Biomedical subjects

A Cossarizza

Publications and source records attributed to A Cossarizza.

At least 37 records · Page 2Linked to original sources

PubMed2000

Discontinuation of primary prophylaxis for Pneumocystis carinii pneumonia and toxoplasmic encephalitis in human immunodeficiency virus type I-infected patients: the changes in opportunistic prophylaxis study.

A multicenter open, randomized, controlled trial was conducted to determine whether primary prophylaxis for Pneumocystis carinii pneumonia and toxoplasmic encephalitis can be discontinued in patients infected with human immunodeficiency virus type 1 (HIV-1) whose CD4+ T cell counts have increased to >200 cells/mm3 (and who have remained at this level for at least 3 months) as a result of highly active antiretroviral therapy (HAART). Patients were randomized to either the discontinuation arm (i.e., those who discontinued prophylaxis; n=355) or to the continuation arm (n=353); the 2 arms of the study were similar in terms of demographic, clinical, and immunovirologic characteristics. During the median follow-ups of 6.4 months (discontinuation arm) and 6.1 months (continuation arm) and with a total of 419 patient-years, no patient developed P. carinii pneumonia or toxoplasmic encephalitis. The results of this study strongly indicate that primary prophylaxis for P. carinii pneumonia and toxoplasmic encephalitis can be safely discontinued in patients whose CD4+ T cell counts increase to >200 cells/mm3 during HAART.

AIDS-Related Opportunistic Infections↗

PubMed2000

Opposite role of changes in mitochondrial membrane potential in different apoptotic processes.

We have studied the role of changes in mitochondrial membrane potential (DeltaPsi) in two widely-used models of apoptosis, such as dexamethasone-treated rat thymocytes and U937 human cells treated with tumor necrosis factor-alpha and cycloheximide. To dissipate DeltaPsi, we used low concentrations of valinomycin, unable per se to induce apoptosis, and demonstrated that the decline in DeltaPsi exerts opposite effects in the two models. Indeed, in U937 cells, depolarization of mitochondria increased apoptosis, which decreased in rat thymocytes. This leads to the suggestion that disruption of DeltaPsi plays opposite roles depending on the experimental model. In U937 cells, the drop of DeltaPsi is a possible contributory cause for the apoptotic process; in rat thymocytes, it could be a limiting factor. We propose that these opposite effects could be due to the different ATP requirement of each apoptotic pathway.

Adenosine Triphosphate↗

PubMed2000

Deregulation of the CD95/CD95L system in lymphocytes from patients with primary acute HIV infection.

OBJECTIVE: To analyze the role of CD95/CD95 ligand (CD95L) expression and functionality in peripheral blood lymphocytes (PBL) during primary, acute HIV syndrome (AHS) and in the subsequent period. PATIENTS: Twelve patients were studied during the acute phase of the viral infection and most were followed for some months. METHODS: Cell culture and cytotoxicity assays based upon 51Cr release and flow cytometry were used to evaluate cell killing via CD95 molecule, flow cytometry to assess surface antigens, enzyme-linked immunosorbent assay (ELISA) for the determination of soluble CD95 and CD95L plasma levels, quantitative competitive (QC) reverse transcription polymerase chain reaction (RT-PCR) with an original RNA competitor for the analysis of CD95L mRNA expression and QC RT-PCR for determining plasma viral load. RESULTS: The analysis of PBL during this phase revealed that almost all cells, including CD8 T cells with a virgin phenotype, B lymphocytes and natural killer cells displayed CD95 molecules on the plasma membrane. Activation of CD95 on the surface of isolated lymphocytes by anti-CD95 monoclonal antibodies or binding to CD95L induced rapid apoptosis. However, CD95L mRNA was not expressed in PBL from these patients and was poorly inducible. Soluble CD95 was found in the plasma of all patients, but only in a few at high levels, even some months after seroconversion. In contrast, soluble CD95L was detected in only one patient, this occurring after the symptomatic period. For 10 of the 12 patients, expression of CD95 on the cell membrane or in the plasma did not correlate with the plasma viral load, which varied widely from patient to patient. Further, plasma levels of soluble CD95 were not altered by decreased lymphocyte activation or by efficient antiretroviral therapy. CONCLUSIONS: In patients experiencing an acute, primary HIV infection, a prolonged deregulation of the CD95/CD95L system may exist, which is probably not entirely related to virus production but may contribute to the pathogenesis of the disease. The hypothesis can be put forward that a complex balance exists between proapoptotic events (increase in CD95 expression), probably triggered by the host as a method to limit viral production, and antiapoptotic events (decrease in CD95L expression) probably triggered by the virus as a way to increase its production and survival.

Apoptosis↗

PubMed2000

Do men and women follow different trajectories to reach extreme longevity? Italian Multicenter Study on Centenarians (IMUSCE).

Gender accounts for important differences in the incidence and prevalence of a variety of age-related diseases. Considering people of far advanced age, demographic data document a clear-cut prevalence of females compared to males, suggesting that sex-specific mortality rates follow different trajectories during aging. In the present investigation, we report data from a nationwide study on Italian centenarians (a total of 1162 subjects), and from two studies on centenarians living in two distinct zones of Italy, i.e., the island of Sardinia (a total of 222 subjects) and the Mantova province (Northern Italy) (a total of 43 subjects). The female/male ratio was about 2:1 in Sardinia, 4:1 in the whole of Italy, and about 7:1 in the Mantova province. Thus, a complex interaction of environmental, historical and genetic factors, differently characterizing the various parts of Italy, likely plays an important role in determining the gender-specific probability of achieving longevity. Gender differences in the health status of centenarians are also reported, and an innovative score method to classify long-lived people in different health categories, according to clinical and functional parameters, is proposed. Our data indicate that not only is this selected group of people, as a whole, highly heterogeneous, but also that a marked gender difference exists, since male centenarians are less heterogeneous and more healthy than female centenarians. Immunological factors regarding the age-related increase in pro-inflammatory status, and the frequency of HLA ancestral haplotypes also show gender differences that likely contribute to the different strategies that men and women seem to follow to achieve longevity. Concerning the different impact of genetic factors on the probability of reaching the extreme limits of the human life-span, emerging evidence (regarding mtDNA haplogroups, Thyrosine Hydroxilase, and IL-6 genes) suggests that female longevity is less dependent on genetics than male longevity, and that female centenarians likely exploited a healthier life-style and more favorable environmental conditions, owing to gender-specific cultural and anthropological characteristics of the Italian society in the last 100 years.

Aged↗

PubMed2000

A cytofluorimetric study of T lymphocyte subsets in rat lymphoid tissues (thymus, lymph nodes) and peripheral blood: a continuous remodelling during the first year of life.

We previously demonstrated that the rat thymus undergoes a progressive remodelling long before the appearance of typical signs of involution [Quaglino, D., Capri, M., Bergamini, G., Euclidi, E., Zecca, L., Franceschi, C., Pasquali Ronchetti, I., 1998. Age-dependent remodelling of rat thymus. Morphological and cytofluorimetric analysis from birth up to one year of age. Eur. J. Cell. Biol. 76, 156-166]. To focus better on the complex remodelling that occurs in the rat immune system during the first year of life, we analysed the phenotype profile of thymocytes, and T lymphocytes from mesenteric lymph nodes and peripheral blood of the same animals by flow cytometry. Two experimental sets were performed simultaneously using the same animal strain, but starting and ending the study at different ages (15 days up to 300 days in the first experimental set, and 90 days up to 360 days of life in the second). In the rat these ages appear to be crucial not only for developmental, maturative and early involutional processes of the thymus, but also of the entire immune system. The main findings were the following: (1) in the thymus, CD8(-)CD4(-) cells increased, CD5(+)alphabeta TCR(-) and CD8(+)CD4(+) thymocytes decreased, while the most mature cell subset appeared well preserved with ageing; (2) in the lymph nodes, T helper and T cytotoxic lymphocytes decreased in the most aged animals. Memory/activated CD4(+)CD45RC(-) T cells decreased, while naive/resting CD4(+)CD45RC(+) cells increased in the youngest animals and decreased in the oldest. CD8(+)CD45RC(-) and CD8(+)CD45RC(+) lymphocytes showed a complex age-dependent trend, and (3) in peripheral blood, minor modifications were evident, such as an age-dependent increase in the alphabeta TCR(+)CD25(+) cell subset. Some of these changes were related to the developmental process, while others could likely be interpreted as early signs of immunosenescence. The role of these modifications in immune system is discussed within the framework of the remodelling hypothesis of immunosenescence. The age-dependent changes in these three lymphoid compartments, however, appear to be different and only partially overlapping, thus suggesting that the maturational, developmental and ageing processes have distinct characteristics in the central and peripheral lymphoid organs.

Age Factors↗

PubMed2000

Hemopoiesis in healthy old people and centenarians: well-maintained responsiveness of CD34+ cells to hemopoietic growth factors and remodeling of cytokine network.

In vitro hemopoiesis and hemopoietic cytokines production were evaluated in 9 centenarians (median age 100.5 years, age range: 100-104 years), 10 old people (median age: 71 years, age range: 66-73 years), and 10 young people (median age: 35 years, age range: 30-45 years), all carefully selected for their healthy status. The main findings were the following: (i) a trend towards a decreased absolute number of CD34+ progenitor cells in the peripheral blood of old people and centenarians, in comparison to young subjects; (ii) a well-preserved capability of CD34+ cells from old people and centenarians to respond to hemopoietic cytokines, and to form erythroid (BFU-E), granulocyte-macrophagic (CFU-GM), and mixed colonies (CFU-GEMM) in a way (number, size, and morphology) indistinguishable from that of young subjects; (iii) an age-related decreased in vitro production of granulocyte-macrophagic colony-stimulating factor (GM-CSF) and a decreased production of interleukin-3 (IL-3) in centenarians by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC); (iv) a linear increase of the serum level of stem cell factor (SCF), measured in the above-mentioned subjects and in 65 additional subjects, including 4 centenarians. These data suggest that basal hematopoietic potential is well preserved in healthy centenarians, and that the hemopoietic cytokine network undergoes a complex remodeling with age.

Adult↗

PubMed2000

Ceramide 2 (N-acetyl sphingosine) is associated with reduction in Bcl-2 protein levels by Western blotting and with apoptosis in cultured human keratinocytes.

BACKGROUND: Ceramides produced by sphingomyelin hydrolysis activate a cycle that is followed by three different major cellular responses: downregulation of cell proliferation, induction of cell differentiation and apoptosis. In the skin, the generation of intracellular ceramide may also provide a link between an extracellular signal and the induction of the apoptosis programme for the elimination of damaged cells. OBJECTIVES: We investigated the effect of ceramides capable of entering cells on cultured keratinocytes. METHODS: Human keratinocytes from neonatal skin were cultured in serum-free medium with or without increasing concentrations of ceramide 2 (CER-2; N-acetyl sphingosine) (5, 10, 20 and 40 micromol L-1). Proliferative effects were studied either by cell counts or by 3H-thymidine incorporation and flow cytometric analysis. Apoptosis was studied by TUNEL staining and Western blot analysis of Bcl-2 protein. RESULTS: Cell counts and DNA synthesis were reduced in a dose-dependent manner following CER-2 treatment. TUNEL staining showed CER-2-induced apoptosis at 48, 72 and 96 h. Western blot analysis showed that CER-2 induces downregulation of Bcl-2 at 24-96 h. CONCLUSIONS: These results demonstrate that CER-2 inhibits cell proliferation and induces apoptosis, possibly via a Bcl-2-dependent mechanism.

Apoptosis↗

PubMed2000

Quantitation of CD95 and CD95L mRNA expression in chronic and acute HIV-1 infection by competitive RT-PCR.

Human immunodeficiency virus-type 1 (HIV-1) infection is characterized by increased immune cell apoptosis. Apoptosis can be triggered by signals that arise from within the cell, or by signals that are elicited by binding of extracellular "death ligands" to their "death receptors," most of which belong to the tumor necrosis factor (TNF)-receptor family, such as CD95 (Fas/Apo-1). In immune cells the oligomerization of CD95, induced by its ligand CD95L, and the recruitment of different intracytoplasmic molecules that in turn activate FLICE/caspase 8 are crucial. To study the role of CD95/CD95L interactions during HIV-1 infection, we developed an original method based upon quantitative-competitive (QC) RT-PCR that allowed us to quantify the amounts of mRNA coding for the total (tCD95) and membrane (mCD95) forms of CD95. We first studied the expression of different forms of CD95 mRNA in a classical model of chronic HIV infection using two infected cell lines of different origin--lymphocytic (ACH-2) or monocytic (U1). We have shown that infected cells of monocytic origin preferentially produce the "protective" (soluble) form of CD95, and no detectable CD95L mRNA, while lymphoid cells produce more mRNA for the membrane form of CD95 (which triggers apoptosis) along with low but detectable amounts of CD95L mRNA. One can hypothesize that a complex balance exists between pro-apoptotic events, perhaps triggered by the host to limit viral production, and anti-apoptotic events likely triggered by the virus to increase its production and survival. In cells of monocytic origin, which act as a reservoir for the virus, the anti-apoptotic molecules are favored; in cells of lymphocytic origin, molecules with an apoptotic meaning are prevalent.

Apoptosis↗

PubMed1999

Differential down-regulation of CD95 or CD95L in chronically HIV-infected cells of monocytic or lymphocytic origin: cellular studies and molecular analysis by quantitative competitive RT-PCR.

We analysed the expression of CD95/CD95L in two widely used models for studying the cellular effects of chronic infection with human immunodeficiency virus type 1 (HIV-1), i.e. ACH-2 cells, derived from the lymphocytic cell line A301, and U1, derived from monocytic U937 cells. A301 and ACH-2 mounted the same amount of plasma membrane CD95, while U1 had a consistent decrease in CD95 expression. Using different antibodies, we failed to detect the plasma membrane form of its ligand, CD95L, but we could see the intracellular presence of that molecule in A301 cells and, to a lesser extent, in ACH-2 cells, but not in U937 or U1 cells. To confirm the cytofluorimetric data and quantify the expression of CD95L at the RNA level, we developed a quantitative competitive RT-PCR assay. The HUT78 cell line had about 50,000 copies mRNA/1000 cells, three times more after induction with a phorbol ester and ionomycin. ACH-2 expressed about 400- (basal) or 10- (induced) fold less CD95L mRNA than the parental cell line A301; U937 and U1 were below the limit of detection. In cells of lymphoid origin (ACH-2) chronic HIV infection inhibits the expression of CD95L, the phenomenon occurring at the transcriptional level. In cells of monocytic origin (U1) the infection decreases the plasma membrane expression of CD95. This suggests that HIV could trigger different anti-apoptotic strategies which likely depend upon the cell line which is infected. In monocytic cells which act as a viral reservoir, the expression of the molecule whose binding triggers apoptosis decreases, while in lymphoid cells, capable of exerting cytotoxicity, the expression of a molecule which induces apoptosis is reduced.

Apoptosis↗

PubMed1999

Apoptotic features of peripheral blood granulocytes and monocytes during primary, acute HIV infection.

Apoptosis plays a major role during HIV infection, including the primary, acute HIV syndrome (AHS), during which such phenomenon is massive. We asked whether apoptosis involved not only peripheral blood lymphocytes, but also monocytes (PBM) and granulocytes (PBG). Thus, we studied cells from different patients during the acute phase of the viral syndrome. The CD95 molecule was expressed at high density on the PBM and PBG surface during AHS. Culturing PBG for a few hours resulted in a significant membrane expression of phosphatidylserine, consistent with apoptosis. However, cells maintained for hours plasma membrane integrity and showed no relevant changes in mitochondrial membrane potential. The overexpression of CD95 was not associated with high plasmatic levels of sCD95 and, together with apoptosis and its related markers decreased after a few weeks of highly active antiretroviral therapy. During AHS, a deregulation of the CD95 system occurs in monocytes and granulocytes, is related to a high propensity of PBG to undergo apoptosis, and may contribute to the pathogenesis of the disease. Antiretroviral treatment resulted not only in a decrease of virus production, but also in a reduced PBG tendency to undergo spontaneous apoptosis. Even if the mechanism(s) responsible for this phenomenon remains to be elucidated, our data suggest a possible (indirect?) action of antiretroviral therapies on PBG and PBM which could explain, at least partially, the rescue of natural immunity and the reduced use of granulocyte-colony stimulating factor during such treatments.

Acute Disease↗

PubMed1999

Cell adhesion and the immune system: a case study using earthworms.

In the earthworm's immune system, cell adhesion, which occurs by putative receptors on leukocytes, is essential after recognition of self vs. non-self. Confrontation with foreign antigens is a normal event in the environment, replete with microbial pathogens that pose a threat to survival. To better understand what happens when an effector cell first recognizes a foreign target followed by its adhesion to it, isolated leukocytes, in sufficient quantities to be subjected to various analyses, have been extremely beneficial. In vitro approaches when accompanied by biochemical, immunological, and molecular technologies, have opened up new vistas concerning the immune response of earthworms and other invertebrates. The most recent discovery includes the preliminary identification of cell differentiation (CD) markers that play vital roles in recognitive and adhesive events. Certain leukocyte effectors show characteristics of natural killer (NK) cells that may act differently depending upon their source, whether autogeneic, allogeneic, xenogeneic, or expressed under normal or varying environmental conditions including exposure to xenobiotics. At the level of earthworm evolution, there is apparently a dissociation of phagocytosis from the process of killing by NK-like effectors. There are at least three future challenges. First, it is essential to determine the precise nature of the CD markers with respect to their molecular structure. Second, once their molecular and biochemical characteristics have been defined, the role of these markers in cellular and humoral mechanisms must be clarified in order to define effector cell products and resulting immune responses. Third, there is a need to differentiate between the several lytic factors that have been found in earthworms with respect to molecular structure, and biochemical and functional characterization.

Animals↗

PubMed1999

Long-term immunologic effects of thymectomy in patients with myasthenia gravis.

BACKGROUND: Thymectomy (Tx) is a common therapeutic option to treat myasthenia gravis (MG), but its effects on the immune system are still obscure in humans. OBJECTIVE: We sought to evaluate long-term immunologic effects of therapeutic Tx in patients with MG. METHODS: T- and B-cell subsets and T-cell repertoire were analyzed in 35 patients with MG, 16 with previous Tx (at least 8 years before), 6 with recent (<1 year) Tx, and 13 without Tx, as well as in 32 healthy subjects used as normal control subjects. Serum immunoglobulins and a variety of autoantibodies were also measured. A subsequent 3-year clinical follow-up was performed to verify the possible appearance of systemic autoimmune diseases. RESULTS: The long-term thymectomized (Txd) patients had mild T-cell lymphopenia and an expansion of some Vbeta families among circulating CD4+ and CD8+ T cells. They displayed a normal number of total B and CD5+ B-circulating lymphocytes, but they also displayed a polyclonal increase in serum IgM and IgG associated with the presence of high levels of a variety of organ- and nonorgan-specific autoantibodies, including anti-dsDNA and anticardiolipin, without clinical evidence of autoimmune disease. These serologic abnormalities were not detectable in both non-Txd and recently Txd patients. After 3 years, 2 long-term Txd patients had systemic lupus erythematosus and an undifferentiated connective tissue disease. CONCLUSIONS: The association between MG and laboratory findings of systemic autoimmune disease may be in part related to Tx rather than to MG. Tx may represent a risk for the development of systemic autoimmune disorders over years in patients with MG.

Adult↗

PubMed1998

Apoptosis-like, reversible changes in plasma membrane asymmetry and permeability, and transient modifications in mitochondrial membrane potential induced by curcumin in rat thymocytes.

Curcumin (diferuoylmethane) is a natural compound with anticarcinogenic activities which is able to exert either proapoptotic or antiapoptotic effects in different cell types. This paper focuses on the sequence and extent of primary events induced by curcumin, in comparison with those occurring during dexamethasone-induced apoptosis in rat thymocytes. It also presents annexin VI-FITC as a new probe for studying membrane asymmetry. Curcumin readily penetrates into the cytoplasm, and is able to accumulate in membranous structures such as plasma membrane, endoplasmic reticulum and nuclear envelope. Curcumin-treated cells exhibit typical features of apoptotic cell death, including shrinkage, transient phosphatidylserine exposure, increased membrane permeability and decrease in mitochondrial membrane potential. However, nuclei morphology, DNA fragmentation, the extent and time-course of membrane changes are different from those observed during dexamethasone-induced apoptosis, suggesting that, despite many similarities, the mode of action and the events triggered by curcumin are different from those occurring during typical apoptosis.

Animals↗

PubMed1998

Toxin-induced activation of Rho GTP-binding protein increases Bcl-2 expression and influences mitochondrial homeostasis.

It is now well established that apoptosis plays a pivotal role in several physiological and pathological situations. Consequently, the mechanisms controlling the cell fate are currently the subject of intense investigation. In this work, we report that an Escherichia coli protein toxin (Cytotoxic Necrotizing Factor 1, CNF1) which activates the Rho GTP-binding protein and prevent apoptosis in epithelial cells was able to: (i) influence the mitochondrial homeostasis and (ii) modulate the expression of proteins belonging to the Bcl-2 family. In particular, the content of the antiapoptotic products Bcl-2 and Bcl-XL resulted to be increased in treated cells, whereas the expression of the proapoptotic protein Bax remained unaltered. CNF1 induces cell spreading via activation of Rho and cell spreading has been reported to promote cell survival. Cytochalasin B, which provokes most of the morphological changes typical of CNF1, including cell spreading, but without the involvement of Rho, was unable to counteract apoptosis. Altogether our results suggest a link between the Rho GTP-binding protein and the regulation of the mitochondrial homeostasis via an effect on the antiapoptotic proteins of the Bcl-2 family.

Apoptosis↗

PubMed1998

Decrease in mitochondrial energy coupling by thyroid hormones: a physiological effect rather than a pathological hyperthyroidism consequence.

The effect of the in vivo thyroid status on mitochondrial membrane potential (delta psi(m)) in isolated rat hepatocytes was studies by means of a cytofluorimetric technique and the delta psi(m)-specific probe JC-1. It is shown that the delta psi(m) level decreases in the order hypothyroid > euthyroid > hyperthyroid. Polarographic measurement of the hepatocyte respiratory rates revealed an opposite trend of values: the highest respiratory rate in hepatocytes from hyperthyroid animals, the lowest in those from hypothyroid ones. This means that mitochondrial energy coupling is highest in hypothyroid hepatocytes and lowest in hyperthyroid hepatocytes. 6-Ketocholestanol added to hepatocytes failed to counterbalance the uncoupling effect of thyroid hormones on delta psi(m) and respiration rate. Under the same conditions, 6-ketocholestanol appeared to be effective in recoupling of respiration uncoupled by low concentrations of the artificial protonophore FCCP. The mechanism and possible physiological functions of the thyroid hormone-induced decrease in mitochondrial energy coupling are discussed.

Animals↗

PubMed1998

Mitochondrial membrane potential and DNA stainability in human sperm cells: a flow cytometry analysis with implications for male infertility.

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-1 and DNA stainability with propidium iodide (PI). JC-1 staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-1 staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.

Adult↗

PubMed1998

Age-related modifications of the human alphabeta T cell repertoire due to different clonal expansions in the CD4+ and CD8+ subsets.

We have studied the effects of a life-long antigen stimulation on the clonal heterogeneity of human peripheral T cell subsets, as defined by their CD45 isoform expression. CD4+ or CD8+ T cells were obtained from healthy donors ranging in age from 20 to 100 years, and sorted into CD45RA+ and CD45RO+ populations. A modified PCR-heteroduplex analysis was then used to directly compare the TCR Vbeta clonal make up of either compartment pair. We find that the CD4+ T cell repertoire remains largely polyclonal throughout life, since CD4+ expanded clones are rare and accumulate predominantly in the CD45RO+ compartment of exceptionally old donors (100 years old). In contrast, the CD8+ T cell subset contains expanded clones which are already detectable in young adults and become very frequent in 70- to 75-year-old donors in both CD45RA+ and CD45RO+ compartments analyzed. Interestingly, some expanded clones are detectable in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments of either CD4+ or CD8+ T cells. These results indicate that the age-dependent accumulation of expanded clones starts earlier and is more pronounced in the CD8+ than in the CD4+ T cell subset, reinforcing the concept that clonal expansion in the two subsets is controlled by substantially different mechanisms. Furthermore, whereas the finding of expanded CD45RO+ T cell clones is explained by antigen-driven proliferation, the detection of expanded clones in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments would support the hypothesis of reversion from the CD45RO+ to the CD45RA+ phenotype after antigen encounter.

Adult↗

PubMed1998

Use of flow cytometry as a tool to study mitochondrial membrane potential in isolated, living hepatocytes.

The present paper describes the possibility of determination of mitochondrial membrane potential (Deltapsi) in isolated hepatocytes making use of a Deltapsi-sensitive dye, i.e., the lipophilic cationic probe 5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) and of cytofluorimetry. The validity of the method was proved by treating hepatocytes with FCCP (decrease of Deltapsi) and subsequent addition of 6-ketocholestanol (increase of Deltapsi). The results indicate that the proposed method may be used in laboratory practice.

Animals↗