Giorgia Pantano

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.

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 &ggr; (IFN&ggr;) 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-&agr; chemotaxis (P < 0.001) and (2) induced CD4+ T-cell IFN&ggr; 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 IFN&ggr; production, and favor a CD69+ subset expansion, suggesting that CD4+ T cells play an important role in pancreatic cancer immune evasion.

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.

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

PurposeThe aim of the present study was to evaluate the in vivo immunomodulatory effects of an acute short-term estradiol (E2) increase on serum levels of B cell-activating factor (BAFF), immunoglobulins (Ig), anti-nuclear antibodies (ANA), and the peripheral B cell phenotype.MethodsWe 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, βhCG 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 E2 and BAFF, BAFF/E2 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 E2 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.ResultsWith the exception of E2 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.ConclusionsDespite several mechanistic and clinical studies supporting a stimulatory role of E2 on autoimmunity, the acute increase of E2 during COS for infertility treatment does not seem to have a major impact on the immune system.

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 carotids 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.

Polyclonal and monoclonal B lymphocytes response in HCV-infected patients treated with direct-acting antiviral agents

Hepatitis C virus (HCV) chronic infection can be associated with extrahepatic manifestations such as mixed cryoglobulinaemia and lymphoproliferative disorders that are endowed with increased rates of morbidity and all‐cause mortality. In this study, we used flow cytometry to evaluate the effect of interferon‐free antiviral treatment on peripheral blood lymphocytes in HCV‐infected patients with or without associated lymphoproliferative disorders. Flow cytometry analysis of peripheral blood lymphocytes was performed at baseline and at the end of treatment. In HCV‐infected patients with lymphoproliferative disorders, we evaluated immunoglobulin (Ig) light chain κ/λ ratio variations as a measure of monoclonal B‐cell response to antiviral therapy. Healthy volunteers were enrolled as controls. A total of 29 patients were included, nine with and 20 without lymphoproliferative disorders. Sustained virological response was achieved in 29 of 29 patients. We observed a significant reduction in the B‐cell compartment (39% global reduction) in eight of nine HCV‐infected patients with lymphoproliferative disorders after viral clearance. We recognized the same trend, even if less pronounced, in HCV‐infected patients without lymphoproliferative disorders (9% global reduction). Among HCV‐infected patients with lymphoproliferative disorders, three showed an improvement/normalization of the immunoglobulin light chain ratio, whereas in the remaining six patients monoclonal B cells persisted to be clonally restricted even 1 year after the end of treatment. Our data show that DAAs treatment can be effective in reducing the frequency of pathological B cells in the peripheral blood of HCV‐infected patients affected by HCV‐associated lymphoproliferative disorders; however, monoclonal populations can persist after viral eradication.

A case of eosinophilic disorder that evolved in acute lymphoblastyc leukemia

A 45-year-old male blood donor was found to have leukocytosis (14.85 10/L, normal range 4.40–11.00) with an absolute eosinophil count of 9.55 10/L (normal range 0.00–0.50). ADVIA 2120i (Siemens Healthcare Diagnostics, Milan, Italy) analysis showed that eosinophils had normal mieloperoxidase activity (Image 1A left). In the peripheral blood film, the eosinophils showed morphological abnormalities, including nonlobed nuclei and partially agranular defects with a clear area of cytoplasm. (Image 1A right). Ten days later, a repeated CBC showed absolute eosinophilia (5.99 10L), a slightly reduced Hb (128 g/L vs the initial 139 g/L, normal range 140–175 g/L) and morphologically abnormal eosinophils (Image 1B). Total IgE levels were within the normal range, fecal eosinophils, and parasites were negative. Patient was lost to follow-up and presented 6 months later complaining of fever and night sweats: WBC count was normal (4.6 10L) as was the eosinophil count (0.02 10L); Hb had decreased to 101 g/L, platelets to 79 10/L, and neutrophils to 0.90 10L (normal range 1.80–7.80). Peripheral blood film examination showed the presence of agranular blasts, with a feature of lymphoblast (20%) (Image 1C). Flow cytometric immunophenotyping confirmed the presence of Image 1. (A) On the left, ADVIA 2120i cytograms (“Perox” i.e., peroxidase and “Baso” i.e., basophils channels). In the first plot, forward light scattering (x) and intensity of peroxidase activity (y) are plotted with the expandend eosinophil cluster showing normal peroxidase activity (white arrow). In the second plot for the basophil/lobularity channel eosinophils are clustered in the typicall “belly” next to the separation threshold. On the right two panels, dysmorphic eosinophils from the peripheral blood of the patient. (B) Peripheral eosinophils with nuclear and cytoplasmatic abnormalities as described in the text