Methods and Means for Monitoring Disruption of Tissue Homeostasis in the Total Body

Abstract

The invention relates to the field of medical diagnostics. Provided are methods and kits for determining the health status of a subject, for early detection of tissue damage, for early diagnosis and monitoring of a disease, and/or for evaluation of treatment effectiveness in a subject using circulating tissue macrophages (CTM) as a mirror of disrupted tissue homeostasis and disease.

1 . A method for determining the health status of a subject, for early detection of tissue damage, for early diagnosis and monitoring of a disease, and/or for evaluation of treatment effectiveness in a subject using circulating tissue macrophages as a mirror of disrupted tissue homeostasis and disease, the method comprising the steps of:
 a) providing a biological test sample from the subject which contains circulating tissue macrophages (CTM);   b) staining said CTM with a panel of differentially-labeled distinct antibodies against the backbone markers CD14, CD16 and CD300e, and preferably furthermore HLADR, for the identification and enumeration of different CTM subsets;   c) fixation, permeabilization and staining of the CTM using one or more detecting antibodies directed against one or more epitopes on at least one protease-induced protein fragments derived from intracellular degradation of a non-CTM protein by individual CTM in their tissues of origin, thereby identifying at least one subset of circulating tissue-specific macrophages (CTSM);   d) multiparameter flow-cytometric analysis of said stained CTM and CTSM by gating for the backbone markers CD14, CD16 and CD300e to determine the amount of signals of each distinct labeled antibody associated with individual cells;   e) determining the relative and absolute number of individual cells within each CTM subset and each specific subset of CTSM that express each of the measured intracellular epitopes;   f) calculating (i) the relative and absolute number of cells within each CTM subset and each specific subset of CTSM which each originate from different normal and altered tissues as defined by a set of individual protease-induced protein fragments evaluated, and ii) the amount of antibody-related signal associated to every individual intracellular peptide evaluated to obtain a test CTSM staining profile, and;   g) comparing the test CTSM staining profile with a normal CTSM staining profile for each tissue evaluated, wherein an aberrant test staining profile is indicative of tissue damage, an altered tissue homeostasis, the presence of a disease, and/or treatment effectiveness versus resistance.   
     
     
         2 . A method according to  claim 1 , wherein step b) comprises staining with antibodies directed at markers CD300e, CD14, CD16, and HLADR. 
     
     
         3 . A method according to  claim 1 , wherein step b) furthermore comprises staining with antibodies directed at one or more of the markers CD45, CD64 and CD36. 
     
     
         4 . A method according to  claim 1 , wherein step d) comprises performing a gating strategy based on the cell surface expression of CD300e, CD14 and CD16, preferably CD300e, CD14, CD16 and HLADR, in combination with side scatter (SSC) analysis. 
     
     
         5 . A method according to  claim 4 , wherein the gating strategy is composed of (i) an inclusion step to include both classical monocytes and CTMs based on CD300e expression, preferably CD300e and HLADR expression, in combination with side scatter (SSC) analysis, and (ii) a subsequent subset identification step to discriminate the classical CD14high/CD16− monocytes from CTM's and to identify subsets within the CTM population. 
     
     
         6 . A method according to  claim 5 , comprising either one of the following:
 (i) staining for at least CD300e, CD14 and CD16, and gating on a combination of SSC plus CD300e+ cells to select cells with low to intermediate SSC that express CD300e;   (ii) staining for at least CD300e, CD14, CD16 and HLADR, and gating on a combination of side scatter (SSC) plus CD300e+ and HLADR+ cells to select cells with low to intermediate SSC that simultaneously co-express CD300e and HLADR; or   (iii) staining for at least CD300e, CD14, CD16, CD45 and HLADR, and gating on a combination of SSC plus CD300e+, CD45+ and HLADR+ cells, preferably gating on cells with CD45 and low to intermediate SSC that simultaneously co-express CD300e and HLADR.   
     
     
         7 . A method according to  claim 5 , wherein the subset identification step comprises the identification of classical (CD14high/CD16−) monocytes and two main CTM subsets identified as CD14high/CD16low to CD14high/CD16high, CD14low/CD16high, and CD14−/CD16high to CD14−/CD16low cells. 
     
     
         8 . A method according to  claim 5 , wherein the CTM subset identification step comprises:
 gating on CD64low representing late stage CTM versus CD64high representing classical monocytes plus early stage CTM, followed by   discriminating between monocytes and CTM via selection for all other early CTM cells (vs. classical monocytes) as those events carrying a CD16+/CD14+ phenotype and defining the whole CTM compartment as CD6410 and as CD16+/CD14+/CD64high, followed by   further subdividing the selected CTM based on the expression levels of CD64, CD14 and CD16 into distinct functional or maturation-associated compartments, preferably wherein the distinct compartments are CD64high/CD14high/CD16low to CD64high/CD14high/CD16high, CD64high/CD14low/CD16high, CD64low/CD14−/CD16high and CD64low/CD14−/CD16low.   
     
     
         9 . A method according to  claim 8 , wherein the step of further subdividing the selected CTM is based on CD64, CD14, CD16 and CD36, preferably comprising the identification of at least one the following CTM subsets: from CD64high/CD36high/CD14high/CD16low to CD64high/CD36high/CD14high/CD16high, CD64high/CD36high/CD14low/CD16high, CD64low/CD36high/CD14−/CD16high and CD64low/CD14−/CD16low/CD36− to low cells. 
     
     
         10 . A method according to  claim 1 , wherein the biological sample comprises peripheral blood, ascitic fluid, pleural effusion, cerebrospinal fluid, bone marrow, lymph node, lymph fluid, synovial fluid, or a single cell suspension prepared from a solid tissue. 
     
     
         11 . A method according to  claim 1 , wherein step c) comprises intracellular staining of:
 a. one or more epitopes of a single protease-induced protein fragment derived from an intracellularly processed tissue-associated protein;   b. one or more epitopes of two or more distinct protease-induced protein fragments derived from one intracellularly processed tissue-associated protein;   c. one or more epitopes of two or more distinct protease-induced protein fragments derived from two or more intracellularly processed proteins derived from normal cells from a single organ or tissue;   d. one or more epitopes of two or more distinct protease-induced protein fragments derived from two or more intracellularly processed proteins derived from abnormal cells from a single organ or tissue;   e. one or more epitopes of two or more distinct protease-induced protein fragments derived from two or more intracellularly processed proteins derived from normal and abnormal cells from a single organ or tissue, including a combination of at least one antibody against peptide epitopes of a normal protein and at least one antibody against peptide epitopes from an aberrant protein, and;   f. one or more epitopes of two or more distinct protease-induced protein fragments derived from two or more intracellularly processed proteins derived from normal or abnormal cells from two or more organs or tissues.   
     
     
         12 . A method according to  claim 1 , wherein the at least one detecting antibodies allows for detection of one or more peptide epitopes derived from an aberrant protein, preferably wherein the aberrant protein is selected from the group consisting of oncogenic proteins, mutated proteins, fusion proteins, proteins derived from an allergen and proteins derived from a pathogen like a virus, a bacterium, a parasite or a fungus. 
     
     
         13 . A method according to  claim 1 , wherein the panel of differentially-labeled antibodies comprises a combination of compatible fluorochromes selected from fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridin chlorophyll protein (PerCP), allophycocyanin (APC), alexa fluor 488, alexa 647, alexa 710, alexa fluor 405, cyanin 5 (Cy5), Cyanin 5.5 (Cy5.5), pacific blue (PacB), horizon violet 450 (HV450), pacific orange (PacO), brilliant violet (BV), HV500, 00515, Krome Orange, quantum dots and conjugates thereof coupled with PE, to APC or to PerCP (e.g. PE/Cy5, PE/Cy5.5, PE/Cy7, PerCP/Cy5.5, APC/Cy7, PE-Texas Red, APCCy750) or any additional compatible fluorochrome or fluorochrome tandem. 
     
     
         14 . A diagnostic kit comprising in a first container differentially-labeled antibodies against a set of backbone markers comprising differentially-labeled antibodies, wherein the backbone markers are CD300e, CD14 and CD16 and preferably also HLADR, and optionally instructions for use of the kit for identification and enumeration of at least one, preferably at least two, more preferably at least three, CTM subsets. 
     
     
         15 . A diagnostic kit according to  claim 14 , wherein the first container comprises antibodies directed at markers CD300e, CD14 and CD16; preferably CD300e, CD14, CD16, and HLADR, more preferably furthermore comprising at least one antibody selected from the group of antibodies directed at marker CD36, CD64 or CD45. 
     
     
         16 . Kit according to  claim 15 , wherein the first container comprises antibodies against CD300e, CD14, CD16, HLADR, CD45, CD64 and CD36. 
     
     
         17 . Kit according to  claim 14 , further comprising a second container comprising at least one detecting antibody directed against one or more epitopes on at least one protease-induced protein fragment derived from intracellular degradation of a non-CTM protein by individual CTM in their tissues of origin. 
     
     
         18 . Kit according to  claim 17 , wherein the second container comprises at least one detecting antibody directed against one or more protease-induced protein fragment(s) derived from intracellular degradation of an epithelium protein, preferably a breast epithelium protein, an esophagus epithelium protein, a gastric epithelium protein, a pancreas epithelium protein, a colon epithelium protein, a rectum-sigmoid epithelium protein, a thyroid epithelium protein, a lung or bronchus epithelium protein, a prostate epithelium protein, a bladder epithelium protein, a cervix epithelium protein, an uterus epithelium protein, melanoma protein (e.g. Melan-A), kidney-glomerulus protein, or against another epithelial cell marker, optionally further comprising instructions for use in early diagnosis and monitoring of malignancies 
     
     
         19 . A diagnostic kit according to  claim 17 , wherein the second container comprises at least one detecting antibody allowing for detection of one or more epitopes derived from intracellular degradation of brain or nerve specific proteins, preferably selected from the group consisting of Apoε4, amyloid precursor protein (APP), GFAP, and myelin, optionally furthermore comprising instructions for use in early diagnosis and monitoring of Alzheimer's disease, glioma, or Multiple Sclerosis. 
     
     
         20 . A diagnostic kit according to  claim 17 , wherein the second container comprises at least one detecting antibody allowing for detection of one or more epitopes derived from intracellular degradation of heart muscle specific proteins (e.g. troponine or CK-MB), kidney/glomerulus-specific proteins, liver-specific proteins, or lung-specific proteins, optionally furthermore comprising instructions for use in the assessment of post-transplant organ survival and transplant rejection processes in a patient which has undergone, respectively, a heart, kidney, liver or lung transplantation.