WO2017059427A1

WO2017059427A1 - Methods for monitoring and determining the prognosis of strokes, peripheral vascular disease, shock, and sickle cell disease and its complications

Info

Publication number: WO2017059427A1
Application number: PCT/US2016/055171
Authority: WO
Inventors: Zenaide M.n. QUEZADO; Luis E. ALMEIDA; Nicholas A. SPORNICK
Original assignee: Children's National Medical Center
Priority date: 2015-10-02
Filing date: 2016-10-03
Publication date: 2017-04-06

Abstract

This invention provides a method for the diagnosis and monitormg of Sickle Cell Disease and other conditions that involve the occlusion of blood vessels by detecting alterations in the level of activated caspases, especially a pi 7 fragment of caspase 3 in blood plasma. Methods for treating subjects identified as having elevated pi 7 levels are also described.

Description

TITLE

METHODS FOR MONITORING AND DETERMINING THE PROGNOSIS OF STROKES, PERIPHERAL VASCULAR. DISEASE, SHOCK, AND SICKLE CELL

DISEASE AND ITS COMPLICATIONS

Cross-reference to Related Application. This application claims priority, including priority under 35 U.S.C. § 1 19(e), to U.S. Provisional Application No. 62/236,400, filed October 2, 2015, the disclosure of which is hereby incorporated by reference in its entirety.

Field of the Invention. The present invention relates to the fields of diagnosis and therapy of Sickle Cell Disease and vascular occlusive diseases by detection of the level of caspase 3 activation, especially the pl7 fragment of caspase 3, a cysteine-aspartic protease.

Description of the Related Art.

Sickle cell disease (SCD) is the most common inherited hematologic disorder and is caused by a single-base mutation in the sixth codon for the β-globin chain, which generates the mutant sickle hemoglobin¹'².

Upon deoxygenation, sickle hemoglobin polymerizes, which results in rigidity and distortion of red cell morphology into a characteristic sickle shape. Polymerization of sickle hemoglobin and sickling of red blood ceils is associated with various biochemical and pathological events including hemolysis, increased expression of inflammatory mediators, endothelial dysfunction, and obstruction of blood flow. Consequently, recurrent tissue damage due to hypoxia, ischemia, and reperfusion injury will occur³'⁴.

Clinical ly, patients with SCD can have a spectrum of manifestations that includes chronic hemolysis, anemia, recurrent painful vaso-occlusive episodes, and varying degrees of dysfunction of almost every major organ. Consequently, over time, SCD patients will develop significant co-morbidities including strokes, acute chest syndrome, recurrent vaso- occlusive episodes, pulmonary hypertension, kidney failure, bone infarcts, which will inevitably lead to reduction in life expectancy.

There is evidence to suggest that complications of SCD and its associated chronic inflammatory state are related to recurrent ischemia, resulting from vaso-occiusion, and reperfusion injury ⁴. Researchers suggest that endothelial dysfunction, acute chest syndrome, arterial vascuiopathy, and pain associated with inflammatory mediators derived from vaso- occlusive phenomenon are examples of SCD-associated ischemia and reperfusion injury ⁴.

While it is well established that tissue hypoxia secondary to vaso-occlusion and anemia may result in necrosis and ongoing inflammation, the potential role of hypoxia stimulated apoptosis, or programmed cell death, remains largely unknown. Apoptosis is a normally occurring phenomenon, which is very important during normal development. In some instances, apoptosis is protective, such as when it prevents potentially malignant cells from becoming established. However, in some conditions apoptosis can accentuate tissue damage and result in organ dysfunction. One example where apoptosis appear to play a role in organ damage and can accentuate tissue damage is during myocardial infarction.

Researchers have shown that during myocardial infarction, both necrosis and apoptosis occur and in fact, apoptosis significantly contributes to the size of cardiac muscle damage in a setting of myocardial infarct ⁵.

Once apoptosis ensues, it leads to shrinkage and self-phagocytosis of cells without significant inflammation in the surrounding tissue. Two main pathways lead to the induction of apoptosis. The extrinsic pathway, which can be activated by several ligands (tumor necrosis factor and CD95L) of the various cell death-receptors, leads to activation of caspase 8⁶. The second, the intrinsic pathway is activated by a number of stimuli including reactive oxygen species (which are extensively generated during ischemia reperfusion injury), radiation and chemotherapeutic compounds, which in turn lead to mitochondria-mediated apoptosome formation and activation of caspase 9 ⁶. Activation of both the extrinsic (caspase 8) and intrinsic (caspase 9) pathways are followed by activation of caspase 3, a key effector caspase, and ultimately to destruction of the cell. Given that inflammatory caspases (caspase 1, caspase 4, caspase 5, caspase 1 1-14), apoptosis initiator caspases (caspase 2, caspase 8, caspase 9 and caspase 10), and apoptosis effector caspases (caspase 3, caspase 6, and caspase 7) are key elements of the apoptosis pathway, they are potential targets for pharmacological therapy for conditions associated with increased activation of the apoptosis pathway.

Caspase structure, activation, substrates, and functions, including apoptosis, are described by Nicholson, Cell Death Differentiation 6:0128-1042 (1999) and by Eamshaw, et ai., Annu. Rev. Biochem. 68:383-424 (1999), and by Rotonda, et ai., Nature Struc, Biol. 3(7):619-625 (1996); each of which are incorporated by reference. A representative amino acid sequence for Caspase 3 is shown in Fig. 6 and is incorporated by reference to

http://_www.nextprot.org/db/entry/NX_P42574/sequence (last accessed September 15, 2016). The shaded portion of this sequence denotes the residues of the pi 7 sequence.

Caspase 3, a cysteine-aspartic acid protease, is a pivotal enzyme for the execution of the cell apoptosis pathway, it is one of the effector caspases⁷. Caspase 3 exists as an inactive enzyme, procaspase 3, which can be activated by various processes including ischemia. When activated, caspase 3 consists of a heterotetramer composed of two heterodimers, subunits pi 7 and pi 2. Once activated, caspases, including caspase 3, an effector caspase, are strictly regulated by a group of proteins called inhibitors of apoptosis. These intracellular proteins are naturally occurring, evolutionarily conserved, and directly inhibit caspase 3 activity by binding its catalytic active site⁸. Prior attempts to correlate disease states with caspase 3 activity have been unsuccessful because strict biological regulation makes it difficult if not impossible to detect activated caspase 3 in blood or blood components in humans.

Activated caspase 3 is a heterodimer formed of two dimers each comprising a pl2 and pi 7 monomer. The pi 2 and pi 7 monomers are formed by proteolytic cleavage of procaspase 3. The pi 7 has been detected in the plasma of subjects during myocardial infarction⁹'¹⁰. While pl7 levels were tentatively coincident with cardiac myocyte apoptosis and

hypothesized to increase by escape from apoptotic cells⁹, pi 7 levels have not been previously correlated with Sickle Cell disease or Sickle Cell trait, both of which have a pathobiologv that is distinct from that of myocardial infarction.

The pathobiology of vaso-occlusion in sickle cell disease is very different than that taking place in ST segment elevation myocardial infarction (STEMI). In sickle cell disease, vaso-occlusion results from break down of sickling red blood ceils, activation of adhesion molecules, and endothelial dysfunction. In contrast, acute STEMI myocardial infarction (heart attack) results from coronary atherosclerotic plaque rupture with formation of a superimposed thrombus, which in turn blocks the blood supply to the heart muscle initiating death of virtually all the heart muscle supplied by the affected artery.

BRIEF SUMMARY OF THE INVENTION

The invention encompasses a method for diagnosing, prognosing, or monitoring Sickle Cell Disease or a disease, disorder, or condition associated with occlusion of blood vessels by detecting the level of a pi 7 fragment of caspase 3 in blood plasma or another biological sample from a subject. It further involves selecting and treating subjects having elevated pi 7 levels for such a condition.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Homozygous sickle cell mice have significant increases in caspase 3 activity in plasma. Caspase 3 activity levels were measured using a fluorimetric assay (Sigma- Aldrich, St Louis, MO, USA) according to the manufacturer protocol. Control: control mice. Hetero: Mice heterozygous for sickle hemoglobin and model sickle cell trait, which is seen in humans. Homo: Mice homozygous for sickle cell hemoglobin, which mirror human sickle cell disease

FIG. 2A and 2B. Sickle cell mice from two strains, Townes (FIG. 2A) and BERKs (FIG. 2B) have increased caspase 3 activity in kidney medulla. Caspase 3 activity levels were measured using a fluorimetric assay (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer protocol. Control/^'C57: control mice. Hetero: Mice heterozygous for sickle hemoglobin and model sickle cell trait, which is seen in humans. Homo: Mice homozygous for sickle cell hemoglobin, which mirror human sickle cell disease

FIG. 3A and 3B. Sickle ceil mice from two strains, Townes (FIG. 3A) and BERKs (FIG. 3B) have increased caspase 3 activity in the liver. Caspase 3 activity levels were measured using a fluorimetric (Sigma-Aidrich, St Louis, MO, USA) according to the manufacturer protocol. Control/C57: control mice. Hetero: Mice heterozygous for sickle hemoglobin and model sickle cell trait, which is seen in humans. Homo: Mice homozygous for sickle cell hemoglobin, which mirror human sickle cell disease

FIG. 4A and 4B. Sickle cell mice from the Townes strain have elevated caspase 3 activity in the heart (FIG. 4A) and lung (FIG. 4B). Caspase 3 activity levels were measured using a fluorimetric assay (Sigma-Aldrich, St Louis, MO, USA) according to the

manufacturer's protocol. Control : control mice. Hetero: Mice heterozygous for sickle hemoglobin and model sickle cell trait, which is seen in humans. Homo: Mice homozygous for sickle cell hemoglobin, which mirror human sickle cell disease

FIG. 5. During baseline conditions, sickle cell disease patients have elevated levels of pi 7, a proteolytic fragment of caspase 3, in plasma. These findings show that there is increase in caspase activity in patients with sickle cell disease. PI 7 levels were measured using a sandwich ELISA kit from Cell Signaling (Danvers, MA, USA) according to the manufacturer's protocol.

FIG. 6A and 6B. Caspase 3 activity was assayed in stored blood transfusion bags (n ^:= 8) after 2 days with vehicle (AS3) or 30 μΜ nitrite (diluted in AS3). Protease inhibitors were included. FIG. 6A: RBC Caspase 3 activity compared between samples with vehicle and samples with vehicle + 30 μΜ nitrite. FIG. 6B: RBC Caspase 3 activity assayed after 40 to 180 days. As shown by FIG. 6B Caspase 3 activity in red blood cells that have been stored for longer than 40 days was significantly increased compared to blood stored for less than two weeks

FIG. 7A and 7B. FIG. 7A: Caspase 3 and pl7 fragment amino acid sequence. The underlined portion denotes the pi 7 fragment.

Available at: http://_www.nextprot.org/db/entry/NX_P42574/sequence (last accessed September 30, 2016).

FIG. 7B. Caspase 3 pi 7 fragment.

DETAILED DESCRIPTION OF THE INVENTION

The following examples are offered by way of illustration, and not by way of limitation.

The inventors have shown that in mice with sickle cell disease there is evidence of increased activation of the apoptosis pathway.

Example 1. Elevation of Caspase 3 Activity in Plasma

The inventors showed that caspase 3 activity is elevated in the plasma (FIG. 1 ). Example 2. Caspase 3 Activation in Kidney

In addition, the inventors found in two strains of sickle cell mice that in the kidneys, there is increased activation of caspase 3 in the medulla (FIG. 2).

Example 3. Increased Caspase 3 level in Liver in Sickle Cell Mice

The inventors also found that in two strains of sickle cell mice that there is increased activation of caspase 3 in the liver (FIG. 3).

Example 4. Increased Caspase 3 level in Heart and Lungs of Sickle Cell Mice There is increased activation of caspase 3 in the heart and lungs (FIG. 4).

Example 5, Increased level of Caspase 3 pi 7 Fragment in Human Sickle Cell Patients Levels of caspase 3 activation in the plasma of sickle cell patients are not measurable. Caspase 3 activity cannot be measured in human plasma given that active caspase 3 is quickly degraded. The inventors instead, determined the presence of pi 7 fragment, a surrogate marker for activated caspase 3, The inventor found that, i 7, the proteolytic fragment of activated caspase, was present in plasma and was significantly higher in the plasma of patients with sickle cell disease compared to control subjects (FIG. 5).

Example 6. Increased Caspase 3 activity in red blood cells, which had been stored for over 40 days compared to recently collected (2 day old) red blood cells (FIG. 6). Patients with sickle cell disease often require transfusion of blood products. For instance, in patients with sickle cell who present with brain strokes, the risk of stroke recurrence is significantly reduced by the use of chronic blood transfusion (Helton et al. 2014)¹². In addition to sickle cell patients, trauma victims, patients who undergo surgical procedures, patients with malignancies who undergo anti-cancer treatment, often need blood transfusion. The transfusion of red blood ceils can indeed be a needed life-saving procedure and it is performed to ensure delivery of oxygen to tissues and to maintain stable hemodynamics and replace lost blood. However, when patients receive massive blood transfusion, a number of complications may occur including coagulopathy, hemolysis, and transfusion-related lung injury. Caspase activity in stored blood bags was measured and showed that after 40 days of blood storage, there is significantly increased caspase activation compared with red blood cells that have been recently col lected. This aspect of the invention refers to a method to identify the degree of caspase activation in stored blood transfusion bags, which, if elevated, could be associated with increased complications after blood transfusion of said transfusion bags.

In the United States, nearly 14 million units of whole blood and packed red blood cells are transfused each year. While there is no substitute for blood transfusion in the setting of massive hemorrhage or anemia, the transfusion of blood components has been associated with a number of complications (Koch et al. 2008)¹⁴. These complications range from transfusion reactions, hemolysis, organ injury, to increased mortality (Koch et al. 2008)ⁱ⁴. Further, there is clinical evidence to suggest that the transfusion of old blood (stored for over two weeks) to patients undergoing cardiac surgery, increases the rate of in-hospital mortality, need for mechanical ventilation, the incidence of renal failure and of sepsis and septicemia (Koch et al. 2008)¹⁴. Many believe that these complications, are at least partially due to changes in the blood products, which take place during blood storage. After collection of blood from donors, red cells are refrigerated and preserved in special bags until they are transfused or discarded after certain periods of time. During storage, red blood cells undergo a series of changes that are associated with what is called "storage lesions" (Koch et al.

2013)ⁱ³. Storage lesions results from changes to red cell cellular membrane that results in decreases in survivability and increases in blood cellular components breakdown. In addition, there is accumulation of cytokines, lipids, enzymes, and active caspase as we have recently shown. Therefore, in order to minimize transfusion-related complications, clinicians prefer to transfuse blood products that were stored for shorter periods of time. In addition, while transfusion of younger blood is preferred as indicated in clinical sUidies, the supply of blood available for transfusion is l imited as it depends on voluntary blood donations. Currently, blood units that are older than 40 days are discarded, which results in a waste of blood products. Therefore, a method that allows for improvement in storage methods that can decrease storage lesions by inactivating or removing active caspases, will greatly increase blood availability.

While not being bound to any particular theory or mechanism of action, the inventors suspect that pi 7 levels are elevated due to activation of an apoptosis pathway involving caspase 3 activation in subjects having sickle cell disease. Such an elevation may be due to organ dysfunction associated with sickle ceil disease, endothelial damage, or some other pathological feaUire of sickle cell disease. The pathology of occlusive diseases and disorders, such as strokes, cerebrovascular accidents, ischemia, and reperfusion injuries, involves the occlusion of blood vessels and ischemia of ceils distal to the distribution of the occluded blood vessel . Occlusions, which trigger activation of an apoptosis pathway involving caspase 3, would result in degradation of activated caspase 3 and elevation of pi 7 levels. Thus the pathophysiology and assessment of these conditions, including hemodynamic shock due to hypovolemia, sepsis, toxins, or overwhelming infection would be indicated by elevated pi 7 levels, which indicates that there has been prior activation of procaspase 3.

Particular embodiments of the invention include methods for diagnosing, monitoring, and/or prognosing occlusive diseases or disorders, including - sickle cell disease and its complications. Such methods may involve qualitative or quantitative detection of caspase 3, or its fragments, such as the pi 7 fragment in biological samples.

Such samples may be obtained from subjects suspected of having such a disorder or disease and analyzed for the presence of caspase 3, its pi 7 fragment or other detectable fragments and compared to a control value from a subject not having such a disorder or disease. Patients having SCD may also be monitored by obtaining one-time or multiple samples at different points in time, such as longitudinal and repeated measurements of the p 17 fragment in a blood, plasma, or serum sample from: patients having sickle ceil disease who are in vaso-occlusive crisis; with sickle cell disease who are developing liver disease; patients with sickle cell disease who are developing renal dysfunction; patients with sickle cell disease who are developing heart dysfunction; patients with sickle cell disease who are developing pulmonary hypertension; patients with sickle cell disease who are developing acute chest syndrome; patients with sickle cell disease who are developing acute strokes; or patients with sickle cell disease who are developing hemolysis.

These one-time, repeated, or longitudinal analytic methods may also be applied to monitor and determine the prognosis of patients with: peripheral vascular disease; patients with peripheral vascular disease who are developing limb ischemia; or patients with peripheral vascular disease who underwent corrective surgery.

These one-time, repeated, or longitudinal analytic methods may also be applied to monitor and determine the prognosis of patients with: at risk of devel oping strokes or transient ischemic attacks; patients developing strokes and receiving thrombolytic therapy; or patients admitted to emergency rooms with the possible diagnosis of stroke. These one-time, repeated, or longitudinal analytic methods may also be applied to monitor and determine the prognosis of patients with: developing or at risk of developing shock, such as hypovolemic, septic, or toxic shock; or patients admitted to emergency rooms or intensive care units with the possible diagnosis of shock.

Patients or other subjects having above normal caspase 3 activity, p l7 or other caspase 3 fragment levels, at thus having or at risk of having or developing an occlusive disease or disorder, may be selected for treatment with a caspase 3 inhibitor. Such a method involves administering to a subject in need of therapy a caspase inhibitor, said administering being effective to decrease caspase activity in the subject thereby treating the subject.

Subjects benefiting from such a method of treatment include those with occlusive diseases, disorders or conditions, including subjects having sickle cell disease, subjects who have had strokes, subjects having a peripheral vascular disease, and subjects in a state of hemodynamic shock.

Other aspects of the invention include methods for evaluating the freshness or suitability of whole blood or other blood products for medical procedures, such as blood transfusion, or for treating subjects having higher than normal levels of activated caspase. Sickle cell disease often requires transfusion of blood products. For instance, in patients with sickle cell who present with brain strokes, the risk of stroke recurrence is significantly reduced by the use of chronic blood transfusion, (Helton, 2014V². In addition to sickle cell patients, trauma victims, patients who undergo surgical procedures, patients with

malignancies who undergo anti-cancer treatment often need blood transfusion.

The transfusion of red blood ceils can indeed be a needed l ife-saving procedure as its primary goal is to ensure delivery of oxygen to body tissues and maintain stable

hemodynamics. However, when patients receive massive blood transfusion, a number of complications may occur including coagulopathy, hemolysis, and transfusion-related lung injury.

The inventors measured caspase activity in stored blood bags and showed that after 40 days of blood storage there is significantly increased level of caspase activation compared with recently collected blood. The higher level of active caspase in stored and transfused blood increases the risk of caspase-activation associated complications. The inventors disclose herein methods for selecting blood for transfusion that decrease the risk of such complications by selecting blood with a lower active caspase level.

Representative, but non-limiting, embodiments of the invention include the following;

A method for diagnosing, prognosing, or monitoring sickle ceil disease or another a disease, disorder, or condition associated with occlusion of blood vessels, comprising detecting the concentration or activity of at least one activated caspase, a subunit thereof, or a proteolytic fragment thereof in a biological sample from a subject and selecting a subject having sickle cell disease or another disease, disorder, or condition associated with occlusion of bl ood vessels, when the concentration or activity of the at least one activated caspase, subunit thereof, a proteolytic fragment thereof, or oligomer of the caspase or its subunits or fragments is elevated in comparison to said level in a control subject who does not have the disease, disorder, or condition associated with occlusion of blood vessels.

Caspase activity, such as Caspase 3 activity, may be measured by methods known in the art or the concentrations of caspase(s), activated caspase(s), caspase subunits, caspase fragments, or oligomers of these caspase products may be determined by methods known in the art. Caspases, including caspase 3 and its fragments, such as the p i 7 fragment, are detected and analyzed using methods commonly used to detect proteins and enzymes or analyze enzymatic reactions. These include, for example, SDS-PAGE, spectroscopy, HPLC analysis, autoradiography, chemiluminescence, ELISA, sandwich ELISA, chromogenic reactions, and immunochemistry (e.g., blotting, precipitating, etc.). One example of such a method is given by Agosto, et al., J Am Coil Cardiol 201 1 ;57:220-1 and is incorporated by reference. Caspase activity, such as caspase 3 activity, may also be measured using a commercial assay, such as that described by Sigma Technical Bulletin for a fluormetric

Caspase 3 Assay Kit which is incorporated by reference (available at

http://www.sigm aaldrich orn/co

last accessed September 30, 2016) or by PathScan^® Cleaved Caspase 3 (Asp 175) Sandwich Eiisa Kit (rev. 10/21/13) (available at http://www.cellsignal.com/products/elisa-kits/cleaved- caspase3-aspl75-sandwich-eiisa-kit/7190; last accessed September 30, 2016) which is incorporated by reference.

This method may be used to detect a disease, disorder, or condition associated with occlusion of blood vessels, such as ischemia, a reperfusion injury, stroke, peripheral vascular disease, hypovolemic or septic shock, sickle cell disease, sickle ceil trait. When the disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease, then the sample may be obtained from a subject developing or having vaso-occlusive crisis, from a subject developing or having liver disease, from a subject developing or having renal dysfunction, from a subject developing or having heart dysfunction, from a subject developing or having pulmonary hypertension, from a subject developing or having acute chest syndrome, from a subject developing or having acute stroke(s), from a subject developing or having hemolysis, from a subject developing or having limb ischemia, from a subject who has undergone corrective surgery, from a subject undergoing thrombolytic therapy, or from a subject suspected of having a stroke who has been admitted to a hospital or emergency room. The same or similar samples may be obtained from subjects not having sickle cell disease or traits, such as from subjects having other diseases, disorders, or conditions associated with occlusion of blood vessels. However, advantageously, subjects having sickle cell disease or subjects heterozygous for sickle cell trait can be treated.

In some embodiments of this method, the concentrations of, or caspase activities of the pi 2 and/or pi 7 fragments of caspase 3 or heterodimers of heterotetramers thereof may be determined. In other embodiments the levels or concentrations of caspase(s), including inactive caspase(s), including caspase 3, compared to those in a normal or control sample or subject may be determined.

Biological samples for this method and other methods disclosed herein include whole blood, diluted whole blood, stored or banked whole blood, plasma, serum, CSF, synovial fluid, lymph, tissue fluid, saliva, mucus, urine, feces, red or white blood cells, platelets or other cellular or non-cellular components of blood, endothelium or other ceils expressing caspase(s), such as caspase 3. In another related embodiment of this method, may further comprise selecting a subject having an elevated concentration of a caspase subunit, caspase fragment or oligomer thereof, or an elevated level of caspase activity in a blood sample, for example, an elevated level of activated caspase 3 or elevated caspase activity attributable to the presence of activated caspase 3 or another caspase, wherein the elevated concentration or activity is determined by reference to a control value, for example, from a normal subject not having a disease, disorder, or condition associated with occlusion of blood vessels. In this

embodiment, the subject may be treated with drugs or other medicaments or therapies for treatment of diseases, disorders, or conditions associated with occlusion of blood vessels.

Treatments include administering an inhibitor of production, activation or activity of caspases such as those inhibiting the activity of caspase 3, its processing or activation by caspases 8, 9 or 10, or by inhibition of its proteolytic activity, or its substrate or target recognition or affinity. Caspase inhibitors and methods for inhibiting caspase activity are known in the art and include those described by Wang, et al., U.S. Patent No. 6,495,522, by Brenchley, et al., U.S. Patent No. 7,960,415, by Mortimer, et al., U.S. 2004/0019017, by Lavrik, et al., J. Clin. Invest. 1 15(10):2665 (2005), by Hotchkiss, et al., Nature Rev. 6:813 (2006), and by Kudelova, et al., J. Physiol. Pharm. 66(4): 473-482 (2015) which are each incorporated by reference. A caspase inhibitor that selectively or preferentially inhibits a particular caspase, such as caspase 3, may be selected for use in conjunction with the invention as disclosed herein, or a less selective or pan-caspase inhibitor may be selected. Such caspase inhibitors may be selected to be reversible or irreversible caspase inhibitors and may comprise compounds mimicking a peptide backbone, including peptidomimetics, and non-peptidic caspase inhibitors including molecule caspase inhibitors. A number of caspase inhibitors have been generated and some have been examined in humans with various inflammatory conditions^{1 1}. These are examples of caspase inhibitors that could be shown effective in ameliorating the vasoocclusive episodes, acute chest syndrome, strokes, and kidney dysfunction in sickle cell disease ^u . These include Z-VAD-FMK, MX 1 122, YVAD- CMK, IDN-6556, VX-166, Boc-Asp-FMK, LEHD-CHQ, Z-LEHD-FMK, M826, VRT- 018858, Q-VD-OPH, TRP601 , IDN-5370, Z-Asp-CMK, Ac-YVAD-CMK, YVAD-CHO, VX-740, M-867, VX-166, Z-DEVD-FMK, and Ac-DMQD-CHO.

A subject to be treated with the methods of the present invention can be one that has been diagnosed with sickle cell disease, for example by detection of activated caspase 3 or its pi 7 fragment in the blood, plasma or serum. Sickle ceil disease is characterized by red blood cells that assume an abnormal rigid sickle shape and is caused by a genetic mutation in the β-globin hemoglobin gene. Sickle cell disease encompasses a group of symptomatic disorders and is generally defined by the presence of hemoglobin S. The genotype of patients with sickle cel l disease is typically HbSS, but other hemoglobin variants can cause symptomatic sickle cell disease, including HbSC, HbSD, HbSE, and sickle beta thalassemia.

Administration of a caspase 3 or other caspase inhibitors to the subject having sickle cell disease can be at any time during the progression of the disease. For example, in some aspects, treatment with the caspase 3 inhibitor will occur while the subject is experiencing a symptom of the disease such as severe pain, acute chest syndrome, acute stroke, or vaso- occlusive episodes.

In other aspects, treatment with the caspase 3 or other caspase inhibitor will be preventative in nature and will be administered to a subject having sickle cell disease prior to experiencing one or more symptoms of the disease. Such subjects may have experienced symptoms in the past but are being treated with the caspase 3 or other caspase inhibitor in order to reduce the severity or incidence of future symptoms of the disease. Accordingly, in some aspects, the caspase 3 or other caspase inhibitors will be administered to the subject while the subject is not experiencing noticeable symptoms of the disease, such as, for example, sickle ceil crisis, whereas, in other aspects, the caspase 3 or other caspase inhibitor will be administered to the subject while the subject is experiencing noticeable symptoms of the disease, such as, for example, sickle ceil crisis.

Suitable caspase 3 and other caspase inhibitors include those that can be safely- administered to a mammal in an amount effective to prevent, reduce the severity of, or treat sickle cell disease, vascular obstruction or vaso-occlusion, and/or inflammation in a human or other mammal in need thereof.

A caspase or caspase 3 inhibitor can be selected for parenteral, topical, oral, nasal (or othenvise inhaled), rectal, or local administration such as by mtracerebroventricular pump, by- aerosol administration, or transdermal iy, for prophylactic and/or therapeutic treatment of one or more of the pathologies/indications described herein (e.g., to mitigate the onset, progression, or severity of Sickle Cell Disease).

In some embodiments the caspase or caspase 3 inhibitor can be formulated as an injectable or for delivery via an implanted catheter, and the like. A pharmaceutical composition containing the caspase 3 or other caspase inhibitor can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectables, implantable sustained-release formulations, lipid complexes, etc.

The caspase or caspase 3 inhibitors of this invention are typically combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the caspase 3 inhibitor. Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protection and uptake enhancers such as l ipids, compositions that reduce the clearance or hydrolysis of the caspase or caspase 3 inhibitor or exeipients or other stabilizers and/or buffers.

Another embodiment of the invention constitutes a method for monitoring or prognosing progression or regression of a disease, disorder, or condition associated with occiusion of blood vessels comprising quantitatively detecting the concentration or activity of caspase 3 or another caspase, a subunit thereof, a proteolytic fragment thereof, or oligomers of caspase products in biological samples over at least two different points in time; selecting a subject whose disease, disorder, or condition associated with occlusion of blood vessels has progressed when caspase product concentration or activity increases between the points in time; and selecting a subject whose disease, disorder, or condition associated with occiusion of biood vessels has regressed when caspase product concentration or activity decrease between the points in time.

Another embodiment of the invention is a method for diagnosing a disease, disorder, or condition associated with occiusion of blood vessels comprising detecting the level of mRNA encoding caspase 3 or another caspase, a subunit thereof, or a proteolytic fragment thereof in a biological sample from a subject, and selecting a subject having a disease, disorder, or condition associated with occlusion of blood vessels when the level of mRNA encoding caspase 3 or another caspase, subunit thereof, or a proteolytic fragment thereof is elevated in comparison to said level in a control subject who does not have the disease, disorder, or condition associated with occlusion of blood vessels.

The invention also is directed to a method for monitoring or prognosing a disease, disorder, or condition associated with occlusion of blood vessels has progressed or regressed comprising quantitatively detecting mRNA encoding caspase 3 or another caspase, subunit thereof, or proteolytic fragment thereof in biological samples over at least two different points in time, selecting a subject whose disease, disorder, or condition associated with occlusion of blood vessels has progressed when the quantity of mRNA encoding caspase 3 or another caspase, a subunit thereof, or a proteolytic fragment thereof has increased between the points in time; and selecting a subject whose disease, disorder, or condition associated with occlusion of blood vessels has regressed when the quantity of mRNA encoding caspase 3 or another caspase, a subunit thereof, or a proteolytic fragment thereof has decreased between the points in time.

In yet another embodiment the invention is directed to a method for identifying a compound or other agent that reduces the severity of a disease, disorder, or condition associated with occlusion of blood vessels by contacting blood, vascular or other somatic cell or cell-line with a test compound, and quantitatively detecting the concentration of, or activity of a caspase 3 or another caspase (e.g., the level of caspase 3 activity in a blood sample), produced by the cell compared to a control cell not contacted with said compound or agent: selecting a test compound that decreases the concentration or activity of caspase 3 or other caspase, compared to a control that has not been contacted with the test compound; or, alternatively, contacting blood or another blood product, vascular or other somatic cell or cell-line with a test compound, and quantitatively detecting mRNA encoding caspase 3 or another caspase, subunit thereof, or proteolytic fragment thereof transcribed or translated by the cell compared to a control cell not contacted with said compound or agent; selecting a test compound that decreases the amount of mRNA encoding caspase 3, subunit thereof, or proteolytic fragment thereof, transcribed or translated by the cell compared to a control that has not been contacted with the test compound. As used herein, the term "caspase" includes the caspase itself as well as subunits or caspase fragments, such as fragments exhibiting measurable caspase activity.

In another embodiment, the invention is directed to a method for reducing the severity of, treating, or preventing sickle cell disease or at least one symptom or sign thereof comprising administering an inhibitor of caspase 3 or another caspase to a subject in need thereof. Such an inhibitor may inhibit caspase 3 activity such as an inhibitor from the group consisting of VX-740, VX-765, GS-9450, and ZIDN-6556.

Another embodiment of the invention is a method for selecting a suitable blood product to be transfused or otherwise administered to a subject comprising detecting the activation level of at least one caspase in a blood product and selecting a blood product for transfusion or administration to a subject that has no more caspase activity than freshly drawn blood from a healthy donor. Blood products include cellular, non-cellular components of blood, such as red or white blood cells, platelets, platelet rich plasma, plasma including fresh frozen plasma, blood fractions and isolated components of blood, such as antibodies or blood factors such as coagulation factors. Whole blood, diluted whole blood, or whole blood or other blood products treated with agents to preserve it, prevent its coagulation or lysis, or otherwise facilitate its transfer and storage are also included.

This method of selecting a blood product determines the level of caspase or caspase 3 activity in a blood product based on the level of active caspase in that product.

Advantageously, the seiected blood product will contain a level of caspase, such as caspase 3, activity that is no greater than that found in a blood of a normal healthy donor, for example, 0 pmols Amc/min/g Hb of caspase activity. However, selections may also be made using other caspase activity values within the range of 0, 0.5, 1 , 1 .5, 2, 2.5, 3, 3,5, 4, 4.5, 5, 5.5 to 6 pmols Amc/min/gHb, preferably within the range of 0, 1 , 2 or 3 pmols Amc/min/gHb, or any intermediate value or subrange within these ranges. When a non-zero value for caspase activity is employed, blood product samples may be compared and the sample with the lower caspase activity selected for the transfusion or other administration of the blood product to a subject. Such a selection may be based on the level of caspase 3 activation in a blood product sample which may also range from 0, 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 to 6 pmols Amc/min/gHb or any intermediate value within this range.

In a related embodiment, the invention is directed to a method for storing a blood product or for maintaining red blood cell integrity comprising admixing a blood product with at least one caspase inhibitor or contacting it with an agent that adsorbs or otherwise removes or neutralizes active caspase. The caspase may be caspase 3 and the blood product may comprise whole blood or diluted or treated whole blood. Caspase activity, such as caspase 3 activity, in this embodiment may also range from 0 to 6 pmols Amc/min gHb or any intermediate value or subrange within this range; preferably, caspase activity will range from 0 to 3 pmols AMC/min/gHb.

Another embodiment involves a method for reducing the severity of at least one complication of a blood product transfusion or other administration of a blood product to a subject comprising administering at least one caspase inhibitor to said subject prior to, concurrently with, or after transfusion or administration of a blood product. Such

complications include, but are not limited to, those selected from the group consisting of febrile transfusion reaction, transfusion related acute lung injury, thrombotic complications, and immunological activation or suppression due to accumulation of bioactive substances in the transfusion product. In this embodiment, the caspase inhibitor may inhibit caspase 3 or some other caspase. The term caspase 3 as used herein also includes subunits or fragments of caspase 3 that exhibit caspase activity. The blood product may be whole blood, diluted or treated whole blood or other blood products disclosed herein. Caspase activity, such as caspase 3 activity, in this embodiment may also range from 0 to 6 pmols Amc/min/gHb or any intermediate value or subrange within this range; preferably, caspase activity will range from 0 to 3 pmols Amc/min/gHb.

Another embodiment of the invention is a method for extending or prolonging the shelf-life of whole blood, red blood cells, white blood cel ls, platelets, platelet-rich plasma, or other blood products, such as those disclosed herein, by admixing the blood product with at least one caspase inhibitor or contacting it with an agent that adsorbs or otherwise removes or neutralizes caspase thus extending, prolonging the storage time of shelf-l ife of said blood product. In one embodiment the caspase inhibitor will inhibit caspase 3. The blood product may be whole blood, diluted or treated whole blood.

This method may be used to extend the shelf life of blood by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 , 12, 13, 14 days or more compared to otherwise identical blood stored without a caspase or caspase 3 inhibitor. For example, the shelf life of whole blood may be stored for 40 or more days by incorporating a caspase inhibitor, such as one that inhibits caspase 3 into it. Caspase activity, such as caspase 3 activity, in such blood or blood products will be less than otherwise identical blood or blood products stored without the caspase inhibitor and preferably have no caspase activity, but may have a reduced caspase activating ranging from 0 to 3 pmols Amc/min/gHb.

Another embodiment of the invention is a method for maintaining the integrity or viability of non-adherent cells that circulate in the blood comprising admixing a sample comprising said non-adherent cells with at least one caspase inhibitor or contacting it with an agent that adsorbs or otherwise removes or neutralizes caspase, such as caspase 3, thereby maintaining the integrity or viability of said ceils. Non-adherent cel ls include blood cells, platelets and other somatic ceils that circulate in the blood. These include fetal ceils, placental cells, tumor or cancer cells, transformed cells, or metastatic cells. Preferably, the level of caspase activation attained ranges from 0 to 3 Amc/min/gHb, preferably 0

Amc/min gHb. An agent that neutralizes inhibits or removes caspase 3 activity may be employed in this method. The sample containing non-adherent cells may be whole blood, diluted whole blood or treated whole blood or in some cases other blood products described herein that retain non-adherent cells. REFERENCES

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Claims

Claim 1. A method for diagnosing, prognosing, or monitoring sickle cell disease or another a disease, disorder, or condition associated with occlusion of blood vessels, comprising:

detecting the concentration of or activity of at least one activated caspase, a subunit thereof, or a proteolytic fragment thereof in a biological sample from a subject, and

selecting a subject having sickle cell disease or another disease, disorder, or condition associated with occlusion of blood vessels, when the concentration or activity of the at least one activated caspase, subunit thereof, or a proteolytic fragment thereof is elevated in comparison to said level in a control subject who does not have the disease, disorder, or condition associated with occlusion of blood vessels.

Claim 2, The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease.

Claim 3. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell trait.

Claim 4. The method of claim I , wherein said disease, disorder, or condition associated with occlusion of blood vessels is a reperfusion injury.

Claim 5. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is ischemia.

Claim 6. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is stroke.

Claim 7. The method of claim I , wherein said disease, disorder, or condition associated with occlusion of blood vessels is peripheral vascular disease.

Claim 8, The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is hypovolemic or septic shock.

Claim 9. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease and the sample is obtained from a subject developing or having vaso-occlusive crisis.

Claim 10. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of bl ood vessel s is sickle cell disease and the sample is obtained from a subject developing or having liver disease.

Claim 1 1. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease and the sample is obtained from a subject developing or having renal dysfunction.

Claim 12. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease and the sample is obtained from a subject developing or having heart dysfunction.

Claim 13. The method of claim 1, wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease and the sample is obtained from a subject developing or having pulmonary hypertension.

Claim 14. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease and the sample is obtained from a subject developing or having acute chest syndrome.

Claim 15. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease and the sample is obtained from a subject developing or having acute stroke(s).

Claim 16. The method of claim 1, wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease and the sample is obtained from a subject developing or having hemolysis.

Claim 17. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease and the sample is obtained from a subject developing or having limb ischemia.

Claim 18. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of bl ood vessel s is sickle cell disease and the sample is obtained from a subject who has undergone corrective surgery.

Claim 19. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease and the sample is obtained from a subject undergoing thrombolytic therapy.

Claim 20. The method of claim 1 , wherein said disease, disorder, or condition associated with occlusion of blood vessels is sickle cell disease and the sample is obtained from a subject suspected of having a stroke who has been admitted to a hospital or emergency room.

Claim 21. The method of claim 1 , wherein said at least one caspase is caspase 3 or a caspase 3 subunit, or an oligomer thereof.

Claim 22. The method of claim 1 , wherein said at least one caspase is a pi 7 fragment of caspase 3.

Claim 23. The method of claim 1 , wherein said caspase is a heterodimer or heterotetramer of pi 2 and pi 7 fragments of caspase 3.

Claim 24. The method of claim 1 , wherein said biological sample is blood.

Claim 25. The method of claim 1 , wherein said biological sample is plasma or serum.

Claim 26. The method of claim 1 , wherein said sample is CSF, lymph, or tissue fluid.

Claim 27. The method of claim 1 , further comprising: selecting a subject having an elevated concentration or activity of at least one caspase, and

treating said subject for a disease, disorder, or condition associated with occlusion of blood vessels.

Claim 28, The method of claim 1 , wherein said subject has sickle cell disease.

Claim 29. The method of claim 3 , wherein said subject is heterozygous for sickle cell trait.

Claim 30. A method for monitoring or prognosing a disease, disorder, or condition associated with occlusion of blood vessels has progressed or regressed comprising:

quantitatively detecting an elevated concentration or activity of at least one caspase, subunit thereof, or proteolytic fragment thereof in biological samples over at least two different points in time,

selecting a subject whose disease, disorder, or condition associated with occlusion of blood vessels has progressed when the concentration or activity of the caspase, a subunit thereof, a proteolytic fragment, or oligomer thereof has increased between the points in time; and selecting a subject whose disease, disorder, or condition associated with occlusion of blood vessels has regressed when the concentration or activity of at least one caspase, a subunit thereof, a proteolytic fragment thereof, or oligomer thereof has increased between the points in time.

Claim 31. The method of claim 30, wherein the at least one caspase is caspase 3.

Claim 32. A method for diagnosing a disease, disorder, or condition associated with occlusion of blood vessels comprising:

detecting the amount of mRN A encoding at least one caspase, a subunit thereof, or a proteolytic fragment thereof in a biological sample from a subject, and

selecting a subject having a disease, disorder, or condition associated with occlusion of blood vessels when the level of mRNA encoding caspase, subunit thereof, or a proteolytic fragment thereof is elevated in comparison to said level in a control subject who does not have the disease, disorder, or condition associated with occlusion of blood vessels.

Claim 33. The method of claim 32, wherein the at least one caspase is caspase 3.

Claim 34. A method for monitoring or prognosing a disease, disorder, or condition associated with occlusion of blood vessels has progressed or regressed comprising:

quantitatively detecting mRNA encoding at least one caspase, subunit thereof, or proteolytic fragment thereof in biological samples over at least two different points in time, selecting a subject whose disease, disorder, or condition associated with occlusion of blood vessels has progressed when the quantity of mRNA encoding at least one caspase, a subunit thereof, or a proteolytic fragment thereof has increased between the points in time; and selecting a subject whose disease, disorder, or condition associated with occlusion of blood vessels has regressed when the quantity of mRNA encoding at least one caspase, a subunit thereof, or a proteolytic fragment thereof has decreased between the points in time.

Claim 35. The method of claim 34, wherein the at least one caspase is caspase 3.

Claim 36. A method for identifying a compound or other agent that reduces the severity of a disease, disorder, or condition associated with occlusion of blood vessels comprising:

contacting blood, vascular or other somatic cell or cell-line with a test compound, and quantitatively detecting the concentration or activity of at least one caspase, subunit thereof, proteolytic fragment thereof, or oligomer thereof produced by the cell compared to a control cell not contacted with said compound or agent;

selecting a test compound that decreases the concentration or activity of the at least one caspase, subunit thereof, proteolytic fragment thereof, or oligomer thereof produced by the cell compared to a control that has not been contacted with the test compound; or

(a) contacting blood, vascular or other somatic cell or cell-line with a test compound, and

quantitatively detecting mRNA encoding the at least one caspase, subunit thereof, proteolytic fragment thereof, or oligomer thereof transcribed or translated by the ceil compared to a control cell not contacted with said compound or agent;

selecting a test compound that decreases the amount of mRNA encoding the at least one caspase, subunit thereof, proteolytic fragment thereof, or oligomer thereof, transcribed or translated by the ceil compared to a control that has not been contacted with the test compound.

Claim 37, The method of claim 36, wherein the at least one caspase is caspase 3.

Claim 38. A method for reducing the severity of, treating, or preventing sickle cell disease or at least one symptom or sign thereof comprising administering an inhibitor of activity of at least one caspase to a subject in need thereof

Claim 39. The method of claim 38, wherein the at least one caspase is caspase 3.

Claim 40. The method of claim 38, wherein said caspase 3 inhibitor is at least one selected from the group consisting of VX-740, VX-765, GS-9450, and ZIDN-6556.

Claims 41 , A method for selecting a blood product to be transfused or otherwise administered to a subject comprising detecting the activation level of at least one caspase in a blood product and selecting a blood product for transfusion or administration to a subject that has no more caspase activity than freshly drawn blood of a healthy donor.

Claim 42. The method of claim 41 , wherein the blood product has no more than 0 pmols Amc/min/g Hb of caspase activity.

Claim 43. The method of claim 41 , wherein the blood product is selected to have no more than 3 pmols Amc/min/gHb.

Claim 44. The method of claim 41 , wherein the at least one caspase is caspase 3.

Claim 45. The method of claim 41, wherein the blood product comprises whole blood or a blood product comprising red blood cells, white blood cells, fresh frozen plasma, platelets, platelet rich plasma, and/or coagulation factors.

Claim 46. A method for storing a blood product or for maintaining red blood cell integrity comprising admixing a blood product with at least one caspase inhibitor or contacting it with an agent that adsorbs or otherwise removes or neutralizes active caspase.

Claim 47. The method of claim 46, wherein the caspase is caspase 3.

Claim 48, The method of claim 46, wherein the blood product comprises whole blood.

Claim 49. The method of claim 46, wherein caspase activation in said product is reduced to no more than 0 to 3 pmols Amc/min/gHb.

Claim 50. A method for reducing the severity of at least one complication of a blood product transfusion or other administration of a blood product to a subject comprising administering at least one caspase inhibitor to said subject prior to, concurrently with, or after transfusion or administration of a blood product.

Claim 51 . The method of claim 50, wherein the at least one complication is selected from the group consisting of febrile transfusion reaction, transfusion related acute lung injury, thrombotic complications, and immunological activation or suppression due to accumulation of bioactive substances in the transfusion product.

Claim 52. The method of claim 50, wherein the caspase inhibitor inhibits caspase 3.

Claim 53. The method of claim 50, wherein the blood product comprises whole blood.

Claim 54. The method of claim 50, wherein caspase activation in said blood product is no more than 0 to 3 pmols Amc/min/gHb.

Claim 55. A method for extending or prolonging the shelf-life of whole blood, red blood cells, white blood cells, platelets, platelet-rich plasma, or other blood product comprising admixing the blood product with at least one caspase inhibitor or contacting it with an agent that adsorbs or otherwise removes or neutralizes caspase thus extending, prolonging the storage time of shelf-life of said blood product.

Claim 56. The method of claim 55, wherein the caspase inhibitor inhibits caspase 3.

Claim 57. The method of claim 55, wherein the blood product comprises whole

Claim 58, The method of claim 55, wherein the shelf life of said blood product is extended beyond 40 days or wherein caspase activation in said product is no more than 0 to 3 pmols Amc/min/gHb.

Claim 59, A method for maintaining the integrity or viability of non-adherent cells that circulate in the blood comprising admixing a sample comprising said non-adherent cells with at least one caspase inhibitor or contacting it with an agent that adsorbs or otherwise removes or neutralizes caspase thereby maintaining the integrity or viability of said cells.

Claim 60, The method of claim 59, wherein caspase activation in said sample is no more than 0 to 3 pmols Amc/mia-'gHb.

Claim 61. The method of claim 59, wherein the at least one caspase inhibitor inhibits caspase 3,

Claim 62. The method of claim 59, wherein the blood product sample comprises whole biood.

Claim 63. The method of claim 59, wherein the non-adherent cells are fetal cells or cells associated with the placental.

Claim 64. The method of claim 59, wherein the non-adherent cells are tumor or cancer ceils.

Claim 65. The method of claim 59, wherein the non-adherent cells are metastasizing cells.