Fabio Malacarne

Serum From Patients With Severe Heart Failure Downregulates eNOS and Is Proapoptotic Role of Tumor Necrosis Factor-α

BACKGROUNDnCytokine activation and endothelial dysfunction are typical phenomena of congestive heart failure (CHF). We tested the hypothesis that incubating human umbilical vein endothelial cells with serum from patients with CHF will downregulate endothelial constitutive nitric oxide synthase (eNOS) and induce apoptosis.nnnMETHODS AND RESULTSnWe studied 21 patients with severe CHF. Levels of tumor necrosis factor-alpha (TNF-alpha) and several neuroendocrine parameters were assessed. eNOS was measured by Western Blot analysis and apoptosis by optical microscopy and flow cytometry. We observed (1) eNOS downregulation (difference versus healthy subjects at 24 hours [P<0.05] and 48 hours [P<0.001]), (2) nuclear morphological changes typical of apoptosis; and (3) a high apoptotic rate with propidium iodide (increasing from 2.1+/-0.4% to 11.3+/-1.2% at 48 hours; P<0.001 versus healthy subjects) and annexin V. An anti-human TNF-alpha antibody did not completely counteract these effects. A strong correlation existed between eNOS downregulation and apoptosis (r = -0.89; P<0.001).nnnCONCLUSIONSnSerum from patients with severe CHF downregulates eNOS expression and increases apoptosis. High levels of TNF-alpha likely play a role, but they cannot be the only factor responsible.

Segregation of type 1 cytokine production in human peripheral blood lymphocytes: Phenotypic differences between IFN-γ and IL-2-producing cells in the CD8+ T cell subset

T cell clones are classified as type 0, 1 or 2 depending on the lymphokines they produce. However, it has remained unclear whether single cells of a given type produce one or several cytokine species. Flow cytometric analysis of peripheral blood lymphocytes (PBL) obtained from 20 healthy donors for the production of the type 1 cytokines IFN‐γ and IL‐2 revealed very few cells that co‐expressed both cytokines independently of the mitogenic stimulus used for PBL activation. Similarly, kinetic studies of cytokine synthesis indicated a low percentage of IFN‐γ/IL‐2 double‐positive T cells at all time points. Reverse transcription‐PCR analysis of sorted IL‐2‐ and IFN‐γ‐positive T cells showed the presence of IL‐2‐ or IFN‐γ‐specific mRNA only in those cells expressing the corresponding cytokine. This segregation of the two type 1 cytokines was lost in long‐term cultured T cells and in T cell clones. A high percentage of cells expressing only IL‐2 or IFN‐γ was observed even when the production of these cytokines was evaluated on CD4+ and CD8+ subsets. Moreover, in some healthy individuals, IFN‐γ and IL‐2 production by CD8+ T cells was related to CD8+ expression levels and cell size, i.u2009u2009e. IL‐2‐expressing cells were generally smaller with more intense CD8+ staining as compared with IFN‐γ‐producing T cells. These data indicate that activated T lymphocytes are strongly committed in vivo to produce IFN‐γ or IL‐2 and emphasizes the independent regulation of the two cytokine genes.

Ontogeny of CD40 expression by activated peripheral blood lymphocytes in humans

The CD40 ligand (CD40L) is a molecule expressed by activated T cells which plays a critical role in the regulation of B-cell responses, including differentiation into Ig-producing cells. Using the specific monoclonal antibody TRAP1 we have evaluated the ontogeny of CD40L expression in 97 normal individuals between birth and 50 years of age. The expression of CD40L is a function of age; it is severely reduced at birth, progressively increases during the first months of life, and reaches a plateau in the second decade. This progressive attainment of the ability to express CD40L is due to a process of maturation of the CD4 + subset, being significantly correlated with the expression of the CD45RO antigen.

CD4+ cells from patients with Common Variable Immunodeficiency have a reduced ability of CD40 ligand membrane expression after in vitro stimulation

Background: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production. This has been variably attributed to intrinsic B‐cell defects or to T‐cell disfunctions. Recently, it has been reported that the expression of the CD40 Ligand (CD40L), a T‐cell surface molecule that plays a critical role in the cell‐contact‐mediated helper signals provided to B‐cells, is defective in a subset of patients with CVID.

Naive CD4+ T Lymphocytes Express High Levels of Bcl-2 after Highly Active Antiretroviral Therapy for HIV Infection

The mechanism causing the increasing number of peripheral T cells after highly active antiretroviral therapy (HAART) is still unclear. The bcl-2 oncogene prevents spontaneous apoptosis (SA) in lymphocytes. Spontaneous apoptosis could be a determinant of HIV immunodeficiency and can be reversed by HAART including protease inhibitors (PI-HAART). The aims of our study were to measure Bcl-2 protein expression in memory (CD45RO+) and naive (CD45RO-) CD4+ and CD8+ T lymphocytes of HIV+ patients and to correlate it with efficacy of PI-HAART. Forty-nine HIV+ patients (cases) and 26 HIV- individuals (controls) were evaluated. Patients receiving PI-HAART, and who had undetectable HIV plasma viral load (VL-, n = 21), had higher levels of Bcl-2 than did VL+ patients (n = 28), both in CD4+ cells (p < 0.0001) and in CD8+ cells (p < 0.001). VL+ patients had lower Bcl-2 levels than did controls in CD8+ cells (p = 0.02), but not in CD4+ cells (p > 0.05). Interestingly, VL- patients had higher Bcl-2 expression than did controls both in CD4+ cells (p < 0.0001) and in CD8+ cells (p = 0.03). In a subcohort of the same patients, Bcl-2 was significantly higher in VL- patients (n = 10) than in controls (n = 12), both in naive CD4+ cells (p < 0.0001) and in naive CD8+ cells (p = 0.01). Naive CD4+ cells had higher Bcl-2 expression in VL- than in VL+ patients (p = 0.01). In a subsequent longitudinal study of nine HIV patients, naive CD4+ cells increased after effective PI-HAART (p = 0.03), which paralleled an increase in Bcl-2 expression in the same cells (p = 0.02). In conclusion, upregulation of bcl-2 could be a mechanism of immune reconstitution of naive CD4+ T cells induced by PI-HAART.