Methods of treating sickle cell disease
1. A method of treating a sickle cell disease subject in vaso-occlusive crisis comprising administering an E-selectin inhibitor that interacts with a calcium-binding lectin domain of E-selectin in an amount effective to treat the subject.
The present invention relates to methods of treating sickle cell disease comprising reducing, in a subject in need of such treatment, the adherence between sickle RBCs and leukocytes. It is based, at least in part, on the discovery that leukocytes play a direct role in the initiation of venular occlusion. The present invention further provides for methods for identifying agents which decrease SS-RBC/leukocyte adherence and for animal models which may be used to further elucidate the mechanism of vaso-occlusion in sickle cell crises.
|Methods of treating hemophilia or von willebrand disease with p-selectin|
Patent #US 7,387,777 B2
Current AssigneeCENTER FOR BLOOD RESEARCH INC.
Sponsoring EntityCENTER FOR BLOOD RESEARCH INC.
|Methods of detecting and treating vaso-occlusive crisis in sickle cell disease|
Patent #US 5,669,396 A
Current AssigneePresident and Fellows of Harvard College
Sponsoring EntityPresident and Fellows of Harvard College
- 1. A method of treating a sickle cell disease subject in vaso-occlusive crisis comprising administering an E-selectin inhibitor that interacts with a calcium-binding lectin domain of E-selectin in an amount effective to treat the subject.
This application is a continuation of U.S. patent application Ser. No. 10/123,373 filed Apr. 15, 2002 now abandoned, which claims priority to Provisional U.S. Patent Application No. 60/283,776 filed Apr. 13, 2001, incorporated by reference in their entireties herein.
This invention was made with government support under Grant Nos. HL28381, DK56638 and HL19278 awarded by the National Institutes of Health. The government has certain rights in the invention.
The present invention relates to methods for treating sickle cell disease in which adherence between sickled erythrocytes and leukocytes is inhibited. It is based, at least in part, on the discovery that, in animal models of sickle cell disease, sickled erythrocyte/leukocyte adhesion plays a direct role in the initiation of vaso-occlusion, the cause for sickle cell crises in humans.
Half a century ago, Linus Pauling first showed that sickle cell anemia is a molecular disease (Pauling, 1949; for full citations see list in Section 8, infra). It was later demonstrated that the disease originated from a missense mutation within the β-globin gene, leading to the substitution of valine for glutamic acid on the outer surface of the globin molecule. This amino acid substitution renders the sickle cell hemoglobin (“HbS”) less soluble and prone to polymerization upon deoxygenation (Hoffman, 2000). Erythrocytes (red blood cells, “RBC”) carrying polymerized HbS are thus less deformable and may obstruct microvessels. This vascular occlusion, producing tissue ischemia and infarction, represents a major cause of morbidity and mortality among sickle cell disease patients. Despite recent therapeutic advances with the use of hydroxyurea and butyrate (Charache, 1995; Atweh, 1999) many patients remain severely symptomatic and thus, may benefit from alternate therapeutic modalities.
Over the years, it has become clear that the clinical manifestations of sickle cell disease extend far beyond the homozygous globin mutation. Seminal findings were the discovery that sickle (“SS”) RBCs, unlike normal RBCs, could adhere to stimulated endothelium in vitro and that SS-RBCs'"'"' adhesion correlated with the clinical severity of sickle cell disease(Hoover, 1979; Hebbel, 1980 (a) and (b)). Subsequent studies have recognized the importance of plasma factors in SS-RBC adhesion to the endothelium (Wautier, 1983; Mohandas, 1984) and revealed that the deformable “low-density” cells were more adherent than the dense sickle-shaped cells (Mohandas, 1985; Barbarino, ). Other elegant studies by Kaul and coworkers subsequently showed using a rat mesocecum ex vivo perfusion model that SS-RBCs adhered exclusively in venules (mostly small post-capillary and collecting venules) and confirmed that adhesion was density-class dependent (light-density reticulocytes and young discocytes being most adherent; Kaul, 1989). Collectively, these observations lead to the current multistep model, shown in
Multiple adhesion molecules have been shown to participate in SS-RBC/endothelium interactions (
Several mouse strains expressing HbS have been generated in the last decade. These transgenic strains have been used to study the pathophysiology of sickle cell disease in vivo, and may be divided into two broad categories: i) transgenic mice expressing both the endogenous murine and human globin genes, and (ii) transgenic mice expressing exclusively human globin genes (Nagel, 1998). So-called “SAD” mice represent one example of transgenic animal models for sickle cell disease in which the human β-globin transgene contains three natural mutations that enhance Hb sickling: HbS, HbS-Antilles and Hb D Punjab (hence the acronym “SAD”). RBCs from SAD mice carry approximately 19% human hemoglobin. Although associated with a significant perinatal mortality (when a SAD mouse is bred with a wild-type animal, the frequency of SAD offspring is about 30%, rather than the expected 50%), adult SAD transgenic mice are relatively healthy, suffering neither anemia nor reticulocytosis unless exposed to hypoxemic conditions (Trudel, 1991; Trudel, 1994). Transgenic “knock-outs” (hereinafter referred to as “sickle cell” or “SS” mice) were developed by sequential breeding of mice deficient in α and β globins with transgenic animals expressing both human a and βs globins; such SS mice are genetically identified as Tg(Hu-miniLCRα1GγAγδβS)mα−/−β−/−. These animals display a drastic phenotype characterized by severe anemia with high reticulocyte counts, splenomegaly and evidence of end-organ damage (Paszty, 1997; Ryan, 1997). Although the hematological and histological pictures in SS mice resemble that of patients, the phenotype in mice is more severe and their viability is reduced. When a male SS mouse is bred with a mouse heterozygous for β-globin expression (Tg(Hu-miniLCRα1GγAγδβS)mα−/−β−/+), less than 10% of the offspring exclusively express human globins, instead of the expected 50%. The reduced viability of SS mice has hampered the progression of in vivo studies and the development of useful models to evaluate the mechanisms of vaso-occlusion.
It had been noted, prior to the present invention, that sickle cell patients with leukocyte counts greater than 15,000/microliter have an increased risk of death (Platt, 1994), that lower neutrophil counts were associated with a lower crises rate in sickle cell patients treated with hydroxyurea (Churache, 1996) and that treatment with granulocyte colony stimulating factor (“G-CSF”, which increases leukocyte counts) induced a sickle cell crisis (Abboud, 1998). Schwartz, 1985, reported increased adherence of sickle RBCs to cultured peripheral blood monocytes in vitro, wherein irreversibly sickled RBCs and deoxygenated RBCs were most adherent and adhesion appeared to correlate with the exposure of phosphatidylserine to the outer membrane leaflet. Hofstra et al., 1996, reported that, in vitro, SS-RBCs can bind activated neutrophils in a static in vivo adhesion assay, an interaction which was more pronounced in the presence of autologous sickle cell plasma. Binding of SS-RBCs to activated neutrophils was partially inhibited by RGDS peptides and human IgG, suggesting than one or more integrin(s) and neutrophil Fc receptors may be involved. SS-RBC adhesion also induced an oxidative burst characterized by the production of free radicals by activated neutrophils (Id.) Further, it had been noted that anti-inflammatory agents such as methylprednisolone may be effective in decreasing the duration of sickle cell crisis episodes (Griffin, 1994). A recent study using a sickle cell mouse model indicated that the inflammatory response (number of adherent and emigrated leukocytes and oxidant production) resulting from hypoxia and reoxygenation was increased in sickle cell transgenic mice compared to control animals (Kaul, 2000).
Prior to the present invention, however, it had not been appreciated which of the many potential aspects of the inflammatory response was directly associated with vaso-occlusion.
The present invention relates to methods of treating sickle cell disease comprising reducing, in a subject in need of such treatment, the adherence between sickled RBCs and leukocytes. It is based, at least in part, on the discovery that leukocytes play a direct role in the initiation of venular occlusion. The present invention further provides for methods for identifying agents which decrease SS-RBC/leukocyte adherence and for animal models which may be used to further elucidate the mechanism of vaso-occlusion in sickle cell crises.
For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections:
- (a) methods of treating sickle cell disease;
- (b) methods of identifying agents useful in treating sickle cell disease; and
- (c) animal model systems.
The present invention provides for methods of treating sickle cell disease in which venular occlusion by sickle erythrocytes (“SS-RBCs”, which contain HbS and may be in the sickled or in a discoid conformation) adherent to leukocytes is decreased. The phrase “method of treating” sickle cell disease is used herein to indicate decreasing the occurrence and/or severity of any one or more of the following signs and symptoms: pain, anemia, infection, stroke, tissue damage, visual impairment, bone infarction, jaundice, and gall stones, and the manifestations of “sickle cell crisis”.
The methods of the present invention may intervene in the process by which SS-RBC adhere to leukocytes and initiate venular occlusion at the point where a SS-RBC adheres to a leukocyte and/or the point at which a leukocyte and/or the SS-RBC/leukocyte complex binds to the venule endothelium. Such methods may be directed at the cellular level (for example, decreasing the number of leukocytes) or may be directed at the molecular interactions between the SS-RBC and leukocyte or between the leukocyte or the SS-RBC/leukocyte complex and the endothelial cell.
The recruitment of leukocytes into inflamed tissue has been well characterized at the molecular level. It is now recognized that leukocyte extravasation represents a multi-step process initiated by leukocyte tethering and rolling along the vessel wall of post-capillary venules. The tethering and rolling steps are largely mediated by selectins and their ligands. Rolling on selectins and their ligands allows leukocytes to interact with chemokines on the surface of the activated endothelium. These chemokines may activate the leukocyte and change the conformation of β2 integrins into a high-affinity state, allowing firm adhesion and subsequent diapedesis via the interactions of integrins and immunoglobulin superfamily members (reviewed in Springer, 1995; Frenette, 1996; Vestweber, 1999). The selectin family consists of three members containing a functional calcium-binding lectin domain. Two selectins are expressed by endothelial cells (P- and E-selectins) and one is found on most leukocytes (L-selectin) (Kansas, 1996). Genetic analyses using knockout experiments have shown distinct functions for each selectin (Frenette, 1997; Robinson et al., 1999). While mice lacking a single selectin gene have mildly aberrant phenotypes, animals deficient in both endothelial selectins (P/E−/−) show virtually no leukocyte rolling even after cytokine-induced (tumor necrosis factor alpha; “TNF-α”) inflammation (Frenette et al., 1996; Bullard et al., 1996). The profound defect in leukocyte adhesion and extravasation in P/E−/− mice, reminiscent of mice lacking all β2 integrins, such as mice which are CD18−/− (Wilson et al., 1993; Scharffetter-Kochanek et al., 1998) suggested that overlapping function of the two endothelial selectins is as important for leukocyte adhesion in vivo as are β2 integrins. In addition to four β2 integrins (αLβ2(LFA-1), αMβ2 (Mac-1), αχβ2 and αDβ2) leukocytes express other integrins such as αVβ3 and β1 (on lymphocytes and monocytes but not neutrophils; Carlos, 1994).
Accordingly, the present invention provides for methods of decreasing vaso-occlusion associated with sickle cell disease by inhibiting SS-RBC/leukocyte/endothelial adhesion along any one or several steps in the adhesion process.
Such methods may, for example, but not by way of limitation, inhibit the binding between leukocytes and endothelial P- and/or E-selectin or the binding of leukocyte L-selectin to the endothelium. Such binding may be inhibited, for example, using an immunoglobulin specific for a selectin molecule, such as a P-, E-, and/or L-selectin molecule, or a fragment or derivative of such immunoglobulin. Alternatively, such binding may be inhibited using a non-inmunoglobulin molecule which interacts with the calcium-binding lectin domain of the selectin molecule, including molecules which interfere with calcium binding to the site.
In other non-limiting embodiments, vaso-occlusion in a sickle cell patient may be decreased by inhibiting the interaction of leukocytes or RBC/leukocyte complexes with cytokines on the surface of activated endothelium. As a non-limiting specific example, an agent which inhibits TNF-α, such as an anti-TNF immunoglobulin, fragment or derivative thereof, may be administered.
In further non-limiting embodiments, vaso-occlusion in a sickle cell patient may be decreased by inhibiting the binding between one or more elements selected from the group consisting of leukocytes, SS-RBC/leukocyte complexes, and endothelial cells, via a β2 integrin molecule. Thus, binding (i) among leukocytes, or (ii) among SS-RBC/leukocyte complexes, or (iii) between a SS-RBC/leukocyte complex and a leukocyte, or (iv) between an endothelial cell and a SS-RBC/leukocyte complex, or (v) between an endothelial cell and a leukocyte, may be inhibited, for example, by an agent which interferes with binding of a β2 integrin molecule, where a β2 integrin molecule participates,directly or indirectly, in the binding between partners. For example, a leukocyte may be bound to another leukocyte indirectly by binding to an endothelial cell, and an endothelial cell may be bound to another endothelial cell indirectly via a plurality of adherent SS-RBC/leukocyte complexes.
In specific non-limiting examples, binding to αLβ2(LFA-1), αMβ2 (Mac-1), αχβ2, and/or αDβ2 may be inhibited. Such inhibition may be achieved, for example, using an immunoglobulin molecule, or a fragment or derivative thereof, which specifically binds to the integrin.
In related embodiments, vaso-occlusion in a sickle cell patient may be decreased by inhibiting the change in the conformation of β2 integrins into a high-affinity state. Such inhibition may be effected by an immunoglobulin molecule, fragment or derivative thereof or by a small non-immunoglobulin molecule.
In additional non-limiting embodiments, vaso-occlusion in a sickle cell patient may be decreased by inhibiting binding among or between elements selected from the group consisting of an endothelial cell, a platelet, a leukocyte, and a SS-RBC/leukocyte complex by inhibiting binding via a β3 integrin, for example, αIIbβ3 or αVβ3 integrin. By inhibiting binding via a β3 integrin, binding between an endothelial cell and either a leukocyte, or a SS-RBC/leukocyte complex, or a platelet, or a SS-RBC/leukocyte/platelet complex, or a platelet/SS-RBC complex, may be inhibited. Such inhibition may be achieved, for example, using an immunoglobulin molecule or a fragment or derivative thereof which binds to a β3 integrin. Non-limiting examples of antibodies which bind to αVβ3 integrin include the murine monoclonal antibody 7E3 (deposited with the American Type Culture Collection at ATCC HB 8832), the humanized chimeric equivalent of 7E3, c7E3, the Fab fragment of c7E3 (which is sold commercially as ReoPro®), and the monoclonal antibody LM609 and chimeric equivalents. 7E3, c7E3, and Fab 7E3 also bind to αIIbβ3. Where c7E3 or ReoPro is used, the dosage may be, in specific non-limiting embodiments, between 0.1-0.3 mg/kg, and preferably 0.25 mg/kg. Preferably, after 0.25 mg/kg is administered, the patient may further receive intravenous infusion of 0.125 m/kg/min for a therapeutically effective period of time.
In further non-limiting embodiments, vaso-occlusion in a sickle cell patient may be decreased by inhibiting binding between one or more elements selected from the group consisting of leukocytes, SS-RBC/leukocyte complexes, and endothehal cells, via β1 integrins. Such inhibition may be achieved using an immunoglobulin molecule, or a fragment or derivative thereof, which specifically binds to the β1 integrin.
In further non-limiting embodiments, vaso-occlusion in a sickle cell patient may be decreased by inhibiting the binding of leukocytes or SS-RBC/leukocyte complexes to von Willebrand factor (vWf). Such inhibition may be achieved using an immunoglobulin molecule, or a fragment or derivative thereof, which specifically binds to the vWf
In further non-limiting embodiments, vaso-occlusion in a sickle cell patient may be decreased by inhibiting the binding of leukocytes or SS-RBC/leukocyte complexes to thrombospondin. Such inhibition may be achieved using an immunoglobulin molecule, or a fragment or derivative thereof, which specifically binds to the thrombospondin.
In further non-limiting embodiments, vaso-occlusion in a sickle cell patient may be decreased by inhibiting the binding of leukocytes or RBC/leukocyte complexes to a molecule, such as, but not limited to, ICAM-1, VCAM-1, or their ligands CD18 and α4β1. Such inhibition may be achieved using an immunoglobulin molecule, or a fragment or derivative thereof, which specifically binds to the endothelial adhesion molecule.
The present invention provides for methods of identifying agents useful in treating sickle cell disease which comprise determining whether a test agent is able to modulate the adhesion of SS-RBC to leukocytes and thereby to venular endothelium. Such methods may be practiced in vitro or in vivo. Examples of in vitro studies may include assays which test for SS-RBC/leukocyte binding by, for example, co-precipitation or co-sedimentation, or by retention on a solid matrix.
Alternatively, the effectiveness of the test agent at inhibiting adhesion may be evaluated in vivo. For example, but not by way of limitation, the test agent may be evaluated using intravital microscopy, using techniques as set forth in Example Section 6 below.
The ability of a test agent to inhibit the binding of a SS-RBC to a leukocyte, and/or inhibit the binding of a SS-RBC/leukocyte complex or a leukocyte to an endothelium or to endothelial cells, indicates that the test agent may be useful in the treatment of sickle cell disease. In certain although not all circumstances, it may be desirable to determine that the test agent selectively blocks adhesion of sickled rather than non-sickled erythrocytes; in such circumstances, the amount of available oxygen may be decreased or increased to maximize or minimize, respectively, the formation of SS-RBC.
Because many of the molecules involved in the adhesion pathway are important to normal biological function, it may be desirable to select for agents which have a short half life for administration during sickle cell crises, or which change conformation and become more active at lower oxygen tensions.
The present invention further provides for animal model systems which are designed to lack one or more element of the adhesion pathway, including, for example, those elements set forth in Section 5.1, supra. Such animals may be transgenic animals, including, but not limited to, transgenic mice, lacking or, alternatively, overexpressing a gene encoding a protein selected from the group consisting of a selectin, such as P-, E- or L-selectin; a chemokine, such as TNF-α; a β2 integrin, such as αLβ2(LFA-1), αMβ2 (Mac-1), αχβ2, and αDβ2; a β3 integrin, for example, αVβ3; a β1 integrin; vWf, thombospondin, ICAM-1, VCAM-1, CD18 and α4β1.
Materials and Methods. Sickle cell breeding pairs were obtained from Dr. Mohandas Narla at the Lawrence Berkeley Institute, and were maintained according to Dr. Narla'"'"'s instructions. “Heterozygotes”, referred to herein as “SA” mice, express the sickle transgene, are deficient in a globin and heterozygous for the β-globin locus, and are genetically Tg(Hu-miniLCRα1GγAγδβS)mα−/−β−/+. Female SA mice were bred with male sickle cell mice, which express exclusively human globins, and are genetically Tg(Hu-miniLCRα1GγAγδβS)mα−/−β−/−. After the progeny from these breedings were weaned, a drop of blood was obtained from a tail biopsy to permit phenotyping by hemoglobin electrophoresis. To generate large numbers of male sickle cell mice, a bone marrow transplantation strategy was used which aimed at reconstituting the entire blood compartment of several recipient mice from precursors obtained from a single sickle cell mouse. Fresh femoral bone marrow cells were obtained from one female sickle cell and one “heterozygous” control mouse (derived from the same genetic background as the sickle animals; Paszty, 1997). Wild-type male C57B1/6 recipient mice were lethally irradiated with 1200cGy, in two split doses, and injected, under a sterile hood, with bone marrow nucleated cells from SS or SA animals, at a dose of 1.5×106 cells per recipient. Following the procedure, transplanted animals were transferred into a sterile cage containing sterile food and water (see Frenette, 1998, Frenette, 2000). Since the life-span of the normal mouse RBC is approximately 55 days (Hoffman-Fezer, 1993), mice were allowed to recover for at least two months prior to evaluation for engraftment and chimerism.
Between 8 and 12 weeks after transplantation, blood was obtained from a small tail incision and hemoglobin was separated on a polyacrylamide gel containing urea and Triton-X-100 (Alter et al., 1980); the results are shown in
The cremasteric microcirculation of the highly chimeric animals was evaluated using intravital microscopy. The surgical preparation of the cremaster muscle itself induces inflammatory stimuli leading to leukocyte rolling and progressive recruitment of adherent leukocytes. More severe inflammation may be induced by administering TNF-α, an inflammatory cytokine which induces P- and E-selectin-mediated leukocyte rolling (Frenette, 1996; Bullard, 1996). Because inflammation is clinically known to trigger sickle cell crises, chimeric mice were treated with murine recombinant TNF-α (0.5 micrograms intrascrotally) 3.5 hours prior to preparing the cremaster muscle for intravital microscopy (Frenette, 1996; Frenette, 1998; Ley, 1995; Bullard, 1996). While treatment with TNF-α was tolerated well in SA-WT controls, SS-WT mice died during or soon after surgery. However, 55 transplants survived the surgery when not pre-treated with the cytokine or when treated with a half dose of TNF-α (however, the half-dose did not produce meaningful inflammation, as assessed by the lack of leukocyte rolling velocities). The following protocol was therefore designed to induce a progressive inflammatory response in SS and SA transplants (
As depicted diagrammatically in
Results and Conclusions. Vaso-occlusion is a major cause of morbidity and mortality in sickle cell disease. To better understand the pathophysiology of vaso-occlusion in vivo, intravital microscopy was performed in (1) C57B1/6 wild-type (“WT”) mice; (2) mice exclusively expressing sickle cell hemoglobin (“SS”; [Tg(Hu-miniLCRα1GγAγδβS)mα−/−β−/−]); and (3) lethally irradiated WT mice transplanted with bone marrow from either SS mice or mice heterozygous for sickle hemoglobin (“SA”;human βS/mouse β[Tg(Hu-miniLCRα1GγA65 δβS)mα−/−β−/+].
In the transplant recipients, three months after transplantation, SS bone marrow recipients had >96% donor hemoglobin and displayed severe anemia (hematocrit 21±3%; n=10, p<0.05), high reticulocyte counts and splenomegaly, by comparison, heterozygous bone marrow recipients had nearly normal hematocrits (32±3%; n=7) and only a slight increase in spleen weight (ratio: 5±1).
The cremasteric muscle of the mice was then surgically dissected, and “small” (15-20 μm) and “large” (30-50 μm) venules were visualized between 15-90 minutes after surgery and after treatment with Tumor Necrosis Factor cc (“TNF-α”; 0.5 μg/mouse). The surgery itself produced an inflammatory response leading to leukocyte adhesion, and this response was accentuated by TNF-α treatment.
Although occasionally direct interaction between SS-RBCs and the vasculature was observed, the most striking finding was that numerous SS-RBCs interacted with adherent leukocytes in venules activated by surgery alone and these interactions were increased after TNF α administration. On average, 17±5 SS-RBCs interacted with adherent leukocytes per minute over 100 μm venular length in SS-BMT mice (n=34 venules in 5 mice) (
After TNF-α, RBC/leukocyte interactions increased (or persisted) in SS-WT mice (but were not increased in SA-WT animals) and lead to a progressive reduction in blood flow in the cremaster microvasculature.
These observations suggest a critical role for SS-RBC/leukocyte interactions in initiating vaso-occlusive episodes in sickle cell mice. They are in accord with the documented correlation between low leukocyte counts and reduced painful crises in hydroxyurea-treated patients as well as in in vitro studies of SS-RBC/leukocyte interactions by Hofstra et al.
To further evaluate the role of adherent leukocytes in sickle cell disease, bone marrow from mice exclusively expressing sickle cell hemoglobin (“SS”;[Tg(Hu-miniLCRα1GγSγδβS)mα−/−β−/−]) was transplanted into mice lacking both P- and E-selectins (P/E−/−). P/E−/− mice have severe defects in leukocyte rolling and adhesion in inflamed venules. Experimental data (
Consistent with reduced numbers of adherent leukocytes in SS-P/E −/− mice, the total number of erythrocyte/leukocyte interactions was significantly reduced in P/E−/− mice harboring SS-RBCs (0.4±0.3/min/100 μm; n=23 in 3 mice; p=0.01). It is interesting to note that the remaining adherent leukocytes present in endothelial selectin-deficient venules could still interact with SS-RBCs, suggesting that P/E selectins are not necessary for SS-RBC/leukocyte interactions.
Unlike SS-WT mice which for the most part died during the intravital experiment, all SS-P/E−/− mice survived the entire experiment. These results strongly support a role for adherent leukocytes in initiating vasoconstriction by interacting with circulating sickle erythrocytes, and indicate that P- and E-selectin deficiencies protect SS mice from vaso-occlusion. Moreover, the data suggest that the absolute number of interacting leukocytes in a given venule, rather than the rate of interactions per leukocyte, appear to be a critical factor in venular occlusion.
Determination of blood counts and assessment of spleen weight/body weight ratios among various transplanted and non-transplanted groups revealed several abnormalities. The preliminary blood counts were done after TNF-α treatment, except 4 SS bone marrow donor mice (Table 1, 4th row), which were performed at baseline conditions. In addition to being severely anemic, these resting SS mice exhibited severe leukocytosis, in contrast to WT or SA mice which showed a mild leukocytosis after TNF-α (normal WT WBC counts are ˜3 to 5×103/μl). WBCs were lower after TNF-α administration in SS mice, possibly resulting from increased adhesion to the vessel wall during inflammation. Both SS-WT and SA-WT chimeras displayed blood counts similar to their non-transplanted donor counterparts, suggesting that this transplantation model reproduced very well the phenotype of sickle cell mice.
Leukocytosis was also more severe in P/E−/− mice expressing SS hemoglobin. Interestingly, the blood from TNF-α treated SS mice (and from the chimeras generated by transplantation) contained much fewer platelets suggesting platelet consumption during the vaso-occlusive process. Although this might suggest a role for platelets in vaso-occlusion, the fact that a similar reduction in platelet numbers is seen in SS⇒P/E−/− mice (and that SS⇒P/E−/− mice are protected) argues that platelets may not be necessary for vaso-occlusion. The lower spleen weight in transplanted mice compared to their non-transplanted controls likely results from the fact that transplanted animals have had sickle cell disease for only a few weeks.
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Various publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.