Biochemical SocietyTransactions ( 1 992) 20 377s
Purine nucleotide content in human T4 and T8 lymphocyte subpopulations from normal subjects and AIDS patients TABUCCHI A., CARLUCCI F., CONSOLMAGNO E., RE M.C.*, MONARI P.*. MARINELLO E., PAGAN1 R. Institute of Biochemistry and Enzymology University of Siena Institute of Microbiology - University of Bologna - (Italy) In previous studies we analyzed the purine nucleotide content of peripheral blood lymphocytes (PBL) from healthy subjects and AIDS patients [l-41. Healthy subjects showed low nucleoside monophosphate content, high ADP, GDP, GTP, NAD and even higher ATP levels. The values and their dispersion agreed well with data reported in the literature [5-71. In asymptomatic HIV-1 seropositive subjects, NAD was unchanged and the concentration of IMP enhanced, while diand tri-phosphates were significantly depressed. In ARC patients, the pattern of monophosphates was unchanged with respect to healthy subjects; NAD was lower, ADP and GDP were cloae to normal values, ATP and GTP were higher than in seropositive and normal lymphocytes. In AIDS patients, NAD was enhanced, monophosphates did not show important variations, but di- and triphosphates were significantly higher than in healthy subjects. In order to understand if these variations were related to the staging of the disease or to an altered ratio of the two major subpopulations, we directly analyzed the nucleotide content of T4 and T8 cells. 20 ml of fresh EDTA-treated venous blood was obtained from healthy volunteer donors and sick patients. Lymphocytes were separated by Ficoll density gradient centrifugation and washed with PBS. The T4 and T8 cells were isolated using Dynabeads M-450 CD4 and Dynabeads M-450 CD8 (Dynal Oslo, Norway) respectively, magnetizable polystyrene beads coated with monoclonal antibody specific for CD4 and CD8 antigens of human T-lymphocytes [81. We firt isolated the T4 cells. Rosetting was performed with continuous gentle agitation (4°C) of a suspension of 5 x 106 cells incubated with 15 x 106 beads. The rosetted cells were separated from nonrosetted cells by holding the tube in a magnetic device for 180 a . The non-bound cells (CD4-) were decanted, collected by centrifugation and incubated with CD8 beads. by a similar procedure. After isolation, T4 and T8 cells were resuspended in PBS to a x 106 /ml. final concentration of 5 Nucleotides were dissolved by PCA treatment and analyzed by HPLC, as previously reported [l].Table 1 shows T4 and TB nucleotide levels in a healthy subject and in the two different categories of patients.T4 and T8 cells showed a similar pattern of nucleotides in the case of the healthy subject. T8 cells from all HIV patients showed enhanced NAD and triphosphates levels; diphosphates did not seem to vary significantly. Di- and tri-phosphates increased significantly in T4 cells at the early stages of the disease; also monophosphates showed the same tendency, but with an irregular pattern. The present results confirm the increase in nucleotide content
found in our preliminary studies on revealing a general increase in nucleotide pattern.
PBL, the
Table 1. T4 and TB nucleotide levels in healthy subjects and AIDS patients. Values, expressed as mol/lO~ cells, represent a typical experiment. Nucleotides:
Cells
T8
T4
NAD AMP IMP (;Mp
ADP GDP
ATP GTP
A
B
C
100
250 62
20
10 112 10 303 48 577 91
49
220 756 157 1400 419
93 536 20 718 160
1780 410
A
B
C
30 105 11 16 424 52 390 53
110 39 40 4 123 20 473 120
52 60
52 5 220
24 907 177
A: Healthy subject
B: Asymptomatic subject C: Symptomatic subject The altered pool observed in the case of asymptomatic and symptomatic patients could be ascribed to accelerated synthesis related to impaired activation of the cells. The present study suggests that such alterations are already evident at an early stage of the disease, even if literature reports that the percentage of the cells infected by the virus seems to be very low [S]. On the other hand the results could be related to defective activation and modification of humoral relationships induced by HIV C101. The observed variations so clear that nucleotide pool are determination may be regarded as a diagnostic tool. This work was financed by the Italian Secretariat for Health, National Institute of Health, IV AIDS Project 1991, Rome, Italy. REFERENCES 1. Tabucchi, A.,Carlucci, F., Ramazzotti, E., Re,M.C., Marinello, E., Rubino, M., Pagani, R. (1991) Biomed. & Pharmacother. 45, 25-29. 2. Carlucci, F., Tabucchi, A., Consolmagno, E., Tagliaferri, P., Porcelli, B., Marinello, E., Leoncini, R., Pagani, R. (1992) Biomed. & Pharmacother. 46, 7-12. 3. Tabucchi, A., Carlucci, F., Rubino, M., Marinello,E., Ramazzotti,E., Re,M.C., Pagani, R. (1991) Giorn.It.Chim.Clin. 16, 231-237. 4. Tabucchi, A., Carlucci, F., Marinello, E., Re, M.C., Bisozzi, L., Cacopardo, F., Pagani, R. (1991) Rec.Progr.Med. 82, 581-584. 5. Peters, G.J., De Abreu, R.A., Oosterhof, A., Veerkamp, J.H. (1983) Biochim. Biophys. Acta 759, 7-15. 6. Marijnen, Y.M.T., De korte, D., Van A.H., Ross, D. (1989) Biochim. Gennip, Biophys. Acta 1012, 148-155. 7.De Korte,D., Haverkort,W.A., Van Gennip, A . H., Ross, D.(1985)Anal.Biochem.l47, 197-209 8. Brinchmann,J.E.,Vartdal, F.,Gaudernack, G., Markussen, G.,Funderud, S.,Ugelstadt, J., Thorsby, E.(1988)Clin.Exp.Immunol 71,182-186. 9. Fauci, A.S. (1988) Science 239, 617-622. 10. Nokta, M. & Pollard, R. (1991) Virology 181, 211-217.
Purine nucleotide content in human T4 and T8 lymphocyte subpopulations from normal subjects and AIDS patients TABUCCHI A., CARLUCCI F., CONSOLMAGNO E., RE M.C.*, MONARI P.*. MARINELLO E., PAGAN1 R. Institute of Biochemistry and Enzymology University of Siena Institute of Microbiology - University of Bologna - (Italy) In previous studies we analyzed the purine nucleotide content of peripheral blood lymphocytes (PBL) from healthy subjects and AIDS patients [l-41. Healthy subjects showed low nucleoside monophosphate content, high ADP, GDP, GTP, NAD and even higher ATP levels. The values and their dispersion agreed well with data reported in the literature [5-71. In asymptomatic HIV-1 seropositive subjects, NAD was unchanged and the concentration of IMP enhanced, while diand tri-phosphates were significantly depressed. In ARC patients, the pattern of monophosphates was unchanged with respect to healthy subjects; NAD was lower, ADP and GDP were cloae to normal values, ATP and GTP were higher than in seropositive and normal lymphocytes. In AIDS patients, NAD was enhanced, monophosphates did not show important variations, but di- and triphosphates were significantly higher than in healthy subjects. In order to understand if these variations were related to the staging of the disease or to an altered ratio of the two major subpopulations, we directly analyzed the nucleotide content of T4 and T8 cells. 20 ml of fresh EDTA-treated venous blood was obtained from healthy volunteer donors and sick patients. Lymphocytes were separated by Ficoll density gradient centrifugation and washed with PBS. The T4 and T8 cells were isolated using Dynabeads M-450 CD4 and Dynabeads M-450 CD8 (Dynal Oslo, Norway) respectively, magnetizable polystyrene beads coated with monoclonal antibody specific for CD4 and CD8 antigens of human T-lymphocytes [81. We firt isolated the T4 cells. Rosetting was performed with continuous gentle agitation (4°C) of a suspension of 5 x 106 cells incubated with 15 x 106 beads. The rosetted cells were separated from nonrosetted cells by holding the tube in a magnetic device for 180 a . The non-bound cells (CD4-) were decanted, collected by centrifugation and incubated with CD8 beads. by a similar procedure. After isolation, T4 and T8 cells were resuspended in PBS to a x 106 /ml. final concentration of 5 Nucleotides were dissolved by PCA treatment and analyzed by HPLC, as previously reported [l].Table 1 shows T4 and TB nucleotide levels in a healthy subject and in the two different categories of patients.T4 and T8 cells showed a similar pattern of nucleotides in the case of the healthy subject. T8 cells from all HIV patients showed enhanced NAD and triphosphates levels; diphosphates did not seem to vary significantly. Di- and tri-phosphates increased significantly in T4 cells at the early stages of the disease; also monophosphates showed the same tendency, but with an irregular pattern. The present results confirm the increase in nucleotide content
found in our preliminary studies on revealing a general increase in nucleotide pattern.
PBL, the
Table 1. T4 and TB nucleotide levels in healthy subjects and AIDS patients. Values, expressed as mol/lO~ cells, represent a typical experiment. Nucleotides:
Cells
T8
T4
NAD AMP IMP (;Mp
ADP GDP
ATP GTP
A
B
C
100
250 62
20
10 112 10 303 48 577 91
49
220 756 157 1400 419
93 536 20 718 160
1780 410
A
B
C
30 105 11 16 424 52 390 53
110 39 40 4 123 20 473 120
52 60
52 5 220
24 907 177
A: Healthy subject
B: Asymptomatic subject C: Symptomatic subject The altered pool observed in the case of asymptomatic and symptomatic patients could be ascribed to accelerated synthesis related to impaired activation of the cells. The present study suggests that such alterations are already evident at an early stage of the disease, even if literature reports that the percentage of the cells infected by the virus seems to be very low [S]. On the other hand the results could be related to defective activation and modification of humoral relationships induced by HIV C101. The observed variations so clear that nucleotide pool are determination may be regarded as a diagnostic tool. This work was financed by the Italian Secretariat for Health, National Institute of Health, IV AIDS Project 1991, Rome, Italy. REFERENCES 1. Tabucchi, A.,Carlucci, F., Ramazzotti, E., Re,M.C., Marinello, E., Rubino, M., Pagani, R. (1991) Biomed. & Pharmacother. 45, 25-29. 2. Carlucci, F., Tabucchi, A., Consolmagno, E., Tagliaferri, P., Porcelli, B., Marinello, E., Leoncini, R., Pagani, R. (1992) Biomed. & Pharmacother. 46, 7-12. 3. Tabucchi, A., Carlucci, F., Rubino, M., Marinello,E., Ramazzotti,E., Re,M.C., Pagani, R. (1991) Giorn.It.Chim.Clin. 16, 231-237. 4. Tabucchi, A., Carlucci, F., Marinello, E., Re, M.C., Bisozzi, L., Cacopardo, F., Pagani, R. (1991) Rec.Progr.Med. 82, 581-584. 5. Peters, G.J., De Abreu, R.A., Oosterhof, A., Veerkamp, J.H. (1983) Biochim. Biophys. Acta 759, 7-15. 6. Marijnen, Y.M.T., De korte, D., Van A.H., Ross, D. (1989) Biochim. Gennip, Biophys. Acta 1012, 148-155. 7.De Korte,D., Haverkort,W.A., Van Gennip, A . H., Ross, D.(1985)Anal.Biochem.l47, 197-209 8. Brinchmann,J.E.,Vartdal, F.,Gaudernack, G., Markussen, G.,Funderud, S.,Ugelstadt, J., Thorsby, E.(1988)Clin.Exp.Immunol 71,182-186. 9. Fauci, A.S. (1988) Science 239, 617-622. 10. Nokta, M. & Pollard, R. (1991) Virology 181, 211-217.
