Essential requirement of reduced glutathione (GSH) for the anti-oxidant effect of the flavonoid quercetin.

We have analyzed the anti- or pro-oxidant effects of the flavonoid quercetin (QU) by evaluating, in U937 cell line, hydrogen peroxide (H(2)O(2)), superoxide anion reduced glutathione (GSH) content, mitochondrial membrane potential, DNA content, phosphatidylserine exposure on the outer face of the plasma membrane and cell viability. Polychromatic flow cytometry was used to evaluate in the same cells several functional parameters. For short periods of treatment QU exerted an anti-oxidant effect (decrease in H(2)O(2) levels), whereas for long periods it showed a pro-oxidant activity (increase in ).

Characterization of cells with different mitochondrial membrane potential during apoptosis.

Background: Until now, the simultaneous analysis of several parameters during apoptosis, including DNA content and mitochondrial membrane potential (DeltaPsi), has not been possible because of the spectral characteristics of the commonly used dyes. Using polychromatic flow cytometry based upon multiple laser and UV lamp excitation, we have characterized cells with different DeltaPsi during apoptosis.

Methods: U937 cells were treated with the flavonoid quercetin (Qu) and stained with JC-1 to detect DeltaPsi, propidium iodide (PI) for cell viability, Hoechst 33342 for DNA content, Annexin V conjugated with Alexa Fluor-647 for detection of phosphatidilserine (PS) exposure, marker of early apoptosis, or Mitotracker Deep Red for the determination of mitochondrial mass.

Effect of treatment interruption monitored by CD4 cell count on mitochondrial DNA content in HIV-infected patients: a prospective study.

Background: HIV infection per se and HAART can alter mitochondrial functionality, leading to a decrease in mitochondrial DNA content.

Objective: To evaluate whether treatment interruption monitored by CD4 cell count can restore mitochondrial DNA content in peripheral blood lymphocytes.

Methods: Mitochondrial DNA content was measured in platelet-free CD4 and CD8 T cells by real-time polymerase chain reaction; flow cytometry was used to identify and quantify activated CD4 and CD8 T lymphocytes.

Genetic polymorphisms of Fas (CD95) and Fas ligand (CD178) influence the rise in CD4+ T cell count after antiretroviral therapy in drug-naïve HIV-positive patients.

Fas and Fas ligand (FasL) are the main genes that control cell death in the immune system. Indeed, they are crucial for the regulation of T lymphocyte homeostasis because they can influence cell proliferation. A strong debate exists on the importance of Fas/FasL system during HIV infection, which is characterized by the loss of CD4+ T cells directly, or indirectly, caused by the virus.

Altered mitochondrial RNA production in adipocytes from HIV-infected individuals with lipodystrophy.

Background: Damage to mitochondria (mt) is a major side effect of highly active antiretroviral therapy (HAART) that includes a nucleoside reverse transcriptase inhibitor (NRTI). Such damage is associated with the onset of lipodystrophy in HAART-treated HIV+ patients. To further investigate mt changes during this syndrome, we analysed the expression of mtRNA in adipocytes from lipodystrophic HIV+ patients taking NRTI-containing HAART and compared it with similar cells from healthy individuals.

Mitochondrial membrane potential and nucleosidic inhibitors of HIV reverse transcriptase: a cytometric approach.

Mitochondrial toxicity is a relevant side effect of anti-HIV antiretroviral therapy. Adequate experimental models and advanced technologies are crucial to investigate properly mitochondrial toxicity. Functional flow cytometry allows a rapid and sensitive evaluation of several parameters on single cells, and is an excellent tool to investigate the impact of antiviral drug on mitochondrial activity.

Biological importance of the two Toll-like receptors, TLR2 and TLR4, in macrophage response to infection with Candida albicans.

The aim of this study was to assess the role of TLR2, TLR4 and MyD88 accessory molecule in the effector and secretory response of macrophages to viable microbial agents. Using TLR-deleted macrophage cell lines generated from the bone marrow of genetically engineered mice (TLR4 gene-deficient, MyD88- and TLR2-knockout mice) and wild-type control mice, we found that TLR2-deleted macrophages exhibit increased ability to contain Candida albicans infection compared to TLR2+/+ counterpart. In contrast, both MyD88-/- and TLR4-/- macrophages retain levels of functional activity comparable to that of the respective wild-type MyD88+/+ and TLR4+/+ controls.

Changes in mitochondrial RNA production in cells treated with nucleoside analogues.

Background: To investigate mitochondrial (mt) toxicity of antiretroviral drugs further, we developed a novel real-time PCR-based assay for the quantification of mtRNA. We analysed the effects of stavudine (d4T), didanosine (ddl) and zidovudine (AZT) on the production of mtRNAs in different human cell lines and compared the production with the amount of mtDNA present in the same cells.

Materials And Methods: HUT78, CEM and U937 cells were exposed to different nucleoside reverse transcriptase inhibitors (NRTIs) for 7 days.

CD4 cell-monitored treatment interruption in patients with a CD4 cell count > 500 x 106 cells/l.

Background: Little is known about CD4 cell count changes in patients with high CD4 cell counts who interrupt antiretroviral therapy, especially in those with a nadir of 250-350 x 10 cells/l.

Methods: Data derived from 139 patients from seven prospective cohorts who had > 12 months highly active antiretroviral therapy (HAART), CD4 cell count nadir of > 250 x 10 cells/l and at pre-interruption of > 500 x 10 cells/l. Endpoint was time to CD4 cell count < 350 x 10 cells/l or reinitiation of treatment.

Direct analysis of thymic function in children with Down's syndrome.

Background: Down's syndrome (DS) is characterized by several immunological defects, especially regarding T cell compartment. DS is considered the best example of accelerated ageing in humans. Direct observations of the thymus have shown that in DS this organ undergoes severe histological and morphological changes.

HIV type 1 protease inhibitors enhance bone marrow progenitor cell activity in normal subjects and in HIV type 1-infected patients.

HIV-1 protease inhibitors (PIs) may improve hematopoietic functions owing to their direct effects on bone marrow (BM) progenitor cells. In this study we investigated this hypothesis evaluating the effect of adding ritonavir (RTV) and indinavir (IND) on hematopoietic colony formation assays by colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, on apoptosis, on cytokine production and stromal cells, in subjects with HIV-1 infection, and in seronegative controls. After PI addition, CFC and LTC-IC assays in HIV-1-infected patients showed levels of colony growth significantly higher than those observed at baseline; the same PI activity on colony formation was observed in healthy subjects.

Balanced regulation of mRNA production for Fas and Fas ligand in lymphocytes from centenarians: how the immune system starts its second century.

Background: The functionality of the immune system during aging is crucial for protection against the most common age-related diseases. Apoptosis plays a central role in the senescence of the immune system, as evidenced by the increased plasma membrane expression of a key molecule like Fas protein. We analyzed the mRNA levels of different forms of Fas (total [tFas] and membrane [mFas]) and of its ligand (FasL) in peripheral blood lymphocytes from centenarians, the best example of successful aging, who were compared with young and middle-aged donors.

The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans.

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells.

Decreased apoptosis of bone marrow progenitor cells in HIV-1-infected patients during highly active antiretroviral therapy.

Impaired haematopoiesis during HIV-1 infection may be caused by the overproduction of inflammatory cytokines by immune cells at the bone marrow level inducing Fas-mediated apoptosis of stem progenitors. In this study, we evaluated the effects of highly active antiretroviral therapy on apoptosis of CD34+ stem cells derived from the bone marrow of HIV-1-infected patients, and observed decreased Fas expression on progenitor cells, in parallel with the diminution of TNF-alpha levels and the amelioration of clonogenic parameters.

Highly active antiretroviral therapy restores CD4+ Vbeta T-cell repertoire in patients with primary acute HIV infection but not in treatment-naive HIV+ patients with severe chronic infection.

In drug-naive HIV+ patients, we analyzed the effects of highly active antiretroviral therapy (HAART) on the reconstitution of the T-cell receptor (TCR) repertoire. We followed 2 groups of patients for 1 year: 18 individuals who experienced acute HIV infection and 24 patients who had HIV infection for many years but never took HAART. They were compared with 10 healthy controls who were longitudinally analyzed for the same period.

Discontinuation of maintenance therapy for cryptococcal meningitis in patients with AIDS treated with highly active antiretroviral therapy: an international observational study.

We conducted a retrospective, multicenter study evaluating the safety of discontinuing maintenance therapy for cryptococcal meningitis after immune reconstitution. Inclusion criteria were a previous definitive diagnosis of cryptococcal meningitis, a CD4 cell count of >100 cells/microL while receiving highly active antiretroviral therapy (HAART), and the subsequent discontinuation of maintenance therapy for cryptococcal meningitis. The primary end point was relapse of cryptococcal disease.

Different sensitivity to apoptosis in cells of monocytic or lymphocytic origin chronically infected with human immunodeficiency virus type-1.

Apoptotic death of CD4+ T lymphocytes is a major cause of the immunodeficiency caused by human immunodeficiency virus (HIV), but it is still unclear how this process precisely occurs. To characterize a potentially useful cellular model, we have analyzed the tendency of chronically HIV-infected CD4+ human cell lines of different origin to undergo apoptosis. We studied ACH-2 and U1 lines, derived from the CD4+ T-cell A301 and the promonocytic U937 cell lines, respectively, and induced apoptosis via several stimuli that trigger different pathways.

Increased mitochondrial DNA content in peripheral blood lymphocytes from HIV-infected patients with lipodystrophy.

We have evaluated mitochondrial (mt) DNA content in CD4 and CD8 peripheral blood lymphocytes (PBLs) from HIV-infected patients taking highly active antiretroviral therapy (HAART) who display different types of adipose tissue alterations. A cross-sectional study was performed in a total of 23 patients with lipodystrophy (LD): nine patients with fat accumulation, six patients with fat loss, eight patients with combined form, who were compared to 11 individuals infected by HIV without LD (HIV-positive) and 10 seronegative controls (CTRL). PBLs were obtained by standard methods, that is, gradient density centrifugation on Ficoll, and CD4 or CD8 cells were positively isolated by magnetic sorting to eliminate the contamination of platelets.

MDR1 C3435T genetic polymorphism does not influence the response to antiretroviral therapy in drug-naive HIV-positive patients.

P-glycoprotein, a membrane-localized protein transporter, codified by the MDR1 gene, influences the response to pharmacological treatments, including antiretroviral drugs. MDR1 polymorphism C3435T is correlated with the functionality of the protein. We investigated the influence of this polymorphism in the reconstitution of the peripheral CD4 T cell pool in 149 drug-naive HIV-positive patients starting highly active antiretroviral therapy.

Tests for mitochondrial function and DNA: potentials and pitfalls.

Purpose Of Review: In the past few years, mitochondria have been carefully studied to ascertain whether and how in patients affected by HIV antiretroviral therapy is able to alter their functionality and exert a toxic effect on immune cells, as well as on cells present in other districts.

Recent Findings: A variety of in-vivo and ex-vivo models have been developed to investigate the functionality of mitochondria and DNA during a variety of physiopathological situations, including HIV infection and its treatment. Numerous technologies are available to study at the single-cell or at the single-organelle level a variety of parameters, such as membrane potential, the activity of respiratory chain enzymes, and DNA content or its sequence.

Development of real time PCR assays for the quantification of Fas and FasL mRNA levels in lymphocytes: studies on centenarians.

Apoptosis plays a central role in the homeostasis of the immune system. During aging, there is an altered regulation of pivotal molecules that are responsible for the regulation of this type of cell death, such as those of the Fas/FasL system. Understanding the regulation of these genes can help to clarify, at least in part, the age-related changes that occur in immune cells.

Placenta and cord blood mitochondrial DNA toxicity in HIV-infected women receiving nucleoside reverse transcriptase inhibitors during pregnancy.

Recent studies of pregnant women and animal models have raised concerns regarding potentially serious mitochondrial toxicity-related side effects in infants born to mothers who received nucleoside reverse transcriptase inhibitors (NRTIs) during their pregnancy to prevent HIV-1 perinatal transmission. The aim of this study was to assess mitochondrial DNA (mtDNA) content of cord blood and placenta in HIV-infected pregnant women receiving NRTI compared with HIV-negative women, hypothesizing that placenta and cord blood mtDNA copies per cell would be decreased in women on NRTI therapy. Immediately following delivery, placenta and cord blood were obtained from eight HIV-infected pregnant women on NRTIs and five HIV-negative women.