Kit for monitoring, detecting and staging gvhd

Abstract

The present invention provides a method of diagnosing and treating Graft versus host disease (GVHD), said method comprising: a. obtaining a body fluid or tissue sample from said mammal, throughout the year post-transplant; b. detecting the expression level, phosphorylation level, or a combination thereof, of biomarkers in said body fluid or tissue sample; c. diagnosing said mammal with Graft versus host disease (GVHD) when the expression level, phosphorylation level, or a combination thereof of said biomarkers in said body fluid or tissue sample are detected with a deviation of at least about 25% from standard measurements taken from healthy subjects and/or from measurements taken from said mammal prior to said transplant; and, d. administering an effective amount of at least one immunosuppressant or steroid or a combination thereof to the diagnosed mammal.

1 . A method of diagnosing and treating Graft versus host disease (GVHD) in a mammal including a human undergoing transplant, said method comprising:
 a. obtaining a body fluid or tissue sample from said mammal, throughout the year post-transplant;   b. detecting the expression level, phosphorylation level, or a combination thereof, of biomarkers selected from the group consisting of: total glycogen synthase kinase-3β (GSK-3β), serine 9 phosphorylated GSK-3β (p-GSK-3β ser 9), proteins associated with the regulation of GSK-3β, and any combination thereof in said body fluid or tissue sample;   c. diagnosing said mammal with Graft versus host disease (GVHD) when the expression level, phosphorylation level, or a combination thereof of said biomarkers in said body fluid or tissue sample are detected with a deviation of at least about 25% from standard measurements taken from healthy subjects and/or from measurements taken from said mammal prior to said transplant; and   d. administering an effective amount of at least one immunosuppressant or steroid or a combination thereof to the diagnosed mammal.   
     
     
         2 . The method according to  claim 1 , wherein said detection is done by protein analysis. 
     
     
         3 . The method according to  claim 1 , wherein an array of antibodies is used for said detection. 
     
     
         4 . The method according to  claim 3 , wherein said array of antibodies are bound to a glass slide or equivalent substrate, or a chip thereby providing a protein chip analysis tool. 
     
     
         5 . The method according to  claim 3 , wherein said biomarkers are selected from the group consisting of Total GSK-3β, ser 9 phosphorylated GSK-3β, GSK-3β ser 9 phosphorylation PKC Activation, b-catenin, Dvl, Akt, Erk, P38 MAPK, GSK-3β Tyrosine 216 Phosphorylation, Serum Albumin, Fas, Bcl family, Cyctochrome C, caspases, Nf-kB, CD25, Cyclin D1, PCNA and p27, phosphorylation sites on GSK-3β and any combination thereof. 
     
     
         6 . The method according to  claim 1 , wherein said detection is done using a diagnostic kit having a protein chip analysis tool, or any other protein analysis analytical tool. 
     
     
         7 . The method according to  claim 1 , further comprising at least one of the following steps:
 a. measuring at least one parameter selected from a group consisting of GSK-3β expression levels and phosphorylation levels, GSK-3β activation levels, GSK-3β inactivation levels, serine 9 phosphorylated GSK-3β expression, expression levels and phosphorylation levels of proteins associated with regulating the activation status and function of GSK-3β, phosphorylation sites on the GSK-3β and p-PKC expression;   b. measuring at least one activation indicator selected from the group consisting of over-expression of GSK-3β, tyrosine 216 phosphorylation and any combination thereof;   c. determining the expression, phosphorylation and/or inactivation state of GSK-3β by measuring at least one inactivation indicator selected from the group consisting of 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 expression levels or through a protein marker specific for inactivation of the GSK-3β through activated PKC, and any combination thereof; and   d. measuring expression, phosphorylation and/or activation of GSK-3β by measuring at least one activation indicator selected from the group consisting of over-expression of GSK-3β, tyrosine 216 phosphorylation and any combination thereof.   
     
     
         8 . The method according to  claim 1 , comprising additional steps of measuring the levels of changes in expression levels and phosphorylation levels for pathways and proteins associated with regulating the activation status and function of GSK-3β, phosphorylation sites on the GSK-3β, apoptosis markers, proliferation markers, and other biological and biochemical molecules. 
     
     
         9 . The method according to  claim 1 , comprising additional steps of:
 a. measuring the level of GSK-3β inactivation in said sample by at least one assay selected from the group consisting of measuring β-catenin levels in the absence of serine 9 phosphorylation, measuring stimulation levels at the site on the GSK-3β stimulated through the activated Wnt pathway, measuring a protein marker that is unique to the inactivation of GSK-3β through the activated Wnt pathway and measuring a protein marker that is unique to the inactivation of GSK-3β through the activated PKC; and   b. comparing the result of said inactivation levels with a predetermined normal inactivation value obtained from healthy subjects and time 0 from said patient,   
       wherein an increased deviation of at least about 25% from normal inactivation values indicates the presence of GVHD. 
     
     
         10 . The method according to  claim 1 , comprising at least one additional step selected from the group consisting of;
 a. measuring GSK-3β inactivation indicators;   b. measuring GSK-3β activation indicators;   c. combining measurements of activation and inactivation indicators;   d. measuring changes in expression levels and phosphorylation levels for pathways and proteins associated with regulating the activation status and function of the GSK-3β;   e. measuring changes in phosphorylation of the phosphorylation sites on the GSK-3β;   f. measuring changes in expression levels or phosphorylation levels for GSK-3β upstream and downstream proteins and pathways and on the GSK3 GSK-3β molecule itself,   
       wherein an activation to inactivation ratio is obtained, characteristic of GVHD. 
     
     
         11 . A method of diagnosing and treating Graft versus host disease (GVHD) in a mammal including a human undergoing transplant, said method comprising:
 a. obtaining a body fluid or tissue sample from said mammal, throughout the year post-transplant;   b. detecting the level of biomarkers selected from the group consisting of: total glycogen synthase kinase-3β (GSK-3β), serine 9 phosphorylated GSK-3β (p-GSK-3β ser 9), and any combination thereof in said body fluid or tissue sample;   c. diagnosing said mammal with Graft versus host disease (GVHD) when the level of said biomarkers in said body fluid or tissue sample is detected with a decreased deviation of at least about 25% from standard measurements taken from healthy subjects and/or from measurements taken from said mammal prior to said transplant; and   d. administering an effective amount of at least one immunosuppressant or steroid or a combination thereof to the diagnosed mammal.