WO2014087435A1 - Méthode de diagnostic de maladies auto-immunes - Google Patents

Méthode de diagnostic de maladies auto-immunes Download PDF

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Publication number
WO2014087435A1
WO2014087435A1 PCT/IT2012/000367 IT2012000367W WO2014087435A1 WO 2014087435 A1 WO2014087435 A1 WO 2014087435A1 IT 2012000367 W IT2012000367 W IT 2012000367W WO 2014087435 A1 WO2014087435 A1 WO 2014087435A1
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stem cells
vitro method
cells
previous
mesenchymal stem
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PCT/IT2012/000367
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English (en)
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Cristina ZANINI
Marco FORNI
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Euroclone S.P.A.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism

Definitions

  • the present invention relates to a method for the diagnosis of autoimmune diseases which comprises the use of substrates in place of the animal tissue commonly used.
  • Autoimmune diseases constitute a group of pathologies characterised by an alteration of the immune system which entails the development of cellular and humoral responses directed against components of the organism (self) .
  • the immune system distinguishes between the organism's own antigens, denominated self and those which are exogenous ⁇ non- self) thanks to a process known as "immunological tolerance”.
  • MHC II major histocompatibility complex
  • the genetic component also plays an important role in autoimmune disease, mainly by the association with the human leukocyte antigen (HLA, the equivalent of MHC for humans) .
  • HLA human leukocyte antigen
  • One example of genetic control of tolerance breakdown consists of the Autoimmune Regulator gene (AIRE) found on chromosome 21q22.3. Mutations in the AIRE gene significantly reduce the repertoire of self genes against which the immune system does not react; as a result, as soon as the T lymphocytes are liberated by the thymus into the circulation stream, numerous sensitisation processes of the immune system against self components take place [Matsumoto M. , 2009]. As regards classification, autoimmune diseases divide into two groups: organ-specific and systemic.
  • Hashimoto's thyroiditis primary biliary cirrhosis, ce.liac disease and diabetes mellitus type I.
  • systemic autoimmune diseases directed against autoantigens of various tissues are rheumatoid arthritis, systemic erythematous lupus, scleroderma and Goodpasture syndrome.
  • the concentration of anti-insulin autoantibodies is, at the moment of diagnosis, inversely proportional to the age of the patient. It is believed that during the cell-mediated destruction of the a cells of the islets of Langhera.ns, the endogenous insulin alters its three-dimensional conformation, thereby becoming antigenic and inducing an immune response [Martin S. e coll. 2001] . Moreover, a high level of antibodies in the serum of young patients is correlated with increased aggressiveness of the disease [Patel P., Macerollo A., 2010].
  • ICA Pancreatic islet cell autoantibodies
  • the ICA are class G organ-specific antibodies, identified for the first time in 1974. They are detected by means of an indirect immunofluorescence test of sections of human pancreatic tissue. The fluorescence generally affects all the islet cells, but in some preparations may colour only the ⁇ cells.
  • One of the antigens which these autoantibodies are directed against is a transmembrane protein of the islet cells which has a domain very similar to that of a tyrosine phosphatase present in the cytoplasm of T lymphocytes _ [Levi-Marchal C. e coll., 1995]. These antibodies may be present in a very high percentage (about 80%) in diabetic patients and their frequency falls over time.
  • Anti-insulin antibodies do not take part in the immunofluorescence reaction associated with anti-ICA antibodies. They may however be identified in the pre-clinical stage of diabetes, especially in paediatric subjects and have a high predictive value for a rapid development of the disease [Martin S. e coll. 2001, Jaeger C, 2008].
  • the presence of IAA in the serum is an index of the destruction process of a cells typical of DMT 1.
  • the IAA are fundamental for determining the risk of DMT 1, in that their concentration is very high in patients who develop the disease during infancy and they are the first autoantibodies which are seen before the pathological signs of DMT 1.
  • the prevalence of IAA is inversely proportional to the age at which the disease is diagnosed [Cambuli V.M., 2010].
  • IAA Instruction Manual MEDIPAN - CentAK® IAA RT, July 20th 2009] .
  • Celiac disease is an autoimmune disease characterised by intestinal malabsorption, by a characteristic intestinal lesion and by an intense lymphocyte infiltration which suggests a priority role of the immune system in the pathogenesis of the disease. It mainly affects the intestinal mucosa, while the sub-mucosa and muscular tunica are not affected. The lesions may only affect the duodenum and proximal jejunum, or the entire small intestine, though more serious in the proximal sections.
  • the transglutaminase enzyme is the antigenic target of autoantibodies found in the serum of patients suffering from MC. The main function of this enzyme is the deamination of the glutamine residues of the gliadin peptides and their conversion into glutamic acid.
  • This modification makes the gliadin negatively charged, and this causes its bonding with the antigens HLA-DQ2/DQ8, with the consequent presentation of the new peptides to the T lymphocytes.
  • the activation of an immune response of type T and B leads to the synthesis of anti-tTG class A autoantibodies and of pro-inflammatory cytokines, giving rise to a chronic inflammation with consequent destruction of the intestinal mucosa [Lindfors K. E coll. , 2009].
  • the anti-tTG class A autoantibodies are a highly sensitive and specific marker for the diagnosis of MC, while the type G autoantibodies are used at the moment in which the subject potentially suffering from MC presents a class A immunoglobulin deficit.
  • the anti-tTG antibodies recognise the same antigen as the anti-endomysial (EMA) autoantibodies, from which they differ only in the revelation method used.
  • the anti-tTG antibodies are dosed using an ' immunochemical and thereby quantitative type method
  • the EMA are . evaluated using indirect immunofluorescence (IFA) by means of measurement of the ⁇ fluorescence with a fluorescent light optic microscope, on cryostatic sections of monkey oesophagus.
  • IFA indirect immunofluorescence
  • the EMA test is highly specific, but less sensitive than immunochemical tests for tTG, and is consequently preferably used as a confirmation test [Caglar E. e coll. , 2009].
  • transglutaminase In healthy subjects, transglutaminase has been found in all the layers of the wall of the small intestine; the enzyme is expressed in _ the sub-mucosa; while only a small fraction is located in the epithelium.
  • the expression of tTG In subjects suffering from MC, the expression of tTG has been reported in specific districts of the small intestine, such as at the ciliated border and cytoplasm of the enterocytes, as well as in the extra cellular matrix.
  • the autoantibodies anti-tTG and EMA are synthesised by the B lymphocytes at the level of the intestinal mucosa of subjects suffering from MC not following a gluten-free diet.
  • the anti-tTG IgA may be found . to be positive without the EMA being positive.
  • the SMA Smooth Muscle Autoantibodies
  • SMA smooth Muscle Autoantibodies
  • the SMA are autoantibodies present in 85% and more of patients with chronic active hepatitis and in about 50% of patients with primary biliary cirrhosis. Their presence may sometimes be found in celiac . patients too .
  • the ANA have an important role in the medical sphere - despite being present in minimal amounts and at low intensity in many healthy individuals, they tend to increase considerably in the blood of subjects suffering from systemic autoimmune diseases.
  • cryostatic sections of animals are used for the anti-tissue antibodies.
  • as of now primate tissue is indispensable.
  • the anti-endomysial antibodies used to diagnose celiac disease the third distal of the oesophagus of the monkey is used, while for example for the paraneoplastic syndromes of the central nervous system, the cerebellum of the monkey is used.
  • the present invention exploits the potential of stem cells, differentiated and manipulated ad hoc if necessary, in place of animal tissues and cryostatic section technologies. Compared to sacrificing animals so as to freeze the tissue thereof and preparing cryostatic sections to use as diagnostic substrates, the culture of cells appears a less wasted, more flexible method easily applicable to the preparation of diagnostic kits.
  • Figure 1 show the results of chondrocyte differentiation (A: enlargement 4X; B: enlargement 10X) ;
  • Figure 2 show the results of osteoblast differentiation (A: enlargement 4X; B: enlargement 10X) ;
  • Figure 3 show the results of myogenic differentiation (A: positive for myogenin with nuclear contrast DAPI; B: myogenin only; C: DAPI only) ;
  • Figure 4 shows the results of maturation of the (3 cells after culture of HI-MSC cells in suspension;
  • Figure 5 shows the results of the immunochemical assay on differentiated pancreas cells from pancreas MSC (A: negative control, B: after incubation with the pool of positive serums for IAA, C: after incubation with the pool of positive serums for ICA) ;
  • Figure 6 shows the results of the immunochemical assay, on differentiated pancreas cells from the bone marrow (A: after incubation with the pool of positive serums for IAA, B: after incubation with the pool
  • a first object of the invention is represented by an in vitro method for the diagnosis of autoimmune diseases which comprises the use of stem cells.
  • embryonic, tumour, mesenchymal, etc. stem cells are used and preferably are mesenchymal stem cells.
  • stem cells are differentiated into other types of cells.
  • the method may also be used for the prognosis of autoimmune diseases .
  • a further object of the invention is represented by a kit for the diagnosis and/or prognosis of autoimmune diseases .
  • Stem cells are. defined as such on account of their plastic capacity to differentiate into different lines or to identically regenerate themselves.
  • embryonic stem cells inasmuch as pluripotent, may originate all the different tissues of the organism. Their use is limited by ethical issues, although cells of embryonic derivation but partially commissioned, can be derived from amniotic liquid and from the umbilical cord.
  • tumour stem cells The study of stem cells has shown that the propagation and dissemination of tumours in the organism depends on sub-populations of tumour cells with stem properties, in other words capable of auto- renewal and favouring the recurrence of the tumour.
  • stem properties in other words capable of auto- renewal and favouring the recurrence of the tumour.
  • literature has reported their presence in almost all types of leukaemia and solid human tumours (Dalerba et al, Ann. Rev. Med, 2007) .
  • the neuroglioblastoma is an embryonic tumour of the central nervous system which appears in children and only rarely in adults. The presence of tumour stem cells in this tumour has been demonstrated and described in literature (Raso A, Negri F, Neutopath Appl Neurobiol 2008 Jun; 34(3): 306-15).
  • MSCs Mesenchymal stem cells
  • BM bone marrow
  • the collection of a sample of bone marrow is a somewhat invasive procedure and in addition it has been shown that the number and differentiating potential of the MSCs from bone marrow decreases proportionally with age.
  • MSCs are myelar cells
  • UC-MSC umbilical cord
  • pancreatic islets HI-MSC- human islet mesenchymal stem cell
  • DP-MSC dental pulp
  • the mesenchymal stem cells obtained from BM-MSC, dental pulp PD-MSC and umbilical cord tissue have been isolated according to one of the methods reported in Chapter 1, Mesenchymal Stem Cell Assays and Applications, Methods in Molecular Biology, vol. 698, Springer-Verlag, 2011; or in Salvade et al., TISSUE ENGINEERING: Part C, Volume 15, Number 00, 2009; or in Rodriguez-Lozano.
  • MSCs may comprise peripheral blood, adipose tissue, amniotic liquid, renal glomerulus, liver and stratified human epithelium.
  • the autoimmune diseases for which the present method may be used comprise: celiac disease, paraneoplastic syndromes, diabetes mellitus I, Hashimoto's thyroiditis, rheumatoid arthritis, systemic erythematous lupus, scleroderma, Goodpasture syndrome, autoimmune hepatopathy, anti-phospholipid antibody syndrome, anticoagulant lupus.
  • the method according to the invention is aimed at the diagnosis of diabetes mellitus type I and celiac disease.
  • the method described herein is also useful for the prognosis of said diseases, meaning that the method provides useful information for assessing and predicting whether the subject will develop the disease over time.
  • the method comprises the determination of the presence of specific antibodies in a sample of serum of a subject on whom one wishes to perform diagnosis or prognosis.
  • the method proceeds by placing the serum sample in contact with a preparation of mesenchymal stem cells for a sufficient length of time to permit the formation of specific antigen- antibody complexes.
  • the incubation may be protracted by 10-60 minutes, preferably 20-40 minutes and generally 30 minutes, after which washing is performed to remove any non-bonded components. ' The presence of antibodies is determined by indirect immunofluorescence.
  • a secondary antibody is added (anti-IgG or anti IgA) marked with fluorescein which forms an immunocomplex with, the antigen-antibody complex.
  • the MSCs are used in the form of a preparation.
  • the cells are mesenchymal stem cells on a solid support and to such purpose a normal laboratory slide may be used.
  • methylcellulose or fibronectin may be used.
  • the mesenchymal stem cells used in the diagnostic/prognostic method are obtained from one of the sources mentioned above or ⁇ sourced commercially.
  • the diagnostic method according to the present invention may use stem cells obtained by differentiating mesenchymal stem cells.
  • MSC cells of the peripheral blood, adipose tissue, amniotic liquid, renal glomerulus, liver, stratified human epithelium, pancreatic islets and dental pulp can be differentiated into various cytotypes: osteoblasts, chondrocytes, myocytes, adipocytes, . pancreatic ⁇ cells, etc.
  • pancreatic ⁇ cells are obtained from MSCs isolated from pancreas islets, from bone marrow or from dental pulp.
  • kits to be used for the diagnosis or prognosis of autoimmune diseases which comprises a preparation of mesenchymal stem cells is described.
  • the autoimmune diseases diagnosed/ prognosticated are those mentioned above.
  • the cells are immobilised on a solid support, for example consisting of a normal laboratory slide.
  • the adhesion of the cells to the support is promoted by the use of methylcellulose or fibronectin, as for example performed when operating ' with pancreatic cells.
  • the kit according to the invention comprises anti-IgG antibodies appropriately marked for their determination.
  • these are fluoroscein-marked antibodies.
  • lxlO 5 mesenchymal stem cells were expanded with a serum-free expansion medium (Euromed Mesenchymal medium MSC) and brought into confluence over a period of 1-2 weeks. They are then placed in a medium comprising suitable differentiation factors (such as for example, the Euromed Human Adipogenic differentiation kit) . . The differentiation into adipocytes is observed after 2-3 weeks (Concise Review: Adipose Tissue-Derived Stromal Cells—Basic and Clinical Implications for Novel Cell-Based Therapies. Stem Cells. 2007 ; 25; 818-827 ) .
  • the MSC cells are induced to chondrogenic differentiation in pellets.
  • 2.5x105 of MSC are placed in a 15 ml bottle in 1 ml of serum-free expansion medium (Euromed Mesenchymal medium MSC) and grown for 1-2 weeks. They are subsequently centrifuged at 150 x g for 5 min.. at room temperature, aspirating and eliminating the supernatant (the operation should be repeated twice) .
  • 5 ml of complete medium comprising suitable differentiation factors (such as the Chondrogenic differentiation kit) is added to the pellet thus formed according to the methods reported in literature (Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, et al. (2006) Minimal criteria for.
  • the MSC cells are differentiated into osteoblasts using a . medium comprising suitable differentiation factors (such as for example EUROMED Human Osteogenic) placing 1X105 MSC cells in a T25 for about 2-3 we.eks (Barker E, Shiga A, Davies JE : Growing human bone in vitro. In: Bone Engineering. Emsquared Inc. Toronto, pp 63-77, 1999) .
  • the cells are then coloured with Alzarin Red to identify the calcium deposits present in the extracellular matrix during mineralisation of the bone ( Figure 2).
  • MSC bone marrow
  • BM-MSC bone marrow
  • dental pulp 10 15 MSC cells isolated from the pancreas, bone marrow (BM-MSC) and dental pulp, were put on plates in a T75 bottle and placed in a serum-free culture medium called EuroMed Human Mesenchymal Stem Cell Kit (ECM0888K) for 1-2 weeks; once 60% confluence was reached, the medium was replaced with another containing DMEM, glucose in a variable concentration from 4.5.
  • ECM0888K EuroMed Human Mesenchymal Stem Cell Kit
  • GLP-1. glucagon-like peptide I
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • ⁇ -cellulin nicotinamide
  • glutamine IX glutamine IX
  • the IFA analysis of serums containing EMA, IAA and ICA, SMA and ANA were performed at a referral laboratory using highly automated methods and, subsequently, the same samples were manually measured at the EuroClone laboratory (Molecular Biotechnology Center, Turin) .
  • the serums containing ICA and anti-insulin IAA are diluted in PBS at a proportion of 1:2.
  • IFA on differentiated pancreatic cells starting from MSC from the pancreas and MSC from the bone marrow. IFA performed on positive serums for EMA, AA, ICA
  • pancreatic cell populations were incubated with a negative control belonging to the kit marketed by INOVA diagnostic Inc., with a pool of positive serums for IAA and with a pool of positive serums for ICA.
  • the differentiated pancreatic cells starting from pancreas MSC and incubated with the negative control INOVA diagnostic Inc. did not show fluorescence (Figure 5A) despite showing a slightly positive background probably on account of molecular interactions with the fibronectin.
  • pancreatic cells starting from bone marrow MSC and incubated with the negative control INOVA diagnostic Inc., did not show positivity, despite a slight fluorescence of the background.
  • Umbilical cord mesenchymal cells were sown in concentrations of 2xl0 4 in each well of the slides Lab-Tek® Chamber-SlideTM 2 well glass slide (code 177380, NUNC) . After 48 hours in the incubator at 37 °C at 5% C0 2 , the cells achieved confluence, the culture medium was aspirated and washing performed with PBS. Subsequently the UC-MSCs on the slide were fixed with cold acetone for 5 minutes.
  • the serums of patients with celiac disease were diluted 1:5. 100 ⁇ ] _, of diluted serum are placed in each well of every slide; . these are left to incubate for 30 minutes, after which the slide is washed with PBS to remove the aspecific bonds, immersed for 5 minutes in a Coplin tray containing PBS, and incubated for 30 minutes with 100 of secondary antibody fluorescein-marked anti-IgA. After the incubation time is up, the slide is washed in PBS, mounted and observed under the fluorescence microscope with the filter regulated to the wavelength of the fluorescein.
  • Figure 1 shows positivity of fibrils referable to filaments of actin and rod cells corresponding to the anti-endomysial antibodies.
  • Serums ' of patients with autoimmunity of various origin were diluted 1:40 to verify anti-nuclear positivity.
  • 100 ⁇ , of diluted serum are placed in each well of every slide; these are left to incubate for 30 minutes, after which the slide is washed with PBS to remove the aspecific bonds, immersed for 5 minutes in a Coplin tray containing PBS, and incubated for 30 minutes with 100 ⁇ i of secondary antibody fluorescein-treated total anti-IgG.
  • Figure 2 shows the positivity with aspects of nuclear mitotic apparatus (NUMA) and high granular positivity (APG)
  • Figure 3 shows anti-nuclear positivity with high positivity of the SPECKLED type.
  • the serum containing EMA is diluted 1: 2.5 with PBS and subsequently a volume of 100 L is placed in the well of the slide made previously.
  • the serum is left to incubate for 30 minutes and after such time the slide is washed with PBS to remove the fraction of antibodies which have not bonded, immersed for 5 minutes in a Coplin tray containing PSB and subsequently incubated with 100 ⁇ L of secondary fluorescein-marked secondary antibody (anti -IgA 1:40 for EMA) for 30 minutes in a humidity chamber in the dark.
  • the slide is washed following the procedure described in the washing step above, a slide cover applied to it and fixed with glycerol and subsequently read under the fluorescence microscope with the filter regulated to the wavelength of the fluorescein .
  • the innovation described in this patent application permits the replacement of the use of human and animal tissue with a set of adequately manipulated stem cells.
  • This technology therefore makes it possible to avoid sacrificing animals, thereby offering numerous advantages both of an economic and ethical nature.
  • it makes it possible to simplify the procedures and operations for preparing diagnostic substrates.
  • it makes the reading of the results simpler in that advanced skills in the morphological field no longer need to be availed of.
  • Another considerable advantage is related to the possibility of automating the diagnostic procedure thus developed.

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Abstract

La présente invention concerne une méthode de diagnostic et/ou de pronostic de maladies qui comprend l'utilisation de cellules souches mésenchymateuses.
PCT/IT2012/000367 2012-12-06 2012-12-06 Méthode de diagnostic de maladies auto-immunes WO2014087435A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016157142A1 (fr) * 2015-04-02 2016-10-06 Stegi-Ra Trust Composition destinée à être utilisée pour traiter une maladie cœliaque

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016157142A1 (fr) * 2015-04-02 2016-10-06 Stegi-Ra Trust Composition destinée à être utilisée pour traiter une maladie cœliaque

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