Patent Application
20150099645
The present invention is in the field of diagnostic and therapeutic of pathological cell proliferation which is associated with variety of pathological conditions. More specifically, the invention is adapted to measure and regulate the expression and/or activation and inactivation levels of Glycogen Synthase Kinase 3β (GSK-3β) as a marker of T-cell proliferation.
Glycogen synthase kinase 3β (GSK-3β) is a serine/threonine kinase that was first isolated and purified as an enzyme capable of phosphorylating and inactivating the enzyme glycogen synthase. GSK-3β is now known to act as a downstream regulatory switch that determines the output of numerous signaling pathways initiated by diverse stimuli. Among these pathways GSK-3β is also suggested to play a role in cellular proliferation and apoptosis. The ability to detect the activation state of the GSK-3β and if necessary to regulate it, would allow a greater understanding and ability to treat and diagnose conditions related to cell proliferation and apoptosis.
In many conditions related to the immune response, including tissue rejection, inflammation, infection and Graft-versus-host disease (GVHD), there is a marked change in cell proliferation, in particular an increase in T-cell proliferation. Other conditions in which cell proliferation is a key factor includes cancer. A method of detecting, understanding and controlling these pathological states would greatly improve the diagnosis and treatment of these conditions.
The incidence of GVHD, for example, has increased substantially over recent years. The number of allogeneic hematopoietic stem cell transplants (HSCT) that occur annually continues to increase each year. Clinical observations along with current diagnostic techniques are time-consuming and require invasive tissue sampling, involving tissue biopsies obtained from an involved organ in order to confirm and diagnose diseases such as GVHD. Graft versus Host Disease (GVHD) is a life-threatening complication of HSCT occurring in 30 to 80% of patients (Sullivan K M 2004, Srinivasan R et al 2006).
Presently, the only way to diagnose GVHD is with biopsies of the affected tissue during phase 3 of the disorder, when T cells are already attacking the host tissues and there are observable clinical symptoms (Srinivasan R et al 2006, Jacobsohn D A and Vogelsang G B 2007).
Although biopsies are the only way to properly diagnose cell proliferation in diseases today, the diagnosis is determined only after T cells are already attacking the host tissues and the disease has reached phase 3 of the disorder, where clinical symptoms are apparent. This is time-consuming, invasive to the patient, and there is no guarantee that an accurate diagnosis can be rendered.
Biopsy procedures to procure tissue are thus invasive and incur delays of 24 to 48 hours before a diagnosis can be rendered. Furthermore, an unequivocal diagnosis is not always possible with biopsies, and diagnosis from clinical symptoms is not reliable, given that other post-transplant conditions may present in a similar manner. Therefore, currently, there is no available treatment that can effectively detect this disease before the onset of clinical symptoms.
There is therefore a long-felt need to provide a rapid, reliable, relatively non-invasive method, for detecting cell proliferation and other pathological syndromes in the early stages of the disease process.
The present invention provides a method for detecting T-cell proliferation in a subject comprising the steps of; (a) obtaining a sample from the subject; and (b) profiling at least one parameter selected from the group consisting of: the activation level of GSK-3β, the expression level of GSK-3β and the activation or expression level of related members of pathways in which GSK-3β plays a part; wherein a significant deviation from normal values indicates cellular proliferation.
It is another object of the current invention to disclose the method as defined in any of the above further comprises steps of profiling the expression level of p-GSK-3β or proteins associated with GSK-3β related pathways.
It is another object of the current invention to disclose the method as defined in any of the above, wherein a decrease in the expression level of serine 9 phosphorylated GSK-3β (p-GSK-3β) or GSK-3β or proteins associated with GSK-3β related pathways indicate increased T-cell proliferation.
It is another object of the current invention to disclose the method as defined in any of the above, further comprises steps of profiling the inactivation levels of GSK-3β or proteins associated with GSK-3β related pathways.
It is another object of the current invention to disclose the method as defined in any of the above further comprises steps of measuring protein kinase C (PKC) activity.
It is another object of the current invention to disclose the method as defined in any of the above further comprises steps of measuring protein kinase C (PKC) expression.
It is another object of the current invention to disclose the method as defined in any of the above, wherein a decrease in PKC expression indicates an increase in cell proliferation.
It is another object of the current invention to disclose the method as defined in any of the above further comprises steps of inhibiting PKC activity thereby significantly decreasing T-cell proliferation.
It is another object of the current invention to disclose the method as defined in any of the above further comprises steps of profiling GSK-3β related pathways selected from a group consisting of: total Gsk3β, ser9 phosphorylated Gsk3β, b-catenin, Dvl, tyr216 phosphorylated Gsk3β P38 MARK, Erk, PI-3K/Akt, PKC and any combination thereof.
It is another object of the current invention to disclose the method as defined in any of the above further comprises steps of inhibiting GSK-3β related pathways selected from a group consisting of: P38 MARK, Erk, PI-3K/Akt, PKC and any combination thereof.
It is another object of the current invention to disclose the method as defined in any of the above, wherein a decrease in cell proliferation is indicated when P38 MAPK, Erk and PI-3K/Akt, are inhibited and PKC is unaffected.
It is another object of the current invention to disclose the method as defined in any of the above, wherein inhibition of cell proliferation is indicated when P38 MAPK, Erk and PI-3K/Akt, are unaffected and PKC is inhibited.
It is another object of the current invention to disclose the method as defined in any of the above, wherein upregulation of cell proliferation is indicated when P38 MAPK, Erk and PI-3K/Akt, are inhibited and PKC is activated.
It is another object of the current invention to disclose the method as defined in any of the above, further comprises steps of measuring the phosphorylation levels of GSK-3β or proteins associated with GSK-3β related pathways.
It is another object of the current invention to disclose the method as defined in any of the above further comprises steps of identifying phosphorylation sites of GSK-3β or proteins associated with GSK-3β related pathways.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of measuring phosphorylation of at least one of GSK-3β phosphorylation sites selected from a group consisting of: Serine 9, Thr324, Ser215, Ser219, Thr309, Thr356, Thr390, Thr392, Ser389 and any combination thereof.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of measuring auto-phosphorylation of at least one of GSK-3β phosphorylation sites selected from a group consisting of: Serine 9, Thr324, Ser215, Ser219, Thr309, Thr356, Thr390, Thr392, Ser389 and any combination thereof.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of sampling the spleen.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of sampling the blood.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of sampling a body fluid or tissue.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of comparing the result of GSK-3β levels with a predetermined normal value obtained from healthy subjects.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of comparing the result of GSK-3β status with a predetermined normal value obtained from healthy subjects, wherein a deviation of at least about 25% from normal values indicates the presence of pathological T-cell proliferation.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of detecting disorders associated with apoptosis, cell differentiation, protein synthesis and any combination thereof.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of detecting disorders associated with apoptosis using annexin-V or anti-annexin-V mAb as a marker.
It is another object of the current invention to disclose the method as defined in any of the above further comprising the steps of profiling a predetermined disorder associated with pathological T-cell proliferation by the steps of: (a) preparing an array with predetermined antibodies or antigens recognizing GSK-3β or proteins associated with GSK-3β related pathways; (b) incubating the antibodies with a biological sample; (c) adding at least one labeled analyte or agent producing a signal; (d) detecting and analysing the signal produced by the analyte or agent using at least one fingerprinting or profiling technique; wherein a significant deviation from normal values indicates cellular proliferation.
It is another object of the current invention to disclose the method as defined in any of the above further comprising the steps of detecting and analysing the signal produced by the analyte or agent using at least one fingerprinting or profiling technique selected from a group consisting of: Western blot, ELISA, immunohistochemistry, FACS, Mass spectrometry or any other conventionally used fingerprinting or profiling technique.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of detecting pathological T-cell proliferation associated with Graft-versus-host disease (GVHD).
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of detecting GVHD at an early stage before appearance of pathological GVHD symptoms.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of selecting GVHD symptoms from a group comprising Hunched posture, Closed eyes, Reduced activity, Diarrhea, Weight loss and any combination thereof.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of detecting GVHD during phase 2 of a disease.
It is another object of the current invention to disclose the method as defined in any of the above further comprising steps of detecting GVHD at an early stage between days 4 and 5 after transplant.
It is another object of the current invention to disclose a method of detecting disorders associated with pathological T-cell proliferation, in a subject comprising the steps of; (a) obtaining a sample from a subject; (b) measuring the level of GSK-3β inactivation by measuring at least one parameter selected from a group consisting of: (i) serine 9 phosphorylation; (ii) the Wnt pathway activation; (iii) activated PKC and any combination thereof; comparing the result of the inactivation levels with a predetermined normal inactivation value obtained from healthy subjects; wherein a significant deviation from normal inactivation values indicates cellular proliferation.
It is another object of the current invention to disclose the method as defined in any of the above, wherein the step of measuring is carried out by measuring the serine 9 phosphorylation.
It is another object of the current invention to disclose the method as defined in any of the above, wherein the step of measuring the levels of GSK-3β inactivation, is carried out in a way selected form a group consisting of: (a) measuring β-catenin levels in the absence of serine 9 phosphorylation; (b) measuring the site of stimulation on the GSK-3β once stimulated through the activated Wnt pathway; (c) measuring a protein marker that is unique to the inactivation of GSK-3β through the activated Wnt pathway and any combination thereof.
It is another object of the current invention to disclose the method as defined in any of the above, wherein the step of measuring the levels of GSK-3β inactivation, is carried out in a way selected form a group consisting of: (a) measuring the site of stimulation on the GSK-3β once stimulated through activated PKC; (b) measuring a protein marker that is unique to the inactivation of GSK-3β through the activated PKC; and any combination thereof.
It is another object of the current invention to disclose a method of monitoring and regulating the GSK-3β activation state, comprising the steps of; (a) obtaining a biological sample from a subject; (b) determining at least one of: the expression level of GSK-3β, the activation level of GSK-3β, the expression or activation level of proteins associated with GSK-3β related pathways; (c) comparing the levels to control levels determined from healthy subjects; wherein levels with pre-specified characteristics is used for the diagnosis of pathological cell proliferation.
It is another object of the current invention to disclose a diagnostic kit for the detection of a disorder associated with pathological cell proliferation in a subject wherein the kit comprising profiling means adapted for profiling the status of GSK-3β or proteins associated with GSK-3β related pathways, in a biological sample derived from the subject.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the status of GSK-3β or related proteins is selected from a group consisting of: (a) activation; (b) over-activation (c) expression levels; (d) inactivation; (e) over-inactivation and any combination thereof.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the activation status of GSK-3β or related proteins (proteins associated with GSK-3β related pathways) is a determinant of the cell proliferation or cell apoptosis.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the activation status of GSK-3β or related proteins (proteins associated with GSK-3β related pathways) is a determinant of apoptosis.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the expression level of GSK-3β or related proteins (proteins associated with GSK-3β related pathways) is a determinant of the cell proliferation or cell apoptosis.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the status is an indicator for the best treatment program.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the profiling means are adapted to detect the activation status of GSK-3β or related proteins (proteins associated with GSK-3β related pathways).
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the profiling means are adapted to detect the expression level of GSK-3β or related proteins (proteins associated with GSK-3β related pathways).
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the activation status of GSK-3β or at least one related protein (proteins associated with GSK-3β related pathways) is a marker for cell proliferation.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the activation status of GSK-3β is determined by profiling markers for the Wnt pathway or the actual site on the GSK-3β being affected by the activated Wnt pathway.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the kit comprises profiling means for determining the activation status of GSK-3β is determined by profiling the status of at least one protein selected from a group including: total Gsk3β, PKC, b-catenin, Dvl, Erk, P38 MAPK, PI-3K/Akt, tyr216 phosphorylated GSK-3β, serine 9 phosphorylated GSK-3β and any combination thereof.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the kit comprises profiling means for profiling the activation status of GSK-3β is determined by measuring PKC activation sites on GSK-3β.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the kit comprises profiling means for profiling over-activation of GSK-3β determined by measuring tyrosine 216 phosphorylation.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein over-activation of GSK-3β is determined by measuring the expression levels of GSK-3β.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the kit comprises profiling means for profiling or measuring phosphorylation of at least one of GSK-3β phosphorylation sites selected from a group comprising: Serine 9, Thr324, Ser215, Ser219, Thr309, Thr356, Thr390, Thr392, Ser389 and any combination thereof.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the kit comprises profiling means for profiling or measuring auto-phosphorylation of at least one of GSK-3β phosphorylation sites including: Serine 9, Thr324, Ser215, Ser219, Thr309, Thr356, Thr390, Thr392, Ser389 and any combination thereof.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the kit comprises profiling means for profiling or measuring GSK-3β related pathways including total Gsk3β, ser9 phosphorylated Gsk3β, b-catenin, Dvl, tyr216 phosphorylated Gsk3β P38 MARK, Erk, PI-3K/Akt, PKC and any combination thereof.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the presence of over-inactivated GSK-3β as indicated by abnormal levels of phosphorylated serine 9 provides a positive diagnosis of pathological cell proliferation.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the pathological cell proliferation is a marker for at least one pathological condition associated with cell proliferation, tissue rejection, cancer, wound healing, tissue regeneration, aging, skin care, obstetrics and any phenomena associated with abnormal levels of cellular proliferation or apoptosis.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the kit is adapted for detecting disorders associated with apoptosis using annexin-V protein or anti-annexin-V mAb as a marker.
The diagnostic kit according to It is another object of the current invention to disclose the diagnostic kit as defined in any of the above 5, wherein the pathological cell proliferation is detectable during phase 2 of a GVHD disease, prior to appearance of disease symptoms.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the GVHD symptoms include hunched posture, closed eyes, reduced activity, diarrhea and weight loss.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the protein marker that is unique to the inactivation of GSK-3β is selected from a group comprising abnormal levels of phosphorylated serine 9 GSK-3β, abnormal levels of markers for Wnt activity including but not limited to abnormal levels of β-catenin expression, abnormal levels of stimulation at the site on the GSK-3β affected by the activated Wnt pathway, abnormal levels of protein markers unique to the inactivation of GSK-3β through the activated Wnt pathway, b-catenin, Dvl, tyr216 phosphorylated Gsk3β P38 MARK, Erk, PI-3K/Akt, PKC and any combination thereof.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, comprising profiling means for detection of pathological or normal physiological conditions that are also characterized by increased cell proliferation, decreased cell apoptosis and an immune response.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the results provided by the kit indicate the pathways to be modulated to control the activation status and/or expression level of GSK-3β, thereby regulating T-cell proliferation.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the kit is adapted for the detection and quantification of the activation or expression status of GSK-3β or related proteins (proteins associated with GSK-3β related pathways), further wherein the activation or expression status is an indicator for the progression/presence of pathological T-cell proliferation.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein the pathological T-cell proliferation is diagnosed by determining the activation or expression status of GSK-3β or related proteins (proteins associated with GSK-3β related pathways) in samples from a subject selected from a group consisting of: (a) blood; (b) spleen; (c) biological fluid; (d) tissue and any combination thereof.
It is another object of the current invention to disclose a diagnostic kit for the detection of T-cell proliferation, provided with the means for detecting the presence of inactivated GSK-3β as indicated by abnormal levels of stimulation at the site on the GSK-3β affected by activated PKC, or as indicated by abnormal levels of a protein marker that is unique to the inactivation of GSK-3β.
It is another object of the current invention to disclose the diagnostic kit as defined in any of the above, wherein provided with the means for detecting the presence of over-inactivated GSK-3β as indicated by abnormal levels of phosphorylated serine 9 and abnormal levels of markers for Wnt activity including but not limited to abnormal levels of β-catenin expression, abnormal levels of stimulation at the site on the GSK-3β affected by the activated Wnt pathway, and abnormal levels of protein markers unique to the inactivation of GSK-3β through the activated Wnt pathway; and by abnormal levels of markers for activated PKC inactivation of the GSK-3β including but not limited to abnormal levels of stimulation at the site on the GSK-3β affected by the activated PKC, and abnormal levels of protein markers unique to the inactivation of GSK-3β through activated PKC; in combination, thereby providing a positive diagnosis of cell proliferation.
It is another object of the current invention to disclose a method of regulating T-cell proliferation in a patient, comprising the steps of monitoring the activation or expression status of GSK-3β or related proteins (proteins associated with GSK-3β related pathways) and modulating according to the needs of the patient.
It is another object of the current invention to disclose a method of diagnosing a disorder associated with pathological T-cell proliferation in a subject comprising the steps of: (a) measuring GSK-3β inactivation status or indicators thereof; (b) measuring GSK-3β activation status or indicators thereof; (c) combining measurements of activation and inactivation status or indicators thereof; wherein an activation to inactivation ratio is obtained characteristic of the disorder associated with pathological T-cell proliferation.
It is another object of the current invention to disclose the method as defined in any of the above, wherein the step (a) of determining inactivation is done by measuring at least one inactivation indicator selected from the group including serine 9 phosphorylation, Wnt activation as determined by β-catenin levels or through a protein marker specific for inactivation of the GSK-3β through the activated Wnt pathway or through the site on the GSK-3β that the activated Wnt pathway affects to stimulate inactivation of the GSK-3β; PKC inactivation sites on GSK-3β or through a protein marker specific for inactivation of the GSK-3β including Dvl, tyr216 phosphorylated Gsk3β P38 MARK, Erk, PI-3K/Akt, PKC, or any combination thereof.
It is another object of the current invention to disclose a protein chip comprising an array of bound antibodies, biomarkers or antigens useful for determining the expression levels of proteins associated with T-cell proliferation in a given sample.
It is another object of the current invention to disclose the protein chip as defined in any of the above, comprising antibodies, biomarkers or antigens characteristic of the activation state and/or expression level of GSK-3β, related pathways, apoptosis markers, proliferation markers, and other biological and biochemical molecules associated with cell proliferation.
It is another object of the current invention to disclose the protein chip as defined in any of the above, wherein the antibodies, biomarkers or antigens characteristic of the activation state and/or expression level of GSK-3β, related pathways, apoptosis markers, proliferation markers, and other biological and biochemical molecules associated with cell proliferation are detectable before the appearance of GVHD symptoms in the subject.
It is another object of the current invention to disclose the protein chip as defined in any of the above, wherein the antibodies, biomarkers or antigens are selected from the group comprising, but not limited to, total GSK-3β, ser 9 phosphorylated GSK-3β, ser 9 phosphorylated GSK-3β, PKC, phosphorylated PKC, b-catenin, Dvl, Akt, PI-3K/Akt Erk, P38 MAPK, Tyrosine 216 phosphorylated GSK-3β, Serum Albumin, markers for apoptosis comprising Fas, Bcl family, Cyctochrome C and caspases, markers for immune activity comprising Nf-kB and CD25, markers for proliferation comprising Cyclin D1, PCNA and p27), and any combination thereof.
It is another object of the current invention to disclose a method of regulating the activation status and/or expression level of the GSK-3β in a in a subject comprising the steps of; (a) obtaining a blood sample from a patient or subject; (b) determining the activation status or expression level of GSK-3β or related proteins, (c) regulating the activation status or expression level of GSK-3β or related proteins according to the results of step (b); and (d) obtaining a normal activation status and/or expression level of GSK-3β or related proteins; wherein regulation of the activation status and/or expression level of the GSK-3β or related proteins is adapted for controlling a disorder associated with pathological T-cell proliferation such as GVHD.
It is another object of the current invention to disclose a diagnostic kit for the detection and quantification of at least one of: the activation status of GSK-3β, the expression status of GSK-3β and the activation or expression status of proteins associated with GSK-3β related pathways, wherein the status is an indicator of cell proliferation or apoptosis levels, further wherein the status is an indicator for the best treatment program.
It is another object of the current invention to disclose a regulatory and therapeutic kit, wherein the therapeutic molecule is GSK-3β or related proteins (proteins associated with GSK-3β related pathways).
It is another object of the current invention to disclose the kit as defined in any of the above, wherein the therapeutic kit is useful in applications selected from a group consisting of: GVHD, tissue rejection, cancer, wound healing, tissue regeneration, aging, skin care, obstetrics, autoimmune diseases and any phenomena associated with abnormal levels of cellular proliferation or apoptosis.
The invention is herein described, by way of example only, with reference to the accompanying drawings. The particulars shown are by way of example only and for the purposes of illustrative discussion of the preferred embodiments of the present invention only. The description taken with the drawings should make apparent to those skilled in the art how several forms of the invention may be embodied in practice.
In the drawings:
The following description is provided, alongside all chapters of the present invention, so as to enable any person skilled in the art to make use of said invention and sets forth the best modes contemplated by the inventor of carrying out this invention. Various modifications, however, is adapted to remain apparent to those skilled in the art, since the generic principles of the present invention have been defined specifically to provide means and methods for detecting T-cell proliferation in a subject.
The present invention solves many of the problems of current methods and systems for monitoring patients suffering from conditions associated with pathological cell proliferation and pathological cell apoptosis such as Graft-versus-host disease (GVHD).
The term “expression level” refers hereinafter to the amount of a protein or the amount of a protein that underwent a post-translational modification (e.g. phosphorylation).
The terms “GSK-3β related proteins” or “GSK-3β related members” refer hereinafter to proteins associated with GSK-3β related pathways, including antibodies, antigens or biomarkers associated with GSK-3β, proteins upstream of GSK-3β and GSK-3β substrates and downstream products i.e. described inter alia hereinafter.
It is therefore a preferred embodiment of the present invention to provide means and methods capable of detecting the expression level of glycogen synthase kinase 3β (GSK-3β) and related pathways. In this embodiment the expression and or activation status of GSK-3β and/or proteins associated with related pathways are surprisingly found to an indicator of pathological cell proliferation and medical disorders associated with pathological cell proliferation such as GVHD in a patient.
Thus in one embodiment, a main concept of the present invention is monitoring the different aspects of GSK-3β.
A further embodiment of the invention is the identification of phosphorylation sites of GSK-3β and their affect on the activation state of the aforementioned molecule.
A further embodiment of the invention is the monitoring of T-cell proliferation and T-cell apoptosis to monitor T-cell proliferation the and thus detecting early onset of GVHD and preventing the need for the invasive biopsy and surgery procedures. The methods and kits provided by the present invention enable a prompt early diagnosis, followed by timely initiation of appropriate immunosupressive therapy, which is vital for the reduction of morbidity and mortality associated with GVHD.
A further embodiment of the invention is the use of fingerprinting technology to diagnose multiple disorders associated with abnormal levels of cell proliferation. Such a fingerprinting technology is preferably based upon custom made protein chip and unique software.
According to a main embodiment of the invention, a diagnostic kit is provided that is adapted for the detection of a disorder associated with pathological cell proliferation, wherein the diagnostic molecule is GSK-3β and/or at least one protein associated with GSK-3β related pathways.
According to a further embodiment of the invention the diagnostic kit as described above comprises profiling means adapted for profiling the status of GSK-3β and/or related proteins (proteins associated with GSK-3β related pathways) in a biological sample derived from a subject or patient.
According to a further embodiment of the invention a diagnostic kit as described above is useful for the early detection of GVHD. GVHD serves herein as an example for a cell proliferative disorder that is firstly shown by the present invention to be associated with GSK-3β status and/or related pathways thereof.
It is herein acknowledged that GVHD has 3 molecular phases:
Molecular phase 1: activation of antigen presenting cells (APCs).
Molecular phase 2: T-cell activation, an increase in T-cell proliferation, T-cell differentiation and T cell migration.
Molecular phase 3: T cells target the tissue for destruction
Thus phase two of the disease is characterized by several activities on the cellular/molecular level including increased T-cell proliferation.
It is emphasised that GVHD development is not evident until clinical symptoms appear (phase three), when the patient's life is already in danger.
Thus advantages of a diagnostic kit as provided by the present invention may include:
A main aspect of the invention is the ability to identify, monitor and then regulate the change in the GSK-3β molecule. This process comprises the flowing steps:
(a) Detecting a change in GSK-3β activation and/or expression state and/or proteins associated with related pathways thereof, relative to control values in a biological sample derived of a subject or a patient;
(b) Monitoring and regulating the different pathways that affect the activation and/or expression state of GSK-3β;
(c) Regulating cellular proliferation and apoptosis in the subject or patient.
The following examples are specific to particular diseases, however, it will become apparent to anyone skilled in the art, after studying the present invention and examples described herein, other diseases and syndromes to which, with no undue burden can be predictably and reasonably diagnosed or treated. The disclosure shows by example, that activation or expression levels of GSK-3β or related members of pathways in which GSK-3β plays a part are related to T-cell proliferation. Since T cell proliferation is a significant phase, stage or process in many diseases, preparation of specific diagnostic kits, research tools treatment protocols, drug screening protocols, new drugs will now become predictable using the methodologies of the present invention. These methodologies are taught by the disclosure and examples described herein, and broadly speaking include:
The deviation indicates an abnormality related to the disease in question.
According to one embodiment of the invention, it is demonstrated for the first time that GSK-3β regulates GVHD through the regulation of T-cell proliferation.
According to a further embodiment, the present invention shows that GSK-3β regulates T-cell proliferation through changes in the activation and/or expression state of GSK-3β and through specific substrates and their particular products. The function of GSK-3β is determined by its substrates and their particular products; the activation and/or expression state of GSK-3β initiates its function.
According to a further embodiment, the present invention identifies the expression and/or activation state (activation/inactivation, and specific changes in the activation state) and substrates with their particular products that regulate T-cell proliferation in association with the onset and progression of medical disorders associated with abnormal cell proliferation such as the GVHD disorder. It is herein shown for the first time that GSK-3β is connected to the onset and progression of GVHD.
According to a further embodiment of the present invention, a protein chip based on GSK-3β expression and/or activation state and associated substrates/products is provided.
Such a protein chip is adapted to monitor changes in the expression and/or activation state of GSK-3β and to monitor changes in the substrates of GSK-3β and their particular products. This allows for example, monitoring the progression of the state of the transplant and detecting the early onset of GVHD through monitoring changes in T-cell proliferation.
In a further embodiment, a protein chip based on GSK-3β activation state and associated substrates/products is provided. Such a kit is capable of monitoring changes in the activation state of GSK-3β and monitoring changes in the substrates of GSK-3β and their particular products. This allows monitoring the progression of the state of the transplant and detects the early onset of GVHD through monitoring changes in T-cell proliferation associated with changes in the GSK-3β activation state and GSK-3β substrates and their particular products which are associated with the onset and progression of GVHD. A protein chip (i.e. antibody microarray/protein microarray/biomarker microarray/antigen microarray) is used as a diagnostic tool
According to a further embodiment, the present invention shows that inactivation of GSK-3β stimulates an increase in T-cell proliferation and a decrease in T-cell apoptosis, while activation of GSK-3β stimulates a decrease in T-cell proliferation and an increase in T-cell apoptosis.
The technology provided by the present invention allows monitoring increases in T-cell proliferation which can be utilized as an indicator for the early onset of GVHD. The detection of the onset of GVHD is made during molecular phase 2 of the disorder which is associated with an increase in T cell proliferation.
The present invention gives detection and diagnosis manners of cell proliferation which can give a solution of problems of current methods and systems for monitoring patients suffering from conditions associated with pathological cell proliferation and pathological cell apoptosis.
It is therefore a preferred embodiment of the present invention to disclose a diagnostic kit, capable of detecting the expression and/or activation status of glycogen synthase kinase 3β (GSK-3β). In this embodiment the GSK-3β level of expression, activation and inactivation is an indicator of cellular activity in the patient.
GSK-3β plays a central role in regulating the cellular cycle, cellular proliferation and apoptosis through changes in its activation state. Determining the activation state of GSK-3β is therefore a useful tool for detecting and regulating both normal (physiological) and abnormal (pathological) cellular proliferation and apoptosis. GSK-3β is now known to act as a downstream regulatory switch that determines the output of numerous signaling pathways initiated by diverse stimuli (Frame and Cohen 2001, Grimes and Jope 2001, Woodgett 2001 and Doble and Woodgett 2003).
Thus in this embodiment the products produced through the activation or inactivation of the GSK-3β include both proliferatory and apoptotic factors. The activation status of the GSK-3β, the expression levels of the GSK-3β or the site on the GSK-3βresponsible for activating or inactivating of the GSK-3β, affect cellular proliferation and/or apoptosis based on the products produced depending upon the conformation change of the GSK-3β.
Gsk3β has sites which are phosphorylated by PKC, therefore, they may play a role in Gsk3β regulation. These sites can be potentially used to monitor the activation state of Gsk3β, which can be used for diagnostic and therapeutic purposes. PKC works through Gsk3β to regulate T-cell proliferation. Therefore PKC can potentially be used to help more accurately assay Gsk3β activity levels, T-cell proliferation, PKC inhibition and inhibition of other pathways which regulate GSK3beta. This suggests that PKC inhibition and inhibition of other pathways which regulate GSK3beta activity play an important role in regulating proliferation. Further more, PKC inhibition and inhibition of other pathways which regulate GSK3beta activity play an important role in regulating cellular proliferation in the human body which suggests importance in developing diagnostics to identify and monitor normal and abnormal cellular proliferation in the human body and developing pharmaceutical to regulate normal and treat abnormal pathological levels of cellular proliferation in the body. PKC works through the GSK3beta to regulate T-cell proliferation. PKC regulates proliferation either working independently but synergistically with the GSK-3beta, or directly by regulating the activity/activation state of the GSK3beta. Through monitoring the activation state of the GSK3beta and the expression levels of the GSK3beta, one can identify, monitor and quantitate changes in cellular proliferation and apoptosis. Once one identify how the activation state the GSK3beta has been altered one can then regulate changes in cellular proliferation and apoptosis through regulating changes in the activation state of the GSK3beta.
In a further embodiment of the present invention, GSK-3β inactivation may also occur through the Wnt signaling pathway. Wnt activation inactivates the GSK-3β through an inactivation site independent of serine 9 phosphorylation and is identified and quantitated through measuring changes in non-phosphorylated β-catenin levels.
In a further embodiment activation of the GSK-3β is either through dephosphorylation or through phosphorylation of tyrosine 216 (Y216). GSK-3β phosphorylation can be identified and quantitated through changes in the molecular weight of the GSK-3β using methods described forthwith in the description. In one aspect of this embodiment the diagnostic kit of the present invention uses antibodies to identify and quantify GSK-3β phosphorylation levels.
The activation status of GSK-3β is an indicator of cell proliferation and cell apoptosis in the patient. It is therefore a preferred embodiment of the present invention that GSK-3β will be used as diagnostic and/or therapeutic molecule incorporated into a diagnostic and/or therapeutic kit for detection and monitoring of cell proliferation.
It is a further embodiment of the present invention that inactivation of GSK-3β through the serine 9 position increases T-cell proliferation. In another aspect of the present invention, inhibition of serine 9 phosphorylation in GSK-3β decreases T-cell proliferation. Thus in this embodiment T-cell proliferation and apoptosis can be regulated through activation or inactivation of GSK-3β by phosphorylation or dephosphorylation of the serine 9 position of GSK-3β.
It is a preferred embodiment of the diagnostic kit of the present invention to characterize GSK3β activity by detecting and quantifying GSK3β activation and inactivation at the aforementioned sites. Also in this embodiment, the diagnostic kit is able to detect and quantify GSK3β inactivation through activated protein kinase C (PKC) and GSK3β inactivation through activated Wnt.
Thus it is a preferred embodiment of the present invention to identify the site on the GSK-3β that is affected by the activated Wnt pathway and the site on the GSK-3β that is stimulated by activated PKC.
Another preferred embodiment of the present invention is to identify GSK-3β as a central molecule for the identification and regulation of normal and abnormal levels of cellular proliferation and apoptosis. In this embodiment regulation of normal and abnormal level of cellular proliferation and apoptosis is achieved through regulating the activation state of the GSK-3β.
Abnormal cell proliferation and apoptosis is a feature of pathological conditions such as Graft Versus Host Disease (GVHD) and other conditions related to immune responses in which there is proliferation of T-cells as part of the patient's response to a foreign body or bodies. In this embodiment and other preferred embodiments the diagnostic molecule and kit has also applications in GVHD, tissue rejection, cancer and any other phenomena or condition in which there is a change in cellular proliferation and/or apoptosis.
It is a further embodiment of the present invention that regulation of GSK-3β activation status by identification and quantification of inactivation and activation sites on GSK-3β and their associated pathways is adapted to regulate normal physiological and abnormal pathological levels of cellular proliferation and apoptosis. In this embodiment GSK-3β contains a plurality of activation and inactivation sites that can be identified and quantified by the diagnostic kit of the present invention.
A diagnostic kit of identifying cell proliferation is been preformed in at least phase 2 of a disease by identifying and quantifying the GSK-3β activation status, is a further embodiment of the present invention. In this embodiment, the diagnostic kit measures a range of parameters associated with GSK-3β and/or related proteins, activation and expression states and associated pathways. In a further embodiment, the aforementioned measurements are compared to the deviation from standard measurements taken from healthy subjects in order to determine the extent of cell proliferation and apoptosis. Furthermore in this embodiment the diagnostic kit and methods of the present invention will be able to detect T cell proliferation and apoptosis by determining how the GSK-3β regulates the aforementioned proliferation and apoptosis. In this aspect of the preferred embodiment the information provided by the diagnostic kit is used for determining the most effective treatment for a pathological conditions via the T cells through regulating activation and/or inactivation sites on the GSK-3β.
It is a further preferred embodiment of the present invention that the diagnostic kit is adapted to detect protein profiles specific and uniquely characteristic to pathological conditions and other unique protein profiles characteristic of related disorders so as to be able to differentiate between said related disorders and to make a correct and accurate diagnosis of GVHD. In this embodiment the unique protein profile prevents the diagnosis of false positive abnormal T-cell proliferation pathologies such as GVHD.
It is therefore a preferred embodiment of the present invention that GSK-3β acts as a regulatory and therapeutic molecule in a therapeutic or pharmaceutical kit in applications not limited to GVHD, tissue rejection, cancer, wound healing, tissue regeneration, aging, skin care, obstetrics and any phenomena associated with abnormal levels of cellular proliferation and/or apoptosis.
It is an embodiment of the present invention that a diagnostic kit capable of detecting and quantitating the activation status of the GSK-3β (GSK-3β activation levels and GSK-3β inactivation levels) is able to diagnose cell proliferation which occurs during phase II of a disease. The novel diagnostic kit is able to diagnose cell proliferation at a relatively early stage before extensive tissue damage occurs, in a quick and relatively non-invasive manner.
In a further embodiment of the present invention, the diagnostic kit for cell proliferation is able to determine the activation status of GSK-3β as measured by examining GSK-3β expression, the activation sites of the GSK-3β and the inactivation sites of the GSK-3β.
In another preferred embodiment of the present invention, the diagnostic and therapeutic kit is incorporated in an implant type device inserted into the patient's body at a site where constant monitoring and treatment of GSK-3β fluctuations is possible. In this embodiment the diagnostic implant is provided with the means to communicate GSK-3β fluctuations wirelessly and at frequent intervals, for example with radio-frequency identification (RFID) tags and readers or from a remote location using wireless internet technology. Thus for example a patient, having undergone a transplant operation may be released from a hospital to their own home with said diagnostic implant inserted and constantly transmitting data to doctors and medical staff at a remote location, any changes would be flagged up early and the patient then recalled for further monitoring. Also in this embodiment the diagnostic implant may further incorporate a therapeutic portion, in which the implant is able to calculate and dose the correct amount of GSK-3β upon detection of abnormal GSK-3β activity or to wirelessly receive dosing instructions from a medical professional monitoring the diagnostic implant.
In another embodiment of the invention, a protein chip utilizing an array of antibodies bound to a glass slide is used to determine the expression levels of different GVHD relevant proteins in a given sample.
In another embodiment of the invention, a diagnostic kit based on a protein chip is used to diagnose GVHD before the appearance of clinical symptoms. In certain embodiments the kit contains a custom-made protein chip comprising antibodies which are adapted to monitor the activation state and/or expression level of the GSK-3β, related pathways, apoptosis markers, proliferation markers, and other biological and biochemical molecules. Such molecule candidates can be pre determined and analysed by western blot, FACS, and other fingerprinting techniques.
In another embodiment another plat form is the substrate of the GSK which determines the function of the GSK-3beta. GSK-3beta has at least 50 substrates which have been identified. The functions of the substrates are based on the products they produce. The function of the different substrates can be mapped out and connect to the products they produce. Another aspect that may need to be considered in the future is that the affect of the substrate may be determined by the cell/tissue type were it is located. The cell/tissue type may effect what products are being produced by the substrate in the cell/tissue in which it is located. By knowing the substrates that are present in your system, cellular function the GSK3beta is playing in the system is been diagnosed.
The diagnostic kit then can be matched to the activation state of the GSK3b, to the substrate, to the products, to the disorder, that is being produced. There may be multiple substrates that can trigger the same disorder or a closely related disorder. It may allow differentiation between similar disorders that was either very difficult or impossible prior to the plat form technology. It is a way to categorize disorders based on their substrates and products.
In another embodiment another plat form is finger printing. Every disorder has a specific finger print. Something unique to that particular disorder therefore each diagnostic kit has a specific finger printer for a specific disorder.
The protein level is examined with Mass Spectrometry so that all the proteins are presented in the system. Changes in protein expression and changes in phosphorylation of specific proteins are assessed. The RNA level is examined with RNA arrays and/or microRNA arrays to assess gene expression. The DNA level is examined, for example with SNP arrays to assess mutations.
The finger print could be on one level of complexity, multiple levels of complexity or involve all levels of complexity. It could be based on increased expression, decreased expression, or whether or not it is or is not expressed. It could be a combination of all these factors at a single time point or changes observed at multiple points over time.
Another fingerprinting technique that can be used to study T-cell proliferation, Gsk3β and PKC in living cells is western blotting. Western blotting confirms the effectiveness of the inhibitors and activators by detecting the following proteins in a given sample. Using a fingerprinting technique (i.e. Western blot), the levels of proteins, antigens or biomarkers, exemplified in the list below can be determined:
1) Alzheimer's Disease (56, 57, 58, 59, 64)
More specifically, the following disorders can be diagnosed and/or prevented by the methods and kits or means provided by the present invention:
Diseases Associated with Cell Differentiation:
Cancer—Abnormal differentiation or de-differentiation of cells is often seen in tumors. Cancer is often diagnosed too late. Very difficult to treat, and treatment regimens are harsh. Affects many people, comes in many different types.
Myelodysplastic Syndrome (MDS)—A very rare disease (orphan drug) caused by exposure to radiation or toxins, characterized by impaired differentiation of blood cells. MDS Affects mostly people over 50 or 60. Causes severe anemia, and often leads to blood cancer (AML). Important to distinguish it in diagnosis from other anemias; blood counts and bone marrow counts are used in diagnosis.
Fibrodysplasia ossificans progressiva (FOP)—A very rare disease (orphan drug) in which muscle tissue turns into bone. The disease appears in early childhood, causes severe handicap. Injuries or minor trauma causes muscle to turn into bone throughout childhood. It is diagnosed by x-rays or other scans. Biopsies make the condition worse. Not many good treatment options are available. The disease is caused by a dominant mutation.
Diseases Associated with Protein Synthesis:
Cancer—Abnormal protein synthesis is often seen in cancer. Cancer is often diagnosed to late. Very difficult to treat, and treatment regimens are harsh. Affects many people, comes in many different types.
Diseases Associated with Cytoskeleton/Microtubules:
Liver disease/cirrhosis—Many different types of liver disease are characterized by changes in the microtubules and cytoskeleton. Scarring and damage to the liver is often irreversible. Disease is often diagnosed by blood tests (liver enzymes) and/or biopsy. Liver disease is somewhat hard to treat and can often lead cancer.
Parkinson's disease—Debilitating neurodegenerative condition. Some research shows that changes proteins (Parkin) which interact with the microtubules play a role in some cases. Parkinson's is impossible to treat, most treatment options only slow down the disease, at best. Diagnosis is done by MRI and clinical manifestations (not a good candidate for us, because you can't do brain biopsies).
Alzheimer's disease—Debilitating neurodegenerative condition. Some research shows that changes proteins (Tau) which interact with microtubules play a role in some cases. Alzheimer's is impossible to treat, most treatment options only slow down the disease, at best. Diagnosis is done by MRI and clinical manifestations (not a good candidate for us, because you can't do brain biopsies).
Diseases Associated with Proliferation/Apoptosis:
Cancer—caused by increased proliferation and decreased apoptosis; also abnormal differentiation or de-differentiation of cells is often seen in tumors, and abnormal protein synthesis is often seen in cancer. Cancer is often diagnosed too late. Very difficult to treat, and treatment regimens are harsh. Affects many people, comes in many different types.
Diseases Associated with Proliferation (Rare Diseases/Orphan Drugs):
Synovial osteochondromatosis (SOC)—Rare disease (orphan drug) of the joints that affects adults, usually beginning between age 20-40. The disease is caused by proliferation or changes in the synovial fluid of the joints. The diagnosis of the disease is done by MRI or other imaging. The treatment is done by surgery. The molecular mechanisms are not understood.
Lymphangiomyomatosis (LAM)—Rare disease (orphan drug) involving benign proliferation of cells in the lungs. Almost exclusively affects women of child-bearing age. Treatment is often hormonal therapy. The disease is sometimes not diagnosed until late stages, because of misdiagnosis in the early stages.
Diseases Associated with Apoptosis (Common):
AIDS—One of the biggest killers in the world. It occurs as a result of HIV infection, and it causes apoptosis of CD4+T cells. The HIV disease is very difficult to treat.
Mono (infectious mononucleosis)—A common viral infection which causes T cells to induce apoptosis of infected B cells. Most patients recover on their own within a few weeks, but there is no way to speed this up or alleviate symptoms. The disease spreads very easily by contact with saliva or other body fluids.
Diseases Associated with Apoptosis (Rare Diseases/Orphan Drugs):
Whipple's disease—a rare (orphan drug), chronic bacterial infection that can be deadly if untreated. Usually affects adult men, more common in farmers/people who work the land. Causes apoptosis of macrophages, and other T-cells attack the intestinal tracts. There are good diagnostic tools available (PCR-based blood test or histology), but often takes a while to diagnose, because there are symptoms affecting several organs but they take years to develop/progress. The conventional treatment is by long-term antibiotics.
Autoimmune lymphoproliferative syndrome (ALPS)—A very rare disease (orphan drug) in which there is a defect in lymphocyte (T-cells and B-cells) apoptotic pathways (usually Fas), causing increased lymphocyte survival. This is non-malignant proliferation, but can lead to autoimmune disorders, secondary cancers. Treatment is harsh, similar to that of autoimmune disorders. Diagnosis is by blood test that has several criteria.
Systemic capillary leak syndrome (SCLS)—A very rare disease (orphan drug) involving recurring episodes which fix themselves, but can also be fatal. Very difficult to diagnose, because often initially resembles a cold or flu. The disease is caused by a sudden leakage of blood plasma into body cavities and muscles, which causes a sharp drop in blood pressure and pain/swelling of hands, feet, and other organs. After a while, the fluid returns to the blood vessels. Not well understood, though some studies show apoptosis is involved. The disease is difficult to diagnose. Treatment involves management by steroids (cannot be cured, though it can go into remission for many years).
Diseases Associated with Migration and/or Proliferation (Autoimmune):
Atheroscelrosis—A very common risk factor in the population for many fatal conditions (heart attack, stroke). The disease is caused by an immune response to injury, cholesterol, and plaque buildup in the arteries. Diagnostic techniques, such as stress tests can detect narrowing of the arteries (indicating severe build-up); earlier diagnosis is done by blood test and/or imaging but are not widespread. Prevention (healthy lifestyle) is the best cure. Other common treatments use oral medication or surgery.
Type I Diabetes—Diabetes is one of the most common conditions. Type I is caused when the body's immune system attacks the pancreatic cells which produce insulin. Treatment is done by management of insulin levels, by insulin injections and diet regulation.
Rheumatoid arthritis—Autoimmune disorder mainly affecting the joints, appearing often in people aaged 40-50. Diagnosed by clinical symptoms, blood tests, and imaging. Treatment is harsh, by steroids and/or immunosuppressants, among others.
Lupus (SLE) (and other autoimmune disorders)—A rare multi-systemic autoimmune disorder, affecting adults, and more common in women, usually appearing between age 20 and 40. Diagnosed by clinical symptoms, blood tests, and imaging. Treatment is harsh, by steroids and/or immunosuppressants, among others.
Sjogren's Syndrome—A rare autoimmune disorder in which the body's immune system attacks exocrine glands (tears, saliva, etc); occurs in a dults, but is much more common in women. This autoimmune disorder can be diagnosed by blood tests, tear tests, and/or gland imaging. Treatment is aimed at managing symptoms.
Scleroderma—A rare multi-systemic autoimmune disorder, affecting adults. Diagnosed by clinical symptoms, blood tests, and imaging. Treatment is harsh, by steroids and/or immunosuppressants, among others; better treatment options are needed.
Inflammatory Bowel Disease (Crohn's disease and ulcerative colitis)—These rare gastrointestinal disorders and are characterized by migration of T-cells to the gut and they attack the gastrointestinal tract. These disorders are usually diagnosed by colonoscopy and blood tests for inflammatory markers. Treatment is harsh, by steroids and/or immunosuppressants, among others.
Multiple Sclerosis—A rare disease in which the body's immune system attacks the neurological system, causing a neuro-muscular “attacks”. It usually occurs in adults, and most patients eventually lose the ability to walk, among other motor functions. Diagnosis is done by biopsy, MRI, and/or electrophysiological tests. There is no cure, but several treatment options can be helpful, though better options are needed.
Diseases Associated with Migration and Proliferation (Non-Autoimmune):
Langerhans cell histiocytosis—a very rare disease that usually occurs in children between ages 1-15, and even less frequently in adults. The disease involves proliferation and migration of different blood cell types and subsequent migration/invasion in other organs. The diagnosis of this disease is usually done by biopsy. Treatment is very aggressive (steroids, immunosuppressants, chemotherapy, radiation).
Examples of antibodies, antigens or biomarkers used in the diagnostic kit of the present invention may include:
1. GSK-3β Activation State:
1. GSK-3β Antibodies:
2. Proteins Upstream of GSK-3β:
3. Monitoring of Other Cellular Processes:
To demonstrate that the levels of GSK-3β and proteins from related pathways, can be used as markers for pathological conditions associated with T-cell proliferation, such as GVHD diagnosis, at an early disease stage.
Mouse Model:
Eight- to 10-week-old male C57BL/6 and Balb/c mice were purchased from the Harlan Animal Farm. The animals were maintained in a pathogen-free animal facility at the Hadassah-Hebrew University Medical School, Jerusalem, Israel. Animals were treated according to the standards of the Animal Ethics Committee, Hebrew University Medical School Animal Care Facilities. Mice were harvested and samples were collected from blood and spleen on days 0, 1, 3, 4, 5, & 6 post-transplant.
Splenocyte Collection:
Mice were sacrificed at the indicated time points and spleens were harvested. Splenocytes were produced by passing spleens through a mesh screen in PBS, and processed with erythrocyte lysis buffer, and washed with cold PBS. Donor splenocytes were processed the same way.
Mouse Splenocyte Transplant:
Mice are divided into 3 groups (Untreated, Syngeneic and Allogeneic) per time point with 6-12 mice per group. The untreated group mice (without irradiation) were injected with plain medium. The syngeneic group irradiated (600 rads) Balb/c mice were injected with splenocytes (30×10̂6) from matched donor Balb/c mice. The allogeneic group irradiated (600 rads) Balb/c mice were injected with splenocytes (30×10̂6) from unmatched donor C57Bl/6 mice. An additional 2-3 mice in each group were injected with 2.0 mg/kg LiCl (dissolved 1:2 LiCl:PBS), once a day from day 1 to day 5 post-transplant, sacrificed at days 3 and 5.
FACS analysis of Apoptosis: Splenocytes were washed twice with cold FACS buffer (1×PBS/0.5% BSA/0.02% sodium azide), and incubated on ice for 30-45 min with APC-conjugated anti-CD3. FITC-conjugated anti-annexin-V mAb was used as a marker of early stages of apoptosis. To differentiate apoptotic cells from necrotic ones, splenocytes were co-stained with PI and annexin-V-FITC in a PI kit (MBL), according to the manufacturer's protocol. Flow cytometry was performed on a FACScan, and data were analyzed using CellQuest software (Becton Dickinson). A total of 5×105 CD3+ cells were counted for each sample.
FACS Analysis of Proliferation:
Splenocyte were washed with cold PBS, and fixed with 95% ethanol overnight at 20° C. On the following day, splenocytes were washed twice with FACS buffer (PBS supplemented with 0.5% BSA and 0.01% sodium azide), and incubated on ice for 30-45 min with APC-conjugated anti-CD3. Before flow cytometry, splenocytes were resuspended in PBS containing 5 mg/ml propidium iodide (PI) and 10 μg/ml RNase A (Sigma-Aldrich). Fluorescence-labeled cells were analyzed on a FACScan, using a CellQuest software package (Becton Dickinson).
Western Blot:
Splenocytes were lysed in NP-40 lysis buffer (0.5% Nonidet P-40, 50 mmol/L Tris-HCl [pH 8.0], 100 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) for 20 to 30 minutes on ice. Protein concentration was determined by Bradford reagent (Bio-Rad) Lysates were mixed with 2× Laemmli sample buffer at 1:1 ratio, heated for 5 minutes at 95° C., and equal amounts of protein were loaded onto 10% SDS-polyacrylamide gel electrophoresis (PAGE). Following electrophoresis, gels were blotted onto nitrocellulose membranes (Bio-Rad), blocked with 5% milk/PBS, and probed overnight with primary Abs. After extensive washing, blots were incubated with horseradish peroxidase-conjugated matching secondary Abs (Bio-Rad), and developed with enhanced chemiluminescent substrate (Sigma-Aldrich) before exposure to X-ray film (Fuji Film). Membranes were subsequently incubated with a-tubulin or GAPDH to ensure equal protein concentrations in each sample. Films were scanned and protein bands were quantitated using ImageJ software (NIH).
MLR:
Splenocytes were harvested from untreated Balb/c and C57BL/6 mice. C57BL/6 cells were incubated in a flat bottom 96 well plate for 96 h with either irradiated (1500 rads) or not irradiated Balb/c cells as indicated, at the indicated concentrations. XTT reagent for proliferation assay was added 24 h before reading. Plate was read at 620 nM.
Antibodies and Reagents:
Anti-mouse CD3 mAb were purchased from R&D Systems. For Western blot analysis, anti-Gsk-3β Ab, anti-p-Gsk-3β (ser9) Ab, anti-p-PKC Ab, and and anti-a-tubulin Ab were purchased from Cell Signaling. Anti-mouse GAPDH Ab were purchased from Sigma-Aldrich. XTT kit, PBS and cell medium were purchased from Biological Industries (Israel). All other reagents were purchased from Sigma-Aldrich unless otherwise indicated. Disposables were purchased from Corning.
Samples were collected from blood and spleen on days 0, 1, 3, 4, 5, & 6 post-transplant, mainly from the following 3 groups (at least 3 mice per group):
The results obtained show that GVHD mice have significantly lower levels of p-GSK-3β (phosphorylated in Ser-9) and total GSK-3β than both syngeneic and untreated control mice. This effect has been observed over time and is most significant on days 4-5 post transplant, both in the spleen and the blood samples. The main significance of these results is that the present invention provides for the first time a tool to diagnose and predict pathological conditions of T-cell proliferation, such as the diagnosis of GVHD effects in a subject, before emergence of the symptoms of the disease.
1. Spleen Results
a. Total GSK-3β and p-GSK-3β Expression Levels
The allogeneic mice developed GVHD symptoms by day 6 post-transplant. The symptoms of GVHD seen in the allogeneic mice included, but were not limited to, ruffled fur, hunched posture, closed eyes, reduced activity, diarrhea and weight loss. As shown in
Reference is now made to
The results have been normalized such that the values for the untreated control mice were set as 100%. Each parameter was compared to the values of the untreated mice.
As shown in
More particularly, a prolonged decrease in the level of p-GSK-3β and total GSK-3β over time was exclusively detected in the spleen of the allogeneic group mice. It should be mentioned that in the Syngeneic group, the splenocyte population has not recovered enough to collect cells, until day 5.
As shown in
To summarise the results described hereinabove, there is a prolonged decrease of total GSK-3β and p-GSK-3β over time in the spleen exclusively seen in the allogeneic group. The fact that in the syngeneic group, the splenocyte population has not been recovered enough to collect cells until day 5, while in the allogeneic mice there were enough splenocytes already on day 3, may be a result of the immune response characteristic of GVHD.
Once severe symptoms have appeared (days 5-6), levels of p-GSK-3β and total GSK-3β in allogeneic mice maintained lower than other groups. It is noted that the fact that in the syngeneic mice, the expression level of GSK-3β appears to approach almost normal by day 6, may reflect the effect of recovery from irradiation.
In the allogeneic group, p-GSK-3β and total GSK-3β decrease before the appearance of clinical symptoms (symptoms begin on day 5) and maintain lower than other groups throughout the disease course.
b. PKC Expression Level
Protein Kinase C (PKC) is upstream of GSK-3β in several pathways. Particularly, it is capable of phosphorylating GSK-3β directly. It is shown by the results of the present invention that activated PKC levels are significantly lower in splenocytes of both the syngeneic and allogeneic mice.
It is noted that the results described herewith have been normalized such that values for the control mice were set at 100%. Each parameter was compared to the value of untreated (control) mice independently of other parameters.
As shown in
c. T-Cell (CD3+) Proliferation Level
Total T cells (CD3+) were collected from spleens of mice of the 3 groups of the GVHD model as described above. Cells were stained for CD3 and counted by Flow Cytometry.
Reference is now made to
In order to explore the effect of GSK-3β activation state on T-cell proliferation, the GVHD model mice were treated with Lithium chloride (LiCl). Lithium chloride (LiCl) is known to affect GSK-3β activation state by regulating its phosphorylation state. Lithium chloride (LiCl) treatments were given to a separate group of allogeneic mice. The data obtained shows that the amount of T-cells increased after LiCl treatments on day 3 post-transplant. LiCl is a known inhibitor of GSK-3β, suggesting that the increase in T cells is due to increased inhibition of the GSK-3β.
Thus, according to certain embodiments of the invention, it is concluded that by monitoring and/or affecting GSK-3β expression and/or activation state, and/or related proteins thereof, early diagnosis and prevention of GVHD is achieved.
d. Mixed Lymphocyte Reaction
Splenocytes were collected from spleens of C57Bl/6 and Balb/c mice and cultured ex-vivo and assayed for proliferation by XTT (tetrazolium salt). The C57Bl/6 and Balb/c cells were cultured ex-vivo together to mimic a GVHD reaction. The cells were cultured in 3 groups, having different cell concentrations:
In order to test the effect of GSK-3β activation state on cell proliferation, Lithium Chloride (a known inhibitor of GSK-3β) was added at different concentrations. As shown in
e. Cell Cycle Analysis
Splenocytes were collected from mice of the GVHD model groups. T cells (CD3+) were analyzed by flow cytometry (FACS). Cell Cycle analysis was performed by detecting proliferating cells in each group.
Reference is now made to
The spleen results described hereinabove demonstrate that the decrease in the total GSK-3β and the p-GSK-3β in the spleens of allogeneic mice can be detected as early as day 3, before the appearance of observable symptoms. In view of these results it is a main aspect of the present invention that total GSK-3β, p-GSK-3β and/or proteins associated or related to GSK-3β protein are utilized as markers to identify and diagnose the onset of GVHD or any other medical condition associated with cell proliferation in mice and potentially in humans.
The results disclosed by the present invention formed the basis to the development of a novel diagnostic kit that can predict the onset of GVHD or any other medical condition associated with cell proliferation in subjects. According to one embodiment, the diagnostic kit utilizes protein chip technology to monitor the changes in the GSK-3β and/or at least one related protein thereof expression levels. In a further embodiment, the diagnostic kit utilizes changes in the phosphorylation or activation state of GSK-3β and/or at least one related protein thereof to predict the onset of GVHD or any other medical condition associated with cell proliferation.
It is further demonstrated by the experiment that once the mice have developed severe GVHD (i.e. day 5), a significant increase in T cell numbers post-transplant has been seen. In addition, it is shown by the present invention that LiCl, a known GSK-3β inhibitor, affect T-cell proliferation ex-vivo and possibly in vivo. In view of these results it is herein concluded that regulation of GSK-3β has a therapeutic use in GVHD or any other medical condition associated with cell proliferation.
2. Blood Results
Reference is now made to
The graphic results shown in
More specifically, the results obtained show that there is a decrease in total GSK-3β and p-GSK-3β in the syngeneic mice, which seems to be later than the decrease observed in the allogeneic mice. In the allogeneic group, there is a decrease in the levels of total GSK-3β and p-GSK-3β over time, this decrease is greater in the allogeneic group versus the syngeneic group.
It can be concluded from the results obtained by the blood experiment that a decrease in total GSK-3β and the p-GSK-3β is seen in the blood in mice of the allogeneic group, which is more pronounced than both the control and syngeneic mice.
It is further indicated that the decrease is most noticeable on days 3 and 4, which are at an early stage of GVHD. It is noted that at this stage of the disease T-cell proliferation is pronounced.
Thus based on the results described herein, a diagnostic kit is provided, which enables the use of blood samples to detect the early onset of GVHD and/or any other medical disorder associated with cell proliferation. According to certain embodiments, such a kit may utilize protein chip technology to monitor the changes in the GSK-3β expression levels and/or changes in the activation state of the GSK-3β to predict the onset of GVHD and/or any other medical disorder associated with cell proliferation. According to further embodiments, such a kit may additionally include GSK-3β related proteins, and by monitoring their expression levels and/or activation (i.e. phosphorylation) state the onset of GVHD and/or any other medical disorder associated with cell proliferation is predicted.
It is indicated that the blood results in mice reflects the proliferation in the spleen, meaning that the cells in the syngeneic mice on days 3, 4, and maybe 5 are cells that existed in the periphery before the transplant. However, in the allogeneic mice, the cells from day 4 are already new cells which have emerged from the spleen.
It should be emphasized that the fact that blood samples could be obtained from mice is a good indication of the ability to collect usable blood samples from humans.
The unique data presented herewith correlates GSK-3β directly to the progression and onset of the GVHD disorder which has never been done before. The correlation between GSK-3β expression and/or activation state and GVHD progression is associated with the effect on cell proliferation. Thus by monitoring and regulating GSK-3β and/or proteins related to GSK-3β (i.e. upstream or downstream GSK-3β), medical disorders associated with cell proliferation, such as GVHD can be early detected and inhibited before the emergence of undesirable symptoms.
According to a main aspect of the invention, the changes seen in the GSK-3β or related proteins expression levels and activation state can be utilized as markers for diagnosing the GVHD and/or any other medical disorder associated with cell proliferation.
According to a further main embodiment, the early onset of GVHD is detected prior to observable clinical symptoms through monitoring changes of the GSK-3β expression levels and/or changes in the activation state of the GSK-3β protein or related proteins thereof. Using GSK-3β and/or proteins associated with GSK-3β, as markers allow the diagnosis of GVHD or any other medical disorder associated with cell proliferation prior to patients demonstrating observable symptoms and prior to the T cells attacking the host body.
It is within the scope of the present invention, that by regulating GSK-3β expression in a time-course study of an acute GVHD mouse model, GSK-3β manipulation is shown to prevent GVHD and/or any other medical disorder associated with cell proliferation onset.
In another embodiment of the invention, a diagnostic kit based on a protein chip is used to diagnose GVHD and/or any other medical disorder associated with cell proliferation before the appearance of clinical symptoms. In certain embodiments the kit contains a custom-made protein chip comprising antibodies which are adapted to monitor the activation state and/or expression level of the GSK-3β, related proteins associated with related pathways such as apoptosis markers, proliferation markers, and other biological and biochemical molecules related to GSK-3β. Such molecule candidates can be pre determined and analysed by western blot, FACS, and other conventional fingerprinting techniques.
It is herein acknowledged that every disorder has a unique fingerprint. Finding the fingerprint for GVHD and/or any other disorder associated with cell proliferation allows us to provide a diagnostic kit with a set of proteins that are specifically and uniquely correlated with a particular disorder or medical condition associated with cell proliferation, for example GVHD.
Reference is now made to
As shown in
It is also within the scope of the present invention that clinical trials are performed to determine the efficacy of the diagnostic kit (i.e. protein chip prototype) to predict the development of GVHD in human transplant patients.
In order to determine the fingerprint for GVHD, samples form humans are collected from sick and healthy people, and analyzed by mass spectrometry to identify the following:
Human samples were collected using a CRA (clinical research associate) to recruit patients for a clinical trial (including Helsinki approval and negotiations with medical centers). The blood samples were collected from patients who have undergone bone marrow transplants.
In a preferred embodiment, a protein chip is utilized for analysis of the human samples to demonstrate its efficacy in predicting acute GVHD onset.
In a specific embodiment, the present invention provides a protein chip utilizing a large number of antibodies bound to a glass slide which can be used to determine the expression levels of different proteins in a given sample. One example is the development of a diagnostic kit based on a protein chip that can diagnose GVHD before the appearance of clinical symptoms
It is well within the scope of the present invention that the aforementioned kit contains a custom-made protein chip containing antibodies which are capable of monitoring the activation state and expression level of the GSK-3β, related pathways, apoptosis markers, proliferation markers, and others (taken from western blot, FACS, and fingerprinting data).
It is emphasised that the principal of the invention, namely GSK-3β and/or related proteins as diagnostic tool for abnormal cell proliferation, demonstrated the GVHD model, can be used to provide customized kit and method to detect and predict any other medical disorder associated with cell proliferation and/or the GSK-3β molecule.
The kit analysis results are preferably compared against the diagnostic outcome (according to current/updated medical standards) for each patient.
In certain embodiments of the invention, the kit is calibrated by assaying human samples by western blot and/or other fingerprinting technique common in the relevant art.
To demonstrate Gsk-3β phosphorylation sites by PKC, thus allowing understanding the mechanisms of Gsk3β regulation used herein for diagnostic and therapeutic applications.
PKC and Gsk3β were incubated together in a test tube. For analysis of phosphorylated amino acids on Gsk3β, two samples were subjected to mass spectrometry:
Nine phosphorylation sites were found on the Gsk3β incubated with PKC (experimental sample).
One of these sites (Thr324) was presented in the sample which contained Gsk3β alone (control sample), indicating that this site was previously phosphorylated or auto-phosphorylated.
The additional 8 sites on Gsk3β are shown by the current results to be phosphorylated directly by PKC.
Table 1 below summarizes the phosphorylation results of Gsk3β, showing 8 sites that are potentially phosphorylated by PKC in vivo. These phosphorylation sites are important in regulating Gsk3β.
As shown in Table 1, three new phosphorylation sites were identified that have not been discovered previously, including a potential auto-phosphorylation site. More specifically, the 3 sites that were discovered as new phosphorylation sites of Gsk3β are Thr324, Thr309 and Thr356. In addition there are discovered sites which are with unknown function, namely, Ser215, Ser219, Thr392 and Ser389.
In a further embodiment of the invention, mass spectrometry is utilized to assay Gsk3β phosphorylation sites in human T-cells and thus assessing the effect of PKC on phosphorylation of the Gsk3β in human T cells.
To determine the effect of PKC on Gsk3β and proliferation in human T-cells, in order to better understand the regulatory mechanisms of Gsk3β in T-cell proliferation. This data enables the development of tools as described inter alia to diagnose and treat medical disorders associated with cell proliferation and/or apoptosis such as GVHD.
In the following described experiment, these four major pathways were inhibited which directly phosphorylate Gsk3β in human T cells, preferably, Jurkat cells:
According to a certain embodiment, the following experimental groups were used:
Reference is now made to
As shown in
None:
Untreated control group, the values of which is set as 100%.
Inhibitors Group:
Inhibition of 3 different pathways, namely p38, Erk, and PI-3K pathways, significantly reduced cell proliferation in comparison to the control group. These results indicate that lower levels of Gsk3β phosphorylation is correlated with decreased cell proliferation.
PKC Group:
Activation of PKC, which is shown by the present invention to phosphorylate Gsk3β (Example 2) restores the cells to a “normal” proliferation level, which is similar to the control, despite the presence of other inhibitors. These results indicate that Gsk3β phosphorylation is correlated with an increase in proliferation.
PKC Inhibited Group:
When all of the 4 major pathways are inhibited (and thus Gsk3β is not phosphorylated), there is almost no cell proliferation. These results indicate that Gsk3β regulates T-cell proliferation.
Of the above described experiments it can be concluded that:
The above described studies provide a better understanding of the role of Gsk3β in T-cell proliferation and subsequently in early diagnosis and prevention of diseases associated with T-cell proliferation such as GVHD development.
In a further aspect of the invention, the affect of the newly identified phosphorylation sites on the Gsk3β on the activation state of the Gsk3β, is determined.
In a specific embodiment, Gsk3β activity is determined by assaying the levels of Gsk3β substrates.
A description of a GVHD diagnostic kit is provided as an example of a diagnostic kit prepared by the method and guidance of the present invention that can be tailored to diagnose any pathological disorder associated with cell proliferation or apoptosis that is herein shown for the first time to be linked with Gsk3β and related proteins.
The advantages of such a GVHD diagnostic kit:
A kit based on the platform technology as provided by the present invention can be applied for the diagnosis and/or prevention of pathological disorders associated with T-cell proliferation, for example: autoimmune disorders, inflammatory disease, tissue rejection, cancer, etc.
Diagnostic kits based on the technology of the present invention can be used in companion diagnosis approaches, by preferably performing the following steps: (1) Monitoring changes in activation state of Gsk3β and related proteins allows diagnosing other disorders. (2) Unique protein chips can be developed based on the platform technology of the present invention, which are custom-designed for each disorder. (3) Additional software packages are developed for analysis of each individual disease. (4) Disease treatments are screened using protein chips to ensure the drug is effectively targeting the molecular causes of the disease. (5) The understanding of the molecular pathways involved in each disease allows developing new specialized drugs.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL13/50237 | 3/14/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61610495 | Mar 2012 | US |
