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HEMATOPOIESIS
From the Departments of Immunology and Hematology,
University of Cape Town, Cape Town, South Africa;
Departments of Cell Biology and Medicine and Kaplan Cancer Center, New
York University Medical Center, New York, NY; Center for Cancer Biology
and Nutrition, Institute for Biosciences and Technology, Texas A & M
University, System Health Science Center, Houston; Zeria Pharmaceutical
Company, Saitama, Japan; Department of Transfusion
Medicine, Columbia University, New York, NY; Department of Cell
Differentiation, Kumamoto University, Kumamoto,
Japan; Department of Medicine, University of Minnesota
Medical School, Minneapolis; Department of Pathology, Weill Medical
College of Cornell University, New York, NY.
Recent experiments show that hematopoietic progenitor cell
populations contain endothelial precursor cells. We have isolated a
population of CD34+ cells that expresses fibroblast growth
factor receptor-1 (FGFR-1) and that differentiates into endothelial
cells in vitro. We find that 4.5% ± 2.1% of CD34+
cells isolated from bone marrow, cord blood, and mobilized peripheral blood express FGFR-1 and that viable CD34+FGFR+
cells are small, with little granularity, and express both primitive hematopoietic and endothelial markers on their surface. The primitive hematopoietic markers AC133, c-kit, and Thy-1 are coexpressed by 75%,
85%, and 64% of CD34+FGFR+ cells,
respectively. Most of the CD34+FGFR+ cells also
express antigens found on endothelial cells, such as CD31, vascular
endothelial growth factor receptor-2, and the endothelial-specific cell
surface marker, vascular endothelial cadherin (VE-cadherin),
whereas 56% to 60% of the cells express Tie, Tek, and the
endothelial-specific marker, P1H12. The
CD34+FGFR+ population is enriched in cells
expressing endothelial-specific antigens compared with the
CD34+ population. Isolated
CD34+FGFR+ cells grow slowly in culture, are
stimulated by fibroblast growth factor-2 and vascular endothelial
growth factor, and give rise to cells that express von Willebrand
factor and VE-cadherin and that incorporate acetylated low-density
lipoprotein. These experiments show that FGFR-1 is expressed by a
subpopulation of CD34+ cells that give rise to endothelial
cells in vitro, indicating that this population contains endothelial
stem/progenitor cells.
(Blood. 2002;100:3527-3535) The CD34+ population contains
hematopoietic stem cells as well as endothelial stem/progenitor cells
that can differentiate into endothelial cells. A small number of
CD34+ hematopoietic progenitor cells express receptors for
fibroblast growth factors,1-5 and fibroblast growth
factor-2 (FGF-2) has both synergistic and direct effects on progenitor
cell proliferation.6-10 FGF-2 also promotes the
self-renewal11 and proliferation12 of primitive
hematopoietic cell lines, indicating its relevance in early hematopoiesis.
Hematopoietic and endothelial cells have a close association during
ontogeny. A common embryonic precursor, the hemangioblast, that gives
rise to both hematopoietic and endothelial cells, has been identified
within the Tek+ fraction of the aorta-gonad-mesonephros
(AGM) region of the embryo,13 and FGF-2 has been shown to
stimulate the proliferation of hemangioblasts.14 In
addition, FGF-2 acts as a potent angiogenic cytokine, stimulating endothelial proliferation15 and inducing
angiogenesis.16-18 FGF-2 and its receptors are therefore
relevant in hemangioblast, early hematopoietic, and endothelial cell biology.
Hematopoietic stem cells have been isolated from
CD34+ cells obtained from bone marrow, cord blood, and
peripheral blood.19-22 CD34+ cells have also
been shown to contain endothelial stem/precursor cells.23-29 Human CD34+ cells isolated from
peripheral blood are incorporated into the endothelium of ischemic
blood vessels of recipient animals.23,24 They also promote
neovascularization of ischemic myocardium, improve cardiac
function,25 and accelerate the restoration of blood flow
in diabetic mice undergoing neovascularization.26
CD34+ cells purified from umbilical blood give rise to von
Willebrand factor (VWF)-expressing endothelial cells in
vitro,27 whereas bone marrow-derived CD34+
cells are found lining the surfaces of vascular prostheses in sex-mismatched canine grafts.28 Furthermore, endothelial
cells of donor origin have been cultured from the peripheral blood of human subjects who had previously received sex-mismatched allogeneic bone marrow transplants, thereby definitively identifying the existence
of circulating progenitor endothelial cells.30,31
Because hematopoietic and endothelial cells share many cell surface
markers, including CD34,21,32 it is important to establish whether circulating cells that give rise to endothelium are endothelial precursors or merely mature endothelial cells derived from vessel walls. The primitive hematopoietic cell surface marker,
AC133,33-37 is not expressed on differentiated endothelial
cells29,38 and therefore coexpression of AC133 with
endothelial markers can be used to distinguish endothelial precursors
from mature endothelial cells. Human AC133+ cells isolated
from mobilized peripheral blood have been shown to differentiate into
endothelial cells in vitro and to form new blood vessels in vivo when
injected with tumor cells into immunodeficient mice.39
We have isolated a subpopulation of CD34+ cells from bone
marrow, cord blood, and mobilized peripheral blood that expresses FGFR-1 and that coexpresses a number of antigens found on primitive hematopoietic cells, including AC133. This population is selectively enriched for the expression of antigens specific for endothelial cells
(P1H1230,40 and vascular endothelial
cadherin41-45 [VE-cadherin]). The isolated
CD34+FGFR+ cells grow slowly in culture, are
stimulated by FGF-2 and vascular endothelial growth factor (VEGF), and
give rise to cells that express VWF and VE-cadherin and that
incorporate acetylated low-density lipoprotein (ac-LDL). These data
indicate that the CD34+FGFR+ population
contains endothelial stem/progenitor cells.
Cell preparation
Samples were enriched for CD34+ cells by immunomagnetic
separation, using either CD34 magnetically activated cell sorter
(MACS) microbeads, magnetic columns, and the MiniMACS system
(Miltenyi Biotec, Auburn, CA) or the Dynal CD34 Progenitor Cell
Selection System (Dynal AS, Oslo, Norway). Separation of
CD34+ cells was carried out according to the
manufacturers' recommendations. In some experiments, lineage depletion
was performed to obtain lineage-negative (Lin Antibodies and reagents
Cell staining and flow cytometry MACS-selected or Dynal-selected CD34+ cells or Lin cells were resuspended in fluorescence-activated cell
sorting (FACS) buffer that comprised PBS supplemented with BSA (0.1%),
sodium azide (0.01%), and aprotinin (20 µg/mL). Fc receptors and
nonspecific binding of immunoglobulins to cell surfaces were blocked
with human IgG and either mouse or goat IgG, where appropriate. In most
experiments, staining of FGFR+ cells was performed by using
directly labeled FGFR-1-APC, although FGFR-1-FITC was used in some
cases. In a few experiments unconjugated FGFR-1 was used together with
either goat antimouse-PE or goat antimouse IgG2a-PE as secondary
antibodies. Cells were incubated with appropriate antibodies for 30 minutes on ice, using FACS buffer to wash the cells between staining
steps. Cells were analyzed on a FACSCalibur flow cytometer (Becton
Dickinson), equipped with an argon laser to excite FITC, PE, and
RPE-CY5 fluorochromes and a helium-neon diode, with time delay adjusted
according to manufacturer's recommendations, for excitation of APC.
CD34+ or Lin selected cells
(30 000-150 000) were analyzed with the use of CellQuest software
(Becton Dickinson). The dye 7-aminoactinomycin D (7-AAD), at a final
concentration of 1 µg/mL, was added 5 minutes before flow cytometry
to identify dead cells. This task was done to ensure analysis of viable
CD34+FGFR+ cells.48
For the detection of VWF expression, cells were fixed in 2%
paraformaldehyde for 10 minutes at 37°C and 10 minutes at 4°C, permeabilized with 9:1 methanol/PBS for 20 minutes at
Culture of CD34+FGFR+ cells CD34-enriched cells were incubated with antibodies to CD34 and FGFR-1 and sorted on an Epics Elite Cell Sorter (Beckman Coulter, Fullerton, CA) into CD34+FGFR+ and CD34+FGFR populations. Sorted cells were
seeded at 1000 cells/well in 96-well plates in long-term culture medium
(Myelocult H5100; StemCell Technologies) containing 12.5% horse serum
and 12.5% FCS, in the presence or absence of FGF-2 (10 ng/mL) or FGF-2
and VEGF (10 ng/mL). Cells were fed twice weekly by replacing half the
medium with fresh medium. Growth was assessed by determining the number of viable cells per well present after various times in culture.
Purified CD34+FGFR+Lin Cultured CD34+FGFR+ cells were also examined for concomitant incorporation of DiI-ac-LDL and expression of VWF. Cells were incubated with DiI-ac-LDL (10 µg/mL) for 4 hours at 37°C,49 washed twice with PBS to remove free DiI-ac-LDL, and fixed with 3% paraformaldehyde in PBS. Cells were subsequently permeabilized with ice-cold acetone/methanol (50/50), treated with VWF antibodies (see above), and examined by fluorescent microscopy for VWF and evidence of DiI-ac-LDL uptake. Sorted CD34+FGFR+ and
CD34+FGFR Statistical analysis Data are expressed as the mean ± SD. When comparing the data from 2 populations, paired 2-tailed Student t tests were used to determine levels of significance. A P value of < .05 was considered statistically significant.
Phenotype of the CD34+FGFR+ population Purified CD34+ cells (Figure 1A) were examined for evidence of FGFR-1 expression. CD34+ cells were incubated with 7-AAD to ensure that only viable cells (R2, Figure 1B) that excluded 7-AAD were analyzed for expression of FGFR-1 and other antigens. CD34+ cells from bone marrow, umbilical cord blood, and mobilized peripheral blood contained a distinct population of FGFR-1-expressing cells (R3, Figure 1C). These cells comprised 4.5% ± 2.1% of the total CD34+ population (Table 1) and have low forward (FSC) and side scatter (SSC) properties (Figure 1D), indicating that they are small cells with little granularity. It is important to ensure the viability of the CD34+FGFR+ cells, because substantial numbers of these small cells are found in a region of the FSC/SCC dotplot that is often excluded from FACS analysis, as this region may contain nonviable cells.
To determine the phenotypic nature of the
CD34+FGFR+ population, we examined viable
CD34+FGFR+ cells isolated from mobilized
peripheral blood for the expression of the following 10 antigens: CD38,
AC133 (CD133), Thy-1, c-kit, CD31, Tie, Tek, vascular endothelial
growth factor receptor-2 (VEGFR-2/KDR), P1H12, and VE-cadherin (Table
2, Figure
2). This examination was done by using
4-color flow cytometry: 3 different fluorochromes were used for the
staining of CD34, FGFR-1, and one of each of the 10 antigens, and the
dye, 7-AAD, comprised the fourth color. The number of
CD34+FGFR+ cells obtained in each experiment
limited the number of antigens examined per sample. Most of the
CD34+FGFR+ cells expressed cell surface
antigens that are present on primitive hematopoietic cells, such as
AC13333-37 (75%, Table 2, Figure 2B), c-kit50
(85%, Table 2, Figure 2C), and Thy-151 (64%, Table 2,
Figure 2D). In addition, substantial numbers of
CD34+FGFR+ cells expressed the
endothelial-specific markers VE-cadherin41-45 (88%, Table
2, Figure 2I) and P1H1230,40 (58%, Table 2, Figure 2J). A
number of antigens that are expressed by both hematopoietic and
endothelial cells were also present on the
CD34+FGFR+ population: CD3152,53
(84%, Table 2, Figure 2E), Tie54,55 (56%, Table 2, Figure
2H), Tek56,57 (60%, Table 2, Figure 2L), and
KDR58,59 (76%, Table 2, Figure 2M). CD38 was found on 95%
of CD34+FGFR+ cells (Table 2, Figure 2G).
(Percentages provided here are rounded; for more precise data, refer to
Table 2.) Isotypic control antibodies were used to indicate
the specificity of the staining (Figure 2A,F,K).
CD34+FGFR+ cells isolated from bone marrow and
cord blood samples showed a pattern of expression of hematopoietic and
endothelial markers similar to that found on cells isolated from
mobilized peripheral blood (data not shown). These
results demonstrate that substantial numbers of viable
CD34+FGFR+ cells express antigens that are
found on both early hematopoietic and endothelial cells.
To determine whether CD34+FGFR+ cells
coexpressed both primitive and endothelial antigens on the same cells,
we excluded the dye, 7-AAD, from analysis so that we could
simultaneously examine the cells for the expression of AC133 and
VE-cadherin. We found that 74% of CD34+FGFR+
cells coexpress AC133 and VE-cadherin (Table
3). We also determined whether
CD34+FGFR+ cells demonstrated concomitant
expression of Thy-1 with either Tek or VE-cadherin. We found that 65%
of CD34+FGFR+ cells express both Thy-1 and
VE-cadherin, whereas 57% express Thy-1 and Tek (Table 3). Because the
majority (68%) of cells that expressed AC133 also expressed
Thy-1 (Table 3), this finding indicates that substantial numbers of
CD34+FGFR+ cells coexpress AC133, Tek,
VE-cadherin, and Thy-1.
To confirm the coexpression of hematopoietic and endothelial markers on
viable cells, we examined cells that had been depleted of lineage
markers (ie, Lin
We compared the levels of expression of endothelial and hematopoietic antigens on CD34+FGFR+ cells with that of the CD34+ population to determine if any of these antigens were preferentially expressed on CD34+FGFR+ cells. We found that CD38, AC133, c-kit, and CD31 are expressed to a similar extent by CD34+ and CD34+FGFR+ cells (Table 2). In contrast, substantially increased numbers of CD34+FGFR+ cells expressed Thy-1, Tie, Tek, P1H12, VE-cadherin, and KDR compared with the CD34+ population (Table 2). Thy-1 is expressed by 64% of CD34+FGFR+ cells compared with 12% of the CD34+ cells (Table 2). This finding indicates that there is a 5.3-fold enrichment of Thy-1+ cells in the CD34+FGFR+ subset as compared with the whole CD34+ population (P < .001, Table 2). The differences in expression of the endothelial-specific markers, VE-cadherin and P1H12, between these 2 populations are even more striking, with a 10- to 14-fold increase in expression by the CD34+FGFR+ population compared with the CD34+ population (P < .001, Table 2). Similar significant increases in expression (12- to 16-fold) of Tie (P < .001), Tek (P < .001), and KDR (P = .013) were noted by the CD34+FGFR+ cells compared with the CD34+ cells (Table 2). The increased numbers of cells expressing the endothelial-specific markers, P1H12 and VE-cadherin, as well as the antigens Tie, Tek, and KDR, found within the CD34+FGFR+ population compared with that of the CD34+ population, indicates that the CD34+FGFR+ subset is enriched for cells that express endothelial antigens. These results show that approximately 4.5% of CD34+ cells from bone marrow, cord blood, and mobilized peripheral blood express FGFR-1 and that they simultaneously express antigens found on both primitive hematopoietic and endothelial cells. The expression of a number of antigens present on endothelial cells (Tie, Tek, KDR, P1H12, VE-cadherin) is enriched in the CD34+FGFR+ population as compared with the CD34+ population. As 2 of these antigens (VE-cadherin, P1H12) are endothelial cell specific, the FGFR-1+ cells are likely to contain endothelial stem/progenitor cells. Growth characteristics of CD34+FGFR+ cells To determine whether CD34+FGFR+ cells respond to endothelial growth factors and give rise to endothelial cells, we isolated and cultured them under conditions that promote endothelial cell growth. CD34+FGFR+ and CD34+FGFR cells isolated on the FACS were
cultured in the absence and presence of FGF-2 alone or FGF-2 and VEGF
together. In the absence of growth factors both populations had minimal
growth (Table 4). An increase in the
numbers of CD34+FGFR+ cells (2.5- to 6.9-fold)
was noted when these cells were cultured with FGF-2 or with FGF-2 and
VEGF, whereas substantially less growth was found when
CD34+FGFR cells were cultured under similar
conditions (Table 4). These data demonstrate that
CD34+FGFR+ cell growth is stimulated by factors
known to promote endothelial cell proliferation.
We next determined whether CD34+FGFR+
cells could differentiate into endothelial cells as evidenced by the
expression of the endothelial-specific markers VWF and VE-cadherin and
the incorporation of ac-LDL. CD34+FGFR+ cells
were cultured in fibronectin-coated wells in the presence of
endothelial growth factors and examined for evidence of VWF expression.
CD34+FGFR+ cells cultured under these
conditions grew in small groups (Figure 4A), beadlike strings
(Figure 4B), or clusters (Figure 4C) of small round cells (~6 µm in
diameter), similar in size to freshly isolated cells (Figure 4D).
Cultured cells displayed VWF expression (Figure 4E), whereas no
staining was found in wells that received control antibodies (Figure
4F). HUVECs (Figure 4G,H) and cells of the erythroleukemic cell line,
K562 (Figure 4I,J), were used as positive (Figure 4G) and negative
(Figure 4I) controls, respectively, to ensure the specificity of the
VWF staining. These data indicate that
CD34+FGFR+ cells give rise to cells that
express VWF.
To confirm the endothelial nature of the cultured CD34+FGFR+ cells, they were also examined for their ability to incorporate DiI-ac-LDL. Because VWF is expressed only by megakaryocytic cells and endothelial cells60 and because ac-LDL is only taken up by endothelial cells and macrophages,49 we examined cultured CD34+FGFR+ cells for the presence of these characteristics. Dual fluorescence (Alexa Fluor 488 = green fluorescence, DiI = red fluorescence), indicating simultaneous VWF expression (Figure 4K) and ac-LDL incorporation (Figure 4L), confirmed the endothelial nature of the cells. To ensure the absence of endothelial cells in the original cell population, freshly isolated CD34+FGFR+ cells were examined by FACS analysis for the expression of VWF and their ability to incorporate DiI-ac-LDL. We show that freshly isolated CD34+FGFR+ cells do not express VWF (Figure 4M) or incorporate DiI-ac-LDL (Figure 4N). These data confirm that CD34+FGFR+ cells are able to give rise to endothelial cells. Because OP9 cells have been shown to promote the differentiation of
progenitor cells to mature endothelial cells,13 we also cultured CD34+FGFR+ cells on OP9 feeder layers.
Phase contrast microscopy shows that sorted
CD34+FGFR+ cells adhered to the OP9 feeder
layer in the presence of FGF-2 and VEGF (Figure 4O). The expression of
VE-cadherin in wells seeded with CD34+FGFR+ and
CD34+FGFR Our results show that the CD34+FGFR+ population grows slowly in culture in an FGF-2- and VEGF-dependent manner, indicating that these cells are stimulated by angiogenic factors. The cultured CD34+FGFR+ cells give rise to cells that incorporate ac-LDL and express VWF, whereas freshly isolated cells do not. This finding indicates that the CD34+FGFR+ population contains cells that differentiate into endothelial cells.
We show that endothelial precursor cells reside within a population of FGFR+ progenitor cells. FGFR-1 is expressed on a small population of CD34+ cells (Table 1, Figure 1) that coexpresses primitive hematopoietic and endothelial cell surface antigens (Tables 2 and 3, Figures 2 and 3) and gives rise to endothelial cells in culture (Figure 4). The CD34+FGFR+ population is considerably enriched (10- to 16-fold) with respect to the expression of many antigens that are found on endothelial cells (VE-cadherin, P1H12, Tie, Tek, KDR) compared with the CD34+ population (Table 2). The CD34+FGFR+ population also contains 5-fold more cells expressing Thy-1, a primitive hematopoietic marker,51 than the CD34+ population, indicating that Thy-1 is also expressed on most endothelial progenitor cells. Most CD34+FGFR+ cells express AC133, a primitive hematopoietic antigen33 that is not expressed on mature endothelial cells.29,38 Because VE-cadherin and P1H12 are expressed exclusively on endothelial cells and because Thy-1 and AC133 are expressed on early hematopoietic cells, the CD34+FGFR+ population that coexpresses these markers is likely to represent an endothelial stem/progenitor cell population. Cultured CD34+FGFR+ cells respond to angiogenic cytokines (Table 4), maintain their expression of VE cadherin (Figure 4P), and give rise to cells that express VWF (Figure 4K) and incorporate ac-LDL (Figure 4L). The freshly isolated CD34+FGFR+ population does not express VWF (Figure 4M) or incorporate ac-LDL (Figure 4N), indicating that the CD34+FGFR+ population contains cells that differentiate into endothelial cells. We have combined the results from 4-color fluorescence staining
(Table 3, Figure 3B) with those of the incidence of expression of
antigens on the CD34+FGFR+ cells (Table 2)
to obtain the following phenotype to characterize endothelial progenitor cells:
CD34+FGFR+CD38+VE-cadherin+c-kit+CD31+KDR+AC133+
(Figure 5). As fewer cells express Thy-1,
Tie, Tek, and P1H12, it is possible that these antigens may be
expressed on endothelial progenitor cells of different stages of
maturity in a manner analogous to the expression of lineage-specific
markers on hematopoietic cells as they mature along different lineages.
Cells that have the capacity to develop into endothelial cells have recently been isolated from the circulation.23,27-31,39,61,62 Previous studies indicating the presence of endothelial precursor cells in cord blood or peripheral blood have used either unfractionated mononuclear cells or samples enriched for CD34+,23,27,29,61,63 AC133+,39 P1H12+,30 or CD14+ cells.62 In these studies the phenotype of the circulating endothelial precursors was determined by either single-color flow cytometry alone or together with immunocytochemisty, or alternatively by 2-color flow cytometry. Our data indicate that endothelial precursor cells can be isolated and identified by the expression of FGFR-1 and provides the first detailed description of the phenotype of these cells by using FACS isolation and 4-color flow cytometry. The identification of progenitor cells that express FGFR-1 as well as primitive hematopoietic and endothelial cell antigens and that give rise to endothelial cells in vitro is not surprising, as hematopoietic and endothelial systems have a close association during ontogeny. A common embryonic precursor, the hemangioblast, that gives rise to both hematopoietic and endothelial cells has been identified,13 and experiments using murine embryonic stem cell lines carrying mutations for FGFR-1 show that FGF-2-mediated signaling is essential for hemangioblast proliferation.14 FGFRs are expressed on pluripotent human embryonic stem cells and embryoid bodies64 as well as on CD34+ hematopoietic cells1-5 and on leukemic cell lines, such as K562, U937, Hel, Daudi, MO7E, HL60, Jurkat, Molt 3, and TF-1.65-67 FGF-2 has been shown to promote the self-renewal of a multipotent hematopoietic cell line11 and the growth of a primitive erythroid cell line.12 FGF-2 also stimulates the growth of early hematopoietic progenitors in synergy with other cytokines.7-10 In addition to its effects on embryonic and hematopoietic cells, FGF-2 also acts as a potent angiogenic cytokine. It stimulates endothelial cell proliferation15 and is a potent inducer of angiogenesis.16-18 FGF-2 is able to induce uncommitted mesoderm to differentiate into endothelial precursors (angioblasts) in the quail embryo68,69 and is required for the organization of vascular endothelial cells into functional networks in the murine embryo.70 FGF-2 has also been shown to induce the development of blood islands and endothelial cells from dissociated quail epiblasts,71 and FGF-2 stimulates the growth of both endothelial and hematopoietic cells from embryonic avian mesoderm.72 Furthermore, experiments using 3-dimensional spheroid models of endothelial cell differentiation have shown that FGF-2 is a survival factor for immature endothelial cells, inhibiting the apoptosis of these cells.73 There is therefore considerable evidence that indicates that FGF-2 is a relevant cytokine that affects endothelial cells as well as hemangioblasts and early hematopoietic cells. Although regarded as an endothelial-specific marker of mature endothelial cells, VE-cadherin is also expressed by embryonic cells that have hemangioblast potential.74,75 Because FGFR-1 and FGF-2 are required for hemangioblast proliferation,14 it is possible that CD34+FGFR+ cells that express VE-cadherin may have hemangioblast potential. It is currently unknown whether hemangioblasts exist in postnatal life. In summary, we show that the FGFR+ progenitor population contains endothelial precursor cells. These cells may be of significant clinical benefit in treating a number of disorders such as cardiovascular disease, diabetes, and cancer. Endothelial stem cells have been used to repair sites of vascular injury,76 to improve cardiac function,25 and to enhance the blood flow in diabetic mice after hindlimb ischemia.26 CD34+FGFR+ cells from syngeneic individuals may also be useful for delivering angiogenic or antitumor agents to the rapidly expanding vascular bed associated with tumors.
This paper is dedicated to the memory of Eugene B. Dowdle, a mentor, friend, and collaborator of P.E.B., S.C., and E.L.W.
Submitted August 15, 2001; accepted July 15, 2002.
Supported by the Cancer Association of South Africa (CANSA), the University of Cape Town Staff Research Fund, and the National Institutes of Health DK48728.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Patricia E. Burger, Department of Immunology, H53-22 Old Main Bldg, Groote Schuur Hospital, Observatory 7925, Cape Town, South Africa; e-mail: pburger{at}uctgsh1.uct.ac.za.
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Abstract
Introduction
20°C,
