Showing papers by "Marcello Pinti published in 2008"

OPA1 mutations associated with dominant optic atrophy impair oxidative phosphorylation and mitochondrial fusion.

Abstract: Dominant optic atrophy (DOA) is characterized by retinal ganglion cell degeneration leading to optic neuropathy. A subset of DOA is caused by mutations in the OPA1 gene, encoding for a dynamin-related GTPase required for mitochondrial fusion. The functional consequences of OPA1 mutations in DOA patients are still poorly understood. This study investigated the effect of five different OPA1 pathogenic mutations on the energetic efficiency and mitochondrial network dynamics of skin fibroblasts from patients. Although DOA fibroblasts maintained their ATP levels and grew in galactose medium, i.e. under forced oxidative metabolism, a significant impairment in mitochondrial ATP synthesis driven by complex I substrates was found. Furthermore, balloon-like structures in the mitochondrial reticulum were observed in galactose medium and mitochondrial fusion was completely inhibited in about 50% of DOA fibroblasts, but not in control cells. Respiratory complex assembly and the expression level of complex I subunits were similar in control and DOA fibroblasts. Co-immunoprecipitation experiments revealed that OPA1 directly interacts with subunits of complexes I, II and III, but not IV and with apoptosis inducing factor. The results disclose a novel link between OPA1, apoptosis inducing factor and the respiratory complexes that may shed some light on the pathogenic mechanism of DOA.

Altered Clonogenic Capability and Stromal Cell Function Characterize Bone Marrow of HIV-Infected Subjects with Low CD4+ T Cell Counts Despite Viral Suppression during HAART

Abstract: BACKGROUND Inflammatory cytokines in bone marrow may impair hematolymphopoiesis in human immunodeficiency virus (HIV)-infected subjects who do not experience reconstitution of CD4(+) T cells despite suppression of virus replication while receiving highly active antiretroviral therapy (HAART) (immunological nonresponders). METHODS Bone marrow samples from 12 immunological nonresponders receiving HAART were studied and compared with samples from 11 immunological responders. The mean CD4(+) T cell count (+/- standard deviation) was 174 +/- 68 cells/mm(3) and plasma HIV RNA levels had been <50 copies/mL for at least 1 year for individuals enrolled in the study. The clonogenic capability of bone marrow samples was evaluated using the colony forming cell assay and the long-term culture-initiating cell assay. CD34(+) cells from the colony forming cell assay were pooled for real-time polymerase chain reaction analysis of Fas and Fas ligand. Bone marrow cytokine production (interleukin-2 and tumor necrosis factor-alpha) and stromal interleukin-7 levels were analyzed by enzyme-linked immunosorbent assay in both groups. Flow cytometric analysis of CD4(+) and CD8(+) T cell subsets was performed. RESULTS A reduced clonogenic capability and a decrease in the level of more primitive progenitor cells were observed in parallel with lower production of interleukin-2 and increased tumor necrosis factor-alpha levels. A significant upregulation of Fas and Fas ligand on CD34(+) cells and a higher stromal interleukin-7 production were observed. Impairment of the naive T cell compartment and persistent T cell activation were observed in peripheral blood. CONCLUSIONS Samples from immunological nonresponders show reduced growth of in vitro colonies and an altered cytokine production in bone marrow. The cytokine pattern observed and the altered Fas and Fas ligand pathway may determine stem cell apoptosis and low CD4(+) cell recovery. These features, which are similar to those observed in HIV-infected subjects before starting therapy, persist despite treatment.

Homeostatic Cytokines and Expansion of Regulatory T Cells Accompany Thymic Impairment in Children with Down Syndrome

Abstract: Down syndrome (DS), the most common chromosomal abnormality in humans, is characterized by precocious immunologic aging that results, among other things, in alterations of B and T lymphocyte subsets and natural killer cells, defective phagocytosis, and chemotaxis of polymorphonuclear leukocytes. We studied 30 children affected by DS, compared them to 29 healthy controls, and evaluated the functionality of the thymus (by measuring the amount of lymphocytes that express the signal-joint T cell receptor rearrangement excision circles [sj-TREC+]), the plasma levels of interleukin (IL)-7 and IL-15, the proliferative T cell response to these cytokines, the expression of the α chain of the IL-7 receptor (CD127), the extrathymic differentiation of T lymphocytes, and the presence of natural regulatory T cells (Tregs) in peripheral blood. We found that DS children had a significantly lower number of sj-TREC+ lymphocytes, the levels of which were strongly correlated with age. We found higher plasma levels o...

Resistance of mtDNA-depleted cells to apoptosis.

Abstract: Cells lacking mitochondrial genome (defined as ρ0) are useful models in studies on cancer, aging, mitochondrial diseases and apoptosis, but several of their functional aspects have been poorly characterized. Using different clones of ρ0 cells derived from the human osteosarcoma line 143B, we have tested the effects of different apoptogenic molecules such as staurosporine (STS), doxorubicin, daunomycin and quercetin, and have analyzed apoptosis, mitochondrial membrane potential (MMP), levels of oxygen free radicals, reduced glutathione (GSH) content, and expression of P-glycoprotein (P-gp). When compared to parental cells, ρ0 cells resulted much less sensitive to apoptosis. MMP was well maintained in ρ0 cells, and remained unchanged after adding apoptogenic agents, and did not change after treatment with molecules able to depolarize mitochondria such as valinomycin. After adding STS, the production of reactive oxygen species was similar in both cell types, but ρ0 cells maintained higher levels of GSH. In ρ0 cells, P-gp was strongly over-expressed both at mRNA and protein level, and its functionality was higher. The resistance to apoptosis of ρ0 cells could be not only due to an increased scavenger capacity of GSH, but also due to a selection of multidrug resistant cells that hyperexpress P-gp. © 2008 International Society for Advancement of Cytometry

Mitochondrial DNA Haplogroups and Highly Active Antiretroviral Therapy–Related Lipodystrophy

Abstract: Background. The combination of different point mutations in mitochondrial DNA (mtDNA), which are defined as haplogroups, may cause modification in organelle function and may be involved in several pathologies. We analyzed the distribution of mtDNA polymorphisms in human immunodeficiency virus (HIV)-infected patients with lipodystrophy, a relevant adverse event caused by highly active antiretroviral therapy, and their correlation with metabolic and viroimmunologic parameters. Methods. The frequency of the 9 most common European haplogroups was investigated in 346 white, HIV-infected patients with lipodystrophy. Haplogroups were identified on the basis of classic methods. Statistical analysis was performed with use of 1-way analysis of variance, the Χ 2 test, and principal-components analysis. Results. The distribution of mtDNA haplogroups among patients with lipodystrophy was similar to that among the general European population. We found no differences between patients with different haplogroups with regard to viroimmunologic results (plasma HIV load, CD4 + T cell count, and nadir CD4 + T cell count), glucose data (glucose, insulin, C-peptide, and glycosylated hemoglobin concentrations and insulin resistance), lipid data (levels of triglycerides, total cholesterol, high- and low-density lipoproteins, and apolipoprotein Al and B), acid-base balance parameters (lactate level and anion gap), or anthropometric measures (weight, body mass index, and waist-to-hip ratio). No differences were observed in trunk fat levels, leg-fat ratio (which was determined by dual-energy x-ray absorptiometry), or exposure to different drug classes. Principal-components analysis confirmed that the spatial distribution of patients belonging to a given haplogroup was not influenced by different clinical parameters. Conclusions. Our study indicates that, in HIV-infected patients with lipodystrophy, mtDNA haplogroups are not related to major metabolic changes or to particular viroimmunologic features.

Effects of the change from Stavudine to tenofovir in human immunodeficiency virus-infected children treated with highly active antiretroviral therapy: studies on mitochondrial toxicity and thymic function.

Abstract: Background:Changing from drugs that have significant mitochondrial toxicity to less toxic compounds may be of benefit in human immunodeficiency virus (HIV)-positive patients who receive highly active antiretroviral therapy. Few data on mitochondrial toxicity of antiviral drugs are available in HIV-p

Plasma HIV load and proviral DNA decreases after two standard antiretroviral regimens in HIV-positive patients naïve to antiretrovirals.

Abstract: (i) To compare early decrease of HIV plasma viral load (pVL) after two standard combinations of highly active antiretroviral therapy (HAART) (ii) To evaluate variations of proviral HIV-DNA load on conditions of sustained pVL undetectability Two different sub-studies of a multicentre prospective randomized controlled trial which compared two first-line HAART (ie, zidovudine+lamivudine+lopinavir/ritonavir versus tenofovir+lamivudine+ efavirenz) Only patients enrolled at the coordinating centre (University of Brescia) were included in the two sub-studies In the first sub-study, we calculated pVL decrease with respect to baseline at any of the following time-points: days 1, 3, 7, 14 and 28 Decreases of the pVL were compared between the two treatment groups In the second sub-study, we analyzed variation of proviral HIV-DNA load in CD4+ T-cells from baseline to week 52 only in patients who maintained the same treatment regimen and had sustained undetectable pVL In either studies, linear regression analysis was used to investigate what factors could influence variations of pVL and of proviral HIV-DNA load (i) 64 patients were studied A significant decrease of pVL was found from day 3 on, without statistically significant differences between the two study groups However, after adjusting for possible confounders, tenofovir+lamivudine+efavirenz resulted to be associated with greater pVL decreases (ii) 45 patients were studied Mean proviral HIV-DNA load decreased from 1,610 (95%CI: 879-2,341) to 896 (95% CI 499-1,293) copies/10(6) cells (P=005) Linear regression analysis showed that the decrease of proviral DNA load during follow-up was independently and inversely correlated with age Further studies are needed to compare pVL decay between antiretroviral regimens and assess whether proviral HIV-DNA load is a surrogate marker of treatment effectiveness

Physiology and Immunology of the Thymus Gland

Abstract: The thymus is a gland located in the upper anterior portion of the chest cavity just behind the sternum. Under the evolutionary pressure exerted by the emergence of adaptive immunity and its inherent risk to form receptors that recognize self molecules, this gland appeared about 500 million years ago as a novel structure that had the role of instructing T-cells in order to prevent autoimmunity and orchestrate self-tolerance. The thymus has thus become a crucial lymphoid organ in which cells arriving from the bone marrow undergo a finely tuned process of selection, based on the specificity of T-cell receptors (TCRs), and differentiate into mature T-cells. The development of thymocytes involves a stringent selection in which only 1–3% of these cells succeed in survival and can leave the gland to colonize the periphery and give origin to effective immune cells [1]–[3]. During the maturation in the thymus, T-cells are first positively selected for “usefulness” [positive selection, driven by the affinity of the clonotypic TCR for Major Histocompatibility Complex (MHC) molecules] and then negatively selected against autoreactivity (negative selection, driven by the recognition of self peptides on self MHC by the TCR, which triggers the process of cell death).