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Use of flow cytometry to monitor the expression of HIV p24 antigen in PBMC obtained from HIV-seropositive individuals undergoing antiviral chemotherapy
153 Intravenous Dextran Sulfate (DS) Increases Circulating HIV Antigen Levels in Patients with ARC or AIDS. C. Flexner, P. Barditch-Crovo, D. M. Kornhauser, L. Nerhood, C. Hendrix, K. Lorentsen, H. Farzadegan*, B. G. Petty, and P. S. Lietman, Division of Clinical Pharmacology, The Johns Hopkins University School of Medicine, and * The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland, USA. Polysulfated polysaccharides have been proposed as potential antiviral drugs because of potent in vitro activity against HIV, herpesviruses, and other enveloped viruses. In order to investigate the potential anti-HIV activity of a prototypical polysulfated polysaccharide, we administered the maximally-tolerated dose of dextran sulfate (a continuous intravenous infusion adjusted to maintain an activated partial thromboplastin time [APTT] of 65 to 80 seconds [8 subjects] or 50 to 65 seconds [2 subjects]) to patients with ARC or AIDS for up to 14 days. Circulating HIV antigen (p24) levels increased significantly in all eight subjects receiving drug for six or more days (median proportional increase 73.5%, range 32-130%). This increase was highly significant when compared to a large cohort of untreated historical controls (Fisher's exact test, p <.001). DS infusion may have increased the production or reduced the clearance of virus or p24 protein. Attempts to uncover a methodological artifact have failed to explain this result. Plasma DS concentrations achieved with this protocol were 2.4 to 21.6 mcg/ml (median 6.8 mcg/ml), which are 20- to 200-fold greater than the in vitro IC50 for free HIV infectivity. Finally, the drug was toxic, producing profound but reversible thrombocytopenia in all subjects receiving drug for greater than three days, and extensive but reversible alopecia in five subjects. We feel that DS is unlikely to have a practical role in the treatment of symptomStic HIV infection.
154 U s e of F l o w C y t o m e t r y to M o n i t o r the E x p r e s s i o n of H I V p24 A n t i g e n in P B M C O b t a i n e d from HIV-seropositive Individuals Undergoing Antiviral Chemotherapy. J McSharry, S Remick, S Szebenyi, D Herman, J Bills, J Slaga, P Gorman, A Ogden-McDenough, E Dickerson a n d J L e h m a n . Albany Medical College, Albany, New York, USA A novel technique involving indirect immunofluorescence in conjunction with flow cytometry was developed to d e t e c t and quantitate the n u m b e r of p24 a n t i g e n positive PBMC obtained from individuals infected with HIV (McSharry et al. , J. C l i n . M i c r o biol. 64:724, 1990) . This procedure is r a p i d (24 to 48 h r s ) , sensitive and quantitative and the p e r c e n t of p24 a n t i g e n positive PBMC reflects disease progression. This technique is b e i n g u s e d to m o n i t o r the e f f e c t of a n t i v i r a l chemotherapies, including Z D V , D D C a n d D D I , on the e x p r e s s i o n of p24 a n t i g e n in P B M C f r o m AIDS patients. 25 p a t i e n t s have been entered on a d o u b l e - b l i n d comparative t r i a l of Z D V vs D D C ; 10 p a t i e n t s on a d o u b l e - b l i n d comparative t r i a l of Z D V vs D D I ; 4 p a t i e n t s on an o p e n - l a b e l ZDV vs D D C c o m p a r a t i v e study; and another 193 p a t i e n t s are f o l l o w e d , the m a j o r i t y of w h o m are r e c e i v i n g ZDV off study. The median CD4 l y m p h o c y t e c o u n t at e n t r y in t h e s e p a t i e n t s w a s 75 p e r ul ( r a n g e 5 - 3 3 1 p e r ul). The median percent of p24 a n t i g e n positive P B M C at e n t r y w a s 8 . 4 5 % ( r a n g e 4 . 5 - 1 8 . 6 % ) . A patient is c o n s i dered positive for p24 a n t i g e n employing this technique when greater t h a n or e q u a l to 4% of the P B M C are p o s i t i v e . Patients continue to be f o l l o w e d prospectively and this technique m a y be a b l e to d i f f e r e n t i a t e antiviral activity of the v a r i o u s nucleoside analogues currently under investigation. (Supported in p a r t by N I H G r a n t # A 1 3 0 8 8 3 and AmFAR Project Grant # 600027-9-CT) .
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154 U s e of F l o w C y t o m e t r y to M o n i t o r the E x p r e s s i o n of H I V p24 A n t i g e n in P B M C O b t a i n e d from HIV-seropositive Individuals Undergoing Antiviral Chemotherapy. J McSharry, S Remick, S Szebenyi, D Herman, J Bills, J Slaga, P Gorman, A Ogden-McDenough, E Dickerson a n d J L e h m a n . Albany Medical College, Albany, New York, USA A novel technique involving indirect immunofluorescence in conjunction with flow cytometry was developed to d e t e c t and quantitate the n u m b e r of p24 a n t i g e n positive PBMC obtained from individuals infected with HIV (McSharry et al. , J. C l i n . M i c r o biol. 64:724, 1990) . This procedure is r a p i d (24 to 48 h r s ) , sensitive and quantitative and the p e r c e n t of p24 a n t i g e n positive PBMC reflects disease progression. This technique is b e i n g u s e d to m o n i t o r the e f f e c t of a n t i v i r a l chemotherapies, including Z D V , D D C a n d D D I , on the e x p r e s s i o n of p24 a n t i g e n in P B M C f r o m AIDS patients. 25 p a t i e n t s have been entered on a d o u b l e - b l i n d comparative t r i a l of Z D V vs D D C ; 10 p a t i e n t s on a d o u b l e - b l i n d comparative t r i a l of Z D V vs D D I ; 4 p a t i e n t s on an o p e n - l a b e l ZDV vs D D C c o m p a r a t i v e study; and another 193 p a t i e n t s are f o l l o w e d , the m a j o r i t y of w h o m are r e c e i v i n g ZDV off study. The median CD4 l y m p h o c y t e c o u n t at e n t r y in t h e s e p a t i e n t s w a s 75 p e r ul ( r a n g e 5 - 3 3 1 p e r ul). The median percent of p24 a n t i g e n positive P B M C at e n t r y w a s 8 . 4 5 % ( r a n g e 4 . 5 - 1 8 . 6 % ) . A patient is c o n s i dered positive for p24 a n t i g e n employing this technique when greater t h a n or e q u a l to 4% of the P B M C are p o s i t i v e . Patients continue to be f o l l o w e d prospectively and this technique m a y be a b l e to d i f f e r e n t i a t e antiviral activity of the v a r i o u s nucleoside analogues currently under investigation. (Supported in p a r t by N I H G r a n t # A 1 3 0 8 8 3 and AmFAR Project Grant # 600027-9-CT) .
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