Alberto Degrassi

Age-associated changes in CD8+ and CD16+ cell reactivity: clonal analysis.

A cloning technique was used to estimate the frequency of proliferating T cell precursors, the growth capacity of clone‐forming cells and the functional activity of clones established in vitro from peripheral blood lymphocytes of young and old people. The mean frequency of proliferating precursors was lower in the elderly as was the proliferative capacity of CD8+ clones. In contrast, CD4+ and CD16+ clones showed a proliferation similar to that obtained from young subjects. When the clones were examined for their functional activity, CD4+ clones from both groups failed to show any cytolytic activity, while CD8+ clones exerted cytolysis against K562 and in antibody‐dependent cell‐mediated cytotoxicity but this function was reduced in clones derived from old subjects. Similarly, CD16+ clones from the elderly showed a decreased activity at some effector‐to‐target cell ratios. We conclude that the impaired functional activity (T or NK‐dependent) found in the peripheral blood of aged subjects persists after in vitro selection when these cells are analysed at clonal level.

A fluorescent in situ hybridization method in flow cytometry to detect HIV-1 specific RNA

In HIV+ patients, the presence of HIV-RNA in plasma and circulating cells has been reported to be a marker of progression but the percentage of transcriptionally active infected cells remains unclear. We have developed a reliable fluorescent in situ hybridization method for the detection of HIV specific RNA by flow cytometry. The procedure was applied to a panel of chronically infected cell lines and to an acutely infected cell line mimicking normal peripheral blood lymphocytes in susceptibility to HIV-1. The cells were fixed in suspension and hybridized by means of an HIV-1 genomic probe labeled with digoxigenin-11-dUTP. An FITC-labeled anti-digoxigenin antiserum was then applied and the resulting fluorescence signals were analyzed both by flow cytometry and confocal microscopy. Different procedures for double staining HIV-RNA together with virus induced proteins or surface markers were also developed. Flow cytometric detection of in situ hybridization offers the possibility of analyzing thousands of cells in a few seconds and of collecting multiparametric information at the single cell level, thus providing a potential tool for detecting the rare HIV-RNA expressing cells in peripheral blood samples.

Temporary neurological improvement in a patient with acute or chronic liver failure treated with a bioartificial liver device.

Temporary neurological improvement in a patient with acute or chronic liver failure treated with a bioartificial liver device

Cellular response and anti-HBs synthesis in vitro after vaccination with yeast-derived recombinant hepatitis vaccine

Two groups of subjects receiving two different doses of yeast-derived recombinant hepatitis B surface antigen (rHBsAg) (10 micrograms Gen-HB-Vax, Merck Sharp and Dohme and 20 micrograms Engerix-B, Smith Kline and French) were investigated for in vitro specific humoral and cellular response to the native protein. In vitro proliferative response was dependent on the following critical variables: (1) antigen-specific precursor lymphocytes were present in the peripheral blood for a very short time; (2) the number of circulating specific precursors was dependent on the dose of HBsAg used for vaccination; (3) the presence of antigen-presenting cells was necessary to obtain a blastogenic response in vitro. In vitro proliferation was enhanced by the addition of recombinant interleukin 2 (rIL-2). Spontaneous and stimulated (anti-CD3, pokeweed mitogen) anti-HBs antibody production in vitro was obtained in only eight out of 20 subjects after the fourth boost. Although a different immunogenicity of the two vaccines cannot be excluded, these data strongly suggest that T and B cells responsive to HBsAg present different kinetics of recirculation in the peripheral blood, depending on the antigen dose used for immunization.

In vitro Toxicity of 2′,3′-Dideoxy-3′-Thiacytidine (BCH189/3TC), a New Synthetic Anti-HIV-1 Nucleoside

2′,3′-Dideoxy-3′-thiacytidine (BCH189 or 3TC, IAF BioChem International Inc., Montreal, Canada) is a new synthetic anti-HIV-1 dideoxynucleoside with a sulphur atom in the heterocycle ring. Preclinical studies showed that this compound at 5–10 μM displays potent anti-retroviral activity in vitro against HIV-1 infected T (H9 and MT4) and monocyte/macrophage (U937) cell lines, associated with low acute and chronic myelotoxicity in animals. The present study evaluates the in vitro toxicity of BCH189 in comparison with 3′-azido-2′,3′-dideoxythymidine (AZT) on peripheral blood lymphocytes (PBL) from normal subjects and asymptomatic HIV-1 seropositive patients. BCH189 and AZT were used at concentrations ranging from 0.01 to 50 μM, and their action was analysed by: (1) proliferation assay using two activation pathways (concanavalin A and anti-CD3 monoclonal antibody), (2) natural killer (NK) activity, and (3) IL2 receptor (CD25) expression after stimulation with concanavalin A (ConA). Exposure of PBL for 72 h to the two drugs resulted in decreased DNA synthesis only in cells treated with AZT. In particular, BCH189 did not modify significantly cellular proliferation, while a significant inhibition of PBL from normal subjects and HIV-1 positive patients, was observed at AZT concentrations ranging between 5 to 50 μM. Both 2′,3′-dideoxy-3′-thiacytidine (BCH189) and AZT had no effect on NK cytolytic activity. BCH189, at various doses, did not interfere with IL2 receptor (CD25) expression on CD3 positive cells, while AZT, only at very high concentration (50μM), caused a decrease in the number of CD25 positive cells. These studies suggest that BCH189 is less toxic in vitro to lymphocytes than AZT and further support its use in clinical trials on HIV-1 infected subjects as a good alternative to AZT.

Lymphocyte clones from old subjects: growing rate and functional activity

Old subjects exhibit a decline in circulating T cells and an impaired proliferative response to mitogens, plus a relative increase in cells with NK phenotype not associated with a concomitant increase in their cytolitic activity.In the present study a limiting dilution assay was used to evaluate the phenotype, the functional activity and the proliferative capacity of clones obtained from peripheral blood lymphocytes of old and young subjects. CD5+ CD8+ clones from old people showed a significant impairment in their proliferative capacity and a decreased lytic activity against K562 and P815-IgG cell lines.

PROLIFERATIVE CAPACITY OF LYMPHOCYTE CLONES FROM OLD SUBJECTS

ABSTRACT Many parameters have been used to evaluate immune response in elderly people: the number of peripheral T lymphocytes is decreased and a reduced response to mitogens has been demonstrated. The aim of the study was to evaluate at a clonal level the proliferative capacity of clones derived from peripheral blood lymphocytes of a group of young and old healthy people. CD5+CD8+ clones from old people showed a significant impairment in their growing rate, on the contrary the proliferative capacity of CD5+CD4+ and CD5+CD16+ clones was similar between old and young subjects.

Phenotype and Function of Lymphocyte Clones from Old and Young Subjects

Alterations of the immune system during human aging affect different lymphocyte functions. A progressive modification in the distribution of lymphocyte subpopulations in peripheral blood has been described, the major finding being a decrease in circulating T cells after adulthood (Kay and Makinodan, 1981).

Thymopentin Therapy in Elderly Patients with Chronic Bronchitis

Aging has been reported to be the most common immune deficiency syndrome (Kay and Makinodan, 1981), since major pathological events and diseases are associated with advancing age. The decline in immune responsiveness affects both the T- and B-cell compartment, although T-cell functions seem to be the most impaired (Facchini et al., 1986, 1987). In particular, a reduced number of T-cell subsets (Abo et al., 1981) has been reported during human aging together with a depleted production of lymphokines as interleukin-2 (IL-2) and its receptor (IL-2R) (Gillis et al., 1981; Facchini et al., 1983).