Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study.

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p < 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large (> 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

Peripheral imbalanced TFH/TFR ratio correlates with intrathecal IgG synthesis in multiple sclerosis at clinical onset.

BACKGROUND: Alteration of T-follicular helper (TFH) and regulatory (TFR) subpopulations may contribute to the development of auto-reactive B-cell. OBJECTIVE: To investigate whether changes in TFH and TFR subsets are associated with abnormal IgG synthesis in blood and cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. METHODS: Paired blood and CSF samples were obtained from 31 untreated relapsing-remitting multiple sclerosis (RRMS) patients at diagnosis. Peripheral blood TFH (CD3+CD4+CXCR5+CD25-CD127+), TFR (CD3+CD4+CXCR5+CD25+CD127dim), conventional T-Helper (TH, CD3+CD4+CXCR5-CD25-CD127+), and regulatory T-cells (T-Reg, CD3+CD4+CXCR5-CD25+CD127dim) were analyzed in all RRMS patients and in 13 healthy controls (HCs). Qualitative and quantitative intrathecal IgG synthesis was evaluated in RRMS patients, who were then further subclassified according to the presence of IgG oligoclonal bands in blood and/or CSF. RESULTS: Compared to HC, RRMS had lower TFR percentage ( p < 0.01) and higher TFH/TFR ratio ( p < 0.001). In RRMS, TFH/TFR ratio correlated with both qualitative ( r = 0.56, p < 0.005) and quantitative intrathecal IgG synthesis (IgG Index: r = 0.78; IgGLoc: r = 0.79; IgGIF: r = 0.76, all p < 0.001). Patients with the highest TFH/TFR ratios had higher percentages of circulating B-cells (36.1 ± 35.2%, p < 0.05). CONCLUSION: In RRMS, increased TFH/TFR ratio associates with abnormal IgG production in blood and CSF, suggesting that antibody-producing cells, derived from deregulated peripheral germinal center reaction, colonize the CNS.

Thoughts modulate the expression of inflammatory genes and may improve the coronary blood flow in patients after a myocardial infarction.

BACKGROUND: Mental stress is one of the main risk factors for cardiovascular disease. Meditation and music listening are two techniques that are able to counteract it through the activation of specific brain areas, eliciting the so-called Relaxing Response (RR). Epidemiological evidence reveals that the RR practice has a beneficial prognostic impact on patients after myocardial infarction. We aimed to study the possible molecular mechanisms of RR underlying these findings. METHODS: We enrolled 30 consecutive patients after myocardial infarction and 10 healthy controls. 10 patients were taught to meditate, 10 to appreciate music and 10 did not carry out any intervention and served as controls. After training, and after 60 days of RR practice, we studied the individual variations, before and after the relaxation sessions, of the vital signs, the electrocardiographic and echocardiographic parameters along with coronary flow reserve (CFR) and the carotid's intima media thickness (IMT). Neuro-endocrine-immune (NEI) messengers and the expression of inflammatory genes (p53, Nuclear factor Kappa B (NfKB), and toll like receptor 4 (TLR4)) in circulating peripheral blood mononuclear cells were also all observed. RESULTS: The RR results in a reduction of NEI molecules (p < 0.05) and oxidative stress (p < 0.001). The expression of the genes p53, NFkB and TLR4 is reduced after the RR and also at 60 days (p < 0.001). The CFR increases with the relaxation (p < 0.001) and the IMT regressed significantly (p < 0.001) after 6 months of RR practice. CONCLUSIONS: The RR helps to advantageously modulate the expression of inflammatory genes through a cascade of NEI messengers improving, over time, microvascular function and the arteriosclerotic process.

Acute immunomodulatory changes during controlled ovarian stimulation: evidence from the first trial investigating the short-term effects of estradiol on biomarkers and B cells involved in autoimmunity.

PURPOSE: The aim of the present study was to evaluate the in vivo immunomodulatory effects of an acute short-term estradiol (E(2)) increase on serum levels of B cell-activating factor (BAFF), immunoglobulins (Ig), anti-nuclear antibodies (ANA), and the peripheral B cell phenotype. METHODS: We conducted, at the Infertility Center of the University of Padua, a prospective case-control study on a cohort of infertile normo-responder women (group-A, 63 patients) undergoing controlled ovarian stimulation (COS) compared with an age-matched cohort of normo-ovulatory healthy women (group-B, 39 patients). Three serial blood sample assays were conducted in both groups, at T0, hypothalamic suppression; T1, ovulation induction; and T2, ßhCG test in group A, and at T0, 2nd day; T1, 14th day; and T2, 21st day of cycle in group B, and serum levels of E(2) and BAFF, BAFF/E(2) ratio, circulating IgM, IgG, and IgA, ANA titer, and peripheral B cell phenotype were measured. We compared group-A versus group-B in terms of absolute and E(2) normalized values of BAFF at baseline (T0) to verify for possible differences between healthy and infertile women, at T1 to verify for possible differences occurring after spontaneous ovulation versus COS, and at T2 to evaluate differences in serum BAFF levels between pregnant versus non-pregnant patients (considering only group-A) and between non-pregnant women after spontaneous versus COS cycles (group-B versus group-A). In group-A, we also evaluated IgM, IgG, IgA levels, ANA titer, and peripheral B cell phenotype at T0 versus T1 versus T2. RESULTS: With the exception of E(2) levels at T1 (as expected), no significant differences were found between the two groups for all outcome measures. In group-A, BAFF at T0 positively correlated with IgM levels; marginal zone CD19+/CD27+/IgD+ memory B cell compartment tended to be expanded at T1 when compared with T0. CONCLUSIONS: Despite several mechanistic and clinical studies supporting a stimulatory role of E(2) on autoimmunity, the acute increase of E(2) during COS for infertility treatment does not seem to have a major impact on the immune system.

Circulating Stem Cells Associate With Adiposity and Future Metabolic Deterioration in Healthy Subjects.

CONTEXT: Obesity and metabolic syndrome are associated with mild leukocytosis, but whether hematopoietic stem/progenitor cells (HSPCs) play a role in metabolic deterioration is unknown. OBJECTIVE: Our objective was to analyze the cross-sectional and longitudinal associations between CD34(+) HSPCs, adiposity, and metabolic syndrome features. DESIGN: This is a cross-sectional study on 242 participants, 155 of whom were followed and included in a longitudinal assessment. SETTING: This study took place in a tertiary referral center for metabolic diseases. PARTICIPANTS: Healthy working individuals attending a cardiovascular screening program (total n = 3158) and having a baseline measure of circulating CD34(+) cells participated. MAIN OUTCOME MEASURES: We collected demographic and anthropometric data, cardiovascular risk factors, and metabolic syndrome parameters. RESULTS: Participants (34.7% males, mean age 45.9 ± 0.5 years) were free from diabetes and cardiovascular disease. Cross-sectionally, absolute CD34(+) cell counts were directly correlated with body mass index and waist circumference, inversely correlated with high-density lipoprotein cholesterol and the quantitative insulin sensitivity check index, and were higher in individuals with the metabolic syndrome. The hematopoietic component contributed most to the association of CD34(+) cells with adiposity. During a 6.3-year follow-up, high absolute levels of CD34(+) cells were associated with increasing waist circumference, declining quantitative insulin sensitivity check index and high-density lipoprotein cholesterol, and with incidence of metabolic syndrome. Relative CD34(+) cell counts showed weaker associations with metabolic parameters than absolute levels, but were longitudinally associated with increasing waist circumference and metabolic syndrome development. CONCLUSIONS: A mild elevation of circulating CD34(+) progenitor cells, reflecting expansion of HSPCs, is associated with adiposity and future metabolic deterioration in healthy individuals.

Biological variability of lymphocyte subsets of human adults' blood.

BACKGROUND: Alterations in lymphocyte subpopulations are present in several immune diseases, and clinicians and researchers recognise the importance of investigating the distribution and changes in lymphocyte subsets over relatively long periods of time in order to evaluate the effectiveness of treatment and follow the course of disease. Yet further insight is required on the biological variability (BV) of lymphocyte subsets, which is crucial to the correct interpretation of longitudinal changes and provides essential information for setting desirable quality specifications and defining the usefulness of reference values. METHODS: Four-colour-flow cytometry was used to investigate the BV of lymphocyte populations (LP) in the peripheral blood of 20 healthy adults recruited from our laboratory staff and followed for three months. The total lymphocyte count was measured, and the relative frequencies determined for T-cells (CD3+), T-helper cells (CD3+CD4+), cytolytic T-cells (CD3+CD8+), B-cells (CD3-CD19+), NK-cells (CD3-CD16+/56+), non-MHC restricted cytolytic T-cells (CD3+CD56+) and activated T-cells (CD3+HLA-DR+). RESULTS AND CONCLUSIONS: Data on the components of BV were applied to set quality specifications for allowable precision, bias and total error. Analytical performances were established, and they were more than desirable for all the markers considered in our study. By comparing within-subject and between-subjects BV, we were able to define the uselessness of reference ranges in the evaluation of changes in CD serial results. Data on within-subject BV and analytical precision were thus used to determine the reference change values, in order to identify the significance of changes in serial results. The findings made in the present study provide further evidence of the relevance of BV in the evaluation of immunological markers of LP.

Pancreatic cancer alters human CD4+ T lymphocyte function: a piece in the immune evasion puzzle.

OBJECTIVES: To verify whether the dysregulation of CD4 T cells concurs in worsening the outcome of pancreatic cancer, we compared the effects of pancreatic cancer and other gastrointestinal cancer cell-conditioned media on the (1) proliferation, migration, and differentiation of CD4 T cells and (2) expansion of CD4 memory (CD45RO), naive (CD45RA), activated (CD69), and regulatory (CD25) subsets. METHODS: After culture of CD4 T cells in control, pancreatic (BxPC3, Capan1, MiaPaCa2), or gastrointestinal cancer (AGS, HepG2, HT29) cell-conditioned media, we evaluated proliferation, migration, interferon γ (IFNγ) production, and CD45RA, CD45RO, CD69, and CD25 membrane expression in control and conditioned CD4 T cells. RESULTS: Only pancreatic cancer-conditioned media (1) inhibited CD4 T-cell proliferation (P < 0.001) and migration under human stromal cell-derived factor-α chemotaxis (P < 0.001) and (2) induced CD4 T-cell IFNγ production (P < 0.05) and the expansion of the CD69-positive subset (P < 0.001) with respect to the control, with no changes being found in the CD45RA, CD45RO, and CD25 subsets. CONCLUSIONS: The in vitro findings achieved in the present study demonstrate that pancreatic cancer cells inhibit CD4 T-cell proliferation and migration, induce IFNγ production, and favor a CD69 subset expansion, suggesting that CD4 T cells play an important role in pancreatic cancer immune evasion.

Extraction methods of red blood cell membrane proteins for Multidimensional Protein Identification Technology (MudPIT) analysis.

Since red blood cells (RBCs) lack nuclei and organelles, cell membrane is their main load-bearing component and, according to a dynamic interaction with the cytoskeleton compartment, plays a pivotal role in their functioning. Even if erythrocyte membranes are available in large quantities, the low abundance and the hydrophobic nature of cell membrane proteins complicate their purification and detection by conventional 2D gel-based proteomic approaches. So, in order to increase the efficiency of RBC membrane proteome identification, here we took advantage of a simple and reproducible membrane sub-fractionation method coupled to Multidimensional Protein Identification Technology (MudPIT). In addition, the adoption of a stringent RBC filtration strategy from the whole blood, permitted to remove exhaustively contaminants, such as platelets and white blood cells, and to identify a total of 275 proteins in the three RBC membrane fractions collected and analysed. Finally, by means of software for the elaboration of the great quantity of data obtained and programs for statistical analysis and protein classification, it was possible to determine the validity of the entire system workflow and to assign the proper sub-cellular localization and function for the greatest number of the identified proteins.

Antioxidant defences in cybrids harboring mtDNA mutations associated with Leber's hereditary optic neuropathy.

Oxidative stress and imbalance between free radical generation and detoxification may play a pivotal role in the pathogenesis of Leber's hereditary optic neuropathy (LHON). Mitochondria, carrying the homoplasmic 11778/ND4, 3460/ND1 and 14484/ND6 mtDNA point mutations associated with LHON, were used to generate osteosarcoma-derived cybrids. Enhanced mitochondrial production of reactive oxygen species has recently been demonstrated in these cybrids [Beretta S, Mattavelli L, Sala G, Tremolizzo L, Schapira AHV, Martinuzzi A, Carelli V & Ferrarese C (2004) Brain 127, 2183-2192]. The aim of this study was to characterize the antioxidant defences of these LHON-affected cells. The activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutases (SOD) and catalase, and the amounts of glutathione (GSH) and oxidized glutathione (GSSG) were measured in cybrids cultured both in glucose-rich medium and galactose-rich medium. The latter is known to cause oxidative stress and to trigger apoptotic death in these cells. In spite of reduced SOD activities in all LHON cybrids, and of low GPx and GR activities in cells with the most severe 3460/ND1 and 11778/ND4 mutations, GSH and GSSG content were not significantly modified in LHON cybrids cultured in glucose medium. In contrast, in galactose, GSSG concentrations increased significantly in all cells, indicating severe oxidative stress, whereas GR and MnSOD activities further decreased in all LHON cybrids. These data suggest that, in cells carrying LHON mutations, there is a decrease in antioxidant defences, which is especially evident in cells with mutations associated with the most severe clinical phenotype. This is magnified by stressful conditions such as exposure to galactose.