5th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV 8–11 July 2003, Le Meridien Montparnasse, Paris, France

THE ROLE OF TUMOUR NECROSIS FACTOR ALPHA AND ANTIRETROVIRALS IN ADIPOCYTE APOPTOSIS IN VITRO

Antiviral Therapy 2003; 8:L23 (abstract 26)

P Domingo, F Vidal, S Veloso, MA Sambeat, J Sirvent, J Vendrell, X Matias-Guiu, C Richart and JC Domingo
Hospital de la Santa Creu i Sant Pau, Barcelona, Spain

BACKGROUND: Subcutaneous adipocyte apoptosis is a prominent feature in patients with HIV-associated fat redistribution syndromes (FRS). Tumour necrosis factor alpha (TNF-α), which is elevated in patients with FRS, is known to potentially promote preadipocyte and adipoyte apoptosis.

METHODS: MTT method was used to assess the cellular viability of 3T3-L1 adipocytes in presence of antiretroviral drugs [stavudine (d4T), lamivudine (3TC) and indinavir, either alone or in combination]. DNA fragmentation associated with apoptotic cell death was studied by TUNEL (TdT-mediated dUTP nick-end labelling) assay. Adipocyte cells (1×10⁶/ml) placed in 6-well plates were processed according to the ApoAlert DNA fragmentation kit protocol supplied by the manufacturer (Clontech Laboratories, Inc., Palo Alto, CA, USA). Briefly, cells were incubated with TdT digoxigenin- labelled dUTP followed by incubation with FITC-labelled anti-digoxigenin antibody. The FITC-labelled- positive cells were identified by flow cytometry. A total of 10,000 cells per sample were analysed and FACS analysis was performed using a FACScan flow cytometer (Becton Dickinson) equipped with a 488 nm Argon laser. CellQUEST software (Becton Dickinson) was used for data analysis.

RESULTS: The adipocyte apoptotic index did not significantly increase with respect to controls, in the presence of indinavir, d4T+3TC and d4T+3TC+indinavir, either in the absence or presence of TNF-α. However, it significantly increased for indinavir (P=0.0001), d4T+3TC (P=0.01) and d4T+3TC+indinavir (P=0.0001) when TNF-α was added with respect to antiretroviral drugs either alone or combined. The apoptotic index induced by d4T+3TC+indinavir combined with TNF-α was significantly greater than that induced by indinavir (P=0.03) and d4T+3TC (P=0.0001), when combined with TNF-α.

CONCLUSIONS: Our results suggest that TNF-α is the driving force in subcutaneous adipocyte apoptosis, which occurs in the setting of FRS in HIV-1-infected patients on highly active antiretroviral therapy.

Presenting author: P Domingo

2003-07-08
26

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