WO2007142769A1 - Methods for treating diseases involving stress-related diseases and conditions - Google Patents

Abstract

The present invention provides a unique mode of action for Extracorporeal Photopheresis (ECP). ECP may act through modulation of cellular stress responses characteristic of evolutionary conserved mechanism for responding to environmental stress (i.e. responses to infection and varied nutrient availability, obesity, xenobiotics). A specific pathway, the hexosamine metabolic pathway appears to modulate this stress response by-altering cellular O-linked glycoprotein composition.

Description

Methods for treating diseases involving stress-related diseases and conditions BACKGROUND OF THE INVENTION

Cells and organisms respond to environmental stresses in an evolutionarily conserved manner as a mechanism to maintain homeostasis. Aberrant or prolonged stress responses underlie more than 100 serious diseases (Xu et al. (2005)) and impact the success of many medical procedures.

Cellular stress responses characteristic of evolutionarily conserved mechanism for responding to environmental stress (i.e. responses to infection and varied nutrient availability, obesity, xenobiotics) include those which involve the so-called "heat shock proteins" (HSPs). HSPs form a family of highly conserved proteins that are widely distributed throughout the plant and animal kingdoms. Although HSPs were originally identified in cells subjected to heat stress, they have been found to be associated with many other forms of stress, such as infections, and are thus more commonly known as "stress proteins" (SPs).

The biological function of a protein depends on its three dimensional structure, which is determined in large part by its amino acid sequence but also by the environment. In fact, protein conformation governs its interaction with other factors, which can participate in the regulation of protein function. The failure of polypeptides to adopt and maintain their proper structure through proper protein folding is a major threat to cell function and viability. Consequently, elaborate systems have evolved to protect cells from the deleterious effects of misfolded proteins.

Consequently, abnormalities of protein folding, oligomerization, aggregation or deposition may play an important role in the pathophysiology of a diverse set of chronically progressive degenerative disorders. Non-limiting examples of such diseases include the following: Parkinson's Disease (PD), diffuse Lewy body dementia (DLBD), multiple system atrophy (MSA), dystrophia myotonica, dentatorubro-pallidoluysian atrophy (DRPLA), Friedreich's ataxia, fragile X syndrome, fragile XE mental retardation, Machado-Joseph Disease, spinobuibar muscular atrophy (also known as Kennedy's Disease), spinocerebellar ataxia, Huntington's disease (HD), familial encephalopathy with neuroserpin inclusion bodies (FENIB)₁ Pick's disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis/parkinsonism dementia complex, Amyotrophic Lateral Sclerosis (ALS), Down's syndrome, Age-Related Macular Degeneration, Cataract, and Wilson's Disease. In many cases, genetic mutations underlying the familial forms of these diseases have been identified. In most cases, however, the initiating idiopathic event facilitating or triggering the conformational transition of the target protein is unknown but ultimately results in the abnormal processing, misfolding, oligomerization or aggregation of protein, which triggers cellular toxicity. As part of a cellular detoxification, defense strategy or as an attempt to degrade the abnormally folded proteins, such abnormal proteins often accumulate in various types of cellular inclusions or aggresomes. These inclusions or aggresomes have thus become one of the pathological hallmarks of this diverse set of degenerative conditions. To date, such therapeutic agents and completely effective treatments for these diseases are not available.

Light irradiation or phototherapy has been widely used in the chemical and biological sciences for many years. Ultraviolet (UV) light irradiation of blood was used in the 1930's, 40's, and 50's for the treatment of many conditions. These conditions included bacterial diseases such as septicemias, pneumonias, peritonitis, wound infection, viral infections including acute and chronic hepatitis, poliomyelitis, measles, mumps, and mononucleosis. Phototherapy or light irradiation also includes the processes of exposing photoactivatable or photosensitizable targets, such as cells, blood products, bodily fluids, chemical molecules, tissues, viruses, and drug compounds, to light energy, which induces an alteration in or to the targets. In recent years, the applications of phototherapy are increasing in the medical field. These applications include the inactivation of viruses contaminating blood or blood products, the preventive treatment of platelet-concentrate infusion-induced all immunization reactions, and the treatment of both autoimmune and T cell mediated diseases because numerous human disease states, particularly those relating to biological fluids such as blood, respond favorably to treatment by visible or UV light irradiation.

Irradiation applications may also include the irradiation sterilization of fluids that contain undesirable microorganisms, such as bacteria or viruses. Light irradiation may also be effective to eliminate immunogenicity in cells, inactivate or kill selected cells, inactivate viruses or bacteria, or activate desirable immune responses. Phototherapy may be used as an antiviral treatment for certain blood components or whole blood. For example, a pathogenic virus in a donated platelet concentrate may be inactivated by UV light exposure. WO 97/36634.

Although light irradiation may be effective by itself, without the introduction of outside agents or compounds, it may also involve the introduction of specific agents or catalysts, such as, for example, photoactivatable drugs. In a particular application, it is well known that a number of human disease states may be characterized by the overproduction of certain types of leukocytes, including lymphocytes, in comparison to other population of cells which normally comprise whole blood. Excessive abnormal lymphocyte populations result in numerous adverse effects in patients including the functional impairment of bodily organs, leukocyte mediated autoimmune diseases and leukemia related disorders many of which often ultimately result in fatality. Indeed, uses of these photoactivatable drugs may involve treating the blood of a diseased patient where specific blood cells have become pathogenic as a consequence of the disease state. The methods generally may involve treating the pathogenic blood cells, such as lymphocytes, with a photoactivatable drug, such as a psoralen, which is capable of forming photoadducts with lymphocyte DNA when exposed to UV radiation.

Photopheresis using a psoralen such as methoxsalen may cause an immunization against the abnormal (cancerous, in the case of CTCL) T cells. During photopheresis, methoxsalen enters the white blood cell nuclei and intercalates in the double-stranded DNA helix. In an extracorporeal circuit, long wave ultraviolet light is directed at the leukocyte- enriched blood volume. The methoxsalen, responding to the ultraviolet energy, links to the thymidine base in the DNA helix. This results in the cross-linking of thymidine bases which prevent the unwinding of the DNA during transcription. Ultraviolet A light (UVA) damages abnormal T-cells rendering them more immunogenic. Other psoralens or psoralen derivatives may act via another pathway. Nevertheless, after cells are photoactivated, reinfusion of these altered T-cells causes an immunological reaction that targets T cells carrying the same surface antigens. Edelson (1991) Ann NY Acad Sci 636:154-64. This results in the production of a highly specific immune response against the abnormal cells (either a cancer clone or perhaps T cells which express viral antigens on their surface). It is estimated that approximately 25-50% of the total peripheral blood mononuclear cell 5 compartment is treated per photopheresis session (2 consecutive days schedule). SUMMARY OF THE INVENTION

The present invention provides a unique mode of action for Extracorporeal Photopheresis (ECP). ECP may act through modulation of cellular stress responses characteristic of evolutionarily conserved mechanism for responding to environmental stress (i.e. responses to infection and varied nutrient availability, obesity, xenobiotics). A specific pathway, the hexosamine metabolic pathway appears to modulate this stress response by altering cellular O- linked glycoprotein composition. The data presented herein support the claim that this pathway acts as a sensor of apόptotic cell death, and can take part in the stress response that ensues after ECP. Given that only two enzymes are known to affect this pathway, and that this pathway has been implicated in the onset and pathologies of diabetes, this pathway provides new targets for ECP therapy. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the clinical procedure for extracorporeal photopheresis.

Figure 2 depicts bar graphs showing that ECP ameliorates inflammation in a mouse asthma model.

Figure 3 depicts graphs showing that ECP protects NOD/LtJ mice from diabetes and its complications.

Figure 4 depicts a bar graph showing that ECP blunts the diabetogenic effect of STZ in a mouse model of T1 DM.

Figure 5 depicts the immunological mechanism of action of ECP.

Figure 6 is a bar graph showing cytokine secretion pattern of dendritic cells co-cultured with ECP-treated cells.

Figure 7 is a bar graph showing T reg generated in vitro using AC to suppress T cell proliferation in a MLR.

Figure 8 is a series of bar graphs showing the effect of ECP regimen on acute phase proteins.

Figure 9 is a bar graph showing that ECP modulates insulin and glucagons in a mouse model of pulmonary inflammation.

Figure 10 depicts bar graphs showing ECP effects on biomarkers over time (fold change from baseline v time).

Figure 11 is a bar graph showing ECP-induced leptin secretion in a mouse model of pulmonary inflammation.

Figure 12 is a graph showing the time course of biomarkers in healthy mice.

Figure 13 is a series of bar graphs showing cell dose-response relationships in ECP-treated healthy mice. Figure 14 is a series of bar graphs showing dose-dependent effect on DC mTOR activity after engulfment of AC is dependent on DC maturation state.

Figure 15 is a bar graph showing the effect of O-GlcNAc inhibition on AC tolerogenesis in a MLR.

Figure 16 shows that Alloxan reverses ECP effects in MLR by stimulating T cell proliferation.

Figure 17 shows ECP pre-conditions animals and cells against chemical stressors.

Figure 18 shows ECP treatment induces TSP-1 secretion in PBMC.

Figure 19 shows TSP-1 blockade attenuates ECP tolerogenic effects in MLR.

@ DETAILED DESCRIPTION OF THE INVENTION

Many of the most prevalent chronic diseases, including diabetes are driven by maladaptive responses to stress. Stress, here defined broadly as a state of threatened homeostasis, is provoked by an array of stimuli including psychological, environmental, and physiological demands. Extracorporeal photopheresis (ECP) has now been shown to be a therapy that can modulate stress responses, and facilitate adaptation. This process enhances cell and tissue repair and speeds the resolution of disease pathology. This therapy thus offers a safe, novel approach to treating currently intractable and typically fatal diseases. ECP is a safe and effective immunomodulatory therapy with demonstrated utility in numerous inflammatory conditions. The therapeutic benefit of ECP appears to arise from the induction of immune tolerance, in part by the induction of regulatory T cells (Treg). The present invention provides signaling pathways that modulate ECP function in vitro, as confirmed by in vivo models of inflammatory disease, including T1DM.

In animal models and patients, better outcomes typically occur after numerous ECP treatments. Indeed, for several diseases, it is rare that a response is seen until after several treatment cycles.

In contrast to immunosuppressive treatments, patients receiving ECP show no increased rate of infection or cancer. Results from animal models show that immune responses are preserved, arising with essentially equal speed and intensity as untreated controls, but they resolve more quickly with ECP treatment. Treating cells and animals with drugs from widely disparate classes potentiates ECP in vitro and in vivo. Synergies are seen with drugs affecting systems critical to cellular stress responses and distinct signaling pathways.

Gene expression profiling of ECP showed that ECP elicits changes in transcript abundance of stress-responsive genes, including many that have been implicated in stereotypical, programmed stress responses.

Consistent with our gene array data, two specific control points for immune tolerance, one known and one novel, are modulated by ECP. Both pathways, the hexosamine biosynthetic pathway (HBP) and the mTOR-signaling cascade, are implicated in nutrient sensing and diabetes. Both normally respond to low-level stress to render cells more tolerant to subsequent challenges, suggesting an ECP-induced stress conditioning effect.

ECP is protective under conditions of acute stress, improving clinical outcomes after myeloablative bone marrow transplant, protecting ceils in culture from pro-apoptotic insults, and arresting the progression of diabetes in genetic and chemically induced mouse models.

These observations are consistent with stress-induced conditioning responses, best characterized by ischemic preconditioning. Pre-conditioning elicits adaptive functional and structural changes that reduce or prevent damage from subsequent stressors. Epigenetic changes resulting from such "stress conditioning" alter gene expression patterns, affecting both the acute stress response and its resolution, and establishing durable protection. The present invention demonstrates that ECP can elicit such a response, bolstering cellular and organismal capacity for stress. In treating pre-existing disease, ECP elicits a more rapid resolution of pathology via a form of post-conditioning, where physiological changes elicited by the conditioning regimen enable repair and a more rapid return to homeostasis.

Examples of stress conditioning of immune responses have been reported. Experimental data show that conditioning with exogenous stressors is possible, but the options are inherently risky. For example, regimens of ischemia, irradiation, hyper- or hypothermia, anaesthesia, endotoxin-, adjuvant-, or pathogen exposure, and relatively toxic drugs have shown efficacy in animal models and a few clinical trials.

The means by which conditioning can be safely achieved has eluded investigators and clinicians for decades. It has now been shown that ECP provides the means to achieve this goal. The present invention shows that that the stress response can be safely modulated, revealing a novel treatment approach and new drug targets for diseases affecting millions of people worldwide and demonstrates a new link between stress responses and the immune system. Apoptotic cell (AC) infusion, as in ECP, is known to induce T_reg and tolerogenic dendritic cells but beyond the obligate clearance of AC (efferocytosis), the induction mechanism is unclear. The present invention shows that several classes of compounds, notably Rapamycin, antioxidants, and Celastrol, were synergistic with AC in vitro. These drugs modify stress responses, and the present invention shows that efferocytosis can as well. Indeed, AC infusion altered the expression profiles of stress-responsive and metabolic genes in vivo and in dendritic cells in vitro. One example is the extracellular protein thrombospondin-1. TSP-1 is stress responsive, (Zebo et al. (2005); and Favier et al. (2005)) is released by both apoptotic and phagocytic cells, and has been shown to generate tolerogenic APCs and Tregs in vitro through specific receptor interactions. Krispin et al. (2006); and Grimbert et al. (2006). Confirmation of these findings in vitro, shows that the effect is independent of the method of induction of apoptosis. Metabolic control genes were examined as well. Since stressors have significant effects on the immune system, evinced by changes in gene expression and energy metabolism, (Fox et al. (2005)) serum markers of energy metabolism are herein shown to change in response to ECP. Indeed, the concentrations of insulin, glucagon, and IGF- 1 were modulated by ECP in a dose- and time-dependent manner in treated mice. Given that animals with dysregulated metabolism can have abnormal preconditioning responses, (Katakam et al. (2006)) we examined gene expression in relevant pathways, including enzymes of the hexosamine biosynthetic pathway (HBP). O-GlcNAcase expression is responsive to efferocytosis, and its inhibition or that of the counter-regulatory HBP enzyme O-GlcNAc transferase, significantly alters tolerogenesis in vitro. Kudlow (2006); and Dauphinee et al. (2005). Specifically, PUGNAc and Streptozotocin, O-GlcNAcase inhibitors, potentiated the effect of AC, while OGT inhibition with Alloxan completely reversed it. The HBP is stress- responsive, and is implicated in neutrophil chemotaxis. Zachara et al. (2004); and Kneass et al. (2005). The simultaneous modulation of immune tolerance and stress responses by the HBP was heretofore unknown, and has far-reaching implications, including new insights into common pathologies in metabolic, neurological, and cardiovascular disorders. Lehman et al. (2005); Katakam et al. (2006); Mattson et al. (1999); and Hashizume et al. (2006). The terms "subject" or "patient" are used interchangeably and refer to an animal, preferably a mammal and more preferably a human.

A "cell population" generally includes a cell type found in blood. The term may include one or more types of blood cells, specifically, red blood cells, platelets, and white blood cells. A cell population may comprise subtypes of white blood cells, for example, T-cells, dendritic cells, B-cells, etc. In one embodiment, a cell population may comprise a mixture or pool of cell types. Alternatively, a cell population may comprise a substantially purified type of cells, for example, T cells or dendritic cells (DCs).

"ECP procedure" or "ECP" refers to extracorporeal photopheresis, also known as extracorporeal phototherapy. It is a treatment of a population of cells that has been subjected to UVA light and a photoactive ble compound. Preferably the population of cells is from an organ or tissue; more preferably, the population of cells is a portion of blood; and most preferably, the population of cells is a buffy coat. ECP is sometimes used to refer to a process in which a cell population has been subjected to an apoptosis-inducing procedure with UVA light in the presence of a DNA cross linking agent such as a psoralen (preferably, 8-MOP).

In the most preferred embodiment of the invention, ECP is used. Methods of using ECP to induce apoptosis and use of the cells are described in US20050163778. This involves a photoactivatable compound added to a cell population ex vivo. The photosensitive compound may be administered to a cell population comprising blood cells following its withdrawal from the subject, recipient, or donor, as the case may be, and prior to or contemporaneously with exposure to ultraviolet light. The photosensitive compound may be administered to a cell population comprising whole blood or a fraction thereof provided that the target blood cells or blood components receive the photosensitive compound. In another embodiment, a portion of the subject's blood, recipient's blood, or the donor's blood could first be processed using known methods to substantially rerhove the erythrocytes and the photoactive compound may then be administered to the resulting cell population comprising the enriched leukocyte fraction.

In an alternative embodiment, the photoactivatable compound may be administered in vivo. The photosensitive compound, when administered to a cell population comprising the subject's blood, recipient's blood, or the donor's blood, as the case may be, in vivo may be administered orally, but also may be administered intravenously and/or by other conventional administration routes. The oral dosage of the photosensitive compound may be in the range of about 0.3 to about 0.7 mg/kg, more specifically, about 0.6 mg/kg. When administered orally, the photosensitive compound may be administered at least about one hour prior to the photopheresis treatment and no more than about three hours prior to the photopheresis treatment.

Photoactivatable compounds for use in accordance with the present invention include, but are not limited to, compounds known as psoralens (orfurocoumarins) as well as psoralen derivatives such as those described in, for example, US4321919 and US5399719. Preferred compounds include 8-methoxypsoralen; 4,5'8-trimethylpsoralen; 5-methoxypsoralen; 4- methylpsoralen; 4,4-dimethylpsoralen; 4-5'-dimethylpsoralen; 4'-aminomethyl-4,5\8- trimethylpsoralen; 4'-hydroxymethyl-4,5',8-trimethylpsoralen; 4',8-methoxypsoralen; and a 4¹- (omega-amino-2-oxa) alkyl-4,5'8-trimethylpsoralen, including but not limited to 4'-(4-amino-2- oxa)butyl-4,5',8-trimethylpsoralen. In one embodiment, the photosensitive compound that may be used comprises the psoralen derivative, amotosalen (S-59) (Cerus, Corp., Concord, CA). In another embodiment, the photosensitive compound comprises 8-methoxypsoralen (8 MOP).

The cell population to which the photoactivatable compound has been added is treated with a light of a wavelength that activates the photoactivatable compound. The treatment step that activates the photoactivatable compound is preferably carried out using long wavelength ultraviolet light (UVA), for example, at a wavelength within the range of 320 to 400 nm. The exposure to ultraviolet light during the photopheresis treatment preferably is administered for a sufficient length of time to deliver about 1-2 J/cm² to the cell population.

Extracorporeal photopheresis apparatus useful in the methods according to the invention include those manufactured by Therakos, Inc., (Exton, Pa.) under the name UVAR™ A description of such an apparatus is found in US4683889. The UVAR™ System uses a treatment system and consists of three phases including: 1 ) the collection of a buffy-coat fraction (leukocyte-enriched), 2) irradiation of the collected buffy coat fraction, and 3) reinfusion of the treated white blood cells. The collection phase has six cycles of blood withdrawal, centrifugation, and reinfusion steps. During each cycle, whole blood is centrifuged and separated in a pheresis bowl. From this separation, plasma (volume in each cycle is determined by the UVAR™ instrument operator) and 40 ml buffy coat are saved in each collection cycle. The red cells and all additional plasma are reinfused to the patient before beginning the next collection cycle. Finally, a total of 240 ml of buffy coat and 300 ml of plasma are separated and saved for UVA irradiation.

The irradiation of the leukocyte-enriched blood within the irradiation circuit begins during the buffy coat collection of the first collection cycle. The collected plasma and buffy coat are mixed with 200 ml of heparinized normal saline and 200 mg of UVADEX™ (water soluble 8- methoxypsoralin). This mixture flows in a 1.4 mm thick layer through the PHOTOCEPTOR™ Photoactivation Chamber, which is inserted between two banks of UVA lamps of the PHOTOSETTE™ PHOTOSETTE™ UVA lamps irradiate both sides of this UVA-transparent PHOTOCEPTOR™ chamber, permitting a 180-minute exposure to ultraviolet A light, yielding an average exposure per lymphocyte of 1-2 J/cm². The final buffy coat preparation contains an estimated 20% to 25% of the total peripheral blood mononuclear cell component and has a hematocrit from 2.5% to 7%. Following the photoactivation period, the volume is reinfused to the patient over a 30 to 45 minute period. US20030181305 describes another system for use in ECP administration. US5951509; US5985914; US5984887; US4464166; US4428744; US4398906; US4321919; WO 97/36634; and WO 97/36581 also contain description of devices and methods useful in this regard.

Another system that may be useful in the methods of the present invention is described in US6793643. That system includes an apparatus by which the net fluid volume collected or removed from a subject may be reduced during ECP. The effective amount of light energy that is delivered to a cell population may be determined using the methods and systems described in US6219584.

A variety of other methods for inducing apoptosis in a cell population are well known and may be adopted for use in the present invention. One such treatment comprises subjecting a cell population to ionizing radiation (κ-rays, x-rays, etc.) and/or non-ionizing electromagnetic radiation including ultraviolet light, heating, cooling, serum deprivation, growth factor deprivation, acidifying, diluting, alkalizing, ionic strength change, serum deprivation, irradiating, or a combination thereof. Alternatively, apoptosis may be induced by subjecting a cell population to ultrasound.

Yet another method of inducing apoptosis comprises the extracorporeal application of oxidative stress to a cell population. This may be achieved by treating the cell population, in suspension, with chemical oxidizing agents such as hydrogen peroxide, other peroxides and hydroperoxides, ozone, permanganates, periodates, and the like. Biologically acceptable oxidizing agents may be used to reduce potential problems associated with residues and contaminations of the apoptosis-induced cell population so formed.

In preparing an apoptosis-induced cell population, care should be taken not to apply excessive levels of oxidative stress, radiation, drug treatment, etc., because otherwise there may be a significant risk of causing necrosis of at least some of the cells under treatment. Necrosis causes cell membrane rupture and the release of cellular contents often with biologically harmful results, particularly inflammatory events, so that the presence of necrotic cells and their components along with the cell population comprising apoptotic cells is best avoided. Appropriate levels of treatment of the cell population to induce apoptosis, and the type of treatment chosen to induce apoptosis are readily determinable by those skilled in the art.

One process according to the present invention involves the culture of cells from the subject, or a compatible mammalian cell line. The cultured cells may then be treated extracorporeally to induce apoptosis and to create a cell population therein. The extracorporeal treatment may be selected from the group consisting of antibodies, chemotherapeutic agents, radiation, extracorporeal photopheresis, ultrasound, proteins, and oxidizing agents. The cells, suspended in the subject's plasma or another suitable suspension medium, such as saline or a balanced mammalian cell culture medium, may then be administered to the patient.

Methods for the detection and quantitation of apoptosis are useful for determining the presence and level of apoptosis in the preparation to be administered to the subject in the present invention. The number of apoptotic cells in a cell population required to obtain the required clinical benefit in a subject may vary depending on the source of cells, the subject's condition, the age and weight of the subject and other relevant factors, which are readily determinable by well-known methods. Preferably, the number of apoptotic cells that are administered to a patient are 0.1 to 50 billion, more preferably 1 to 10, and most preferably 2.5 to 7.5 billion.

In one embodiment, cells undergoing apoptosis may be identified by a characteristic "laddering^' of DNA seen on agarose gel electrophoresis, resulting from cleavage of DNA into a series of fragments. In another embodiment, the surface expression of phosphatidylserine on cells may be used to identify and/or quantify an apoptosis-induced cell population. Measurement of changes in mitochondrial membrane potential, reflecting changes in mitochondrial membrane permeability, is another recognized method of identification of a cell population. A number of other methods of identification of cells undergoing apoptosis and of a cell population, many using monoclonal antibodies against specific markers for a cell population, have also been described in the scientific literature.

Modification of ECP therapy to optimize the effect of ECP on the stress. response, perhaps via the hexosamine pathway would offer new therapeutic opportunities. By optimizing ECP to best modulate the immune system via this "integrated stress response" (ISR, also known as the "unfolded protein response", "endoplasmic reticulum (ER) stress" and other names) expands ECP into previously unexamined disease states in which this stress response is believed to play a pathobiological role. These diseases include pathologies of most organ systems, which are currently intractable and typically fatal. This mode of action involves conditioning cells to tolerate stress through the single or repeated administration of an adequate number of apoptotic cells, which serves as a low-level stressor to the immune system. Previous studies have shown that other stressors can induce such preconditioning, but these stressors would likely be unsafe as treatments. Therefore ECP, a safe and effective marketed therapy, provides a method to specifically modify the stress response and alter disease progression or susceptibility.

This stress response is induced by numerous stimuli that evoke signals indicative of ER stress by placing demands on the ER that it is not prepared to handle at the time of the stimulus. These stressors include conditions where a loss of host/patient cellular control of protein expression occurs. This includes numerous cellular signaling events that lead to ER dysfunction (i.e., a rate of protein translation that exceeds the cellular capacity to modify or otherwise properly process proteins). It appears that this stress response conditioning is induced not only in the cells that phagocytose the apoptotic cell, but also as a result of signaling from those cells to other tissues in a manner that propagates signal(s) that an increased stress is present. Such a response requires an adaptive change by affected tissues to properly recover from the initiating or consequent stresses under which the system is likely to suffer. Therefore, this invention extends the use of ECP into infectious diseases and related therapies including viral infection, (i.e. pathological or therapeutically-induced double stranded RNA exposure), into cancer therapy where transformation both induces ER stress and overrides the normal cell death and reparative responses, into renal diseases associated with protein mis-folding or overexpression, and fibrotic diseases where various stimuli induce the overexpression of extracellular matrix proteins and cell overgrowth.

This invention expands the use of existing therapeutic regimes into disease states including protein aggregation diseases including, without limitation, Alzheimer's disease, Parkinson's Disease, Huntington's chorea; and wasting diseases including, without limitation, the spongiform encephalopathies of Prion infection and inherited variant Creutzfe Id t- Jacob disease ("vCJD"). Metabolic and immune stress-related diseases that involve insulin resistance and defects in secretory cells including diabetes, Metabolic Syndrome and other obesity-related pathologies have been related to the unfolded protein response, and is thus tractable to ECP therapy. This includes, without limitation, atherosclerosis and related cardiovascular diseases where cellular overproduction of proteins leads to pathological deposition of proteins and their aggregates, and diseases of any of the secretory cells that overproduce proteins as part of their normal or pathological natural histories. Cells often considered among this secretory category include, without limitation, pancreatic beta cells, prostate tissues, immune cells that secrete significant amounts of immunoglobulins (i.e. plasma cells) or other proteins (i.e. dendritic cell interferon gamma production), and mucosal secretory cells (i.e. mucin-secreting cells in the gastrointestinal tract and airways).

Cells that are stimulated to alter their phenotype to produce increased amounts of protein can be affected by this stress, and diseases where a dysfunction in this production capacity results from a pathological response to that stress provide additional target tissues for which ECP provides therapeutic benefit. This includes, without limitation, enzyme-producing cells of the liver and stomach, peptide secreting neurons and neuroendocrine tissues, and cells that are stimulated to secrete extracellular matrix and tissue remodeling proteins in response to injury (i.e. in wound healing, response to radiation damage).

Any cell exposed to temperature stress or oxidative stress can require a proper stress response, and ECP conditioning can now benefit many such cases, since such conditioning can provide resistance to damage, or to enhance the repair of damage resulting from such stresses. Therefore various exposures to environmental insults that yield reactive oxygen species, peroxynitrites, or other oxidative stresses are a target of ECP therapy. This including, without limitation, poisoning (i.e. with agrochemicals like paraquat), radiation exposure, extreme exercise, chemotherapy, anaesthesia, hyperbaric exposures, genetic preconditions that elicit reactive oxygen species (i.e., those produced by the platelet derived growth factor ("PDGF") receptor signaling system), and trauma.

Alterations in cellular metabolism that result from stress responses are known to affect cellular transport of small molecules in addition to altering protein trafficking. Gene expression profiles show that ECP beneficially alters the expression of multi-drug resistance ("MDR") transporters. Optimized ECP alters the expulsion of drugs from the cell by limiting or altering the expression of MDR pumps and transporters, thereby affecting drug resistance (i.e. in cancer chemotherapy or antibiotic/antiviral treatments). In other cases ECP can be optimized to enhance the uptake of drugs into the cell.

A widely accepted hypothesis posits that the process of aging is driven by the cumulative effect of oxidative stress on an organism. Caloric restriction has been shown to extend lifespan in part by altering cellular responses to oxidative stress or the generation of that stress by normal or excess metabolism. Given that the stress response induced by ECP involves mechanisms similar to those induced by caloric restriction, ECP modifies the effects of aging.

Existing methods can be used to enable this optimization of ECP. First, the integrated stress response induces alternative splicing in several known transcripts, including transcripts directly involved in the reparative process and translational arrest characteristic of this stress response, and the apoptotic alternative to repair that ensues when the cell is unable to overcome the ER stress.

Alternative splicing of Xbp-1 mRNA has been shown to occur in the stress response, and can be measured by any method known in the art including, without limitation, quantitative PCR using peripheral blood or fine tissue biopsies. Alternatively, monitoring mTOR activity serves as a biomarker of the stress response, since the stress responses characterized to date signal via the mTOR pathway. The phosphorylation of S6 kinase, the immediate downstream substrate of mTOR has been used as a pharmacodynamic marker to monitor immunosuppression in patients undergoing Rapamycin treatment. Although published reports describe a Western blot method, reagents are available that have been demonstrated useful in the flow cytometric analysis mTOR activity in cell lines. Monitoring mTOR activity in peripheral blood is possible using methods known in the art, including, without limitation, flow cytometry and immunohistochemistry. As shown in the Examples provided herein, monitoring the response to different ECP conditions using biomarkers of the stress response and apoptosis provides a method to determine the effectiveness of ECP in stress-related diseases and conditions. By altering the cell dose and dosing regimen using these biomarkers, it is possible to deliver ECP with a previously unattainable clinical precision. This maximizes the therapeutic potential of ECP as a stand-alone therapy and allows the optimization of the combination of ECP with other immunomodulatory pharmacotherapies and treatment regimes.

A Biomarker is any indicia of the level of expression of an indicated Marker gene. The indicia can be direct or indirect and measure over- or under-expression of the gene given the physiologic parameters and in comparison to an internal control, normal tissue or another carcinoma. Biomarkers include, without limitation, nucleic acids (both over and under-expression and direct and indirect). Using nucleic acids as Biomarkers can include any method known in the art including, without limitation, measuring DNA amplification, RNA, micro RNA, loss of heterozygosity (LOH), single nucleotide polymorphisms (SNPs, Brookes (1999)), microsatellite DNA, DNA hypo- or hyper-methylation. Using proteins as Biomarkers includes any method known in the art including, without limitation, measuring amount, activity, modifications such as glycosylation, phosphorylation, ADP-ribosylation, ubiquitination, etc., or imunohistochemistry (IHC). Other Biomarkers include imaging, cell count and apoptosis Markers.

A Marker nucleic acid corresponds to the sequence designated by a SEQ ID NO when it contains that sequence. A gene segment or fragment corresponds to the sequence of such gene when it contains a portion of the referenced sequence or its complement sufficient to distinguish it as being the sequence of the gene. A gene expression product corresponds to such sequence when its RNA, mRNA, miRNAorcDNA hybridizes to the composition having such sequence (e.g. a probe) or, in the case of a peptide or protein, it is encoded by such mRNA. A segment o •r f _*ragment of a gene expression product corresponds to the sequence of such gene or gene expression product when it contains a portion of the referenced gene expression product or its complement sufficient to distinguish it as being the sequence of the gene or gene expression product.

The inventive methods, compositions, articles, and kits of described and claimed in this specification include one or more Marker genes. "Marker" or "Marker gene" is used throughout this specification to refer to genes and gene expression products that correspond with any gene the over- or under-expression of which is associated with a stress- or inflammatory-related response.

The present invention further provides microarrays or gene chips for performing the methods described herein.

The present invention further provides diagnostic/prognostic portfolios containing reagents suitable for measuring Biomarkers such as isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes as described herein where the combination is sufficient to measure or characterize gene expression in a biological sample.

Any method described in the present invention can further include measuring expression of at least one gene constitutively expressed in the sample.

The invention further provides a method for providing direction of therapy by identifying a stress- or inflammatory-related response according to the methods described herein and identifying the appropriate treatment therefor.

The invention further provides a method for providing a prognosis by identifying a stressor inflammatory-related response according to the methods described herein and identifying the corresponding prognosis therefor.

The invention further provides a method for finding Biomarkers comprising determining the expression level of a Marker gene, measuring a Biomarker for the Marker gene to determine expression thereof, analyzing the expression of the Marker gene according to the methods described herein and determining if the Marker gene is effectively specific for a stress- or inflammatory-related response. '

The invention further provides kits, articles, microarrays or gene chip, diagnostic/prognostic portfolios for conducting the assays described herein and patient reports for reporting the results obtained by the present methods.

The mere presence or absence of particular nucleic acid sequences in a tissue sample has only rarely been found to have diagnostic or prognostic value. Information about the expression of various proteins, peptides or mRNA, on the other hand, is increasingly viewed as important. The mere presence of nucleic acid sequences having the potential to express proteins, peptides, or mRNA (such sequences referred to as "genes") within the genome by itself is not determinative of whether a protein, peptide, or mRNA is expressed in a given cell. Whether or not a given gene capable of expressing proteins, peptides, or mRNA does so and to what extent such expression occurs, if at all, is determined by a variety of complex factors. Irrespective of difficulties in understanding and assessing these factors, assaying gene expression can provide useful information about the occurrence of important events such as a stress- or inflammatory-related response, and other clinically relevant phenomena. Relative indications of the degree to which genes are active or inactive can be found in gene expression profiles.

Preferred methods for establishing gene expression profiles include determining the amount of RNA that is produced by a gene that can code for a protein or peptide. This is accomplished by reverse transcriptase PCR (RT-PCR), competitive RT-PCR, real time RT-PCR, differential display RT-PCR, Northern Blot analysis and other related tests. While it is possible to conduct these techniques using individual PCR reactions, it is best to amplify complementary DNA (cDNA) or complementary RNA (cRNA) produced from mRNA and analyze it via microarray. A number of different array configurations and methods for their production are known to those of skill in the art and are described in for instance, 5445934; 5532128; 5556752; 5242974; 5384261 ; 5405783; 5412087; 5424186; 5429807; 5436327; 5472672; 5527681 ; 5529756; 5545531; 5554501 ; 5561071; 5571639; 5593839; 5599695; 5624711 ; 5658734; and 5700637.

Microarray technology allows for measuring the steady-state mRNA or miRNA level of thousands of genes simultaneously providing a powerful tool for identifying effects such as the onset, or modulation of a stress- or inflammatory-related response. Two microarray technologies are currently in wide use, cDNA and oligonucleotide arrays. Although differences exist in the construction of these chips, essentially all downstream data analysis and output are the same. The product of these analyses are typically measurements of the intensity of the signal received from a labeled probe used to detect a cDNA sequence from the sample that hybridizes to a nucleic acid sequence at a known location on the microarray. Typically, the intensity of the signal is proportional to the quantity of cDNA, and thus mRNA or miRNA, expressed in the sample cells. A large number of such techniques are available and useful. Preferred methods can be found in 6271002; 6218122; 6218114; 6004755; and Keene et al. (2006) RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts Nature Protocols 1 :302-307. Analysis of the expression levels is conducted by comparing such signal intensities. This is best done by generating a ratio matrix of the expression intensities of genes in a test sample versus those in a control sample. For instance, the gene expression intensities from a diseased tissue can be compared with the expression intensities generated from normal tissue of the same type. A ratio of these expression intensities indicates the fold-change in gene expression between the test and control samples.

The selection can be based on statistical tests that produce ranked lists related to the evidence of significance for each gene's differential expression between factors related to a stress- or inflammatory-related response. Examples of such tests include ANOVA and Kruskal- Wallis. The rankings can be used as weightings in a model designed to interpret the summation of such weights, up to a cutoff, as the preponderance of evidence in favor of one class over another. Previous evidence as described in the literature may also be used to adjust the weightings.

Gene expression profiles can be displayed in a number of ways. The most common is to arrange raw fluorescence intensities or ratio matrix into a graphical dendogram where columns indicate test samples and rows indicate genes. The data are arranged so genes that have similar expression profiles are proximal to each other. The expression ratio for each gene is visualized as a color. For example, a ratio less than one (down-regulation) appears in the blue portion of the spectrum while a ratio greater than one (up-regulation) appears in the red portion of the spectrum. Commercially available computer software programs are available to display such data including "GeneSpring" (Silicon Genetics, Inc.) and "Discovery" and "Infer" (Partek, Inc.).

In the case of measuring protein levels to determine gene expression, any method known in the art is suitable provided it results in adequate specificity and sensitivity. For example, protein levels can be measured by binding to an antibody or antibody fragment specific for the protein and measuring the amount of antibody-bound protein. Antibodies can be labeled by radioactive, fluorescent or other detectable reagents to facilitate detection. Methods of detection include, without limitation, enzyme-linked immunosorbent assay (ELISA) and immunoblot techniques.

The gene expression profiles of this invention can also be used in conjunction with other non-genetic diagnostic methods useful in diagnosis, prognosis, or treatment monitoring. For example, in some circumstances it is beneficial to combine the diagnostic power of the gene expression based methods described above with data from conventional Markers such as serum protein Markers. In one such method, blood is periodically taken from a patient and then subjected to an enzyme immunoassay for a serum Markers such as albumin. When the concentration of the Marker suggests the likelihood of a stress- or inflammatory-related response, a sample source amenable to gene expression analysis is taken. This approach can be particularly useful when other testing produces ambiguous results.

Kits made according to the invention include formatted assays for determining the Biomarker expression. These can include all or some of the materials needed to conduct the assays such as reagents and instructions and a medium through which Biomarkers are assayed.

Articles of this invention include representations of the Biomarker expression useful for treating, diagnosing, prognosticating, and otherwise assessing diseases. These profile representations are reduced to a medium that can be automatically read by a machine such as computer readable media (magnetic, optical, and the like). The articles can also include instructions for assessing the gene expression profiles in such media. For example, the articles may comprise a CD ROM having computer instructions for comparing gene expression profiles of the portfolios of genes described above. The articles may also have gene expression profiles digitally recorded therein so that they may be compared with gene expression data from patient samples. Alternatively, the profiles can be recorded in different representational format. A graphical recordation is one such format. Clustering algorithms such as those incorporated in "DISCOVERY" and "INFER" software from Partek, Inc. mentioned above can best assist in the visualization of such data.

Different types of articles of manufacture according to the invention are media or formatted assays used to reveal gene expression profiles. These can comprise, for example, microarrays in which sequence complements or probes are affixed to a matrix to which the sequences indicative of the genes of interest combine creating a readable determinant of their presence. Alternatively, articles according to the invention can be fashioned into reagent kits for conducting hybridization, amplification, and signal generation indicative of the level of expression of the genes of interest for predicting or monitoring a stress- or inflammatory-related response. The following Examples are provided to illustrate but not limit the invention. All references cited herein are hereby incorporated herein by reference. Example 1

Extracorporeal photopheresis is an innovative immune cell therapy approved in the United States for the palliative treatment of cutaneous T cell lymphoma (CTCL) and in Europe for the treatment of CTCL and immune-mediated conditions, including graft vs. host disease, systemic sclerosis, rheumatoid arthritis, Crohn's disease, HIV, and solid organ transplantation. Su ri et al. (2006). Therakos photopheresis employs a closed-loop, sterile, patient-connected, point of care apheresis device that withdraws and isolates approximately 3-5% of circulating peripheral blood mononuclear cells (PBMC). The collected cells are treated ex vivo with the UV sensitizing agent 8-methoxypsoralen (UVADEX®), which is then activated by exposure to UV-A and returned promptly to the patient (See Figure 1 ). ECP treated cells subsequently die via apoptosis (a form of programmed cell death). The intravenous reinfusion of apoptotic cells (AC) has been shown to down-regulate immune responses through the modulation of cytokines, the generation of tolerogenic dendritic cells (DCs), and the generation of Treg. Meloni et al. (2007). Example 2 The anti-inflammatory action of ECP in mouse models

We have shown that ECP reduces inflammatory processes associated with OVA-induced airway hyper-responsiveness in mice. As shown in Figure 2, increasing doses of ECP-treated cells have a dose-dependent ability to reduce airway constriction (left panel) and inflammatory pathology (immune cell infiltrates, right panel) after antigenic challenge of OVA-sensitized mice. ECP dose and treatment frequency have been studied in both of these mouse models. Collectively, the data demonstrate that increasing ECP cell dose and treatment frequency is associated with better outcomes, especially if ECP is given before the establishment of significant immune responses. Example 3 ECP in mouse models of Diabetes

The effectiveness of ECP in mouse models of diabetes has been reported. Marks et al. (1991). The former studies showed a reduced incidence of diabetes in NOD/Lt mice and cyclophosphamide-treated NOD/Wehi using weekly delivery of ECP-treated cells. Our experience with ECP in murine models of diabetes is similar. NOD/LtJ mice treated once weekly with 10 million cells, beginning at 11 weeks of age, after pancreatic damage is established, and mice begin to show evidence of frank diabetes. Turley et al. (2003). Nevertheless, ECP treated animals have lower blood glucose levels than untreated NOD controls (Figure 3, top panel). The inset in this figure shows that the rate of diabetes in untreated animals at 20 weeks of age is 60%; while only two of nine (22%) of the ECP treated animals are diabetic. ECP treated animals are healthier in other respects. Polydipsia and polyurea are reduced or absent on ECP treated mice, and ECP-treated animals gain weight normally. Untreated NOD/LU mice, however, show a pronounced weight loss (Figure 3, lower panel, ~8% average loss from peak average weight). Example 4

We performed studies to examine the ability of ECP to prevent pancreatic damage in a multiple low-dose Streptozotocin (STZ) model of T1 DM. As in the NOD model, blood glucose levels are lower than controls in mice treated with 10 million ECP-treated cells on days -7, -3 and 0 or days -3, 0, and +2 relative to administration of the first dose of STZ. The results of this experiment show that ECP can provide substantial protection from chemically induced diabetes (p≡0.1 , n=5-7). Figure 4. Example 5 Induction of immune tolerance by ECP

Apoptosis of cells is an extremely common event, even in healthy organisms, and serves important functions in development and the maintenance of tissues. Krysko et al. (2006). Apoptosis and other forms of programmed cell death are distinguished in many ways from the necrotic death that can result from damage or infection. One such distinction is critically important with regard to the immune system. Cell death that involves the release of otherwise intracellular contents, as in necrosis, tends to evoke an inflammatory response. This process, where "danger signals" lead to inflammation, is contrasted by the homeostatic system whereby AC are cleared by phagocytes without eliciting an inflammatory response. Matzinger (2002). In fact, the clearance of AC elicits the opposite response, immune tolerance. The mechanisms that drive this process are still unclear, but recent data highlights two key components of tolerance induction.

AC are cleared by the patient's resident antigen presenting cells (APC, e.g., iDC and macrophages). This process rapidly clears AC, and leads to the engagement of cell surface receptors that signal to actively inhibit DC maturation, and reduce the expression of co- stimulatory molecules and pro-inflammatory cytokines. Mahnke et al. (2003); and Morelli et al.

(2003). Also, there is an increase in anti-inflammatory cytokine production and induction of Treg, all of which mediate immune tolerance.

These biological properties of apoptotic cells are routinely exploited therapeutically with ECP, limiting DC activation/maturation and thereby down-regulating detrimental T cell responses. The current hypothesis explaining the mechanism of action of ECP is outlined in Figure 5. ECP is believed to elicit immunomodulatory effects, enlisting endogenous systems to effect repair and resolution.

Example 6

In vitro studies of the MOA of ECP

Evidence in support of this mechanism is found in studies examining the modulation of DC Function by ECP-treated cells in vitro. Quiescent iDC stimulated to mature in vitro with CD40 ligand produce reduced levels of IL-12 when co cultured with ECP-treated cells and stimulated with bacterial lipopolysaccharide (LPS, Figure 6, left panel). Similar results are seen for other pro-inflammatory cytokines including IL-1β, IL-6, and TNFα. Conversely, the addition of ECP-treated cells to activated DC cultures stimulates increased production of the anti- inflammatory cytokine TGFβ (Figure 6, right panel). Thus the anti-inflammatory effect is double- edged, altering both pro- and anti-inflammatory processes. Example 7 In vivo studies

A model of ECP has been developed in mice, allowing the combination of live animal studies with in vitro models. For example, APC isolated from the spleens of mice administered ECP-treated AC intravenously demonstrate a reduced capacity to stimulate a MLR when co- cultured with allogeneic T cells. Similarly, allogeneic human T cells do not proliferate as robustly when immature DCs and T cells are co-cultured with ECP-treated cells. Thus AC "feeding" induces a tolerogenic phenotype in DCs, reducing their capacity to stimulate T cell responses (see figures 14 and 15).

There is evidence in mice and humans that AC infusion induces Treg. Maeda and co- workers showed in a model of contact hypersensitivity that immune modulation of ECP is mediated by Treg. Maeda et al. (2005). Kleinclauss and colleagues reported that intravenous delivery of apoptotic cells induces TGFβ-dependent Treg cell expansion in a murine model of graft-versus-host disease. Kleinclauss et al. (2006). In humans, up-regulation of CD4⁺CD25⁺ cells is seen in solid organ transplant patients treated with ECP. Lamioni et al. (2005

We have shown that ECP-treated cells can drive Treg generation in vitro. Strobl et al. (2006).

Treg maintain peripheral tolerance by actively inhibiting immune responses. Tang et al. (2006).

Co-culture of ECP-treated cells with naϊve human T cells for 6-8 days leads to generation of a T cell population that is capable of suppressing T cell proliferation and IFNγ production in a MLR.

This is illustrated in figure 7. ECP-generated Treg express an anergic phenotype, which can be reversed by addition of IL-2.

Example 8

Systemic stress responses and ECP-induced immune tolerance

T1 DM results from the autoimmune destruction of insulin-producing pancreatic β cells by T cells and macrophages that infiltrate pancreatic islets. Barker (2006). A key factor in this destruction of β cells is the breakdown of immune tolerance. Currently, there are a number of immunosuppressive therapies being tested that target diabetogenic T cell responses. Cyclosporin, hOKT_3Ti (Ala-Ala), anti-CD3, and Rapamycin, which target T cells, have shown efficacy in clinical studies of patients with T1DM. Staeva-Vierira et al. (2007). Many of these strategies, however, induce chronic immunosuppression or have associated toxicities that limit their use. Therapies that induce tolerance and specifically control self-reactive T cells are an attractive option for prevention of T1DM. Battaglia et al. (2006). In particular, therapeutic strategies that target the action of T reg in vivo offer an attractive option for therapy.

It has now been shown that ECP acts as a low-level environmental stressor, invoking a response akin to an acute phase response. The therapeutic effect of ECP in this scenario arises from a form of "stress conditioning," where ECP induces adaptive responses to prevent additional damage and aid in the recovery. Despite common disease-causing end points in type-1 and -2 diabetes (i.e., β cell apoptosis) different factors drive the final outcome in each manifestation of diabetes. Eizirik et al. (2001); and Cnop et al. (2005). Environmental stressors play a role in both diseases, suggesting that bolstering stress responsiveness might alter sub cellular pathogenic processes. Dahlquist (2006); Ludvigsson (2006); and Knip et al. (2005). ECP is thus affective in treating diseases that arise from maladaptation to sustained stress, including diabetes. Environmental and cellular stressors have significant impacts on immune function. The central nervous system (CNS) has been shown to modulate the immune response, pancreatic function, and the susceptibility of cells to diabetogenic agents, demonstrating the close connection between systemic stress responses and cellular effects. Flesner (2005); Ader et al. (1992); Morrell et al. (1988); and Coskun et al. (2004). Links between the immune- and central nervous systems are well established, and are most often illustrated by suppressive effects of chronic stress on immunity. Forgotten, however, are the hard-wired connections of the "inflammatory reflex," whereby infection and tissue damage acutely activate components of the fight or flight response, temporarily enhancing innate immunity. Baumann et al. (1994); and Baumann et al. (1990). This interplay between major regulatory systems offers opportunities for malfunction and disease, but also for intervention and cure. In this section we present evidence that ECP can modulate systemic stress response systems and thereby alter immune function.

The archetypal systemic stress response involves the activation of the Hypothalamic- Pituitary-Adrenal (HPA) axis and other neuroendocrine pathways. These are well-understood processes, leading to the predictable systemic responses of the fight or flight response. Most commonly viewed as a response to external stimuli (e.g., combat or escape from predation) the activation of these pathways is essentially identical when the stressor arises from within. For example, the activation of neuroendocrine stress responses by psychological perceptions (and misperceptions) can generate the same physiological reaction as the hypothetical lion in the bushes, and impact the immune system in the same manner. Rohleder et al. (2006); Jara et al. (2006); Leonard (2005); and Gold et al. (2005).

Stress and inflammation are energy intensive processes. We examined the impact of ECP treatment on endocrine markers of energy metabolism in mice. We performed multiplex immunoassays on the Luminex platform, examining 59 analytes concurrently. Consistent with our stress-conditioning hypothesis, circulating concentrations of numerous analytes is affected by ECP in a dose-dependent manner that varies with the treatment schedule. The bulk of this work was done in a mouse model of pulmonary inflammation, whereby ECP "pre-treatment" was tested as a prophylactic for the prevention of inflammation from subsequent antigenic challenges.

In this study animals were sensitized and later challenged with OVA to elicit an asthma- like response. Physiological testing and tissue collection were performed 72 hours after the inflammatory challenge {i.e., 96 hours after the last dose of ECP-treated cells). Various ECP regimens were tested for their ability to reduce inflammation. As shown previously in figure 2, ECP leads to a cell dose-dependent reduction in airway hyper-responsiveness and lung inflammation in this model. Figure 8 shows results from a separate experiment. Once weekly doses of 50 million ECP-treated cells were given at varying times during the 22 days between sensitization and challenge. Here, all three ECP regimens were effective to different extents as prophylactic therapies, reflected in several different measures of physiology and inflammatory pathology.

Unexpectedly, ECP-induced changes in the profile of inflammatory biomarkers resembled an acute phase response (APR). Serum C-reactive protein (CRP), growth hormone (GH), von Willebrand factor (vWF), and haptoglobin were all altered in an ECP-dependent fashion. Not shown, but also affected by ECP are the positive acute phase proteins (APP) leptin, leukemia inhibitory factor (LIF), insulin, and fibrinogen, which are upregulated in response to tissue damage, infection, and other stressors. Baumann et al. (1994). Leptin, in addition to its roles in modulating fat mass and satiety, is a putative pro-inflammatory adipokine. Materese et al. (2005). Additional pro-inflammatory, vasoactive, and hemostatic proteins known to be altered by systemic inflammatory responses showed ECP-responsiveness, including tissue factor (TF), factor VII (F7), monocyte chemotactic protein-1 (MCP-1).

It is apparent from these data that all ECP regimens affected these APP, and that the timing of ECP administration is critical. Four treatments of equal cell dose had a greater impact on biomarkers when given on consecutive days just prior to OVA challenge than when spread out over the three-week period between sensitization and challenge. Remarkably, treating the mice on the day of OVA challenge and the next two days had the greatest impact on most of these ECP biomarkers. Interestingly, biomarker profiles of earlier regimens, where up to 6 doses of ECP were given no later than 11 days after sensitization, were indistinguishable from the once-weekly regime regardless of the number of treatments. Again, those profiles resembled an APR. Similar cell dose- and regimen-dependent effects on serum biomarkers were reproduced in other mouse models of inflammatory disease. The diversity of those models, which included contact hypersensitivity, low dose LPS, allogeneic tracheal ring transplant, and even healthy mice, suggests that this is a generalized phenomenon resulting from ECP rather than the inflammatory insult of a given model. An APR would be expected to increase metabolic demands, so we focused on relevant APP biomarkers that reflect increased metabolism. Richardson et al. (2003). Insulin, which is known to modulate the expression of APP, and glucagon, another APP, were measured in pulmonary inflammation model. Campos et al. (1992). Compared to the untreated mice, serum insulin and glucagon were reduced in ECP-treated mice 72 hours after intranasal OVA challenge (Figure 9, n=18-21 , p<0.01 and 0.05, respectively).

72 hours after OVA challenge, ECP-induced biomarker profiles are not different from the negative control, while untreated animals show elevations in both insulin and glucagon. These biomarker profiles, occurring simultaneously with treatment-related reductions in inflammation, led us to propose that 72 hours post challenge samples reflect the recovery phase of treatment groups in this model, with ECP treated animals recovering more quickly than controls. This theory was tested directly by sampling at earlier time points in the same model (Figure 10).

These results show that ECP induces increases in circulating insulin (left panel) and glucagon (right panel) when compared to negative controls. These analytes rise in tandem in both positive control and ECP-treated animals relative to negative control animals. As seen in figure 9, both biomarkers return to baseline by 72 hours in ECP-treated mice, but no indication of resolution is seen in untreated control animals at that time.

This simultaneous rise in two pancreatic hormones with opposing effects on energy metabolism at first appeared contradictory. Insulin is widely held to be an anabolic hormone, while glucagon is typically viewed as a driver of catabolism. Reports from the early 1970's demonstrated synergy between insulin and glucagon in aiding the recovery of rats from partial hepatectomy, and subsequent studies showed increased plasma concentrations of epinephrine, norepinephrine, glucagon, and corticosterone after endotoxin infusion or cecal ligation and puncture, two models of sepsis. Bucher et al. (1973). Increases in plasma glucagon in those models was later correlated with the appearance of TNF-α, IFN-γ, and IL-6, leading to the suggestion that glucagon may alter hepatic glucose metabolism to manage simultaneous needs for hyperglycemia and increased tissue glucose uptake during inflammation. Bucher et al. (1975).

LPS and cytokines are also known to increase leptin secretion(54). We examined serum leptin and saw it increase, albeit with a later peak that remains elevated for at least a week in this model (Figure 11). These ECP-dependent effects on serum leptin also resemble an APR. We next examined the leptin profile in the same samples depicted in figure 10 to determine the time course of leptin release. Again, leptin is increased over baseline in both the untreated and ECP-treated mice. Consistent with published profiles, leptin peaks later in the APR than early response genes like IL-1β and TNFα. Bornstein et al. (1998); and Granowitz et al. (1999).

Therefore, these endocrine effects as solely a reflection of the energy consumed during inflammation and its resolution, in other words, rather than a mechanism-based result, these findings are merely an indirect consequence of ECP activity. Although insulin is purportedly an anti-inflammatory hormone, the increases in GH and leptin seemed inconsistent with an antiinflammatory therapeutic response. Viardot et al. (2007). This prompted us to consider an alternative hypothesis.

Considering these data, we proposed that ECP itself induces a mild, transient proinflammatory effect, not unlike an APR, that might serve as a therapeutic "conditioning stress." This was addressed experimentally. ECP treated cells were infused into healthy mice and the same biomarkers were examined. Results in figure 12 show that, insulin, leptin, and glucagon are elevated in ECP-treated mice compared to negative controls. The time-course of these changes shows that the peak concentrations appear 24 hours after treatment, and resolve almost completely by 48 hours. This is different from the time course seen in inflammatory models, where ECP-induced changes peak 48 hours after the last ECP treatment.

Cortisol (CORT, corticosterone in rodents) is another acute phase reactant that is correlated with leptin in the APR. Faggioni et al. (1999). CORT was also measured in this experiment, and rises in tandem with the other three biomarkers. Corticosteroid release like that shown here is a classic sign that the fight or flight response has been activated. Harbuz (2002). CORT thus provides a crucial link to systemic stress responses and the central nervous system, suggesting a mechanism whereby infusion of AC into the peripheral circulation affects many disparate tissues and diseases.

To strengthen the link between these markers and the MOA of ECP, we next examined biomarker dose-response relationships in healthy mice. Figure 13 shows the effect of infusing different numbers of AC on CORT (left panel), insulin (middle), and leptin, (right panel). All samples were collected 24 hours after ECP. As occurs with other mechanism-proximal biomarkers, these hormones change in proportion to exposure, here reflected in the cell dose. Colburn (2003). We have thus provided evidence that ECP induces systemic stress responses in mice that are reminiscent of the classic fight or flight and acute phase responses. In the next section we link ECP to cellular stress responses and present a mechanistic model for ECP-induced stress conditioning. Example 9 Molecular and cellular evidence linking ECP to stress responses

Inherent in the idea that a systemic stressor can effect changes in organs, tissues, and cells is a mechanism by which systemic stress induces those reparative responses. Abundant evidence shows that stress hormones affect energy metabolism in response to fight or flight stimuli, re-apportioning ready sources of energy away from less essential systems. These systemic responses to catecholamines and other stress mediators arise from receptor-mediated events at the cellular level, leading to adaptive changes at the organismal level.

Individual cells respond to local environmental changes in a manner that mirrors systematic stress responses. Cells make fundamental changes in their energy use and resource distribution to best adapt to a given challenge with what can be described as a sub-cellular fight or flight response. The former strategic path leads to cellular adaptation, whereby structural and functional changes "join in battle" to meet newly altered environmental demands. The latter (flight) elicits cellular alarm signals, leading to localized or systemic inflammation. In addition to repair programs, these responses can elicit the apoptotic death of cells that fail to adequately adjust. Marciniak et al. (2004). This form of "flight" makes their resources available to the organism for the restorative process.

The systemic version of this response leaves room for short-term error, but cells are extremely conservative in this regard. Even slight delays in passing damage response checkpoints causes apoptosis in normal cells. This has been called the Samurai law of cell biology, where "it's better to be dead than wrong". Skulachev (2001). To maintain homeostasis, these repair and adaptive responses to ECP must occur in every affected cell, and arise after the detection of stress-induced environmental changes. Failure to meet either of these two homeostatic endpoints is an underlying cause of diabetes and other chronic diseases. Marciniak et al. (2006).

The process of protein translation, particularly ribosome biogenesis and peptide elongation, is among the most energy intensive processes in a cell. To engage a stress and attempt repair, a cell typically arrests the translation of "non-essential" proteins, shifts towards catabolic energy use, and builds increased biosynthetic capacity. Given the frugality of nature, systems that carry out this function in the face of normal environmental fluctuations in resources seem likely to serve additional roles in maintaining homeostasis in the face of stressors. At least three nutrient-sensitive systems have been identified that fulfill this role. The ER-dependent unfolded protein response (UPR) and ER-independent responses mediated by the mammalian target of Rapamycin (mTOR) and the hexosamine biosynthetic pathway (HBP) are activated in response to diverse stressors. In this section we describe these pathways and introduce evidence implicating them in ECP activity. The Unfolded protein response

Protein-folding diseases like T1 DM result from dysfunctions in cellular stress responses, especially the evolutionarily conserved "unfolded protein response" (UPR). Harding et al. (2004). This stereotypical program, sometimes called the endoplasmic reticulum (ER) stress response, is driven by ER-resident proteins and typically enables a return to cellular homeostasis. This process, when repeated among the affected cells of a given tissue, can drive the recovery of that tissue from environmental insults. The UPR plays a role in healthy tissues as well, since external signals that increase demands on a normal cell can initiate a UPR. For example, acute binding of antigen to the B cell receptor activates the cell and drives its development and expansion into antibody-secreting plasma cells. This requires a major "re-tooling" of the biosynthetic machinery of the cell, and serves as a stressor that initiates the UPR. Iwakoshi et al. (2003).

The UPR is a rapid response system, and mediates the phosphorylation of the eukaryotic initiation factor 2α (EIF2α) to arrest translation. Harding et al. (2003). This component of the translational machinery is necessary for the initiation of "cap-dependent translation," which drives the production of most cellular proteins. Kozak (1999). A subset of genes are translated by a cap-independent system using internal ribosomal entry sites (IRES) to translate mRNA. Hellen et al. (2001). Such IRES translation is found in genes that drive the UPR, typically encoding biosynthetic enzymes, transmembrane channels and transporters that increase the availability of raw materials, proteins involved translational and post-translational quality control, and antioxidant defense systems. Komar et al. (2005); and Lin et al. (2007). At least four distinct pathways lead to phosphorylation of EIF2α to initiate different components of the UPR. For example, up-regulating antioxidant capacity and certain downstream UPR response genes is driven by a process known as the integrated stress response (ISR), and mediated by activating transcription factor 4 (ATF4). PERK, is an EIF2α- inactivating kinase that drives the inhibition of cap-dependent translation, leading to decreased demands for ATP in translation and folding of nonessential proteins. IRE1 and ATF6 initiate additional UPR responses to up-regulate specific genes involved in protein secondary structure modulation and the degradation of misfolded translation products. Extreme acute ER stress or prolonged activation of these pathways, as in overloaded or otherwise stressed pancreatic β cells, leads to ER-dependent apoptosis by several different pathways. Szegezdi et al. (2006). Failure of these reparative and apoptotic events in the face of continued ER stress may be an underlying factor in the pathology of UPR diseases. One objective of this proposal is to better understand the role that the UPR plays in the modulation of immune tolerance in vitro.

The HBP and mTOR are nutrient- and environmental stress sensors that mediate UPR-independent cellular stress responses

Cap-dependent translation is also regulated by another stress responsive signaling pathway, independent of UPR signals. Significantly, most of these alternative effects on translation are mediated by mTOR, also known as the Target of Rapamycin, a phosphatidylinositol 3-kinase-related protein kinase central to the control of cell growth and proliferation. Sarbassov et al. (2005). mTOR lies downstream of numerous nutrient and growth factor-sensing signaling pathways, and controls critical components of the protein biosynthetic machinery to alter cell size and division, ribosome biogenesis, and nutrient utilization. Phosphorylation of key components of translation initiation complexes by mTOR allows cap- dependent translation to proceed.

The nutrient- and stress-sensitive nature of mTOR signaling plays a key role in ensuring that cellular processes appropriately match their environment. Nutrient-sensitive pathways are known to alter mTOR activity, or the localization of its targets, adding another level of complexity to translational control. Upstream signals include Akt, a survival kinase effector of insulin and growth factor receptors, and AMPK, a sensor of energy availability in the cell. Reioling et al. (2006). Figure 14 shows evidence linking AC engulfment to mTOR signaling. DC were co- cultured with AC at different AC: DC ratios (denoted along the abscissa). Cells were incubated for various times, and S6 kinase phosphorylation, a measure of mTOR activity, was measured by flow cytometry. Hinton et al. (2004). Both panels show a cell dose-dependent affect in iDC and mDC. Interestingly, there is a significant DC maturation state-dependent difference in these responses.

The UPR and mTOR pathways are directly affected by nutrient availability and indirectly affected by stress. Carreterp eta; (2007). The hexosamine biosynthetic pathway is a third such system. The HBP is activated in direct proportion to the intracellular concentration of glucose. Love et al. (2005). The end result of HBP activation, an increase in the modification of proteins with O-GlcNAc, results in symptoms associated with diabetes. Amdra;o eta; (2007). A cell can thus sense and respond to the availability of intracellular glucose and to a variety of cellular stressors using this pathway. One consequence of HBP activation is adaptation. The HBP drives this response, and mediates stress conditioning. Stress conditioning via nutrient sensing pathways

Stressors and glucose increase HBP activity and increase resistance to subsequent stress. Zachara et al. (2004). Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the first and rate-limiting enzyme in the HBP, and plays a role in glucose and satiety sensing. Cooksey et al. (2002). GFAT-mediated generation of UDP-N-Acetylglucosamine (UDP-GIcNAc) provides this essential precursor for post-translational N- and O-l inked glycosylation of proteins. Buse (2006). One such reaction involves the addition of the monosaccharide GIcNAc to serine and thereonine resides of mature proteins. This is driven by O-GlcNAc transferase (OGT) a key enzyme in the HBP, and can both alter protein tertiary structure and the limit accessibility of those Ser/Thr residues and neighboring sites to protein kinases. Yang (2005). These events can cause modulate the participation of OGT substrates in certain signaling cascades and alter their localization in the cell. The latter consequence is due to the alteration of protein-protein interactions, notably with lectins and cellular chaperones. Guinez et al. (2004). This can affect their ability to participate in gene regulation and/or translation. Prominent examples of this are seen in the inhibitory effects of HBP activation on proteasomal activity and on the localization of various transcription factors. Gao et al. (2003). Hyperglycemia increases HBP activity, and increases the O-GlcNAc content of NeuroDI , a pancreatic transcription factor. This affects the localization of NeuroDI , thus increasing insulin gene transcription. Although a direct link between the HBP and the UPR has not been described, the HBP can alter translation, at least indirectly. The HBP has been shown to alter signaling downstream of mTOR, affecting the cellular localization of alpha4. This protein is an mTOR substrate that complexes with and regulates certain phosphatases to modulate p70S6 kinase and components of the JAK/STAT signaling pathway. Dauphinee et al. (2005). Since alpha4 lacks a nuclear localization signal, its distribution between the cytoplasm and nucleus is controlled by protein- protein interactions. Disruption of these interactions occurs with the modifications resulting from HBP activation, causing alpha4 to remain cytosolic and unable to modulate translation.

These nutrient-sensing systems are sensitive to stress and, like the UPR, can alter cellular functions to match the demand for and availability of resources. In another similarity with ER stress-responsive systems, transient modulation of mTOR and the HBP protects against subsequent stressors. For example, a variety of stressors alter HBP activity in vitro, and pharmacological mimics of HBP activation have an adaptive conditioning effect. Rapamycin has recently been shown to induce a preconditioning effect as well, protecting against subsequent pro-apoptotic stressors, most likely through its effects on p70S6 kinase and autophagy. Ravikumar et al. (2006). This is especially relevant in diabetes, which is linked to both the HBP and mTOR activity. Marshall (2006). The mTOR pathway occupies a critical control point for nutrient-mediated signals and plays a role in pancreatic development and function. Bussier et al. (2006).

Further evidence linking nutrient/stress responses to immune function was obtained using the MLR. We used drugs known to inhibit the two key enzymes in the HBP, OGT and O- GlcNAc-selective N-acetyl-beta-D glucosaminidase (O-GlcNAcase), which add and remove the O-GlcNAc moiety from proteins, respectively. We examined MLR in the presence of either Streptozotocin (STZ) or PUGNAc, two antibiotics known to inhibit O-GlcNAcase and increase the amount of cellular proteins with the O-GlcNAc modification. Arias et al. (2004). In both cases, the inhibitors potentiated ECP, increasing the tolerogenic nature of AC. This occurs at drug concentrations well below the toxic range for DC and T cells. Figure 15 shows results using PUGNAc. This drug has no appreciable effect on the MLR itself across all doses tested, reflected in the absence of inhibition of proliferation in the assay. When AC are added, however, ECP-induced "tolerogenesis" is increased by up to about 50% above this typical AC response (~30% inhibition, see OmM PUGNAc result). This finding was reproduced using STZ, another inhibitor of O-GlcNAcase.

Further evidence implicating the HBP in immune function is seen when the opposing enzyme, OGT, is inhibited using Alloxan (AIIx, figure 16). Alloxan has an opposite effect on the MLR compared to PUGNAc, and affects mDC and iDC differentially. Remarkably, AIIx reproducibly potentiates proliferation in the MLR when iDC are used as APC (left panel). Alloxan has essentially no effect on the MLR when using mDC (not shown). Additional preliminary data analyzing alamar blue reduction as a measure of viability showed Allx-induced increased proliferation of T cells in culture across the ranges tested (figure 16 right panel). See Table 1

Table 1: List of transcripts most affected by ECP

Change Gene Description/Annotation down CD 126 Il6ra interleukin 6 receptor, alpha down Ornithine decarboxylase antizyme 2 Oaz2 inhibitor of cell growth and proliferation down RAS-like, estrogen-regulated, growth-inhibitor Rerg inhibitor of cell growth and proliferation down Aminoadipate-semialdehyde synthase Aass Lysine catabolism down GATA binding protein 5 Gataδ Transcription factor down Growth arrest specific 1 Gas1 Cell cycle inhibitor down Sodium/glucose co transporter Slc5a1 Glucose transport down deoxyribose-phosphate aldolase-like Dera glycolytic enzyme down Meningioma expressed antigen 5 (hyaluronidase)Mgea5 O-GlcNAcase up cytohesin Pscd3 Golgi structure and function up Ataxia telangiectasia and rad3 related Atr DNA damage responsive PI3K up non-SMC condensin Il complex, subunit H2 Ncaph2 epigenetics up proteasome subunit, alpha type 6 Psmaδ Proteasome up Pentatricopeptide repeat domain 2 Ptcd2 Stress responsive gene of unknown function up GRP94 Hsp90b1 Heat shock protein/antigen presentation up Embryonic large molecule derived from yolk sac elys Transcription factor up Rearranged L-myc fusion sequence RIf transcriptional regulation up Splicing factor 3b, subunit 3 Sf3b3 spliceosome up Set domain containing 4 Setd4 epigenetics: lysine methyltransferase

Seeking additional insights into the cellular and molecular MOA of ECP, we used Affymetrix gene expression arrays and standard bioinformatics approaches to examine mRNA profiles in various tissues from mouse models of inflammation. In secondary lymphoid organs, we observed significant changes in expression within a set of genes that often change as part of programmed cellular stress responses. As illustrated in Table 1 , 96 hours after the last AC infusion, ECP altered the expression of genes involved in metabolic pathways, genes involved in protein synthesis and processing, and stress-responsive pathways (e.g., O-GlcNAcase). This further suggests that stress response pathways are modified by ECP, but additional experiments will be needed to assess the role of individual genes in the ECP response. Example 10 Therapeutic stress conditioning

Protein folding diseases include the most prevalent chronic diseases and are associated with high degrees of morbidity and mortality. Xu et al. (2005). Effective treatment for most of these conditions is limited, owing in large part to our poor understanding of their underlying etiologies. It is increasingly apparent that many of the debilitating and ultimately fatal sequelae of protein folding diseases include an inflammatory component. Yoshida (2007). Existing therapies are typically aimed at limiting inflammation without altering its underlying cause. Prominent examples of this approach include steroids, NSAIDs, and anti-cytokine therapies. In the case of UPR diseases and their complications.

Newer strategies will likely target underlying causes of that inflammation (e.g., prevention of protein aggregates or acceleration of their clearance). Boyce et al. (2005). Most, if not all of these therapies, will employ exogenous therapeutic compounds or biological materials that may have associated systemic toxicity. ECP offers a novel approach in that it uses endogenous resources to fundamentally alter both the disease-causing dysfunction and its resultant inflammation. We hypothesize that ECP can both prevent and resolve complications of stress- related UPR diseases through its ability to modify stress response pathways.

Increasing the stress capacity of a cell allows it to repair and clear its damaged cellular components, and can help resolve disease. Badin et al. (2006). This has been demonstrated using over-expressed heat shock proteins or "chemical chaperones" to aid in the secretion of mis-folded proteins, and by chemically modifying the cellular milieu to improve ER function. Imaizumi et al. (2001 ). Whereas these strategies have shown success in protein over- expression systems and in cell lines, they typically must be optimized for a given mis-folded protein. Stress conditioning regimens offer a more global approach, enlisting a variety of cellular repair systems, simultaneously relieving different types of cellular stress.

Simultaneous manipulation of cellular stress response systems is an established experimental therapy. Two decades of research has seen the publication of well over 4000 papers on ischemic conditioning alone. Transient ischemia is the most commonly studied conditioning regimen, and experiments have shown efficacy in cardiovascular medicine, neurology, and solid organ transplantation. Kuntscher et al. (2005). Those studies show that ischemia-reperfusion (I/R) injury can be dramatically reduced by pre- or post-treatment with brief periods of ischemia and recovery. This approach is still experimental, but its value is supported by epidemiology, since a reduction in tissue damage after stroke or myocardial infarction is seen if preceded by transient ischemic attacks or unstable angina, respectively. Liberato et al. (2005). Conditioning regimens also have therapeutic value when heterologous stressors are employed, suggesting that any therapeutic conditioning regimen might offer protection from a diverse array of stressors. Remarkably, it has also been shown that conditioning of a distant organ or tissue can confer protection to an entire organism (an effect known as remote conditioning). Conditioning has been examined in clinical trials in cardiac surgery, but its uptake by physicians has been very limited, most likely due to the inherent danger of existing conditioning agents and methods. ECP Acts as a Stress Conditioning Regimen

Clinical experience showing the timing of treatment responses, the preservation of immune function, and the generation of Treg in patients suggested that ECP offers a novel therapeutic approach to mitigating inflammation. The preliminary data described here suggest to us that the therapeutic action of ECP elicits an adaptive response by acting as a low-level stressor. Pharmacological and biochemical evidence implicates systemic and sub-cellular stress response systems, some novel, that modulate immune tolerance. ECP-induced "stress- conditioning" thus takes advantage of a well-described adaptive phenomenon, habituation, of cells, organs, and organisms. Conditioning confers protection to subsequent, more intense stressors, and can elicit long lasting, protective changes in the structure and function of cells and tissues. These lasting changes, in addition to protecting from subsequent stress, can drastically affect disease progression and accelerate repair processes.

Certain predictions arise from this hypothesis, including increased cellular and organismal resistance to a variety of stressors. We have demonstrated that ECP protects mice from antigenic challenge in pulmonary models of asthma, contact hypersensitivity, and organ transplant. Resistance to chemical stress, specifically the diabetogenic effects of STZ (figure 17, left panel) is a dramatic extension of the therapeutic utility of ECP. At the cellular level, we demonstrate that DC pretreated with AC tolerate Anisomycin-induced ribotoxic stress better than untreated controls (figure 17, right panel). ECP thus demonstrates the characteristics that define preconditioning regimens. Of note, the NOD/LtJ data presented above shows that ECP is protective even after significant pancreatic damage has occurred. Those experiments offer an example of post-conditioning using ECP.

The above phenomena, when considered together, point to the potential value of stress conditioning as a new therapeutic approach for UPR diseases. If, as we believe, ECP is a stress conditioning therapy, it will have invaluable utility in preventing and treating stress-induced diseases. This novel concept arose after we recognized connections that link several otherwise disparate fields of research. Specifically, recent publications link stress conditioning at the cellular level with the cellular and systemic stress responses described above. In one study, for example, macrophage cell lines were pre-conditioned against a variety of stressors, enhancing survival and preserving function in culture. Lu et al. (2004). This combined with the UPR-HPA axis connection mentioned above suggests that modifying cellular stress responses can, in addition to resolving underlying causes of pathology, protect from subsequent challenges. Conditioning effects can be mediated by immune cells

It is well established that immune cells are pathogenic mediators of ischemic injury; however, their role in ischemic preconditioning is not clear. Adoptive transfer of spleen cells from ischemically pre-conditioned mice into T cell deficient recipients protects recipient mice from subsequent ischemic injury. Ascon et al. (2006). These studies strongly suggest that immune cells are mediators of protective ischemic preconditioning responses. The protective cell was not identified in this study. However, as mentioned above, there is precedent for generation of Treg as a result of systemic stress responses. MacConmara et al. (2006). It is therefore reasonable to propose that a regulatory T cell population arose after the initial ischemia, allowing transferred Treg to confer protection in the recipients.

Example 11

Thrombospondin-1 links stress responses, Treg, and ECP

Thrombospondin-1 (TSP-1) a matricellular protein that contributes to both tissue structure and cell function, is massively up-regulated in most cells in response to injury or tissue damage, growth factors, nutritional conditions, and various environmental stressors. Murphy-Ullrich (2001 ). Matricellular proteins, including TSP-1 , are thought to provide contextual information to cells about their environment. This is relevant in immune function, since TSP-1 is a potent antiinflammatory protein that is secreted by and modulates antigen-presenting cells and T cells. Doyen et al. (2003). The immunomodulatory properties of TSP-1 , combined with the fact that TSP-1 secreted from cells in response to stress, positions this molecule between stress and immunity. Here we show that TSP-1 is a potential effector for ECP.

TSP-1 binds to CD36 on APC and to CD47 and α4β1 on T cells. Chen et al. (2000). TSP- 1 enhances the ability of phagocytes to engulf apoptotic cells and inhibits their maturation and ability to produce pro-inflammatory cytokines. In T cells, TSP-1 has been shown to down regulate TCR signal transduction as well as responsiveness to IL-12. Li et al. (2002). TSP-1 is a major physiological activator of TGFβ and recently was shown to induce a Foxp3⁺ Treg phenotype in activated human CD4^*CD25^" T cells. Grimbert et al. (2006) Treg induction can contain and resolve inflammation and its damage. In TSP-1 null mice, delayed wound healing, reduced anti-inflammatory responses, and decreased levels of TGFβ are seen. Agah et al. (2002).

In the pancreas, islet cells express TSP-1 , and TSP-1 null mice demonstrate inflammation, islet cell hyperplasia, and acinar hypoplasia, all of which were attributed to a lack of TGFβ activation. Crawford et al. (1998). The pancreas of TSP-1 null mice systemically treated with a TSP-1 -derived peptide reverted to a more normal phenotype. These observations suggest that TSP-1 is an important regulator of pancreatic development and may be important for immune and exocrine processes within this microenvironment. Interestingly, hyperglycemia was recently linked to the expression of TSP-1 in the vasculature of Zucker diabetic rat. Raman et al. (2007). The up regulation of TSP-1 was directly linked to the HBP, and TSP-1 expression was proportional to glucose concentrations. Given these data, it is a strong possibility that TSP- 1 -modulating therapies would interfere with the progression of pathology in diseases like diabetes.

We, and others, have shown that TSP-1 is secreted in large quantities from apoptotic cells. As shown in Figure 18, a cell dose-dependent increase in TSP-1 is seen after ECP- treatment of PBMC. Monocytes secrete the bulk of the TSP-1 produced in ECP-treated PBMC suspensions, and blocking TSP-1 activity reduces the ability of ECP to drive Treg generation. This is consistent with our own, additional unpublished results showing that monocytes are essential for in vitro generation of Treg.

Figure 19 shows that, as in figure 7, T cells isolated from co-cultures of ECP-treated PBMC with naive T cells display a tolerogenic phenotype. If, however, this co-culture includes neutralizing anti-CD36 or anti-CD47 monoclonal antibodies, alone or in combination, the tolerogenic phenotype is blunted. We are currently investigating whether TSP- 1 is necessary for mediating the immunological effects driven by ECP in vivo, and we propose to investigate whether TSP- 1 plays a role in ECP protection of β cell function in models of diabetes.

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Sequences

H6ra attagcctgtccgcctctgcgggaccatggagtggtagccgaggaggaagcatgctggccgtcggctgcgcgctgctggctgccctgctggccgcgccgggagc ggcgctggccccaaggcgctgccctgcgcaggaggtggcgagaggcgtgctgaccagtctgccaggagacagcgtgactctgacctgcccgggggtagagc cggaagacaatgccactgttcactgggtgctcaggaagccggctgcaggctcccaccccagcagatgggctggcatgggaaggaggctgctgctgaggtcggt gcagctccacgactctggaaactattcatgctaccgggccggccgcccagctgggactgtgcacttgctggtggatgttccccccgaggagccccagctctcctgc ttccggaagag∞ccctcagcaatgttgtttgtgagtggggtcctcggagcaccccatccctgacgacaaaggctgtgctcttggtgaggaagtttcagaacagtcc ggccgaagacttccaggagccgtgccagtattcccaggagtcccagaagttctcctgccagttagcagtcccggagggagacagctctttctacatagtgtccatgt gcgtcgccagtagtgtcgggagcaagttcagcaaaactcaaacctttcagggttgtggaatcttgcagcctgatccgcctgccaacatcacagtcactgccgtggc cagaaacccccgctggctcagtgtcacctggcaagacccccactcctggaactcatctttctacagactacggtttgagctcagatatcgggctgaacggtcaaag acattcacaacatggatggtcaaggacctccagcatcactgtgtcatccacgacgcctggagcggcctgaggcacgtggtgcagcttcgtgcccaggaggagttc gggcaaggcgagtggagcgagtggagcccggaggccatgggcacgccttggacagaatccaggagtcctccagctgagaacgaggtgtccacccccatgc aggcacttactactaataaagacgatgataatattctcttcagagattctgcaaatgcgacaagcctcccagtgcaagattcttcttcagtaccactgcccacattcct ggttgctggagggagcctggccttcggaacgctcctctgcattgccattgttctgaggttcaagaagacgtggaagctgcgggctctgaaggaaggcaagacaag catgcatccgccgtactctttggggcagctggtcccggagaggcctcgacccaccccagtgcttgttcctctcatctccccaccggtgtcccccagcagcctggggt ctgacaatacctcgagccacaaccgaccagatgccagggacccacggagcccttatgacatcagcaatacagactacttcttccccagatagctggctgggtgg caccagcagcctggac

Oaz2 gcggcggcggcggcggcggcggtgacggccgggaagggtcagttggaggcaggcgctcgctgaggcaaaaggaggcgctcggcccgcggcctgacagg gacttagc∞gcagagatcgaccccgcgcgcgtgaccccacacccacccactcatccatctatccactccctgcgccgcctcctcccaccctgagcagagccgc cgaggatgataaacacccaggacagtagtattttgcctttgagtaactgtccccagctccagtgctgcaggcacattgttccagggcctctgtggtgctcctgatgccc ctcacccactgtcgaagatccccggtgggcgagggggcggcagggatccttctctctcagctctaatatataaggacgagaagctcactgtgacccaggacctcc ctgtgaatgatggaaaacctcacatcgtccacttccagtatgaggtcaccgaggtgaaggtctcttcttgggatgcagtcctgtccagccagagcctgtttgtagaaat cccagatggattattagctgatgggagcaaagaaggattgttagcactgctagagtttgctgaagagaagatgaaagtgaactatgtcttcatctgcttcaggaagg gccgagaagacagagctccactcctgaagaccttcagcttcttgggctttgagattgtacgtccaggccatccctgtgtcccctctcggccagatgtgatgttcatggtt tatcccctggaccagaacttgtccgatgaggactaatagtcatagaggatgctttacccaagagccacagtgggggaagaggggaagttaggcagccctggga cagacgagagggctcctcgctgtctagggaaggacactgaggggctcagggtgagggttgcctattgtgttctcggagttgactcgttgaaattgttttccataaaga acagtataaacatattattcacatgtaatcaccaatagtaaatgaagatgtttatgaactggcattagaagctttctaaactgcgctgtgtgatgtgttctatctagcctag gggaggacattgcctagagggggagggactgtctgggttcaggggcatggcctggagggctggtgggcagcactgtcaggctcaggtttccctgctgttggctttct gttttggttattaagacttgtgtattttctttctttgcttcctgtcaccccaggggctcctgagtataggcttttcagtccctgggcagtgtccttgagtlgttttttgacactcttacc tgggcttctctgtgtgcatttgcgtctggcctggagtaagcaggtccgacccctcgttctttacagcttagtgttattctggcatttggttaagctggcttaatcttgtttaatgtt atcagtacattttaaataggggcattgaaatttactcccaccaccagggcttttttgggggatgcctgggcctttaaaacactagccaaactctaattaattctcaaatc actgccaggagttcttgctcctggctgcaggcccaggccccaaggtctccttcttggggtcacaaacagcagtaaggaagaggaatatatagcaactcagggcct gggaattgtggggcaatccgttcttagggactggatacttctggctggctgagtatagtactagctgcctccccaccaggttccgagtagtgtctgagactctgctctgc agggcctagggtagcgctgggagtgtagaagtggcctgcccttaactgttttcactaaacagctttttctaaggggagagcaagggggagagatctagattgggtg agggggacggggatgtcagggaggcaagtgtgttgtgttactgtgtcaataaactgatttaaagtt

Rerg cgccgggccactggcacttgcttctgcggcgagtcccacccacgaccgcagcccagcaactcgcaaacgcaacctgaagcctgggctgcgcagtgtgggagg gcttcgcgatcttgggggacccattccgaacttgcagaggaccgtagctctcctggcctggagagtgtgaacaggattgtggactcttccaagattcacaatgatatg gtgaatccaaagactggaaccaaaaagatttactcagtgctttagttttaacaacagtaaattgtctaccaacacccatcatggctaaaagtgcggaggtcaaactg gcaatatttgggagagcaggcgtgggcaagtcagctcttgtagtgagatttctgaccaaacggttcatctgggaatatgatcccaccctcgaatcaacctaccgaca ccaagcaaccatcgatgatgaagttgtttccatggagatactagacactgctggtcaggaagataccattcagagggaggggcacatgcgatggggggaaggc tttgtgctggtctacgacattactgaccgaggaagttttgaggaagtgctgccacttaagaacatcctagatgagatcaaaaagcccaagaatgtgactctcatcttg gttggaaacaaagctgacttggaccactccaggcaggttagcacagaagaaggagagaagctggccacagaattggcttgtgctttttacgagtgctctgcctgc actggagaagggaacatcacagagatattctatgaattgtgtcgagaggtgcgtcgccggaggatggtgcagggcaagacgaggcgacgcagctccaccacg catgtcaagcaagccattaacaagatgctcaccaaaatcagtagttaggcagcccagctgaggtggaccaactaattggaaacactcttccccttctgttcccctttc aaaaataaaacaaaatattgcattctttgtttggattctgagaaatgtctgggcttcccattgtttctggcctctaataggttgggaagttttagcgtgttttatgcaatttcagt gctaacaatttcttcctttcctgcttgaataagatacactctaatggcatttgaacatgtaatcaccagagattctgaaatgactggtttatgttaagctatttttaggcatctt caccttgctttaagtaggttgaagtttttgcaaaggcatttaaaaattcaatttcttgtcagatactacaaataattttcttaaaagtctaagatagcagaaaatacagtaa aaacacaggagaagaagctgagctattggaacaggaaatagaaggaactctagtttctgtttgaagtgaggattttctgaattatctaatatcatctaggttttctttaa aattttattttgttcttcagttcaagcatcttctcactaatgtttttcactataacagagaattcatttcaatttgagttggttctctcaatgatctattgatcattacaccctaactct ccttccttggctcaaacaatattttccctataacaaaggcaataggacacaaaattcacatcctgctgggccttttttcatcaagtcagggtgatataaaaacattgga agtcttttcaccaaaccctgactttattgaatgctagtagaagatgtagaattagagacatctgatttgtttatcaccttagcagaaaaaccacagtccaaaagacaag caaattaagaatggagcttaaccatgcctccattgggaagtctagactttgagccaggtacagtaagaaaaattagcctctgattcattaagtttgccacatgacttatt ttgatattttggatacattaactcacttaggagaattcagaaaagaatgggtgattaaagttcattacagctgaataaatgtgtctaaaacagactcttgtattctgaaag tacagtctacaactgataaaaccttatgattcttttctcccccattatgcccctatatatatcaagatttgggtactttattttagtagaaaatatatatcttttacatatgtatgta tttataaatgcatagatatatgtataaaaatttgtaagcgttagcggcattaattcaccaatgcatttggacaacttgatgtaactgactttattttatgtgactataataaaa agcataattttctcattctgtcaaaaaaaaaaaaaaaaaaaaaaaa

Aass gactcggaagattcgaggcggcgggggacaagtcggcgccccagagcggacgagtcaccaggtgtcaagatgctgcaagtacataggactggactgggca ggctgggggtcagcctctccaagggtcttcaccacaaagctgtgttggccgtccggagggaggatgtgaacgcctgggagagaagggccccgctagctcccaa gcacatcaaaggcatcaccaatctgggatacaaggtcttgatacagccttcgaatcggcgggccattcatgataaggactatgtcaaagctggtggcattcttcag gaggatatttctgaagcttgtctaattttaggagttaaaagacctccagaggaaaaattaatgtccaggaagacttatgcatttttctcccacacaataaaagctcagg aggccaatatgggcttgttggatgagattctaaaacaggaaattcgccttattgattatgagaaaatggtggatcatagaggagtacgggtagtggcatttggacagt gggctggtgtggcaggaatgatcaacattttacatggaatgggtttaaggctccttgctttgggacatcacacaccttttatgcacattggcatggctcataactacagg aatagcagtcaggctgtgcaagctgtccgtgatgctggctatgaaatatctttgggtttgatgcctaagtcaataggacccttaacatttgtgttcacaggaactggtaa tgtttctaagggagcccaagcaatctttaatgagctaccttgtgaatatgtggagccccatgaattaaaagaagtttcccaaactggagacctcagaaaagtgtatg ggacggtgttaagtcgtcatcatcatcttgtcaggaaaacagatgctgtgtatgatcctgcagagtatgacaaacatccggagcgctacataagtcgttttaatactga tattgcaccctatacaacttgcttaartaatggaatctactgggaacaaaacactcctcgcctcctaacccgccaagatgctcagagtctcctggctccgggcaagtt ctcacctgctggtgtggaaggctgccctgcattaccacacaaactcgtggcaatatgtgacatttcagctgacacaggagggtctatagagtttatgactgagtgtac aacaatagagcatcccttttgcatgtatgatgcagaccagcatattattcatgacagtgttgaaggctcggggatcctgatgtgttccattgacaatttgccggcacag ctcccaattgaagctacagaatgctttggagacatgctttacccttatgttgaagaaatgatattatcagacgcgacacagcctcttgaaagtcagaatttttctcctgtg gtgagagatgcagtgattacatccaacggtacattacctgataaatataaatatatccagacactccgggagagcagggaacgtgctcagtcactttcaatgggca ccaggagaaaggttttggttcttggatctggctacatatctgagcctgtattagaatatttatcaagagatggcaatatagaaataacagtaggatctgacatgaaga atcaaattgaacagttaggcaagaaatataatattaatcctgttagcatggacatttgtaaacaagaagagaagctgggcttcttggtggcaaaacaggatcttgtc atcagcttgttgccttatgtattgcaccctcttgtggccaaggcctgcatcacaaacaaagttaacatggtcactgcaagctacatcacaccagcactaaaagaattg gaaaagagtgtggaagatgctggcatcacaatcattggtgaattgggattggaccctggtctggatcacatgttagcaatggaaacaatagataaagccaagga agtgggagccacgattgaatcatatatttcctactgtggtgggcttccagcccctgaacattcaaacaatccattgagatataaatttagctggagtccagtgggagttt tgatgaatgtaatgcagtctgccacctatctgctcgatggaaaggttgtgaatgttgcaggaggcatctcctttcttgatgccgttacgtccatggatttttttccaggatta aatttggaaggctatcctaacagagacagtacgaaatatgctgagatttatggcatttcttctgctcacactttgttgcgggggacactgagatataagggatatatga aagctttgaatggatttgtaaaattaggtcttataaacagagaagcgcttcctgcctttagacctgaggccaaccctctcacctggaaacaactcctctgtgacctagtt gggatttcaccctcctctgagcatgatgtgttgaaggaagctgttcttaagaaactaggaggagacaatacccagttggaggctgctgaatggttgggcttacttggg gatgaacaagttcctcaggcagagtccattctggatgccctctccaagcatttggtcatgaagctttcctatggtcctgaagaaaaagatatgattgtgatgagagac agctttggaatcagacatccttctggacatttagaacataaaacgattgatcttgtggcttatggggacatcaatggcttttcagccatggctaaaaccgtggggttacc caccgccatggcagccaaaatgttgcttgatggtgaaattggagccaaaggcctaatggggcccttttcaaaggagatctatggaccaatattggagcgaattaaa gcagaaggcattatatatactacacagagtacaattaaaccataattgggaattatattttgtttttttcttcccaggcaatacacctctgaacatgtgtgtgataaatggg tttgctaatgtgctgttttaaagtataaagcataatatgttttggttaacacaatgtactttttgaactataaatctttattttaatatggaaatgtttggaacaggagatgcaa gccactaacagagaactttaataattctaccctgtattttataaatacgtatgtgaaagtgatgaa

Gataδ α^ccgccaccgccgtgc∞tgccgccctccctgcccgctggtcaagaccacgcctgggaggatgtaccagagcctggcgctggccgcgagcccccgccagg

∞gcctacgα^actc^ggctccttcctgcac^ctccgggcgccggctctccgatgmgtgccgα^gcgcgc^tcccctcgatgctgtαΛacctgtccgggtgtg agccgagcccgcagccccccgagctcgctgcgcgccccggctgggcgcagacagccaccgcggattcgtcggccttcggcccgggcagtccgcacccccca gccgcgcacccgcccggggccaccgccttccctttcgcgcacagcccctcggggcccggcagcggcggcagcgcggggggccgagacggcagtgcctacc agggcgcgctgttgcctcgagaacagttcgcggccccgcttgggcggccggtggggacctcgtactccgccacctacccggcctacgtgagccccgacgtggc ccagt∞tggactgc^ggcccttcgatggcagcgtcctgcacggcctcccaggccgcaggcccaccttcgtgtccgacttcttggaggagttcccgggtgagggt cgtgagtgtgtcaactgcggggccctgtccacaccgctgtggcgccgagacggcaccggccactacctgtgcaatgcctgcggcctctaccacaagatgaatgg cgtcaaccggccgctcgttcggcctcagaagcgcctgtcctcgtcccgccgcgccggcctctgctgcaccaactgccacacgaccaacaccacgctgtggcggc ggaactcggagggggagcccgtgtgcaatgcctgcggcctctacatgaagctgcacggggtgccgcggcctctggctatgaagaaagaaagcatccagacac ggaagcggaagccaaagaccatcgccaaggccaggggctcctcaggatccacaaggaatgcctcggcctccccatctgctgtcgccagcactgacagctcag cagccacttcgaaagccaag∞cagcctggcgtccccagtgtgccctgggwx^gcatggccccccaggcctctggccaggaggatgactctcttgcccccgg ccacttggagttcaagttcgagcctgaggactttgccttcccctccacggccccaagcccccaggctggcctcaggggggctctgcgccaagaggcctggtgtgc gctggccttggcctaggtccccaggccagcccatgtcaggggaacagcctggaacagaccacccactgagtcacctccgtgcctgcittgctccagcacagcag agaccagcaggccccccaacccagagactgggtctgctggagtctccacacagtggtggggaggccttctggacagacggcagtcgggccccagagcaaga aggctggtgagggaagggctcagcttcccaccccacgtacagcaagggactccccaggtgcggcccaaggctccggaccacactggccccctgcggcggag gccaacgcagggcaccaccaccaccaacttgaattccgtcatcaatgctcaccgtcaatatgtttacaagttgtagcagttgggggaaaacagtcaacctcccagt gtaaaaccaagattcccagtgaagcacctgaggccaagcaggggagaggaatgaggggagcagctggacatgggcctcctgaggcctcggggctgtccttc attgcccacatggatagacggagctgtggtgcagagaacttttcccgcaacaggtgcaggactgccagggatcggagtgcgggccgcgcacggtgccaggatt ccgccgaggggaagccgctcacattgcagtcatcacagacttacgcacttgtttggacagtttttccagaggggatgggaaagggccttgttctagctgaatctgtgt atcatgaccatttctgacaggcagaatgaattgtctggtagccctgtcctgacccatccaagcgctgttggggctggtggigacgtggtcacatgtcctggcatatctg gggαacgcagWagtctcttgtcccaggagaattgttagtgacccctctttctcttgcaagccccctccacactgggttggatgataccttaatgagtgacgctggcg agaggcaccctacccgacgcagctgtgaatggccggtgatgtatgtcaggaggccacagggagcggaggagcggggcaggcagccacaggggccctgcg gggagcacatcctcgcctccgtccggctgctgcccttcaacaacaagccctgatttttccagcaatgccagaaacctggattttaagtcttccaatttgattcaaaaat atttttaacattgtgagccagctagacccccagtgcaccaccccatattgaaaaacagttgtctggcatcagcttcaggagcgggtccggtcattctgaaactgtccct ccagaggttcttccagccccacttctatgcgatgtcatcttttctaaaagagacaaatgaagccacagggaaagtgaaataaagccttgaacctcaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaa Gas1 agcagccggcacggggacagccggccgcacaacggatctgcaggcgcggagcaaaatgcacccgccgcgccgcgcggtcctgcagccccgccacggcc cc^cggc∞gcacccccccggggcga∞gtgagcctctcccgccaccaccgggggccgagcggagggctctcgggtgggagagcgggaccagatctcga cagctgttcatttccaggaagccaccgcagccagagcgaaaggggaccttctgccaccagcggggcatcagccagcggcgcgcatggatttatgaagacactc atgcaagaagtgggcaggacttggacaaacttttccaccggctccgcgtccgccgctccccgcgcctcgtctcctttcccctcctctcccggcggccgccgctgccc gcgatggtggccgcgctgctgggcggcggcggcgaggcccgcggggggacagtgccgggcgcctggctgtgcctgatggcgctgctgcagctgctgggctcg gcgccgcggggatcggggctggcgcacggccgccgcctcatctgctggcaggcgctgctgcagtgccagggggagccggagtgcagctacgcctacaacca gtacgccgaggcgtgcgcgccggtgctggcgcagcacggcgggggcgacgcgcccggggccgccgccgccgctttcccggcctcggccgcctctttctcgtcg cgctggcgctgcccgagtcactgcatctcggccctcattcagctcaaccacacgcgccgcgggcccgccctggaggactgtgactgcgcgcaggacgagaact gcaagtccaccaagcgcgccattgagccgtgcctgccccggacgagcggcggcggcgcgggcggccccggcgcgggcggggtcatgggctgcaccgagg cccggcggcgctgcgaccgcgacagccgctgcaacctggcgctgagccgctacctgacctactgcggcaaagtcttcaacgggctgcgctgcacggacgaat gccgcaccgtcattgaggacatgctggctatgcccaaggtggcgctgctcaacgactgcgtgtgcgacggcctcgagcggcccatctgcgagtcggtcaaggag aacatggcccgcctgtgcttcggcgccgagctgggcaacggccccggcagcagcggctcggacgggggcctggacgactactacgatgaggactacgatga cgagcagcgcaccgggggcgcgggtggtgagcagccgctggacgacgacgacggcgtcccgcacccaccgcgcxxgggcagcggcgctgctgcatcggg cggccgcggggacctgccctatgggcctgggcgcaggagcagcggcggcggcggccgcttggcgccccggggcgcctggaccccactcgcctccatcttgct gctgctgcttggg∞gctctWagccctcgcgcccc∞gccgttggctgc^ggagagcccgc^tcccactcccgtgctcgcctcgaccccgcgccgggcacctgt ggcttgggacagatagaagggatggttggggatacttcccaaaactttttccaagtcaacttggtgtagccggttccccggccacgactctgggcacttcccctgaa gctcctctccggagcttgacttcttggacctcctcccccgccccaattccaagctccagaaactcccaactcgtctgccgtccagaaagctagctgcagtgttcagga cgtccgggaggaagcaagcatgtgggggacagaacagtagtcctggactcgaaagggaaggtgctgaccagtggggccttagcaatttgaagggttgggaag gaggaattatatttgcaaaggggctgtctattagcatatttcctttgagggggcaaaaaaaagtgccagtatcgacttttacagattgtggccagtgaggatattataat cctatgtaaacagaaaagtcccacttaccgattcattctttcactgtttgtatctgcgcccagaattctcagtgacgtgggggtgagggtgggtggcgattgccttagag ggaacccctaaattggttttggataagtttgagcccttgaccttaatttcattgctaccactctgatctcttagcacatttcttaggattaagggtccaaaaatgctgatcta aggggttgccatggtgttgaacaatgcaactttttatttaaaaaagctctgcactgccatgtatgaaagtctctttatgatgtttgtttttttgtcatttttgttctttacatcaaga aattttatgtttaaatatgcggagaatgtatattgcctctgctcctatcagggttgctaaaccctggtacatcgtatataaaatgtattaaaactggggtttgttaccagttgc tgtactttgtatatagaatttttataaattgtatgcttcagaaataatttatttttaaaaagaaattaaaagttttaaactcacatccatattacacctttcccccctgaaatgtat agaatccatttgtcatcaggaatcaaaacccacagtccattgtgaagtgtgctatatttagaacagtcttaaaatgtacagtgtattttatagaattgaagttaacattctt attttcaagagaatttatggacgttgtagaaatgtacaaatgcatttccaaactgccttaaacgttgtatttttatagacatgtttttttaaaaatcctaagtttttaaataacta tggatttgtgtattttttttggttatttgttttattaaaacatgtacatcagtaaagagttttaaacaatga Slc5a1 cgctgccaccatggacagtagcacctggagccccaagaccaccgcggtcacccggcctgttgagacccacgagctcattcgcaatgcagccgatatctccatc atcgttatctacttcgtggtagtgatggccgtcggactgtgggctatgttttccaccaatcgtgggactgttggaggcttcttcctggcaggccgaagtatggtgtggtgg ccgattggagcctccctctttgctagtaacattggaagtggccactttgtggggctggccgggactggggcagcttcaggcatcgccattggaggctttgaatggaat gccctggttttggtggttgtgctgggctggctgtttgtccccatctatattaaggctggggtggtgacaatgccagagtacctgaggaagcggtttggaggccagcgga tccaggtctacctttcccttctgtccctgctgctctacattttcaccaagatctcggcagacatcttctcgggggccatattcatcaatctggccttaggcctgaatctgtattt agccatctttctcttattggcaatcactgccctttacacaattacagggggcctggcggcggtgatttacacggacaccttgcagacggtgatcatgctggtggggtctt taatcctgactgggtttgcttttcacgaagtgggaggctatgacgccttcatggaaaagtacatgaaagccattccaaccatagtgtctgatggcaacaccacctttca ggaaaaatgctacactccaagggccgactccttccacatcttccgagatcccctcacgggagacctcccatggcctgggttcatctttgggatgtccatccttaccttg tggtactggtgcacagatcaggtcattgtgcagcgctgcctctcagccaagaatatgtctcacgtgaagggtggctgcatcctgtgtgggtatctaaagctgatgccc atgttcatcatggtgatgccaggaatgatcagccgcattctgtacacagaaaaaattgcctgtgtcgtcccttcagaatgtgagaaatattgcggtaccaaggttggct gtaccaacatcgcctatccaaccttagtggtggagctcatgcccaatggactgcgaggcctgatgctatcagtcatgctggcctccctcatgagctccctgacctcca tcttcaacagcgccagcaccctcttcaccatggacatctacgccaaggtccgcaagagagcatctgagaaagagctcatgattgccggaaggttgtttatcctggt gctgattggcatcagcatcgcctgggtgcccattgtgcagtcagcacaaagtgggcaactcttcgattacatccagtccatcaccagttacttgggaccacccattgc ggctgtcttcctgcttgctattttctggaagagagtcaatgagccaggagccttttggggactgatcctaggacttctgattgggatttcacgtatgattactgagtttgctta tggaaccgggagctgcatggagcccagcaactgtcccacgattatctgtggggtgcactacttgtactttgccattatcctcttcgccatttctttcatcaccatcgtggtc atctccctcctcaccaaacccattccggatgtgcatctctaccgtctgtgttggagcctgcgcaacagcaaagaggagcgtattgacctggatgcggaagaggag aacatccaagaaggccctaaggagaccattgaaatagaaacacaagttcctgagaagaaaaaaggaatcttcaggagagcctatgacctattttgtgggctag agcagcacggtgcacccaagatgactgaggaagaggagaaagccatgaagatgaagatgacggacacctctgagaagcctttgtggaggacagtgttgaac gtcaatggcatcatcctggtgaα^tggctgtcttttgccatgcatatmgcctgagtcctaccttttgctgtagatttacx^tggctggactcttactcaccttcctttagtctc gtcctgtggtgttgaagggaaatcagccagttgtaaattttgcccaggtggataaatgtgtacatgtgtaattataggctagctggaagaaaaccattagtttgctgtta atttatgcatttgaagccagtgtgatacagccatctgtacctactggagccgcagaagggagtccactcagtcacatccagaaaaaggcagactaagaatcaga agccatgtgattgatgtctgacgtgagtctgtctcaggtagattccgggtgtcagtgtggtttataatccttgaatattgttttagaaactttggtctccctggttcctgccactt ttcctgtccgtcctcctccccattttttttttaaaagaaagctgttttcccctc

Dera cccgggcccgcgactccgccccgggagcgggcaggggcggggcgagcgctccagctggcgggaaggaggaagggccgggcgcggcgcagaggcggg cgcctaccagccggcagctccggagctgcccgcgccatgtccgcgcacaatcggggcaccgagctcgaccttagctggatctccaaaatacaagtgaatcacc cggcagttctgaggcgtgcggaacaaatccaggctcgcagaaccgtgaaaaaggagtggcaggctgcttggctcctgaaagctgttacctttatagatcttactac actttcaggtgatgatacatcttccaacattcaaaggctctgttataaagccaaatacccaatccgggaagatctcttaaaagctttaaatatgcatgataaaggcatt actacagccgccgtttgtgtttatcccgcccgggtgtgtgatgctgtaaaagcactcaaggctgcaggctgtaatatccctgtggcatcagtggccgctggatltccag ctggacagactcatttgaagacacgattagaagagatcagattggctgtggaagatggagctacagaaatcgacgtggtaattaacagaagcttggtgctgacag gccagtgggaagccctgtatgatgagattcgtcagtttcgcaaggcctgtggggaggctcatcttaaaactatattagcgacaggagaacttggaactcttactaatg tctataaagccagtatgatagcaatgatggcaggatcagattttattaagacctctactggaaaagaaacagtaaatgccaccttcccggtagctatagtaatgctgc gggccattagagatttcttctggaaaactggaaacaagatagggtttaaaccagcaggaggcatccgcagtgcaaaggattcccttgcttggctctctcttgtaaag gaggagcttggagatgagtggctgaagccagaactctttcgaataggtgccagtactctgctctcggacattgagaggcagatttaccatcatgtgactggaagat atgcagcttatcatgatcttccaatgtcttaaatcagtcaccagttccagaaaagttctttacgacaatgtttaaaaattatttttctacgtaattgctaaaattatttaattaa aaaattgggcagtaggtaactggcattcctctctttaaaatttctaccgaacttaatggaatggaaaaagcaaactcatccacatgtggtactcatttcaggcacatct gaaatgatcttaattactagaagatcfgcactattaactttgtgaagagtttctcctaaaaactttaagtaaaatgttaatggtagctttgataacatcaaattctaaggga gaaaaaaacaatattaaaccgcccaagcagtgtgccctagcagaggaaaatgcaacatctcgcaagcgctgctgtaacgacttcaggagtcactgattcagca ctaatttcctgctgtgaaaactcatctttcatttttgccgtggataggcgcttttattaattgttgtcctaatgaaatttctgacattgtcatatacaacgatgaatatcattaaa atttttaaaataataaagttcctatagtttatttttttaaaaaacttaaaaattgttacaatacataatgaaaaaataatccattaaacataaaaagaggtttgatcagtga aaaaaaaaaaaaaaaaa

Mgea5 aggcagcagcagagggagagctcggggcttggaggggaaacagcggaagacctaagattatcgggagggcagcagaggcagagaacgaggacaggac ccttggccgtcttcttccagggaacgagaggtcacagcctcgctctccgcttatgcttctggcgccccagcttaaagccgaggctgcggctgacaaagggctcgcg ccggtgccgccgcccttctcatccgggcattcgggtccctgcgagaaggagggggaaggacagagggggaggggaaggagccggaggggcgcacacttgg agctgaagccctctccagggctccgggccggtgccccaacggacagaggtcgaggaggacccgcagaggtggcagcggccgggggcaggaggatggtgc agaaggagagtcaagcgacgttggaggagcgggagagcgagctcagctccaaccctgccgcctctgcgggggcatcgctggagccgccggcagctccggc acccggagaagacaaccccgccggggctgggggagcggcggtggccggggctgcaggaggggctcggcggttcctctgcggtgtggtggaaggattttatgg aagaccttgggttatggaacagagaaaagaactctttagaaggctccagaaatgggaattaaatacatacttgtatgccccaaaagatgactacaaacataggat gttttggcgagagatgtattcagtggaggaagctgagcaacttatgactctcatctetgctgcacgagaatatgagatagagttcatctatgcgatctcacctggattg gatatcactttttctaaccccaaggaagtatccacattgaaacgtaaattggaccaggtttctcagtttgggtgcagatcatttgctttgctttttgatgatatagaccataat atgtgtgcagcagacaaagaggtattcagttcttttgctcatgcccaagtctccatcacaaatgaaatctatcagtacctaggagagccagaaactttcctcttctgtcc cacagaatactgtggcactttctgttatccaaatgtgtctcagtctccatatttaaggactgtgggtgaaaagcttctacctggaattgaagtgctttggacaggtcccaa agttgtttctaaagaaattccagtagagtccatcgaagaggtttctaagattattaagagagctccagtaatctgggataacattcatgctaatgattatgatcagaag agactgtttctgggcccgtacaaaggaagatccacagaactcatcccacggttaaaaggagtcctcactaatccaaattgtgaatttgaagccaactacgttgctat ccacacccttgccacctggtacaaatcaaacatgaatggagtgagaaaagatgtagtgatgactgacagtgaagatagtactgtgtccatccagataaaattaga aaatgaaggcagtgatgaagatattgaaactgatgtactctatagtccacagatggctctaaagctagcattaacagaatggttgcaagagtttggtgtgcctcatca atacagcagtaggcaagttgcacacagtggagctaaagcaagtgtagttgatgggactcctttagttgcagcaccctctttaaatgccacaaccgtagtaacaaca gtttatcaggagcccattatgagccagggagcagccttgagtggtgagcctactactctgaccaaggaagaagaaaagaaacagcctgatgaagaacccatgg acatggtggtggaaaaacaagaagaaacggaccacaagaatgacaatcaaatactgagtgaaattgttgaagcgaaaatggcagaggaattgaaaccaatg gacactgataaagagagcatagctgaatcaaaatccccagagatgtccatgcaagaagattgtattagtgacattgcccccatgcaaactgatgaacagacaaa caaggagcagtttgtgccaggtccaaatgaaaagcctttgtacactgcggaaccagtgaccctggaggatttgcagttacttgctgatctattctaccttccttacgag catggacccaaaggagcacagatgttacgggaatttcaatggcttcgagcaaatagtagtgttgtcagtgtcaattgcaaaggaaaagactctgaaaaaattgaa gaatggcggtcacgagcagccaagtttgaagagatgtgtggactagtgatgggaatgttcactcggctctccaattgtgccaacaggacaattctttatgacatgtac tcctatgtttgggatatcaagagtataatgtctatggtgaagtcttttgtacagtggttagggtgtcgtagtcattcttcagcacaattcttaattggagaccaagaaccctg ggcctttagaggtggtctagcaggagagttccagcgtttgctgccaattgatggggcaaatgatctcttttttcagccacctccactgactcctacctccaaagtttatac tatcagaccttattttcctaaggatgaggcatccgtgtacaagatttgcagagaaatgtatgacgatggagtgggtttaccctttcaaagtcagcctgatcttattggag acaagttagtaggagggctgctttccctcagcctggattactgctttgtcctagaagatgaagatggcatatgtggttatgccttgggcactgtagatgtgacccccttta ttaaaaaatgtaaaatttcctggatccccttcatgcaggagaagtataccaagccaaatggtgacaaggaactctctgaggctgagaaaataatgttgagtttccat gaagaacaggaagtactgccagaaactttccttgctaatttcccttctctgataaagatggacattcacaaaaaagtaactgacccaagtgtggccaaaagcatga tggcttgcctcctgtcttcactgaaggctaatggctcccggggagctttctgtgaagtgagaccagatgataaaagaattctggaattttacagcaagttaggatgtttt gaaattgcaaaaatggaaggatttccaaaggatgtggttatacttggtcggagcctgtgacatttgttgacactgtgaactgtccaaaagtctcttaactgcaccttgtg aatggtagttgaggtcttcatacagttcagcctctagaatggtaacaaatcagccaattggattcgaaacaaagaagactatgtaaaactcacccatcacactttga gactactcactggttggaagaatatagtattgcagcaaatcctgtatgaaagagagatgtgggcttcctttttgagtcttgtgttaggtgctgagaccttttacatgggctt atacagggagagagtcttcaataaatgtagtcagcactattttctgcatccagtgtggttgcgtttctcacctgagagtaatcaagataacatctgtcatcttccttggttta ttgagtgaaatgcctctcagtcttaggggacatggcagagatgaaagaaagaaagagtgggtttcagaagtgtcagggtggagtgattccaagtgggatggttgtg gcattagtttaagctgaataaataatttcaatttggggcagttattctgctttttgtaaagccgtggccaattgtctcctgtaatgactgttggttcaggcatgttgtactttgta gggacaaatgtgcatttgtttgtggcaaaagcctacaattgacaaacttgtaaatttctttgtatataaactagctgtaacctgactatcctttgtgtttactgtttttgtaaattt ttttcctctataaatgaaagggtgttggttcagaatggcactttgaataatgtaaaccagtgaaaagtggattttctttacttttgtctttgggtttggggttgtttttgttctttttga agttttattatttttaaagtgcctcccacctaggcgtaggccatgaccatttggggtacgagagcctaattttgtaggacttaatctgttgaaaagtgcagttacttctgga aattaacctcaatattaggtcagcatgtgaaatgttggatttgacatgtcaggtagggttcagggactgattggtcccatttgccctcaggtcagttgtttaatctcaaga

∞tgttactactgattttattaaatcagagtctttaattcttgcatgmgtatctaamctgaacgaatgagcacactttaaccagttatttacagttacctttttcctttaaccgg attgtgaaagcttcatgtattttaatttagattctgtgtttttaagggttctgagcatgaagctggcagatagtcggcaggactcattttttcatcatggctggctgatttctcca tagattgataacagtattttgttatcttgcttctctgtagttttgcatcagctgtttaactttgagctgagtgaggggagaggggtaaagagaaagaaacttaagttttctttc acagaactccaccattgtgggctttgagagagccctaaagcattgtacctagtggtacctagtgacttccaaccaaagcctttgagtatgcactaaataggtgagaa gaaaggagagaaggtttttaggttagaaacctttaaccgatagaaggatatggtatgttgtaaagctggaaccaagtttgcatttttgagggcttgagatgaaggga agactcttaccagatagtaagacagctgagttttcctcagttttctcgtcttaacactagtggacaattctagcattttgtttggaggatttcagagttaacctcatggaattc aggattttttagcaagtttgcttttggttttatcttggcttttagtaatcatgttggctggtctggtcacaggtgactgtgaaacagatgcccctggtcttgctttcatcactctag gatcatgaagtgctatgctatttcctggttatgaatattaaggttggaattacatttttattgattgtttggatcagagctcagttcctgtagaaaacgaactgtaaaagacc atgcaagaggcaaaataaaacttgaagtg

Pscd3 tgaggagccgcccggtcgcctgcgcgctccctccggcggcgtccccagcccgcggcccctctgctgccggcccccggctcgccggctgcgggagtggcctcaa gatggatgaagacggcggcggcgagggtggtggcgtgcctgaagacctctcattagaagagagagaagaacttctagacattcgtcgaagaaaaaaggaact tattgatgacattgagaggctgaaatatgaaattgcagaggtgatgacagagatcgacaatctaacttccgtagaggagagcaaaacgactcagaggaacaaa cagatagccatgggaagaaagaaattcaacatggatcccaaaaagggaattcagtttctaatagaaaatgacctgctacagagttccccagaagacgtcgccc agttcctttataaaggagaaggcctaaataagaccgtcattggggactacctgggtgaaagggatgaatttaatattaaagttcttcaagcctttgttgaactccatga gtttgctgatctcaaccttgtacaagccttaaggcagttcttatggagcttcaggctgcccggggaggcgcagaagattgatcgcatgatggaggctttcgcttctcgct actgcctgtgcaaccccggggtcttccagtccacagacacgtgctacgtgctgtcattcgccatcatcatgctcaacaccagcctccacaaccacaacgtgcgtga caagcccacggcagaacggttcatcgccatgaaccgcggcatcaacgagggcggggacctccctgaggagctgctgaggaatttgtatgagagcattaagaa cgagccatttaagatcccggaggacgacgggaacgacctgacccacaccttcttcaaccccgaccgcgagggctggctcctgaagctgggagggcgtgtgaa gacctggaagcgccggtggttcatcctgaccgataactgcctctattactttgaatacacaacagataaggagcccaggggaatcatcccgttggaaaacctcag catcagggaggtggaggacccccggaaacccaactgttttgagctctacaatcccagccacaaagggcaggtcatcaaggcctgtaagactgaggccgacgg ccgcgtggtagaggggaaccatgtggtgtaccggatctcagccccgagcccggaggagaaggaggagtggatgaaatccatcaaagccagtatcagcagag atcccttctatgacatgttggcaacgaggaaacgaaggattgccaataaaaaatagctttcctggctaaaagacccaggtaaaagacccaaccccagcagaaa gacaccgcgggcggcccctccgcggaaggcgtggcagggaggcagtcgccctgcggtgcaagctgctgctccagagcataccgtggcccaggtggtatcccc aaggcctcgtgccgtggctggggtcctgggaggtggtcgccctgcagtgcaagctgctgctccagagcgtaccgtggcccagactgatcctcgaggcctcctgcc gtggctggggtcatggtcggctgcgcatgtccagaagcatttccttcctgcgaccatcccggcgcccctagggggagaagccaggacagcagcttccgctgtctc cacagcagacacgggacggattccacagacgggagcctcattcgtaccatgccaaacgcattcactcggggcagtattaaccgttctagaaagccactgttttat agcaaaacaggaaaggaaaagctaccagttttttattcagaatttttctcagatatataggattatagcttttatatgcctttttatattctgaaattataacaaaagatactt tctaacagtagtatttttagaatggcagctataaagttaactcctggacacaagtatatactgtgcactgaaaaaatatccatctacacagcacccaaggggagggc tgggggcaccggcacgggggcagcgtgcagccctgccctgtcaggctgtcagacaagccccggggggcagcaggtgggctcgggacgggctgggggagg gacggccatggcacttgggggctccagggtgactcccatgaggcctcccttcaaccaggctttttggccccacaaatactttaagcaaatcattaaaattataacag ttaatggtttgggggtgtttaggctgtaactgctaactcctaggaaacagccttttccctggacacagatggtccatacgctgagccacgtgaaactgctgatgttttgttt agatgcacacacatggcagcgtttcatacaggtcagcaggttagaccggcttttgaccatattcatcgctatttaaaacctgtggcaaaatgaacgcttattttacaga ctttctaatttgac∞gaWcttaatgaatagacacagaattaactaaaaacagtctcaccccatgtagtgcgccgtgtcctgagagaggtgccctccctacgagga gggaagaacaggccctggggtgcagaggcccggcacgtagagaacccagatagacgccggtggtggaactggtcaaactccacgcccgcctgggaggttgt caggttgctgtggatgtaaggataggaggtgcccagtgctccgctcaaggaaggctggatctgggccccacctacagagagggctcagggctggaccggggg cattgtgtgcttgggccgacccgggccggtggcagacgctgttctctgtcgggagatttgcgtccccaggaccctgttacacagtgggctgttgggttggtggctggct tttcctctatggacttcctcttcctgccccacctgcataggcacacacaccttgaatctgcaccctctggagggcatctgtactcctgtgcaaaatgcxx^gtccagag acaaaacctcagactttgtgcacctaggtttccttctcagcagcggagactgttctttgagttgccttgaagtggaggccgagcggctgcgggcccttcgcctccctgc ggctgaccttgatgtagctttaagtcacactagactgcagaggggtccgaggccagaaacccctgtcctgcatcagactttcattcccacgttcttaggctttgttactg atacctcaaatcggaagttttagttctgagaaaggcaagtcagcgttcttgaaatgcctgactggtagatatgcaactctggcctccagtcttccatgaaaataaatgc tgc^tggacccccaαx^gaccacacactgacggccggctccggcggtgcccacccctcaggctggcccggcacccaagactggccacagccagctctgtca gcatgttgtgctcggacaagctgtttccttcttctgaccaacccaggtgtgacctggggatgcagagctttctgttttgggtgttgggagaagcagcaggaaggagtcg ccagatgatcaagctccccctttgctgtcatctgtgaatgagcttcgccaggtggtgggcacctgggagccatgcagaggctgtggtgctgagttagactccaggta ctttgtggtcaaaggaaatcgcctagctccaggctgtgttaggacagtattagcatgaaggctgtgcgaccatcatgcctgctgatccttgaggcaggcctggtccag aaaactctgggtcagtgactgcgcagggccagccgctaccaggacggccctgaaacaggacacatctgttttttgtccctcaccctgggcaggccgcgtcacaat cacagtcctcctcctccccaccctgacgtctgagcgcagggcttgaattgttagtcccaactctggccaaagatacttttttccagagacagaggccaggaggcagt gaggggagccccgcggggaggcggcggcgactgccacagcccttccagcctgtcttgctggccgccctggttcatatttgagtttaattgtactgaccctggaccc agataagcagcaactttgtgtctttggggtcacagaacattttggggcagtttaatgtggtaccaaactgaaaataggagctatttatagatggagcagcacttagtgc ttcatagaaagcaatgcctatttttaaagttacaaacgcagatatctacatagatatgctttgctgagaagttaggtctgtggtagaccagaaaccacaaattgacttttt ttcttagaaaatatttctatttgcggtaaatatagtaatatgtaaataatgtacatctgttgatttctggagtgtctgttattcaatgatgtatatactcccacagctcgcatgaa ggaacagcctctattgatacttggttgtaaagtgaagtaagattggagggtggatggctgtcagagctcttgcagatactgtgttcactaaataaaaatcacatgtatt gttaaaaaaaaaaaaaaaaa

Atr gcctccacacggctccgtcgggcgccgcgctcttccggcagcggtacgtttggagacgccgggaacccgcgttggcgtggttgactagtgcctcgcagcctcagc atgggggaacatggcctggagctggcttccatgatccccgccctgcgggagctgggcagtgccacaccagaggaatataatacagttgtacagaagccaagac aaattctgtgtcaattcattgaccggatacttacagatgtaaatgttgttgctgtagaacttgtaaagaaaactgactctcagccaacctccgtgatgttgcttgatttcatc cagcatatcatgaaatcctccccacttatgtttgtaaatgtgagtggaagccatgagcgcaaaggcagttgtattgaattcagtaattggatcataacgagacttctgc ggattgcagcaactccctcctgtcatttgttacacaagaaaatctgtgaagtcatctgttcattattatttctttttaaaagcaagagtcctgctatttttggggtactcacaa aagaattattacaactttttgaagacttggtttacctccatagaagaaatgtgatgggtcatgctgtggaatggccagtggtcatgagccgatttttaagtcaattagatg aacacatgggatatttacaatcagctcctttgcagttgatgagtatgcaaaatttagaatttattgaagtcactttattaatggttcttactcgtattattgcaattgtgtttttta gaaggcaagaactcttactttggcagataggttgtgttctgctagagtatggtagtccaaaaattaaatccctagcaattagctttttaacagaactttttcagcttggag gactaccagcacaaccagctagcacttttttcagctcatttttggaattattaaaacaccttgtagaaatggatactgaccaattgaaactctatgaagagccattatca aagctgataaagacactatttccctttgaagcagaagcttatagaaatattgaacctgtctatttaaatatgctgctggaaaaactctgtgtcatgtttgaagacggtgtg ctcatgcggcttaagtctgatttgctaaaagcagctttgtgccatttactgcagtatttccttaaatttgtgccagctgggtatgaatctgctttacaagtcaggaaggtctat gtgagaaatatttgtaaagctcttttggatgtgcttggaattgaggtagatgcagagtacttgttgggcccactttatgcagctttgaaaatggaaagtatggaaatcatt gaggagattcaatgccaaactcaacaggaaaacctcagcagtaatagtgatggaatatcacccaaaaggcgtcgtctcagctcgtctctaaacccttctaaaaga gcaccaaaacagactgaggaaattaaacatgtggacatgaaccaaaagagcatattatggagtgcactgaaacagaaagctgaatcccttcagatttcccttga atacagtggcctaaagaatcctgttattgagatgttagaaggaattgctgttgtcttacaactgactgctctgtgtactgttcattgttctcatcaaaacatgaactgccgta ctttcaaggactgtcaacataaatccaagaagaaaccttctgtagtgataacttggatgtcattggatttttacacaaaagtgcttaagagctgtagaagtttgttagaat ctgttcagaaactggacctggaggcaaccattgataaggtggtgaaaatttatgatgctttgatttatatgcaagtaaacagttcatttgaagatcatatcctggaagat ttatgtggtatgctctcacttccatggatttattcccattctgatgatggctgtttaaagttgaccacatttgccgctaatcttctaacattaagctgtaggatttcagatagcta ttcaccacaggcacaatcacgatgtgtgtttcttctgactctgtttccaagaagaatattccttgagtggagaacagcagtttacaactgggccctgcagagctcccat gaagtaatccgggctagttgtgttagtggattttttatcttattgcagcagcagaattcttgtaacagagttcccaagattcttatagataaagtcaaagatgattctgacat tgtcaagaaagaatttgcttctatacttggtcaacttgtctgtactcttcacggcatgttttatctgacaagttctttaacagaacctttctctgaacacggacatgtggacct cttctgtaggaacttgaaagccacttctcaacatgaatgttcatcttcicaactaaaagcttctgtctgcaagccattccttttcctactgaaaaaaaaaatacctagtcc agtaaaacttgctttcatagataatctacatcatctttgtaagcatcttgattttagagaagatgaaacagatgtaaaagcagttcttggaactttattaaatttaatggaa gatccagacaaagatgttagagtggcttRagtggaaatatcaagcacatattggaatccttggactctgaagatggatttataaaggagctttttgtcttaagaatgaa ggaagcatatacacatgcccaaatatcaagaaataatgagctgaaggataccttgattcttacaacaggggatattggaagggccgcaaaaggagatttggtac catttgcactcttacacttattgcattgtttgttatccaagtcagcatctgtctctggagcagcatacacagaaattagagctctggttgcagctaaaagtgttaaactgca aagttttttcagccagtataagaaacccatctgtcagtttttggtagaatcccttcactctagtcagatgacagcacttccgaatactccatgccagaatgctgacgtgc gaaaacaagatgtggctcaccagagagaaatggctttaaatacgttgtctgaaattgccaacgttttcgactttcctgatcttaatcgttttcttactaggacattacaagt tctactacctgatcttgctgccaaagcaagccctgcagcttctgctctcattcgaactttaggaaaacaattaaatgtcaatcgtagagagattttaataaacaacttca aatatattttttctcatttggtctgttcttgttccaaagatgaattagaacgtgcccttcattatctgaagaatgaaacagaaattgaactggggagcctgttgagacaaga tttccaaggattgcataatgaattattgctgcgtattggagaacactatcaacaggtttttaatggtttgtcaatacttgcctcatttgcatccagtgatgatccatatcagg gcccgagagatatcatatcacctgaactgatggctgattatttacaacccaaattgttgggcattttggctttttttaacatgcagttactgagctctagtgttggcattgaa gataagaaaatggccttgaacagtttgatgtctttgatgaagttaatgggacccaaacatgtcagttctgtgagggtgaagatgatgaccacactgagaactggcctt cgattcaaggatgattttcctgaattgtgttgcagagcttgggactgctttgttcgctgcctggatcatgcttgtctgggctcccttctcagtcatgtaatagtagctttgttac ctcttatacacatccagcctaaagaaactgcagctatcttccactacctcataattgaaaacagggatgctgtgcaagattttcttcatgaaatatattttttacctgatcat ccagaattaaaaaagataaaagccgttctccaggaatacagaaaggagacctctgagagcactgatcttcagacaactcttcagctctctatgaaggccattcaa catgaaaatgtcgatgttcgtattcatgctcttacaagcttgaaggaaaccttgtataaaaatcaggaaaaactgataaagtatgcaacagacagtgaaacagtag aacctattatctcacagttggtgacagtgcttttgaaaggttgccaagatgcaaactctcaagctcggttgctctgtggggaatgtttaggggaattgggggcgataga tccaggtcgattagatttctcaacaactgaaactcaaggaaaagattttacatttgtgactggagtagaagattcaagctttgcctatggatlattgatggagctaacaa gagcttaccttgcgtatgctgataatagccgagctcaagattcagctgcctatgccattcaggagttgctttctatttatgactgtagagagatggagaccaacggccc aggtcaccaattgtggaggagatttcctgagcatgttcgggaaatactagaacctcatctaaataccagatacaagagttctcagaagtcaaccgattggtctggag taaagaagccaatttacttaagtaaattgggtagtaactttgcagaatggtcagcatcttgggcaggttatcttattacaaaggttcgacatgatcttgccagtaaaatttt cacctgctgtagcattatgatgaagcatgatttcaaagtgaccatctatcttcttccacatattctggtgtatgtcttactgggttgtaatcaagaagatcagcaggaggttt atgcagaaattatggcagttctaaagcatgacgatcagcataccataaatacccaagacattgcatctgatctgtgtcaactcagtacacagactgtgttctccatgct tgaccatctcacacagtgggcaaggcacaaatttcaggcactgaaagctgagaaatgtccacacagcaaatcaaacagaaataaggtagactcaatggtatct actgtggattatgaagactatcagagtgtaacccgttttctagacctcataccccaggatactctggcagtagcttcctttcgctccaaagcatacacacgagctgtaa tgcactttgaatcatttattacagaaaagaagcaaaatattcaggaacatcttggatttttacagaaattgtatgctgctatgcatgaacctgatggagtggccggagtc agtgcaattagaaaggcagaaccatctctaaaagaacagatccttgaacatgaaagccttggcttgctgagggatgccactgcttgttatgacagggctattcagct agaaccagaccagatcattcattatcatggtgtagtaaagtccatgttaggtcttggtcagctgtctactgttatcactcaggtgaatggagtgcatgctaacaggtccg agtggacagatgaattaaacacgtacagagtggaagcagcttggaaattgtcacagtgggatttggtggaaaactatttggcagcagatggaaaatctacaacat ggagtgtcagactgggacagctattattatcagccaaaaaaagagatatcacagctttttatgactcactgaaactagtgagagcagaacaaattgtacctctttca gctgcaagctttgaaagaggctcctaccaacgaggatatgaatatattgtgagattgcacatgttatgtgagttggagcatagcatcaaaccacttttccagcattctc caggtgacagttctcaagaagattctctaaactgggtagctcgactagaaatgacccagaattcctacagagccaaggagcctatcctggctctccggagggcttt actaagcctcaacaaaagaccagattacaatgaaatggttggagaatgctggctgcagagtgccagggtagctagaaaggctggtcaccaccagacagccta caatgctctccttaatgcaggggaatcacgactcgctgaactgtacgtggaaagggcaaagtggctctggtccaagggtgatgttcaccaggcactaattgttcttc aaaaaggtgttgaattatgttttcctgaaaatgaaaccccacctgagggtaagaacatgttaatccatggtcgagctatgctactagtgggccgatttatggaagaaa cagctaactttgaaagcaatgcaattatgaaaaaatataaggatgtgaccgcgtgcctgccagaatgggaggatgggcatttttaccttgccaagtactatgacaa attgatgcccatggtcacagacaacaaaatggaaaagcaaggtgatctcatccggtatatagttcttcattttggcagatctctacaatatggaaatcagttcatatatc agtcaatgccacgaatgttaactctatggcttgattatggtacaaaggcatatgaatgggaaaaagctggccgctccgatcgtgtacaaatgaggaatgatttgggt aaaataaacaaggttatcacagagcata∞aactatttagctccatatcaatttttgactgctttttcacaattgatctctcgaatttgtcattctcacgatgaagtttttgttgt cttgatggaaataatagccaaagtatttctagcctatcctcaacaagcaatgtggatgatgacagctgtgtcaaagtcatcttatcccatgcgtgtgaacagatgcaa ggaaatcctcaataaagctattcatatgaaaaaatccttagagaagtttgtiggagatgcaactcgcctaacagataagcttctagaattgtgcaataaaccggttga tggaagtagttccacattaagcatgagcactcattttaaaatgcttaaaaagctggtagaagaagcaacatttagtgaaatcctcattcctctacaatcagtcatgata cctacacttccatcaattctgggtacccatgctaaccatgctagccatgaaccatttcctggacattgggcctatattgcagggtttgatgatatggtggaaattcttgctt ctcttcagaaaccaaagaagatttctttaaaaggctcagatggaaagttctacatcatgatgtgtaagccaaaagatgacctgagaaaggattgtagactaatgga attcaattccttgattaataagtgcttaagaaaagatgcagagtctcgtagaagagaacttcatattcgaacatatgcagttattccactaaatgatgaatgtgggatta ttgaatgggtgaacaacactgctggtttgagacctattctgaccaaactatataaagaaaagggagtgtatatgacaggaaaagaacttcgccagtgtatgctacc aaagtcagcagctttatctgaaaaactcaaagtattccgagaatttctcctgcccaggcatcctcctatttttcatgagtggtttctgagaacattcxxtgatcctacatca tggtacagtagtagatcagcttactgccgttccactgcagtaatgtcaatggttggttatattctggggcttggagaccgtcatggtgaaaatattctctttgattctttgact ggtgaatgcgtacatgtagatttcaattgtcttttcaataagggagaaacctttgaagttccagaaattgtgccatttcgcctgactcataatatggttaatggaatgggtc ctatgggaacagagggtctttttcgaagagcatgtgaagttacaatgaggctgatgcgtgatcagcgagagcctttaatgagtgtcttaaagacttttctacatgatcct cttgtggaatggagtaaaccagtgaaagggcattccaaagcgccactgaatgaaactggagaagttgtcaatgaaaaggccaagacccatgttcttgacattga gcagcgactacaaggtgtaatcaagactcgaaatagagtgacaggactgccgttatctattgaaggacatgtgcattaccttatacaagaagctactgatgaaaa cttactatgccagatgtatcttggttggactccatatatgtgaaatgaaattatgtaaaagaatatgttaataatctaaaagtaatgcatttggtatgaatctgtggttgtatc tgttcaattctaaagtacaacataaatttacgttctcagcaactgttatttctctctgatcattaattatatgtaaaataatatacattcagttattaagaaataaactgctttctt aataaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

Ncaph2 røgcaaaatggcgccagaactagtggcgggctgaggacgccgtacccctcggaaggcagccctgcggtccctttgccgcccgttccctcccggacatggagga cgtggaggcgcgcttcgcccacctcttgcagcccatccgcgacctcaccaagaactgggaggtggacgtggcggcccagctgggcgagtatctggaggagctg gatcagatctgcatttcttttgacgaaggcaagaccacaatgaacttcattgaggcagcgttgttgatccagggctctgcctgcgtctacagtaagaaggtggaatac ctctactcactcgtctaccaggcccttgatttcatctctggaaagaggcgggccaagcagctctcttcggtgcaggaggacagggccaatggggttgccagctccg gggtcccccaggaggcagagaatgagttcctgtcgctggatgacttccctgactcccggactaacgtggatctcaagaatgatcagacgcccagtgaggtcctca tcatccccctcctgcccatggccctggtggcccctgatgaaatggagaagaacaacaatcccctgtacagccgtcagggtgaggtcctggccagccggaaggat ttcaggatgaacac^tgcgttccc∞ccccagaggggccttcatgttggagccagagggcatgtcccccatggaaccagcgggcgtttcccccatgccagggac ccagaaggacaccgggaggactgaggagcagccaatggaagtttccgtgtgcaggagccctgtcccagcactcggctlctcccaggagccaggcccctctcc agaaggcccgatgcccctgggtgggggcgaggacgaggatgcagaggaggcagtagagcttcctgaggcctcggcccccaaggccgctctggagcccaag gagtccaggagcccgcagcaggtgggacccacatggaggcctgcagaacctgagctgtgaactggcaaccctggctctggggccgagtcaccttgcacaagg aggacagtggtatggccttggccccagaαactggtctggggcagaagcccacctgtcttgcagcccgtcctgcaaccagcccttttgaagagcagcttctgtgttc ctcccctctctgagcagaactgatgctcctcagagtagtgggctggcgtccaaggatttgagccctgtcgagctcacggcaacctgggatggccgccggttgccaa ggcgcctctctgcagtcgggctggtaggagggagtgtctggaggccattgctgcctccctcaacccccggggtcaactgtacccagcctagagccaagaaatcct tcctttttattcattaaaacaaaatcaacctgaaaaaaaaaaaaaaaaaa

Psmaδ cttccgggaggtgcttgtgtgcctggtgcgggagctacggggcccagggattgtgtttaaagtagtgcttgtaccaacatgtcccgtggttccagcgccggttttgacc gccacattaccattttttcacccgagggtcggctctaccaagtagaatatgcttttaaggctattaaccagggtggccttacatcagtagctgtcagagggaaagact gtgcagtaattgtcacacagaagaaagtacctgacaaattattggattccagcacagtgactcacttattcaagataactgaaaacattggttgtgtgatgaccgga atgacagctgacagcagatcccaggtacagagggcacgctatgaggcagctaactggaaatacaagtatggctatgagattcctgtggacatgctgtgtaaaag aattgccgatatttctcaggtctacacacagaatgctgaaatgaggcctcttggttgttgtatgattttaattggtatagatgaagagcaaggccctcaggtatataagtg tgatcctgcaggttactactgtgggtttaaagccactgcagcgggagttaaacaaactgagtcaaccagcttccttgaaaaaaaagtgaagaagaaatttgattgg acatttgagcagacagtggaaactgcaattacatgcctgtctactgttctatcaattgatttcaaaccttcagaaatagaagttggagtagtgacagttgaaaatccta aattcaggattcttacagaagcagagattgatgctcaccttgttgctctagcagagagagactaaacattgtcgttagtttaccagatccgtgatgccacttacctgtgt gtttggtaacaacaaaccaacatcatggaggtccctggattgaaaaaggagcctctcccactcctcctaccaccgaagtggttaggactctatataaataaaaaca aggcttttggaaaataaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

Ptcd2 agtagttggtatggtccgagacagtatggctgctgcatttcggccctcgaatcgagttctcctgcaggcgctgcagattttggtgtatcctggggtgggaggctccggc tctgtcagctgccgctgccctctcggagctaaaagatacctacttacagataatgtggtgaaattaaaagaatttcaacaaaagaaagtggctgttgcatgtaatcttt ctggcactaaagaaacgtattttagaaacttgaaaaagaaactgacccagaacaagctcatcttgaagggggagttgataaccttactacatttgtgtgagtctcgg gaccatgtggaactggctaaaaatgtcatttacaggtaccatgcagagaacaaaaatttcactttgggggagtataaatttggaccgctttttgtgaggttgtgttacga gttggatctcgaggaatctgcagtggagctcatgaaagaccagcatttacgaggtttcttctcagactccacatcattcaatattttgatggatatgttatttatcaaaggc aaaiataaaagtgctttgcaagtattgatagagatgaaaaaccaagatgtgaagttcaccaaagatacctatgttcttgcttttgcaatttgctacaaactgaatagcc ctgagtctttcaaaatctgtactacattaagagaagaagctctactcaaaggagaaattctctccaggagagcatcctgtttcgctgtggcattagctctgaatcagaa tgagatggcaaaagctgtgtccattttttctcaaatcatgaatccagaaagcatagcctgcattaatttaaatattataatccatatccagtcaaatatgttggaaaacct gataaagactctaaaaaatgctgcagaaggaaatttatcaaaatttgtgaaaagacatgtgttctcggaggaagtgctggccaaagtgagggaaaaagtgaagg atgtgcctgcccttgtggccaaatttgatgagatctatgggacactgcacatcactggccaggtcaccactgattctttggatgctgtgctctgccacacccccaggga caggaaatct∞cacgttgctattaaacaagaggatggtcagccgtcgcaccttα^gccactcagccagtccctgttggctgagtaaccctggtttcagtccacct atggatctgaggggcctgcttctagtgagttattacctttcctaagaagccaggtatcgcacttcagcagacagtgtgctgacacttggtcttctcctgaaattcccaaat tcactgaatggtaccatgccgatctctgagaagttatgttgcaccactgtgaaggtctagatgcaagcttggctccctcagaaaggcgcttcccttttgcatggctgag gatccttgaaggaacctggtcagtctccggttcagcttccgacaccagagtggaacccagtaagcaccatcaggaatgaatttcactacaagtgtggataactctg attttcaaaggagtagttacttgcaaattacatccttgctgaattcaggaggtatgaaaccctattttaccatgttagaaaacagcccaggattttctcattgctctgccat catatatgtctatgacttgagcccttatttttccatctgcaaaacaataatgcctatgtgtctttgcatatagatttgaaatcttcattcaaggtttagtaggatcatattttctca aaaataagagaaataaggttcataaggaaacttgctgggattgtggttgttttgttttctcagcagcacaaacaaaaccagaatttagcctttaggactgctgagtaa gccaaatttaaatgactactgctttgttcatgggtaagccatgtgcttttcaaaataagtgccactaaaaaccacataatgctttggtttctatgtggataataaatatttag tcctatagtttatcttatttgttaatgatttttctctcttgaatgcctcatattaaaaaaaaatgtgccatgagagcagttcaaagctgcttcatacttatggtctcaaatatagt atgtgttgaatttta

Hsp90b1 gtgggcggaccgcgcggctggaggtgtgaggatccgaacccaggggtggggggtggaggcggctcctgcgatcgaaggggacttgagactcaccggccgc acgccatgagggccctgtgggtgctgggcctctgctgcgtcctgctgaccttcgggtcggtcagagctgacgatgaagttgatgtggatggtacagtagaagagga tctgggtaaaagtagagaaggatcaaggacggatgatgaagtagtacagagagaggaagaagctattcagttggatggattaaatgcatcacaaataagaga acttagagagaagtcggaaaagtttgccttccaagccgaagttaacagaatgatgaaacttatcatcaattcattgtataaaaataaagagattttcctgagagaact gatttcaaatgcttctgatgctttagataagataaggctaatatcactgactgatgaaaatgctctttctggaaatgaggaactaacagtcaaaattaagtgtgataag gagaagaacctgctgcatgtcacagacaccggtgtaggaatgaccagagaagagttggttaaaaaccttggtaccatagccaaatctgggacaagcgagttttt aaacaaaatgactgaagcacaggaagatggccagtcaacttctgaattgattggccagtttggtgtcggtttctattccgccttccttgtagcagataaggttattgtca cttcaaaacacaacaacgatacccagcacatctgggagtctgactccaatgaattttctgtaattgctgacccaagaggaaacactctaggacggggaacgaca attacccttgtcttaaaagaagaagcatctgattaccttgaattggatacaattaaaaatctcgtcaaaaaatattcacagttcataaactttcctatttatgtatggagca gcaagactgaaactgttgaggagcccatggaggaagaagaagcagccaaagaagagaaagaagaatctgatgatgaagctgcagtagaggaagaagaa gaagaaaagaaaccaaagactaaaaaagttgaaaaaactgtctgggactgggaacttatgaatgatatcaaaccaatatggcagagaccatcaaaagaagt agaagaagatgaatacaaagctttctacaaatcattttcaaaggaaagtgatgaccccatggcttatattcactttactgctgaaggggaagttaccttcaaatcaatt ttatttgtacccacatctgctccacgtggtctgtttgacgaatatggatctaaaaagagcgattacattaagctctatgtgcgccgtgtattcatcacagacgacttccatg atatgatgcctaaatacctcaattttgtcaagggtgtggtggactcagatgatctccccttgaatgtttcccgcgagactcttcagcaacataaactgcttaaggtgatta ggaagaagcttgttcgtaaaacgctggacatgatcaagaagattgctgatgataaatacaatgatactttttggaaagaatttggtaccaacatcaagcttggtgtga ttgaagaccactcgaatcgaacacgtcttgctaaacttcttaggttccagtcttctcatcatccaactgacattactagcctagaccagtatgtggaaagaatgaagga aaaacaagacaaaatctacttcatggctgggtccagcagaaaagaggctgaatcttctccatttgttgagcgacttctgaaaaagggctatgaagttatttacctcac agaacctgtggatgaatactgtattcaggcccttcccgaatttgatgggaagaggttccagaatgttgccaaggaaggagtgaagttcgatgaaagtgagaaaact aaggagagtcgtgaagcagttgagaaagaatttgagcctctgctgaattggatgaaagataaagcccttaaggacaagattgaaaaggctgtggtgtctcagcg cctgacagaatctccgtgtgctttggtggccagccagtacggatggtctggcaacatggagagaatcatgaaagcacaagcgtaccaaacgggcaaggacatc tctacaaattactatgcgagtcagaagaaaacatttgaaattaatcccagacacccgctgatcagagacatgcttcgacgaattaaggaagatgaagatgataaa acagttttggatcttgctgtggttttgtttgaaacagcaacgcttcggtcagggtatcttttaccagacactaaagcatatggagatagaatagaaagaatgcttcgcct cagtttgaacattgaccctgatgcaaaggtggaagaagagcccgaagaagaacctgaagagacagcagaagacacaacagaagacacagagcaagacg aagatgaagaaatggatgtgggaacagatgaagaagaagaaacagcaaaggaatctacagctgaaaaagatgaattgtaaattatactctcaccatttggatc ctgtgtggagagggaatgtgaaatttacatcatttctttttgggagagacttgttttggatgccccctaatccccttctcccctgcactgtaaaatgtgggattatgggtcac aggaaaaagtgggttttttagttgaattttttttaacattcctcatgaatgtaaatttgtactatttaactgactattcttgatgtaaaatcttgtcatgtgtataaaaataaaaa agatcccaaat elys tgcggctcgagggggccagcgctgacggtggcgggtacggcaggctcgcgggcgccgggcttcgttacataatctcggaccggaggagcggtggcacatggc ggcggaacggcgctgtggaagtatgcgagacttaagagctcaagtgactagtggtctcctgccatttccagaagtgactcttcaagcccttggagaagacgaaat aacattagaatctgtgcttcgtggaaagtttgctgcggggaaaaatggacttgcttgcttggcttgtggtccacaacttgaggtagtaaactctataacaggagagcg attgtctgcttacagattcagtggagtcaatgaacagcctcctgtagttttagctgtgaaagaattctcttggcagaagagaactggattattaataggattggaagaa acagaagggagtgttctctgtctttatgaccttggaatatcaaaagtagttaaagcagttgttcttcctggaagggtaacagctattgaacctataattaatcatggagg agccagtgcaagcactcagcatttacatccaagtctgcgatggctttttggagtggcagctgtggtcactgatgttggacagatccttcttattgacctatgtttggatga cttgtcatgcaatcaaaatgaagttgaagcatcagatcttgaagttctaactggtatcccagctgaagtaccacacattagagaaagtgtgatgagagaagggcgc catctgtgtttccagttagtaagtccaacaggaacagccgtttcaactcttagttacataagcaggacaaatcagcttgctgcaggtttttctgatggctatctagcacttt ggaacatgaaaagcatgaaaagagaatattacatacaattggaaagtggacaagttcctgtatatgctgtcacttttcaagaacctgagaatgatcgtcggaattgc tgctacttgtgggctgttcagtctacacaagatagtgaaggggatgttttgagtttgcatctgctgcagctggcctttggtaatagaaagtgtttggcatcaggacaaatc ttatatgaggggttagaatactgtgaagaaagatacaccctggacctgacaggtggcatgttccctttgaggggacagacgagtaataccaaattgttgggatgcc agagtatagagaaatttcgatctcatggtgacagggaggaaggcgtgaatgaagctctatcgcctgacactagtgtttcagtctttacctggcaggtgaatatatatg gacagggaaagccttctgtatatttggggctttttgatataaatcgttggtatcatgcacaaatgccagattcgttaaggtcaggagaatatctacataattgctcttatttt gcactgtggtcattggagtctgttgtaagtaggacttctccacatggcatcttggatatattagtacatgagagaagtttaaatagaggagtccctccttcatatccacct cccgagcagttttttaatccaagcacttataattttgatgccacttgtttgttaaactcgggagttgttcatttaacttgtactggctttcagaaggagactttgacttttttaaa gaaatcaggtccatcactcaatgaactcattcctgatggttataatcgatgtcttgtagctggccttctttccccaagatttgttgatgttcagccttccagtttaagccaag aagaacagttagaagctatattgtcagcagcaattcagactagttccctgggacttttgactggttatatccgaagatggataacagaagaacaaccaaattctgcc actaatttgcgctttgttcttgaatggacgtggaataaagtggttctcacaaaagaggaatttgacagactatgtgtgccattatttgatggttcgtgtcatttcatggatcc acaaactatacagtctatccagcaatgctatttgcttcttagcaatcttaatatagtcttgagctgttttgcatcagaagcccgagagatcgctgagagaggactgatag acttaagcaataagtttgtggtttcccacctcatctgtcagtatgcacaagtggttctttggttctctcattctgggcttttaccagaaggcatagatgattctgtgcagttgtc aaggttatgctacaactaccctgtaattcagaactactacaccagtcgtcgacagaagtttgagcgtttatcaagagggaagtggaatcccgattgcttgatgattgat ggactggtttctcagttaggagagcgaattgagaagttgtggaaacgagatgaaggaggcacaggaaaatatcctcctgctagtctgcatgcagtacttgatatgta cctattagacggcgttactgaagcagccaaacactctattaccatttatttgctacttgatattatgtattcctttcccaacaaaacagacactcccattgaatctttcccaa ctgtatttgccatttcttggggccaagttaaacttattcaggggttttggttgatagatcataatgactatgagagtggtttggatcttttgtttcatccagctactgcaaaacc tttgtcatggcaacattcaaagattattcaggcattcatgagtcagggcgagcacagacaagccctcagatatattcagacaatgaagccaacagtgtccagtggt aacgatgttatccttcacctcactgttttgctttttaataggtgtatggttgaagcctggaattttttgcggcaacattgcaataggttgaatatagaggagttactgaagca catgtatgaagtctgtcaggaaatgggcttgatggaagatttactgaagttaccatttacagacactgagcaggaatgtttagtgaaatttttgcagtccagtgccagc gttcagaatcatgaattccttttagtgcaccatttgcagcgtgccaattatgtgcctgccttgaagctgaaccaaactctgaagattaatgttatgaatgatcgtgatcctc gtttgcgggagagatcactggctcgaaattctatattagaccagtatggaaaaatccttcctagagtccatcgaaaattagccattgaacgagctaagccttatcatct gtcaacatcatcagtttttcgattagtttctagacccaaaccattatcagcagttccaaagcaagttgtaacaggaactgtgttgacaagatctgttttcatcaacaatgt gttatctaaaattggagaagtttgggcaagcaaagaacctataaatagcaccacacctttcaatagttctaaaatagaagaaccatctcctatagtgtattcgctccc agctccagagctgcctgaggcattttttggaacaccaatttcaaaagcatcacaaaaaatttctagactgctagatttggttgttcagcctgtcccccggccttctcagt gttcggagtttattcagcaaagctccatgaaatctcctttgtacctagtatcccgttcactgccctcaagttcgcaattaaaaggatcgcctcaggccatctccagggctt cagaattacatttgcttgaaactcctcttgtagttaagaaagctaaaagtttggccatgtcagttactacttctggattttctgagttcactcctcagtccatcctgaggtcta ctcctcgatcaacacctttagcatctccctctccatcacctggaaggtctcxtcaacgacttaaagaaactagaatttcatttgtggaagaagatgtccacccaaaatg gattcctggggctgcagatgatagcaaattagaagtatttactacacctaaaaaatgtgcagttccagtggaaactgaatggccgaagagcaaagataggacca catcttttttcctgaacagccctgaaaaggagcatcaagaaatggatgaggggtcacaaagtttagagaaactggatgtgagcaaaggaaacagcagtgtttcaa tcacatccgatgagactaccttagagtatcaggatgcaccgtcaccggaagaccttgaagagactgttttcacggcctctaagcccaaaagctcttccactgcacta actactaatgtaactgaacaaactgaaaaggatggagataaagatgtatttgcatcagaagtaactccttcagacctacagaaacaaatgggcaatttagaagat gcagaaacaaaggatctcttagttgcagcagaggcattttcagaattgaatcacttaagcccggttcaaggaactgaagcttctctttgtgcaccatcagtctatgaa gggaaaatcttcacccagaagtccaaggtaccagtgttggacgaaggattaacatctgttgaaacctacacccctgcaattagagcaaatgacaataaatctatg gctgatgtccttggtgatggtggaaactcctcgctcactatctctgaaggtcctattgtctctgagcgcaggcttaaccaggaagtagcgctgaacttaaaagaagat catgaagtagaagttggtgtactaaaagaaagtgttgacttaccagaagaaaagcttccaatttctgacagccctcctgatactcaagaaattcatgtgattgaaca agaaaagcttgaagctcaagattcaggagaagaggctaggaatctttcatttaatgagttatatccctctggaacacttaagcttcagtacaattttgatactattgacc aacagnttgtgacttagctgataacaaagacactgctgaatgtgacattgctgaagtagatggggaactttttgtggctcaaagcaactttaccttgatattggaaggt gaagaaggagaagttgagccaggtgattttgcatcatctgatgtgttacctaaagcagctaacacagcaactgaagaaaaacttgtatgcagtggggaaaatgat aatcatggacaaattgcaaatttgccatctgccgtaactagtgaccaaaagtcccaaaaagtagacactttaccatatgtgcctgaacctattaaagtagcaattgc agaaaatttactagatgtaattaaagacacaagaagtaaagaaattacttcagatacaatggaacagtccattcatgaaacaatacctttagtgagccaaaacata atgtgtcccactaaattggtcaaatctgcatttaagactgctcaggaaacaagcacaatgactatgaatgtcagccaggttgatgacgtggtttcctccaaaactcgt acgagaggtcaacgtatccaaaacgtgaatgtcaaatcagcacaacaggaagcatcagcagatgttgctactcctaagatgccagggcagtcagtcaggaag aaaactaggaaggcaaaagaaatttctgaagcttctgaaaacatctattctgatgtcagaggactatttcagaaccagcaaatacctcaaaattctgttacgcctag gagaggaaggagaaagaaagaagttaatcaggacatactagaaaacaccagttctgtggaacaagaattacagatcactacaggtagggaatcaaaaagat taaaatcatctcagctgttggaaccagcagttgaagaaactactaaaaaagaagttaaggtttcatctgttacaaaaaggactcctagaagaattaaaagatctgta gaaaatcaggaaagtgttgaaattataaatgatctaaaagttagtacggtaacaagtcctagcagaatgatcagaaaattgagaagtactaatttagatgcttctga aaatacaggaaataagcaagatgataaatccagtgacaagcagctgcgtattaaacatgttagaagggtcagagggagagaagttagtccatcagatgtgaga gaagactccaaccttgagtcatctcagttgactgttcaagcagaatttgatatgtctgccatacctagaaaacgtggtagaccaagaaaaatcaatccatctgaaga tgtaggatctaaggctgttaaggaagagagaagccccaagaagaaagaagctcccagcattagaaggagatctacaagaaataccccagctaaaagtgaaa atgttgatgttggaaaaccagctttaggaaaatccattttagtgccaaacgaggaactttcgatggtgatgagctctaagaaaaaacttacaaaaaagactgaaag tcaaagccaaaaacgttcattgcactcagtatcagaagaacgcacagatgaaatgacacataaagaaacaaatgagcaggaagaaagattgctcgccacag cttccttcactaaatcatcccgcagcagcaggactcggtctagcaaggccatcttgttgccggacctttctgaaccaaacaatgagcctttattttctccagcgtcaga agttccaaggaaagcaaaagctaaaaaaatagaggttcctgcacagctgaaagaattagtttcggatttatcttctcagtttgtcatctcacctcctgctttaaggagc agacaaaaaaacacatccaataagaacaagcttgaagatgaactgaaagatgatgcacaatcagtagaaactctgggaaagccaaaagcgaaacgaatca ggacgtcaaaaacaaaacaagcaagcaaaaacacagaaaaagaaagtgcttggtcacttcctcccatagaaattcggctgatttcccccttggctagcccagct gacggagtcaagagcaaaccaagaaaaactacagaagtgacaggaacaggtcttggaaggaacagaaagaaactgtcttcctatccaaagcaaattttacg cagaaaaatgctgtaatttcttgggaagattttaatgtacacctatttgtaaagtcatcagaatagtgtggattattaaatatctagtttggaagaaaataatttatataaat tattgtaaatttttatgtaaacagaaggtcttcaataagtaaagtaactccatatggagtgattgtttcagtccaggcaatttttctattttatattaagacttcatacatttatat atgtaaatatggcttattaatggaatgttaaataaaatgtatacttctcaaaaaaaaaaaaaaaaaaaaaaaaaaa

RIf gccgtgggaagatggcggacggaaagggagacgccgccgctgtcgccggggctggggctgaggctccggcggtagcgggagccggagatggagtcgaga ctgagtccatggttcggggtcatcgccccgtatctccagcgccgggagcctcgggactgcggccgtgtctgtggcagctggagacagagctgagggagcaaga ggtgtcggaggtctcatctttgaactactgccggagcttctgccagactttattgcaatatgcaagcaacaagaatgcatcagaacatattgtgtatcttctggaggtat atcgacttgccatccaaagctttgccagtgcacgtccatacttaactactgaatgtgaagatgtcctcttagtgcttggcagattagtactgagttgtttcgaattactgctt tcagtgtctgaaagtgaactgccatgtgaagtctggctaccattccttcagtctctacaggagtcacatgatgcattattggaatttgggaataataacctacaaatattg gttcatgttaccaaggaaggggtgtggaaaaacccagttcttcttaaaattctgtctcaacagccagtagaaacggaggaagtcaataaattgattgcacaagaag gaccttcctttctgcaaatgcgaataaaacatttgttgaaatctaactgcatcccccaggctactgctttatcaaaactatgtgcagaatctaaagaaatttcaaatgtgt catcttttcagcaagcctatatcacatgtttatgttctatgctccctaatgaagatgctattaaggagattgcaaaggtcgactgcaaggaagtactagacatcatttgta atctggaatctgaggggcaggataacacagcatttgttctttgtacgacttaccttacccagcagctccaaactgcaagtgtatattgttcttgggaactgactcttttttg gagtaaactgcaaagaagaattgacccttctttagatacttttttggagcgctgtcgtcagtttggtgtcatagctaaaacgcagcagcatttattttgcctcattagagtt atacaaactgaagcacaagatgctggtcttggggtgtcaattttactgtgtgtcagagctcttcaactcagatcaagtgaagatgaggaaatgaaggcatcagtttgt aaaacaattgcctgtcttttaccagaagatttagaagttagacgagcctgtcagcttacagaattcttaattgaacccagtttggatggatttaatatgttagaagaacta tatttgcaaccagatcaaaaatttgatgaagaaaatgcaccggttccaaattctcttcgatgtgagctcttactagctttaaaagcccactggccttttgatcctgagtttt gggactggaaaactttaaaacgacactgccaccaacttttaggacaagaagcctcagattctgatgatgatttaagtggctatgaaatgtccattaatgacacagat gttttagagtcatttctcagtgactatgatgagggtaaagaagataaacaatatagaagaagagatttgacagatcagcataaggagaaaagagacaaaaaacc tattggctcttctgaaagatatcagaggtggcttcagtacaagtttttctgtttgttatgtaagcgggaatgtatagaggctagaattcttcatcattctaagatgcatatgg aagatggaatttacacctgtccagtttgtattaaaaaatttaagagaaaagaaatgtttgttcctcatgtgatggagcatgttaaaatgccaccaagcagaagggacc gctctaaaaagaaattactgttaaaaggctctcaaaagggtatttgtcctaagagcccctctgcaatcccagagcaaaaccattcattgaatgaccaagccaaag gagagtctcatgaatacgtcacattcagcaaattagaagattgccacctgcaagacagagatttgtatccatgtcccggtacagactgttcccgtgtgtttaagcaatt taaatacttaagtgtgcatcttaaagctgaacaccaaaataatgatgaaaatgccaagcactacttggatatgaaaaatagaagagagaagtgtacttactgtcga cgacattttatgtctgcttttcaccttcgagagcacgaacaagtgcattgtgggcctcagccttatatgtgtgtatctatagattgctatgctaggtttggatcagtaaatga actacttaaccataaacaaaagcatgacgatctgcgttacaaatgtgaattaaatggctgtaatattgttttcagtgacttgggacagctttaccaccatgaagcacaa cactttagggatgcatcttacacatgcaacttccttggctgtaaaaagttctaHactccaaaattgaataccagaatcacctctcaatgcataatgttgaaaattcaaat ggagacataaagaaatcagtgaaacttgaggagtctgcaacaggtgaaaagcaagattgtattaatcagccccatctacttaaccaaactgataaatcacattta cctgaagatcttttctgtgcagaatcagctaattctcaaatagatacagaaactgcagaaaacctgaaagaaaacagtgacagtaattctagtgatcagttaagtca tagctcttcagcttcaatgaatgaagagctaattgacacactagatcactctgaaactatgcaggatgtattgttatctaatgagaaagtctttgggccctccagtttaaa agaaaaatgttccagtatggcagtttgttttgacgggactaagtttacctgtggttttgatggctgtggttccacatacaaaaatgcaagaggaatgcagaaacatttac ggaaggttcatccataccatttcaagcccaaaaagataaagacgaaagatctgtttccctctttgggtaatgaacataatcagacaactgaaaagttggatgcaga acctaaaccctgctcagatacaaacagtgactccccagatgaaggtctagatcacaatattcacattaaatgtaaacgagaacatcaaggttattcctcagaatcct ccatttgtgcttctaaaaggccctgtacagaggataccatgttggaacttctgttacgcttgaaacatttaagcttgaaaaactcaataacacatggatctttctcagggt cattgcaggggtacccatccagtggtgctaagtctcttcagtcagtttcatctatctcagaccttaattttcagaatcaagatgaaaacatgccaagtcagtaccttgca cagttggcggctaagccgtttttctgtgagcttcaaggatgcaaatatgaatttgtgaccagagaggctctgttaatgcattatcttaaaaagcataattattcaaaaga aaaagtccttcagttaaccatgttccaacatcggtattccccatttcagtgtcatatttgccaaaggtcatttacaagaaaaacacaccttaggattcattataaaaata aacatcaaattggcagtgacagagcaactcacaaactattagataatgaaaagtgtgatcatgaaggcccatgttcagtagataggttgaaaggtgattgttctgca gaacttggaggtgatcccagtagtaactctgagaaaccacactgtcatcctaaaaaggatgaatgtagttctgaaacagatttggaatcatcttgtgaagaaacag aaagtaaaacatctgacatttcatcaccaataggcagccatagagaagaacaagaaggaagagagggcagaggtagcaggcgaactgttgctaaaggaaat ctgtgttatattttgaataaataccacaaaccattccattgtattcataaaacttgcaactcctcattcaccaatctaaaaggcttaattcgccattacagaactgtacatc agtacaacaaagaacagttatgtttggagaaagacaaagcaagaaccaaaagggaacttgtcaaatgtaaaaagatatttgcttgcaaatataaggaatgtaat aaacgcttcctgtgttccaaagctcttgctaagcactgtagtgattctcataacctagaccatattgaagagcctaaagtactttccgaagctggatctgcagcaaggtt ttcttgtaaccagcctcagtgccctgctgttttttatacattcaacaagttgaagcaccacttgatggaacagcataatattgaaggggaaatacattcagattatgaaat tcattgtgatcttaatggctgtggccagattttcacccatcgcagtaattactcacaacatgtatattaccgacataaagactattatgatgatttgtttagaagccagaaa gtagcaaatgagagactactaaggagtgaaaaggtatgtcaaacagctgatactcaggggcatgaacatcagaccaccaggagatcatttaatgctaagtctaa aaaatgtggcttaatcaaagaaaagaaagccccaataagttttaaaaccagagctgaggccctccatatgtgtgtggagcactctgagctctctctgtacccctgca tggttcaaggatgcttatctgtggtgaagttggagagcagcattgtgaggcattacaaacgcactcatcagatgagtagtgcctatttagagcaacagatggagaat cttgttgtttgcgttaagtacggtaccaaaattaaggaggaacccccttctgaagcagatccctgtataaagaaagaagaaaatagaagctgtgaatcagagcgc acagaacacagccattccccgggtgacagtagtgcacccatccagaacactgattgctgtcattcaagtgaaagggatggaggtcagaaagggtgcatagaaa gcagctcagtatttgatgcagatactctgctctacaggggaactttgaaatgtaatcatagttccaaaaccacttccctagaacagtgtaatatagttcagcctcctcct ccttgtaaaatagaaaattccatacctaatcccaatgggactgaaagtgggacttatttcacaagtttccagctgcctttaccaaggatcaaagaatcagaaactag gcagcatagttcagggcaagaaaacactgtaaaaaatccaacccatgtcccaaaagagaattttaggaaacattcacagccccggtcatttgatttgaagactta caaacctatgggatttgaatcttcatttctgaaatttattcaggaaagtgaagagaaagaagatgattttgatgattgggagccttcagagcacttaacattaagtaatt cttcacagtccagtaatgatttaacagggaatgttgtggcaaataatatggtgaatgacagtgaacctgaagttgacatacctcattcttccagtgactctacaattcat gagaacctgactgcaatcccacctttaatagtagctgaaacaacaacagttccttccttggaaaacctgagggttgtattggacaaagcattaacagactgtggag agcttgccttaaaacagcttcattatcttcggccagtggtggttcttgaaagatctaagttttccacaccaattttagacttatttccaacaaaaaagacagatgagctttg tgtaggaagttcataagtagcaattttgttttagtaacagactggctccaacactgcaacatggggacatttgccaactcgaacaaaggctgagaagcagccacac cgttgtttagggtagaataggctgtgtatttacatgaatgtataatatctatgtcagcagtattggctgagtccattagctctccagttggtttaatgattgggtttatttttgtttg tttgtttattaaaaaaatggaactgtacacttgtttggtgctaattaatacatcaaaatatactggggcttcctttttcaaattaagtgtgcatgattgtatatggaacaaata ctaaggtcccagggtgggagggctagggaaagggatatggagttcttacttgacttgaatgtgcacctgagggtgctttgtgtaatatattgtacactacagcatcttat attttttgagttgagtttcaataaattacaatttttcacc

Sf3b3 gccgcgcgccaccagaatgtccctgtcttgaggtctaatggcggacgccagtatgttggagttggtggtggcttaagttttgaagggaggtagcatccgttggatatc cacaccatccttctcgctgcaggctttcttggactccgtactgttggtgtaaccaaggcctggaggtctgggtggctcaggtttcxtgcagccatgtttctgtacaacttaa ccttgcagagagccactggcatcagctttgccattcatggaaacttttctggaaccaaacaacaagaaattgttgtttcccgtgggaagatcttggagctgcttcgccc agaccccaacactggcaaagtacataccctactcactgtggaagtattcggtgttatccggtcactcatggcctttaggctgacaggtggcaccaaagactacattg tagttggcagtgactctggtcgaattgttattttggaataccagccatctaagaatatgtttgagaagattcaccaagaaacctttggcaagagtggatgccgtcgcat cgttcctggccagttcttagctgtggatcccaaagggcgagccgttatgattagtgccattgagaaacagaaattggtgtatattttgaacagagatgctgcagcccg acttaccatttcatctcccctggaagcccacaaagcaaacactttagtgtatcatgtagttggagtagatgtcggatttgaaaatccaatgtttgcttgtctggaaatgga ttatgaggaagcagacaatgatccaacaggggaagcagcagctaatacccagcagacacttactttclatgagctagaccttggtttaaatcatgtggtccgaaaa tacagtgaacctttggaggaacacggcaacttccttattacagttccaggagggtcagatggtccaagtggagtactgatctgctctgaaaactatattacttacaag aactttggtgaccagccagatatccgctgtccaattcccaggaggcggaatgacctggatgaccctgaaagaggaatgatπttgtctgctctgcaacccataaaac caaatcgatgttcttctttttggctcaaactgagcagggagatatctttaagatcactttggagacagatgaagatatggttactgagatccggctcaaatattttgatact gtacccgttgctgctgccatgtgtgtgcttaaaacagggttcctttttgtagcatcagaatttggaaaccattacttatatcaaattgcacatcttggagatgatgatgaag aacctgagttttcatcagccatgcctctggaagaaggagacacattcttttttcagccaagaccacttaaaaaccttgtgctggttgatgagttggacagcctctctccc attctgttttgccagatagctgatctggccaatgaagataclccacagttgtatgtggcctgtggtaggggaccccgatcatctctgagagtcctaagacatggacttg aggtgtcagaaatggctgtttctgagctacctggtaaccccaacgctgtctggacagtgcgtcgacacattgaagatgagtttgatgcctacatcattgtgtctttcgtg aatgccac∞tagtgttgtccattggagaaactgtagaagaagtgactgactctgggttcctggggaccaccccgaccttgtcctgctccttattaggagatgatgcct tggtgcaggtctatccagatggcattcggcacatacgagcagacaagagagtcaatgagtggaagacccctggaaagaaaacaattgtgaagtgtgcagtgaa ccagcgacaagtggtgattgccctgacaggaggagagctggtctatttcgagatggatccttcaggacagctgaatgagtacacagaacggaaggagatgtca gcagatgtggtgtgcatgagtctggccaatgtaccccctggagagcagcggtctcgcttcctggctgtggggcttgtggacaacactgtcagaatcatctccctggat ccctcagactgtttgcaacctctaagcatgcaggctctcccagcccagcctgagtccttgtgtatcgtggaaatgggtgggactgagaagcaggatgagctgggtg agaggggctcgattggcttcctatacctgaatattgggctacagaacggtgtgctgctgaggactgtcttggaccctgtcactggggatttgtctgatactcgcactcg gtacctggggtcccgtcctgtgaagctcttccgagtccgaatgcaaggccaggaggcagtattggccatgtcaagccgctcatggttgagctattcttaccaatctcg cttccatctcaccccactgtcttacgagacactggaatttgcatcgggttttgcctcggaacagtgtcccgagggcattgtggccatctccaccaacaccctacggattt tggcattagagaagctcggtgctgtcttcaatcaagtagccttcccactgcagtacacacccaggaaatttgtcatccaccctgagagtaacaaccttattatcattga aacggaccacaatgcctacactgaggccacgaaagctcagagaaagcagcagatggcagaggaaatggtggaagcagcaggggaggatgagcgggag ctggccgcagagatggcagcagcattcctcaatgaaaacctccctgaatccatcttiggagctcccaaggctggcaatgggcagtgggcctctgtgatccgagtg atgaatcccattcaagggaacacactggaccttgtccagctggaacagaatgaggcagcttttagtgtggctgtgtgcaggttttccaacactggtgaagactggtat gtgctggtgggtgtggccaaggacctgatactaaacccccgatctgtggcagggggcttcgtctatacttacaagcttgtgaacaatggggaaaaactggagtttttg cacaagactcxtgtggaagaggtccctgctgctattgccccattccaggggagggtgttgattggtgtggggaagctgttgcgtgtctatgacctgggaaagaagaa gttactccgaaaatgtgagaataagcatattgccaattatatctctgggatccagactatcggacatagggtaattgtatctgatgtccaagaaagtttcatctgggttc gctacaagcgtaatgaaaaccagcttatcatctttgctgatgatacctacccccgatgggtcactacagccagcctcctggactatgacactgtggctggggcagac aagtttggcaacatatgtgtggtgaggctcccacctaacaccaatgatgaagtagatgaggatcctacaggaaacaaagccctgtgggaccgtggcttgctcaat ggggcctcccagaaggcagaggtgatcatgaactaccatgtcggggagacggtgctgtccttgcagaagaccacgctgatccctggaggctcagaatcacttgt ctataccaccttgtctggaggaattggc^tccttgtgccattcacgtcxx^tgaggaccatgacttcHccagcatgtggaaatgcacctgcggtctgaacatccccct ctctgtgggcgggaccacctcagctttcgctcctactacttccctgtgaagaatgtgattgatggagacctctgtgagcagttcaattccatggaacccaacaaacaa aagaacgtctctgaagaactggaccgaaccccacccgaagtgtccaagaaactcgaggatatccggacccgctacgccttctgagccctcctttcccggtgggg cttgccagagactgtgtgttttgtttcccccaccaccatcactgccacctggcttctgccatgtggcaggagggtgactggataattaagactgcattatgaaagtcaa cagctctttcccctcagctcttctcctggaatgactggcttcccctcaaattggcactgagatttgctacacttctccccacctggtacatgatacatgacccxaggttcc agtgtagaacctgagtcccccattccccaaagccatccctgcattgatatgtcttgactctcctgtctacttttgcacacacccttaatttttaattggttttcttgtaaataca gttttgtacaatgttatctctgtgggaggaaggaggcaggctgtggtgggactgggtagggtatagtatcactcctgagttccactgctctagaatctaaccagaaata gaaacctagtttttaaggtgaaaaaaaaaaaaaaaaaa

Setd4 gttgcccgggtaacgggagcagagtcggcgggattgagcatttccacagaaattacagttttgtcctttttgaaaaaatagaactgtatttcagaaaaaagaaacta cagttttagcatgcagaaaggaaaagggagaacaagccggatcagaagacgaaaactctgcggaagttctgaatcaagaggagtgaatgagagccacaagt ctgaatttatagagctgaggaagtggctgaaagctaggaagtttcaagattcaaacttagcgcctgcttgttttccaggtacaggaagagggctgatgagtcaaaca tccctgcaggagggacagatgattatttcgttgcctgagagttgcxtgctcaccacggacacagtgattcgaagctacttaggggcatacattactaagtggaagcc tcctccatctcctctgctggcgctgtgcacctttttagtttcagaaaagcatgctgggcaccgatctctttggaagccttacctggagattttacccaaggcgtatacctgc cctgtttgtttggagccggaagtggtgaaccttcttcccaaatctttaaaagcaaaggctgaagagcagagagcccacgtgcaggagttctttgcttcctccagagac tttttctcttctctgcagcctctgtttgcggaggctgttgacagcatcttcagctacagtgccctgctgtgggcttggtgcaccgtcaacaccagagccgtgtacctgagg cccaggcagcgggaatgcctttctgcagagccggacacctgtgcactcgctccgtacctggacctgctgaatcatagcccacatgtccaggtaaaagcagcgttt aatgaagaaactcattcttacgaaattagaacgacttcacgttggagaaagcatgaagaggtattcatctgttacggccctcacgataatcaacggctgttcctgga atacggatttgtttctgtccataatcctcatgcttgtgtttatgtctcaagaggttggaatcaactttgttcttaacattaacactatataatttttttccccatttggagatgtgtat tttcagttttaataaaaatatcaaaaccttaaaaaaaaaaaaaaaaaa

Claims

Claims

1. A method of stress conditioning comprising:

. a) identifying a stressful event to which a subject will be or has been subjected, and b) conducting ECP according to a schedule and with a dose effective to ameliorate the effect of the stressful event.

2. The method of claim 1 wherein the stressful event is a trauma or toxic exposure.

3. The method of claim 1 wherein the trauma is ischemia, stroke, decompression, hyperbaric therapy (oxidative stress), accidental, surgery, burn, smoke, fire, battle, intense exercise, sleep deprivation, abnormalities of nutrition, malnutrition, space travel, torture, disaster management, viral infection, bacterial infection, cancer or organ transplantation.

4. The method of claim 1 wherein the toxin exposure is chemotherapy, radiation, poison, pharmaceutical side effects, environmental toxins, ultrasound, laser light or antibiotics.

5. The method of claim 1 further comprising measuring a biomarker wherein ECP delivery is adjusted based upon the level or metabolism of said biomarker.

6. The method of claim 4 wherein the biomarker is selected from the group consisting of the genes listed in Table 1.

7. The method of claim 1 further comprising administering to said subject a substance or environmental signal that affects the mTOR pathway.

8. The method of claim 8 wherein Rapamycin is administered to said subject.

9. The method of claim 7 wherein the substance or environmental signal is administered in amounts and at intervals sufficient to induce sub-cellular stress in the subject.

10. The method of claim 9 wherein the substance is lycopene or celestral.

11. A method of monitoring conditioning efficacy comprising identifying differential modulation of a biomarker selected from the group consisting of those in Table 1.

12. The method of claim 11 wherein the pattern of the modulation of said group of biomarkers is compared to patterns of biomarker modulation known to be efficacious...

13. A kit for conducting an assay according to claim 1 comprising: biomarker detection reagents.

14. A microarray or gene chip for performing the method of claim 1