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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Plasma matrix metalloproteinase-9 better predicts outcome than N-terminal protype-B natriuretic peptide in patients with systolic heart failure and a high prevalence of coronary artery disease.

Metalloproteinases have been proposed as biochemical markers of left ventricular (LV) remodeling in systolic heart failure (HF). However, their role in the prognostic stratification of these patients remains controversial. In the present study, we aimed at investigating the value of plasma metalloproteinases-3 and -9 in comparison with N-terminal protype-B natriuretic peptide in patients with systolic HF. One hundred and 27 consecutive patients hospitalized for systolic HF (LV ejection fraction < 45%) were enrolled. Coronary artery disease (CAD) was the aetiology in 67% of the study patients. Plasma metalloproteinases-3 and -9 and N-terminal protype-B natriuretic peptide levels were assessed. A complete echocardiographic and Doppler examination was also performed. Follow-up period was 24-15 months. On univariate analysis, a number of measurements predicted cardiac events in the following order of power: NYHA class >2, LV ejection fraction < 25%, metalloproteinases-9 > 238 ng/ml, mitral E wave deceleration time < 150 ms, N-terminal protype-B natriuretic peptide > 1586 pg/ml and metalloproteinases-3 > 15 ng/ml. However, on multivariate analysis the only independent variables of cardiac events were NYHA class (OR=2.26, p=0.059) and plasma metalloproteinases-9 (OR=2.00, p=0.029). On Kaplan-Meier survival analysis, patients with elevated levels of metalloproteinases-9 exhibited a significantly worse event free-survival at 45 months than those without (21% vs. 54%, log-rank: 13.93, p=0.0002). A worse survival was also observed in patients with elevated N-terminal protype-B natriuretic peptide levels with respect to those without (18% vs. 46%, log-rank: 9.11, p=0.025). Our results demonstrated the value of plasma metalloproteinases-9 levels for prognostication of patients with systolic HF and a high prevalence of CAD.

The methodological approach for the generation of human dendritic cells from monocytes affects the maturation state of the resultant dendritic cells.

Dendritic cells (DCs) are effective as antigen-presenting cells in the immune system and are present at two functional stages depending on their maturation state. For experimental investigation of this concept, CD14(+) monocytes from blood are isolated and cultured to generate in vitro the DCs needed for functional analysis. For positive selection of CD14(+) monocytes we compared two immunomagnetic bead technologies: MACS Separation, created by Miltenyi Biotec, and EasySep Selection, created by StemCell Technologies. The monocytes provided dendritic cells for their functional analysis. Lipopolysaccharide was added to cultured DCs to induce maturation. Although both systems generated DCs from the positively selected CD14(+) cells, there were certain differences between them. Morphological, phenotypic, and functional analysis showed that MACS-selection provided DCs that have typical features corresponding to day 6 or 7 of maturation. EasySep-DCs exist in a partially-mature state from day 6 onward, even without the addition of a maturation stimulus. The reason behind this partial maturation is possibly based on the dextran-coated beads that are associated with the EasySep product. Both methods provide pure and viable DCs, but we would recommend using the MACS system for obtaining DCs suitable for functional studies.

Deficiency and supplementation of PUFA in the diet have similar effects on the age-associated changes in rat-plasma cholesterol levels.

Levels of plasma cholesterol, particularly LDL cholesterol, increase with increasing age in humans and rodents. Feeding a fish oil-rich diet may exert hypocholesterolemic effects. The aim of this work was to examine the effects of a life-long administration of a PUFA-enriched diet and of a PUFA-deficient diet in male Sprague-Dawley rats on the age-associated increases in plasma cholesterol and triglycerides. Diet had small effects on body-weight, and had dramatic effects on liver phospholipids-fatty acids. Surprisingly, both diets counteracted the age-associated changes in plasma cholesterol and triglycerides similarly and benefits were already visible in adult rats.

TNF and sTNFR1/2 plasma levels in ALS patients.

The involvement of the immune system has been hypothesized in the pathogenesis of amyotrophic lateral sclerosis (ALS). In this study a significantly higher level of TNF-alpha and its soluble receptors, TNF-R1 and TNF-R2, has been found in plasma of patients affected by the sporadic form of ALS compared to normal subjects. The genetic analysis of the polymorphisms of TNF-alpha, TNF-R1 and TNF-R2 showed no statistically significant differences in alleles and genotype frequencies between patients and controls. These data suggest a participation of the immune system in response to as far unknown intracellular signals.

A case report of long-term remission of ulcerative colitis after lymphocyto-plasmapheresis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease of the colorectum, with mucosal infiltration by activated leukocytes, which are the result of complex interactions between lymphocytes, antigen, and dendritic cells (DCs). We carried out lymphocyto-plasmapheresis (LCPA) in a UC patient with the aim of removing lymphocytes, DCs and inflammatory cytokines (ICKs). A 42-year-old female with UC in moderate activity phase was submitted to 5 weekly LCPA treatments. Before and after LCPA we monitored: (i) the percentage of T, B, NK lymphocytes, monocytes, and peripheral blood lymphoid and myeloid DCs; (ii) the T lymphocyte subpopulations; (iii) the ICKs; and (iv) the immune complexes (IC). We achieved the interruption of all pharmacological therapies, and so far the clinical and histological remission has lasted for 24 months. The flow cytometric assessment of the leukocyte subpopulations did not show any relevant variation of their numbers after LCPA, while TNFalpha, IL-6, IL-12 and serum IgG-C1q ICs decreased. In the present case, the contemporary depletion of plasma, lymphocytes and DCs, allowed LCPA to emerge as an efficient alternative to UC pharmacological therapy without affecting the number of white blood cells, DCs and leukocyte subpopulations that were assessed. Further studies are needed both to address LCPA mechanism of action and optimal apheresis protocol, and to compare this form of therapy to a placebo control group.