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AIDS:
Research Letters

Changes in apoptosis after interruption of potent antiretroviral therapy in patients with maximal HIV-1-RNA suppression

Grelli, Sandroa; Di Traglia, Loidea; Matteucci, Claudiaa; Lichtner, Miriamc; Vullo, Vincenzoc; Di Sora, Fiorellad; Lauria, Filippod; Montella, Francescod; Favalli, Cartesioa; Vella, Stefanoe; Macchi, Beatricebf; Mastino, Antoniog

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aDepartment of Experimental Medicine and Biochemical Sciences and bDepartment of Neuroscience, University of Rome ‘Tor Vergata', Rome, Italy; cDepartment of Infectious and Tropical Disease, University of Rome ‘La Sapienza', Rome, Italy; dS. Giovanni Hospital, Rome, Italy; eIstituto Superiore di Sanità, Rome, Italy; fIRCCS, S. Lucia, Rome, Italy; and gDepartment of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina, Italy.

Sponsorship: This study was supported by grants from the Italian Ministry of Health, AIDS Project, from MURST, National Project ‘Cell Death Signalling and Viral Infections', co-ordinated by Professor E. Garaci, and from the CNR, Special Project ‘Biology of Ageing'.

Received: 15 January 2001; accepted: 30 January 2001.

Potent antiretroviral therapy has considerably changed the natural history of HIV infection throughout the western world [1,2]. A sustained reduction of plasma viral loads is indeed associated with progressive immune reconstitution, even in patients who start therapy in a fairly advanced stage of the disease. A number of studies have also shown that potent antiretroviral therapy is associated with the inhibition of either spontaneous or activation-induced cell death, as an early response to treatment [3]. Whether the suppression of viral replication is directly responsible for the inhibition of apoptotic T cell death during antiretroviral therapy is an area of current investigation [4,5]. Our recent results have demonstrated that spontaneous and anti-Fas-induced apoptosis were inversely correlated with an increase in the CD4 cell count and with significant suppression of plasma viraemia [6].

Concerns about adherence and toxicity over the long term have led to a reassessment of the risk–benefit ratio of starting antiretroviral treatment early in the course of the infection. Moreover, because an increased proportion of patients undergo discontinuation of treatment as a result of several factors [7], many have started to investigate the strategy of structured treatment interruptions in patients with full HIV-RNA suppression, with the aim of reducing exposure to antiretroviral drugs. Viral rebound occurs in most, but not all cases. Although not usually associated with the selection of resistance mutations [8], it is often associated with an abrupt decrease in CD4 cell levels [9]. The full virological and immunological long-term consequences of structured treatment interruptions are not fully understood [10].

In order to understand the actual relationship between modulation of the apoptotic response in HIV-infected individuals and the immunological and virological response to treatment, we analysed the apoptotic pattern in three patients who achieved and maintained viral suppression for a significant length of time but experienced HIV plasma rebounds during treatment interruption. Patients were enrolled within the framework of a longitudinal open study, which included a larger cohort of HIV-1-infected patients, focused on the significance of apoptosis in peripheral blood mononuclear cells (PBMC) during potent antiretroviral therapy. Details of this study and results obtained at 6 months after the start of treatment have already been published [6]. Data reported in this letter refer to 96 weeks of follow-up. Blood samples were drawn after informed consent was obtained from each patient. PBMC were isolated from the heparinized blood of the patients and cultured for 48 h, before spontaneous apoptosis was evaluated by multiparameter flow cytometry analysis using a method previously described [11]. HIV-RNA levels were determined by a quantitative reverse transcription–polymerase chain reaction assay (Roche Molecular System, Branchburg, NJ, USA). CD4 cell counts were determined by immunofluorescence and flow cytometry analysis, according to standard procedures.

Patient 1, classified by the criteria of the Centers for Disease Control and Prevention (CDC) as A1, underwent antiretroviral therapy with didanosine, stavudine and ritonavir. After 20 weeks, the patient discontinued the protease inhibitor (PI) because of toxicity, and continued with the two nucleoside analogues for a few months. After 16 weeks from the discontinuation of the PI, the patient was switched to zidovudine, lamivudine and efavirenz. At the time of enrolment the CD4 T cell count was 663 cells/mm3, his CD8 cell count was 1089 cells/mm3, and his plasma-RNA level was 5.6 log10 copies RNA/ml. During the first 16 weeks of treatment, patient 1 responded to the therapy with a remarkable decrease in viral RNA in the plasma (Fig. 1a). The susceptibility to spontaneous apoptosis was strongly inhibited with respect to the value detected in the period before treatment (Fig. 1b). Surprisingly, the level of total CD4 cells slightly decreased to below the pre-therapy baseline (Fig. 1c). After 20 weeks of therapy, the PI was discontinued by the patient (who continued with the two nucleoside analogues only). One month after discontinuation, viral RNA was detected above the pre-treatment level and remained high for 2 months. In parallel, susceptibility to spontaneous apoptosis, which was still low one month after the discontinuation, returned dramatically to the pre-therapy baseline a month later, whereas CD4 cells continued to increase slightly. After 4 months of therapy with nucleoside inhibitors only, i.e. at 36 weeks from the beginning of treatment, the patient again started a potent regimen, in which the PI was substituted with a non-nucleoside reverse transcriptase inhibitor. One month after starting the new treatment, viral RNA was undetectable and spontaneous apoptosis gradually decreased again, reaching stable low levels after 56 weeks of observation.

Fig. 1
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Patient 2, classified as CDC A2, started therapy with didanosine, stavudine and nevirapine. After 20 weeks didanosine was substituted with lamivudine. At week 46 the patient experienced a major episode of non-compliance. After 18 weeks from the interruption of therapy the same regimen was started again. At enrolment in this study, the CD4 T cell count of the patient was 264 cells/mm3, his CD8 cell count was 776 cells/mm3 and his plasma-RNA level was 5.6 log10 copies RNA/ml. During the first 32 weeks of treatment, patient 2 responded to therapy with a decrease in viral load (Fig. 1d), accompanied by a consistent increase in CD4 cells (Fig. 1f), and a slow but clear inhibition of spontaneous apoptosis with respect to the pre-treatment level (Fig. 1e). After 46 weeks of therapy, treatment was interrupted on the decision of the patient. Viral load was already increased at 48 weeks of therapy, and achieved a high level at 64 weeks of therapy, although the detectable range was below the pre-treatment baseline. In parallel, spontaneous apoptosis, which was remarkably inhibited before treatment interruption, peaked at 64 weeks. Subsequently, the patient responded to the resumption of potent antiretroviral therapy, and apoptosis decreased in line with the viral load. Conversely, the CD4 cell level continued to increase, independently of the variations in viral load and apoptosis.

Patient 3, classified as CDC B2, started therapy with lamivudine, stavudine and nelfinavir. At week 61 he also stopped therapy because of non-compliance. After 11 weeks from interruption, the same regimen was restarted. At the time of enrolment in this study, the CD4 T cell count of the patient was 334 cells/mm3, his CD8 cell count was 1395 cells/mm3, and his plasma RNA level was 5.7 log10 copies RNA/ml. In this patient the discontinuation of therapy also caused a remarkable increase in viral load (Fig. 1g). Viral load rebound was followed by a prompt rebound in apoptosis level (Fig. 1h), whereas no modification in CD4 cell count was observed (Fig. 1). After the resumption of potent therapy, viral load progressively decreased (Fig. 1g) and apoptosis levels returned to lower values (Fig. 1h), whereas CD4 cell counts slowly continued to decrease (Fig. 1i).

Case reports presented in this letter show that viral rebounds caused by treatment interruption were invariably associated with a marked increase in apoptosis levels. After treatment re-institution, the successful virological response was followed by a new decrease in apoptosis. However, the increase in spontaneous apoptosis was not associated with a parallel decrease in the total number of CD4 cells. Rather, the increase in apoptosis was concomitant with an increase in the percentage of CD4+Fas+CD45R0+ cells (data not shown). Overall, our data contrast the hypothesis that the limited decrease in CD4 cells during viral rebound is caused by the persistence of low levels of apoptosis. Moreover, our results also suggest that the absence of a decrease in the CD4 cell count is not caused by a slow turnover of CD4 cells at the peripheral level. The partly reconstituted immune system of the host may thus be able to absorb the impact of a renewed increase in T cell apoptosis by replacing dead cells with mobilized cells or with newly originated lymphocytes. On the other hand, enhanced susceptibility to apoptosis associated with an increase in viral load could be caused by host cell antiviral mechanisms that attempt to control viral spread [12], thus contributing temporarily to the maintenance of immune cell homeostasis. However, these compensation phenomena could occur at the point when: (i) the acceleration in cell death persists for a limited duration in time; (ii) the immunological status of the patient at the moment of treatment interruption, and particularly his CD4 cell count, was relatively conserved. This is indeed the case with our patients. Presumably, the case of patients on virological failure and who undergo treatment interruption while waiting for a salvage regimen is quite different, as described in a recent study [9].

In conclusion, despite the limited number of patients examined, our data strongly suggests that viral load rebounds have an immediate impact on the levels of spontaneous apoptosis in the PBMC of HIV-infected patients. This finding should be considered when designing and evaluating structured treatment interruption strategies.

Sandro Grellia

Loide Di Tragliaa

Claudia Matteuccia

Miriam Lichtnerc

Vincenzo Vulloc

Fiorella Di Sorad

Filippo Lauriad

Francesco Montellad

Cartesio Favallia

Stefano Vellae

Beatrice Macchibf

Antonio Mastinog

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References

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4. Dockrell DH, Badley AD, Algeciras-Schimnich M. et al. Activation-induced CD4+ T cell death in HIV-positive individuals correlates with Fas susceptibility, CD4+ T cell count, and HIV plasma viral copy number. AIDS Res Hum Retroviruses 1999, 15: 1509 –1518.

5. Roger PM, Breittmayer JP, Arlotto C. et al. Highly active anti-retroviral therapy (HAART) is associated with a lower level of CD4+ T cell apoptosis in HIV-infected patients. Clin Exp Immunol 1999, 118: 412 –416.

6. Grelli S, Campagna S, Lichtner M. et al. Spontaneous and anti-Fas induced apoptosis in lymphocytes from HIV-infected patients undergoing highly active anti-retroviral therapy. AIDS 2000, 14: 939 –949.

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11. Matteucci C, Grelli S, De Smaele E, Fontana C, Mastino A. Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry. Cytometry 1999, 35: 145 –153.

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© 2001 Lippincott Williams & Wilkins, Inc.

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