|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2180-2188
By
From the Cerus Corp, Concord, CA.
A photochemical treatment (PCT) process using a novel psoralen and
long wavelength ultraviolet light (UVA, 320-400 nm) has been developed
to inactivate bacteria and viruses in platelet concentrates. This study evaluated the efficacy of PCT for
inactivation of leukocytes that contaminate platelet preparations.
Three psoralens, 8-methoxypsoralen (8-MOP), 4
TRANSFUSION OF CELLULAR blood products is
associated with a number of adverse immune reactions.
Transfusion-associated graft-versus-host disease (TA-GVHD) has been
well documented for severely immunocompromised patients1-3
as well as immunocompetent patients who have received blood from donors
homozygous for shared HLA haplotypes.4-7 T cells
contaminating cellular blood components have been implicated as the
initiating agents for TA-GVHD.8,9 There is no effective therapy for TA-GVHD which is 80% to 90% fatal.10 At
present, the primary prophylactic measure is irradiation of cellular
blood products using a gamma source.11,12 Based on more
than 30 years of clinical practice, gamma radiation has been shown to
be effective in reducing the incidence of TA-GVHD. The effective
clinical dose of gamma radiation has been determined to be 2,500 cGy
based on a clonogenic expansion assay using limiting dilution analysis (LDA).8,13 This dose is required for the inactivation of
>5 log10 of T cells in cellular blood products.
While gamma irradiation is efficacious in reducing the incidence of
TA-GVHD, transfusion-associated viral and bacterial diseases remain a
persistent problem for blood products.14 To reduce the
risks of viral and bacterial diseases associated with platelet transfusion, a photochemical treatment (PCT) process using psoralens and long wavelength ultraviolet radiation (UVA 320-400 nm) has been
developed.15-17 Psoralens are planar, aromatic molecules
that can bind reversibly to nucleic acids by
intercalation.18 On illumination with UVA, intercalated
psoralens form covalent monoadducts and interstrand crosslinks with RNA
and DNA. In the absence of repair, the psoralen-modified genomes of
viruses and bacteria are inactivated because replication cannot occur.
Because platelets do not have nuclei, they are unaffected by treatment
with psoralens and UVA. Novel psoralens have been synthesized to
maximize viral inactivation efficiency.19-22 PCT with a
novel psoralen, S-59, has been shown to be effective in inactivating a
broad spectrum of viruses and bacteria without adversely affecting in
vitro or in vivo platelet function.15,23
This report describes the additional benefits derived from a PCT
process that is antiviral and antibacterial. Because psoralens are
nucleic acid specific reagents, contaminating nucleated leukocytes in
platelet concentrates are susceptible targets for inactivation. In this
study, the inactivation of leukocytes by PCT with psoralens is
evaluated using four independent but complimentary biological and
molecular assays. Evidence is presented to demonstrate that (1) T cells
in platelet concentrates are extremely susceptible to PCT inactivation;
(2) cytokine synthesis by PCT-inactivated leukocytes is inhibited
during platelet storage because PCT is performed before storage; (3)
leukocyte genomic DNA is heavily modified by psoralens after PCT; and
(4) psoralen-DNA adducts block DNA polymerase activity and inhibit the
polymerase chain reaction (PCR). Three psoralens, 8-methoxypsoralen
(8-MOP), 4 Preparation of Platelet Concentrates in 35% Plasma
Random donor platelet concentrates.
Five freshly drawn ABO- matched random donor platelet concentrates
(Alameda Contra Costa Medical Association, Oakland, CA) were pooled and
transferred to sterile 50 mL polypropylene centrifuge tubes in 30 mL
aliquots. After centrifugation (3,000g for 6 minutes at
22°C), the supernatant plasma concentration was adjusted to 35% by
removing 65% of the total volume and replacing it with a platelet
additive solution (PAS III) and then resuspending the pelleted
material. PAS III is a modified platelet synthetic medium (115 mmol/L
Na chloride, 30 mmol/L Na acetate, 10 mmol/L Na citrate, and 26 mmol/L
Na phosphate) similar to that used by others.25 The pH of
PAS III is 7.2 and the osmolarity is 300 mOsm/L. The platelet counts of
concentrates ranged between 1.5 to 2.5 × 106/µL.
Single donor plateletpheresis units.
Single donor plateletpheresis units were collected using a CS3000 Blood
Cell Separator (Baxter-Fenwal, Round Lake, IL) equipped with a PLT30
collection chamber with a modified procedure (Sacramento Blood Center,
Sacramento, CA). The platelets (2.5 to 5.0 × 1011)
were collected in approximately 105 mL of plasma. After resuspension, 195 mL of PAS III was added. The final platelet concentrate in approximately 300 mL was transferred into a 1-L PL2410 plastic container (Baxter-Fenwal) and placed on a reciprocal platelet shaker
(Helmer Labs, Nobelsville, IN) for storage with temperature control
(20°C to 24°C) until use. Concurrent plasma (100 to 150 mL) was
also collected and centrifuged (3,000g for 10 minutes at
22°C) to make platelet poor plasma (PPP). The platelet counts of SDP
concentrates were similar to random donor platelet concentrates.
Characterization of Platelet Concentrates
Preparation of Peripheral Blood Mononuclear Cells (PBMCs)
Preparation of Psoralen Stock Solutions Stock aqueous solutions of AMT (HRI Associates, Concord, CA), 8-MOP (Sigma), and S-59 (Cerus Corp, Concord, CA) were optically measured with a Shimadzu UV160U spectrophotometer (Shimadzu Scientific Instruments, Pleasanton, CA). The concentration was calculated using the absorbance at 250 nm and the extinction coefficient of 26,900 M 1cm1 for S-59, 25,000 M1cm1 for AMT, and 22,900 M1cm1 for 8-MOP. S-59 and AMT are water
soluble. In aqueous medium, 8-MOP saturates at approximately 30 µg/mL. Concentrated stock solutions of 8-MOP were made in dimethyl
sulfoxide (DMSO) (Research Industries Corp, Salt Lake City, UT). The
structure and synthesis of the novel psoralen S-59 are as described
previously.24 S-59 is an amino alkylated psoralen
formulated as a hydrochloride salt; it is an odorless, white
microcrystalline powder, with solubility greater than 50 mg/mL in 0.9%
NaCl. Solid S-59 is stable at high temperature. Aqueous solutions of
S-59 show good long-term stability and can be terminally sterilized by
autoclaving.
UVA Illumination After adding psoralens at the indicated concentrations in platelet concentrates, they were illuminated with UVA on a modified Baxter Ultraviolet Irradiation System (Model #4R4440; Baxter-Fenwal) while being mixed. This illumination device is air-cooled, mounted on a reciprocal platelet shaker (Helmer Labs), and capable of maintaining the temperature rise of the platelet concentrate to less than 1°C per Joule/cm2 during the course of the illumination. There are two opposing banks of seven F15T12-BL fluorescent lamps (Spectronics, Westbury, NY) mounted approximately 8 inches from each other. Platelet samples were placed in the center on a piece of glass between the two banks of lights. The output of this device was approximately 15 to 20 mW/cm2 permitting delivery of 3 Joules/cm2 in approximately 3 to 4 minutes.UVA Dosimetry Chemical actinometry was used to normalize UVA doses for platelet concentrates with volumes less than 300 mL. The configuration that has been validated for viral and bacterial inactivation while preserving in vitro and in vivo platelet function is 3 Joules/cm2 UVA with 150 µmol/L S-59 for a 300 mL platelet concentrate.15 To deliver an equivalent dose of UVA for a 20 mL platelet concentrate, only 1.9 Joules/cm2 was required. LDA experiments with 30 mL platelet concentrates received 1.0 Joule/cm2 of UVA, which is equivalent to a dose of 1.4 Joules/cm2 for a 300 mL platelet concentrate.Limiting Dilution Analysis (LDA) Preparation of allostimulator cells.
Whole blood was drawn into two 10 mL ACD collection tubes (Vacutainer,
Rutheford, NJ) from each of 10 volunteers. The PBMCs were isolated by
Ficoll density gradient centrifugation as described above. After two
washings (250g, 20 min, 22°C) in PBS, the final PBMC pellets
were pooled in RPMI 1640 (Mediatech) supplemented with 2.0 mmol/L
L-glutamine (Sigma), 50 µg/mL penicillin, 50 U/mL streptomycin (GIBCO
Life Technologies, Baltimore, MD), and 20% fetal bovine serum (FBS)
(Hyclone, Logan, UT) (RPMI/20% FBS) and counted with the Hematology
Analyzer. DMSO was added to a final volume of 10%. Aliquots of 2.0 × 106 cells/mL were placed into 1.5 mL sterile cryotubes
(Sarstedt, Newton, NC) and frozen at Plating of allostimulator cells. On the day of each assay, the required number of allostimulator cells was thawed and washed with 6 to 7 volumes of RPMI/20% FBS. The cells were pooled, resuspended in 10 to 15 mL of RPMI/20% FBS, and irradiated with 5,000 cGy of gamma using a Nordion Gamma Cell-1000 irradiator (Alameda Contra Costa Medical Association). After gamma-irradiation, the allostimulator cells were centrifuged at 250g for 10 minutes at 22°C and resuspended in 2× T-cell medium (80% RPMI/20% FBS, 20% TCGF [Cellular Products Inc, Buffalo, NY], 100 U/mL rIL-2 [Cellular Products Inc], and 16 µg/mL PHA-M [Sigma]). To each well of the 96-well flat bottom tissue culture plates (VWR Scientific, Foster City, CA) 1.0 × 105 cells in 100 µL of 2× T-cell medium were plated. Isolation of leukocytes from platelet concentrates. The leukocytes from a platelet concentrate sample were pelleted (350g for 5 minutes at 22°C) and resuspended in 20 mL of PBS. This cell suspension was underlayed with 20 mL of 1-Step Platelets (Accurate Chemical and Scientific, Westbury, NY) and centrifuged at 350g for 15 minutes at 22°C. The resulting cell pellet was washed with 40 mL PBS (250g for 10 minutes at 22°C), resuspended in 20 mL PBS, and underlayed with 10 mL of Ficoll (Sigma). After centrifuging at 400g for 30 minutes at 22°C, the buffy coat (8 to 10 mL) was removed and diluted 3 to 4× with PBS. The cells were recovered by centrifugation (250g for 20 minutes at 22°C) and washed twice with 40 mL of PBS (250g for 10 minutes at 22°C). The final cell pellet was resuspended in 2 to 10 mL of RPMI/20% FBS for plating. Plating of control samples. Leukocytes from control platelet concentrates that contained no psoralen and were not UVA illuminated were diluted in RPMI/20% FBS to achieve the following concentrations: 3,000, 1,000, 333, 110, and 37 cells/mL. Leukocytes from platelet concentrates treated with UVA alone (1.0 Joule/cm2 or 4.0 Joules/cm2) and from platelet concentrates treated with S-59 alone (1.0 µmol/L or 0.05 µmol/L) were diluted in RPMI/20% FBS to achieve the following concentrations: 10,000, 1,000, 100 cells/mL. Aliquots of 100 µL of each dilution were plated in ten replicates into wells containing 1.0 × 105 allostimulator cells plated previously as described above. To the allostimulator cells control wells, 100 µL of RPMI/20% FBS was added. Plating of treated samples. Leukocytes from platelet concentrates (30 mL) treated with low doses of psoralen and 1 Joule/cm2 UVA were serially diluted (1:10) in RPMI/20% FBS to achieve the following range of concentrations: 106 to 101 cells/mL. For each dilution 100 µL was plated in each of 10 wells containing 1.0 × 105 allostimulator cells. Two experiments were performed with two independent sources of platelet concentrate. Leukocytes from platelet concentrates (300 mL) treated with 150 µmol/L S-59 and 3 Joules/cm2 UVA were adjusted to 106 cells/mL with RPMI/20% PBS and 100 µL aliquots were plated into each of 110 wells containing 1.0 × 105 allostimulator cells. Four replicate experiments were performed using four independent 300 mL units of single donor platelet concentrate. Incubation and feeding. Cells were incubated at 37°C for 3 weeks in a humidified 5% CO2 incubator (Forma Scientific, Marietta, OH). After 3 days, 1 week, and 2 weeks, cells in each well were fed with 25 µL of media consisting of 50% FBS, 50% TCGF, 500 U/mL rIL-2, and 80 µg/mL PHA-M. Data analysis. LDA plates were scored visually after 3 weeks using an inverted microscope (Model # CK2, Olympus, Japan). Wells with at least one T-cell clone were scored positive. Wells without a T-cell clone were scored negative. The T-cell frequencies were calculated by minimum chi-square analysis based on a Poisson distribution.13,26 The log10 T-cell reduction was calculated as log10 (fcontrol/ftreated), where fcontrol is the T-cell frequency of the control platelet concentrate and ftreated is the T-cell frequency of the photochemically treated platelet concentrate. Mean and standard deviations were calculated using standard methods. Measurement of Cytokine Levels These experiments were performed with random donor platelet concentrates with higher levels of contaminating leukocytes. The leukocyte level was quantified using a procedure as previously described.27 If necessary the platelet concentrates were supplemented with leukocytes isolated from a buffy coat unit so that the final pooled leukocyte count was 4.33 × 106/mL for all of the samples.
Measurement of Psoralen-DNA Adduct Formation The platelet concentrates were spiked with 3H-labeled psoralens (HRI Associates) to achieve a final specific radioactivity for each psoralen of 5 mCi/mmol. Platelet concentrates were treated with 10 µmol/L, 75 µmol/L, 100 µmol/L and 150 µmol/L S-59; 150 µmol/L AMT; or 75 µmol/L and 150 µmol/L 8-MOP. After photochemical treatment, 1.0 mL samples were centrifuged as described above. The pelleted material was used for measurement of psoralen-DNA adduct formation.PCR Amplification Inhibition Assay Samples containing 1.0 µg DNA obtained from above were amplified for a 242 bp sequence in the HLA-DQ locus using the primer pair
GH26/GH2728 and for a 439 bp sequence in the  -globin
gene using the primer pair PC03/RS42.29 PCR reactions were
set up in 50 µL of 1× Taq Buffer (Perkin Elmer, San Francisco, CA)
containing 200 µmol/L each of dATP, dCTP, dGTP, and dTTP (Perkin
Elmer), 0.5 µmol/L each of the respective primer set and 2.5 U Taq
polymerase (Perkin Elmer). The control DNA sample was serially diluted
(1:10) and then amplified. The treated DNA samples were amplified
undiluted. Amplification was carried out to 35 cycles on a Perkin Elmer
Cetus DNA thermal cycler with the denaturing temperature at 95°C (30 seconds), the annealing temperature at 55°C (30 seconds), and the
extension temperature at 72°C (1 minute). The amplification products
were analyzed by electrophoresis on a 2.5% NuSieve agarose gel (FMC
bioproducts, Rockland, ME). The extent of PCR signal reduction was
estimated by comparing the degree of ethidium bromide staining of the
amplification products of treated samples with the serially diluted
untreated samples.
Photochemical Inactivation of T Cells in Platelet Concentrates PBMCs isolated from freshly prepared whole blood by Ficoll density gradient centrifugation were spiked into each of four full-sized (300 mL) single donor plateletpheresis units to achieve final leukocyte concentrations of 4.8 × 105 to 3.4 × 106/mL. After photochemical treatment with 150 µmol/L S-59 and 1.0 or 3.0 Joules/cm2 UVA, no viable T cells were detected using the LDA assay. The mean ± standard deviation of T-cell inactivation under these conditions was greater than 5.4 ± 0.3 log10 in four replicates (Table 1). These results indicate that photochemical treatment conditions (150 µmol/L S-59 and 3.0 Joules/cm2 UVA) that are virucidal and bactericidal are capable of inactivating high levels of T cells in platelet concentrates.15
Inhibition of Cytokine Synthesis by Photochemical Treatment
Psoralen Modification of Leukocyte Genomic DNA After Photochemical
Treatment
Psoralen-DNA Adducts Inhibit PCR DNA Amplification
To reduce the risk of viral and bacterial disease transmission through
platelet transfusion, a photochemical treatment process using a novel
psoralen, S-59, and UVA was developed.15,19-23 Treatment conditions have been optimized for platelet concentrates to inactivate high levels of a broad spectrum of viruses and bacteria while preserving platelet function.15 This study demonstrates
that contaminating leukocytes in platelet concentrates are inactivated after treatment with psoralens and UVA. More importantly, the virucidal
and bactericidal conditions provide a large margin of safety for
leukocyte inactivation.
Submitted May 12, 1997;
accepted November 6, 1997.
The authors acknowledge the excellent technical assistance of Terri
Anderson in performing the LDA assay. Lainie Corten provided critical
contributions during the development phase of the LDA assay. The
authors also thank Dr John Hearst for his constant encouragement and
valuable suggestions throughout this study. The cooperation of Dr Paul
Holland, Vangie Schoening, and Barbara Evans of the Sacramento Blood
Center, Sacramento, CA and Dr Sherri Evans of the Alameda Contra Costa
Medical Association, Oakland, CA in supplying platelet concentrates for
this study was greatly appreciated. The Alameda Contra Costa Medical
Association also provided services for gamma irradiation of cellular
samples with the Nordion Gamma Cell-1000. David Drothler of the
Children's National Medical Center, Washington DC provided critical
information regarding the LDA assay and the IBM PC software program for
calculation of T-cell frequency from the LDA results.
1.
Anderson KC,
Weinstein HJ:
Transfusion-associated graft-versus-host disease.
N Engl J Med
323:315,
1990[Medline]
[Order article via Infotrieve]
2.
Hathway WE,
Fulginiti VA,
Pierce CW,
Githens JH,
Pearlman DS,
Muschenheim F,
Kempe CH:
Graft-vs-host reaction following a single blood transfusion.
JAMA
201:139,
1967[Medline]
[Order article via Infotrieve]
3.
Greenbaum B:
Transfusion-associated graft-versus-host disease: Historical perspectives, incidence, and current use of irradiated blood products.
J Clin Oncol
9:1889,
1991[Abstract]
4.
Capon SM,
DePond WD,
Tyan DB,
Pepkowtz SH,
Toyoda H,
Cinman AC,
Azer PC,
Goldfinger D:
Transfusion-associated graft-versus-host disease in an immunocompetent patient.
Ann Intern Med
114:1025,
1991
5.
Benson K,
Marks AR,
Marshall MJ,
Goldstein JD:
Fatal graft-versus-host disease associated with transfusions of HLA-matched, HLA-homozygous platelets from unrelated donors.
Transfusion
4:432,
1994
6.
Takahashi K,
Juji T,
Kiyazaki H:
Post-transfusion graft-versus-host disease occurring in non-immunosuppressed patients in Japan.
Transfus Sci
12:281,
1991
7.
Petz LD,
Calhoun L,
Yam P,
Cecka M,
Schiller G,
Faithowicz AR,
Herron R,
Sayah D,
Wallace RB,
Belldegrun A:
Transfusion-associated graft-versus-host disease in immunocompetent patients: Report of a fatal case associated with transfusion of blood from a second-degree relative, and survey of predisposing factors.
Transfusion
33:742,
1993[Medline]
[Order article via Infotrieve]
8.
Kernan NA,
Collins NH,
Juliano L,
Cartagena T,
Dupont B,
O'reilly RJ:
Clonable T lymphocytes in T-cell-depleted bone marrow transplants correlate with development of graft-v-host disease.
Blood
68:770,
1986
9.
Vallera D,
Solderling C,
Carlson G,
Kersey J:
Bone marrow transplantation across major histocompatibility barriers in mice.
Transplantation
31:218,
1981[Medline]
[Order article via Infotrieve]
10. Spitzer TR: Transfusion induced graft-vs-bone disease, in
Burakoff SJ, Deeg HJ, Ferrara J, Atkinson K (eds): Graft-vs.-Host Disease
11. Anderson KC: Clinical indications for blood component
irradiation, in Baldwin ML, Jefferies LC (eds): Irradiation of Blood
Components. Bethesda, MD, American Association of Blood Banks, 1992, p
31
12.
Anderson KC,
Goodnough LT,
Sayers M,
Pisciotto PT,
Kurtz SR,
Lane TA,
Anderson CS,
Silberstein LE:
Variation in blood component irradiation practice: Implications for prevention of transfusion-associated graft-versus-host disease.
Blood
77:2096,
1991
13.
Pelzynski MM,
Moroff G,
Luban NLC,
Taylor BJ,
Quinones RR:
Effect of gamma irradiation on T-cell inactivation as assessed by limiting dilution analysis: Implications for preventing transfusion associated graft vs. host disease.
Blood
83:1683,
1994
14. Dodd RY: Adverse consequences of blood transfusion: Quantitative
risk estimates, in Nance SJ (ed): Blood Supply: Risks, Perceptions and
Prospects for the Future. Bethesda, MD, American Association of Blood
Banks, 1994, p 1
15.
Lin L,
Cook DN,
Wiesehahn GP,
Alfonso R,
Behrman B,
Cimino GD,
Corten L,
Damonte PB,
Dikeman R,
Dupuis K,
Fang YM,
Hanson CV,
Hearst JE,
Lin CY,
Londe HF,
Metchette K,
Nerio AT,
Pu JT,
Reames AA,
Rheinschmidt M,
Tessman J,
Isaacs ST,
Wollowitz S,
Corash L:
Photochemical inactivation of viruses and bacteria in human platelet concentrates using a novel psoralen and long wavelength ultraviolet light.
Transfusion
37:423,
1997[Medline]
[Order article via Infotrieve]
16.
Lin L,
Wiesehahn GP,
Morel PA,
Corash L:
Use of 8-methoxypsoralen and long wavelength ultraviolet radiation for decontamination of platelet concentrates.
Blood
74:517,
1989
17.
Lin L,
Londe H,
Janda JM,
Hanson CV,
Corash L:
Photochemical inactivation of pathogenic bacteria in human platelet concentrates.
Blood
83:2698,
1994
18.
Cimino G,
Gamper H,
Isaacs S,
Hearst J:
Psoralens as photoactive probes of nucleic acid structure and function: Organic chemistry, photochemistry, and biochemistry.
Ann Rev Biochem
54:1151,
1985[Medline]
[Order article via Infotrieve]
19. Wollowitz S, Fang Y, Pu JT, Nerio A, Spielmann HP, Lin L,
Behrman B, Londe H, Alfonso R, Corash L, Isaacs S: Novel psoralens with
enhanced UVA dependent inactivation of human immunodeficiency virus and
reduced mutagenicity in the absence of UVA light. Blood 82(S1):402a,
1993 (abstr)
20. Alfonso R, Londe H, Cook D, Hanson CV, Rheinschmidt M, Behrman
B, Lin L: Inactivation of cell free, cell associated, and proviral
HIV-1 by the novel psoralen S-59 in human platelet concentrates. Blood
84(S1):530a, 1994 (abstr)
21. Corash L, Cook D, Reames A, Arnstein P: Photochemical
decontamination of platelet concentrates prevents transfusion induced
cytomegalovirus infection. Blood 84(S1):503a, 1994 (abstr)
22. Cook D, Lin C, Reames A, Marion PL: Inactivation of duck
hepatitis B virus in human platelet concentrate by the novel psoralen
S-59 and light. Transfusion 34(S):43S, 1994 (abstr)
23. Lin L, Behrman B, Rheinschmidt M, Mayaudon V, Payrat JM, Corash
L: Evaluation of in vitro platelet function in synthetic media
following photochemical decontamination treatment of apheresis platelet
concentrates. Transfusion 34(S):71S, 1994 (abstr)
24. Wollowittz S, Isaacs ST, Rapoport H, Spielman HP, inventors;
Cerus Corporation, assignee. Compounds for the photodecontamination of
pathogens in blood. US Patent 5,399,719. 1995, March 21
25.
Gulliksson H,
Eriksson L,
Hogman CF,
Payrat JM:
Buffy coat derived platelet concentrates prepared from half-strength citrate CPD and CPD whole blood units: Comparison between three additive solutions, in vitro studies.
Vox Sang
68:152,
1995[Medline]
[Order article via Infotrieve]
26.
Taswell C:
Limiting dilution assays for the determination of immunocompetent cell frequencies: I data analysis.
J Immunol
126:1614,
1981[Abstract]
27.
Kao KJ,
Scornik JC:
Accurate quantitation of the low number of white cells in white cell-depleted |
Abstract
Introduction
-aminomethyl
4,5
Abstract
80°C.
and 
), AMT
(
), and 8-MOP (
) on T cells in platelet concentrates was
characterized. Leukocytes from photochemically treated and untreated
pooled random donor platelet concentrates were plated in the LDA assay.
The minimum psoralen concentrations required to inactivate T cells to
the limit of detection by LDA (
) after 1.0 Joule/cm2 UVA
were 0.05 µmol/L S-59, 1.0 µmol/L AMT, and 10.0 µmol/L 8-MOP. The
results in Fig 
), and 8-MOP (
Immunology, Pathophysiology and Treatment. NY, Marcel Dekker,
1990, p 539