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*
James Ewing Laboratory of Developmental Hematopoiesis, Sloan-Kettering Institute for Cancer Research, Divisions of
Hematology/Oncology and
Pulmonary and Critical Care Medicine, The New York Hospital-Cornell Medical Center, New York, NY 10021; and
§
Cardiovascular Research Department, Genentech, Inc., South San Francisco, CA 94080
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gene transfer offers an alternative strategy to the administration of the recombinant TPO protein. This has been achieved in experimental animals using retrovirus vectors to transfer the murine TPO cDNA into bone marrow cells of mice (15) as well as using an adenovirus vector to transfer the human TPO cDNA to mice via various routes of administration (16). Although both strategies are effective and attractive because they circumvent the necessity of ex vivo production and purification of TPO, gene transfer is associated with the risk of uncontrolled stimulation of the megakaryocytic lineage as has been observed with murine megakaryocyte growth and development factor (MGDF) cDNA delivered to the bone marrow (15, 17). We attempted to study the biologic consequences of chronic TPO overexpression by an in vivo transfection strategy. Using i.p. administration of AdTPO to mice, our experimental design differs from the retrovirus vector-based model (17) in several important aspects: First, the transfection of TPO cDNA happens in vivo and without perturbing the bone marrow, which will become one of the read-out tissues for the experimental outcome. Second, the artificial autocrine and paracrine mechanisms of TPO release in the bone marrow microenvironment that were induced by the retroviral transduction of bone marrow cells can be excluded in our approach. Third, the CMV promotor used in our AdTPO construct assures high levels of TPO overexpression at extramedullary sites, which guarantees an optimal experimental challenge for the envisioned outcome parameter. Therefore, the present study may provide a clinically more relevant model for studying the consequences of chronically elevated plasma TPO level.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice matched for age (8 to 10 wk), weight (>20 g), and sex were obtained from Charles River Laboratories (Wilmington, MA) and The Jackson Laboratory (Bar Harbor, ME) and maintained in germfree conditions. Four strains of mice were evaluated, all on BALB/c background, including: 1) immune normal BALB/c (18, 19); 2) nu (T cell defect, with diminished CTL response and impaired Ab production) (20, 21, 22, 23); 3) SCID (T and B cell defect, diminished CTL, low to absent Ab production) (24, 25); and 4) NOD-SCID (also referred to as NOD/LtSz-SCID; T and B cell defect, mononuclear phagocytes diminished in number and function, low to absent Ab production, decreased NK activity) (26, 27). All strains received the AdTPO and the AdNull vector (109 PFU in a volume of 100 µl, single i.p. administration on day 0). Each study group contained three animals.
Adenovirus vectors
AdTPO is an Ad5-derived E1a-, partially E1b-, partially E3-deficient vector with an expression cassette in the E1a region containing the human TPO cDNA driven by the CMV major immediate/early promotor/enhancer (28, 29). The control vector AdNull is similar in design, except that it contains no transgene in the expression cassette (29). All vectors were amplified, purified, and titered as previously described (28, 30).
Peripheral blood counts
Initially every 3 to 4 days and later on a weekly basis, retroorbital blood was collected with capillary pipettes (Unopette, Fisher Scientific, Springfield, MA). Platelets, total white blood cells and granulocytes (polymorphonuclear leukocytes) were counted using a Neubauer hemocytometer. The hematocrit was measured in heparinized microhematocrit capillary tubes (Fisher Scientific). The plasma was collected, stored at -80°C, and assessed later by ELISA for human TPO levels and anti-TPO Abs.
ELISA for human TPO and anti-TPO Abs
Human TPO present in mice serum was assessed according to Emmons et al. (31). Briefly, microtiter wells were incubated overnight with 2 mg/ml rabbit F(ab')2 anti-human Fc (Jackson ImmunoResearch, West Grove, PA) and then for 2 h with 100 ng/ml of chimeric mpl-IgG. Serially diluted plasma samples and standard (recombinant full-length human TPO produced in mammalian cells) were added to wells and incubated for 1 h. Bound TPO was quantitated colorimetrically by addition of biotinylated affinity-purified polyclonal rabbit anti-TPO Abs (Genentech, South San Francisco, CA) followed by streptavidin-conjugated peroxidase. The detection threshold of the ELISA was 0.057 ng/ml.
Abs to murine (m) TPO present in the plasma were tested using a TPO binding ELISA. Briefly, biotinylated mTPO was bound onto a streptavidin-coated microtiter plate, and the plates were incubated with mouse serum from the treated mice. Bound anti-mTPO Abs were then detected with a secondary HRP-conjugated Ab (Genentech). The mTPO-coated microtiter plate (Costar, Cambridge, MA) was developed by incubating wells with 100 µl of streptavidin (250 µg/ml) in PBS containing 0.01% thimerosal for 12 to 72 h at 2 to 8°C followed by three washes with PBS containing 0.5% BSA and 0.01% polysorbate 20 (assay diluent). After washing, 100 µl of biotinylated mTPO (100 µg/ml) in assay diluent were added and incubated with assay diluent for 1 h at room temperature (RT) and subsequently washed 3 times with wash buffer. Samples (mouse plasma) and positive and negative controls (goat anti-mTPO serum and goat nonimmune serum, respectively) in assay diluent were added to appropriate wells and incubated for 2 h at RT with agitation. Bound murine anti-mTPO was detected by adding 100 µl of goat anti-murine IgG-HRP in assay diluent to the wells and incubating for 1 h at RT. The plate was then washed 6 times with wash buffer. One hundred microliters of HRP solution was added and incubated for 10 to 20 min in the dark at RT. The reaction was stopped by adding 100 µl of 4.5 M sulfuric acid. The plate was read at 490 to 492 nm for detection absorbance and 405 nm for reference absorbance. The presence of anti-mTPO Abs was determined by evaluating the optical density ratio (ODR) for each control and sample. ODR = OD of sample well/OD of blank well. An ODR > 1.8 was reported as positive.
Progenitor assay
All animals were killed 80 days after vector administration. One femur per animal was flushed with cold (4°C) IMDM/20% FCS, and the total yield of bone marrow mononuclear cells (MNC) was determined by counting an aliquot in 0.36% acetic acid using a Neubauer hemocytometer. MNCs (105) were plated in duplicate in 1 ml of 0.8% methylcellulose containing 30% FCS, 1% L-glutamine, 2.5% hemin, 0.05 mM 5-ME, rmIL-3 (50 ng/ml; R&D Systems, Minneapolis, MN), rm c-kit ligand (20 ng/ml, Immunex, Seattle, WA) and recombinant human erythropoietin (2 U/ml; Sandoz, Basel, Switzerland) in 35-mm suspension culture dishes (Nunc, Inc, Naperville, IL). Cultures were incubated at 37°C in 100% humidity and 5% CO2 for 7 days. Scoring was performed with an inverted microscope with 40x magnification on day 7. Colonies containing more than 50 cells were counted as burst-forming unit-erythroid (BFU-E), CFU-granulocytic/monocytic (CFU-GM), CFU-granulocytic/erythroid/megakaryocytic/monocytic (CFU-GEMM), and CFU-megakaryocytic (CFU-Mk). CFU-Mk were defined as colonies containing cells with exclusively megakaryocytic features. The total number of colonies per femur was calculated.
Histology
Twelve weeks after vector administration, spleen, liver, and bone marrow (femur, sternum, and thoracic vertebrae) were harvested for histologic evaluation. The tissues were fixed in 4% formalin, paraffin embedded, cut and stained with hematoxylin and eosin, and silver stain according to standard protocols. Quantification of megakaryocytes was performed on bone marrow sections of thoracic vertebrae by counting mature megakaryocytes in 10 high power fields of comparable cellularity.
Statistical evaluation
Data are expressed as mean ± SEM of at least three animals per experiment per group. Statistical significance was determined using the two-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As previously observed for the AdTPO vector (16), a single i.p.
administration of the vector to BALB/c mice resulted in a 3- to 4-fold
increase of platelet levels, peaking at day +7 and returning to
baseline at day +14 (Fig. 1
A). In contrast, all
mice with immune defects demonstrated persistent elevation of platelet
levels for the 80-day duration of the study. The nu mice had
peak platelet levels 4-fold above baseline at day +10, falling to
2-fold above baseline by 3 wk, and sustaining that level for the
remainder of the study. The SCID and NOD-SCID mice responded with a
platelet peak of 12- to 14-fold above baseline 4 to 5 wk after vector
administration. The pattern of thrombocytosis was similar in the two
strains, with a parallel, gradual decrease over the 2 mo following peak
levels. By 77 days, the platelet levels of the SCID and NOD-SCID mice
were similar (p > 0.5), but both were higher
than the platelet levels of the nu mice
(p < 0.05, both comparisons) as well as the
BALB/c mice (p < 0.01). The platelet levels of
all control animals were unaffected throughout the study (Fig. 1B). Despite the fact that platelets reached high
levels in all mice (peak levels BALB/c 6.4 ± 0.5 x
106/µl, nu 5.9 ± 0.6 x
106/µl, SCID 14.5 ± 0.5 x
106/µl, NOD-SCID 16 ± 2.1 x
106/µl) and were maintained >8 x
106/µl for 1 mo in the SCID and NOD-SCID mice; no
lethality nor adverse effects related to thrombocytosis were
observed.
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). Anti-TPO Abs were found only in BALB/c mice (Table I) and first detected on day +7. In these
animals, human TPO (45 ± 15 ng/ml) was detectable only on day +3.
In contrast, nu, SCID, and NOD-SCID mice did not develop
anti-TPO Abs, and human TPO could be detected up to 80 days after
vector administration (Table I). In the SCID animals, sequential
evaluation of human TPO levels throughout the study revealed good
correlation between TPO and platelet levels (Fig. 2). The increasing TPO concentration was
followed by a rapid increase of circulating platelets. On day +21, the
peak of TPO concentration (635 ± 106 ng/ml) was reached. The peak
level of platelets (14.5 ± 0.5 x 106/µl)
followed 7 days later. TPO and platelets dropped gradually in a
parallel fashion with platelet counts decreasing roughly 1 wk following
the drop in TPO concentration. None of the control animals had
detectable levels of human TPO or anti-TPO Abs (not shown).
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Although TPO is considered to be relatively megakaryocyte
specific, the SCID and NOD-SCID mice exhibited a marked granulocytosis
over several weeks (Fig. 3A). Granulocytes
increased 10- to 12-fold above baseline and peaked parallel to the
platelet counts 3 to 5 wk after vector administration. The granulocytes
completely returned to baseline by 6 to 11 wk after vector
administration. In contrast, no significant changes of granulocyte
number was observed in BALB/c and nu mice after AdTPO
treatment or in control mice (Fig. 3B).
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A). In both strains,
this was associated with low levels of circulating reticulocytes (not
shown). The hematocrit nadir (33 ± 4%) was reached 5 wk after
vector administration, coinciding with the peak of platelets and
granulocytes. No significant change in hematocrit was observed in
BALB/c or nu mice after AdTPO treatment, nor in the control
mice (Fig. 4).
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Bone marrow cellularity 80 days after vector administration,
estimated by the yield of MNCs from one femur cavity, was in the normal
range for BALB/c, nu, and NOD-SCID mice independent of
treatment (Table II). However,
AdTPO-treated SCID mice had very poor yields compared with control SCID
mice (p < 0.001, Table II). In terms of
progenitors, AdTPO-treated BALB/c and SCID mice had diminished amounts
of CFUs per femur compared with the respective control mice (BALB/c,
p < 0.05; SCID, p < 0.01; Fig. 5). In contrast, AdTPO-treated
nu mice had increased numbers of CFUs compared with control
nu mice (p = 0.06). As shown in the
clonogenic assay, these differences were mainly due to changes in the
compartments of CFU-GM,-GEMM, and -Mk (Fig. 5).
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Histologic examination revealed significantly more mature
megakaryocytes in the bone marrow of AdTPO-treated SCID and NOD-SCID
animals as compared with the control mice (SCID, p <
0.001, Fig. 6). In contrast, BALB/c mice
had significantly decreased numbers of mature megakaryocytes
(p < 0.001), consistent with the presence of
circulating anti-murine TPO Abs (Table I). The nu mice
had only slightly, not significantly (p = 0.15)
elevated numbers of megakaryocytes as compared with the control.
Histologically, BALB/c, nu, and NOD-SCID mice showed normal
cellularity and architecture of the bone marrow (Fig. 7, A, B, and
D). In contrast, AdTPO-treated SCID mice developed
severe osteomyelofibrosis and osteomyelosclerosis throughout the
hemopoietic tissue (Figs. 7C and 9A).
Reticulin staining confirmed the finding of osteomyelofibrosis
exclusively in SCID mice (Fig. 8,
A–D). As a consequence of osteomyelofibrosis, only
AdTPO-treated SCID mice had extramedullary/extrasplenal hemopoiesis,
with the formation of blood islands and foci of megakaryocytopoiesis in
liver and lung (Fig. 9, B and
D). In the spleen, fibrotic tissue surrounding
islands of hemopoiesis and megakaryocytes was observed (Fig. 9C). Splenomegaly was present also in NOD-SCID mice
treated with AdTPO (not shown), but neither myelofibrosis nor
extrasplenal hemopoiesis were found. No pathologic findings in the bone
marrow and other organs (lung, liver, spleen) were observed in
AdNull-treated mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One major finding of this study is the observation of fibrotic changes in the bone marrow and at extramedullary sites in response to chronic overexpression of TPO exclusively present in SCID mice. Recently, Yan et al. (17) described a mouse model for osteomyelofibrosis using a retrovirus-based transfer of TPO cDNA to bone marrow mononuclear cells. Our experimental design differs in several respects from the highly artificial retrovirus-based model and may reflect more closely the clinical picture of osteomyelofibrosis. 1) In contrast to the retroviral model, which was characterized by a low range of TPO production as measured by moderately elevated platelet counts and no effect on circulating granulocytes, we observed excessive thrombocytosis and granulocytosis as consequence of high level of TPO expression. The hyperproliferative phase followed by marrow fibrosis/failure mimicks realistically the development of osteomyelofibrosis. 2) In our experimental design, the transfer of TPO cDNA does not require manipulations of the bone marrow or bone marrow reinfusion and may therefore facilitate the interpretation of the histologic changes in the bone marrow. 3) The CMV promotor of AdTPO assures high level of expression of TPO at extramedullary sites as shown by sequential determination of TPO plasma levels, which may provide an optimal, strong, and lasting challenge by TPO. 4) Since adenovectors deposit the transgene epichromosomally, there is no risk of integrational mutagenesis as has been debated in retroviral approaches (37). 5) The proposed model is simple and easy to perform.
Surprisingly, we observed myelofibrotic and myelosclerotic changes only in AdTPO-treated SCID mice. NOD-SCID mice with an identical pattern of TPO overexpression and hypermegakaryocytopoiesis in the bone marrow did not develop osteomyelofibrosis. In vivo and in vitro studies have shown that bone marrow megakaryocytes produce TGF-ß1, platelet-derived growth factor, and fibroblast growth factor, which stimulate fibroblasts to proliferate, produce, and secret type IV collagen (17, 38, 39, 40). Normally, these cytokines are packaged into platelets, transported through the blood stream, and released at the site of vascular injury (41). It has been proposed that secondary osteomyelofibrosis is related to ineffective megakaryocytopoiesis, leading to increased apoptosis of megakaryocytes and promegakaryocytes with local release of TGF-ß1, platelet-derived growth factor, and fibroblast growth factor, which then stimulates bone marrow fibroblasts (41, 42). Our observation of the striking difference in the development of osteomyelofibrosis between SCID and NOD-SCID mice suggests an additional factor in the pathogenesis of secondary osteomyelofibrosis. It has been shown that bone marrow monocytes and macrophages of NOD-SCID mice are functionally deficient in terms of IL-1ß secretion, cytokine receptor regulation, and protein kinase C expression (26, 27). Based on our data, we postulate that normal bone marrow monocytes and macrophages, as present in SCID but not in NOD-SCID mice, may be required for generation of secondary osteomyelofibrosis. Further studies are required to support this hypothesis.
From an interventional point of view, the aspects of hematologic responses to high level TPO expression are of greater relevance. The data show marked differences in the extent and magnitude of platelet, granulocyte, and hematocrit responses to AdTPO treatment depending on the immune status of the mice. Immune-competent (BALB/c) and nu mice (T cell deficient) had only transient thrombocytosis of 7 to 10 days with platelet peaks of 4 to 5 times normal value. In contrast, SCID and NOD-SCID mice (both T and B cell deficient) had thrombocytosis for several months, and their platelet peaks were 10 to 12 times above normal values. BALB/c mice had measurable human TPO levels only on day +3 post injection. Compared with BALB/c mice, the peak concentration of human TPO in SCID mice was 14-fold higher and was reached later (day +21). In BALB/c mice, the production of human TPO ceased within 1 wk, whereas nu, SCID, and NOD-SCID mice maintained low levels of human TPO production throughout the study. It has been shown in several animal studies that the stability and level of adenovirally mediated transgene expression is dependent on mouse strain (43), immune status of the host (34, 44), coexpression of adenoviral structure proteins (45), and administered vector dose (46). We show that the expression of TPO, as measured by elevated platelet numbers, can be substantially prolonged (7- to 10-fold) by eliminating the immune functions of the host. On the other hand, in an immunocompetent host, the adenovirally mediated expression of a xenogeneic transgene may induce a humoral immune response against the transgene product, which shortens the transgene expression and may cross-react with the homologous self protein, causing autoimmune phenomenon. This was observed in immune-normal BALB/c mice that developed anti-murine TPO Abs, resulting in chronic thrombocytopenia with depletion of bone marrow megakaryocytes and hemopoietic progenitors 5 wk after administration of AdTPO. In another series of experiments, we showed that BALB/c mice develop immune thrombocytopenia only after treatment with adenovectors expressing human TPO but not after treatment with adenovectors expressing murine TPO (our manuscript in preparation). A similar break of immune tolerance was observed by Tripathy et al. (47) upon infecting BALB/c mice with an adenovector delivering human erythropoietin cDNA. Therefore, the autoantibody production in immunocompetent animals is due to species-specific epitopes in the native protein rather than unspecific adenovirally mediated epitopes. Such transgene-specific immune responses may become therapeutically important in cancer treatment protocols that utilize tumor vaccines. In immunocompetent cancer patients, adenovirally mediated overexpression of a heterologous or mutated tumor Ag may evoke a significant antitumor immune response.
TPO is not the only factor that regulates the circulating platelet level. Clinical studies showed that plasma TPO concentration in patients with thrombocytopenia depends on the pathomechanism of thrombocytopenia: patients with thrombocytopenia, because of peripheral platelet destruction and increased megakaryocytopoiesis in the bone marrow, had low levels of TPO, and patients with bone marrow failure showed high levels of TPO (31). Furthermore, NF-E2 knock-out mice (48), which lack the hemopoietic subunit (p45) of the heterodimeric erythroid transcription factor NF-E2, have severe thrombocytopenia despite normal plasma TPO levels and normal megakaryocyte mass in the bone marrow. In our model, the increase of circulating platelets occurred simultaneously with the increase of TPO plasma concentration. However, platelets continued to increase at least 1 wk past the peak of plasma TPO concentration. Later, platelets dropped markedly delayed to the drop of TPO concentration. By the end of the study, SCID and NOD-SCID mice still had substantially increased numbers of megakaryocytes in the bone marrow along with residual human TPO in plasma. These findings together with clinical and experimental evidence in the literature (31, 48, 49, 50, 51, 52, 53) support the hypothesis that TPO primarily regulates the megakaryocyte mass in the bone marrow and only indirectly influences the level of circulating platelets.
In addition to thrombocytosis, granulocytosis was also observed in mice with prolonged TPO overexpression. In the bone marrow, this was associated with expansion of various hemopoietic progenitor pools, favoring CFU-GM, -GEMM, and -Mk. This pleiotropic effect of TPO was reversed in mice with autoantibodies against TPO. Besides peripheral thrombocytopenia, these mice had significantly lower number of hemopoietic CFUs in the bone marrow as compared with control mice. Although TPO was discovered as a growth factor of the megakaryocytic lineage (1, 2, 4, 8), in vitro data have demonstrated a synergistic stimulatory effect of TPO with c-kit ligand (54, 55), IL-3 (56), and erythropoietin (57) on hemopoietic progenitors. In animal models, pegylated MGDF has led to enhanced recovery of platelets and erythrocytes after myelosuppressive treatment (13, 16), and studies in humans suggest that TPO is effective for mobilizing progenitor cells into the peripheral circulation (49). Taken together, these results identify TPO as a hemopoietic growth factor that exerts multilineage stimulation of progenitors in concert with other factors present in the bone marrow microenvironment. Moreover, recently Young et al. (58) showed that TPO alone is capable of stimulating lineage uncommitted CD34+ progenitor cells. Therefore, TPO may have a stimulatory effect on granulocytic (CFU-GM) and pluripotent (CFU-GEMM) progenitors as well as on megakaryocytic (CFU-Mk) progenitors in vivo. Maximal stimulation may be exerted along the entire megakaryocytic lineage, where TPO controls even end stage maturation of megakaryocytes and formation of proplatelets, as shown by Zucker-Franklin et al. (59). In the myeloid lineage, TPO may mainly stimulate early progenitors and, to a lesser degree, more mature progenitors and myeloid precursors. This may explain the observed close link between TPO concentration and platelet number and the weak correlation between TPO overexpression and granulocytosis. In animals with low level of TPO overexpression (BALB/c and nu mice), we did not see an increase of granulocytes at all, and in animals with chronic, high levels of TPO overexpression, the granulocytic response was delayed and variable.
The unexpected reversible hyporegenerative anemia in animals with excessive thrombocytosis and granulocytosis was most likely due to proliferation of myeloid and megakaryocytic progenitors in a space-limited hemopoietic microenvironment at the expense of erythroid precursors. Blood loss can be excluded as an alternative explanation, since reticulocyte counts were in the normal range and no signs of bleeding were observed.
In conclusion, we show that: 1) prolonged and excessive in vivo overexpression of TPO leads to stimulation of megakaryocytopoiesis and myelopoiesis associated with thrombocytosis and granulocytosis; 2) bone marrow accessory cells, possibly macrophages and monocytes, may play a critical role in mediating secondary osteomyelofibrosis and osteomyelosclerosis in response to increased megakaryocytopoiesis and TPO overexpression; 3) duration and magnitude of expression of TPO cDNA delivered by adenovirus is dependent on the immune status of the host; and 4) in immune normal hosts, the humoral immune response induced by adenovirally mediated xenogeneic transgene may break the immune tolerance for the homologous self protein.
These findings may have important implications for designing new gene therapy protocols utilizing adenovirus vector systems for overexpression of TPO and other hemopoietic cytokines. Our experimental design may prove to be useful in studying the physiology of platelet production as well as the pathophysiology of osteomyelofibrosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Beat M. Frey, Stiftung Zürcher Blutspendedienst SRK, Hirschengraben 60, CH-8001 Zürich/Switzerland. E-mail address:
3 Abbreviations used in this paper: TPO, thrombopoietin; MGDF, megakaryocyte growth and development factor; AdTPO, adenovirus vector expressing the human TPO cDNA; PFU, plaque-forming unit; m, murine (e.g., mTPO); HRP, horseradish peroxidase; RT, room temperature; MNC, mononuclear cell; BFU-E, burst-forming unit-erythroid; CFU-GM, CFU-granulocytic/monocytic; CFU-GEMM, CFU-granulocytic/erythroid/megakaryocytic/monocytic; CFU-Mk, CFU-megakaryocytic; nu, nude strain.
Received for publication April 8, 1997. Accepted for publication October 2, 1997.
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BALB/c; 
Nude; 
SCID; 
NOD-SCID
mice) were treated with 109 PFU AdTPO
(A) or AdNull (B)
i.p. on day 0. Mice with SCID and NOD-SCID showed a dramatic increase
of circulating platelet numbers 4 to 5 wk after AdTPO administration.
Peak levels of platelets were followed by a biphasic decrease during
the second half of the study. On day 80, the platelet levels were still
twofold higher than the pretreatment value. Nude and BALB/c mice showed
maximal platelet levels 7 to 10 days following administration of AdTPO.
Subsequently, the platelets of nude mice dropped to high normal levels
and remained slightly elevated throughout the study. BALB/c mice showed
a complete normalization of the thrombocytosis by day 14. Later on
these animals developed a chronic thrombocytopenia. None of the animals
treated with the control vector AdNull showed changes in platelet
numbers.
