Henrietta Margolis-Nunno

IMPORTANCE OF TYPE I AND TYPE II MECHANISMS IN THE PHOTODYNAMIC INACTIVATION OF VIRUSES IN BLOOD WITH ALUMINUM PHTHALOCYANINE DERIVATIVES

The relative importance of type I and type II mechanisms in the photodynamic treatment of red blood cell concentrates (RBCC) to inactivate viruses was studied using aluminum phthalocyanine tetrasulfonate (AlPcS4), visible light and quenching or enhancing agents of reactive forms of oxygen. Treatment of a human RBCC with 10–13 μM AlPcS4 and 25–26 rnW/cm2 visible light resulted in the rapid and complete inactivation of added vesicular stomatitis virus (VSV). The addition of mannitol, glycerol, reduced glutathione (GSH), or superoxide dismutase (SOD), known quenching agents of type I mechanisms, had little to no effect on the rate of inactivation of VSV. Significant inhibition of VSV kill was observed on addition of tryptophan or sodium aide, known quenchers of type II mechanisms. Additionally, the rate of VSV kill was enhanced in the presence of D2O. Taken together, these results indicate a predominant role of singlet oxygen in the inactivation of VSV on photodynamic treatment of RBCC.

Inactivation by phthalocyanine photosensitization of multiple forms of human immunodeficiency virus in red cell concentrates.

BACKGROUND: The use of phthalocyanines in conjunction with red light has been shown to inactivate model lipid‐enveloped viruses in red cell concentrates. The ability of this treatment to inactivate multiple forms of human immunodeficiency virus (HIV) was evaluated in this study. STUDY DESIGN AND METHODS: The phthalocyanines used were aluminum phthalocyanine tetrasulfonate (AIPcS4) and the silicon phthalocyanines HOSiPcOSi(CH3)2(CH2)3 N(CH3)2 (Pc 4), and HOSiPcOSi(CH3)2 (CH2)3N+(CH3)3I‐(Pc 5). HIV was studied in a cell‐free form, in an actively replicating form, in latently infected cells, and in blood from HIV‐positive patients. RESULTS: All three phthalocyanines inactivate > or = 10(5) infectious doses of cell‐free HIV. However, only Pc 4 effectively inactivated actively replicating HIV and latently infected cells. The latter was about four times as sensitive to inactivation as was actively replicating HIV. Increasing the hematocrit of red cells during treatment decreased the rate of inactivation, especially at lower light doses. Under treatment conditions that completely inactivated the laboratory isolates of HIV, cell‐associated HIV in blood from HIV‐positive patients was also completely inactivated. The polymerase chain reaction signal from the gag gene of HIV was not affected on treatment of cell‐free virus, but it was reduced after treatment of cell‐associated HIV, particularly in some latently infected cell lines. CONCLUSION: Pc 4 and red light are effective in eliminating the infectivity of HIV in red cell concentrates. The usefulness of this approach for blood banking depends on future demonstration of the preservation of red cell circulatory survival and function in vivo.

Elimination of potential mutagenicity in platelet concentrates that are virally inactivated with psoralens and ultraviolet A light

BACKGROUND: For virus sterilization of platelet concentrates (PCs), treatment with aminomethyltrimethyl psoralen (AMT) and long‐wavelength ultraviolet A light (UVA) has shown efficacy. It has been found that treatment with 50 micrograms per mL of AMT and 38 J per cm2 of UVA in the presence of 0.35‐mM rutin efficiently kills viruses while maintaining platelet integrity. There is, however, concern about the mutagenic potential of psoralens and UVA (PUVA)‐treated PCs.

Quencher-enhanced specificity of psoralen-photosensitized virus inactivation in platelet concentrates

BACKGROUND: Treatment with psoralens and UVA (PUVA) has been shown to be efficacious in eliminating the risk of virus transmission by platelet concentrates (PCs). It has previously been demonstrated that, during the inactivation of cell‐free vesicular stomatitis virus (VSV) by aminomethyltrimethylpsoralen (AMT) and UVA in PCs, platelet function could be protected either by oxygen removal before irradiation or by inclusion of a type I free radical quencher, such as mannitol.

Inactivation of Trypanosoma cruzi Trypomastigote Forms in Blood Components with a Psoralen and Ultraviolet A Light

Inactivation of the blood‐borne parasite Trypanosoma cruzi by UVA and 4′‐aminomethyl‐4,5′,8‐trimethylpsor‐alen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 μg/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type II photo‐reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form of T. cruzi, the trypomastigote, was achieved at a UVA (320–400 nm radiation) fluence of 4.2 J/cm2. We note that while the infectivity of the parasite is eliminated at 4.2 J/cmZ the trypomastigote motility continues for at least 16 h post‐treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of 3H‐AMT indicated that at the lethal UVA fluence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology, which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborne T. cruzi, the causative agent of Chagas disease.

Photodynamic decontamination of blood for transfusion

Currently transfused cellular components of blood are not available in a sterile form and carry a small risk of transmitting viral and parasite diseases. Using phthalocyanines and red light, lipid enveloped viruses, e.g., HIV-1, can be inactivated in red blood cell concentrates (RBCC). Under conditions leading to virus sterilization the blood borne parasites Trypanosoma cruzi (Chagas disease) and Plasmodium falciparum (malaria) could be eliminated to undetectable levels (> 4 log10 kill). RBC damage during treatment could be avoided by increasing the light fluence rate to 80 mW/cm2, and by including the free radical scavenger glutathione and the vitamin E derivative Trolox during light exposure. Similar sterilization of platelet concentrates was achieved with the psoralen derivative AMT and UVA light. Platelet damage due to PUVA treatment was avoided by including the plant flavonoid rutin during irradiation. It is concluded that elimination of the risk of transmitting pathogens during blood transfusion is feasible with photochemical treatments.