Laboratory of Clinical Research

Milano, Italy

Laboratory of Clinical Research

Milano, Italy
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Zupa A.,Laboratory of Clinical Research | Improta G.,Laboratory of Clinical Research | Silvestri A.,Italian National Cancer Institute | Pin E.,Italian National Cancer Institute | And 9 more authors.
Journal of Thoracic Oncology | Year: 2012

BACKGROUND:: An understanding of the activated protein signaling architecture in non-small-cell lung cancer (NSCLC) is of critical importance to the development of new therapeutic approaches and identification of predictive and prognostic biomarkers for patient stratification. METHODS:: We used reverse-phase protein microarrays to map the activated protein signaling networks of 47 NSCLC tumors, 28 of which were node negative, which were subjected to tumor cellular enrichment using laser capture microdissection. The phosphorylation/cleavage levels of 111 key signaling proteins and total levels of 17 proteins were measured for broadscale signaling analysis. RESULTS:: Pathway activation mapping of NSCLC revealed distinct subgroups composed of epidermal growth factor receptor (ERBB1), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3), v-erb-a erythroblastic leukemia viral oncogene homolog 4 (ERBB4), v-akt murine thymoma viral oncogene homolog 1- mammalian target of rapamycin (AKT-mTOR), protein kinase, AMP-activated, alpha 2 catalytic subunit (AMPK), and autophagy-related signaling, along with transforming growth factor-beta-signaling protein 1 (SMAD), insulin-line growth factor receptor (IGFR), rearranged during transfection proto-oncogene (RET), and activated CDC42-associated kinase (ACK) activation. Investigation of epidermal growth factor receptor (EGFR)-driven signaling identified a unique cohort of tumors with low EGFR protein expression yet high relative levels of phosphorylated EGFR and high EGFR total protein with low relative levels of phosphorylation. Last, mapping analysis of patients with NSCLC with N0 disease revealed a pilot pathway activation signature composed of linked epidermal growth factor receptor family (HER)-AMPK-AKT-mTOR signaling network along with focal adhesion kinase- LIM domain kinase-1 (FAK-LIMK) and janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways that correlated with short-term survival and aggressive disease. CONCLUSIONS:: Functional protein pathway activation mapping of NSCLC reveals distinct activation subgroups that are underpinned by important therapeutic targets and that patients with early-stage node negative disease and poor prognosis may be identified by activation of defined, biochemically linked protein signaling events. Such findings, if confirmed in larger study sets, could help select and stratify patients for personalized targeted therapies. © 2012 by the International Association for the Study of Lung Cancer.


Gomes L.I.,Laboratory of Clinical Research | Gonzaga F.M.,Laboratory of Clinical Research | Morais-Teixeira E.D.,Laboratory of Clinical Research | de Souza-Lima B.S.,Laboratory of Clinical Research | And 2 more authors.
Experimental Parasitology | Year: 2012

In vitro assays play an important role in the discovery and development of new antileishmanial drugs. The classic macrophage-amastigote models using murine peritoneal macrophages or human-monocyte derived macrophages as host cells are useful for drug screening. A major limitation of these models is the dependence on microscopic counting, a time-consuming and subjective method of analysis. The present study describes a detailed protocol for applying quantitative real-time PCR (qPCR) as an accurate and sensitive tool to assess parasite load in an amastigote-macrophage model. This assay can be performed in a standardized medium-to-high throughput procedure, replacing traditional readout of number of amastigote per macrophages by DNA load measurement. © 2012 Elsevier Inc.


Tartarone A.,Centro Of Riferimento Oncologico Of Basilicata | Lazzari C.,San Raffaele Scientific Institute | Lerose R.,Hospital Pharmacy | Conteduca V.,Centro Of Riferimento Oncologico Of Basilicata | And 5 more authors.
Lung Cancer | Year: 2013

The discovery of several molecular alterations that underlie non-small cell lung cancer (NSCLC) pathogenesis has led to the development of targeted therapies. In particular, gefitinib and erlotinib have become the standard of care in patients harboring epidermal growth factor receptor mutations, while crizotinib showed an impressive efficacy in patients with ALK-positive NSCLC. Nevertheless, the occurrence of clinical resistance limits the long term results of these novel agents. The identification of the molecular mechanisms responsible for acquired resistance to targeted therapy is crucial in order to pursue the creation of rational strategies to overcome resistance. In the current review, we will focus on the acquired resistance mechanisms to EGFR-TKIs and crizotinib and the therapeutic strategies currently under study to overcome resistance. © 2013 Elsevier Ireland Ltd.


Cota G.F.,Laboratory of Clinical Research | Cota G.F.,Eduardo Of Menezes Hospital | de Sousa M.R.,Federal University of Minas Gerais | Fereguetti T.O.,Eduardo Of Menezes Hospital | Rabello A.,Laboratory of Clinical Research
PLoS Neglected Tropical Diseases | Year: 2013

Objective:We conducted a systematic literature review with indirect comparison of studies evaluating therapeutic efficacy and toxicity associated to visceral leishmaniasis (VL) therapy among HIV infected individuals.Main outcome measurements:The outcomes of interest were clinical and parasitological cure, mortality, and adverse events.Methods:PRISMA guidelines for systematic reviews and Cochrane manual were followed. Sources were MEDLINE, LILACS, EMBASE, Web of Knowledge databases and manual search of references from evaluated studies. We included all studies reporting outcomes after VL treatment, regardless of their design. Study quality was evaluated systematically by using the Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomized studies in meta-analyses. Comprehensive Meta-Analysis software v.2.2.048 was used to perform one-group meta-analysis of study arms with the same drug to estimate global rates of success and adverse events with each drug. These estimates were used, when possible, to indirectly compare treatment options, adjusted for CD4 count. Direct comparison was pooled when available.Results:Seventeen studies reporting five treatment regimens and outcome of 920 VL episodes occurring in HIV infected individuals were included. The main outstanding difference in outcome among the treatment regimens was observed in mortality rate: it was around 3 times higher with high-dose antimony use (18.4%, CI 95% 13.3-25%), indirectly compared to lipid formulations of amphotericin B treatment (6.1%, CI 95% 3.9-9.4%). It was observed, also by indirect comparison, higher rates of clinical improvement in study arms using amphotericin B than in study arms using pentavalent antimonial therapy (Sbv). The parasitological cure, an outcome that presented some degree of risk of selection and verification bias, had rates that varied widely within the same treatment arm, with high heterogeneity, hampering any formal comparison among drugs. One direct comparison of amphotericin and antimoniate was possible combining results of two studies and confirming the superiority of amphotericin.Conclusions:Available evidence suggests that amphotericin is superior to antimony treatment. Death rate using antimoniate high dose is unacceptably high. Randomized controlled trials are necessary to compare different formulations and doses of amphotericin, alternative therapies and drug combinations. © 2013 Cota et al.


Quaranta L.,University of Brescia | Riva I.,University of Brescia | Floriani I.C.,Laboratory of Clinical Research
European Journal of Ophthalmology | Year: 2013

Purpose: To evaluate the long-term outcomes of a surgical technique using a sutureless bovine pericardial patch graft for the implantation of an Ahmed glaucoma valve (AGV). Methods: This was a pilot study on patients with primary open-angle glaucoma refractory to repeated surgical filtering procedures. All patients underwent AGV implant technique using a sutureless bovine pericardial patch graft. The pericardial membrane was cut using an ordinary corneal trephine with a diameter of 9.0 or 10.0 mm. The anterior part of the tube was covered with the graft and kept in place with fibrin glue. Subsequently, the cap was stitched all around the tube and the dissected conjunctiva was laid over it. Intraocular pressure (IOP) and complications were evaluated 1 week and 1, 3, 6, 12, and 24 months after surgery. Results: The procedure was used to treat 20 eyes of 20 consecutive patients (12 men and 8 women: mean age [SD] 64.8 [7.8] years). Mean IOP was 28.1 mm Hg (SD 4.9) at baseline and decreased to 14.9 mm Hg (SD 1.5) 24 months after surgery (p<0.001). The overall mean number of topical medications was 3.1 (SD 0.5) at baseline and decreased to 1.4 (SD 0.8) after 24 months (p<0.001). During follow-up, there was no conjunctival erosion, thinning of pericardial patch graft over the tube, or tube exposure; no signs of endophthalmitis were recorded. Conclusions: The results suggest that the sutureless technique using a bovine pericardial graft patch is a safe and rapid procedure for AGV implantation. © 2013 Wichtig Editore.


Bhatnagar I.,Pukyong National University | Bhatnagar I.,Laboratory of Clinical Research | Thomas N.V.,Pukyong National University | Kim S.-K.,Pukyong National University
Anti-Cancer Agents in Medicinal Chemistry | Year: 2013

Cancer has long been an area of extensive research both at the molecular as well as pharmaceutical level. However, lack of understanding of the underlying molecular signalling and the probable targets of therapeutics is a major concern in successful treatment of cancer. The situation becomes even worse, with the increasing side effects of the existing synthetic commercial drugs. Natural compounds especially those derived from plants have been best explored for their anticancer properties and most of them have been efficient against the known molecular targets of cancer. However, advent of biotechnology and resulting advances in medical arena have let to the increasing knowledge of newer carcinogenic signaling agents which has made the anticancer drug discovery even more demanding. The present review aims to bring forward the molecular mediators of cancer and compiles the plant derived anticancer agents with special emphasis on their clinical status. Since marine arena has proved to be a tremendous source of pharmaceutical agents, this review also focuses on the anticancer potential of marine plants especially algae. This is a comprehensive review covering major aspects of cancer mediation and utilization of marine flora for remediation of this deadly disease. © 2013 Bentham Science Publishers.


Cota G.F.,Laboratory of Clinical Research | Cota G.F.,Hospital Eduardo Of Menezes | de Sousa M.R.,Federal University of Minas Gerais | Demarqui F.N.,Federal University of Minas Gerais | Rabello A.,Laboratory of Clinical Research
PLoS Neglected Tropical Diseases | Year: 2012

Background: Human visceral leishmaniasis (VL), a potentially fatal disease, has emerged as an important opportunistic condition in HIV infected patients. In immunocompromised patients, serological investigation is considered not an accurate diagnostic method for VL diagnosis and molecular techniques seem especially promising. Objective: This work is a comprehensive systematic review and meta-analysis to evaluate the accuracy of serologic and molecular tests for VL diagnosis specifically in HIV-infected patients. Methods: Two independent reviewers searched PubMed and LILACS databases. The quality of studies was assessed by QUADAS score. Sensitivity and specificity were pooled separately and compared with overall accuracy measures: diagnostic odds ratio (DOR) and symmetric summary receiver operating characteristic (sROC). Results: Thirty three studies recruiting 1,489 patients were included. The following tests were evaluated: Immunofluorescence Antibody Test (IFAT), Enzyme linked immunosorbent assay (ELISA), immunoblotting (Blot), direct agglutination test (DAT) and polimerase chain reaction (PCR) in whole blood and bone marrow. Most studies were carried out in Europe. Serological tests varied widely in performance, but with overall limited sensitivity. IFAT had poor sensitivity ranging from 11% to 82%. DOR (95% confidence interval) was higher for DAT 36.01 (9.95-130.29) and Blot 27.51 (9.27-81.66) than for IFAT 7.43 (3.08-1791) and ELISA 3.06 (0.71-13.10). PCR in whole blood had the highest DOR: 400.35 (58.47-2741.42). The accuracy of PCR based on Q-point was 0.95; 95%CI 0.92-0.97, which means good overall performance. Conclusion: Based mainly on evidence gained by infection with Leishmania infantum chagasi, serological tests should not be used to rule out a diagnosis of VL among the HIV-infected, but a positive test at even low titers has diagnostic value when combined with the clinical case definition. Considering the available evidence, tests based on DNA detection are highly sensitive and may contribute to a diagnostic workup. © 2012 Cota et al.


PubMed | Laboratory of Clinical Research
Type: Journal Article | Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer | Year: 2012

An understanding of the activated protein signaling architecture in non-small-cell lung cancer (NSCLC) is of critical importance to the development of new therapeutic approaches and identification of predictive and prognostic biomarkers for patient stratification.We used reverse-phase protein microarrays to map the activated protein signaling networks of 47 NSCLC tumors, 28 of which were node negative, which were subjected to tumor cellular enrichment using laser capture microdissection. The phosphorylation/cleavage levels of 111 key signaling proteins and total levels of 17 proteins were measured for broadscale signaling analysis.Pathway activation mapping of NSCLC revealed distinct subgroups composed of epidermal growth factor receptor (ERBB1), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3), v-erb-a erythroblastic leukemia viral oncogene homolog 4 (ERBB4), v-akt murine thymoma viral oncogene homolog 1- mammalian target of rapamycin (AKT-mTOR), protein kinase, AMP-activated, alpha 2 catalytic subunit (AMPK), and autophagy-related signaling, along with transforming growth factor-beta-signaling protein 1 (SMAD), insulin-line growth factor receptor (IGFR), rearranged during transfection proto-oncogene (RET), and activated CDC42-associated kinase (ACK) activation. Investigation of epidermal growth factor receptor (EGFR)-driven signaling identified a unique cohort of tumors with low EGFR protein expression yet high relative levels of phosphorylated EGFR and high EGFR total protein with low relative levels of phosphorylation. Last, mapping analysis of patients with NSCLC with N0 disease revealed a pilot pathway activation signature composed of linked epidermal growth factor receptor family (HER)-AMPK-AKT-mTOR signaling network along with focal adhesion kinase- LIM domain kinase-1 (FAK-LIMK) and janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways that correlated with short-term survival and aggressive disease.Functional protein pathway activation mapping of NSCLC reveals distinct activation subgroups that are underpinned by important therapeutic targets and that patients with early-stage node negative disease and poor prognosis may be identified by activation of defined, biochemically linked protein signaling events. Such findings, if confirmed in larger study sets, could help select and stratify patients for personalized targeted therapies.


PubMed | Laboratory of Clinical Research
Type: Journal Article | Journal: Experimental parasitology | Year: 2012

In vitro assays play an important role in the discovery and development of new antileishmanial drugs. The classic macrophage-amastigote models using murine peritoneal macrophages or human-monocyte derived macrophages as host cells are useful for drug screening. A major limitation of these models is the dependence on microscopic counting, a time-consuming and subjective method of analysis. The present study describes a detailed protocol for applying quantitative real-time PCR (qPCR) as an accurate and sensitive tool to assess parasite load in an amastigote-macrophage model. This assay can be performed in a standardized medium-to-high throughput procedure, replacing traditional readout of number of amastigote per macrophages by DNA load measurement.


PubMed | Laboratory of Clinical Research
Type: Comparative Study | Journal: PLoS neglected tropical diseases | Year: 2012

Human visceral leishmaniasis (VL), a potentially fatal disease, has emerged as an important opportunistic condition in HIV infected patients. In immunocompromised patients, serological investigation is considered not an accurate diagnostic method for VL diagnosis and molecular techniques seem especially promising.This work is a comprehensive systematic review and meta-analysis to evaluate the accuracy of serologic and molecular tests for VL diagnosis specifically in HIV-infected patients.Two independent reviewers searched PubMed and LILACS databases. The quality of studies was assessed by QUADAS score. Sensitivity and specificity were pooled separately and compared with overall accuracy measures: diagnostic odds ratio (DOR) and symmetric summary receiver operating characteristic (sROC).Thirty three studies recruiting 1,489 patients were included. The following tests were evaluated: Immunofluorescence Antibody Test (IFAT), Enzyme linked immunosorbent assay (ELISA), immunoblotting (Blot), direct agglutination test (DAT) and polimerase chain reaction (PCR) in whole blood and bone marrow. Most studies were carried out in Europe. Serological tests varied widely in performance, but with overall limited sensitivity. IFAT had poor sensitivity ranging from 11% to 82%. DOR (95% confidence interval) was higher for DAT 36.01 (9.95-130.29) and Blot 27.51 (9.27-81.66) than for IFAT 7.43 (3.08-1791) and ELISA 3.06 (0.71-13.10). PCR in whole blood had the highest DOR: 400.35 (58.47-2741.42). The accuracy of PCR based on Q-point was 0.95; 95%CI 0.92-0.97, which means good overall performance.Based mainly on evidence gained by infection with Leishmania infantum chagasi, serological tests should not be used to rule out a diagnosis of VL among the HIV-infected, but a positive test at even low titers has diagnostic value when combined with the clinical case definition. Considering the available evidence, tests based on DNA detection are highly sensitive and may contribute to a diagnostic workup.

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