Highly sensitive real-time PCR method to identify species origin in heparinoids.

Heparinoids are the starting material for sulodexide production, a drug used as intravenous anti-coagulant, as an alternative to heparin. The origin determination in the starting material for sulodexide, heparin, and derivatives is crucial for safety (including the impact related to bovine spongiform encephalopathy) and efficacy of the final products. Therefore, European countries have decided to approve the production of heparin only from porcine intestinal mucosa.

Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry.

Reactive oxygen species (ROS) are continuously produced in the cell as a consequence of aerobic metabolism, and are controlled by several antioxidant mechanisms. An accurate measurement of ROS is essential to evaluate the redox status of the cell, or the effects of molecules with the pro-oxidant or antioxidant activity. Here we report a cytofluorimetric technique for measuring simultaneously, at the single-cell level, hydrogen peroxide and superoxide anion, reduced glutathione (a main intracellular antioxidant) and cell viability.

HOXD13 binds DNA replication origins to promote origin licensing and is inhibited by geminin.

HOX DNA-binding proteins control patterning during development by regulating processes such as cell aggregation and proliferation. Recently, a possible involvement of HOX proteins in replication origin activity was suggested by results showing that a number of HOX proteins interact with the DNA replication licensing regulator geminin and bind a characterized human origin of replication. The functional significance of these observations, however, remained unclear.

Quercetin inhibits lymphocyte activation and proliferation without inducing apoptosis in peripheral mononuclear cells.

Toxicity of chemotherapeutic drugs towards normal cells is a serious side effect of cancer treatment. Thus, finding of molecules with low toxicity for normal cells is crucial. Several natural compounds, such as flavonoid quercertin, are receiving a growing attention as "chemopreventers".

Homeostatic cytokines and expansion of regulatory T cells accompany thymic impairment in children with Down syndrome.

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 alpha 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.

Resistance of mtDNA-depleted cells to apoptosis.

Cells lacking mitochondrial genome (defined as rho(0)) 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 rho(0) 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, rho(0) cells resulted much less sensitive to apoptosis.

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.

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-positive children.

Methods: Eighteen HIV-positive children (median age, 10.

Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry.

The analysis of changes in mitochondrial membrane potential (MMP) that can occur during apoptosis provides precious information on the mechanisms and pathways of cell death. For many years, the metachromatic fluorochrome JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide) was used for this purpose. Thanks to new dyes and to the technical improvements recently adopted in several flow cytometers, it is now possible to investigate, along with MMP, a variety of other parameters.

Subject classification obtained by cluster analysis and principal component analysis applied to flow cytometric data.

Background: Polychromatic flow cytometry (PFC) allows the simultaneous determination of multiple antigens in the same cell, resulting in the generation of a high number of subsets. As a consequence, data analysis is the main difficulty with this technology. Here we show the use of cluster analysis (CA) and principal component analyses (PCA) to simplify multicolor data visualization and to allow subjects' classification.

Mitochondrial alterations and tendency to apoptosis in peripheral blood cells from children with Down syndrome.

Different types of cells from subjects with Down syndrome (DS) have an increased susceptibility to cell death. We have studied apoptosis and mitochondrial (mt) membrane potential (DeltaPsi(m)) in peripheral blood mononuclear cells (PBMC) from DS children and age-matched healthy donors after in vitro treatment with apoptogenic molecules, along with mtDNA content. We found that PBMC from DS and healthy controls had a similar tendency to undergo apoptosis and a similar amount of mtDNA.

Protective effect of acetyl-L-carnitine against oxidative stress induced by antiretroviral drugs.

Both HIV infection per se and antiretroviral drugs might contribute to oxidative stress and mitochondrial dysfunctions. In this study we assess zidovudine, stavudine and didanosine on U937 and CEM cell lines. All these drugs induced apoptosis and increased intracellular hydrogen peroxide but not superoxide anions.

Immunophenotype of HIV+ patients during CD4 cell-monitored treatment interruption: role of the IL-7/IL-7 receptor system.

Objective: To investigate immunological changes during CD4-guided therapy interruption in HIV(+) patients who suspended HAART.

Patients: Seventeen patients aged > 18 years, who had received HAART for at least 12 months, and had a pre-interruption CD4+ cell count > 500 cells/microl, interrupted treatment. Median nadir CD4(+) cell count was 288 cells/microl.

Thymic output and functionality of the IL-7/IL-7 receptor system in centenarians: implications for the neolymphogenesis at the limit of human life.

During aging, the thymus undergoes a marked involution that is responsible for profound changes in the T-cell compartment. To investigate the capacity of the thymus to produce new cells at the limit of human lifespan, we analyzed some basic mechanisms responsible for the renewal and maintenance of peripheral T lymphocytes in 44 centenarians. Thymic functionality was analyzed by the quantification of cells presenting the T-cell receptor rearrangement excision circles (TREC).

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.

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.

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.

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.

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.

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.