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. However, no data on its capacity to generate T cells are present in the literature. Here, using a new technology based upon real time PCR, we have investigated the capacity of the thymus to produce and release newly generated T lymphocytes (the so called "recent thymic emigrants", RTE) in children with DS. METHODS: We studied 8 children affected by DS, aged 2–7 years, compared with 8 age- and sex-matched healthy controls. Flow cytometry was used to determine different lymphocytes subsets. Real time PCR with the Taqman system was used to quantify the amount of RTE, i.e. peripheral blood lymphocytes that express the T cell receptor rearrangement excision circles (TREC). RESULTS: In comparison with control children, those with DS had a significant lower number of TREC+ peripheral blood cells. Moreover, in DS children but not in controls, a strong negative correlation between age and the levels of TREC+ cells was found. CONCLUSIONS: The direct measure of thymic output indicates that the impairment of the organ results in a reduced production of newly generated T cells. This observation could suggest that cytokines able to modulate thymic function, such as interleukins, could be useful to improve the functionality of the organ and to treat the immunodeficiency present in DS subjects

Mitochondria and HIV infection: the first decade.

In the last few years, the interactions between mitochondria and infection with the human immunodeficiency virus (HIV) have received careful attention. Starting from the first studies regarding the presence of mitochondrial damage in cardiac tissue from patients who died of AIDS, researchers have investigated different aspects of the interactions between the virus and mitochondria, from acute primary infection to the final stages of the disease. Only recently a significant impulse to this type of research has come from the observation that the so called "highly active antiretroviral therapy" (HAART), a combination of potent antiretroviral drugs such as viral protease inhibitors or nucleoside-analogue reverse-transcriptase inhibitors, is capable of damaging these organelles and cause a clinical syndrome called lipodystrophy. There is still an open debate concerning the exact responsibility of HAART as well as on metabolic pathways and mechanisms that are involved in the onset of lipodystrophy. The hypothesis that drug-induced damage to mitochondrial (mt) DNA is able to alter mitochondria functionality to a similar extent as that occurring in genetic disease affecting mtDNA suggests that mitochondria plays a crucial role in the pathogenesis of this syndrome. In this paper, data concerning the interactions between mitochondria and HIV infection will be reviewed

Polychromatic analysis of mitochondrial membrane potential using JC-1

Dissipation of the mitochondrial membrane potential (m) has been accepted as a hallmark of some apoptotic processes. Depending on the model studied, it can occur during the early stages of cell death or later on, after loss of DNA integrity. Discordant data in the literature could be the consequence of using improper probes such as rhodamine 123 (R123) or DiOC6(3), which are not always appropriate for the study of m. The lipophilic cation JC-1, a specific probe for measuring m, is currently the gold standard. Thanks to recently developed instruments and additional probes for cell surface and intracellular markers, it can be used in polychromatic flow cytometric assays to simultaneously detect m along with other biological parameters

Physiology and immunology of the thymus gland

The thymus is a giand located in the upper anteriorportion of the chest cavity just behind the sternurn,Under the evolutionary pressure exerted by the emergence of adaptive immunityAnd its inherent risk to from receptorsThat recognize self molecules, this gland appeared about 500 milT cells in order to prevent autoimmunity and orchestrate selftolerance.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 thymocitesn 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. Duting maturation in thymus, T cells are first positively selected for uselfulness and then negatively selected against autoreactivity. These intrathymis events are governed by sequential interactions of thymocites with different stromal cell types during the migration through the thymus essentially from the external to the internal part of the thymic lobuli. The mature T cells called na\uefve T cells leave the organ and contribute to the peripheral T cell poll. The complex process of intrathymic maturation of T-lymphocites involvesvarious thymic specific factors and several other molecules. Indeed T cell maturation requires either direct cell-to-cell or paracrine interactions that occur via cytokines or thymic hormones produced by the cells of the thymic microenvironment. For a long toime the functions of the thymus have remained obscure. The first demonstration of its crucial role in the ontogeny and development of the immune system was provided in 1961 when it was shown that mice thymectomized immediately after birth had poorly developed lymphoid tisses impaired immune responses and susceptibility to infections. Althought cells present in the thymus were believed to be immunoincompetent a few years later it was shown that they could proliferate after an antigenic challenge and produce cells unable to synthetize antibodies. Such cells were capable of enabling other lymphocyte to differentiate to antibody-forming cells. This was the first demonstration in mammalians of the existence of two major subsets of lymphocytes now known as T- and B-cells. It required a re-evaluation of many immunological phenomena such as tolerance memory and autoimmunity and it was followed by a huge number of studies elucidating many of the mysteries of the immune system

Complementary and alternative medicine during HIV infection.

According to the Joint United Nations Program of HIV/AIDS (UNAIDS) and the World Health Organization (WHO) (Joint United Nations Program of HIV/AIDS, 2001), as of the end of 2001, there were about 40 million adults and children living with human immunod-eficiency virus (HIV) infection. This total does not include the 20 million people around the world who already died of AIDS. Of the 40 million currently alive, 37.2 are adults, 17.6 are women, and more than 2.7 are children. In 2001, there were 5 million new cases of HIV infection in the world, and 3 million AIDS related deaths. The large majority (almost three quarters) live in Sub-Saharan Africa where the prevalence rate of the infection among adults is 8.4%; more than 55% of infected individuals are women. The second major pocket of HIV infection is in South and Southeast Asia, with more than 6 million people infected. In North America where the epidemic was first described, there are 940,000 individuals who are HIV- , and in Western Europe, 540,000. Furthermore, South America, China, and East-ern Europe are characterized by a rapid increase in infection rates. These dramatic numbers clearly indicate that the fight against HIV/AIDS is an absolute health, social, economical and political priority in all parts of the world

Flow Cytometry as a tool for analyzing invertebrate cells.

Flow cytometry (FCM) is a powerful tool that allows analysis of thousand of cells in a few seconds,at the single cell level. In the last 15 years, researchers have used FCM to investigate the cellularmachinery of invertebrates. Analyses have focused on functions linked to innate immunity, such asphagocytosis and natural killer cell activity, as well as on the sensitivity of invertebrate cells to aparticular stress or to a toxic agent. Further, FCM has been employed to recognize antigens, or atleast immunodominant epitopes, shared in common with mammalian cells, including humanleukocytes. In this review, main studies that have utilized FCM to investigate either phenotype andfunctions of invertebrate cells are reported and discussed

Protective effect of acetyl-L-carnitine on oxidative damage 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. The addition of acetyl-l-carnitine (ALC) was able to prevent the pro-oxidant effect of the drugs tested. Supplementation with ALC, deficient in certain cohorts of HIV-infected individuals, especially on high active antiretroviral therapy regimen, has been associated with favourable effects. These data suggest that one of these effects could be a direct anti-oxidant action

Functional changes during apoptosis revealed by the simultaneous detection of mitochondrial membrane potential and DNA content by JC-1 and Hoechst 33342

ApopThe study of functional changes that occur in intracellular organelles such asmitochondria during cell death/apoptosis are of consistent interest since severalyears. Probes exist that allow the analysis of a variety of mitochondrial (mt)parameters, including mt mass and mt membrane potential (MMP), along withthose that stain DNA and reveal its modifications. The dye JC-1, excited by theargon laser present in almost all flow cytometers, is considered the gold standardfor detecting MMP (Current Protocols in Cytometry, 9.14.1-9.14.7, 2000), andemits in FL-1 (monomers) and FL-2 (aggregates, whose formation is due by ahigh MMP). However, its spectrum of emission does not allow the use of otherdyes excitable by such laser since there is a relevant signal also in FL-3. Thus,until now, the simultaneous study of changes in DNA content and MMP duringapoptosis have been difficult if not impossible, and all data, including ours(Cytometry, 40: 189-197, 2000) have been obtained by indirect measures. In orderto investigate such parameters in the same cell, we took advantage of a novelinstrument, i.e. CyFlow ML by Partec (Germany), equipped with an air-cooledargon ion laser (488 nm, 200 mW), a UV Mercury lamp HBO (100 long life, 100W), a red diode laser (635 nm, 25 mW), a Nd:YAG laser (532 nm, 50 mW) and aCCD camera. Such configuration allowed us to use JC-1, excited by the Ar laser,along with Hoechst 33342 excited by the UV lamp. Apoptosis was induced inU937 or in CEM cell lines by a 48 hour incubation with 50 μM quercetin. Theanalysis of MMP allowed us to identify 3 different populations, which correspondedto three different stages of cell death. The first (about 50% of cells) hada normal MMP (high red and intermediate green fluorescence by JC-1), normalDNA content, FSC and SSC. In the second population (about 20%), MMP waschanging (high red and high green fluorescence), DNA content was normal whileFSC and SSC were those of apoptotic cells. In the third population (about 30%),MMP was consistently decreased (low red and high green fluorescence), DNAcontent was decreased (with the presence of the typical hypodiploic peak), FSCand SSC were altered. Thus, our data indicate that, in this model of apoptosis,cells start to loose MMP (as JC-1 aggregates begin to become monomers, causingan increase in green fluorescence) and change their physical parameter beforethe beginning of loss of nuclear DNA and the appearance of the hypodiploic peak.Studies are in course for the simultaneous detection of changes in mt mass (measuredby Mitotracker Deep Red) or other parameters (such as plasma membranepermeability or phosphatidylserine exposure) with dyes excited by the red laser

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. The staining is performed with the fluorescent dyes 2',7'-dichlorodihydrofluorescein diacetate (H2DCFH-DA), hydroethidine (HE), monobromobimane (MBB) and TO-PRO-3. This analysis is possible with new-generation flow cytometers equipped with several light sources (in our case, four lasers and an UV lamp), which excite different fluorochromes. This approach is extremely useful to study the balance between ROS content and antioxidants in cells receiving different stimuli, and to analyze the relationship between oxidative stress and cell death