Photodynamic inactivation of retrovirus by benzoporphyrin derivative: a feline leukemia virus model J. NORTH,S. FREEMAN,J. OVERBAUGH, J. LEVY,AND R. LANSMAN The ability of a photosensitizer, benzoporphyrin derivative monoacid ring A (BPDMA), and either broad-spectrum (400-1200 nm) or narrow-band (600-700 nm) red light to kill feline leukemia virus (FeLV) and FeLV-infected cat T cells (cell line 3201) was investigated in culture medium containing fetal calf serum and in blood from infected cats. A molecular clone of FeLV, 61 E, is minimally pathogenic and productively infects 3201 cells while causing no change in rate of cell division, viability, or size. Active virus (either free or within infected cells) was quantified by using a limiting dilution assay that involved cocultivation of test samples with naive 3201 cells, after which either the polymerase chain reaction or a reverse transcriptase assay was used to detect the presence of virus. It was shown that 61E-infectedT cells in culture were slightly more sensitive to photodynamic killing than were uninfected cells. Infected cells and free virus were eliminated from whole blood taken from infected cats by using 4 pg per mL of BPD-MA and 40 J per cm2of red light. These results correlate well with previous results with BPD-MA and vesicular stomatitis virus in whole human blood and suggest that this photosensitizer is a promising agent for the elimination of retroviruses that are either free or located within infected cells in blood. TRANSFUSION 1992;32:121-128.

Abbrevlatlons: BPD-MA = benzoporphyrlnderlvatlve monoacld rlng A; FCS = fetal calf serum; FeLV = fellne leukernla vlrus; HIV = human lmmunodeflclencyvlrus; LD- = medlan lethal dose; LDlW = lethal dose at whlch 100 percent of cells are kllled; PBS = phosphate-bufferedsallne; PCR = polymerasechain reactlon; PMS = phenazlne methosulfate; RT = reverse transcrlptase; SDS = sodlurn dodecyl sulfate; TCIU(s) = tlssue culture Infectlous unlt(8); TE = Trls/EDTA buffer; UV = ultravlolet; VSV = veslcular stomatltls vlrus; WBC(s) = whlte cell(s); XTT = 2, 3-bls(2-methoxy+nltro-5-sulfophenyl)d-([phenylamIno]carbonyl)-2H-tetrazoIlurnhydroxlde.

as either free or cell-associated virus. The recent development of adsorption filters to remove white cells (WBCs) from donor blood has the potential to lower the risk of transmission of cell-associated viruses.' However, WBC reduction will not eliminate all risk, as this procedure guarantees the reduction of WBC by only 3 log,,. One approach to virus inactivation in blood or blood components has involved the use of photosensitizer molecules that are activated by ultraviolet (W)light, such as 8-methoxypsoralen,* or visible light. There is considerable documentation of the ability of photosensitizers such as hematoporphyrin derivative, Photofrin, merocyanine 540, and zinc or aluminum phthalocyanine to kill a variety of viruses in either medium or blood when activated by light at the appropriate wavelength^.^-^ The reported drug dosimetry effecting successful virus inactivation varie extensively from one system to another. Enveloped viruses are significantly more sensitive than other viruses to inactivation by photosensitizers. This is thought to be due in part to the affinity of hydrophobic photosensitizer molecules for the lipids and glycolipids that form an integral part of the viral envelope. The formation of singlet oxygen when photosensitizer mol-

BLOODAND BLOOD COMPONENTS are currently regarded as significantly safer for recipients than they were 5 years ago, because of improved methods of screening donor blood for a variety of viruses. Screening procedures at this time essentially are immunologically based and rely on the detection of antibodies specific for a given virus or of viral antigens by an antibody capture assay. While these procedures, instituted in all North American transfusion centers, significantly lower the risk of infection via transfusion, they do not completely eliminate the risk. The majority of viruses considered potentially dangerous in blood and blood components are either enveloped viruses, which may occur as free virions in blood, as do hepatitis viruses, or as cell-associated viruses, as does cytomegalovirus, or retroviruses, which can occur From Quadra Logic Technologies, Inc., Vancouver, BC, Canada, and the Department of Microbiology, University of Washington, Seattle, Washington. Supported by grant STDF-AGAR from the British Columbia Science Council and by National Institutes of Health grant ROI-CA51080 (JO). Received for publication April 8, 1991; revision received July 17, 1991, and accepted July 25, 1991.

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ecules are activated by light is presumed to cause irre-

versible damage to the viral envelope. Benzoporphyrin derivative monoacid ring A (BPDMA) is a photosensitizer that has been studied extensively in this 1aboratoIy and that shows considerable promise as an agent for photodynamic therapy.1°-12 It is a hydrophobic molecule with a strong affinity for the lipoproteins in plasma. We have studied BPD-MA as a potential agent for virus inactivation in blood because it is optimally activated by light at 688 nm, a wavelength that penetrates through blood and plasma with little or no interference from hemoglobin. In a previous study,8 we found that BPD-MA at concentrations between 2 and 4 pg per mL could eliminate 6 to 7 log,, of vesicular stomatitis virus (VSV), an enveloped RNA virus, from whole human blood and under light conditions that did not cause significant hemolysis. On the basis of promising results with VSV, we have investigated the ability of BPD-MA to eliminate retroviral infection in cell culture and in blood. For this purpose, we have used feline leukemia virus (FeLV) as a model for studying the effects of photoinactivation on retrovirus-infected WBCs as well as on free virus. Our findings are reported and discussed herein.

Materials and Methods Cell lines and virus We maintained the feline T-cell line 320113 in Leibovitz’s L15 medium and RPMI 1640, (GIBCO, Grand Island, NY) supplemented with 15 percent fetal calf serum (FCS, GIBCO) in a humidified 2 percent C 0 2 incubator at 37°C. The 3201 cells were productively infected with 61E, a prototype molecular clone that is replication-competent and minimally pathogenic.I4 We stored cell-free culture supernatant harvested from chronically 61E-infected cells at -70°C and used it as a source of stock virus.

Transfection We introduced the 61E virus genome, obtained as a recombinant plasmid, into 3201 cells by using cationic liposomes” (Lipofectin, GIBCOBRL). From 1 to 20 pg of both DNA and Lipofectin was diluted to 50 p L in water and then mixed and allowed to stand at room temperature for 15 minutes. We washed 2 x lo6 3201 T cells twice with FCS-free medium and gently resuspended them in 3 mL of FCS-free medium containing the Lipofectin-DNA complex (100 yL). The cells were incubated in a 60-mm dish at 37°C in a humidified 2 percent CO, incubator. After 18 hours’ incubation, we added 3 mL of medium containing 15 percent FCS to the cells, which were incubated for a further 48 hours before being assayed for transfection efficiency. After transfection, we monitored the presence of replicating 61E virus by detecting Mn2+-dependent reverse transcriptase (RT) activity in tissue culture supernatants. We also monitored the presence of proviral genomes in cellular DNA by polymerase chain reaction (PCR) amplification.

R T activity RT activity was measured in culture supernatants as described previously.16 We added 15 p L of supernatant to a

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cocktail containing poly(A)oligo(dT) primerhemplate (Pharmacia, Piscataway, NJ), MnCI,, and radiolabeled thymidine triphosphate ([“PI TIT, Amersham Corporation, Arlington Heights, IL) and incubated it for 2 hours at 37°C. A 10-pL portion of the cocktail was then spotted onto filter paper (DEN, Whatman LabSales, Hillsboro, OR), dried, and washed three times in saline sodium citrate buffer and then with 95 percent ethanol. We dried the paper and counted its radioactivity in a scintillation counter (LS3801, Beckman Instruments, Palo Alto, CA).

DNA extraction DNA was extracted from 3201 T cells by the method of Miller et al.” Briefly, we resuspended T cells in 3 mL of nucleus lysis buffer (10 mM [lo mmol/L] Tris-HC1, 400 mM [400 mmol/L] NaCI, and 2 mM [2mmol/L] Na2EDTA, pH 8.2). The cell lysates were then digested overnight at 37°C with 0.2 mL of 10 percent sodium dodecyl sulfate (SDS) and 0.5 mL of a protease K solution (1 mL protease K in 1% SDS and 2 mM [2 mmol/L] Na,EDTA). After digestion was complete, we added 1 mL of saturated NaCl (approximately 6 M [6 mol/L]) to each sample, shook the sample vigorously for 15 seconds, and then centrifuged it at 2500 rpm for 15 minutes. The supernatant containing the DNA was removed and the DNA was precipitated with absolute ethanol. We centrifuged the samples at 2500 rpm for 30 minutes and removed the ethanol. The DNA pellet was dissolved in 100 to 200 pL of buffer (TE: 10 w 1 0 mmol/L] Tris-HC1 and 0.2 mM [0.2 mmol/L] Na,EDTA, pH 7.2) for 2 hours at 37°C before PCR amplification.

PCR We detected the presence of exogenous FeLV sequence in cellular DNA with a PCR kit (Perkin Elmer-Cetus, Nonvalk, CT)with recombinant Tuq polymerase. Primers specific to the U3 region of the exogenous FeLV long terminal repeat (LTR) transcription region were used for amplification (Overbaugh J, unpublished data, 1988). The reaction mixture contained 1 p g of genomic DNA, 100 pM of each primer, 10 pL of I O X reaction buffer (500 mM [500 mmol/L] KCI, 100 mM [lo0 mmol/L] Tris-CI [pH 8.31, 15 mM [15 mmol/L] MgCI,, 0.1 percent [wt/vol] gelatin), 500 N ( 5 0 0 pmol/L) deoxynucleotides, 0.5 U (5 pL) of Tuq polymerase, and water to a volume of 100 pL. Amplification was achieved with 30 cycles of denaturation at 9 2 T , annealing at 37”C, and polymerization at 72°C. The products of PCR were demonstrated by electrophoresis of 4 percent agarose gels (NuSieve, FMC BioProducts, Rockland, ME) with TE buffer containing 0.5 pg per mL of ethidium bromide, and bands were visualized by using a W transilluminator.

Photosensitizer BPD-MA was synthesized and purified as described previously. We maintained BPD-MA as a stock solution at 4 mg per mL in dimethyl sulfoxide or at 2.1 mg per mL in human plasma at -70°C. We thawed BPD-MA and diluted it with complete medium or whole blood immediately prior to use.

Experimental conditions in tissue culture medium We dispensed 0.5-mL samples containing lo6of either 61Einfected or uninfected 3201 T cells in culture medium containing 15 percent FCS into 5-mL test tubes (Falcon #2058, Becton Dickinson, Oxnard, CA). Under reduced-light conditions, we added various concentrations of BPD-MA to the

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samples, which we then incubated at 37°C in the dark. After 60 minutes (previously shown to be the optimal time for this condition) and still under reduced-light conditions, we dispensed 100-pL aliquots into 96-well flat-bottom plates (Falcon #3072) in triplicate and then exposed the plates to light for 30 minutes. The light source used for this treatment was a light box containing 16 100-W tungsten light bulbs (spectrum 4001200 nm, General Electric Canada, Oakville, ON). The light passed through a 4-cm thick chamber filled with circulating cooled water and covered by a matted glass plate to disperse the light. The light intensity was 10 mW per cmz as measured by a radiometer (Model TPM, Gentec Inc., Sainte Foy, Pa). Culture supernatants were also treated with BPD-MA in a similar manner. We incubated treated cells for 24 hours under humidified conditions at 37"C, after which we assessed phototoxicity by one of two assays (See below). Appropriate controls consisting of BPD-MA alone (no light exposure) had no effect on virus or cell viability.

Experimental conditions in whole blood Whole blood from cats experimentally infected with 61E was provided by Drs. M. Linenberger and J. Abkowitz (University of Washington, Seattle, WA). We dispensed 1-mL aliquots of whole blood into six-well plates (#25810, Corning Glass Works, Corning, NY)and, under reduced-light conditions, added various concentrations of BPD-MA to the samples. Samples were placed on an orbital shaker (Lab-Line Instruments, Melrose Park, IL) at 100 rpm and incubated at room temperature in the dark for 1 hour before exposure to light. The light source used for this treatment was a light box containing 12 15-W red fluorescent tubes (spectrum 600-700 nm, Philips Electronic Instruments, Somerset, NJ). The light intensity was 2.8 mW per cm2 as measured by a radiometer/ photometer with a silicon detector (IL 1350, International Light, Newbury Port, MA). After treatment, plasma and mononuclear cells were isolated from the whole blood samples. Following centrifugation at 1500 rpm for 10 minutes, the plasma was removed from treated samples. We gently resuspended the remaining cellular fraction in phosphate-buffered saline (PBS) and layered it over 4 mL of a density gradient (Percoll, Sigma Chemicals, St. Louis, MO; specific gravity 1.070 g/mL, Sigma Chemicals, St. Louis, MO). The band containing only mononuclear WBCs was collected after centrifugation at 1600 rpm for 20 minutes. We then washed the cells three times in complete medium to remove the Percoll. We assessed the photoinactivation of free and cell-associated virus with an infectivity assay. Positive infectivity controls consisting of untreated whole blood, plasma, and mononuclear cells were set up in parallel with the same cat blood as was used for photoinactivation.

or drug alone) simultaneously on the same plate and determined the percentage of survival by using control values as 100 percent. Color development constitutes a linear function of cell viability.

Infectivity assay To test for infectious virus in drug- and light-treated samples of viral supernatant, virally infected cells, or blood, we cocultured these samples with uninfected 3201 target cells in tissue culture medium in 24-well plates (#25820, Corning Glass Works). Cultures were incubated in a humidified 2 percent CO, incubator at 37°C. At various times throughout the culture, we mixed the contents of individual wells, removed onehalf the medium containing cells, and replaced it with fresh medium. Samples were centrifuged at 5000 rprn for 5 minutes, and the cell-free supernatant was removed. Both the cell pellet and cell-free supernatant were stored at -70°C; the former was analyzed by PCR amplification, and the latter was analyzed for RT activity. Untreated (control) samples were assayed in parallel.

Results The 61E virus used in these studies is a prototype for the common form of FeLV that was molecularly cloned from proviral DNA from the small intestine of a cat with immunodeficiency." A productively infected cat T-cell line was established by transfection of 61E and subsequent viral spread. We demonstrated viral infection by two independent means, the presence of RT activity in the culture supernatant and the presence of the viral sequence in cellular DNA, detected with PCR. Cell-free culture supernatants harvested from the chronically 61E-infected 3201 T-cell line were titrated in an endpoint di-

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XTT assay Cellular growth or survival after photodynamic treatment was determined with a new a ~ s a y . ~ Briefly, * J ~ 2,3-bis (2-meth-

oxy-4-nitro-5-sulfophenyl)-5-([phenylamino]carbonyI)-2H-tetrazolium hydroxide (x?T, Polysciences, Inc., Wamngton, PA) was prepared at 1 mg per mL in prewarmed (37°C) medium without serum. We prepared phenazine methosulfate (PMS, Sigma St. Louis, MO) at 5 mM (5 mmol/L) in PBS and stored it as a stock solution at 4°C. We added 50 ILLof a mixture of XTT and PMS (final concentration, 50 p,g of Xl'T and 0.38 pg of PMS/well) to each well 24 hours after light treatment. After 4 hours' incubation at 37"C, the contents of each plate were mixed and absorbance at 450 nm was measured with a microplate reader (Titertek, Flow Laboratories, Phoenix, AZ); we made appropriate control tests (cells treated with light alone

Days after infection FIG. 1. Endpoint titration of 61E-containing supernatant. Ten-fold

dilutions of the 61E-infected culture supernatant were added to naive 3201 cells. Starting at Day 3 following coculture, one-half of the culture supernatant was removed daily and frozen; the culture was then made up to full volume with complete medium. At the end of the experiment, culture supernatants were assayed for reverse transcriptase (RT) activity. The results shown here indicate the time at which significant RT activity was first detected for each dilution.

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lution assay to determine the number of tissue culture infectious units (TCIUs) produced by these cells. We added tenfold dilutions of the 61E-containing supernatant to uninfected 3201 cells and assessed the presence of RT activity at various times over an 18-day period. At the lowest dilution tested (l:lO), RT activity reached detectable levels on Day 4. Higher dilutions reached detectable levels at later times. After 9 days, however, no RT activity was detected at the endpoint of lo-, dilution. These results, shown in Fig. 1, demonstrate that the number of infectious units in culture medium can be estimated by determining the time of appearance of detectable enzyme activity following cocultivation of infected material. We detected no RT activity in dilutions greater than lo-, or in control culture supernatant. PCR amplification showed the presence of proviral integration in naive 3201 cells that had been infected with a lo-, dilution of 61E supernatant, but no FeLV DNA material could be detected in cultures exposed to greater dilutions of supernatant (Fig. 2). We have interpreted the observations from both limiting-dilution assays employed here to indicate the presence of about lo-' viral infectious units per mL of infected culture supernatant. Repeated analysis of supernatants has shown that these observations are reproducible from one batch of culture supernatant to another. We conducted experiments to compare the efficacy of BPDMA in the inactivation of naive 3201 T cells to that in 61Einfected cells. Cells cultured in medium containing 15 percent FCS were incubated in the dark with various concentrations of BPD-MA for 60 minutes at 37°C and then exposed to broadspectrum (400-1200 nm) light for 30 minutes (the equivalent of 17.4 J/cm2 of energy). We assessed the photoinactivation of cells by measuring, with the XTT assay, the metabolic activity of surviving cells 24 hours after exposure to light. Control cultures and cells that survived drug and light treatment continued to proliferate and were able to reduce soluble tet-

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razolium salt to soluble X l T formazan, which is orange in color. These cultures yielded high optical densities when absorbance was read at 450 nm. However, cells killed by the treatment did not proliferate; they therefore produced less XTT formazan and thus yielded lower optical densities. The median lethal dose (LD,,) values derived from these photoinactivation experiments were defined as the concentrations of BPD-MA required to reduce the color generated in the XTT reaction in control cultures by 50 percent. The results (Fig. 3) show that, under the conditions discussed, the LD,, for 61E- infected cells is in the range of 20 ng of BPD-MA per mL and 30 ng per mL for uninfected cells. This relatively small difference was reproducible and significant. To determine whether infected cells, destroyed by BPD-MA and light, could still transmit active virus to uninfected cells, we treated 61E-infected 3201 cells with various concentrations of BPD-MA and broad-spectrum light. To serve as a positive control, cultures of infected cells received light treatment alone. Following treatment, aliquots of treated cells (equivalent to 104 infected cells) were cocultured with naive cells using a porous cell culture insert (Transwell chamber, Costar, Cambridge, MA). In this device, light-treated infected cells were separated from target cells by a 0.4-ym filter, which will allow the diffusion of virus but not cells. We removed samples of culture supernatant from the outer chambers containing target cells and assessed them for the presence of RT activily. The inner chambers, containing drug- and/or light-treated cells, were examined microscopically for evidence of cell survival and proliferation. Results are shown in Fig. 4. The levels of BPD-MA and light used in these studies were not sufficient to effect 100 percent killing of infected cells (see Fig. 3 in which the LDlm is in the range of 50 ng/mL); even at the highest doses, only 80 to 90 percent of cells were expected to

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FIG. 2. Amplification of feline leukemia virus (FeLV) long terminal repeat (LTR) sequences by polymerase chain reaction (PCR). The 3201 cells, cocultured with various dilutions of 61E-infected supernatant, were subject to PCR amplification on Day 9 of culture by the use of primers specific to the U3 region of the FeLV LTR. The PCR products were identified by electrophoresisof a 4 percent agarose gel. Lane A, positive DNA control; Lane B, negative (no DNA) control; Lanes C through H, lo-' to dilution of 61E-infected supernatant. Marker size (in base pairs [bp]) is indicated on the left.

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Days after infection infected cats. Whole blood (150 pL), plasma, or 3.75 x 1V white cells was cocultured with 1.75 x 10' naive 3201 T cells in 1 mL of complcte medium. Reverse transcriptase activity in infected cultures was assessed ovcr time as before. Whole blood, 0-0;white cells, *-*; plasma, m-m.

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Days after infection FIG. 7. The effect of various doses of benzoporphyrin derivative monoacid ring A (BPD-MA) and light on feline leukemia virus-infectious units in the blood of 61E-infected cats. Reverse transcriptase activity in 3201 cells cultured with infected blood was determined over time. Control (light only), m-m; 1 pg per mL BPD-MA, 0-0; 2 pg per mL BPD-MA, *-*; 4 pg per mL BPD-MA, x-x.

Discussion We have used the presence of RT activity in the coculture assay as an indication of active FeLV. This procedure allowed us to determine the approximate number of TCIUs transferred to naive target cells as a function of the time after coculture at which RT activity can be detected in culture (as shown in Fig. 1). Culture super-

FIG. 8. The effect of various doses of benzoporphyrin derivative monoacid ring A (BPD-MA) and light on feline leukemia virus-infectious units in white cells of 61E-infected cats. Reverse transcriptase activity in 3201 cells cultured with white cells was determined over time. Control (light only), m-m; 1 pg per mL BPD-MA, 0.0; 2 pg per mL BPD-MA, *-a; 4 pg per mL BPD-MA, x-x.

natants from 61E-infected 3201 cells consistently produced about lo5 infectious units per mL as measured by this assay. When infected cell preparations were examined by PCR for the presence of viral genomic material, this procedure confirmed the results from the RT assay in that viral DNA could be detected in cells that were cocultured with but not in the TCIU dilution of free virus preparations. It was interesting to note that infected cells were slightly more sensitive to photoinactivation than uninfected cells under the conditions described, as 61E-infected 3201 cells do not differ in terms of size, general morphology, or division time from naive cells, and no detectable cytopathologic effect is present. However, virus budding may make the membrane fragile and therefore more sensitive to photodamage. Further studies will investigate this possibility. Even though 61E-infected cells were being killed by BPD-MA and light, it was possible that free virus was released undamaged from dead cells and had the potential to infect naive cells. To investigate this possibility, we treated cells with various doses of BPD-MA and light, transferred them to the inner section of Transwell chambers, and indirectly cocultured them with uninfected 3201 cells that were located outside the chamber. The results (Fig. 4) showed that very low levels of RT activity were recovered from chambers containing cells treated with 20 and 40 ng per mL of BPD-MA, whereas activity comparable to that of the control was seen in chambers containing cells treated with 10 ng per mL of BPD-MA. Microscopic examination of the internal

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PHOTOINACTIVATION OF VIRUSES BY BENZOPORPHYRIN DERIVATIVE

chambers of the culture showed the presence of a few surviving infected cells from preparations treated at 20 and 40 ng per mL, whereas controls and cells treated at 10 ng per mL of BPD-MA showed approximately equivalent numbers of live cells. These are expected results, because the LD,, for infected cells is 20 ng per mL and the LD,,, is in the range of 50 ng per mL. The same number of cells were transferred after drug and light treatment in all cases; if dead cells were capable of releasing active virus, one would expect RT activity levels to be comparable in all cases. As this was not the case, we infer that dead cells do not release infectious virus. This finding is relevant in this system, because we observed, somewhat surprisingly, that free FeLV appears to be considerably more resistant to photodynamic killing than are the host cells (LDlo0in the range of 300 ng to 1 pg/mL in 15% FCS). The finding that free FeLV in culture supernatants required approximately 1 pg of BPD-MA per mL effectively to eliminate infectivity was, however, very similar to the sensitivities we have found for both VSV* and herpes simplex virus (Neyndorff H, King D: unpublished data, 1990). The first series of experiments successfully demonstrated the ability of BPD-MA and broad-spectrum light to photoinactivate both virally infected cells and free virus in tissue culture medium. Subsequent experiments were performed in blood by using a red light that has a strong emission at wave lengths between 600 and 700 nm, which penetrates whole blood without interference from hemoglobin. Under these conditions, high drug doses (4 pg/mL) and long light exposures were required to eradicate infectivity. This treatment regimen did not cause significant hemolysis (approximately 2% during 2 weeks’ storage at 4°C). To evaluate the efficacy of photodynamic therapy with BPD-MA in a clinically relevant setting, we performed experiments on blood drawn from cats infected with 61E. Evaluation of the infected blood using our limiting-dilution RT activity assay (Fig. 6 ) showed that the blood contained between lo4 and lo5 infectious units per mL and that essentially all the infectious material was associated with the WBCs. We were unable to detect any RT activity in cocultures containing the plasma fraction of the infected blood. Results of the studies involving both whole blood and WBCs isolated after treatment showed similar patterns, in that significant reductions of RT activity were effected at 2 pg per mL of BPD-MA and that essentially all activity was eliminated at 4 pg per mL. We chose FeLV as a model in which to investigate the efficacy of BPD-MA in the photoinactivation of retroviruses because the high levels of virus produced would make it a difficult model and therefore a good test of our drug. Using this model, we successfully demonstrated that BPD-MA was effective in eliminating both

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retroviruses and retrovirally infected cells from blood and blood components without causing significant hemolysis. As the level of human immunodeficiency virus (HIV) in human blood is expected to be less than the level of FeLV in cat blood, it is thought that BPD-MA will be a promising photosensitizer for the elimination of HIV from transfusion components. Work is currently underway to address the efficacy of BPD-MA in the photoactivation of HIV in red cell concentrates. It should be emphasized that these studies constitute a preliminary stage in assessing the feasibility of using photosensitizers in the viral inactivation of blood components.

References 1. Kickler TS, Bell W, Ness PM, Drew H, Pall D. Depletion of white cells from platelet concentrates with a new adsorption filter. Transfusion 1989;29:411-4. 2. Lin L, Wiesenhahn GP, Morel PA, &rash L. Use of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates. Blood 1989; 74517-25. 3. Sieber F, Krueger JJ, O’Brien JM, Schober SL, Sensenbrenner LL, Sharkis SJ. Inactivation of Friend erythroleukemia virus and Friend virus-transformed cells by merocyanine 540-mediated photosensitization. Blood 1989;73:345-50. 4. Alter HJ, Creagon RP, Morel PA, et al. Photochemical decontamination of blood components containing hepatitis B and nonA non-B virus. Lancet 1988;2:1446-50. 5. Matthews, JL, Newman, JT, Sogandares-Bernal F. et al. Photodynamic therapy of viral contaminants with potential for blood banking applications. Transfusion 1988;28:81-3. 6. Lytle CD, Carney PG, Felten RP, Bushar HF, Straight RC. Inactivation and mutagenesis of herpes virus by photodynamic treatment with therapeutic dyes. Photochem Photobiol 1989;50:36771. 7. Lewin AA, Schnipper LE, Crumpacker CS. Photodynamic inactivation of herpes simplex virus by hematoporphyrin derivative and light. Proc SOCExp Biol Med 1980;163:81-90. 8. Neyndorff HC, Bartel DL, Tufaro F, Levy JG. Development of a model to demonstrate photosensitizer-mediatedviral inactivation in blood. Transfusion 1990;30:485-90. 9. Horowitz B, Williams B, Rywkin S, et al. Inactivation of viruses in blood with aluminum phthalocyanine derivatives. Transfusion 1991;31:102-8. 10. Richter AM, Kelly B, Chow J, et al. Preliminary studies on a more effective phototoxic agent than hematoporphyrin. J Natl Cancer Inst 1987;79:1327-32. 11. Richter AM, Sternberg ED, Dolphin D, Levy JG. Characterization of benzoporphyrin derivative, a new photosensitizer. Adv Photochemother 1988;997:132-8. 12. Jamieson CHM, McDonald WN, Levy JG. Preferential uptake of benzoporphyrinderivative by leukemic versus normal cells. Leuk Res 1990;14:209-19. 13. Snyder HW, Hardy WD,Zuckerman EE, Fleissner E. Characterisation of a tumour-specific antigen on the surface of feline lymphosarcoma cells. Nature 1978;275:656-8. 14. Overbaugh J, Donahue PR, Quackenbush SL, Hoover CA, Mullins JI. Molecular cloning of a feline leukemia virus that induces fatal immunodeficiency disease in cats. Science 1988;239:90610. 15. Felgner PL, Gadek TR, Holm M, et al. Lipofectin: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A 1987;84:7413-7. 16. Goff S , Traktman P, Baltimore D. Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J Virol 1981;38:239-48.

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17. Miller SA, Dykes DD, Polesky HF. A simple salting procedure for extracting DNA from human nucleated cells. Nucleic Acids Rcs 1988;16:1215. 18. Wcislow OS, Kiser R, Fine DL, Bader J, Shoemaker RH, Boyd MR. New soluble-formazan assay for HIV-1 cytopathic effects: application to high-flux screening for synthetic and natural products for AIDS-antiviral activity. J Natl Cancer Inst 1989;81:57786. 19. Scudicro DA, Shoemaker RH, Paul1 KD, et al. Evaluation of a solublc tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 1988;48:4827-33.

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Janice North, PhD, Scientist, Research and Development, Quadra ., BC, v5z Logic Technologies, I,,~., 520 west 6th A ~ ~vancouver, 4H5, Canada. [Reprint requests] Sally Freeman, BSc, Technician, Research and Development, Quadra Logic Julie Overbaugh. PhD. Research Assistant Professor, Department of Microbiology,kniversityof Washington, Seattle, Washington 98195. Julia G. Levy, PhD, Vice President, Discovery, Quadra Logic Technologies. Robert Lansman, PhD, Scientist, Molecular Biology, Quadra Logic Technologies.

Announcement

Advances in Transfusion Medicine and Immunohematology April 3 and 4,1992

No. 2-1992

Stamford, Connecticut

The fourth annual Symposium on Advances in Transfusion Medicine sponsored by Yale University School of Medicine, Harvard Medical School, and the American Red Cross Connecticut Region has been expanded. The two-day symposium will blend advances in basic science with clinical medicine. Major areas are: P Bone marrow transplantationupdate-1992. A review of bone marrow transplantationfollowed by lectures on the relationship between HLA type and unrelated donor transplantation, advances in bone marrow purging and collection of pheripheral blood stem cells for autologous bone marrow harvest, the status of bone marrow transplantation for solid tumors, and an update on the treatment of graft-versus-hostdisease. P Advances in diagnosis and management of coagulopathies. A review of diagnosis and management of hypercoagulablestates, new approaches to the treatment of hemophilia, the latest advances in virus inactivation of coagulation factors and blood components,a review of the storage of platelets for transfusion, and the treatment of patients with immune thrombocytopenia. P Diagnosisand therapy of immunohematologic disorders. A review of the basic molecularbiology needed to understand the molecular diagnosis of hematopoieticdisease, the technologic methods used including flow cytometry, and the status of biologic response modifiers for the treatment of patients with acquired immune deficiency syndrome. For registration information, contact: The Office of Postgraduate and Continuing Medical Education Yale University School of Medicine Room IE-53 SHM 333 Cedar Street New Haven, CT 06510 (203) 785-4578.