Digital Droplet PCR is a Specific and Sensitive Tool for Detecting IDH2 Mutations in Acute Myeloid LeuKemia Patients.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) interfere with cellular metabolism contributing to oncogenesis. Mutations of IDH2 at R140 and R172 residues are observed in 20% of acute myeloid leukemias (AML), and the availability of the IDH2 inhibitor Enasidenib made IDH2 mutational screening a clinical need. The aim of this study was to set a new quantitative polymerase chain reaction (PCR) technique, the drop-off digital droplet PCR (drop-off ddPCR), as a sensitive and accurate tool for detecting IDH2 mutations. With this technique we tested 60 AML patients. Sanger sequencing identified 8/60 (13.5%) mutated cases, while ddPCR and the amplification refractory mutation system (ARMS) PCR, used as a reference technique, identified mutations in 13/60 (21.6%) cases. When the outcome of IDH2-mutated was compared to that of wild-type patients, no significant difference in terms of quality of response, overall survival, or progression-free survival was observed. Finally, we monitored IDH2 mutations during follow-up in nine cases, finding that IDH2 can be considered a valid marker of minimal residual disease (MRD) in 2/3 of our patients. In conclusion, a rapid screening of IDH2 mutations is now a clinical need well satisfied by ddPCR, but the role of IDH2 as a marker for MRD still remains a matter of debate.

The assessment of minimal residual disease versus that of somatic mutations for predicting the outcome of acute myeloid leukemia patients.

BACKGROUND: In addition to morphological and cytogenetic features, acute myeloid leukemias are characterized by mutations that can be used for target-therapy; also the minimal/measurable residual disease (MRD) could be an important prognostic factor. The purpose of this retrospective study was to investigate if somatic mutations could represent an additional prognostic value in respect of MRD alone. METHOD: At baseline, 98 patients were tested for NPM1, FLT3, and for WT1 expression; 31 for ASXL1, TET2, IDH1, IDH2, N-RAS, WT1, c-KIT, RUNX1, and DNMT3A. The same genes have been also tested after induction and consolidation. RESULTS: Overall, 60.2% of our patients resulted mutated: 24.5% carried mutations of FLT3-ITD, 38.7% of NPM1, 48.4% of c-KIT, 25.8% of N-RAS and 19.3% of IDH2. The probability of achieving a complete response (CR) was higher for younger patients, with low ELN risk score, NPM1-mutated, with low WT1 levels, and without FLT3. The presence of additional mutations represented a poor predictive factor: only 19% of these cases achieved CR in comparison to 43% of subjects without any of it. Concerning survival, it was conditioned by a lower ELN risk score, younger age, reduction > 1 log of the NPM1 mutational burden, disappearance of FLT3 mutations and lower WT1 expression. Regarding the role of the additional mutations, they impaired the outcome of 20% of the already MRD-negative patients. Concerning the possibility of predicting relapse, we observed an increase of the NPM1 mutational burden at the time-point immediately preceding the relapse (about 2 months earlier) in 50% of subjects. Similarly concerning WT1, an increase of its expression anticipated disease recurrence in 64% of cases. CONCLUSIONS: We demonstrated that additional somatic mutations are able to impair outcome of the already MRD-negative subjects. About MRD, we suggest a prognostic role also for the WT1 expression. Finally, we considered as relevant the assessment of NPM1 quantity clearance instead of the presence/absence of mutations alone. Still remains in doubt the utility in terms of long-term prognosis of a baseline more complex mutational screening; we could hypothesize that it would be useful for those patients where other markers are not available or who reached the MRD negativity.

Myelodysplastic syndromes: advantages of a combined cytogenetic and molecular diagnostic workup.

In this study we present a new diagnostic workup for the myelodysplastic syndromes (MDS) including FISH, aCGH, and somatic mutation assays in addition to the conventional cytogenetics (CC). We analyzed 61 patients by CC, FISH for chromosome 5, 7, 8 and PDGFR rearrangements, aCGH, and PCR for ASXL1, EZH2, TP53, TET2, RUNX1, DNMT3A, SF3B1 somatic mutations. Moreover, we quantified WT1 and RPS14 gene expression levels, in order to find their possible adjunctive value and their possible clinical impact. CC analysis showed 32% of patients with at least one aberration. FISH analysis detected chromosomal aberrations in 24% of patients and recovered 5 cases (13.5%) at normal karyotype (two 5q- syndromes, one del(7) case, two cases with PDGFR rearrangement). The aGCH detected 10 "new" unbalanced cases in respect of the CC, including one with alteration of the ETV6 gene. After mutational analysis, 33 patients (54%) presented at least one mutation and represented the only marker of clonality in 36% of all patients. The statistical analysis confirmed the prognostic role of CC either on overall or on progression-free-survival. In addition, deletions detected by aCGH and WT1 over-expression negatively conditioned survival. In conclusion, our work showed that 1) the addition of FISH (at least for chr. 5 and 7) can improve the definition of the risk score; 2) mutational analysis, especially for the TP53 and SF3B1, could better define the type of MDS and represent a "clinical warning"; 3) the aCGH use could be probably applied to selected cases (with suboptimal response or failure).

Matrix metalloproteinases and airway remodeling and function in primary ciliary dyskinesia.

BACKGROUND: The balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) is important in the regulation of airway damage. OBJECTIVE: To evaluate whether they are important in the pathophysiology of primary and secondary ciliary dyskinesia (PCD, SCD). METHODS: We measured sputum bacteriology, lung CT changes, MMPs, TIMPs and lung function in 86 patients (51 PCD, 35 SCD) in a cross-sectional study; the 10 controls studied did not have HRCT or sputum cultures. MMPs, TIMPs and lung function were evaluated longitudinally for up to one year in 38 PCD patients. RESULTS: At baseline, there were no differences in MMPs, TIMPs and MMPs/TIMPs, between PCD and SCD but lower levels were found in controls. There was an association between poorer lung function with increasing levels of MMPs in PCD, while in SCD only MMP-9/TIMP-1 values correlated with FRC z-scores. Levels of MMPs and TIMPs significantly correlated with severity HRCT changes. Longitudinally, there were significant correlations between slope of changes in spirometric parameters and slope of change in sputum MMPs in PCD patients. CONCLUSIONS: In conclusion, we report for the first time that increased MMPs are associated with worse airway damage in PCD and SCD, and thus are potential therapeutic targets.

The Droplet Digital PCR: A New Valid Molecular Approach for the Assessment of B-RAF V600E Mutation in Hairy Cell Leukemia.

Hairy cell leukemia (HCL) is a chronic lymphoproliferative B-cell disorder where the B-RAF V600E mutation has been recently detected, as reported for solid neoplasias but not for other B-cell lymphomas. The digital droplet PCR (dd-PCR) is a molecular technique that, without standard references, is able to accurately quantitate DNA mutations. ddPCR could be an useful instrument for the detection of the B-RAF V600E mutation in HCL, where the minimal residual disease monitoring is fundamental for planning a patients-targeted treatment in the era of new anti-CD20 and anti-RAF compounds. This retrospective study enrolled 47 patients observed at the Hematology Unit of the University of Pisa, Italy, from January 2005 to January 2014: 27 patients were affected by "classic" HCL, two by the variant HCL (vHCL), and 18 by splenic marginal zone lymphoma (SMZL). The aim of the study was to compare dd-PCR to "classic" quantitative PCR (QT-PCR) in terms of sensitivity and specificity and to demonstrate its possible use in HCL. Results showed that: (1) the sensitivity of dd-PCR is about half a logarithm superior to QT-PCR (5 × 10-5 vs. 2.5 × 10-4), (2) the specificity of the dd-PCR is comparable to QT-PCR (no patient with marginal splenic lymphoma or HCL variant resulted mutated), (3) its high sensitivity would allow to use dd-PCR in the monitoring of MRD. At the end of treatment, among patients in complete remission, 33% were still MRD-positive by dd-PCR versus 28% by QT-PCR versus 11% by the evaluation of the B-cell clonality, after 12 months, dd-PCR was comparable to QT-PCR and both detected the B-RAF mutation in 15% of cases defined as MRD-negative by IgH rearrangement. Moreover, (4) the feasibility and the costs of dd-PCR are comparable to those of QT-PCR. In conclusion, our study supports the introduction of dd-PCR in the scenario of HCL, also during the follow-up.

A clinical and laboratory study evaluating the profile of cytokine levels in relapsing remitting and secondary progressive multiple sclerosis.

The main aim of the study was to evaluate levels of cytokines IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, TNF-alfa, TGB-beta1 and IFN-gamma in 30 patients with relapsing remitting (RRMS) compared to 30 secondary progressive multiple sclerosis (SPMS) in a peripheral blood sample. Statistical analysis showed significant higher levels of IL-17 and INF-gamma, which are cytokines with pro-inflammatory properties, and lower levels of TGF-beta1, a molecule with immunosuppressant activity, in RRMS compared to SPMS. These results underline the existence of a different cytokines dysregulation in RRMS compared to SPMS phases with higher pro-inflammatory activity in RRMS.

Innovative approach to interpret the variability of biomarkers after ultra-endurance exercise: the multifactorial analysis.

AIMS: We assessed the inter-relationship that exists between variations of different biochemical and hematological parameters following strenuous endurance exercise in Ironmen by using multiple factor analysis (MFA). MFA was used to estimate the associations among groups of parameters in order to identify concurrent changes in many different biochemical variables. MATERIALS & METHODS: In total, 14 Ironman athletes were followed before and early after a race. MFA was applied to the parameters that showed a significant variation after the race, as we previously described in detail. Specifically, MFA standardizes data in each group and calculates the global axes (GAs), which are the linear combination of original parameters that maximize the global data variance. RESULTS: MFA identified three global axes (GAs) as significant, explaining approximately 62% of the global data variance. The first GA contained NT-proBNP, IL-1ra, IL-6, IL-8 and the oxidative index. The second and third GAs included calcium, creatinine, potassium, uric acid, hemoglobin, hematocrit and glucose. Analysis of the first two GAs showed that changes in the oxidative index were associated with variations in IL-8 and NT-proBNP. CONCLUSION: Among all the variables considered, MFA evidenced a close relationship between variations in oxidative stress, IL-8 and NT-proBNP, which may have a meaning in the mechanisms related to the physiological response after strenuous acute exercise.

Processing large-diameter poly(L-lactic acid) microfiber mesh/mesenchymal stromal cell constructs via resin embedding: an efficient histologic method.

In this study, we performed a complete histologic analysis of constructs based on large diameter ( >100 µm) poly-L-lactic acid (PLLA) microfibers obtained via dry-wet spinning and rat Mesenchymal Stromal Cells (rMSCs) differentiated towards the osteogenic lineage, using acrylic resin embedding. In many synthetic polymer-based microfiber meshes, ex post processability of fiber/cell constructs for histologic analysis may face deterring difficulties, leading to an incomplete investigation of the potential of these scaffolds. Indeed, while polymeric nanofiber (fiber diameter = tens of nanometers)/cell constructs can usually be embedded in common histologic media and easily sectioned, preserving the material structure and the antigenic reactivity, histologic analysis of large polymeric microfiber/cell constructs in the literature is really scant. This affects microfiber scaffolds based on FDA-approved and widely used polymers such as PLLA and its copolymers. Indeed, for such constructs, especially those with fiber diameter and fiber interspace much larger than cell size, standard histologic processing is usually inefficient due to inhomogeneous hardness and lack of cohesion between the synthetic and the biological phases under sectioning. In this study, the microfiber/MSC constructs were embedded in acrylic resin and the staining/reaction procedures were calibrated to demonstrate the possibility of successfully employing histologic methods in tissue engineering studies even in such difficult cases. We histologically investigated the main osteogenic markers and extracellular matrix molecules, such as alkaline phosphatase, osteopontin, osteocalcin, TGF-ß1, Runx2, Collagen type I and the presence of amorphous, fibrillar and mineralized matrix. Biochemical tests were employed to confirm our findings. This protocol permitted efficient sectioning of the treated constructs and good penetration of the histologic reagents, thus allowing distribution and expression of almost all the tested molecules to be revealed. Our results demonstrated that it is possible to perform histologic analyses of large-diameter PLLA-based microfiber scaffold/MSC constructs that face the failure of standard histologic procedures.

Balance of pro- and anti-inflammatory cytokines in cirrhotic patients undergoing liver transplantation.

This study was prospectively aimed at having better information about the natural history of serum cytokines in cirrhotic patients undergoing liver transplant surgery and at assessing their ability to set up an appropriate dynamic relationship between pro-inflammation and anti-inflammation. The levels of six cytokines (TNF-α, IL-1, IL-2, IL-6, IL-8, IL-10) were measured in blood samples collected at different time points before, during and after (48 h) the transplant procedure from the radial artery of 62 consecutive cirrhotic patients who underwent orthotopic liver transplantation. IL-1 always stayed within the normal range; IL-2 showed elevated baseline levels but decreased up to half at the end of the study (p<0.0001). IL-6 peaked at the end of surgery and returned to baseline 48 h afterwards. The same happened to IL-8 concentrations. IL-10 levels shown above the normal threshold at baseline, peaked at the end of surgery (p<0.0001) and were halved at the end of the study (p<0.0001). TNF-α peaked at the end of surgery without, however, being different from baseline levels (p=0.6). The physiologic pattern of cytokine release and their dynamic relationship was found to be preserved with a quick return to a balance between pro-inflammation and anti-inflammation as shown by the IL-6/IL-10 and TNF-α/IL 10 ratios (used to assess the inflammatory balance). A correlation was found between perioperative pro-inflammatory cytokine levels and the severity of the liver disease necessitating OLT. In summary, cirrhotic patients can achieve a balanced inflammatory response to surgery which is considered a primary requirement for uneventful grafts and patients' postoperative recovery.

Neuroendocrine markers and psychological features in patients with irritable bowel syndrome.

BACKGROUND AND AIMS: The key role of the brain-gut axis in the pathophysiology of irritable bowel syndrome (IBS) has been recognized. The aim of this study was to assess the possible association between IBS, neuroendocrine markers, and psychological features. METHODS: One hundred and twenty-five consecutive IBS patients and 105 healthy subjects were enrolled. Plasma serotonin, plasma and urinary cortisol, and plasma neuropeptide Y levels were evaluated. All patients were given a questionnaire to assess IBS symptom severity. In 66 patients, a psychodiagnostic assessment was carried out. RESULTS: A high incidence of specific psychological features, including state anxiety (69.69 %), trait anxiety (54.54 %), obsessions and compulsions (28.78 %), was observed in IBS patients. A positive correlation between neuropeptide Y and state anxiety (r = 0.287, p = 0.024) and simulation/social ingenuity (r = 0.269, p = 0.039) was found in these patients. In diarrhea-predominant IBS, plasma cortisol was linearly related to plasma serotonin (r = 0.5663, p < 0.001). CONCLUSIONS: In IBS patients, a significant correlation was found between specific psychological features and neuroendocrine markers, especially plasma cortisol and neuropeptide Y; in diarrhea-predominant IBS, a correlation between plasma cortisol and serotonin was found, although it needs to be confirmed in more extensive cohorts.

Interactions between immune, stress-related hormonal and cardiovascular systems following strenuous physical exercise.

Physical exercise represents a eustress condition that promotes rapid coordinated adjustments in the immune, stress-related hormonal and cardiovascular systems, for maintaining homeostasis in response to increased metabolic demands. Compared to the tight multisystem coordination during exercise, evidence of between-systems cross talk in the early post exercise is still lacking. This study was aimed at identifying possible interactions between multiple systems following strenuous physical exercise (Ironman race) performed by twenty well-trained triathletes. Cardiac hemodynamics, left ventricle systolic and diastolic function and heart rate variability were measured along with plasma concentrations of immune messengers (cytokines and C-reactive protein) and stress-related hormones (catecholamines and cortisol) both 24h before and within 20 min after the race. Observed changes in antiinflammatory pathways, stress-related hormones and cardiovascular function were in line with previous findings; moreover, correlating parameters' changes (post versus pre-race) highlighted a dependence of cardiovascular function on the post-race biohumoral milieu: in particular, individual post-race variations of heart rate and diastolic function were strongly correlated with individual variations of anti-inflammatory cytokines, while individual baroreflex sensitivity changes were linked to IL-8 increase. Multiple correlations between anti-inflammatory cytokines and catecholamines were also found according with the autonomic regulation of immune function. Observed post-race cytokine and hormone levels were presumptively representative of the increases reached at the effort end while the cardiovascular parameters after the race were measured during the cardiovascular recovery; thus, results suggest that sustained strenuous exercise produced a stereotyped cardiovascular early recovery, whose speed could be conditioned by the immune and stress-related hormonal milieu.

Plasma metalloproteinase-9 and restrictive filling pattern as major predictors of outcome in patients with ischemic cardiomyopathy.

OBJECTIVE: Assessment of plasma matrix metalloproteinase-9 (MMP-9) and Doppler markers of increased left ventricular (LV) filling pressure may be added to risk stratify patients with ischemic cardiomyopathy (IC). Therefore, we aimed at investigating the value of plasma MMP-9 and restrictive filling pattern (RFP) in IC patients. METHODS: Eighty-eight consecutive patients hospitalized for heart failure (LV ejection fraction ≤ 40%) due to IC were enrolled. A complete M-mode and two-dimensional echo-Doppler examination were performed. Patients were defined as having RFP if they had a mitral E wave deceleration time<150 ms. Plasma MMP-9 and N-terminal protype-B natriuretic peptide levels were assessed at the time of the index echocardiogram. The end point was all-cause mortality or hospitalization for worsening HF. Follow-up period was 25 ± 17 months. RESULTS: Median value of MMP-9 was 714 ng/ml. On univariate analysis, a number of measurements predicted the composite end point: NYHA class>2, RFP, MMP-9>60.5 ng/ml, LV ejection fraction<27%, anemia, pulmonary pressure ≥ 35 mm Hg, N-terminal protype-B natriuretic peptide>1742 pg/ml, and glomerular filtration rate<60 ml/min/1.73 m(2). Independent variables of outcome were anemia (HR=1.9, p=0.031), and the combination of plasma MMP-9 and RFP (HR=3.2, p=0.004). On Kaplan-Meier survival curves, patients with elevated MMP-9 levels and RFP had the lowest event-free survival rate (log-rank: 29.0, p<0.0001). The net reclassification improvement showed a significant increase in the prediction model when elevated MMP-9 and RFP were added to the base model that included clinical, biochemical and echocardiographic parameters (p<0.0001). CONCLUSION: MMP-9 levels and RFP have an incremental predictive value to risk classify IC patients.

Early subclinical increase in pulmonary water content in athletes performing sustained heavy exercise at sea level: ultrasound lung comet-tail evidence.

Whether prolonged strenuous exercise performed by athletes at sea level can produce interstitial pulmonary edema is under debate. Chest sonography allows to estimate extravascular lung water, creating ultrasound lung comet-tail (ULC) artifacts. The aim of the study was to determine whether pulmonary water content increases in Ironmen (n = 31) during race at sea level and its correlation with cardiopulmonary function and systemic proinflammatory and cardiac biohumoral markers. A multiple factor analysis approach was used to determine the relations between systemic modifications and ULCs by assessing correlations among variables and groups of variables showing significant pre-post changes. All athletes were asymptomatic for cough and dyspnea at rest and after the race. Immediately after the race, a score of more than five comet tail artifacts, the threshold for a significant detection, was present in 23 athletes (74%; 16.3 ± 11.2; P < 0.01 ULC after the race vs. rest) but decreased 12 h after the end of the race (13 athletes; 42%; 6.3 ± 8.0; P < 0.01 vs. soon after the race). Multiple factor analysis showed significant correlations between ULCs and cardiac-related variables and NH(2)-terminal pro-brain natriuretic peptide. Healthy athletes developed subclinical increase in pulmonary water content immediately after an Ironman race at sea level, as shown by the increased number of ULCs related to cardiac changes occurring during exercise. Hemodynamic changes are one of several potential factors contributing to the mechanisms of ULCs.

ITF2357 interferes with apoptosis and inflammatory pathways in the HL-60 model: a gene expression study.

BACKGROUND: Cytotoxic and pro-apoptotic effects exerted by the histone deacetylase inhibitor ITF2357 have been reported in acute myeloid leukemia HL-60 cells. In the current study, its mechanism of action was investigated at the molecular level. MATERIALS AND METHODS: Cell proliferation was evaluated by methyl thiazol tetrazolium bromide reduction; apoptosis by annexin V, mitochondrial transmembrane potential by tetramethylrhodamine ethyl ester. Functional experiments and gene expression evaluations were performed by flow cytometry, microarray, and quantitative polymerase chain reaction. RESULTS: Significant cell growth inhibition and increased apoptosis were observed. ITF2357 reduced protein levels of BCL-2, MCL-1, and BCL-X, and increased levels of BAK. Exposure to ITF2357 did not abrogate NF-κB DNA binding. After microarray analysis, interleukin-10, interleukin-6, epidermal growth factor, peroxisome proliferator-activated receptor (PPAR), transforming growth factor ß, P38 mitogen-activated protein kinase, aryl hydrocarbon receptor, xenobiotic metabolism, PPAR/retinoic acid receptor, NF-κB, apoptosis, lipopolysaccharide/interleukin-1, G-protein receptor, T-cell receptor, and platelet-derived growth factor were the de-regulated pathways. CONCLUSION: This study shows that ITF2357 influences both proliferation and inflammatory pathways in HL-60 cells; this observation could have possible applications in clinical practice.

Intensive risk-adjusted follow-up with the CEA, TPA, CA19.9, and CA72.4 tumor marker panel and abdominal ultrasonography to diagnose operable colorectal cancer recurrences: effect on survival.

HYPOTHESIS: Intensive risk-adjusted follow-up leads to improved resectability of tumor recurrences and better overall survival among patients who have undergone surgery for colorectal cancer. DESIGN: Long-term observational single-center study. SETTING: University of Pisa, Pisa, Italy. PATIENTS: One hundred eight disease-free patients who had undergone surgery for colorectal cancer were submitted to long-term follow-up with the serum CEA, TPA, CA19.9, and CA72.4 tumor marker (TM) panel and abdominal ultrasonography. MAIN OUTCOME MEASURES: Sensitivities and specificities of TMs, abdominal ultrasonography, and abdominal and chest computed tomography (CT); the median survival among patients operated on and those not operated on and the cumulative 5-year overall survival among the entire group. RESULTS: Twenty-two patients with asymptomatic colorectal cancer recurred 32 times. The CEA, TPA, CA19.9, CA72.4, and TM panel sensitivities were 46.9%, 34.4%, 9.4%, 9.4%, and 81.0%, respectively, and the mean (SD) lead times before confirmation of recurrence were 4.3 (4.8), 4.1 (4.7), 8.3 (10.9), 5.0 (7.0), and 5.3 (5.8) months, respectively. Abdominal and chest CT sensitivities were 100.0%. Among 86 patients without recurrence, specificities of the TM panel and all panel markers were 100.0%, while specificities of abdominal ultrasonography, abdominal CT, and skeletal CT were 99.9%, 99.0%, and 100.0%, respectively. The median survival after first recurrence was 16 months (range, 3-48 months) for 8 patients with recurrence who did not undergo second-line surgery. Among 14 remaining patients who underwent metastasectomy, the median survival after first recurrence was 37 months (range, 12-187 months; P = .03). Among the entire group of 108 patients, the cumulative 5-year overall survival was 88.7%. CONCLUSIONS: Long-term intensive risk-adjusted monitoring using the CEA, TPA, CA19.9, and CA72.4 TM panel and abdominal ultrasonography allows early detection of most recurrences. Patients can then undergo radical metastasectomy, with potentially improved overall survival.

CXCL9 and CXCL11 chemokines modulation by peroxisome proliferator-activated receptor-alpha agonists secretion in Graves' and normal thyrocytes.

CONTEXT: Peroxisome proliferator-activated receptor (PPAR)-α has been shown to exert immunomodulatory effects in autoimmune disorders. However, until now, no data were present in the literature about the effect of PPARα activation on CXCL9 and CXCL11 chemokines in general or on secretion of these chemokines in thyroid cells. OBJECTIVE AND DESIGN: The presence of PPARα and PPARγ has been evaluated by real-time-PCR in Graves' disease (GD) and control cells in primary culture. Furthermore, we have tested the role of PPARα and PPARγ activation on CXCL9 and CXCL11 secretion in GD and control cells after stimulation of these chemokines secretion with IFNγ and TNFα. RESULTS: This study shows the presence of PPARα and PPARγ in GD and control cells. A potent dose-dependent inhibition by PPARα-agonists was observed on the cytokines-stimulated secretion of CXCL9 and CXCL11 in GD and control cells. The potency of the PPARα agonists used was maximum on the secretion of CXCL9, reaching about 90% of inhibition by fenofibrate and 85% by ciprofibrate. The relative potency of the compounds was different with each chemokine; for example, gemfibrozil exerted a 55% inhibition on CXCL11, whereas it had a weaker activity on CXCL9 (40% inhibition). PPARα agonists were stronger (ANOVA, P<0.001) inhibitors of CXCL9 and CXCL11 secretion in thyrocytes than PPARγ agonists. CONCLUSIONS: Our study shows the presence of PPARα in GD and control thyrocytes. PPARα activators are potent inhibitors of the secretion of CXCL9 and CXCL11, suggesting that PPARα may be involved in the modulation of the immune response in the thyroid.

Synergistic antiproliferative effect of arsenic trioxide combined with bortezomib in HL60 cell line and primary blasts from patients affected by myeloproliferative disorders.

Both arsenic trioxide (ATO) and bortezomib show separate antileukemic activity. With the purpose of evaluating whether the combination of ATO and bortezomib would be an option for patients with acute leukemia, we incubated HL60 leukemic cells with ATO alone and in combination with bortezomib. ATO and bortezomib cooperated to induce cell death and to inhibit proliferation and apoptosis in a synergistic way. The combined treatment resulted in a stronger activation of caspase 8 and 9, moderate activation of caspase 3, and increased expression of Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-DR5 receptors. When bortezomib was added, some proapoptotic genes (CARD9, TRAIL) were upregulated, and some antiapoptotic genes (BCL2, BCL3, FLICE) were downregulated. When coincubated, approximately 80% of cells showed altered mitochondrial membrane permeability. Moreover, ATO alone and in combination with bortezomib abrogated DNA-binding activity of nuclear factor kappa beta (NF-kappaB). Gene expression assays showed that more deregulated genes were related to proliferation of leukocytes, tumorigenesis, control of cell cycle, hypoxia and oxidative stress, cytokines, PI3K-AKT, ERK-MAPK, EGF pathways, and ubiquitination. Finally, in three cases of acute myeloid leukemia, the addition of bortezomib to ATO significantly increased cytotoxicity. We conclude that the combination of bortezomib and ATO may be efficacious in the treatment of myeloid disorders.

Standard versus video-assisted thyroidectomy: objective postoperative pain evaluation.

BACKGROUND: This prospective, randomized study was designed to objectively demonstrate that minimally invasive video-assisted thyroidectomy (MIVAT) improves postoperative pain compared with standard thyroidectomy, via the dosage of biochemical mediators measured before and after surgery. METHODS: Forty-nine patients undergoing total thyroidectomy were allotted to MIVAT (n = 23) or traditional thyroidectomy (OPEN) (n = 26) groups. At hospitalization (T0), interleukin (IL)-1, -2, -4, -6, -10, -3, tumor necrosis factor (TNF)-α, TGF-ß, and MCP-1 were measured. The basal pain tolerance also was evaluated by VAS. Blood samples for interleukin measurement and VAS evaluations were obtained from all patients in the recovery room (T1) and 24 h after surgery (T2). RESULTS: At T0, the MIVAT and the OPEN groups were not different in terms of basal pain tolerance and biochemical profile. At T1, VAS scores were significantly higher (p = 0.04), whereas TGF-ß (p = 0.03) and MCP-1 (p = 0.03) levels were significantly lower in the OPEN than in the MIVAT group. No significant difference was demonstrated for other interleukins. A significant inverse relationship between VAS and TGF-ß was demonstrated and confirmed through the correlation (p = 0.003) and regression (p = 0.003, p < 0.0001, R (2) = 0.172) coefficients; the stepwise regression also demonstrated that TGF was the most predictive factor of postoperative pain (p = 0.0038) through an inverse relationship. No statistically significant difference has been demonstrated at T2. CONCLUSIONS: TGF-ß serum levels immediately after surgery seem to correlate with pain evaluation, confirming that reduced postoperative distress is an objective outcome of MIVAT. This result confirms the results of studies based only on subjective pain evaluations.

Circulating matrix metalloproteinase-3 and metalloproteinase-9 and tissue Doppler measures of diastolic dysfunction to risk stratify patients with systolic heart failure.

Abnormal matrix metalloproteinase (MMP) activity and diastolic dysfunction may affect left ventricular (LV) remodeling and prognosis, but it is not known whether the combined evaluation of MMP-3 and MMP-9 and variables of diastolic dysfunction are useful for the risk stratification of patients with systolic heart failure (HF). Therefore, this study was designed to assess the value of combining circulating levels of MMPs and tissue Doppler measures of LV diastolic dysfunction to risk-stratify patients with systolic HF. Consecutive patients with systolic HF due to either ischemic or nonischemic cardiomyopathy (n = 134) and LV ejection fractions <45% were submitted to complete echocardiographic and Doppler examinations. The ratio of mitral E peak velocity and averaged e' velocity (E/e') was calculated. Plasma levels of MMP-3 and MMP-9 were measured at the time of index echocardiography. All-cause mortality was defined as the end point. The mean LV ejection fraction was 28 +/- 9%. There was a total of 32 deaths during follow-up (24 +/- 14 months). Several clinical, biochemical, Doppler, and echocardiographic parameters were associated with the outcome on univariate Cox regression analysis. After statistical adjustment for the potentially confounding factors by multivariate analysis, E/e' (hazard ratio 1.11, p = 0.0028), ejection fraction (hazard ratio 0.92, p = 0.017), and MMP-9 (hazard ratio 1.01, p = 0.027) remained significant independent predictors of the end point. Kaplan-Meier curves showed that survival was worse in patients with E/e' ratios >/=13 and MMP-9 levels >89.9 ng/mL (p <0.0001). In conclusion, the assessment of circulating MMP levels and tissue Doppler measures of LV diastolic dysfunction may improve the prognostic stratification of patients with systolic HF.

Time course of oxidant markers and antioxidant defenses in subgroups of amyotrophic lateral sclerosis patients.

Oxidative stress markers have been found in nervous and peripheral tissues of familial and sporadic amyotrophic lateral sclerosis patients. Here, we evaluated the activity of some antioxidant enzymes glutathione peroxidase, glutathione reductase and Cu-Zn superoxide dismutase in erythrocyte, the marker of non-enzymatic antioxidant response (total antioxidant status), as well as plasma reactive oxygen species, at the enrolment and during disease progression in 88 patients affected by the sporadic form of amyotrophic lateral sclerosis. Our study has been performed along 72 months by grouping the patients according to the ALS functional rating score or rate of disease progression. Our results showed a significant impairment of erythrocytes glutathione peroxidase activity in all groups of patients that remained low during disease time course. SOD1 activity significantly decreased along disease course in subjects with a more impaired functional status. A decreasing activity of all assayed enzymes was found in patients who have a faster disease progression rate. By this work we have the evidence that different ALS phenotypes present with different profile of enzymatic and non-enzymatic antioxidant response.