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    • 专利摘要:
    A method of diagnosing an eye disease having an element of age-related retinopathy with acquired choroidal abnormalities in a subject is described. The method comprises providing a sample to be tested obtained from the subject, determining the level of expression of at least one biomarker in the sample, determining a control level of expression of said at least one biomarker in a control sample obtained from a subject not afflicted with the eye disease, comparing said level of expression with said control level of expression of said at least one biomarker, wherein a level of expression of said at least one biomarker in said sample that is higher than said control level of expression of said at least one biomarker in said control sample is indicative of the presence of said eye disease, and wherein a level of expression of said at least one biomarker in said sample that is equal to or lower than said control level of expression of said at least one biomarker in said control sample is indicative of the absence of said eye disease, and wherein said at least one biomarker is selected from IL-33, regulatory T-cells, and/or T-helper-2-like regulatory T-cells.
    公开号:DK201870463A1
    申请号:DKP201870463
    申请日:2018-07-05
    公开日:2020-01-14
    发明作者:Subhi Yousif；Lykke Sørensen Torben
    申请人:Region Sjælland；
    IPC主号:
    专利说明:

    BIOMARKER IN POLYPOIDAL CHOROIDAL VASCULOPATHY AND USES THEREOF
    TECHNICAL FIELD
    The present invention relates to methods for diagnosing an eye disease having an element of age-related retinopathy with acquired choroidal abnormalities in a subject.
    BACKGROUND
    Age-related retinopathy with acquired choroidal abnormalities is a condition seen in advanced age (>50 years of age) where the retina is anatomically affected due to acquired abnormalities originated from the underlying choroid that are blood vessels.
    Polypoidal choroidal vasculopathy (PCV) is an example of an eye disease having an element of age related retinopathy. It is a chorioretinal disease with characteristic polypoidal aneurysms in the choroid that protrude into the retina. Resulting exudates and retinal damage leads to vision loss. An aneurysm is a localized dilation of an arteria that occurs when part of an artery wall weakens or is injured, allowing it to widen abnormally.
    PCV is frequently associated with recurrent hemorrhagic or exudative pigment epithelium detachment (PED), and with leakage and bleeding from the polypoidal components.
    PCV is an important differential diagnosis to neovascular age-related macular degeneration (AMD), which is the most common reason for irreversible vision loss in developed countries. Although the prevalence of PCV is relatively rare in Caucasian populations, e.g. approx. 0.04 % of the population in Denmark, it is estimated that the prevalence is approximately x 10 times higher in Asian and African populations. PCV is characterized by choroidal-originated polyps in relation to choroidal neovascularization. The visual prognosis in PCV is correlated with the size of the lesions. Larger lesions in patients often results in higher rates of lesion progression, more severe complications and poor visual outcome, while patients having smaller lesions have lower rates of lesion progression and a better visual outcome.
    DK 2018 70463 A1
    Currently, diagnosis of PCV requires intravenous administration of indocyanine-green to perform indocyanine-green retinal angiography. Although indocyanine-green angiography is considered safe for most patients, some patients show reactions to the dye and develop itching, nausea and a rash. Patients with an iodine or shellfish allergy may not be able to have indocyanine-green angiography because of known allergic cross-reaction. Hence, indocyanine green not only requires high expertise, it also requires a costly setting and may be impractical or impossible for some patients.
    Even though neovascular AMD and PCV both are characterized by exudative macular lesions, there are important differences between them. For instance, AMD is associated with drusen-formation, whereas PCV has unique vascular malformations.
    It is important to make the distinction between neovascular AMD and PCV because treatment approaches may vary. Treatment of PCV differs from neovascular AMD in that photodynamic therapy is considered for add-on treatment for polyp closure.
    Current treatment of PCV is based on monthly/bimonthly injections with anti-vascular endothelial growth factor into the eye in addition to laser-based photodynamic therapy to obtain polyp closure. However, regular injections into the eye are highly costly, impractical for the patient, and is not free of adverse events. Laser-based photodynamic therapy for polyp closure leads to retinal lesions in the immediate surroundings and hence cannot be applied without great risk of vision loss if the polyps are in the central foveal area.
    Thus, in view of the above there is a need for new methods for the diagnosis of PCV, methods which are practical and easy to perform and, not causing too many adverse effects on the subject, and which identify potential targets for effective treatment of PVC.
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    SUMMARY OF THE INVENTION
    It is an object of at least certain aspects of the present invention to provide an improvement over the above described techniques and known art; particularly, to achieve a method of diagnosis that provides an easy, correct and patient friendly diagnosis of PCV.
    Accordingly, a first aspect of the present invention provides a method of diagnosing an eye disease having an element of age-related retinopathy with acquired choroidal abnormalities in a subject comprising:
    a. providing a sample to be tested obtained from the subject,
    b. determining the level of expression of at least one biomarker in the sample,
    c. determining a control level of expression of said at least one biomarker in a control sample obtained from a subject not afflicted with the eye disease,
    d. comparing said level of expression with said control level of expression of said at least one biomarker, wherein a level of expression of said at least one biomarker in said sample that is higher than said control level of expression of said at least one biomarker in said control sample is indicative of the presence of said eye disease, and wherein a level of expression of said at least one biomarker in said sample that is equal to or lower than said control level of expression of said at least one biomarker in said control sample is indicative of the absence of said eye disease, and wherein said at least one biomarker is selected from IL33, regulatory T-cells, and/or T-helper-2-like regulatory T-cells.
    According to one aspect the subject is not suffering from diabetes related retinopathy.
    According to one aspect the subject is not suffering from neovascular age-related macular degeneration, defined as acquired choroidal neovascularizations with no polyp-like features.
    According to one aspect the healthy subjects are matched with the subjects by age and/or sex.
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    According to one aspect a level of IL-33 in a plasma sample from the subject that is equal to or above 0.1 pg/ml, such as equal to or above 0.2 pg/ml, such as equal to or above 0.3 pg/ml, such as equal to or above 0.4 pg/ml, such as equal to or above 0.5 pg/ml, such as equal to or above 0.6 pg/ml, such as equal to or above 0.7 pg/ml, such as equal to or above 0.8 pg/ml is indicative of said eye disease.
    According to one aspect a proportion of CD4+ T cells that are regulatory T cells that is equal to or below 8 %, such as equal to or below 7.5 %, such as equal to or below 7 %, such as equal to or below 6.5 %, such as equal to or below 6 %, such as equal to or below 5.5 % is indicative of said eye disease.
    According to one aspect a proportion of CD4+ regulatory T cells that are T-helper-2-like that is equal to or above 40 %, such as equal to or below 45 %, such as equal to or below 50 %, such as equal to or below 55 %, such as equal to or below 60 %, such as equal to or below % is indicative of said eye disease.
    According to one aspect the eye disease having an element of age related retinopathy with acquired choroidal abnormalities is Polypoidal Choroidal Vasculopathy.
    According to one aspect the subject is likely to experience one or more of a change in best-corrected visual acuity, change in visual function, change in retinal thickness, need for therapy in terms of type of therapy and the quantity of therapy, or development of subretinal fibrosis.
    According to one aspect the subject is likely to benefit from treatment with an inhibitor of IL-33 when the level of IL-33 in a plasma sample from the subject is equal to or above 0.1 pg/ml, such as equal to or above 0.2 pg/ml, such as equal to or above 0.3 pg/ml, such as equal to or above 0.4 pg/ml, such as equal to or above 0.5 pg/ml, such as equal to or above 0.6 pg/ml, such as equal to or above 0.7 pg/ml, such as equal to or above 0.8 pg/ml.
    A further aspect of the present invention provides a composition comprising a therapeutically, prophylactic or alleviation effective
    DK 2018 70463 A1 dosage of an IL-33 inhibitor for use in the treatment, prophylaxis or alleviation of an age related retinopathy with acquired choroidal abnormalities in a subject, wherein the patient to receive the composition exhibits a level of IL-33 in a plasma sample from the subject which is equal to or above 0.1 pg/ml, such as equal to or above 0.2 pg/ml, such as equal to or above 0.3 pg/ml, such as equal to or above 0.4 pg/ml, such as equal to or above 0.5 pg/ml, such as equal to or above 0.6 pg/ml, such as equal to or above 0.7 pg/ml, such as equal to or above 0.8 pg/ml.
    According to one aspect the eye disease having an element of agerelated retinopathy with acquired choroidal abnormalities is Polypoidal Choroidal Vasculopathy.
    According to one aspect the IL-33 inhibitor is an antibody or a small organic molecule.
    The anti-IL-33 antibody may for example be selected from any of those described in US2014/0271642 or WO2017124110.
    According to one aspect the composition is formulated for ophthalmic administration.
    A further aspect of the present invention provides a method of treatment, prophylaxis or alleviation of an eye disease having an element of age-related retinopathy with acquired choroidal abnormalities in a subject, wherein a composition comprising an IL-33 inhibitor is provided to the subject.
    According to one aspect the eye disease having an element of age related retinopathy with acquired choroidal abnormalities is Polypoidal Choroidal Vasculopathy.
    A further aspect of the present invention provides a method of treatment, prophylaxis or alleviation of an eye disease having an element of age related retinopathy with acquired choroidal abnormalities in a subject, comprising:
    a. providing a sample to be tested obtained from the subject,
    b. determining the level of expression of at least one biomarker in the sample,
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    c. determining a control level of expression of said at least one biomarker in a control sample obtained from a subject not afflicted with the eye disease,
    d. comparing said level of expression with said control level of expression of said at least one biomarker, wherein a level of expression of said at least one biomarker in said sample that is higher than said control level of expression of said at least one biomarker in said control sample is indicative of the presence of said eye disease, and wherein a level of expression of said at least one biomarker in said sample that is equal to or lower than said control level of expression of said at least one biomarker in said control sample is indicative of the absence of said eye disease, and wherein said at least one biomarker is selected from IL33, regulatory T-cells, and/or T-helper-2-like regulatory T-cells,
    e. providing to the subject, if step d. is indicative of the presence of said eye disease, a composition with a therapeutically, prophylactic or alleviation effective dosage of an IL-33 inhibitor.
    According to one aspect the eye disease having an element of age related retinopathy with acquired choroidal abnormalities is Polypoidal Choroidal Vasculopathy.
    According to one aspect the IL-33 inhibitor is an antibody.
    The anti-IL-33 antibody may for example be selected from any of those described in US2014/0271642 or WO2017124110.
    According to one aspect the method further comprises measuring at least one environmental or lifestyle factor selected from the group consisting of age, smoking history, weight, body mass index, diet, and exercise habits.
    According to one aspect the measuring of the at least one environmental or lifestyle factor is used to pair diseased subjects with control subjects with similar environmental or lifestyle factor profiles.
    According to one aspect the sample or control sample is a tissue sample or a bodily fluid sample.
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    According to one aspect the bodily fluid sample is a blood sample, a plasma sample or a serum sample.
    At least some of the above identified and other objects and advantages that may be apparent from the description have been achieved by the methods and composition in accordance with the above.
    BRIEF DESCRIPTION OF THE DRAWINGS
    These and other aspects, features and advantages of which embodiments of the invention are capable of, will be apparent and elucidated from the following description of embodiments and aspects of the present invention, reference being made to the accompanying drawings, in which
    Figure 1. is a study flow diagram, which shows how patients recruited for the study were checked regarding their eligibility, and then divided into three groups, patients with PCV, patients with neovascular AMD, and healthy controls.
    Figure 2. shows identification and quantification of T helper cell subsets. A: Singlet leukocytes were identified using forward scatter (FSC) area vs. height density plot with contour. B: Lymphocytes were identified using FSC area vs. side scatter (SSC) area density plot with contour on singlet leukocytes. C: CD4+ T cells were identified as CD4+ lymphocytes. D and E: Negative isotype controls were used to distinguish non-specific background signals (1 % was accepted) from
    CD4+ T cells that were CXCR3+ (D) and CCR6+ (E). F: CD4+ T cells were subgrouped into four groups according to their CXCR3 and CCR6 expression: CXCR3+CCR6-, CXCR3-CCR6-, CXCR3-CCR6+, CXCR3+CCR6+, which were assumed to respectively represent the Th1, Th2, Th17, and Th1/Th17 populations. G: Mean and 95% confidence intervals of the distribution of T cell populations in healthy controls.
    Figure 3. shows identification and quantification of regulatory T cells (Treg) and comparison between healthy controls and patients with polypoidal choroidal vasculopathy (PCV) or patients with neovascular age-related macular degeneration (nAMD). A: CD4+ T cells were identified as CD4+ lymphocytes. B: Treg were identified as CD4+ T cells that were CD25high and CD127low. C: Treg levels did not differ significantly between healthy controls and patients with nAMD (P =
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    0.96, independent samples t-test), but were significantly lower in patients with PCV than in healthy controls (P = 0.027, independent samples t-test, signified with *). Whiskers represent mean and 95% confidence interval.
    Figure 4. shows comparison between healthy controls and patients with polypoidal choroidal vasculopathy (PCV) or patients with neovascular age-related macular degeneration (nAMD) in plasma levels of interleukin(IL)-4 and IL-33. A: Plasma IL-4 levels did not differ significantly between the groups. Whiskers represent median and interquartile range. B: Plasma IL-33 levels did not differ significantly between healthy controls and patients with nAMD (P = 0.55, Mann-Whitney U-test), but were significantly higher in patients with PCV than in healthy controls (P = 0.033, Mann-Whitney U-test, signified with *). Whiskers represent median and interquartile range.
    Figure 5. shows identification and quantification of regulatory T helper(Th)-like cells. A: CD4+ T cells were identified as CD4+ lymphocytes. B: Regulatory T cells (Treg) were identified as CD4+ T cells that were CD25high and CD127low. C: Tregs were subgrouped into four groups according to their CXCR3 and CCR6 expression: Treg CXCR3+CCR6-, Treg CXCR3-CCR6-, Treg CXCR3-CCR6+, Treg CXCR3+CCR6+, which were assumed to respectively represent the Th1-like Treg, Th2-like Treg, Th17-like Treg, and Th1/Th17-like Treg populations. D: Mean and 95% confidence intervals of the distribution of Th-like Treg cell populations in healthy controls.
    DETAILED DESCRIPTION OF THE INVENTION
    Aspects of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings.
    The terminology used herein is for the purpose of describing particular aspects of the disclosure only, and is not intended to limit the disclosure. As used herein, the singular forms a, an and the are intended to include the plural forms as well, unless the context clearly indicates otherwise.
    In the drawings and specification, there have been disclosed exemplary aspects of the disclosure. However, many variations and modifications
    DK 2018 70463 A1 can be made to these aspects without substantially departing from the principles of the present disclosure. Thus, the disclosure should be regarded as illustrative rather than restrictive, and not as being limited to the particular aspects discussed above. Accordingly, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation.
    It should be noted that the word “comprising” does not necessarily exclude the presence of other elements or steps than those listed and the words “a” or “an” preceding an element do not exclude the presence of a plurality of such elements.
    EXAMPLES
    Study Design and Ethics
    All aspects of this prospective case-control study follows the principles in the Declaration of Helsinki and ethical approval was obtained by the Regional Committee of Ethics in Research of the Region of Zealand (SJ-379). Prior to participation, participants had the nature of the study explained and gave oral and written informed consent. Participants were recruited consecutively from an outpatient clinic at Department of Ophthalmology, Zealand University Hospital, Denmark. To estimate participants needed for this exploratory study, the power calculation was based on some of the previous experiences with T cell studies of patients with AMD, which is that generally at least 20 individuals is needed to obtain a meaningful sample size.17'18 It was estimated that at least 18 participants in each group were necessary to obtain sufficient power (assuming α=0.05, β=0.8, and σ=2). At least 18 participants were recruited in each group and further recruitment was stopped after a total of 110 participants. Eight were excluded either because ongoing immune response was suspected or because the samples failed flow cytometry, see Fig .1.
    Groups did not differ significantly in demographics, co-morbidities, and lifestyle factors, see Table 1.
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    Table 1. Participant characteristics.
    Healthy controls (n=32)Patients with PCV (n=24)Patients with nAMD (n=45)P-valueDemographicsAge, years, mean (SD)73.472.375.40.24 a (7.9)(7.7)(7.2) Females, n (%)18 (56)16 (67)24 (53)0.56 b Co-morbiditiesHypertension, n (%)11 (34)11 (46)24 (53)0.26 b Cardiovascular disease,9 (20)5 (21)9 (16)0.85 b n (%)Hypercholesterolemia, n12 (38)9 (38)11 (24)0.37 b (%)Type 2 diabetes, n (%)0 (0)3 (13)6 (13)0.057 cLifestyle factorsSmoking, n (%) 0.29 cActive5 (15)8 (33)13 (29) Previous14 (44)12 (50)18 (40) Never13 (41)4 (17)14 (31) Alcohol consumption,4 (2 to4 (1 to4 (1 to0.72 d units, median (IQR)7)14)9) Body mass index, mean25.325.326.10.73 a (SD)(4.4)(3.7)(4.2) Physically active, n22 (69)13 (54)24 (53)0.36 b (%)
    Smoking habits was categorized into one of three groups: active, previous (>100 cigarettes during lifetime and ceased smoking >12 months), or never.
    Alcohol consumption is quantified using units/week (1 unit = 15 mL/12 g pure ethanol). Body mass index is calculated as weight divided by height squared. Being regularly physically active is categorized using a simple question for epidemiological studies previously validated in Danish patients with AMD.
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    Abbreviations: PCV = polypoidal choroidal vasculopathy; nAMD = neovascular age-related macular degeneration; SD = standard deviation; IQR = interquartile range.
    Statistical comparisons are made using (a) one-way analysis of variance, (b) X2-test, (c) Fisher's Exact test because of categories with 4 cases, and (d) Kruskal-Wallis' test.
    Participant Eligibility, Clinical Information, and Retinal Diagnosis
    Participants were recruited if they had either PCV in one or both eyes, neovascular AMD in one or both eyes, or healthy retinas in both eyes (healthy controls). Healthy controls were recruited among biologically unrelated relatives to the patients. The strategy was to better match the control group to the patient groups, as lifestyle factors can potentially influence systemic immunology. 28,29 Participants were included if they fulfilled the following criteria to avoid blurring the results due to a known active ongoing immune activity: no active cancer, no immune disease, no ongoing or chronic infectious disease, and no chemo- or immunotherapy for any reason. Because this study looked at Th cell polarization, participants with asthma where not included in the study. Patients with recent onset of CNV were not included because of acute immune activity.19 Patients in ranibizumab or aflibercept therapy were only included 4 or 8 weeks after last injection, respectively, to avoid potential interaction with the antibodies used for flow cytometry. All participants were interviewed to obtain data on lifestyle and medical history. Lifestyle factors noted were smoking habits, alcohol consumption, body mass index, and physical activity.29 Medical data were crosschecked with the electronic patient record to ensure accuracy. Participants were examined using slit-lamp bio-microscopy, digital fundus photography, and spectraldomain optical coherence tomography (OCT), and retinal angiography (fluorescein and ICGA) where CNV was suspected. The following definition for the groups were used:
    - Healthy controls: Less than 10 small drusen (diameter < 63 μm) and no pigment abnormalities.30
    - Neovascular AMD: Fibrovascular pigment epithelium detachments and choroidal neovascular membranes with subretinal or sub-RPE hemorrhages or fibrosis.30
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    - PCV: One or more polyps in early-phase ICGA with a hypofluorescent halo and with/without BVN. Other stigmata used to support the diagnosis were orange-red focal subretinal polyplike structures, pulsation of polyps on ICGA video, and a protrusion from the choroid elevating RPE from the Bruch's membrane observed on OCT.7
    Blood Sampling
    Venous blood was sampled from antecubital veins in separate ethylenediaminetetraacetic acid (EDTA) and lithium heparin coated tubes. EDTA stabilized blood was used for flow cytometric analyses within 4 hours after blood sampling. One lithium heparin coated tube was used for routine CRP measurement. Other tubes with lithium heparin stabilized blood were centrifuged for 15 minutes at 1500 G after which plasma was isolated and stored at -80° C for later quantification of plasma cytokines.
    Flow Cytometry
    The white blood cell count was obtained using an automated hematology analyzed Sysmex KX-21N™ (Sysmex Corporation, Kobe, Japan) to calculate blood volume necessary to obtain 5·105 white blood cells in each test tube. The red blood cells were lysed in 1 % lysis buffer (Nordic Biosite AB, Täby, Sweden) for 10 minutes in the dark at room temperature. The cells then were washed three times by first centrifuging for 5 minutes at 500 G and then resuspending in an isotonic buffer (BD FACSFlow™, BD Biosciences, Franklin Lakes, NJ,
    USA). Marker-specific monoclonal antibodies were added thereafter to the sample tube and flourochrome-matched negative isotypes were added to a separately prepared tube. Table 2 shows antibodies used for flow cytometry.
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    Table 2. Antibodies used for flow cytometry.
    TargetFluorochromeIsotypeCloneCatalog No.Markers investigatedCD4PeridininChlorophyllIgG2a11830FAB3791C-100 aCXCR3Phcoerythrin-CY7IgG11C6/CXCR3560831 b CCR6FluoresceinIsothiocyanateIgG2bG034E3353412 cCD25Pacific BlueIgG1302627302627 cCD127BrilliantIgG1A019D5351332 c
    Violet 510
    Negative isotypes
    -PeridininChlorophyllIgG2aMOPC-173400250 c-Phcoerythrin-CY7IgG1MOPC-21400126 c -FluoresceinIsothiocyanateIgG2b133303IC0041F a -Pacific BlueIgG1IC0041F400151 c -BrilliantIgG1MOPC-21400172 c
    Violet 510
    Manufacturers: (a) R&D Systems, Minneapolis, MN, USA. (b) BD
    Biosciences, Franklin Lakes, NJ, USA. (c) BioLegend, San Diego, CA, 5 USA.
    Tubes were incubated in the dark at room temperature for 20 minutes, after which the cells were washed and resuspended in an isotonic buffer (BD FACSFlow™, BD Biosciences). Stained cells were analyzed using flow cytometry (BD FACSCanto II™, BD Biosciences) with a sample size gated for 100.000 singlet leukocytes. Kaluza™ analysis software v. 1.5.20365.16139 (Beckman Coulter Inc., Pasadena, CA, USA) was
    DK 2018 70463 A1 used for all flow cytometric analyses. Two independent evaluators performed all analyses completely blinded to each participant's condition and each other. CD4+ T cells were identified and gated based on their CXCR3 and CCR6 expression, see figure 2. Zhang et al. HF, Zhao MG, Liang GB, Yu CY, He W, Li ZQ, Gao X. Dysregulation of studied Th cell subsets in humans and found that CD4+CXCR3+CCR6 cells had characteristics of Th1, CD4+CXCR3“CCR6_ had characteristics of Th2, and that CD4+CXCR3-CCR6+ had characteristics of Th17.31 For the present invention the CD4+CXCR3+CCR6+ cells were also measured, which may reflect the Th1/Th17 cell subset, see figure 2.27 Treg cells were identified as CD4+CD127lowCD25high cells, see figure 3,18 and Th subsets in Treg cells (Th-like Tregs) were determined, see figure 5.27 Non-specific signaling was eliminated using the corresponding negative isotype control at a threshold of 1 %.
    Plasma cytokine assays
    Because of the finding indicating Treg and Th2-like Treg dysfunction in patients with PCV, it was decided to quantify the cytokines interleukin IL-4 and IL-33. IL-4 induces Th polarization towards Th2 in general.24 IL-33 has been implicated in a range of immune-mediated diseases as it causes the differentiation of Treg cells into Th2-like Treg cells.32 IL-4 and IL-33 were quantified using the commercially available U-PLEX Human Assays™ from MSD (U-PLEX, Meso Scale Diagnostics, Rockville, MD, USA). The assays were performed as per the manufacturer's instructions and recommendations, see below. Prepared plates were immediately read on the QuickPlex SQ120™ (Meso Scale Diagnostics), which converts luminescence values to plasma concentrations of the cytokines measured based on the pre-defined concentrations in calibrators. Duplicate measurements allowed samplespecific calculation of the coefficient of variation (CV), which was used as a guide to re-analyze any samples with high CV (> 20 %). Overall quality was satisfactory (CV mean ± SD: IL-4 = 5.6 ± 4.4 %; IL-33 = 5.6 ± 4.5 %). No extreme outliers were found for plasma IL-4. Two cases of extreme outliers (2.3 pg/mL and 8.7 pg/mL) for plasma IL33 were excluded from the analyses.
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    Plasma cytokine assay method
    Each plate was first coated with coupling antibodies. Calibrators prepared using Diluent 43™ (Meso Scale Diagnostics) were added to the first two columns so that they were run in duplicate. Remaining wells were used for our thawed plasma samples prepared using Diluent 43™ (Meso Scale Diagnostics), which were also run in duplicate. After sealing and incubating for 1 hour at room temperature with shaking, plates were washed three times with Phosphate Buffered Saline with Tween (PBS-T), after which detection antibodies prepared in Diluent 3™ (Meso Scale Diagnostics) were added.
    After sealing and incubating for another 1 hour at room temperature with shaking, plates were washed three times with PBS-T. The Read Buffer™ (Meso Scale Diagnostics) was added and the plates were immediately read on the QuickPlex SQ120™ (Meso Scale Diagnostics) using electrochemiluminescence.
    Statistical Analysis
    All statistical analyses were made using SPSS™ v. 23 (IBM Corporation, Armonk, NY, USA). Mean and standard deviation (SD) and parametric tests were used where normal distribution was present, and otherwise median and interquartile range (IQR) and non-parametric tests were used. Categorical variables were presented in numbers and percentages and compared between groups using the χ2 test or Fisher's Exact test when dealing with small categories (n<5). P-values below 0.05 were interpreted as sign of statistical significance. Figures were made using Prism™ v. 7 (GraphPad Software Inc., San Diego, CA, USA).
    Results
    Study Population
    A total of 110 participants were recruited. Nine were excluded either because they were suspected of having an ongoing immune response or because the samples failed flow cytometry, figure 1. Remaining participants were all included for analyses, see table 1. Groups did not differ significantly in demographics, co-morbidities, and lifestyle factors. Because a trend was observed (P = 0.057, Fisher's Exact test) towards higher prevalence of type 2 diabetes as a comorbidity in patients with PCV and patients with neovascular AMD, all
    DK 2018 70463 A1 our findings were compared between those with and without type 2 diabetes. No significant differences were found (P > 0.1 for all comparisons).
    Differences in CD4+ T Helper Cell Polarization
    Compared to healthy controls, fewer Th1/Th17 (CXCR3+CCR6+) cells were observed in patients with neovascular AMD (P = 0.049, independent samples t-test), see table 2. Patients with PCV did not differ significantly from healthy controls, but a trend towards higher Th2 was observed (P = 0.059, independent samples t-test).
    Lower Tregs and Higher Th2-like Tregs in Patients with PCV
    CD25high and CD127low in CD4+ lymphocytes were used to identify and quantify the Treg population, see figure 3. In healthy controls, it was found that mean: 8.7 % (SD: 2.8 %) of CD4+ T cells were Tregs, which was similar to the level in patients with neovascular AMD (mean: 8.7 %, SD: 2.1 %, P = 0.96, independent samples t-test). Patients with PCV had significantly fewer Tregs than healthy controls (mean: 7.3 %, SD: 1.7 %, P = 0.027, independent samples t-test) with a significantly lower distribution variance (F = 4.029, P = 0.050, Levene's test for equality of variances).
    In Tregs, Th-like populations were investigated, see figure 5. Healthy controls and patients with neovascular AMD did not differ significantly in any of the measured populations, see table 3. Patients with PCV had significantly higher Th2-like Treg (P = 0.029, independent samples t-test) and significantly lower Th17-like Treg (P = 0.039, independent samples t-test).
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    Table 3. Comparison between healthy controls and patients with polypoidal choroidal vasculopathy (PCV) or patients with neovascular age-related macular degeneration (nAMD) in regulatory (Treg) T helper(Th)-like cells. All values are presented as % of the total Treg population in means and standard deviations in parentheses.
    Healthy controlsPatients with PCVP-valueaPatients with nAMDPvalueb CD4+CD25hi8hCD127towCXCR3+CCR6-(Th1-likeTreg)2.5 (2.2)2.3 (1.8)0.691.8 (1.6)0.12CD4+CD25hi8hCD127l«CXCR3-CCR6-(Th2-likeTreg)42.6 (13.3)50.5 (13.0)0.02945.4 (14.9)0.39CD4+CD25highCD127l0WCXCR3-CCR6+(Th17-likeTreg)51.4 (12.6)44.6 (11.7)0.03950.4 (14.4)0.72CD4+CD25highCD127lowCXCR3+CCR6+(Th1/Th17-like Treg)3.4 (2.9)2.6 (2.6)0.292.4 (2.1)0.069
    Abbreviations: PCV = polypoidal choroidal vasculopathy; nAMD = neovascular age-related macular degeneration.
    Statistical comparisons are made using independent samples t-test between (a) healthy controls and patients with PCV and between (b) healthy controls and patients with neovascular AMD. Bold P-values indicates statistical significant differences from healthy controls.
    Higher Plasma IL-33 in Patients with PCV
    Because of the findings on Th2-like Treg in patients with PCV, the focus was shifted to plasma levels of cytokines IL-4 and IL-33. Plasma IL-4 did not differ between healthy controls and patients with PCV or neovascular AMD, see figure 4. Patients with PCV had significantly higher and almost twice as high median IL-33, 0.30 pg/mL compared to the healthy controls (median 0.16 pg/mL) (P = 0.037, Mann-Whitney Utest), while patients with neovascular AMD had levels comparable to those in healthy controls (P = 0.55, Mann-Whitney U-test), figure 4. In patients with PCV where a higher IL-33 and Th2-like Treg cells were observed, a correlation trend between higher plasma IL-33 and higher % of Th2-like cells in the Treg population was also found (ρ = 0.4, P = 0.062, Spearman's correlation).
    Discussion
    In line with the previous findings in patients with neovascular AMD,17'33 association between neovascular AMD and lower CD4+CXCR3+ and CD4+CXCR3+CCR6+ was found. CXCR3+ T cells are particularly interesting
    DK 2018 70463 A1 since they migrate towards areas of inflammation,34 CXCR3-CXCL10 interaction activates downstream pathways that inhibits VEGF-induced endothelial motility and tube formation,35 and age-related parainflammation in RPE leads to downregulation of CXCL10 expression.36 Dysregulation of CXCR3-CXCL10 axis have been suggested as a biomarker for AMD specifically and a therapeutic target in diseases with uncontrolled angiogenesis in general. 33'35' 37 Dysregulation of CXCR3 in CD4+ T cells in patients with neovascular AMD may be specific to the CCR6+ subset and reflect a lower systemic Th1/Th17 population important cellular contributors of autoimmunity. Experimental studies of the Th1/Th17 population in patients with AMD are warranted to understand its contribution for CNV development.
    Dysregulation of CXCR3 in CD4+ T cells did not seem to play a significant role in PCV. Instead, the results suggest that PCV is a disease characterized by diminished Tregs, increased Th2-like polarization of the Tregs, and increased plasma IL-33.
    Balancing an effective immune response with self-tolerance and setting the proper magnitude of the immune response is the key role of Tregs.38 Hence, diminished or dysfunctional Tregs are linked to diseases with an autoimmune component, e.g. allergies and asthma. 39'40 This regulatory activity is also present in the retina, where RPE induces Tregs to suppress the intraocular activity of pro-inflammatory leukocytes.41 RPE induced Tregs secrete high levels of immunoregulatory cytokines suppress Th1 and Th17 activity.42 These mechanisms shed explanatory light on the results: well-functioning Treg activity may respond by Th1/Th17 downregulation to the ongoing pro-inflammatory angiogenetic activity in patients with neovascular AMD. Diminished Tregs in patients with PCV allow a different direction of angiogenesis and may explain findings of studies on intraocular cytokine expression in PCV. 43'44 Zhao et al. found significantly higher levels of the proinflammatory IL-1ß in the vitreous of eyes with PCV when compared to controls (eyes with idiopathic epiretinal membrane) and patients with neovascular AMD.43 Sasaki et al. found significantly higher levels of a range of pro-inflammatory cytokines in the aqueous of eyes with PCV, including IL-4 which suggests a local Th2-like response.44
    DK 2018 70463 A1
    Is PCV a Th2-mediated disease Gold-standard diagnosis of PCV requires ICGA and consequently very few epidemiological studies exist; therefore, it is currently not possible to draw clear links to other Th2 diseases. However, it is interesting to note that PCV share similarities with other vasculopathies, e.g. a shift towards Th2-like phenotype has been associated with intracranial aneurysms, wherein degenerative vascular changes are driven by a range of proinflammatory cytokines and infiltration of leukocytes.31 Another interesting observation is that while studies find no link between asthma and CNV in Western populations, the only epidemiological investigation in an Asian (Chinese) population do find a positive association.45 The positive association in Asians and lack of an association in Western populations may be due to an inter-ethnical difference in the prevalence of PCV, which is approximately 10-fold.7 Experimental laser-induced CNVs in mice with asthma confirmed an amplifying role of asthma in VEGF expression and angiogenesis.45
    In 2015, MacDonald et al. reported the first Th2-like Treg associated disease in humans (systemic sclerosis — an autoimmune disease with dysfunctional angiogenesis despite increased VEGF).32 Levels of Tregs were similar in patients with systemic sclerosis when compared to healthy controls, but the Tregs in patients were CXCR3-CCR6- and expressed Th2-associated cytokines. Halim et al. found that Th2-like Tregs have greater migratory ability and a higher viability and blasting capacity through STAT5 phosphorylation27, which promotes angiogenesis.46 Treg polarization into Th2-like phenotype is facilitated by IL-33.32'47 The role of IL-33 in allergic diseases and asthma, as well as the potential of IL-33 as a therapeutical target, is currently under investigation48 ' 49 and may also be relevant for PCV in light of the findings according to the present invention.
    Pachychoroid neovasculopathy is a new clinical definition of CNVdiseases associated with a thick dilated choroid, wherein PCV constitutes a large and important proportion of cases.50 By post-hoc reviewing the PCV cases using the definition by Miyake et al.,51 17 (71 %) patients with PCV could be classified with the diagnosis pachychoroid neovasculopathy. It was found by the present inventors that these patients do not have AMD-like features of lower CXCR3+ and
    DK 2018 70463 A1
    CXCR3+CCR6+, but have lower Tregs than healthy controls (P = 0.014), which were increasingly Th2-like polarized (P = 0.036), and had increased plasma IL-33 levels (median 0.45, IQR: 0.16 to 0.66) at a near-significant level (P = 0.060). Thus, the present invention provides rare immunological insight into pachychoroid neovasculopathy and together with the genetic study by Miyake et al., the collective results suggest that pachychoroid neovasculopathy is etiologically distinct from neovascular AMD.51
    Taken together, it can be concluded that PCV associates with systemic 10 immunological changes that differ from those seen in neovascular AMD.
    Patients with PCV have diminished Tregs that are polarized into a Th2like phenotype wherein IL-33 secretion may play an important role. Shared immunological features with vascular aneurysms, asthma, and autoimmune diseases provide a theoretical framework for possible 15 epidemiological and pathological relationships.
    DK 2018 70463 A1
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    权利要求:
    Claims (23)
    [1] 1. A method of diagnosing an eye disease having an element of agerelated retinopathy with acquired choroidal abnormalities in a subject comprising:
    a. providing a sample to be tested obtained from the subject,
    b. determining the level of expression of at least one biomarker in the sample,
    c. determining a control level of expression of said at least one biomarker in a control sample obtained from a subject not afflicted with the eye disease,
    d. comparing said level of expression with said control level of expression of said at least one biomarker, wherein a level of expression of said at least one biomarker in said sample that is higher than said control level of expression of said at least one biomarker in said control sample is indicative of the presence of said eye disease, and wherein a level of expression of said at least one biomarker in said sample that is equal to or lower than said control level of expression of said at least one biomarker in said control sample is indicative of the absence of said eye disease, and wherein said at least one biomarker is selected from IL33, regulatory T-cells, and/or T-helper-2-like regulatory T-cells.
    [2] 2. The method according to claim 1, wherein the subject is not suffering from diabetes related retinopathy.
    [3] 3. The method according to claims 1 or 2, wherein the subject is not suffering from neovascular age-related macular degeneration, defined as acquired choroidal neovascularizations with no polyp-like features.
    [4] 4. The method according to any one of claims 1-3, wherein the healthy subjects are matched with the subjects by age and/or sex.
    DK 2018 70463 A1
    [5] 5. The method according to anyone of claims 1-4, wherein a level of IL-33 in a plasma sample from the subject that is equal to or above 0.1 pg/ml, such as equal to or above 0.2 pg/ml, such as equal to or above 0.3 pg/ml, such as equal to or above 0.4 pg/ml, such as equal to or above 0.5 pg/ml, such as equal to or above 0.6 pg/ml, such as equal to or above 0.7 pg/ml, such as equal to or above 0.8 pg/ml is indicative of said eye disease.
    [6] 6. The method according to any one of claims 1-5, wherein a proportion of CD4+ T cells that are regulatory T cells that is equal to or below 8 %, such as equal to or below 7.5 %, such as equal to or below 7 %, such as equal to or below 6.5 %, such as equal to or below 6 %, such as equal to or below 5.5 % is indicative of said eye disease.
    [7] 7. The method according to any one of claims 1-6, wherein a proportion of CD4+ regulatory T cells that are T-helper-2-like that is equal to or above 40 %, such as equal to or below 45 %, such as equal to or below 50 %, such as equal to or below 55 %, such as equal to or below 60 %, such as equal to or below 65 % is indicative of said eye disease.
    [8] 8. The method according to any one of claims 1-7, wherein the eye disease having an element of age related retinopathy with acquired choroidal abnormalities is Polypoidal Choroidal Vasculopathy.
    [9] 9. The method according to any one of claims 1-8, wherein the subject is likely to experience one or more of a change in bestcorrected visual acuity, change in visual function, change in retinal thickness, need for therapy in terms of type of therapy and the quantity of therapy, or development of subretinal fibrosis.
    [10] 10. The method according to any one of claims 1-9, wherein the subject is likely to benefit from treatment with an inhibitor of IL33 when the level of IL-33 in a plasma sample from the subject is equal to or above 0.1 pg/ml, such as equal to or above 0.2 pg/ml, such as equal to or above 0.3 pg/ml, such as equal to or above 0.4 pg/ml, such as equal to or above 0.5 pg/ml, such as equal to or above 0.6 pg/ml, such as equal to or above 0.7 pg/ml, such as equal to or above 0.8 pg/ml.
    [11] 11. A composition comprising a therapeutically, prophylactic or alleviation effective dosage of an IL-33 inhibitor for use in the
    DK 2018 70463 A1 treatment, prophylaxis or alleviation of an age related retinopathy with acquired choroidal abnormalities in a subject, wherein the patient to receive the composition exhibits a level of IL-33 in a plasma sample from the subject which is equal to or above 0.1 pg/ml, such as equal to or above 0.2 pg/ml, such as equal to or above 0.3 pg/ml, such as equal to or above 0.4 pg/ml, such as equal to or above 0.5 pg/ml, such as equal to or above 0.6 pg/ml, such as equal to or above 0.7 pg/ml, such as equal to or above 0.8 pg/ml.
    [12] 12. The composition according to claim 11, wherein the eye disease having an element of age-related retinopathy with acquired choroidal abnormalities is Polypoidal Choroidal Vasculopathy.
    [13] 13. The composition according to claims 11 or 12, wherein the IL-33 inhibitor is an antibody or a small organic molecule.
    [14] 14. The composition according to any one of claims 11 to 13, wherein the composition is formulated for ophthalmic administration.
    [15] 15. A method of treatment, prophylaxis or alleviation of an eye disease having an element of age-related retinopathy with acquired choroidal abnormalities in a subject, wherein a composition comprising an IL-33 inhibitor is provided to the subject.
    [16] 16. The method according to claim 15, wherein the eye disease having an element of age related retinopathy with acquired choroidal abnormalities is Polypoidal Choroidal Vasculopathy.
    [17] 17. A method of treatment, prophylaxis or alleviation of an eye disease having an element of age related retinopathy with acquired choroidal abnormalities in a subject, comprising:
    a. providing a sample to be tested obtained from the subject,
    b. determining the level of expression of at least one biomarker in the sample,
    c. determining a control level of expression of said at least one biomarker in a control sample obtained from a subject not afflicted with the eye disease,
    d. comparing said level of expression with said control level of expression of said at least one biomarker,
    DK 2018 70463 A1 wherein a level of expression of said at least one biomarker in said sample that is higher than said control level of expression of said at least one biomarker in said control sample is indicative of the presence of said eye disease, and wherein a level of expression of said at least one biomarker in said sample that is equal to or lower than said control level of expression of said at least one biomarker in said control sample is indicative of the absence of said eye disease, and wherein said at least one biomarker is selected from IL33, regulatory T-cells, and/or T-helper-2-like regulatory T-cells,
    e. providing to the subject, if step d. is indicative of the presence of said eye disease, a composition with a therapeutically, prophylactic or alleviation effective dosage of an IL-33 inhibitor.
    [18] 18. The method according to claim 17, wherein the eye disease having an element of age related retinopathy with acquired choroidal abnormalities is Polypoidal Choroidal Vasculopathy.
    [19] 19. The method according to anyone of claims 17-18, wherein the IL33 inhibitor is an antibody.
    [20] 20. The method according to any one of claims 1-10 or 17-19, further comprising measuring at least one environmental or lifestyle factor selected from the group consisting of age, smoking history, weight, body mass index, diet, and exercise habits.
    [21] 21. The method according to claim 20, wherein the measuring of the at least one environmental or lifestyle factor is used to pair diseased subjects with control subjects with similar environmental or lifestyle factor profiles.
    [22] 22. The method according to anyone of claims 1-10 or 17-21, wherein the sample or control sample is a tissue sample or a bodily fluid sample.
    [23] 23. The method according to claim 22, wherein the bodily fluid sample is a blood sample, a plasma sample or a serum sample.
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    同族专利:
    公开号 | 公开日
    DK179993B1|2020-01-14|
    WO2020007612A1|2020-01-09|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    TWI597051B|2015-04-16|2017-09-01|林伯剛|Use and method for detecting and diagnosis polypoidal choroidal vasculopathy by homocysteine level|
    法律状态:
    2020-01-14| PME| Patent granted|Effective date: 20200114 |
    2020-01-14| PAT| Application published|Effective date: 20200106 |
    2022-02-14| PBP| Patent lapsed|Effective date: 20210705 |
    优先权:
    申请号 | 申请日 | 专利标题
    DKPA201870463A|DK179993B1|2018-07-05|2018-07-05|BIOMARKET IN POLYPOIDAL CHOROIDAL VASCULOPATHY AND APPLICATIONS THEREOF|DKPA201870463A| DK179993B1|2018-07-05|2018-07-05|BIOMARKET IN POLYPOIDAL CHOROIDAL VASCULOPATHY AND APPLICATIONS THEREOF|
    PCT/EP2019/066219| WO2020007612A1|2018-07-05|2019-06-19|Biomarker in polypoidal choroidal vasculopathy and uses thereof|
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