analyses identified 30 proteins exclusive to sophorose and 37 exclusive to cellulose. A correlation of 70.17% was obtained between transcription and secreted protein profiles.
Our data revealed new players in cellulose degradation such as accessory proteins with non-catalytic functions secreted in different carbon sources, transporters, transcription factors, and CAZymes, that specifically respond in response to either cellulose or sophorose.
PMCID: PMC3998047  PMID: 24655731
Trichoderma reesei; RNA-seq; DIGE; Cellulases; Bioethanol
9.  Electrophysiological study of the bulbocavernosus reflex: normative data 
Functional Neurology  2014;28(4):293-295.
In the clinical setting the bulbocavernosus reflex (BCR) is elicited by squeezing the glans penis and digitally palpating the contraction of the bulbocavernosus (BC) muscle. In neurophysiology the BCR is obtained by stimulating the dorsal nerve of the penis or clitoris and by recording the response from BC muscle and it should be performed in selected patients with suspected urinary, bowel, or sexual neurogenic dysfunction. The BCR is considered one of the sacral neurophysiological tests of the greatest clinical utility. Previous normative data were obtained on small samples. The aim of this study was to determine normative values for the BCR in a large sample of men.
We studied a large population (105 men; mean age 53 years, range 19–73 years) without central or peripheral neurological diseases. In each subject the sacral reflex was elicited by electrical stimulation of the base of the dorsum penis and recorded using a surface electrode from the BC muscle. We recorded the latency, calculated at onset, and the maximal amplitude of response, calculated peak to peak.
We were able to detect the BCR in all the men. No correlation between BCR latency and age was found (r=0.136; p=0.160). The mean onset latency value was 33.0±4.85 ms (mean±2SD, range 26.8–39.4). The mean amplitude value was 16.53±12.21 μV (mean±2SD, range 4.2–43.6). Our normative data on the BCR were similar to previously published data.
PMCID: PMC3951258  PMID: 24598398
bulbocavernosus reflex; normative data; sacral reflex testing
10.  The chemotherapy-induced peripheral neuropathy outcome measures standardization study: from consensus to the first validity and reliability findings 
Cavaletti, G. | Cornblath, D. R. | Merkies, I. S. J. | Postma, T. J. | Rossi, E. | Frigeni, B. | Alberti, P. | Bruna, J. | Velasco, R. | Argyriou, A. A. | Kalofonos, H. P. | Psimaras, D. | Ricard, D. | Pace, A. | Galiè, E. | Briani, C. | Dalla Torre, C. | Faber, C. G. | Lalisang, R. I. | Boogerd, W. | Brandsma, D. | Koeppen, S. | Hense, J. | Storey, D. | Kerrigan, S. | Schenone, A. | Fabbri, S. | Valsecchi, M. G. | Mazzeo, A. | Pace, A. | Pessino, A. | Schenone, A. | Toscano, A. | Argyriou, A.A. | Brouwer, B. | Frigeni, B. | Piras, B. | Briani, C. | Dalla Torre, C. | Dominguez Gonzalez, C. | Faber, C. G. | Tomasello, C. | Binda, D. | Brandsma, D. | Cortinovis, D. | Psimaras, D. | Ricard, D. | Storey, D. | Cornblath, D.R. | Galiè, E. | Lindeck Pozza, E. | Rossi, E. | Vanhoutte, E.K. | Lanzani, F. | Pastorelli, F. | Altavilla, G. | Cavaletti, G. | Granata, G. | Kalofonos, H.P. | Ghignotti, I. | Merkies, I.S.J. | Bruna, J. | Hense, J. | Heimans, J.J. | Mattavelli, L. | Padua, L. | Reni, L. | Bakkers, M. | Boogerd, M. | Campagnolo, M. | Cazzaniga, M. | Eurelings, M. | Leandri, M. | Lucchetta, M. | Penas Prado, M. | Russo, M. | Valsecchi, M.G. | Piatti, M.L. | Alberti, P. | Bidoli, P. | Grant, R. | Plasmati, R. | Velasco, R. | Lalisang, R.I. | Meijer, R.J. | Fabbri, S. | Dorsey, S. G. | Galimberti, S. | Kerrigan, S. | Koeppen, S. | Postma, T.J. | Boogerd, W. | Grisold, W.
Annals of Oncology  2012;24(2):454-462.
Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating and dose-limiting complication of cancer treatment. Thus far, the impact of CIPN has not been studied in a systematic clinimetric manner. The objective of the study was to select outcome measures for CIPN evaluation and to establish their validity and reproducibility in a cross-sectional multicenter study.
Patients and methods
After literature review and a consensus meeting among experts, face/content validity were obtained for the following selected scales: the National Cancer Institute-Common Toxicity Criteria (NCI-CTC), the Total Neuropathy Score clinical version (TNSc), the modified Inflammatory Neuropathy Cause and Treatment (INCAT) group sensory sumscore (mISS), the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30, and CIPN20 quality-of-life measures. A total of 281 patients with stable CIPN were examined. Validity (correlation) and reliability studies were carried out.
Good inter-/intra-observer scores were obtained for the TNSc, mISS, and NCI-CTC sensory/motor subscales. Test–retest values were also good for the EORTC QLQ-C30 and CIPN20. Acceptable validity scores were obtained through the correlation among the measures.
Good validity and reliability scores were demonstrated for the set of selected impairment and quality-of-life outcome measures in CIPN. Future studies are planned to investigate the responsiveness aspects of these measures.
PMCID: PMC3551481  PMID: 22910842
assessment; chemotherapy; clinimetrics; peripheral neuropathy; reliability; validity
11.  Psychotherapy versus usual care in pediatric migraine and tension-type headache: a single-blind controlled pilot study 
Despite growing interest in psychotherapy in child and adolescent headache, efficacy studies in this research field have focused mainly on cognitive-behavioral therapies. Whereas relaxation and cognitive-behavioral techniques, in particular, have been found to reduce the intensity and frequency of headache in children and adolescents, data on psychodynamic psychotherapy in this population are lacking.
Our aim was to explore the effectiveness of a brief psychodynamic psychotherapy program in the treatment of idiopathic headache in childhood and adolescence.
Thirty-three newly diagnosed idiopathic headache sufferers aged 6–18 years, consecutively referred to our outpatient services, were randomized to receive either a brief cycle of psychodynamic psychotherapy (eight sessions administered at two-week intervals) or usual care (clinical interview, neurological examination, counselling, symptomatic therapy).
The two groups were evaluated at baseline (T0) and at six months (T1) to be assessed for headache characteristics (i.e. frequency, intensity and duration), quality of life (i.e. the EuroQoL score), patient’s global health status (i.e. the Clinical Global Impression score), and emotional-behavioral symptoms (i.e. Child Behavior Checklist scores).
The two groups were fairly similar with reference to the main demographic and clinical variables. The T0/T1 comparison showed a statistically significant improvement in headache frequency (p = 0.005), intensity (p < 0.001) and duration (p = 0.002), a statistically significant improvement in the CGI score (p = 0.018), and a borderline improvement in the EuroQoL score (p = 0.053) in the group receiving psychotherapy.
According to our pilot findings, a brief psychodynamic psychotherapy program may be more effective than usual care in children and adolescents with idiopathic headache.
PMCID: PMC3899919  PMID: 24444140
12.  Detection of TP53 dysfunction in chronic lymphocytic leukemia by an in vitro functional assay based on TP53 activation by the non-genotoxic drug Nutlin-3: a proposal for clinical application 
TP53 defects, i.e. 17p13 deletion and/or nucleotide mutations, associate with short survival and chemorefractoriness in chronic lymphocytic leukemia (CLL). In this context, since direct sequencing of the TP53 gene does not evaluate TP53 functionality, a functional assessment of TP53 pathway may be of interest to identify high risk CLL. By taking advantage of a training cohort of 100 CLL and a validation cohort of 40 CLL with different patterns of TP53 mutation/deletion by FISH and sequencing, we propose an in-vitro assay in which the modulation of TP53 protein and CDKN1A mRNA were investigated upon 24-hour exposure of CLL cells to Nutlin-3.
The functional assay was set-up on cell lines recapitulating all TP53 genotypes (EHEB, TP53wt/wt; RAJI, TP53mut/wt; MEC-1 and MAVER1, TP53mut/del; HL-60, TP53del/del) and evaluated in two multi-institutional cohorts, purposely enriched in CLL bearing TP53 disruption: a training cohort of 100 cases and a validation cohort of 40 cases, both characterized by FISH and TP53 direct sequencing. Cells were exposed to 10 μM Nutlin-3 for 24 hours; TP53 accumulation was evaluated by Western blotting; TP53 transcriptional activity was determined by quantitative realtime PCR (qRT-PCR) of the TP53 target gene CDKN1A.
According to TP53 protein modulation, in the training cohort we identified: i) 63 cases (51 TP53wt/wt, 12 TP53del/wt) with absence of basal TP53 and induction after treatment (normal pattern); ii) 18 cases (3 TP53mut/wt, 15 TP53mut/del) with high basal TP53 without increase after treatment (mutant pattern); iii) 19 cases (5 TP53mut/wt; 3 TP53mut/del; 11 TP53wt/wt) with basal TP53 that increases upon treatment (intermediate pattern). Evaluation of CDKN1A mRNA levels upon Nutlin-3 exposure showed that the 26 TP53 mutated (TP53mut/del or TP53mut/wt) cases had lower induction levels than the majority (57/63) of cases with normal pattern, and 10/12 cases with intermediate pattern without evidence of TP53 derangement by FISH and sequencing. These results were confirmed in the independent validation cohort of 40 cases (13 TP53wt/wt, 3 TP53del/wt, 12 TP53mut/del, 12 TP53mut/wt).
The proposed functional assay may integrate the conventional analyses for the identification of TP53 dysregulated CLL.
PMCID: PMC4222122  PMID: 24283248
CLL; TP53; Prognosis
13.  Technical Advance: Autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production 
Journal of Leukocyte Biology  2013;94(1):193-202.
A novel method to ultra-purify unstimulated neutrophils by flow sorting and subsequent demonstration of the neutrophils' ability to release IL-1β following inflammatory stimulus.
The technical limitations of isolating neutrophils without contaminating leukocytes, while concurrently minimizing neutrophil activation, is a barrier to determining specific neutrophil functions. We aimed to assess the use of FACS for generating highly pure quiescent neutrophil populations in an antibody-free environment. Peripheral blood human granulocytes and murine bone marrow-derived neutrophils were isolated by discontinuous Percoll gradient and flow-sorted using FSC/SSC profiles and differences in autofluorescence. Postsort purity was assessed by morphological analysis and flow cytometry. Neutrophil activation was measured in unstimulated-unsorted and sorted cells and in response to fMLF, LTB4, and PAF by measuring shape change, CD62L, and CD11b expression; intracellular calcium flux; and chemotaxis. Cytokine production by human neutrophils was also determined. Postsort human neutrophil purity was 99.95% (sem=0.03; n=11; morphological analysis), and 99.68% were CD16+ve (sem=0.06; n=11), with similar results achieved for murine neutrophils. Flow sorting did not alter neutrophil activation or chemotaxis, relative to presorted cells, and no differences in response to agonists were observed. Stimulated neutrophils produced IL-1β, although to a lesser degree than CXCL8/IL-8. The exploitation of the difference in autofluorescence between neutrophils and eosinophils by FACS is a quick and effective method for generating highly purified populations for subsequent in vitro study.
PMCID: PMC3685014  PMID: 23667167
granulocyte; FACS; interleukin-1β
14.  Transcription of N- and O-linked mannosyltransferase genes is modulated by the pacC gene in the human dermatophyte Trichophyton rubrum 
FEBS Open Bio  2012;2:294-297.
In fungi, ambient pH sensing involves the activation of the Pal/PacC signalling pathway. In the dermatophyte Trichophyton rubrum, pH-dependent secretion of keratinases, which are major virulence determinants, is affected by disruption of the pacC gene. Here, the transcription profiling of the genes coding for N- and O-linked mannosyltransferases, enzymes involved in protein glycosylation, was evaluated in T. rubrum in response to disruption of the pacC gene and growth in keratin, glucose, and glucose plus glycine. We show that transcription of these mannosyltransferase genes is affected by nutrients at acidic pH and by PacC.
▸ The PacC/Pal transduction pathway mediates the metabolic response to pH sensing. ▸ pH-dependent secretion of keratinases is modulated by the pacC gene in T. rubrum. ▸ The pacC gene modulates transcription of O- and N-mannosyltransferase genes at acidic pH. ▸ An O-mannosyltransferase gene is preferentially transcribed in keratin at pH 5.0. ▸ An N-mannosyltransferase gene is preferentially transcribed in non-buffered glucose at pH 5.0.
PMCID: PMC3678131  PMID: 23772361
Glycosylation; Enzyme secretion; pH regulation; Transcription profiling
15.  Comparative Genome Analysis of Trichophyton rubrum and Related Dermatophytes Reveals Candidate Genes Involved in Infection 
mBio  2012;3(5):e00259-12.
The major cause of athlete’s foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response.
Athlete’s foot, jock itch, ringworm, and nail infections are common fungal infections, all caused by fungi known as dermatophytes (fungi that infect skin). This report presents the genome sequences of Trichophyton rubrum, the most frequent cause of athlete’s foot, as well as four other common dermatophytes. Dermatophyte genomes are enriched for four gene classes that may contribute to the ability of these fungi to cause disease. These include (i) proteases secreted to degrade skin; (ii) kinases, including pseudokinases, that are involved in signaling necessary for adapting to skin; (iii) secondary metabolites, compounds that act as toxins or signals in the interactions between fungus and host; and (iv) a class of proteins (LysM) that appear to bind and mask cell wall components and carbohydrates, thus avoiding the host’s immune response to the fungi. These genome sequences provide a strong foundation for future work in understanding how dermatophytes cause disease.
PMCID: PMC3445971  PMID: 22951933
16.  Evaluation of a Novel Hexavalent Humanized Anti-IGF-1R Antibody and Its Bivalent Parental IgG in Diverse Cancer Cell Lines 
PLoS ONE  2012;7(8):e44235.
A major mechanism of monoclonal antibodies that selectively target the insulin-like growth factor type 1 receptor (IGF-1R) to inhibit tumor growth is by downregulating the receptor, regardless whether they are capable (antagonistic) or incapable (agonistic) of blocking the binding of cognate ligands. We have developed and characterized a novel agonistic anti-IGF-1R humanized antibody, hR1, and used the Dock-and-Lock (DNL) method to construct Hex-hR1, the first multivalent antibody comprising 6 functional Fabs of hR1, with the aim of enhancing potency of hR1. Based on cross-blocking experiments, hR1 recognizes a region of cysteine-rich domain on the α-subunit, different from the epitopes mapped for existing anti-IGF-1R antibodies, yet hR1 is similar to other anti-IGF-1R antibodies in downregulating IGF-1R and inhibiting proliferation, colony formation, or invasion of selected cancer cell lines in vitro, as well as suppressing growth of the RH-30 rhabdomyosarcoma xenograft in nude mice when combined with the mTOR inhibitor, rapamycin. Hex-hR1 and hR1 are generally comparable in their bioactivities under the in-intro and in-vivo conditions investigated. Nevertheless, in selective experiments involving a direct comparison of potency, Hex-hR1 demonstrated a stronger effect on inhibiting cell proliferation stimulated by IGF-1 and could effectively downregulate IGF-1R at a concentration as low as 20 pM.
PMCID: PMC3432068  PMID: 22952934
17.  Diving Into the Lipid Bilayer to Investigate the Transmembrane Organization and Conformational State Transitions of P-type Ion ATPases 
Current chemical biology  2011;5(2):118-129.
Although membrane proteins constitute more than 20% of the total proteins, the structures of only a few are known in detail. An important group of integral membrane proteins are ion-transporting ATPases of the P-type family, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. There are several crystal structures of the sarcoplasmic reticulum Ca2+ pump (SERCA) revealing different conformations, and recently, crystal structures of the H+-ATPase and the Na+/K+-ATPase were reported as well. However, there are no atomic resolution structures for other P-type ATPases including the plasma membrane calcium pump (PMCA), which is integral to cellular Ca2+ signaling. Crystallization of these proteins is challenging because there is often no natural source from which the protein can be obtained in large quantities, and the presence of multiple isoforms in the same tissue further complicates efforts to obtain homogeneous samples suitable for crystallization. Alternative techniques to study structural aspects and conformational transitions in the PMCAs (and other P-type ATPases) have therefore been developed. Specifically, information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid–protein interactions. Here, we review recent efforts using different hydrophobic photo-labeling methods to study the non-covalent interactions between the PMCA and surrounding phospholipids under different experimental conditions, and discuss how the use of these lipid probes can reveal valuable information on the membrane organization and conformational state transitions in the PMCA, Na+/K+-ATPase, and other P-type ATPases.
PMCID: PMC3117579  PMID: 21691422
Ca2+-ATPase; hydrophobic photo-labeling; lipid-protein interaction; membrane protein; Na+/K+-ATPase; PMCA; P-type ATPase
18.  Transcription of the Hsp30, Hsp70, and Hsp90 heat shock protein genes is modulated by the PalA protein in response to acid pH-sensing in the fungus Aspergillus nidulans 
Cell Stress & Chaperones  2011;16(5):565-572.
Heat shock proteins are molecular chaperones linked to a myriad of physiological functions in both prokaryotes and eukaryotes. In this study, we show that the Aspergillus nidulans hsp30 (ANID_03555.1), hsp70 (ANID_05129.1), and hsp90 (ANID_08269.1) genes are preferentially expressed in an acidic milieu, whose expression is dependent on the palA+ background under optimal temperature for fungal growth. Heat shock induction of these three hsp genes showed different patterns in response to extracellular pH changes in the palA+ background. However, their accumulation upon heating for 2 h was almost unaffected by ambient pH changes in the palA− background. The PalA protein is a member of a conserved signaling cascade that is involved in the pH-mediated regulation of gene expression. Moreover, we identified several genes whose expression at pH 5.0 is also dependent on the palA+ background. These results reveal novel aspects of the heat- and pH-sensing networks of A. nidulans.
PMCID: PMC3156257  PMID: 21553327
Aspergillus nidulans; pH sensing; pal signaling pathway; HSP
19.  50 The Allergic March Resolved at Allergen Component Level 
The allergic march is well known at the level of pattern of sensitisation, but there is no information of its evolution in term of sensitzation to single allergenic molecules. We investigated the evolution of the IgE repertoire by means of a microarray allergen assay.
Serum samples from allergic patients of a wide age range were analyzed by a micorarray chip, which allow to identify in a single assay the presence of specific IgE towards 103 allergenic molecules. Total IgE were also evaluated as an internal control. Patients were stratified in 6 groups according to their age (0–2; 3–5; 6–9; 10–13; 14–17 and >17 years).
Samples from 609 patients were analysed. The behaviour of total IgE according to age strictly paralleled that of the sum of specific IgE. Food-related components were the more frequently recognized in the first ages, whereas specific IgE to plant allergens appeared later. Nonetheless, mite-specific IgE were the most represented in all age classes. Specific IgE against cross-reacting allergens were virtually absent in the first years and tended to appear after the age of 6.
The molecular pattern of allergen recognition according to age well reflects the clinical characteristics of the allergic march.
PMCID: PMC3512585
20.  Reverse left ventricular remodeling is more likely in non ischemic cardiomyopathy patients upgraded to biventricular stimulation after chronic right ventricular pacing 
Chronic right ventricular (RV) apical pacing may lead to left ventricular (LV) dyssynchrony and LV dysfunction. In heart failure due to RV pacing, upgrading to biventricular stimulation (CRT) can improve NYHA Class and LV function. A proportion of patients do not respond to upgrading. Aim was to assess whether etiology of LV dysfunction accounts for responses to CRT in RV-paced patients.
Sixty-two patients treated by CRT, under RV pacing from 50.2 ± 5.4 months, were studied. Cause of LV dysfunction was non-ischemic (NIC) in 28 and ischemic cardiomyopathy (IC) in 34 patients. Clinical and conventional echocardiographic parameters were available within 1 month before RV pacing, within 1 month before CRT and at 12 ± 2 months of follow-up (FU).
Decreased LVEF (from 37.0 ± 8.8 to 25.6 ± 6.1%, p <0.001), increased LV end-systolic dimensions (LVESD) (from 48.1 ± 8.6 to 55.2 ± 7.9 mm, p <0.001) and worsened NYHA Class (from 1.9 ± 1.1 to 3.2 ± .6, p < 0.005) were found before CRT, compared to pre RV-pacing. After CRT, 44/62 patients showed a ≥ 1 NYHA Class improvement; >10% decrease in LVESD was observed in 24 patients: 5 with IC, 19 with NIC (p < .0.001). The association between cause of LV dysfunction with >10% decrease in LVESD remained highly significant (p < 0.001) adjusting for pre-CRT QRS duration, NYHA Class, LVEF, LVESD, treatment or RV pacing duration.
CRT improves functional class even after long-lasting pacing. Reverse remodeling is evident in a small population, more likely with NIC.
PMCID: PMC3265428  PMID: 22177469
congestive heart failure; biventricular stimulation; non-ischemic cardiomyopathy; ischemic cardiomyopathy
21.  Cortico-Cortical Connectivity between Right Parietal and Bilateral Primary Motor Cortices during Imagined and Observed Actions: A Combined TMS/tDCS Study 
Previous transcranial magnetic stimulation (TMS) studies showed functional connections between the parietal cortex (PC) and the primary motor cortex (M1) during tasks of different reaching-to-grasp movements. Here, we tested whether the same network is involved in cognitive processes such as imagined or observed actions. Single pulse TMS of the right and left M1 during rest and during a motor imagery and an action observation task (i.e., an index–thumb pinch grip in both cases) was used to measure corticospinal excitability changes before and after conditioning of the right PC by 10 min of cathodal, anodal, or sham transcranial direct current stimulation (tDCS). Corticospinal excitability was indexed by the size of motor-evoked potentials (MEPs) from the contralateral first dorsal interosseous (FDI; target) and abductor digiti minimi muscle (control) muscles. Results showed selective ipsilateral effects on the M1 excitability, exclusively for motor imagery processes: anodal tDCS enhanced the MEPs’ size from the FDI muscle, whereas cathodal tDCS decreased it. Only cathodal tDCS impacted corticospinal facilitation induced by action observation. Sham stimulation was always uneffective. These results suggest that motor imagery, differently from action observation, is sustained by a strictly ipsilateral parieto-motor cortex circuits. Results might have implication for neuromodulatory rehabilitative purposes.
PMCID: PMC3163809  PMID: 21909322
TMS; tDCS; motor imagery; action observation; connectivity; M1; right PC
22.  Transcription of the Neurospora crassa 70-kDa class heat shock protein genes is modulated in response to extracellular pH changes 
Cell Stress & Chaperones  2009;15(2):225-231.
Heat shock proteins belong to a conserved superfamily of molecular chaperones found in prokaryotes and eukaryotes. These proteins are linked to a myriad of physiological functions. In this study, we show that the N. crassa hsp70-1 (NCU09602.3) and hsp70-2 (NCU08693.3) genes are preferentially expressed in an acidic milieu after 15 h of cell growth in sufficient phosphate at 30°C. No significant accumulation of these transcripts was detected at alkaline pH values. Both genes accumulated to a high level in mycelia that were incubated for 1 h at 45°C, regardless of the phosphate concentration and extracellular pH changes. Transcription of the hsp70-1 and hsp70-2 genes was dependent on the pacC+ background in mycelia cultured under optimal growth conditions or at 45°C. The pacC gene encodes a Zn-finger transcription factor that is involved in the regulation of gene expression by pH. Heat shock induction of these two hsp genes in mycelia incubated in low-phosphate medium was almost not altered in the nuc-1− background under both acidic and alkaline pH conditions. The NUC-1 transcriptional regulator is involved in the derepression of nucleases, phosphatases, and transporters that are necessary for fulfilling the cell's phosphate requirements. Transcription of the hsp70-3 (NCU01499.3) gene followed a different pattern of induction—the gene was depressed under insufficient phosphate conditions but was apparently unaffected by alkalinization of the culture medium. Moreover, this gene was not induced by heat shock. These results reveal novel aspects of the heat-sensing network of N. crassa.
PMCID: PMC2866986  PMID: 19618296
Neurospora crassa; hsp70; Heat shock; Pi sensing; pH regulation; nuc-1; pacC
23.  Lack of neo-sensitization to Pen a 1 in patients treated with mite sublingual immunotherapy 
Some studies reported the possible induction of food allergy, caused by neo-sensitization to cross-reacting allergens, during immunotherapy with aeroallergens, while other studies ruled out such possibility.
The aim of this study was to evaluate the development of neo-sensitization to Pen a 1 (tropomyosin) as well as the appearance of reactions after ingestion of foods containing tropomyosin as a consequence of sublingual mite immunization.
Materials and methods
Specific IgE to Tropomyosin (rPen a 1) before and after mite sublingual immunotherapy in 134 subjects were measured. IgE-specific antibodies for mite extract and recombinant allergen Pen a 1 were evaluated using the immunoenzymatic CAP system (Phadia Diagnostics, Milan, Italy).
All patients had rPen a 1 IgE negative results before and after mite SLIT and did not show positive shrimp extract skin reactivity and serological rPen a 1 IgE conversion after treatment. More important, no patient showed systemic reactions to crustacean ingestion.
Patients did not show neo-sensitization to tropomyosin, a component of the extract (namely mite group 10) administered. An assessment of a patient's possible pre-existing sensitisation to tropomyosin by skin test and/or specific IgE prior to start mite extract immunotherapy is recommended.
Trial Registration
This trial is registered in EudraCT, with the ID number of 2010-02035531.
PMCID: PMC2859740  PMID: 20230633
24.  Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: mean fluorescence intensity T/B ratio versus percentage of positive cells 
ZAP-70 is an independent negative prognostic marker in chronic lymphocytic leukemia (CLL). Usually, its expression is investigated by flow cytometric protocols in which the percentage of ZAP-70 positive CLL cells is determined in respect to isotypic control (ISO-method) or residual ZAP-70 positive T cells (T-method). These methods, however, beside suffering of an inherent subjectivity in their application, may give discordant results in some cases. The aim of this study was to assess the prognostic significance of these methods in comparison with another in which ZAP-70 expression was evaluated as a Mean-Fluorescence-Intensity Ratio between gated T and CLL cells (T/B Ratio-method).
Cytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set).
The optimal prognostic cut-offs for ZAP-70 expression were selected at 11% (ISO-method) or 20% of positive cells (T-method), as well as at 3.0 (T/B Ratio-method) in the test set; these cut-offs yielded 66, 60 and 73 ZAP-70+ cases, respectively. Univariate analyses resulted in a better separation of ZAP-70+ vs. ZAP-70- CLL patients utilizing the T/B Ratio, compared to T- or ISO-methods. In multivariate analyses which included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio method was applied. These findings were confirmed in the validation set, in which ZAP-70 expression, evaluated by the T- (cut-off = 20%) or T/B Ratio- (cut-off = 3.0) methods, yielded 180 or 127 ZAP-70+ cases, respectively. ZAP-70+ patients according to the T/B Ratio-method had shorter TTT, both if compared to ZAP-70- CLL, and to cases classified ZAP-70+ by the T-method only.
We suggest to evaluate ZAP-70 expression in routine settings using the T/B Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B Ratio value as optimal cut-off to discriminate ZAP-70+ (T/B Ratio less than 3.0) from ZAP-70- (T/B Ratio more/equal than 3.0) cases.
PMCID: PMC2846891  PMID: 20211015
25.  Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum 
BMC Microbiology  2010;10:39.
Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries.
The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases.
In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.
PMCID: PMC2831883  PMID: 20144196

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