TSH receptor and IGF1 receptor expression in Circulating Fibrocytes in the pathogenesis of Graves’ Orbitopathy

TSH receptor and IGF1 receptor expression in Circulating Fibrocytes in the pathogenesis of Graves’ Orbitopathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article TSH receptor and IGF1 receptor expression in Circulating Fibrocytes in the pathogenesis of Graves’ Orbitopathy Madhurima Basak, Dipanjan Bhattacharjee, Anish Kar, Sriparna De, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4521745/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Graves’ Orbitopathy (GO), an autoimmune disorder linked to Graves’ Disease (GD), manifests through inflammation in orbital tissues and extraocular muscles (EOMs), driven by key receptors like TSHR and IGF1R. It was observed that a certain individual with GD will develop clinically significant orbitopathy and reason behind this still unclear. This study aimed to elucidate this connection by: i) Assessing IGF1R expression and its correlation with TSHR on circulating fibrocytes. ii) Investigating fibrocyte conversion to fibroblasts upon serum treatment. iii) Analysing cytokine and chemokine expression in fibrocytes post-serum exposure within the Indian population. Methods and Results Flow cytometry analysis of IGF1R in peripheral blood from 30 GO, 30 GD, and 20 healthy controls (HC) revealed significantly elevated IGF1R + fibrocytes in GO (11%) versus GD (2.4%) and HC (0.1%). Immunocytochemistry of TSHR and IGF1R on cultured fibrocytes confirmed colocalization of TSHR and IGF1R on fibrocytes, notably higher in GO. Treating HC-derived fibrocytes with GO patient serum triggered fibroblast transformation, marked by increased fibrotic markers (CD90, alpha SMA). Moreover, sandwich ELISA of cytokines and chemokines like IL-6, IL-8, TNF-α, MCP-1, and HA demonstrated elevated levels of those cytokines and chemokines in GO serum-treated HC-fibrocytes. Conclusion These results highlight the potential pathogenicity of TSHR and IGF1R on fibrocytes in GO, suggesting their role in orbital tissue remodelling and inflammation. The observed receptor colocalization may drive GO pathogenesis, providing insights into targeted therapeutic strategies for this debilitating condition. Fibrocytes Peripheral Blood Mononuclear Cells Insulin Like Growth Factor Receptor Fibroblast Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Graves’ Orbitopathy (GO) is an inflammatory autoimmune disorder in Orbital connective tissues and extra ocular muscles (EOMs) which is closely associated with Graves’ Disease (GD). The thyrotropin receptor (TSHR) present on orbital fibroblast in orbital connective tissue thought to be an important auto antigen which links both conditions [ 1 ][ 2 ]. Thus, GO exhibit expanded volume of the orbital connective tissues, adipose tissues of extraocular muscles due to new fat cell development[ 3 ].Affected orbital tissues show diffuse infiltration of T and B lymphocytes, macrophages (occasional), and proinflammatory cytokines [ 4 ][ 5 ].These cytokines are secreted by peripheral blood mono nuclear cells(PBMCs) infiltrating in orbit together with resident orbital fibroblasts and contribute to the inflammatory basis for active GO[ 4 ][ 6 ][ 7 ][ 8 ][ 9 ]. Apart from TSHR, Insulin like Growth Factor-1Receptor (IGF-1R) present on orbital fibroblast also plays a potent role in orbital tissue remodeling. These two receptors have close physical and functional relationship and they form a physical complex in orbital fibroblasts in GO[ 10 ]. IGF1R mediated cell signaling pathway also stimulates the cell proliferation by TSH or TSH-Receptor auto antibodies (TRAbs)[ 11 ], [ 12 ] .It is observed that orbital fibroblast share some common characteristic features with a group of spindle shape cells found in circulation termed as fibrocytes [ 13 ].Fibrocytes are monocytes derived cells, exhibit hematopoietic marker, progenitor cell marker, tissue trafficking marker (CD34 + , CD45 + ,CXCR4 + respectively) in their cell surface and also found in situ in orbital connective tissues [ 14 ][ 15 ].It is also observed that concentration of these circulating fibrocytes were significantly higher among GO compare to GD and healthy control (HC) [ 14 ], [ 16 ], [ 17 ].These fibrocytes express TSHR on their cell surface and association of TSHR + fibrocytes with various clinical parameters (Age, duration of disease, Clinical Activity Score, Smoking status) among GO reveal its pathogenetic role in progression of orbitopathy[ 14 ], [ 17 ].It is shown that fibrocytes also express IGF1R[ 14 ], [ 18 ], [ 19 ] .So, TSHR and IGF1R signaling in fibrocytes may enhance orbital tissue remodeling in GO. In our previous studies in Eastern India demonstrated the prevalence of GO was 32% among GD [ 20 ].So, It is not clearly understood as to why only certain individual with GD develop clinically significant GO. We had previously observed the concentration of TSHR + circulating fibrocytes among GD and GO in Indian population[ 17 ]. In this present study we have taken a small attempt to highlight- i) Expression of IGF1R and its association with TSHR on circulating fibrocytes in GD, GO and HC ii) In vitro study to detect conversion of fibroblast from fibrocytes after serum treatment. iii) Cytokine and chemokine expression from fibrocytes after serum treatment in GD, GO and HC. 2. Material Methods Study site and sample collection Individuals with Graves’ disease with clinically significant Orbitopathy (GO, n = 30), Graves’ disease without clinically significant Orbitopathy (GD, n = 30) and Healthy Control (HC) subjects (n = 20) aged 20–65 years were recruited from Department of Endocrinology & Metabolism of IPGME&R and SSKM Hospital, Kolkata, India from 2019 to 2021. Patients were evaluated both clinically and radiologically by an endocrinologist. Patients with low level of TSH (Reference range 0.4-4.0 uIU/ml), normal or high levels of FT4 (Reference range 0.80–1.90 uIU/ml), normal or high level of FT3(Reference range 1.50–4.10 uIU/ml) and positive TRAb value (Reference range 1.22 IU/l were included for this study. Patients, who have received anti-thyroid therapy, radiation therapy, immunosuppressive treatment like glucocorticoids, earlier or individuals with other autoimmune diseases, asthma, sinusitis, recent trauma, or HIV infection, malignancy, pregnancy were excluded from this study. Severity of GO was graded based on the European Group of Graves' Orbitopathy (EUGOGO) classification [ 20 ]. All demographic and biochemical data are given in Table 1 . Patients whose TRAb was positive in serum but does not follow EUGOGO guidelines were designated as GD and Patients whose TRAb was positive in serum and follow EUGOGO guidelines were designated as GO. Here clinical activity score (CAS) = 3 was categorized as Moderate GO and CAS > 3 was categorized as active to severe GO. Specifically, 20% of patients were in the active and severe phases, whereas 80% were in the inactive phase. Control subjects were healthy volunteers without symptoms or signs of thyrotoxicosis, normal thyroid function test, TRAb value was within reference range and no history of known autoimmune disease. They were recruited from colleagues, spouse of patients. Clinical evaluation and methods for measuring serum FT3, FT4, TSH-Receptor Antibody (TRAb) were already described in our previously published article [ 17 ] Table 1 Demographic and biochemical data of all study participants Parameters HC (n = 20) GD (n = 30) GO (n = 30) p value Age (Yrs. ± S.D) 32 ± 4 39 ± 11 34 ± 12 0.12 b Sex M = 10 F = 10 M = 09 F = 21 M = 18 F = 12 0.02 a*Ψ Smoker Non-Smoker 8 12 9 21 19 11 0.03 a*Ψ Duration of hyper thyroidism (months) 0 4 (1–24) 6 (1–36) 0.24 c Exophthalmometry in eye (mm) RE LE 14 ± 0.9 14 ± 0.9 16 ± 0.8 16 ± 0.8 24 ± 4 25 ± 4 < 0.0001 *Ψ < 0.0001 *Ψ CAS 0 0 ≥ 1 but ≤ 5 TSH (uIU/ml) 3 ± 0.5 0.02 ± 0.02 0.01 ± 0.01 0.24 cΨ FT3(pg/ml) 1 ± 0.05 11 ± 6 14 ± 8 0.40 cΨ FT4(ng/dl) 2 ± 1 6 ± 5 7 ± 4 0.45 cΨ TRAb (IU/I) 1.21 ± 0.5 20 ± 11 26 ± 14 0.07 cΨ Severity (in GO) Nil Nil Mild = 23 Moderate = 4 Severe = 3 Two tailed unpaired t test; * Statistically significant; Ψ GD vs GO. Abbreviations: CAS-Clinical Activity Score; TSH-Thyroid Stimulating Hormone; FT4-Free T4; FT3-Free T3; TRAb-Thyroid Stimulating Hormone Receptor Antibody; GO-Graves’ Orbitopathy, M- Male, F- Female.RE-Right Eye, LE-Left Eye. a Two-tailed Fisher’s test; b One-way ANOVA; c Bonferroni's Multiple Comparison Test. Immunophenotyping Staining for flow cytometry was performed within 24 h of blood collection. Method for staining was described in our previously published article [ 17 ]. Briefly, a staining buffer (SB) was prepared in 1X Phosphate Buffer Saline (PBS) containing 2% fetal bovine serum (FBS) with 0.1% sodium azide. 200 µl of whole blood were placed in 12X75-mm polypropylene tubes, and 2 ml FACS Lysing solution was added for 10 min at room temperature. Cells were centrifuged at 1800 rpm for 5 min, washed, and resuspended in 100µl SB. The following anti human fluorochrome conjugated monoclonal antibodies were used: CD 34-BB 700 (BD Bioscience, San Jose, CA, USA Catalogue no-746104), CD45 V500 (BD Bioscience, SanJose, CA, USA Catalogue no-560777), CXCR4-BV421, (BD Bioscience, SanJose, CA, USA Catalogue no-562448), CD 221-PE (IGF1R, BD Bioscience, San Jose, CA, USA Catalogue No- 555999) TSHR-Alexa fluor 647 (Novus Biologicals, USA, Catalogue no-NB100-65797AF64). After 30 minutes incubation in the dark at room temperature (RT) the cells were resuspended and washed twice with SB. Collagen -I (Col-I) was present in cytosol. For quantification of Col I, cells were permeabilized with cytofix/ cytoperm for 20 min at RT, washed twice, and resuspended in 100 µl Perm/Wash buffer. After incubation with anti-human FITC conjugated Collagen I (Col I-FITC) in the dark at RT in perm wash buffer, the cells were washed and acquired on FACS Verse Flow cytometer (BD Bioscience, San Jose, CA, USA). At least 1X10 4 events were collected. Fibrocytes were identified from peripheral blood using a multiparametric Flowcytometry technique described previously [ 17 ]. TSHR and IGF1R expression on fibrocytes were measured from the fibrocytes co-expressing TSHR and Col I, IGF1R and Col 1 respectively (Fig. 1a-l). Fibrocyte culture and treatment PBMC were used to culture condition similar to those described by Bucala et al [21]. Here, 7 patients with GO were in active phase (CAS ≥ 3) and remaining (n = 23, CAS < 3) were in inactive phase. Culture was considered for 10 GO (7 active phase + 3 inactive phase) patients,10 GD patients and 10 HC. Clinical data of those 10 GO patients are given in Table 2 . PBMCs were isolated from blood by density gradient centrifugation using HiSep TM (Hi media Laboratories, LS001) according to manufacturer protocol. They were isolated and inoculated in 4-chambered slides (1X 10 6 /well,) in 1 ml Dulbecco’s Modified Eagle Medium (DMEM, Himedia Laboratories cat no AL151A) with 10% FBS (Hi media Laboratories) and 1% penicillin streptomycin antibiotic solution (Hi media Laboratories). The culture was maintained at 37 o C in 5% CO 2 . Table 2: Clinical characteristics of GO (n=10) patients those PBMCs were used in cell culture Patient sl. No. Age (yrs.) / Sex CAS Duration of hyperthyroidism (month) Time since diagnosis of GO (month) Smoking Status Proptosis (mm) LE RE Severity 01 25/M 3 07 05 Yes 22 22 Active and moderate 02 29/M 5 2.5 2.5 Yes 24 25 Active and severe 03 55/M 2 04 04 Yes 20 20 Inactive and mild 04 32/F 3 06 03 No 18 18 Active and moderate 05 48/M 4 03 03 Yes 32 33 Active and severe 06 34/M 2 6 5 Yes 20 20 Inactive and mild 07 48/M 3 4 4 Yes 32 33 Active and moderate 08 40/F 5 2 2 No 32 32 Active and severe 09 56/M 4 1.5 1.5 No 25 25 Active and severe 10 28/M 1 3 3 Yes 22 22 Inactive and mild Here, PBMCs were treated in vitro by 10% serum supplementation. The treatment pattern was depicted in Fig. 2. PBMC of HC was treated by autologous serum as well as GD serum and GO serum separately.GD and GO PBMCs were also treated by autologous serum respectively. After a couple of days (48 hours), the nonadherent cells were removed by gentle aspiration of the media. After 7 days of culture, previous media was discarded, and cells were replenished by fresh media. Prior to collect the supernatant, the cells were starved from serum for 48 hrs. 7 days cultured cells were used to observe fibrotic markers. Cell viability assay PBMCs of HC and patients (both GD and GO) were plated in 96 well plate at density of 1 × 10 4 per each well. Cells were treated with different concentration of patient serum and autologous serum (5%,10%,15% and 20%). We have assessed cell viability in a concentration dependent manner in accordance with days variation. It was inferred from the above findings; significant cell viability was observed at 10% serum treatment. confirmed by MTT (93-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Immunocytochemistry Immunofluorescence staining was performed using 7 days cultured fibrocytes in four chambered slides. At first media was removed and cells were fixed in 2% paraformaldehyde in Phosphate buffer saline (PBS) for 10 minutes at room temperature (RT) and blocked with 5% Bovine Serum Albumin (BSA) in PBS for 45 minutes. Cells were incubated by Mouse anti human TSHR mono clonal antibodies (Catalog no MA3-218, Thermo fisher Scientific), Rabbit anti human IGF1R alpha polyclonal antibodies (Catalog no PA5-79444, Invitrogen), Mouse anti human CD90 monoclonal antibodies (NBP2-52961, Novus biologicals), Rabbit anti human alpha SMA monoclonal antibodies (NBP2-67440, Novus biologicals) at a dilution of 1:100 in PBS containing 5% BSA for 3 hrs at 37 o C. Cells were then stained by goat anti mouse Alexa flour 488 fluorochrome conjugated secondary antibodies in a combination with TSHR monoclonal antibody and CD 90 monoclonal antibody; donkey anti rabbit Alexa flour 647conjugated secondary antibodies in a combination with IGF1R polyclonal antibody and alpha SMA monoclonal antibody. Nucleus were stained using 4′,6-diamidino-2-phenylindole (DAPI) and observed under confocal microscope. Images were acquired from THUNDER Imager 3D Assay, Leica Microsystem. Cytokines and chemokines measurements from cultured fibrocytes Cytokines (IL6, IL8, TNF alpha) and chemokines (Hyaluronic Acid, MCP 1) production of 7 days cultured fibrocytes were observed by sandwich ELISA (G biosciences, St. Louis, U.S.A) according to manufacturer’s protocol. Statistical analysis Statistical analyses specifically two-tailed Fisher’s test, unpaired t-test, one-way ANOVA (Bonferroni’s Multiple Comparison Test) were performed. Calculations were done using GraphPad Prism version 8 ( http://www.grpahpad.com , San Diego, CA) and p value ≤ 0.05 was considered statistically significant. 3. Result Concentration of IGF1R + circulating fibrocytes among GD and GO Fibrocytes display IGF1R (Fig. 1a-l) on its cell surface in peripheral blood (when they were gated from CD 34 + /CD 45 + /Col-1 + cells) among GO, GD, and control. Quantification of IGF-1R + fibrocytes from peripheral blood show that, percentages of IGF-1R + fibrocytes were significantly higher in GO as compared to GD (GO:11 ± 9% vs GD:2.4 ± 0.6%, p < 0.0001) but in control it was almost absent (Fig. 3a). (GD: 2.4 ± 0.6% vs Control: 0.1 ± 0.03%, p = 0.002; GO:11 ± 9% vs Control: 0.1 ± 0.03%, p < 0.0001). TSHR and IGF1R co-expression on fibrocytes among GD and GO A large population of TSHR + fibrocytes also express IGF-1R when they were gated from TSHR + /Col1 + cells. Quantification of TSHR + / IGF-1R + circulating fibrocytes show that, expression of TSHR + / IGF-1R + circulating fibrocytes were significantly higher in GO compare to GD and control (Fig. 3b) [(GD vs Control: TSHR + / IGF1R + fibrocytes., 2.4 ± 0.6% vs 0.1 ± 0.03%, p = 0.003), (GO vs Control: TSHR + / IGF1R + fibrocytes.,11 ± 8% vs 0.1 ± 0.03%, p < 0.0001), (GD vs GO: TSHR + / IGF1R + fibrocytes., 2.4 ± 0.6% vs11 ± 8%, p < 0.0001]. Immunolocalization of TSHR and IGF-1R on fibrocytes among GO TSHR was detected using Alexa flour 488 conjugated secondary antibody against of mouse monoclonal anti human anti TSHR- antibody (green). Detection of IGF-1R alpha was done by Alexa flour 647 conjugated secondary antibody against of rabbit monoclonal anti human anti IGF-1R alpha- antibody (red). Expression of TSHR and IGF1R were almost absent in HC fibrocytes (Fig. 4, Panel I, a-c) and very low to detect in GD (Fig. 4, Panel II, d-f and j). But the expressions were very high in GO as compared to HC and GD (Fig. 3, Panel III, g-i). IGF1R was localized to the periphery of fibrocytes as well as to the perinuclear area. The distribution was identical to the staining pattern of TSHR. The yellow or orange staining present in the merged images (Fig. 4, Panel IV, k) results from the colocalization of TSHR and IGF1R in GO. Evidence for fibroblast transformation from fibrocytes Morphological transformation was observed within the cultured fibrocytes isolated from Healthy Control (HC) (Fig. 5). At first Fibrocytes were differentiated from monocytes (Fig. 5a). These fibrocytes exhibit more cellular processes with prominent oval shape nucleus due to influence of patient’s serum (Fig. 5b and c). More-over, HC-fibrocytes were found to have higher expression of fibroblast specific marker (CD90) and fibroblast activation marker (α -SMA) after stimulation with GO serum and GD serum (Fig. 6a-i). Expression level of those markers were same in between GD serum treated HC fibrocytes and GO serum treated HC fibrocytes (CD 90, p = 0.99; α -SMA, p = 0.06; Fig. 7a and b). But CD90 and α -SMA were not expressed when HC-fibrocytes were treated with autologous serum. CD90 and α -SMA expressions were also seen in autologous serum treated GD fibrocytes and autologous serum treated GO fibrocytes. Autologous serum treated GO -fibrocytes show significant higher expression of fibroblast specific marker CD90(p = 0.01) and fibroblast activation marker α -SMA (Fig. 6m-o and Fig. 7c, d) compare to autologous serum treated GD fibrocytes (p = 0.0003) (Fig. 6j-l and Fig. 7c, d). Expressions of IL-6, IL-8, TNF-α, Hyaluronic Acid (HA), Monocyte chemoattractant protein-1 (MCP1) by cultured fibrocytes Expressions of proinflammatory cytokines like IL-6, IL-8 were markedly elevated in HC-fibrocytes treated with serum from GD patients and GO patients as compared to HC-fibrocytes treated with autologous serum. [(IL-6: autologous serum treated HC fibrocytes; 3.3 ± 0.67 pg/ml vs GO serum treated HC-fibrocytes 39 ± 7 pg/ml, p < 0.0001 and autologous serum treated HC fibrocytes; 3.3 ± 0.67 pg/ml vs GD serum treated HC-fibrocytes 36 ± 8 pg/ml. p = 0.0002, Fig. 8a.) (IL8: autologous serum treated HC fibrocytes 29.6 ± 3 pg/ml vs GO serum treated HC-fibrocytes 83.5 ± 7.1 pg/ml ,p < 0.0001 and autologous serum treated HC fibrocytes 29.6 ± 3 pg/ml vs GD serum treated HC-fibrocytes 82.5 ± 10.1pg/ml, p < 0.0001,Fig. 8c) But the expressions of those cytokines were similar between HC- fibrocytes, treated with GD and GO serum [(IL6: GD serum treated HC-fibrocytes 36 ± 8 pg/ml vs GO serum treated HC-fibrocytes 39 ± 7 pg/ml, p = 0.55) (IL8: GD serum treated HC-fibrocytes 82.5 ± 10.1pg/ml vs GO serum treated HC-fibrocytes 83.5 ± 7.1 pg/ml, p = 0.87)] Interestingly, expressions of those cytokines (IL6 and IL8) were significantly higher in autologous serum treated GO fibrocytes compare to autologous serum treated GD fibrocytes [IL6: autologous serum treated GO fibrocytes 42 ± 14pg/ml vs autologous serum treated GD fibrocytes 29 ± 9pg/ml, p = 0.04, Fig. 8b; IL8: autologous serum treated GO fibrocytes 90 ± 7 pg/ml vs autologous serum treated GD fibrocytes 82 ± 7pg/ml, p = 0.01; Fig. 8d]. Expression of TNF-α was also significantly higher in HC-fibrocytes treated with serum from GD patients and GO patients as compared to HC-fibrocytes treated with autologous serum. [Autologous serum treated HC fibrocytes; 14 ± 2 pg/ml vs GO serum treated HC-fibrocytes 67 ± 12 pg/ml, p = 0.0001 and autologous serum treated HC fibrocytes; 14 ± 2 pg/ml vs GD serum treated HC-fibrocytes 67 ± 15 pg/ml. p = 0.0003, Fig. 9a] But, expression of TNF- α was also same in between HC- fibrocytes, treated with GD and GO serum. [ GO serum treated HC-fibrocytes 67 ± 12 pg/ml vs GD serum treated HC-fibrocytes 67 ± 15 pg/ml. p = 0.9, Fig. 9a]. Surprisingly there were no significant differences of TNF- α expressions were found in between autologous serum treated GO fibrocytes and autologous serum treated GD fibrocytes. [autologous serum treated GO fibrocytes 74 ± 17pg/ml vs autologous serum treated GD fibrocytes 69 ± 15 pg/ml, p = 0.5, Fig. 9b] Chemokines like Hyaluronic acid (HA), Monocyte chemoattractant protein-1 (MCP1) were also markedly elevated in in HC-fibrocytes treated with serum from GD patients and GO patients as compared to HC-fibrocytes treated with autologous serum [(HA: Autologous serum treated HC fibrocytes 29 ± 11 ng/ml vs GO serum treated HC-fibrocytes 136 ± 23 ng/ml, p = 0.02 and autologous serum treated HC fibrocytes 29 ± 11 ng/ml vs GD serum treated HC-fibrocytes 162 ± 36 ng/ml. p = 0.02,Fig. 10a)(MCP1: Autologous serum treated HC fibrocytes 38 ± 5 pg/ml vs GO serum treated HC-fibrocytes 160 ± 10 pg/ml, p < 0.0001 and autologous serum treated HC fibrocytes 29 ± 11 ng/ml vs GD serum treated HC-fibrocytes 162 ± 16 pg/ml. p < 0.0001, Fig. 10c)]. But, no statistically significant changes of expressions were found of those chemokines (HA, MCP1) in between HC- fibrocytes, treated with GD and GO serum [(HA: GO serum treated HC-fibrocytes 136 ± 23 ng/ml vs GD serum treated HC-fibrocytes 162 ± 36 ng/ml, p = 0.2, Fig. 10a) (MCP1: GO serum treated HC-fibrocytes 160 ± 10 pg/ml vs GD serum treated HC-fibrocytes 162 ± 16 pg/ml, p = 0.9,Fig. 10c)]. Expressions of those chemokines were markedly elevated when GO fibrocytes were treated with autologous serum compare to autologous serum treated GD fibrocytes [(HA: autologous serum treated GO fibrocytes 110 ± 5 ng/ml vs autologous serum treated GD fibrocytes 56 ± 17 ng/ml, p < 0.0001, Fig. 10b) (MCP1: autologous serum treated GO fibrocytes 147 ± 5 pg/ml vs autologous serum treated GD fibrocytes 135 ± 3pg/ml, p < 0.0001, Fig. 10d)]. 4. Discussion Uncertainty about the pathogenesis of GO specifically in only proportion of patients with GD developing clinically significant Orbitopathy led to investigate that beside TSHR, IGF1R might play an important role in disease progression. IGF1R belongs to a group of structurally related transmembrane tyrosine kinase receptors that has 1368 amino acids. IGF1works via IGF1 receptor and increases thymic epithelial cell proliferation as well as intra-thymic T cell growth and migration in the thymus. Thus, it influences immune function. T and B lymphocytes, as well as cells from the monocyte lineage, are professional immune cells that express functional IGF1R and react to physiological quantities of the growth factor. IGF1 is crucial for the T and B cell growth processes. It is chemotactic and increase thymidine incorporation in human T cells. IGF1R, which is expressed at a greater level in activated cells, mediates these activities [22,23.]. In this present study, we are reporting the presence of IGF1R on circulating fibrocytes for the first time in GD and GO patients among Indian population. By using flowcytometry we were able to quantify IGF1R expression and co-expression of TSHR -IGF1R among patient group. We found that the percentages of IGF-1R + fibrocytes were significantly higher in GO compared to GD and HC (GO:11 ± 9%, n = 30 vs GD:2.4 ± 0.6% n = 30 vs HC: 0.1 ± 0.03% n = 20 ; p < 0.05 ). Expression pattern was similar with TSHR expression (previously reported) on circulating fibrocytes [ 17 ] and these two receptors co-express in fibrocytes. This co-expression level was also significantly higher in GO compared to GD and HC. Additionally, our recent research indicates that TSHR and IGF-1R co-localize to the cytoplasmic, plasma membrane, and perinuclear compartments of human circulating fibrocytes. Previously, Tsui et al observed the colocalization of TSHR and IGF1R on human orbital fibroblasts and thyrocytes [24] but their colocalization on circulating fibrocytes were not observed before. The possibility of functional interaction between heterotrimeric G proteins and IGF-1 signalling has been suggested by a number of earlier investigations. [25]. On the other hand, a number of prior investigations strongly suggest that IGF-1 may also boost some, but not all, TSHR signalling. For example, Tramontano et al discovered that the effects of IGF-1 and TSH on FRTL-5 cell proliferation and DNA synthesis were synergistic and concentration-dependent [26]. IGF-1 does not appear to have an impact on TSH-dependent cAMP production in FRTL-5 cells [27–30]. Clement and co-workers showed that Increased thyroid gland weight and follicular lumen area are seen in vivo in double-transgenic animals whose thyroids are conditionally overexpressed with both IGF-1 and IGF-1R [31]. Therefore, our results imply that a physical and functional link between TSHR and IGF-1R may mediate features of signalling that have been attributed to each receptor alone. Fibrocytes also transformed into fibroblast when they were triggered by serum. Here, HC-fibrocytes exhibit several processes extending out from cells. The expression of fibroblast specific markers (CD90, α-SMA) were similar when HC-fibrocytes were triggered by GD serum and GO serum but the expression was not observed when HC-fibrocytes were triggered by autologous serum.GD-fibrocytes and GO-fibrocytes were also triggered by autologous serum and the expression of fibrotic markers were markedly higher in GO -fibrocytes compared to GD-fibrocytes and HC-fibrocytes. This finding suggest that patient’s serum plays an important role for the transformation of fibroblast in HC-fibrocytes. Differential expression of fibroblast specific markers (CD90, α-SMA) in fibrocytes among GD and GO suggest that some genetical alteration can be present especially in GO-fibrocytes may be one of the causative factors for orbital tissue remodelling and pathogenesis in GO. The colocalization of TSHR and IGF1R on circulating fibrocytes can induce various immune responses which can initiate the production of proinflammatory cytokines and chemokines (IL-6, IL-8, TNF-α, MCP-1, Hyaluronic Acid). Here, expression of such cytokines and chemokines were significantly higher in HC-fibrocytes with GD and GO treated serum compared to autologous serum treated HC-fibrocytes. But there were no significant changes has been found among HC-fibrocytes + GD Serum group and HC-fibrocytes + GO serum group. But the expression of those cytokines and chemokines were significantly higher in GO-fibrocytes + autologous serum treated group compared to GD-fibrocytes + autologous serum treated group. All of the above evidences suggest that higher expression of TSHR and IGF1R especially their colocalization on fibrocytes can promote pathogenicity in GO. 5. Conclusion The investigation into the pathogenesis of Graves' Orbitopathy (GO) alongside Graves' Disease (GD) has uncovered significant insights into the roles of Thyroid-Stimulating Hormone Receptor (TSHR) and Insulin-like Growth Factor 1 Receptor (IGF1R) in disease progression among the Indian population. The elevated expression of IGF1R on circulating fibrocytes, especially in GO patients, suggests a potential link between IGF1R and GO pathogenicity. Co-expression of TSHR and IGF1R in fibrocytes and their colocalization within cellular compartments further underlines the complexity of signalling pathways implicated in GO. The observed higher percentages of IGF1R + fibrocytes in GO compared to GD and healthy controls (HC), alongside the similar expression patterns with TSHR, point towards a coordinated role of these receptors in GO pathogenesis. The physical and functional link between TSHR and IGF1R may mediate signalling features attributed to each receptor individually, potentially contributing to the development of clinically significant Orbitopathy in GD patients. Moreover, the differential expression of fibroblast-specific markers (CD90, α-SMA) in GO-fibrocytes compared to GD-fibrocytes and HC-fibrocytes suggests genetic alterations specific to GO-fibrocytes, contributing to orbital tissue remodelling and pathogenesis. The colocalization of TSHR and IGF1R on circulating fibrocytes can stimulate immune responses, leading to increased production of proinflammatory cytokines and chemokines. This was evident in the significantly higher expression of such cytokines and chemokines in GO-fibrocytes treated with autologous serum compared to GD-fibrocytes, highlighting the role of patient-specific factors in disease manifestation. In conclusion, the higher expression and colocalization of TSHR and IGF1R, especially on fibrocytes, likely play a pivotal role in promoting pathogenicity in GO. These findings provide valuable insights into potential therapeutic targets for managing Graves' Orbitopathy and advancing our understanding of its underlying mechanisms. Declarations Conflict of interest The authors have no relevant financial or non-financial interests to disclose. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the institutional ethics committee [Institute of Post Graduate Medical Education and Research and SSKM Hospital, Kolkata, West Bengal, India. (Memo No. Inst/ IEC/2016/445)]. Written informed consent was obtained from the parents. Data availability The datasets generated during and/or analysed during the current study are not publicly available due to privacy but are available from the corresponding author on reasonable request. Acknowledgement We are grateful to all the study participants and the members of our research team for their kind support and cooperation. We are thankful to Prof. Simanti Dutta [Nodal Officer, Multi-Disciplinary Research Unit (MRU) IPGME&R] for her guidance in imaging and flowcytometry. Author contribution Conceptualization, Methodology, Formal analysis, Investigation, writing original draft: Madhurima Basak; Formal analysis of flowcytometry data: Dipanjan Bhattacharjee; Formal analysis and clinical data acquisition from patients: Anish Kar; Manuscript editing: Sriparna De, Formal analysis of Imaging: Bidhan Chakraborty ; Initial Conceptualization, Project Administration, Fund acquisition: Madhusudan Das; Supervision: Jyothi Chowdhury; Conceptualization, supervision, Fund acquisition , writing- review and editing: Subhankar Chowdhury. Funding This work was supported by to Department of Science Technology and Biotechnology (DSTBT, West Bengal) Grant number: 460 (Sanc.)/STBT-11012(19)/20/2019-ST SEC dated on 18.08.2021. Dr. Subhankar Chowdhury & Dr. Madhusudan Das have received research support from Department of Science Technology and Biotechnology (DSTBT, West Bengal) References Bahn RS (1998) Thyrotropin Receptor Expression in Graves ’ Orbital Adipose / Connective Tissues: Potential Autoantigen in. 83(3):998–1002 Cai W, ANALYSIS OF HUMAN TSH RECEPTOR GENE AND RNA TRANSCRIPTS IN PATIENTS WITH THYROID DISORDERS (1992), vol. 13, pp. 43–50 Kumar S, Nadeem S, Stan MN, Coenen M, Bahn RS (2011) A stimulatory TSH receptor antibody enhances adipogenesis via phosphoinositide 3-kinase activation in orbital preadipocytes from patients with Graves ’ ophthalmopathy. 155–163. 10.1530/JME-11-0006 Khoo TK, Bahn RS, Access NIHP (2014) vol. 17, no. 10, pp. 1013–1018, 10.1089/thy.2007.0185.Pathogenesis Heufelder AE, Division RSB, Medicine I (1993) Detection and localization of cytokine immunoreactivity in retro-ocular connective tissue in Graves ‘ opthalmopathy, pp. 10–17 Arnold K, Weetman AP (1996) Cell-mediated immunity in thyroid-associated ophthalmopathy. 15(3):159–164 Dejaco C, Grubeck- B (2005) Imbalance of regulatory T cells in human autoimmune diseases. 289–300. 10.1111/j.1365-2567.2005 . 02317. x Pappa A, Lawson JMM, Calder V, Fells P, Lightman S, Tao E (2000) T cells and fibroblasts in a V ected extraocular muscles in early and late thyroid associated ophthalmopathy, pp. 517–522 Flower L, Gray R, Pinkney J, Mohamed-ali V (2003) Stimulation of interleukin-6 release by interleukin-1 b from isolated human adipocytes. 21:32–37. 10.1016/S1043-4666(02)00495-7 Shanli Tsui AS, Naik V, Hoa N, Hwang CJ, Nikoo F, Afifyan AG, HikimGianoukakis RS, Douglas, Smith TJ (2009) Evidence for an association between TSH and IGF-1 receptors. 181(6):4397–4405 Smith TJ, Smith TJ (2019) The insulin-like growth factor-I receptor and its role in thyroid- associated ophthalmopathy. Eye 200–205. 10.1038/s41433-018-0265-2 Iglesias-osma MC et al (2019) The influence of the lack of insulin receptor substrate 2 (IRS2) on the thyroid gland. Sci Rep 2:1–10. 10.1038/s41598-019-42198-7 Douglas KC, Raymond S, Nikoo F, Afifiyan,Catherine J, Hwang and and, Uzma TJS, Haider P, Richards AG, Gianoukakis (2010) Increased Generation of Fibrocytes in, J Clin Endocrinol Metab , vol. 95(1), no. January, pp. 430–438, 10.1038/s41572-020-0184-y Gillespie EF et al (2015) Increased Expression of TSH Receptor by Fibrocytes in Thyroid-Associated Ophthalmopathy Leads to, vol. 97, no. November, pp. 740–746, 10.1210/jc.2011-2514 Smith TJ (2015) TSH-receptor-expressing fibrocytes and thyroid-associated ophthalmopathy. Nat Publishing Group. 10.1038/nrendo.2014.226 Ophthalmopathy T et al (2015) Increased Generation of Fibrocytes in, vol. 95, no. November, pp. 430–438, 10.1210/jc.2009-1614 Basak M, Sanyal T, Kar A, Bhattacharjee P, Das M (2021) Peripheral blood mononuclear cells - Can they provide a clue to the pathogenesis of Graves ’ Orbitopathy ? Endocrine. 10.1007/s12020-021-02865-0 Disease G et al (2011) September., Fibroblasts Expressing the Thyrotropin Receptor Overarch Thyroid and Orbit in Overarch Thyroid and Orbit in Graves ’ Disease, no. 2016, 10.1210/jc.2011-1249 Ophthalmopathy T et al (2015) Increased Generation of Fibrocytes in, vol. 95, no. November, pp. 430–438, 10.1210/jc.2009-1614 Agrawal N, Basak M, Chowdhury S (2020) A CLINICOPATHOGENETIC STUDY ON GRAVES ’ ORBITOPATHY IN A TERTIARY. no July. 10.24327/23956429.ijcmpr202001823 Additional Declarations No competing interests reported. Supplementary Files GRAPHICALABSTRACT.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4521745","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":310740378,"identity":"5b586a38-7d85-4276-9925-665b317e6b24","order_by":0,"name":"Madhurima Basak","email":"","orcid":"","institution":"Institute of Post Graduate Medical Education and Research and SSKM Hospital","correspondingAuthor":false,"prefix":"","firstName":"Madhurima","middleName":"","lastName":"Basak","suffix":""},{"id":310740379,"identity":"46ff46ef-80e0-4b23-b70d-1de0e2eedf67","order_by":1,"name":"Dipanjan 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17:30:29","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":725660,"visible":true,"origin":"","legend":"","description":"","filename":"GRAPHICALABSTRACT.tif","url":"https://assets-eu.researchsquare.com/files/rs-4521745/v1/cfc88aae3c241346b79c73fc.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"TSH receptor and IGF1 receptor expression in Circulating Fibrocytes in the pathogenesis of Graves’ Orbitopathy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGraves\u0026rsquo; Orbitopathy (GO) is an inflammatory autoimmune disorder in Orbital connective tissues and extra ocular muscles (EOMs) which is closely associated with Graves\u0026rsquo; Disease (GD). The thyrotropin receptor (TSHR) present on orbital fibroblast in orbital connective tissue thought to be an important auto antigen which links both conditions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e][\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Thus, GO exhibit expanded volume of the orbital connective tissues, adipose tissues of extraocular muscles due to new fat cell development[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].Affected orbital tissues show diffuse infiltration of T and B lymphocytes, macrophages (occasional), and proinflammatory cytokines [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].These cytokines are secreted by peripheral blood mono nuclear cells(PBMCs) infiltrating in orbit together with resident orbital fibroblasts and contribute to the inflammatory basis for active GO[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e][\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e][\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e][\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Apart from TSHR, Insulin like Growth Factor-1Receptor (IGF-1R) present on orbital fibroblast also plays a potent role in orbital tissue remodeling. These two receptors have close physical and functional relationship and they form a physical complex in orbital fibroblasts in GO[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. IGF1R mediated cell signaling pathway also stimulates the cell proliferation by TSH or TSH-Receptor auto antibodies (TRAbs)[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] .It is observed that orbital fibroblast share some common characteristic features with a group of spindle shape cells found in circulation termed as fibrocytes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].Fibrocytes are monocytes derived cells, exhibit hematopoietic marker, progenitor cell marker, tissue trafficking marker (CD34\u003csup\u003e+\u003c/sup\u003e, CD45\u003csup\u003e+\u003c/sup\u003e,CXCR4\u003csup\u003e+\u003c/sup\u003e respectively) in their cell surface and also found \u003cem\u003ein situ\u003c/em\u003e in orbital connective tissues [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e][\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].It is also observed that concentration of these circulating fibrocytes were significantly higher among GO compare to GD and healthy control (HC) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].These fibrocytes express TSHR on their cell surface and association of TSHR\u003csup\u003e+\u003c/sup\u003e fibrocytes with various clinical parameters (Age, duration of disease, Clinical Activity Score, Smoking status) among GO reveal its pathogenetic role in progression of orbitopathy[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].It is shown that fibrocytes also express IGF1R[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] .So, TSHR and IGF1R signaling in fibrocytes may enhance orbital tissue remodeling in GO.\u003c/p\u003e \u003cp\u003eIn our previous studies in Eastern India demonstrated the prevalence of GO was 32% among GD [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].So, It is not clearly understood as to why only certain individual with GD develop clinically significant GO. We had previously observed the concentration of TSHR\u003csup\u003e+\u003c/sup\u003e circulating fibrocytes among GD and GO in Indian population[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this present study we have taken a small attempt to highlight- i) Expression of IGF1R and its association with TSHR on circulating fibrocytes in GD, GO and HC ii) \u003cem\u003eIn vitro\u003c/em\u003e study to detect conversion of fibroblast from fibrocytes after serum treatment. iii) Cytokine and chemokine expression from fibrocytes after serum treatment in GD, GO and HC.\u003c/p\u003e"},{"header":"2. Material Methods","content":"\u003cp\u003e \u003cb\u003eStudy site and sample collection\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIndividuals with Graves\u0026rsquo; disease with clinically significant Orbitopathy (GO, n\u0026thinsp;=\u0026thinsp;30), Graves\u0026rsquo; disease without clinically significant Orbitopathy (GD, n\u0026thinsp;=\u0026thinsp;30) and Healthy Control (HC) subjects (n\u0026thinsp;=\u0026thinsp;20) aged 20\u0026ndash;65 years were recruited from Department of Endocrinology \u0026amp; Metabolism of IPGME\u0026amp;R and SSKM Hospital, Kolkata, India from 2019 to 2021. Patients were evaluated both clinically and radiologically by an endocrinologist. Patients with low level of TSH (Reference range 0.4-4.0 uIU/ml), normal or high levels of FT4 (Reference range 0.80\u0026ndash;1.90 uIU/ml), normal or high level of FT3(Reference range 1.50\u0026ndash;4.10 uIU/ml) and positive TRAb value (Reference range 1.22 IU/l were included for this study. Patients, who have received anti-thyroid therapy, radiation therapy, immunosuppressive treatment like glucocorticoids, earlier or individuals with other autoimmune diseases, asthma, sinusitis, recent trauma, or HIV infection, malignancy, pregnancy were excluded from this study. Severity of GO was graded based on the European Group of Graves' Orbitopathy (EUGOGO) classification [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. All demographic and biochemical data are given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Patients whose TRAb was positive in serum but does not follow EUGOGO guidelines were designated as GD and Patients whose TRAb was positive in serum and follow EUGOGO guidelines were designated as GO. Here clinical activity score (CAS)\u0026thinsp;=\u0026thinsp;3 was categorized as Moderate GO and CAS\u0026thinsp;\u0026gt;\u0026thinsp;3 was categorized as active to severe GO. Specifically, 20% of patients were in the active and severe phases, whereas 80% were in the inactive phase. Control subjects were healthy volunteers without symptoms or signs of thyrotoxicosis, normal thyroid function test, TRAb value was within reference range and no history of known autoimmune disease. They were recruited from colleagues, spouse of patients. Clinical evaluation and methods for measuring serum FT3, FT4, TSH-Receptor Antibody (TRAb) were already described in our previously published article [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and biochemical data of all study participants\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHC\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGD\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;30)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGO\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;30)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (Yrs. \u0026plusmn; S.D)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003cp\u003eF\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u0026thinsp;=\u0026thinsp;09\u003c/p\u003e \u003cp\u003eF\u0026thinsp;=\u0026thinsp;21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM\u0026thinsp;=\u0026thinsp;18\u003c/p\u003e \u003cp\u003eF\u0026thinsp;=\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.02\u003csup\u003ea*Ψ\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoker\u003c/p\u003e \u003cp\u003eNon-Smoker\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03\u003csup\u003ea*Ψ\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of hyper thyroidism (months)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (1\u0026ndash;24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (1\u0026ndash;36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.24\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExophthalmometry in eye (mm) RE\u003c/p\u003e \u003cp\u003eLE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003csup\u003e*Ψ\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003csup\u003e*Ψ\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCAS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;1 but \u0026le;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTSH (uIU/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.24\u003csup\u003ecΨ\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFT3(pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.40\u003csup\u003ecΨ\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFT4(ng/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.45\u003csup\u003ecΨ\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRAb (IU/I)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.07\u003csup\u003ecΨ\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeverity (in GO)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMild\u0026thinsp;=\u0026thinsp;23\u003c/p\u003e \u003cp\u003eModerate\u0026thinsp;=\u0026thinsp;4\u003c/p\u003e \u003cp\u003eSevere\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eTwo tailed unpaired t test; * Statistically significant; \u003csup\u003eΨ\u003c/sup\u003eGD vs GO.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e\u003cp\u003eAbbreviations: CAS-Clinical Activity Score; TSH-Thyroid Stimulating Hormone; FT4-Free T4; FT3-Free T3; TRAb-Thyroid Stimulating Hormone Receptor Antibody; GO-Graves\u0026rsquo; Orbitopathy, M- Male, F- Female.RE-Right Eye, LE-Left Eye.\u003csup\u003ea\u003c/sup\u003e Two-tailed Fisher\u0026rsquo;s test; \u003csup\u003eb\u0026nbsp;\u003c/sup\u003eOne-way ANOVA; \u003csup\u003ec\u0026nbsp;\u003c/sup\u003eBonferroni\u0026apos;s Multiple Comparison Test.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunophenotyping\u003c/b\u003e \u003c/p\u003e \u003cp\u003eStaining for flow cytometry was performed within 24 h of blood collection. Method for staining was described in our previously published article [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, a staining buffer (SB) was prepared in 1X Phosphate Buffer Saline (PBS) containing 2% fetal bovine serum (FBS) with 0.1% sodium azide. 200 \u0026micro;l of whole blood were placed in 12X75-mm polypropylene tubes, and 2 ml FACS Lysing solution was added for 10 min at room temperature. Cells were centrifuged at 1800 rpm for 5 min, washed, and resuspended in 100\u0026micro;l SB. The following anti human fluorochrome conjugated monoclonal antibodies were used: CD 34-BB 700 (BD Bioscience, San Jose, CA, USA Catalogue no-746104), CD45 V500 (BD Bioscience, SanJose, CA, USA Catalogue no-560777), CXCR4-BV421, (BD Bioscience, SanJose, CA, USA Catalogue no-562448), CD 221-PE (IGF1R, BD Bioscience, San Jose, CA, USA Catalogue No- 555999) TSHR-Alexa fluor 647 (Novus Biologicals, USA, Catalogue no-NB100-65797AF64). After 30 minutes incubation in the dark at room temperature (RT) the cells were resuspended and washed twice with SB. Collagen -I (Col-I) was present in cytosol. For quantification of Col I, cells were permeabilized with cytofix/ cytoperm for 20 min at RT, washed twice, and resuspended in 100 \u0026micro;l Perm/Wash buffer. After incubation with anti-human FITC conjugated Collagen I (Col I-FITC) in the dark at RT in perm wash buffer, the cells were washed and acquired on FACS Verse Flow cytometer (BD Bioscience, San Jose, CA, USA). At least 1X10\u003csup\u003e4\u003c/sup\u003e events were collected. Fibrocytes were identified from peripheral blood using a multiparametric Flowcytometry technique described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. TSHR and IGF1R expression on fibrocytes were measured from the fibrocytes co-expressing TSHR and Col I, IGF1R and Col 1 respectively (Fig.\u0026nbsp;1a-l).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFibrocyte culture and treatment\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePBMC were used to culture condition similar to those described by Bucala \u003cem\u003eet al\u003c/em\u003e [21]. Here, 7 patients with GO were in active phase (CAS\u0026thinsp;\u0026ge;\u0026thinsp;3) and remaining (n\u0026thinsp;=\u0026thinsp;23, CAS\u0026thinsp;\u0026lt;\u0026thinsp;3) were in inactive phase. Culture was considered for 10 GO (7 active phase\u0026thinsp;+\u0026thinsp;3 inactive phase) patients,10 GD patients and 10 HC. Clinical data of those 10 GO patients are given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. PBMCs were isolated from blood by density gradient centrifugation using HiSep TM (Hi media Laboratories, LS001) according to manufacturer protocol. They were isolated and inoculated in 4-chambered slides (1X 10\u003csup\u003e6\u003c/sup\u003e/well,) in 1 ml Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, Himedia Laboratories cat no AL151A) with 10% FBS (Hi media Laboratories) and 1% penicillin streptomycin antibiotic solution (Hi media Laboratories). The culture was maintained at 37\u003csup\u003eo\u003c/sup\u003eC in 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eTable 2: \u003c/strong\u003eClinical characteristics of GO (n=10) patients those PBMCs were used in cell culture\u003c/p\u003e\n\u003ctable style=\"float:;border-collapse:collapse;border:none;margin-:6.75pt;margin-right:6.75pt;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 47.05pt;border: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003ePatient sl. No.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.3pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eAge (yrs.) / Sex\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eCAS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.95pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:125%;'\u003e\u003cspan style='font-size:13px;line-height:125%;font-family:\"Times New Roman\",serif;'\u003eDuration of hyperthyroidism\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e(month)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.7pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:125%;'\u003e\u003cspan style='font-size:13px;line-height:125%;font-family:\"Times New Roman\",serif;'\u003eTime since\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:125%;'\u003e\u003cspan style='font-size:13px;line-height:125%;font-family:\"Times New Roman\",serif;'\u003ediagnosis of GO\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e(month)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.25pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eSmoking Status\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 89.25pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:125%;'\u003e\u003cspan style='font-size:13px;line-height:125%;font-family:\"Times New Roman\",serif;'\u003eProptosis (mm)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:125%;'\u003e\u003cspan style='font-size:13px;line-height:125%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eLE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;RE\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.5pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eSeverity\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 47.05pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;background: rgb(231, 230, 230);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(231, 230, 230);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e25/M\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e07\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e05\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.25pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eYes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(231, 230, 230);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e22 \u0026nbsp; \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(231, 230, 230);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(231, 230, 230);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eActive and moderate\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 47.05pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;background: rgb(174, 170, 170);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(174, 170, 170);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e29/M\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e2.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e2.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.25pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eYes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(174, 170, 170);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e24 \u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(174, 170, 170);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(174, 170, 170);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:125%;'\u003e\u003cspan style='font-size:13px;line-height:125%;font-family:\"Times New Roman\",serif;color:black;'\u003eActive\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eand severe\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 47.05pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e55/M\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e04\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e04\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.25pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eYes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e20 \u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eInactive and mild\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 47.05pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e04\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e32/F\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e06\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.25pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eNo\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e18 \u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(231, 230, 230);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e18\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eActive and moderate\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 47.05pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;background: rgb(174, 170, 170);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New 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style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times 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style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eActive and severe\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 47.05pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e06\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e34/M\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.25pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eYes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e20 \u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eInactive and mild\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 47.05pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e07\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e48/M\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.25pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eYes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e32 \u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(231, 230, 230);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e33\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eActive and moderate\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 47.05pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;background: rgb(174, 170, 170);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e08\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(174, 170, 170);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e40/F\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.25pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eNo\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(174, 170, 170);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e32 \u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(174, 170, 170);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e32\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(174, 170, 170);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eActive and severe\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 47.05pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e09\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e56/M\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e1.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e1.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.25pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eNo\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e25 \u0026nbsp;\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(231, 230, 230);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003e25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;background: rgb(208, 206, 206);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;color:black;'\u003eActive and severe\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 47.05pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e28/M\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76.95pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.25pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eYes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e22 \u0026nbsp; \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003e22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 48.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;line-height:normal;'\u003e\u003cspan style='font-size:13px;font-family:\"Times New Roman\",serif;'\u003eInactive and mild\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u003cbr\u003e\n \u003cp\u003eHere, PBMCs were treated \u003cem\u003ein vitro\u003c/em\u003e by 10% serum supplementation. The treatment pattern was depicted in Fig.\u0026nbsp;2. PBMC of HC was treated by autologous serum as well as GD serum and GO serum separately.GD and GO PBMCs were also treated by autologous serum respectively.\u003c/p\u003e \u003cp\u003eAfter a couple of days (48 hours), the nonadherent cells were removed by gentle aspiration of the media. After 7 days of culture, previous media was discarded, and cells were replenished by fresh media. Prior to collect the supernatant, the cells were starved from serum for 48 hrs. 7 days cultured cells were used to observe fibrotic markers.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell viability assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePBMCs of HC and patients (both GD and GO) were plated in 96 well plate at density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e per each well. Cells were treated with different concentration of patient serum and autologous serum (5%,10%,15% and 20%). We have assessed cell viability in a concentration dependent manner in accordance with days variation. It was inferred from the above findings; significant cell viability was observed at 10% serum treatment. confirmed by MTT (93-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunocytochemistry\u003c/b\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence staining was performed using 7 days cultured fibrocytes in four chambered slides. At first media was removed and cells were fixed in 2% paraformaldehyde in Phosphate buffer saline (PBS) for 10 minutes at room temperature (RT) and blocked with 5% Bovine Serum Albumin (BSA) in PBS for 45 minutes. Cells were incubated by Mouse anti human TSHR mono clonal antibodies (Catalog no MA3-218, Thermo fisher Scientific), Rabbit anti human IGF1R alpha polyclonal antibodies (Catalog no PA5-79444, Invitrogen), Mouse anti human CD90 monoclonal antibodies (NBP2-52961, Novus biologicals), Rabbit anti human alpha SMA monoclonal antibodies (NBP2-67440, Novus biologicals) at a dilution of 1:100 in PBS containing 5% BSA for 3 hrs at 37\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003cp\u003eCells were then stained by goat anti mouse Alexa flour 488 fluorochrome conjugated secondary antibodies in a combination with TSHR monoclonal antibody and CD 90 monoclonal antibody; donkey anti rabbit Alexa flour 647conjugated secondary antibodies in a combination with IGF1R polyclonal antibody and alpha SMA monoclonal antibody. Nucleus were stained using 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) and observed under confocal microscope. Images were acquired from THUNDER Imager 3D Assay, Leica Microsystem.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytokines and chemokines measurements from cultured fibrocytes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCytokines (IL6, IL8, TNF alpha) and chemokines (Hyaluronic Acid, MCP 1) production of 7 days cultured fibrocytes were observed by sandwich ELISA (G biosciences, St. Louis, U.S.A) according to manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eStatistical analyses specifically two-tailed Fisher\u0026rsquo;s test, unpaired t-test, one-way ANOVA (Bonferroni\u0026rsquo;s Multiple Comparison Test) were performed. Calculations were done using GraphPad Prism version 8 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.grpahpad.com\u003c/span\u003e\u003cspan address=\"http://www.grpahpad.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, San Diego, CA) and p value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Result","content":"\u003cp\u003e \u003cb\u003eConcentration of IGF1R\u003c/b\u003e \u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ecirculating fibrocytes among GD and GO\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFibrocytes display IGF1R (Fig.\u0026nbsp;1a-l) on its cell surface in peripheral blood (when they were gated from CD 34\u003csup\u003e+\u003c/sup\u003e/CD 45\u003csup\u003e+\u003c/sup\u003e/Col-1\u003csup\u003e+\u003c/sup\u003e cells) among GO, GD, and control. Quantification of IGF-1R\u003csup\u003e+\u003c/sup\u003e fibrocytes from peripheral blood show that, percentages of IGF-1R\u003csup\u003e+\u003c/sup\u003e fibrocytes were significantly higher in GO as compared to GD (GO:11\u0026thinsp;\u0026plusmn;\u0026thinsp;9% vs GD:2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) but in control it was almost absent (Fig.\u0026nbsp;3a). (GD: 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% vs Control: 0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03%, p\u0026thinsp;=\u0026thinsp;0.002; GO:11\u0026thinsp;\u0026plusmn;\u0026thinsp;9% vs Control: 0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTSHR and IGF1R co-expression on fibrocytes among GD and GO\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA large population of TSHR\u003csup\u003e+\u003c/sup\u003e fibrocytes also express IGF-1R when they were gated from TSHR\u003csup\u003e+\u003c/sup\u003e/Col1\u003csup\u003e+\u003c/sup\u003e cells. Quantification of TSHR\u003csup\u003e+\u003c/sup\u003e/ IGF-1R\u003csup\u003e+\u003c/sup\u003e circulating fibrocytes show that, expression of TSHR\u003csup\u003e+\u003c/sup\u003e/ IGF-1R\u003csup\u003e+\u003c/sup\u003e circulating fibrocytes were significantly higher in GO compare to GD and control (Fig.\u0026nbsp;3b) [(GD vs Control: TSHR\u003csup\u003e+\u003c/sup\u003e/ IGF1R\u003csup\u003e+\u003c/sup\u003e fibrocytes., 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% vs 0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03%, p\u0026thinsp;=\u0026thinsp;0.003), (GO vs Control: TSHR\u003csup\u003e+\u003c/sup\u003e / IGF1R\u003csup\u003e+\u003c/sup\u003e fibrocytes.,11\u0026thinsp;\u0026plusmn;\u0026thinsp;8% vs 0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), (GD vs GO: TSHR\u003csup\u003e+\u003c/sup\u003e / IGF1R\u003csup\u003e+\u003c/sup\u003e fibrocytes., 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% vs11\u0026thinsp;\u0026plusmn;\u0026thinsp;8%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001].\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunolocalization of TSHR and IGF-1R on fibrocytes among GO\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTSHR was detected using Alexa flour 488 conjugated secondary antibody against of mouse monoclonal anti human anti TSHR- antibody (green). Detection of IGF-1R alpha was done by Alexa flour 647 conjugated secondary antibody against of rabbit monoclonal anti human anti IGF-1R alpha- antibody (red). Expression of TSHR and IGF1R were almost absent in HC fibrocytes (Fig.\u0026nbsp;4, Panel I, a-c) and very low to detect in GD (Fig.\u0026nbsp;4, Panel II, d-f and j). But the expressions were very high in GO as compared to HC and GD (Fig.\u0026nbsp;3, Panel III, g-i). IGF1R was localized to the periphery of fibrocytes as well as to the perinuclear area. The distribution was identical to the staining pattern of TSHR. The yellow or orange staining present in the merged images (Fig.\u0026nbsp;4, Panel IV, k) results from the colocalization of TSHR and IGF1R in GO.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvidence for fibroblast transformation from fibrocytes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMorphological transformation was observed within the cultured fibrocytes isolated from Healthy Control (HC) (Fig.\u0026nbsp;5). At first Fibrocytes were differentiated from monocytes (Fig.\u0026nbsp;5a). These fibrocytes exhibit more cellular processes with prominent oval shape nucleus due to influence of patient\u0026rsquo;s serum (Fig.\u0026nbsp;5b and c). More-over, HC-fibrocytes were found to have higher expression of fibroblast specific marker (CD90) and fibroblast activation marker (α -SMA) after stimulation with GO serum and GD serum (Fig.\u0026nbsp;6a-i). Expression level of those markers were same in between GD serum treated HC fibrocytes and GO serum treated HC fibrocytes (CD 90, p\u0026thinsp;=\u0026thinsp;0.99; α -SMA, p\u0026thinsp;=\u0026thinsp;0.06; Fig.\u0026nbsp;7a and b). But CD90 and α -SMA were not expressed when HC-fibrocytes were treated with autologous serum.\u003c/p\u003e \u003cp\u003eCD90 and α -SMA expressions were also seen in autologous serum treated GD fibrocytes and autologous serum treated GO fibrocytes. Autologous serum treated GO -fibrocytes show significant higher expression of fibroblast specific marker CD90(p\u0026thinsp;=\u0026thinsp;0.01) and fibroblast activation marker α -SMA (Fig.\u0026nbsp;6m-o and Fig.\u0026nbsp;7c, d) compare to autologous serum treated GD fibrocytes (p\u0026thinsp;=\u0026thinsp;0.0003) (Fig.\u0026nbsp;6j-l and Fig.\u0026nbsp;7c, d).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cb\u003eExpressions of IL-6, IL-8, TNF-α, Hyaluronic Acid (HA), Monocyte chemoattractant protein-1 (MCP1) by cultured fibrocytes\u003c/b\u003e \u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eExpressions of proinflammatory cytokines like IL-6, IL-8 were markedly elevated in HC-fibrocytes treated with serum from GD patients and GO patients as compared to HC-fibrocytes treated with autologous serum. [(IL-6: autologous serum treated HC fibrocytes; 3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 pg/ml vs GO serum treated HC-fibrocytes 39\u0026thinsp;\u0026plusmn;\u0026thinsp;7 pg/ml, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and autologous serum treated HC fibrocytes; 3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 pg/ml vs GD serum treated HC-fibrocytes 36\u0026thinsp;\u0026plusmn;\u0026thinsp;8 pg/ml. p\u0026thinsp;=\u0026thinsp;0.0002, Fig.\u0026nbsp;8a.) (IL8: autologous serum treated HC fibrocytes 29.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3 pg/ml vs GO serum treated HC-fibrocytes 83.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1 pg/ml ,p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and autologous serum treated HC fibrocytes 29.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3 pg/ml vs GD serum treated HC-fibrocytes 82.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1pg/ml, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001,Fig.\u0026nbsp;8c)\u003c/p\u003e \u003cp\u003eBut the expressions of those cytokines were similar between HC- fibrocytes, treated with GD and GO serum [(IL6: GD serum treated HC-fibrocytes 36\u0026thinsp;\u0026plusmn;\u0026thinsp;8 pg/ml vs GO serum treated HC-fibrocytes 39\u0026thinsp;\u0026plusmn;\u0026thinsp;7 pg/ml, p\u0026thinsp;=\u0026thinsp;0.55) (IL8: GD serum treated HC-fibrocytes 82.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1pg/ml vs GO serum treated HC-fibrocytes 83.5\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1 pg/ml, p\u0026thinsp;=\u0026thinsp;0.87)]\u003c/p\u003e \u003cp\u003eInterestingly, expressions of those cytokines (IL6 and IL8) were significantly higher in autologous serum treated GO fibrocytes compare to autologous serum treated GD fibrocytes [IL6: autologous serum treated GO fibrocytes 42\u0026thinsp;\u0026plusmn;\u0026thinsp;14pg/ml vs autologous serum treated GD fibrocytes 29\u0026thinsp;\u0026plusmn;\u0026thinsp;9pg/ml, p\u0026thinsp;=\u0026thinsp;0.04, Fig.\u0026nbsp;8b; IL8: autologous serum treated GO fibrocytes 90\u0026thinsp;\u0026plusmn;\u0026thinsp;7 pg/ml vs autologous serum treated GD fibrocytes 82\u0026thinsp;\u0026plusmn;\u0026thinsp;7pg/ml, p\u0026thinsp;=\u0026thinsp;0.01; Fig.\u0026nbsp;8d].\u003c/p\u003e \u003cp\u003eExpression of TNF-α was also significantly higher in HC-fibrocytes treated with serum from GD patients and GO patients as compared to HC-fibrocytes treated with autologous serum. [Autologous serum treated HC fibrocytes; 14\u0026thinsp;\u0026plusmn;\u0026thinsp;2 pg/ml vs GO serum treated HC-fibrocytes 67\u0026thinsp;\u0026plusmn;\u0026thinsp;12 pg/ml, p\u0026thinsp;=\u0026thinsp;0.0001 and autologous serum treated HC fibrocytes; 14\u0026thinsp;\u0026plusmn;\u0026thinsp;2 pg/ml vs GD serum treated HC-fibrocytes 67\u0026thinsp;\u0026plusmn;\u0026thinsp;15 pg/ml. p\u0026thinsp;=\u0026thinsp;0.0003, Fig.\u0026nbsp;9a]\u003c/p\u003e \u003cp\u003eBut, expression of TNF- α was also same in between HC- fibrocytes, treated with GD and GO serum. [ GO serum treated HC-fibrocytes 67\u0026thinsp;\u0026plusmn;\u0026thinsp;12 pg/ml vs GD serum treated HC-fibrocytes 67\u0026thinsp;\u0026plusmn;\u0026thinsp;15 pg/ml. p\u0026thinsp;=\u0026thinsp;0.9, Fig.\u0026nbsp;9a].\u003c/p\u003e \u003cp\u003eSurprisingly there were no significant differences of TNF- α expressions were found in between autologous serum treated GO fibrocytes and autologous serum treated GD fibrocytes. [autologous serum treated GO fibrocytes 74\u0026thinsp;\u0026plusmn;\u0026thinsp;17pg/ml vs autologous serum treated GD fibrocytes 69\u0026thinsp;\u0026plusmn;\u0026thinsp;15 pg/ml, p\u0026thinsp;=\u0026thinsp;0.5, Fig.\u0026nbsp;9b]\u003c/p\u003e \u003cp\u003eChemokines like Hyaluronic acid (HA), Monocyte chemoattractant protein-1 (MCP1) were also markedly elevated in in HC-fibrocytes treated with serum from GD patients and GO patients as compared to HC-fibrocytes treated with autologous serum [(HA: Autologous serum treated HC fibrocytes 29\u0026thinsp;\u0026plusmn;\u0026thinsp;11 ng/ml vs GO serum treated HC-fibrocytes 136\u0026thinsp;\u0026plusmn;\u0026thinsp;23 ng/ml, p\u0026thinsp;=\u0026thinsp;0.02 and autologous serum treated HC fibrocytes 29\u0026thinsp;\u0026plusmn;\u0026thinsp;11 ng/ml vs GD serum treated HC-fibrocytes 162\u0026thinsp;\u0026plusmn;\u0026thinsp;36 ng/ml. p\u0026thinsp;=\u0026thinsp;0.02,Fig.\u0026nbsp;10a)(MCP1: Autologous serum treated HC fibrocytes 38\u0026thinsp;\u0026plusmn;\u0026thinsp;5 pg/ml vs GO serum treated HC-fibrocytes 160\u0026thinsp;\u0026plusmn;\u0026thinsp;10 pg/ml, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and autologous serum treated HC fibrocytes 29\u0026thinsp;\u0026plusmn;\u0026thinsp;11 ng/ml vs GD serum treated HC-fibrocytes 162\u0026thinsp;\u0026plusmn;\u0026thinsp;16 pg/ml. p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;10c)].\u003c/p\u003e \u003cp\u003eBut, no statistically significant changes of expressions were found of those chemokines (HA, MCP1) in between HC- fibrocytes, treated with GD and GO serum [(HA: GO serum treated HC-fibrocytes 136\u0026thinsp;\u0026plusmn;\u0026thinsp;23 ng/ml vs GD serum treated HC-fibrocytes 162\u0026thinsp;\u0026plusmn;\u0026thinsp;36 ng/ml, p\u0026thinsp;=\u0026thinsp;0.2, Fig.\u0026nbsp;10a) (MCP1: GO serum treated HC-fibrocytes 160\u0026thinsp;\u0026plusmn;\u0026thinsp;10 pg/ml vs GD serum treated HC-fibrocytes 162\u0026thinsp;\u0026plusmn;\u0026thinsp;16 pg/ml, p\u0026thinsp;=\u0026thinsp;0.9,Fig.\u0026nbsp;10c)].\u003c/p\u003e \u003cp\u003eExpressions of those chemokines were markedly elevated when GO fibrocytes were treated with autologous serum compare to autologous serum treated GD fibrocytes [(HA: autologous serum treated GO fibrocytes 110\u0026thinsp;\u0026plusmn;\u0026thinsp;5 ng/ml vs autologous serum treated GD fibrocytes 56\u0026thinsp;\u0026plusmn;\u0026thinsp;17 ng/ml, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;10b) (MCP1: autologous serum treated GO fibrocytes 147\u0026thinsp;\u0026plusmn;\u0026thinsp;5 pg/ml vs autologous serum treated GD fibrocytes 135\u0026thinsp;\u0026plusmn;\u0026thinsp;3pg/ml, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;10d)].\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eUncertainty about the pathogenesis of GO specifically in only proportion of patients with GD developing clinically significant Orbitopathy led to investigate that beside TSHR, IGF1R might play an important role in disease progression. IGF1R belongs to a group of structurally related transmembrane tyrosine kinase receptors that has 1368 amino acids. IGF1works via IGF1 receptor and increases thymic epithelial cell proliferation as well as intra-thymic T cell growth and migration in the thymus. Thus, it influences immune function. T and B lymphocytes, as well as cells from the monocyte lineage, are professional immune cells that express functional IGF1R and react to physiological quantities of the growth factor. IGF1 is crucial for the T and B cell growth processes. It is chemotactic and increase thymidine incorporation in human T cells. IGF1R, which is expressed at a greater level in activated cells, mediates these activities [22,23.]. In this present study, we are reporting the presence of IGF1R on circulating fibrocytes for the first time in GD and GO patients among Indian population. By using flowcytometry we were able to quantify IGF1R expression and co-expression of TSHR -IGF1R among patient group. We found that the percentages of IGF-1R\u003csup\u003e+\u003c/sup\u003e fibrocytes were significantly higher in GO compared to GD and HC (GO:11\u0026thinsp;\u0026plusmn;\u0026thinsp;9%, \u003cem\u003en\u0026thinsp;=\u0026thinsp;30\u003c/em\u003e vs GD:2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% \u003cem\u003en\u0026thinsp;=\u0026thinsp;30\u003c/em\u003e vs HC: 0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03% \u003cem\u003en\u0026thinsp;=\u0026thinsp;20\u003c/em\u003e; \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Expression pattern was similar with TSHR expression (previously reported) on circulating fibrocytes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and these two receptors co-express in fibrocytes. This co-expression level was also significantly higher in GO compared to GD and HC. Additionally, our recent research indicates that TSHR and IGF-1R co-localize to the cytoplasmic, plasma membrane, and perinuclear compartments of human circulating fibrocytes. Previously, Tsui \u003cem\u003eet al\u003c/em\u003e observed the colocalization of TSHR and IGF1R on human orbital fibroblasts and thyrocytes [24] but their colocalization on circulating fibrocytes were not observed before. The possibility of functional interaction between heterotrimeric G proteins and IGF-1 signalling has been suggested by a number of earlier investigations. [25]. On the other hand, a number of prior investigations strongly suggest that IGF-1 may also boost some, but not all, TSHR signalling. For example, Tramontano \u003cem\u003eet al\u003c/em\u003e discovered that the effects of IGF-1 and TSH on FRTL-5 cell proliferation and DNA synthesis were synergistic and concentration-dependent [26]. IGF-1 does not appear to have an impact on TSH-dependent cAMP production in FRTL-5 cells [27\u0026ndash;30]. Clement and co-workers showed that Increased thyroid gland weight and follicular lumen area are seen in vivo in double-transgenic animals whose thyroids are conditionally overexpressed with both IGF-1 and IGF-1R [31]. Therefore, our results imply that a physical and functional link between TSHR and IGF-1R may mediate features of signalling that have been attributed to each receptor alone. Fibrocytes also transformed into fibroblast when they were triggered by serum. Here, HC-fibrocytes exhibit several processes extending out from cells. The expression of fibroblast specific markers (CD90, α-SMA) were similar when HC-fibrocytes were triggered by GD serum and GO serum but the expression was not observed when HC-fibrocytes were triggered by autologous serum.GD-fibrocytes and GO-fibrocytes were also triggered by autologous serum and the expression of fibrotic markers were markedly higher in GO -fibrocytes compared to GD-fibrocytes and HC-fibrocytes. This finding suggest that patient\u0026rsquo;s serum plays an important role for the transformation of fibroblast in HC-fibrocytes. Differential expression of fibroblast specific markers (CD90, α-SMA) in fibrocytes among GD and GO suggest that some genetical alteration can be present especially in GO-fibrocytes may be one of the causative factors for orbital tissue remodelling and pathogenesis in GO.\u003c/p\u003e \u003cp\u003eThe colocalization of TSHR and IGF1R on circulating fibrocytes can induce various immune responses which can initiate the production of proinflammatory cytokines and chemokines (IL-6, IL-8, TNF-α, MCP-1, Hyaluronic Acid). Here, expression of such cytokines and chemokines were significantly higher in HC-fibrocytes with GD and GO treated serum compared to autologous serum treated HC-fibrocytes. But there were no significant changes has been found among HC-fibrocytes\u0026thinsp;+\u0026thinsp;GD Serum group and HC-fibrocytes\u0026thinsp;+\u0026thinsp;GO serum group. But the expression of those cytokines and chemokines were significantly higher in GO-fibrocytes\u0026thinsp;+\u0026thinsp;autologous serum treated group compared to GD-fibrocytes\u0026thinsp;+\u0026thinsp;autologous serum treated group.\u003c/p\u003e \u003cp\u003eAll of the above evidences suggest that higher expression of TSHR and IGF1R especially their colocalization on fibrocytes can promote pathogenicity in GO.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe investigation into the pathogenesis of Graves' Orbitopathy (GO) alongside Graves' Disease (GD) has uncovered significant insights into the roles of Thyroid-Stimulating Hormone Receptor (TSHR) and Insulin-like Growth Factor 1 Receptor (IGF1R) in disease progression among the Indian population. The elevated expression of IGF1R on circulating fibrocytes, especially in GO patients, suggests a potential link between IGF1R and GO pathogenicity. Co-expression of TSHR and IGF1R in fibrocytes and their colocalization within cellular compartments further underlines the complexity of signalling pathways implicated in GO.\u003c/p\u003e \u003cp\u003eThe observed higher percentages of IGF1R\u003csup\u003e+\u003c/sup\u003e fibrocytes in GO compared to GD and healthy controls (HC), alongside the similar expression patterns with TSHR, point towards a coordinated role of these receptors in GO pathogenesis. The physical and functional link between TSHR and IGF1R may mediate signalling features attributed to each receptor individually, potentially contributing to the development of clinically significant Orbitopathy in GD patients.\u003c/p\u003e \u003cp\u003eMoreover, the differential expression of fibroblast-specific markers (CD90, α-SMA) in GO-fibrocytes compared to GD-fibrocytes and HC-fibrocytes suggests genetic alterations specific to GO-fibrocytes, contributing to orbital tissue remodelling and pathogenesis.\u003c/p\u003e \u003cp\u003eThe colocalization of TSHR and IGF1R on circulating fibrocytes can stimulate immune responses, leading to increased production of proinflammatory cytokines and chemokines. This was evident in the significantly higher expression of such cytokines and chemokines in GO-fibrocytes treated with autologous serum compared to GD-fibrocytes, highlighting the role of patient-specific factors in disease manifestation.\u003c/p\u003e \u003cp\u003eIn conclusion, the higher expression and colocalization of TSHR and IGF1R, especially on fibrocytes, likely play a pivotal role in promoting pathogenicity in GO. These findings provide valuable insights into potential therapeutic targets for managing Graves' Orbitopathy and advancing our understanding of its underlying mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the institutional ethics committee [Institute of Post Graduate Medical Education and Research and SSKM Hospital, Kolkata, West Bengal, India. (Memo No. Inst/ IEC/2016/445)].\u0026nbsp;Written informed consent was obtained from the parents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are not publicly available due to privacy but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to all the study participants and the members of our research team for their kind support and cooperation. We are thankful to Prof. Simanti Dutta [Nodal Officer, Multi-Disciplinary Research Unit (MRU) IPGME\u0026amp;R] for her guidance in imaging and flowcytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, \u0026nbsp;Methodology, Formal analysis, Investigation, writing original draft: Madhurima Basak; Formal analysis of flowcytometry data: Dipanjan Bhattacharjee; Formal analysis and clinical data acquisition from patients: Anish Kar; Manuscript editing: Sriparna De, Formal analysis of Imaging: Bidhan Chakraborty ; Initial Conceptualization, Project Administration, Fund acquisition: Madhusudan Das; Supervision: Jyothi Chowdhury; Conceptualization, supervision, Fund acquisition , writing- review and editing: Subhankar Chowdhury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by to Department of Science Technology and Biotechnology (DSTBT, West Bengal) Grant number: 460 (Sanc.)/STBT-11012(19)/20/2019-ST SEC dated on 18.08.2021. Dr. Subhankar Chowdhury \u0026amp; Dr. Madhusudan Das have received research support from Department of Science Technology and Biotechnology (DSTBT, West Bengal)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBahn RS (1998) Thyrotropin Receptor Expression in Graves \u0026rsquo; Orbital Adipose / Connective Tissues: Potential Autoantigen in. 83(3):998\u0026ndash;1002\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai W, ANALYSIS OF HUMAN TSH RECEPTOR GENE AND RNA TRANSCRIPTS IN PATIENTS WITH THYROID DISORDERS (1992), vol. 13, pp. 43\u0026ndash;50\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Nadeem S, Stan MN, Coenen M, Bahn RS (2011) A stimulatory TSH receptor antibody enhances adipogenesis via phosphoinositide 3-kinase activation in orbital preadipocytes from patients with Graves \u0026rsquo; ophthalmopathy. 155\u0026ndash;163. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1530/JME-11-0006\u003c/span\u003e\u003cspan address=\"10.1530/JME-11-0006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhoo TK, Bahn RS, Access NIHP (2014) vol. 17, no. 10, pp. 1013\u0026ndash;1018, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1089/thy.2007.0185.Pathogenesis\u003c/span\u003e\u003cspan address=\"10.1089/thy.2007.0185.Pathogenesis\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeufelder AE, Division RSB, Medicine I (1993) Detection and localization of cytokine immunoreactivity in retro-ocular connective tissue in Graves \u0026lsquo; opthalmopathy, pp. 10\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArnold K, Weetman AP (1996) Cell-mediated immunity in thyroid-associated ophthalmopathy. 15(3):159\u0026ndash;164\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDejaco C, Grubeck- B (2005) Imbalance of regulatory T cells in human autoimmune diseases. 289\u0026ndash;300. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-2567.2005\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2567.2005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 02317. x\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePappa A, Lawson JMM, Calder V, Fells P, Lightman S, Tao E (2000) T cells and fibroblasts in a V ected extraocular muscles in early and late thyroid associated ophthalmopathy, pp. 517\u0026ndash;522\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlower L, Gray R, Pinkney J, Mohamed-ali V (2003) Stimulation of interleukin-6 release by interleukin-1 b from isolated human adipocytes. 21:32\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1043-4666(02)00495-7\u003c/span\u003e\u003cspan address=\"10.1016/S1043-4666(02)00495-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShanli Tsui AS, Naik V, Hoa N, Hwang CJ, Nikoo F, Afifyan AG, HikimGianoukakis RS, Douglas, Smith TJ (2009) Evidence for an association between TSH and IGF-1 receptors. 181(6):4397\u0026ndash;4405\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith TJ, Smith TJ (2019) The insulin-like growth factor-I receptor and its role in thyroid- associated ophthalmopathy. 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November, pp. 430\u0026ndash;438, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1210/jc.2009-1614\u003c/span\u003e\u003cspan address=\"10.1210/jc.2009-1614\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgrawal N, Basak M, Chowdhury S (2020) A CLINICOPATHOGENETIC STUDY ON GRAVES \u0026rsquo; ORBITOPATHY IN A TERTIARY. no July. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.24327/23956429.ijcmpr202001823\u003c/span\u003e\u003cspan address=\"10.24327/23956429.ijcmpr202001823\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fibrocytes, Peripheral Blood Mononuclear Cells, Insulin Like Growth Factor Receptor, Fibroblast","lastPublishedDoi":"10.21203/rs.3.rs-4521745/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4521745/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eGraves\u0026rsquo; Orbitopathy (GO), an autoimmune disorder linked to Graves\u0026rsquo; Disease (GD), manifests through inflammation in orbital tissues and extraocular muscles (EOMs), driven by key receptors like TSHR and IGF1R. It was observed that a certain individual with GD will develop clinically significant orbitopathy and reason behind this still unclear. This study aimed to elucidate this connection by: i) Assessing IGF1R expression and its correlation with TSHR on circulating fibrocytes. ii) Investigating fibrocyte conversion to fibroblasts upon serum treatment. iii) Analysing cytokine and chemokine expression in fibrocytes post-serum exposure within the Indian population.\u003c/p\u003e\u003ch2\u003eMethods and Results\u003c/h2\u003e \u003cp\u003eFlow cytometry analysis of IGF1R in peripheral blood from 30 GO, 30 GD, and 20 healthy controls (HC) revealed significantly elevated IGF1R\u003csup\u003e+\u003c/sup\u003e fibrocytes in GO (11%) versus GD (2.4%) and HC (0.1%). Immunocytochemistry of TSHR and IGF1R on cultured fibrocytes confirmed colocalization of TSHR and IGF1R on fibrocytes, notably higher in GO. Treating HC-derived fibrocytes with GO patient serum triggered fibroblast transformation, marked by increased fibrotic markers (CD90, alpha SMA). Moreover, sandwich ELISA of cytokines and chemokines like IL-6, IL-8, TNF-α, MCP-1, and HA demonstrated elevated levels of those cytokines and chemokines in GO serum-treated HC-fibrocytes.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese results highlight the potential pathogenicity of TSHR and IGF1R on fibrocytes in GO, suggesting their role in orbital tissue remodelling and inflammation. The observed receptor colocalization may drive GO pathogenesis, providing insights into targeted therapeutic strategies for this debilitating condition.\u003c/p\u003e","manuscriptTitle":"TSH receptor and IGF1 receptor expression in Circulating Fibrocytes in the pathogenesis of Graves’ Orbitopathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-20 17:22:14","doi":"10.21203/rs.3.rs-4521745/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c01f22d7-1a14-4ff5-a642-9b886afccc6b","owner":[],"postedDate":"June 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-20T17:22:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-20 17:22:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4521745","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4521745","identity":"rs-4521745","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}